WO2015196192A2 - Methods and compositions relating to dengue virus - Google Patents
Methods and compositions relating to dengue virus Download PDFInfo
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- WO2015196192A2 WO2015196192A2 PCT/US2015/036939 US2015036939W WO2015196192A2 WO 2015196192 A2 WO2015196192 A2 WO 2015196192A2 US 2015036939 W US2015036939 W US 2015036939W WO 2015196192 A2 WO2015196192 A2 WO 2015196192A2
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- seq
- dengue virus
- denv
- antigen binding
- antibody
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/72—Increased effector function due to an Fc-modification
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
- G01N2333/185—Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue
Definitions
- the present invention relates generally to methods and compositions for detection of dengue virus infection in a subject.
- Dengue is a mosquito-borne disease caused by a virus of the Flaviviridae family, genus Flavivirus.
- Four serotypes of dengue virus are known, DENV-1 , DENV-2, DENV-3 and DENV-4.
- Dengue virus infection is one of the most serious mosquito- borne diseases with regard to morbidity and mortality.
- Dengue virus infection has several clinically identifiable phases.
- An incubation phase begins after a bite by a mosquito harboring the dengue virus and lasts from three to fourteen days, typically four to seven days.
- viremia occurs and the dengue virus enters the bloodstream from the site of infection.
- an acute febrile phase occurs, lasting two to seven days, typically three to five days.
- the acute febrile phase is followed by the critical phase, also called the afebrile phase, lasting one to three days, typically about two days.
- the patient will either recover or, occasionally, progress to dengue hemorrhagic shock syndrome.
- An individual may be infected with the different serotypes at different times.
- a first infection is referred to as a primary infection
- a second, third or fourth incidence of infection with a second, third or fourth dengue virus serotype is referred to as a secondary infection.
- Secondary infection has been identified as a risk factor for progression of the disease to dengue hemorrhagic fever.
- Sensitivity of assays to detect dengue virus (DENV) is generally lower for persons with secondary DENV infections than persons with primary infection.
- a plausible explanation for these findings is not an absence of viral proteins, rather a significant proportion of DENV proteins is trapped in immunecomplexes produced by IgG anti-DENV NS l from previous infection(s).
- Heat treatment may not only dissociate the antigen-antibody complexes but may also denature the viral proteins. Since the serum concentration of DENV NS l in acute-dengue patients (primary and secondary DENV infection status) ranges from l Ong/ml to 50 ug/ml, sensitive assays to detect DENV in biological samples are lacking and required.
- Methods for detecting dengue virus infection in a cell or organism include: obtaining a biological sample of the cell or organism; providing one or more antibodies or antigen binding fragments characterized by specific binding to an epitope of dengue virus nonstructural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQY FQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSW SWG (SEQ ID NO:4); RPQPMEHKYSW SWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDL YSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof; and assaying dengue virus non
- the organism is a mosquito.
- the cell is in vitro.
- the organism is a human subject.
- Methods for detecting dengue virus infection in a human subject having or suspected of having a dengue virus infection include: obtaining a biological sample from the human subject; providing one or more antibodies or antigen binding fragments characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l ); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant
- the biological sample obtained from the human subject can be any of: whole blood, plasma, serum, urine, extracellular fluid, cytosolic fluid, and tissue.
- Methods for detecting dengue virus infection in a human subject having or suspected of having a dengue virus infection are provided according to aspects of the present invention which include: obtaining a biological sample from the human subject; providing one or more antibodies or antigen binding fragments characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NOT ); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7): TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof
- Methods for detecting dengue virus infection in a human subject having or suspected of having a dengue virus infection include: obtaining a biological sample selected from the group consisting of: whole blood, plasma, serum, urine, extracellular fluid, cytosolic fluid, and tissue; providing one or more antibodies or antigen binding fragments characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSW SWG (SEQ ID NO: I); VHTWTEQY FQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSW SWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMEL YSW TWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8);
- Assaying dengue virus non-structural protein 1 in the biological sample using the one or more antibodies or antigen binding fragments is by an ELISA or an antigen capture assay according to aspects of the present invention.
- the antigen capture assay is a lateral flow assay.
- Methods for detecting dengue virus infection in a cell or organism are provided according to aspects of the present invention which include: obtaining a biological sample of the cell or organism; providing a first antibody or antigen binding fragment characterized by specific binding to a first epitope of dengue virus nonstructural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof; providing a second antibody or antigen binding fragment characterized by specific binding to a second epitope of dengue virus nonstructural protein 1 selected from the group consisting of: VHTWTEQ
- Methods for detecting dengue virus infection in a cell or organism are provided according to aspects of the present invention which include: obtaining a biological sample of the cell or organism; providing a first antibody selected from the group consisting of: 4G3, 2G2, 1 A6, 3D5, 1 F4 and an antigen binding fragment of any thereof; providing a second antibody selected from the group consisting of: 2G1 and antigen binding fragment thereof; and assaying dengue virus non-structural protein 1 in the biological sample using the first antibody or antigen binding fragment and the second antibody or antigen binding fragment to detect dengue virus non-structural protein 1 in the biological sample, wherein detection of dengue virus non-structural protein 1 is indicative of dengue virus in ection in the cell or organism.
- Methods for detecting dengue virus infection in a cell or organism are provided according to aspects of the present invention which include: obtaining a biological sample of the cell or organism; providing a first antibody selected from the group consisting of: 4G3, 2G2, 1 A6, 3D5, 1 F4 and an antigen binding fragment of any thereof; providing a second antibody selected from the group consisting of: 2G 1 and antigen binding fragment thereof; heating the biological sample to denature any DENV NS1 which may be present in the sample, producing a heat-treated biological sample; and assaying dengue virus non-structural protein 1 in the heat-treated biological sample using the first antibody or antigen binding fragment and the second antibody or antigen binding fragment to detect dengue virus non-structural protein 1 in the biological sample, wherein detection of dengue virus non-structural protein 1 is indicative of dengue virus infection in the cell or organism.
- Methods for detecting dengue virus infection in a human subject are provided according to aspects of the present invention which include: obtaining a biological sample from the human subject; providing a first antibody selected from the group consisting of: mAb 4G3, mAb 2G2, mAb 1A6, mAb 3D5, mAb 1 F4 and an antigen binding fragment of any thereof; providing a second antibody selected from the group consisting of: mAb 2G 1 and antigen binding fragment thereof; and assaying dengue virus non-structural protein 1 in the biological sample using the first antibody or antigen binding fragment and the second antibody or antigen binding fragment to detect dengue virus non-structural protein 1 in the biological sample, wherein detection of dengue virus non-structural protein 1 is indicative of dengue virus infection in the human subject.
- Methods for detecting dengue virus infection in a human subject are provided according to aspects of the present invention which include: obtaining a biological sample from the human subject; providing a first antibody selected from the group consisting of: mAb 4G3, mAb 2G2, mAb 1A6, mAb 3D5, inAb 1F4 and an antigen binding fragment of any thereof; providing a second antibody selected from the group consisting of: mAb 2G1 and an antigen binding fragment thereof; heating the biological sample to denature any DENV NS 1 which may be present in the sample, producing a heat-treated biological sample; and assaying dengue virus non-structural protein 1 in the heat-treated biological sample using the first antibody or antigen binding fragment and the second antibody or antigen binding fragment to detect dengue virus non-structural protein 1 in the biological sample, wherein detection of dengue virus nonstructural protein 1 is indicative of dengue virus infection in the human subject.
- Dengue virus non-structural protein 1 immunoassay devices are provided according to aspects of the present invention which include a solid porous or non-porous support comprising a first antibody or antigen binding fragment characterized by specific binding to a first epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO: 8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof.
- YSWKSWG SEQ
- Dengue virus non-structural protein 1 immunoassay devices are provided according to aspects of the present invention which include a solid porous or non-porous support comprising a first antibody or antigen binding fragment characterized by specific binding to a first epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: I); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof.
- YSWKSWG SEQ ID
- Dengue virus non-structural protein 1 immunoassay devices are provided according to aspects of the present invention which include a solid porous or non-porous support comprising: a first antibody or antigen binding fragment characterized by specific binding to a first epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSW SWG (SEQ ID NO: l), VHTWTEQYKFQADSP (SEQ ID NO:2), VHTWTEQYKFQPESP (SEQ ID NO:3), GPQPMEHKYSWKSWG (SEQ ID NO:4), RPQPMEHKYSWKSWG (SEQ ID NO:5), RPQPTEL YS WKTWG (SEQ ID NO:6), TPQPMELKYSWKTWG (SEQ ID NO:7), TPPVSDLKYSWKTWG (SEQ ID NO:8), YSW SWGKAKIIGAD (SEQ ID NO:9), and a variant of any thereof; and a conjugate
- Dengue virus non-structural protein 1 immunoassay devices are provided according to aspects of the present invention which include a solid porous or non-porous support comprising: a first antibody or antigen binding fragment characterized by specific binding to a first epitope of dengue virus non-structural protein 1 selected from the group consisting of: mAb 4G3, mAb 2G2, mAb 1A6, mAb 3D5, mAb 1F4 and an antigen binding fragment of any thereof; and a conjugate pad comprising a detectably labeled second antibody mAb 2G1 or an antigen binding fragment thereof.
- a solid porous or non-porous support comprising: a first antibody or antigen binding fragment characterized by specific binding to a first epitope of dengue virus non-structural protein 1 selected from the group consisting of: mAb 4G3, mAb 2G2, mAb 1A6, mAb 3D5, mAb 1F4 and an antigen binding fragment of any thereof; and a conjugate pad comprising
- a dengue virus non-structural protein 1 immunoassay device further includes a conjugate pad including a detectably labeled dengue virus non-structural protein 1 .
- a dengue virus non-structural protein 1 immunoassay device according to aspects of the present invention further includes a wicking pad.
- a dengue virus non-structural protein 1 immunoassay device further includes a housing.
- Isolated antibodies or antigen binding fragments thereof characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof, are provided according to aspects of the present invention, wherein the antibody or antigen binding fragment has an attached detectable label.
- Isolated antibodies or antigen binding fragments thereof characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l ); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO: 5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof, are provided according to aspects of the present invention, wherein the antibody or antigen binding fragment is immobilized on a solid or semi-solid support.
- compositions including isolated monoclonal antibodies mAb 4G3, mAb 2G2, mAb 1A6, mAb 3D5, mAb 1 F4 and mAb 2G1 and antigen binding fragments thereof, are provided according to aspects of the present invention.
- compositions including detectably labeled isolated monoclonal antibodies mAb 4G3, mAb 2G2, mAb 1 A6, mAb 3D5, mAb 1 F4 and mAb 2G 1 and antigen binding fragments thereof, are provided according to aspects of the present invention.
- compositions including isolated monoclonal antibodies mAb 4G3, mAb 2G2, mAb 1 A6, mAb 3D5, mAb 1F4 and mAb 2G1 and antigen binding fragments thereof, immobilized on a solid or semi-solid support is detectably labeled are provided according to aspects of the present invention.
- Isolated immunogenic peptides having 25 or fewer amino acid residues and comprising an epitope selected from the group consisting of: YSW SWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHK YS WKS WG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO: 8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof, are provided according to aspects of the present invention.
- the isolated immunogenic peptide is immobilized on a solid or semi-solid support and/or is detectably labeled.
- Immunoassay kits for detecting dengue virus NS 1 in a biological sample include one or more antibodies or antigen binding fragments characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHK Y S WKS WG (SEQ ID NO:5): RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof.
- YSWKSWG SEQ ID NO: l
- Immunoassay kits for detecting dengue virus infection in a human subject having or suspected of having a dengue virus infection, comprising: one or more antibodies or antigen binding fragments characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); PQPTELK Y S WKT WG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSW SWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof.
- YSWKSWG SEQ ID NO: l
- Immunoassay kits for detecting dengue virus NSl in a biological sample include one or more antibodies or antigen binding fragments characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELK Y S W KT WG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof; and a second antibody or antigen binding fragment characterized by specific binding to VHTWTEQYKFQADSP (SEQ ID NO:2); and/or VHTWTEQYKFQPESP(
- Immunoassay kits for detecting dengue virus NS l in a biological sample include one or more antibodies selected from the group consisting of: mAb 1 A6, mAb 1 F4, mAb 3D5, mAb 2G2 and mAb 4G3, and an antigen binding fragment of any one or more thereof; and a second antibody mAb 2G1, or an antigen binding fragment thereof.
- an antibody or antigen binding fragment included in an immunoassay kit for detecting dengue virus NS l in a biological sample is immobilized on a solid or semi-solid support.
- the antibody or antigen binding fragment included in an immunoassay kit for detecting dengue virus NS l in a biological sample is detectably labeled.
- One or more controls is optionally included in an immunoassay kit for detecting dengue virus NS l in a biological sample.
- an NSl protein or peptide from a non-Dengue virus of family Flaviviridae, genus Flavivirus is included, such as an NSl protein or peptide from yellow fever virus and/or West Nile virus.
- Figure 1A is a schematic diagram showing a DENY NSl capture ELISA according to aspects of the present invention
- Figure I B is a graph showing results of a DENV NSl capture ELISA according to aspects of the present invention
- Figure 2 is a showing a comparison of absorbance values between serum samples with heat treatment (HT) and without heat treatment (NHT) prior to testing by the DENV NS 1 capture ELISA assay according to aspects of the present invention
- Figure 3A is a graph showing the effect of heat treatment on detection of artificial immune complexes of DENV1 NSl with 4G3 mAb at different dilutions by an NSl capture ELISA in the presence of normal human serum (NHS) according to aspects of the present invention
- Figure 3B is a graph showing the effect of heat treatment on detection of artificial immune complexes of DEN VI NS l with 4G3 mAb at different dilutions by an NS l capture ELISA without normal human serum (NHS) according to aspects of the present invention
- FIG. 4 is a table which shows affinity constants (KA), isotype and results of two different ELISAs in which the indirect ELISA (iELISA) recombinant proteins were used as coating antigens ( ⁇ g/ml) and in-cell ELISA using dengue virus infected Vero cells were used to show dimeric binding of antibodies.
- the absorbance was measured at 450nm; +, weak affinity; ++, good affinity; +++, strong affinity; and ++++, highest affinity to NSl antigen;
- Figure 5A is a schematic diagram of an immunochromatography device for detection of NS l according to aspects of the present invention.
- Figure 5B is a schematic diagram of an immunochromatography device for detection of NSl according to aspects of the present invention.
- Figure 6 is a table showing variants of peptide epitopes recognized by mAbs 2G 1 , 1A6, 1 F4, 3D5, 2G2 and 4G3;
- Figure 7 is a table showing variants of peptide epitopes recognized by mAbs 2G1 , 1A6, 1F4, 3D5, 2G2 and 4G3;
- Figure 8 is a table showing binding affinities of mAbs 2G1, 1 A6, 1F4, 3D5, 2G2 and 4G3;
- Figure 9 is a graph showing results of competition ELISA analysis for distinct epitope binding of monoclonal antibodies (mAbs); wherein paired mAb competition was performed between biotinylated mAbs (at a fixed optimal dose) and unlabeled mAbs (2-fold serially diluted starting from a saturated concentration); bars represent OD450 values as a measure of binding of biotinylated mAbs to NSl antigen coated on the microtiter plate across the various doses of unlabeled mAbs; dose- dependent binding inhibition was observed when unlabeled mAb 1 F4 was made to compete with biotinylated-mAb B-4G3 that had same binding epitope while no inhibition was observed when the competing mAbs, e.g. 1 F4 vs B-2G1 had distinct non- overlapping binding epitopes;
- Figure 10 is an image showing a Western blot of affinity-purified recombinant NS l of DENV probed with monoclonal antibodies 2G1 , 2G2, 4G3, 1F4 to determine reactivity to both dimeric and monomeric NS l isoforms, DENV serotype cross-reactivity and cross-reactivity with other flaviviruses.
- Reactivity of 2G1 and 2G2 to NS l from all four DENV serotypes is shown in the left column while reactivity of 4G3 and 1F4 is shown in the right column; the superscripts * and ⁇ in DENV NSl indicate that SDS-PGE was performed under heat-denaturing and non-denaturing conditions, respectively; the estimated sizes of monomeric and dimeric NSl forms are about 50 kDA and about 80 kDA.
- YFV yellow fever virus
- WNV West Nile virus
- rE recombinant envelop protein
- Figure 1 I B is a graph showing the reactivity pattern of affinity-purified mAb 2G1 (1 ug/ml) against three-fold serially diluted culture supernatants (native hexameric NSl) from DENV serotypes 1-4 infected Vero cells 5 days post-infection;
- Figure 1 1C is a graph showing the reactivity pattern of affinity purified mAb 1 F4 (serial two-fold dilutions from 250 to 0.012 ng) against the four affinity-purified hexameric recombinant NSl (rNSl) proteins coated on wells of microtiter plate at a concentration of 1 ug/mL;
- Figure 1 ID is a graph showing the reactivity pattern of affinity purified mAb 2G1 (serial two-fold dilutions from 250 to 0.012 ng) against the four affinity-purified hexameric recombinant NSl (rNS l) proteins coated on wells of microtiter plate at a concentration of 1 ug/mL;
- Figure 1 1 E is a graph showing the reactivity pattern of affinity purified mAb 2G2 (serial two-fold dilutions from 250 to 0.012 ng) against the four affinity-purified hexameric recombinant NS1 (rNSl) proteins coated on wells of microtiter plate at a concentration of 1 ug/mL;
- Figure 1 I F is a graph showing the reactivity pattern of affinity purified niAb 4G3 (serial two-fold dilutions from 250 to 0.012 ng) against the four affinity-purified hexameric recombinant NS1 (rNSl ) proteins coated on wells of microtiter plate at a concentration of 1 ug/mL; and
- Figure 12 is a graph showing the effect of heat treatment on detection of artificially immunecomplexed NS1 (1 ug/ml) with rabbit anti-NSl polyclonal antibodies (poly Abs) at different concentrations (2, 1 and 0 ug/ml) by NS1 capture ELISA as described herein; ICF, immunecomplex formation; ICD, immunecomplex dissociation; heat treatment (HT) and no heat treatment (NHT).
- poly Abs rabbit anti-NSl polyclonal antibodies
- RNA Interference Nuts and Bolts of RNAi Technology, DNA Press LLC, Eagleville, PA, 2003; Herdewijn, P. (Ed.), Oligonucleotide Synthesis: Methods and Applications, Methods in Molecular Biology, Humana Press, 2004; A. Nagy, M. Gertsenstein, K. Vintersten, R. Behringer, Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press; December 15, 2002, ISBN- 10: 0879695919; Kursad Turksen (Ed.), Embryonic stem cells: methods and protocols in Methods Mol Biol. 2002;185, Humana Press; Current Protocols in Stem Cell Biology, ISBN: 9780470151808.
- Assays are provided according to aspects of the present invention which detect DENV NS1 even below the lower serum concentration of 50 ug/ml found in acute-dengue patients.
- Assays are provided according to aspects of the present invention which incorporate a heating step effective to both dissociate immunecomplexes in a patient sample which interfere with detection of DENV NS 1, allow for detection of denatured DENV NS 1 and detect DENV NS 1 even below the lower serum concentration of 50 ug/ml found in acute-dengue patients.
- Methods for aiding in the detection and diagnosis of dengue virus infection in a human subject suspected of having a dengue infection include collecting a biological sample from the human subject; heating the biological sample for a time sufficient to irreversibly denature any antibodies present in the biological sample, thereby producing a heat-treated sample; and assaying the heat-treated sample to detect dengue virus non-structural protein 1 (NS 1) in the heat-treated biological sample.
- NS 1 dengue virus non-structural protein 1
- Methods for detecting dengue virus infection in a human subject having or suspected of having a dengue virus infection which include obtaining a biological sample from the human subject; heating the biological sample for a time sufficient to irreversibly denature any antibodies present in the biological sample, producing a heat-treated biological sample; providing one or more antibodies or antigen binding fragments characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and
- Detection of DENV NS 1 in the heat-treated sample of a human subject is indicative of dengue virus infection in the subject.
- Non-heat-treated biological sample is assayed for DENV NS1 according to aspects of the present invention.
- Non-heat-treated samples assayed for DENV NS1 according to aspects of the present invention are samples of whole or fractionated mosquitoes, or in vitro cells, containing or suspected of containing DENV NS 1.
- a biological sample which is heat treated and assayed for DENV NS1 according to processes of the invention may be any biological sample containing or suspected of containing DENV NSI including, whole blood, plasma, serum, urine, extracellular fluid, cytosolic fluid, and tissue.
- a biological sample which is heat treated and assayed for DENV NSI according to processes of the invention is human whole blood, plasma, serum, urine, extracellular fluid, cytosolic fluid, tissue, or a combination of any two or more thereof.
- the biological sample is human urine, whole blood, plasma, serum or a combination of any two or more thereof.
- Heat-treatment of a biological sample to produce a heat-treated biological sample includes heating the biological sample to a temperature in the range of 38°C - 100°C for one or more periods of time in the range of 10 seconds - 24 hours, sufficient to irreversibly denature the 3-D structure of DENV NS I protein in the biological sample while retaining the 2-D structure of epitopes YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TP
- a heat- treated biological sample assayed includes, or is suspected of including DENV NS I protein characterized by irreversibly denatured 3-D structure of DENV NSI protein while retaining the 2-D structure of the DENV NSI protein or at least epitopes YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof.
- heat-treatment of a biological sample to produce a heat-treated biological sample includes heating the biological sample to a temperature in the range of 55°C - 100°C for one or more periods of time in the range of 10 seconds - 8 hours, sufficient to irreversibly denature the 3-D structure of DENV NS1 protein in the biological sample while retaining the 2-D structure of epitopes YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YS WKS WGKAKIIGAD (SEQ ID NO: l); VHT
- heat-treatment of a biological sample to produce a heat-treated biological sample includes heating the biological sample to a temperature in the range of 60°C - 100°C for one or more periods of time in the range of 10 seconds - 4 hours, sufficient to irreversibly denature the 3-D structure of DENV NS1 protein in the biological sample while retaining the 2-D structure of epitopes YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YS WKS WGKAKIIGAD (SEQ ID NO: l); VHT
- heat-treatment of a biological sample to produce a heat-treated biological sample includes heating the biological sample to a temperature in the range of 70°C - 100°C for one or more periods of time in the range of 10 seconds - 4 hours, sufficient to irreversibly denature the 3-D structure of DENV NS1 protein in the biological sample while retaining the 2-D structure of epitopes YSWKSWG (SEQ ID NO: l ); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:
- heat-treatment of a biological sample to produce a heat-treated biological sample includes heating the biological sample to a temperature in the range of 98°C - 100°C for one or more periods of time in the range of >5 minutes - 1 hour, sufficient to irreversibly denature the 3-D structure of DENV NS1 protein in the biological sample while retaining the 2-D structure of epitopes YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:
- an assay method for a DENV NS1 selected from: DENV1 NS1 , DENV2 NS 1 , DENV3 NS1 and DENV4 NS1 is performed including heat-treatment of a biological sample to produce a heat-treated biological sample includes heating the biological sample to a temperature in the range of 70°C - 100°C for one or more periods of time in the range of >5 minutes - 1 hour, sufficient to irreversibly denature the 3-D structure of DENV NS1 protein in the biological sample while retaining the 2-D structure of epitopes YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6)
- an assay method for a DENV NS1 selected from: DENV1 NS1 , DENV2 NS1 , DE V3 NS1 and DENV4 NS1 is performed including heat-treatment of a biological sample to produce a heat-treated biological sample includes heating the biological sample to a temperature in the range of 98°C - 100°C for one or more periods of time in the range of >5 minutes - 1 hour, sufficient to irreversibly denature the 3-D structure of DENV NS1 protein in the biological sample while retaining the 2-D structure of epitopes YSWKSWG (SEQ ID NOT); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6);
- an assay method for a DENV NS 1 selected from: DENVl NS 1 , DENV2 NS 1 and DENV3 NS 1 but not DENV4 NS l is performed including heat-treatment of a biological sample to produce a heat-treated biological sample includes heating the biological sample to a temperature in the range of 70°C - 100°C for one or more periods of time in the range of >5 minutes - 1 hour, sufficient to irreversibly denature the 3-D structure of DENV NSl protein in the biological sample while retaining the 2-D structure of epitopes YSWKSWG (SEQ ID NO: l ); VHTWTEQ Y FQAD S P (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELK Y S WKT WG (
- an assay method for a DENV NSl selected from: DEN VI NSl , DENV2 NS l and DENV3 NSl but not DENV4 NSl is performed including heat-treatment of a biological sample to produce a heat-treated biological sample includes heating the biological sample to a temperature in the range of 98°C - 100°C for one or more periods of time in the range of >5 minutes - 1 hour, sufficient to irreversibly denature the 3-D structure of DENV NS l protein in the biological sample while retaining the 2-D structure of epitopes YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:
- a single mosquito, or multiple pooled mosquitoes are assayed for DENV
- NS l to detect dengue virus by immunoassay according to aspects of the present invention using an antibody or antigen binding fragment thereof characterized by specific binding to a DENV NS l epitope selected from: YSWKSWG (SEQ ID NO:l);
- VHTWTEQYKFQADSP (SEQ ID NO:2)
- VHTWTEQYKFQPESP (SEQ ID NO:3)
- NS1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYS WKS WG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof.
- YSWKSWG SEQ ID NO: l
- VHTWTEQYKFQADSP SEQ ID NO:2
- VHTWTEQYKFQPESP SEQ ID NO:3
- GPQPMEHKYS WKS WG SEQ ID NO:4
- Mosquito biological samples can be prepared by standard methodology, typically by pulverizing mosquitoes to a fine powder in a physiological buffer, followed by an optional centrifugation to remove non-soluble material.
- Mosquito biological samples can be prepared by digestion of mosquitoes with an exoskeleton specific enzyme, such as but not limited to chitinase, and dissolved in a physiological buffer, followed by an optional centrifugation to remove non-soluble material.
- In vitro cell samples such as but not limited to mosquito cell lines such as C6/36 and AP61, can be prepared by standard methodology, such as cell lysis in a physiological buffer, followed by an optional centrifugation to remove non-soluble material.
- Bio samples of in vitro cells or mosquitoes can be heat-treated or non- heat-treated for assays according to aspects of the present invention.
- a control or standard is included in a method for aiding in the detection and diagnosis of dengue virus infection in a human subject suspected of having a dengue infection according to aspects of the present invention.
- a control or standard is included in a method for aiding in the detection and diagnosis in a mosquito or in vitro cell suspected of containing dengue virus according to aspects of the present invention.
- control and standard are familiar to those of ordinary skill in the art and refer to any control or standard that can be used for comparison.
- the control or standard may be determined prior to assay for DENV NS 1 , in parallel, simultaneously, in a multiplex assay or other assay format.
- a control or standard can be a first NS1 level determined by assay in a first sample obtained from a patient.
- a control or standard can be a negative control and/or a positive control.
- Detection of DENV NS 1 in a heat-treated biological sample is accomplished by immunoassay using an antibody or antigen binding fragment thereof characterized by specific binding to a DENV NS1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSW TWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof.
- YSWKSWG SEQ ID NO: l
- VHTWTEQYKFQADSP SEQ ID NO:2
- epitope refers to a portion of an antigen that is bound by an antibody.
- Dengue Virus Non-Structural protein (DENV NS 1) is secreted and found in abundance in serum of patients infected with Dengue virus during the acute-phase of Dengue virus infection.
- Isoforms of NS1 are produced by the four Dengue virus (DENV) serotypes, DEN 1, DENV2, DENV3 and DENV4.
- DENV Dengue virus
- DENV NS 1 includes NS 1 of serotype DENV 1 identified herein as well as homologues and variants thereof.
- DENV NS 1 of serotype DENV2 identified herein is a serotype homologue of NS 1 of DENV 1 .
- Methods and compositions of the present invention are not limited to particular amino acid sequences identified herein and variants of a reference peptide or protein are encompassed.
- Variants of a peptide or protein described herein are characterized by conserved functional properties compared to the corresponding peptide or protein.
- DENV NS 1 epitopes encompasses variant peptides having at least 95%, 96%, 97%), 98%), 99%, or greater identity to the epitopes having the amino acid sequences identified herein.
- DENV NS1 epitopes encompasses variant peptides including the amino acid sequence YSWKSWG or YSWKTWG and having at least 95%, 96%, 97%, 98%, 99%, or greater identity to the epitopes having the amino acid sequences identified herein.
- Percent identity is determined by comparison of amino acid or nucleic acid sequences, including a reference amino acid or nucleic acid sequence and a putative homologue amino acid or nucleic acid sequence. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
- % identity number of identical overlapping positions/total number of positions X 100%.
- the two sequences compared are generally the same length or nearly the same length.
- the determination of percent identity between two sequences can also be accomplished using a mathematical algorithm.
- Algorithms used for determination of percent identity illustratively include the algorithms of S. Karlin and S. Altshul, PNAS, 90:5873-5877, 1993; T. Smith and M. Waterman, Adv. Appl. Math. 2:482-489, 1981 , S. Needleman and C. Wunsch, J. Mol. Biol., 48:443-453, 1970, W. Pearson and D.
- Gapped BLAST are utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402.
- PSI BLAST is used to perform an iterated search which detects distant relationships between molecules.
- the default parameters of the respective programs e.g., of XBLAST and NBLAST.
- a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4: 1 1 -17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 is used.
- the percent identity between two sequences is determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
- nucleic acid or amino acid mutations can be introduced without altering the functional properties of a given nucleic acid or protein, respectively. Mutations can be introduced using standard molecular biology techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis, to produce variants. For example, one or more amino acid substitutions, additions, or deletions can be made without altering the functional properties of a reference protein. Similarly, one or more nucleic acid substitutions, additions, or deletions can be made without altering the functional properties of a reference nucleic acid sequence.
- amino acid similarity When comparing a reference protein to a putative variant, amino acid similarity may be considered in addition to identity of amino acids at corresponding positions in an amino acid sequence. "Amino acid similarity" refers to amino acid identity and conservative amino acid substitutions in a putative variant compared to the corresponding amino acid positions in a reference protein.
- Conservative amino acid substitutions are art recognized substitutions of one amino acid for another amino acid having similar characteristics.
- each amino acid may be described as having one or more of the following characteristics: electropositive, electronegative, aliphatic, aromatic, polar, hydrophobic and hydrophilic.
- a conservative substitution is a substitution of one amino acid having a specified structural or functional characteristic for another amino acid having the same characteristic.
- Acidic amino acids include aspartate, glutamate; basic amino acids include histidine, lysine, arginine; aliphatic amino acids include isoleucine, leucine and valine; aromatic amino acids include phenylalanine, glycine, tyrosine and tryptophan; polar amino acids include aspartate, glutamate, histidine, lysine, asparagine, glutamine, arginine, serine, threonine and tyrosine; and hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine and tryptophan; and conservative substitutions include substitution among amino acids within each group. Amino acids may also be described in terms of relative size; alanine, cysteine, aspartate, glycine, asparagine, proline, threonine, serine, valine are all typically considered to be small.
- a variant can include synthetic amino acid analogs, amino acid derivatives and/or non-standard amino acids, illustratively including, without limitation, alpha- aminobutyric acid, citrulline, canavanine, cyanoalanine, diaminobutyric acid, diaminopimelic acid, dihydroxy-phenylalanine, djenkolic acid, homoarginine, hydroxyproline, norleucine, norvaline, 3-phosphoserine, homoserine, 5- hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, and ornithine.
- the phrase "specific binding" and grammatical equivalents as used herein in reference to binding of an anti-NSl antibody or antigen binding fragment to a specified NSl epitope refers to binding of the anti-NS l antibody or antigen binding fragment to the specified DENV NSl epitope without substantial binding to other non-NS l substances, such as yellow fever virus NSl and/or West Nile virus NS l , present in a sample to be assayed for presence of DENV NSl, wherein the antibody has an affinity constant (KA) greater than 1 X 10 6 for an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSW
- TPQPMELKYSWKTWG SEQ ID NO:7
- TPPVSDLKYSWKTWG SEQ ID NO:8
- YSWKSWGKAKIIGAD SEQ ID NO:9
- antibody and “antibodies” relate to monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, camelized antibodies, single domain antibodies, single-chain Fvs
- antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site.
- Immunoglobulin molecules are of any type (e.g., IgG,
- IgE IgE, IgM, IgD, IgA and IgY
- class e.g., IgGl , IgG2, IgG3, IgG4, IgAl and IgA2
- subclass e.g., IgGl , IgG2, IgG3, IgG4, IgAl and IgA2
- antigen-binding fragment defines a fragment of an antibody that immunospecifically binds to a target antigen.
- Antigen-binding fragment may be generated by any technique known to one of skill in the art.
- Fab and F(ab')2 antigen-binding fragments may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab antigen- binding fragments) or pepsin (to produce F(ab')2 antigen-binding fragments).
- Antigen- binding fragments are also produced by recombinant DNA technologies. Antigen- binding fragments encompassed by the present compositions and methods retain the ability to bind DENV NS1 as described herein.
- Antibodies, antigen-binding fragments and methods for their generation are known in the art, for instance, as described in Antibody Engineering, Kontermann, R. and Dubel, S. (Eds.), Springer, 2001 ; Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988; Ausubel, F. et al., (Eds.), Short Protocols in Molecular Biology, Wiley, 2002; J. D. Pound (Ed.) Immunochemical Protocols, Methods in Molecular Biology, Humana Press, 2nd ed., 1998; B.K.C. Lo (Ed.), Antibody Engineering: Methods and Protocols, Methods in Molecular Biology, Humana Press, 2003; and Kohler, G. and Milstein, C, Nature, 256:495-497 (1975).
- antibodies contain heavy chain polypeptides and light chain polypeptides. Antigen recognition is mediated by variable regions of the heavy and light chains.
- Complementarity determining region refers to polypeptide regions within the variable region of heavy and light chains. Three CDRs (CDR1, CDR2 and CDR3) are present in each light chain variable region (VL) and each heavy chain variable region (VH). The CDRs are generally responsible for specific antigen recognition properties of the antibody or antigen-binding fragment.
- Antibodies according to aspects of the present invention are those identified as mouse monoclonal antibodies (mAbs) 2G1 , 2G2, 4G3, 1A6, 3D5, and 1 F4.
- Figure 4 describes the biological characteristics of these mAbs including isotype, epitope type, DENV group cross-reactivity, non-reactivity to NS l of Yellow Fever virus (YFV) and NS l of West Nile Virus (WNV), and affinity constant.
- YFV Yellow Fever virus
- WNV West Nile Virus
- Hybridomas which produce these antibodies have been isolated including a hybridoma producing mAb 2G1 , a hybridoma producing mAb 2G2, a hybridoma producing mAb 4G3, a hybridoma producing mAb 1A6, a hybridoma producing mAb 3D5 and a hybridoma producing mAb 1F4.
- Mouse monoclonal antibody 4G3 is characterized by specific binding to the DENV NS 1 epitope GPQPMEHKYS WKSWG (SEQ ID NO:4).
- Mouse monoclonal antibody 2G2 is characterized by specific binding to the DENV NS l epitope GPQPMEHKYSWKSWG (SEQ ID NO:4).
- Mouse monoclonal antibody 1A6 is characterized by specific binding to the DENV NS l epitope GPQPMEHKYSWKSWG (SEQ ID NO:4).
- Mouse monoclonal antibody 3D5 is characterized by specific binding to the DENV NSl epitope GPQPMEHKYSWKSWG (SEQ ID NO:4).
- Mouse monoclonal antibody 1F4 is characterized by specific binding to the DENV NS l epitope GPQPMEHKYSWKSWG (SEQ ID NO:4).
- VHTWTEQYKFQADSP SEQ ID NO:2
- VHTWTEQYKFQPESP SEQ ID NO:3
- substitution at one or more amino acids in a CDR is possible while retaining antigen-binding function, particularly for those amino acid residues that do not contact the antigen. Such substitutions may be made at positions identified and known in the art or may be made empirically.
- Detection of DENV NS1 in a heat-treated or non-heat-treated biological sample may include detection of a detectable label directly or indirectly attached to NS1 in the heat-treated or non-heat- treated biological sample.
- Methods of detection of DENV NS1 in a heat-treated or non-heat-treated biological sample includes contacting a sample containing or suspected of containing DENV NS 1 under antigen/antibody binding conditions with a detectably labeled anti-DENV NS 1 antibody wherein the detectable label is directly or indirectly attached to the anti-DENV NS1 antibody. If present, the DENV NS 1 binds to the detectably labeled anti-DENV NS 1 antibody to form a complex such that specific detection of the complex is indicative of DENV NS1 in the sample.
- detectable label refers to any atom or moiety that can provide a detectable signal and which can be attached to a binding agent, such as a primary or secondary antibody or antigen binding fragment, or analyte, such as DENV NS1 .
- detectable labels include fluorescent moieties, chemiluminescent moieties, bioluminescent moieties, ligands, particles, latex particles, luminescent particles, magnetic particles, fluorescent particles, colloidal gold, enzymes, enzyme substrates, radioisotopes and chromophores.
- Such particles can be of any shape, size, composition, or physiochemical characteristics compatible with assay conditions.
- the particles can be microparticles having a diameter of less than one millimeter, for example, a size ranging from about 0.1 to about 1 ,000 micrometers in diameter, inclusive, such as about 3-25 microns in diameter, inclusive, or about 5-10 microns in diameter, inclusive.
- the particles can be nanoparticles having a diameter from about 1 nanometer (nm) to about 100,000 nm in diameter, inclusive, for example, a size ranging from about 10-1,000 nm, inclusive, or for example, a size ranging from 200-500 nm, inclusive.
- Any appropriate method including but not limited to spectroscopic, optical, photochemical, biochemical, enzymatic, electrical and/or immunochemical is used to detect a detectable label in an assay described herein.
- Immunoassays are well-known in the art and include, but are not limited to, enzyme-linked immunosorbent assay (EL1SA) such as but not limited to, antigen capture ELISA, indirect ELISA, fixed cell ELISA; immunochromatography; antigen capture; flow cytometry; immunoblot; immunoprecipitation; immunodiffusion; immunocytochemistry; radioimmunoassay; and combinations of any of these.
- EL1SA enzyme-linked immunosorbent assay
- Immunoassay may include contacting an anti-NS l antibody or antigen binding fragment thereof with a heat-treated or non-heat-treated biological sample, wherein the anti- DENV NS 1 antibody or antigen binding fragment thereof characterized by specific binding to an DENV NS1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2);
- VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEH YSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTEL YSW TWG (SEQ ID NO:6);
- TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, is immobilized on a solid support to detect binding of the anti-NSl antibody or antigen binding fragment thereof with DENV NS1 in the heat-treated or non-heat-treated biological sample.
- a dengue virus non-structural protein 1 immunoassay device which includes a solid or semi-solid porous or non-porous support which includes an antibody or antigen binding fragment characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHK Y S WKS WG (SEQ ID NO:5); RPQPTEL YSWKTWG (SEQ ID NO:6); TPQPMEL YSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or a variant of any thereof.
- YSWKSWG SEQ ID NO: l
- solid support includes both solid supports and semi-solid supports.
- solid porous support includes both solid porous supports and semi-solid porous supports.
- the solid support can be in any of various forms or shapes, including planar, such as but not limited to membranes, silicon chips, glass plates and dipsticks; or three dimensional such as but not limited to particles, microtiter plates, microtiter wells, pins and fibers.
- a solid support for attachment of an anti-NSl antibody or antigen binding fragment characterized by specific binding to an NS 1 epitope selected from:
- YSWKSWG SEQ ID NO: l
- VHTWTEQYKFQADSP SEQ ID NO:2
- VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4);
- TPQPMELKYSWKTWG SEQ ID NO:7
- TPPVSDLKYSWKTWG SEQ ID NO:8
- YSWKSWGKAKIIGAD SEQ ID NO:9
- a solid support to which an anti-NS l antibody or antigen binding fragment is attached is a particle.
- Particles to which an anti- DENV NS 1 antibody or antigen binding fragment characterized by specific binding to a DENV NS 1 epitope selected from: YSWKSWG (SEQ ID NOT); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSW SWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID N0:7); TPPVSDLKYSWKTWG (SEQ ID N0:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, is bound can be any particles to which an anti- DENV NS 1 antibody or antigen binding fragment can be attached and which are stable and insoluble under assay conditions.
- the particles can be of any shape, size, composition, or physiochemical characteristics compatible with assay conditions.
- the particle characteristics can be chosen so that the particle can be separated from fluid, e.g., on a filter with a particular pore size or by some other physical property, e.g., a magnetic property.
- the particles are optionally latex particles, luminescent particles, magnetic particles, or fluorescent particles.
- the particles can be of any shape, size, composition, or physiochemical characteristics compatible with assay conditions.
- the particles can be microparticles having a diameter of less than one millimeter, for example, a size ranging from about 0.1 to about 1 ,000 micrometers in diameter, inclusive, such as about 3-25 microns in diameter, inclusive, or about 5-10 microns in diameter, inclusive.
- the particles can be nanoparticles having a diameter from about 1 nanometer (nm) to about 100,000 nm in diameter, inclusive, for example, a size ranging from about 10-1,000 nm, inclusive, or for example, a size ranging from 200-500 nm, inclusive.
- Particles to which an antibody or antigen binding fragment characterized by specific binding to a DENV NS 1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEH YSWKSWG (SEQ ID NO:4); RPQPMEHKY S WKS WG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKY S WKT WG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, is bound are optionally encoded and distinguishable from other particles based on a characteristic such as color, reflective index and/or an imprinted or otherwise optically detectable pattern.
- the particles may be encoded using optical, chemical, physical, or electronic tags.
- Encoded particles can contain or be attached to, one or more fluorophores which are distinguishable, for instance, by excitation and/or emission wavelength, emission intensity, excited state lifetime or a combination of these or other optical characteristics.
- Optical bar codes can be used to encode particles.
- immunoassay includes assay of NS1 in a heat-treated biological sample by an ELISA technique.
- immunoassay includes assay of DENV NS 1 in a heat-treated or non-heat-treated biological sample by an immunochromatography technique.
- immunochromatography techniques include flowing a sample containing or suspected of containing an analyte of interest along a solid support including an anti-analyte antibody to detect specific binding of the antibody and analyte.
- detection of NS 1 in a heat- treated or non-heat-treated biological sample obtained from a subject includes antigen capture, such as by lateral flow assay.
- a heterogenous phase sample is assayed for DENV NS1 according to aspects of the present invention.
- a heterogenous phase sample is one which contains materials in two or more phases, generally solids or semi-solids and liquids.
- Whole blood is an example of a heterogenous phase sample that is assayed according to aspects of the present invention.
- Crude preparations of tissue or cells containing both solids/semi-solids and liquids, such as pulverized human tissue or pulverized or enzymatically digested mosquito tissue are heterogeneous phase samples assayed according to aspects of the present invention.
- a filtration material also called a separator, separation material or separation membrane, is optionally included in the flow path such that non- solubilized solids/semisolids, such as red blood cells, tissue and mosquito exoskeleton, are retained in the filtration material and the liquid component of the sample flows along the flow path, see for example U.S. 4,933,092.
- assay of DENV NS 1 in a heat- treated or non-heat-treated biological sample is performed by a lateral flow assay.
- a lateral flow assay includes flowing a heat-treated or non-heat-treated biological sample along a solid support including an anti-NS l antibody or antigen binding fragment characterized by specific binding to an NS 1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYS WKT WG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, to detect specific binding of the anti-NS l antibody or antigen binding fragment and DENV NS 1
- the heat-treated or non-heat-treated biological sample may be diluted or processed to purify DENV NS 1 prior to analysis.
- a lateral flow assay includes flowing a heat-treated or non-heat-treated biological sample along a solid support including an anti-NS l antibody or antigen binding fragment characterized by specific binding to an NS 1 epitope selected from: YSWKSWG (SEQ ID NOT); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, in the presence of a competitor to detect competition for binding of the anti- DENV NS1 antibody or antigen binding fragment,
- a lateral flow assay process for detecting DENV NS 1 includes providing: a conjugate pad where detectably labeled anti- NS 1 antibody or antigen binding fragment characterized by specific binding to an NS1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSW SWG (SEQ ID NO:5); RPQPTELK YS WKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYS WKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, or detectably labeled DENV NS 1 is diffusibly bound, the conjugate
- a non-diffusibly bound detection reagent is an anti- DENV NS 1 antibody or antigen binding fragment characterized by specific binding to a DENV NS1 epitope selected from: YSWKSWG (SEQ ID NO: l ); VHTWTEQYKFQADSP (SEQ ID NO:2);
- VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4);
- TPQPMELKYSWKTWG SEQ ID NO:7
- TPPVSDLKYS WKTWG SEQ ID NO:8
- YSWKSWGKAKIIGAD SEQ ID NO:9
- DENV NS1 epitope selected from: YSWKSWG (SEQ ID NOT );
- VHTWTEQYKFQADSP (SEQ ID NO:2)
- VHTWTEQYKFQPESP (SEQ ID NO:3)
- GPQPMEHKYSWKSWG SEQ ID NO:4; RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7);
- the detection reagent is non-competitive with the detectably labeled anti-DENV NS1 antibody or antigen binding fragment.
- a heat-treated or non-heat-treated biological sample is applied to the conjugate pad.
- the heat-treated or non-heat-treated biological sample may be diluted or processed to purify DENV NS 1 prior to application to the conjugate pad.
- the detectable label is detected in the test zone to detect DENV NS 1 in the sample and lower amounts of detected detectable label are indicative of greater amounts of DENV NS 1 in the sample.
- One or more standards may be used to associate an amount of detected detectable label with an amount of DENV NS1 in a sample.
- the conjugate pad is typically blocked to inhibit non-specific binding.
- a non-limiting example of a blocking solution is lOmM Borate, 3% BSA, 1%, PVP-40, 0.25% Triton x-100, pH 8.
- reaction or diluent buffer compatible with the sample, reagents and reaction can be used, including but not limited to phosphate buffered saline, sodium phosphate buffer, potassium phosphate buffer, Tris-HCl buffer, Tricine buffer and other buffers described herein.
- the conjugate pad is disposed adjacent to the solid porous support and the solid porous support is disposed adjacent to the wicking pad.
- Each component, the conjugate pad, the solid support and the wicking pad has a top surface in substantially the same plane as the top surface of each other component.
- the conjugate pad, the solid porous support and the wicking pad may be attached together so that they may be moved as one unit.
- the conjugate pad, the solid porous support and the wicking pad may all be attached to a structural support, such as a backing material for support and so that they may be moved as one unit.
- a lateral flow assay device including 1 ) a conjugate pad where detectably labeled anti-DENV NS 1 antibody or antigen binding fragment characterized by specific binding to an NS1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO: 5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, or detectably labeled NS 1 is diffusibly bound, 2) a solid porous support which allows for
- the term "diffusibly bound” refers to reversible attachment or adsorption of a material to the conjugate pad such that the material moves with the lateral flow when contacted with the heat-treated or non -heat-treated biological sample.
- non- diffusibly bound refers to attachment of a material to the solid support wherein a non- diffusibly bound material is immobilized and therefore does not move with the lateral flow when contacted with the heat-treated or non-heat-treated biological sample.
- test detection zone refers to a region of the solid porous support where the detection reagent is non-diffusibly bound.
- the test detection zone may have any of various shapes and sizes configured to allow for determination of binding of an analyte to the detection reagent.
- the test detection zone is a line of non- diffusibly bound detection reagent referred to as a "test line.”
- control zone refers to a region of the solid porous support where the control reagent is non-diffusibly bound.
- the control zone may have any of various shapes and sizes configured to allow for determination of binding of a control substance to the control reagent.
- control zone is a line of non-diffusibly bound control reagent, referred to as a "control line.”
- a control reagent allows a user to confirm that the immunoassay is working properly.
- a control reagent may be an antibody which specifically binds to the detectably labeled anti-DENV NS 1 antibody or antigen binding fragment characterized by specific binding to a DENV NS 1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof.
- a lateral flow assay device includes 1 ) detectably labeled anti-DENV NS1 antibody or antigen binding fragment characterized by specific binding to an NS 1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, diffusibly bound to the conjugate pad, 2) a solid porous support having a test detection zone including non-diffusibly bound anti-DEN
- a lateral flow assay device includes 1) a detectably labeled DENV NS 1 epitope diffusibly bound to the conjugate pad, 2) a solid porous support having a test detection zone including non-diffusibly bound anti-DENV NS1 antibody or antigen binding fragment characterized by specific binding to a DENV NS 1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO: l); VHTWTEQY
- the detectably labeled DENV NS 1 epitope diffusibly bound to the conjugate pad binds specifically to the anti-DENV NS1 antibody or antigen binding fragment characterized by specific binding to a DENV NS1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELK Y S WKT WG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, non-diffusibly bound to the solid porous support and therefore competes with DENV NS 1
- the conjugate pad is a material to which a detectably labeled anti-NSl antibody or antigen binding fragment characterized by specific binding to a DENV NS1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, may be diffusibly attached including, but not limited to, glass fiber, bound glass fiber, polyester, cellulose and cellulose derivatives include cellulose acetate and nitrocellulose, nylon,
- the solid porous support may be any solid or semi-solid adsorbent porous material suitable for chromatographic applications including, but not limited to, polyvinylidene fluoride, nylon, polyether sulfone, polyester, polypropylene, paper, silica, rayon, cellulose and cellulose derivatives include cellulose acetate and nitrocellulose, woven or non-woven natural or synthetic fibers and porous gels such as agarose, gelatin, dextran and silica gel.
- the solid porous support may be self-supporting, such as a membrane, or may be deposited on a structural support, such as an agarose thin layer deposited on a glass slide. According to aspects of the invention, the solid porous support is a nitrocellulose membrane.
- the wicking pad is an absorbent material that facilitates lateral flow by wicking fluid including, but not limited to, an absorbent synthetic or natural polymer, such as cellulose.
- a structural support to which the conjugate pad, solid porous support, and/or wicking pad are attached can be any material which provides support including, but not limited to, a backing card, glass, silica, ceramic and/or plastic membrane.
- An adhesive may be used to attach the conjugate pad, solid porous support, and/or wicking pad to the structural support.
- a housing may be included to at least partially enclose the conjugate pad, solid porous support, and wicking pad.
- the housing may be configured to include a well for application of the fluid biological sample to the conjugate pad.
- the housing optionally allows the user to directly visualize assay results.
- the housing may include a detection device, such as an optical scanner, for detection of assay results.
- the fluid biological sample flows by capillary action through the wicking pad to a control line and a test line that have anti-DENV NSl antibodies or antigen binding fragments characterized by specific binding to a DENV NSl epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPE8P (SEQ ID NO:3); GPQPMEH YSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTEL YSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSW TWG (SEQ ID NO:8); and YSWKSWG AKIIGAD (SEQ ID NO:9); disposed at a precise concentration determined through validation experiments.
- the control line is an internal quality control that ensures the sample has migrated appropriately
- Figure 5 A is a schematic illustration of a device, 10, for lateral flow assay of NSl according to aspects of the present invention.
- the conjugate pad 20, solid or semisolid porous support 30, and wicking pad 40 are attached and disposed adjacent to one another.
- the conjugate pad 20, solid or semi-solid porous support 30, and wicking pad 40 each have at least a top surface 75 substantially the same plane as each other top surface 75.
- the direction of lateral flow 50 is shown.
- An optional housing, 60 is shown which encloses the conjugate pad 20, solid or semi-solid porous support 30, and wicking pad 40.
- the housing optionally has one or more openings, 70, such as for application of a sample to be assayed for NSl or visualization of test and/or control results.
- the housing includes an opening 80 for insertion and removal of the conjugate pad 20, solid or semi-solid porous support 30, and wicking pad 40.
- a test zone 1 14 and a control zone 1 16 are shown.
- Figure 5B shows the conjugate pad 20, solid or semi-solid porous support 30, and wicking pad 40, test zone 1 14 and a control zone 1 16.
- a first detectably labeled binding agent capable of specific binding to DENV NS1 102 is diffusibly attached to the conjugate pad 20.
- a test sample is added to the conjugate pad which contains or is suspected of containing DENV NS 1 100.
- the DENV NS1 and first detectably labeled binding agent capable of specific binding to DENV NS 1 form a complex 104.
- the complex 104 is moved by lateral flow in the direction of the test zone 1 14 where it is bound to a second binding agent capable of specific binding to DENV NS1 which is non- competing with the first detectably labeled binding agent capable of specific binding to DENV NS1 , forming complex 106.
- Complex 106 is detected in the test zone by detection of the detectable label, thereby quantitating NS1 in the test sample.
- Excess detectably labeled binding agent capable of specific binding to NS1 102 moves by lateral flow to the control zone where it binds to a binding agent 1 2 capable of specific binding to the binding agent capable of specific binding to NS1 102, forming a complex 1 10.
- a filtration material also called a separator, separation material or separation membrane
- a separator is optionally included in the flow path such that non- solubilized solids/semisolids, such as red blood cells, tissue and mosquito exoskeleton, are retained in the filtration material and the liquid component of the sample flows along the flow path, see for example U.S. 4,933,092.
- a separator is optionally included in an immunoassay device, such as but not limited to a lateral flow assay device, according to aspects of the present invention.
- DENV NS1 contained in a heat-treated or non-heat-treated biological sample is optionally purified for assay according to a method of the present invention.
- the term "purified" in the context of a heat-treated or non-heat-treated biological sample refers to separation of DENV NS 1 in the biological sample from at least one other component present in the biological sample.
- DENV NSl is optionally substantially purified from the heat-treated or non-heat-treated biological sample to produce a substantially purified sample for use in an inventive assay.
- substantially purified refers to a desired material separated from other substances naturally present in a sample obtained from the subject so that the desired material makes up at least about 1 - 100% of the mass, by weight, such as about 1 %, 5%, 10%, 25%, 50% 75% or greater than about 75% of the mass, by weight, of the substantially purified sample.
- Sample purification is achieved by techniques illustratively including electrophoretic methods such as gel electrophoresis and 2-D gel electrophoresis; chromatography methods such as HPLC, ion exchange chromatography, affinity chromatography, size exclusion chromatography, thin layer and paper chromatography. It is appreciated that electrophoresis and chromatographic methods can also be used to separate a peptide or peptides from other components in a sample in the course of performing an assay, as in, for example separation of proteins in immunoblot assays.
- electrophoretic methods such as gel electrophoresis and 2-D gel electrophoresis
- chromatography methods such as HPLC, ion exchange chromatography, affinity chromatography, size exclusion chromatography, thin layer and paper chromatography. It is appreciated that electrophoresis and chromatographic methods can also be used to separate a peptide or peptides from other components in a sample in the course of performing an assay, as in, for example separation of proteins in immunoblot
- DENV NS l is isolated and concentrated by absorption of DENV NS l onto a solid substrate.
- DENV NS l is isolated and concentrated by binding to beads or other particles coupled with an anti-DENV NSl antibody or antigen binding fragment characterized by specific binding to a DENV NS l epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof.
- YSWKSWG SEQ ID NO: l
- VHTWTEQYKFQADSP SEQ ID NO:2
- DENV NS l is isolated and concentrated by binding to magnetic beads coupled with an anti-DENV NSl antibody or antigen binding fragment characterized by specific binding to a DENV NSl epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof.
- YSWKSWG SEQ ID NO: l
- VHTWTEQYKFQADSP SEQ ID NO:2
- Isolated immunogenic peptides having 25 or fewer amino acid residues which include an epitope selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO: 8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof are provided according to aspects of the present invention.
- Such peptides are useful as immunogens and as controls in immunoassays, for example.
- One or more amino acid residues extending from the N' and/or C termini of the specified peptides may be those found in DENV NS 1 proteins or may be different.
- one or more amino acid residues extending from the N' and/or C' termini of the specified peptides may be added to aid in isolation of the peptides.
- Isolated immunogenic DENV NS1 peptides consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof are provided according to aspects of the present invention.
- Such peptides are useful as immunogens and as controls in immunoassays, for example.
- immunoassay kits for detecting dengue virus infection in a human subject having or suspected of having a dengue virus infection which include one or more antibodies or antigen binding fragments characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof.
- YSWKSWG SEQ ID NO: l
- immunoassay kits for detecting dengue virus infection in a human subject having or suspected of having a dengue virus infection which include one or more antibodies or antigen binding fragments including a first antibody or antigen binding fragment characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSW SWG AKIIGAD (SEQ ID NO:9); or variant thereof and a second antibody or antigen binding fragment characterized by specific binding to VHTWTEQYKFQADSP (SEQ ID NO:2) and/or VHTWTEQY
- auxiliary components are optionally included in such kits, such as a control DENV NS 1 peptide or DENV NS1 protein, a control NS 1 peptide or NS 1 protein from a non-dengue virus such as yellow fever virus or West Nile virus, a buffer, diluent or a reconstituting agent.
- inventive compositions and methods are illustrated in the following examples. These examples are provided for illustrative purposes and are not considered limitations on the scope of inventive compositions and methods.
- DENV-1 Hawaii
- DENV-2 New Guinea- C
- DENV-3 H87
- DENV-4 H241
- Vero cells ATCC, Manassas, VA, USA were first propagated in M199 medium (Gibco/Life Technologies, Grand Island, NY, USA) supplemented with 5% fetal bovine serum (FBS) in T-25 culture flasks. Cells at 80-90% confluency were washed twice with M199 containing 2.5% FBS and cultured in the same medium for two additional days, and cells were then infected for 24 hours with each DENV serotype at a multiplicity of infection (MOI) of 0.1. Following incubation, culture medium was replaced with serum-free M199 medium and cells were cultured for another four days. Mock-infected cells were included as a control.
- M199 medium Gibco/Life Technologies, Grand Island, NY, USA
- FBS fetal bovine serum
- RNA was extracted from DENV-1 - and DENV-2- virus stocks using QIAmp Viral RNA Minikit (QIAGEN, Gaithersburg, MD, USA), and NS1 sequences were amplified with Superscript III One-Step RT-PCR reaction mix(Invitrogen) and primers (5'- CACCGACTCGGGATGTGTAATTAAC-3 ' (SEQ ID NO: 12) and 5'- CTATGCAGAGACC AATGACTTTACT-3 ' (SEQ ID NO: 13) for DENV1 ; 5'- CACCGATAGTGGTTGCGTTGTGAGC-3 ' (SEQ ID NO: 14) and 5'-
- rNSl protein was prepared from cell lysates and solubilized in 0.5% sodium dodecylsulfate (SDS) PBS buffer and purified by Precision Antibody (PA), Custom Antibody Generation Services (Columbia, MD, USA).
- SDS sodium dodecylsulfate
- Hybridoma cell lines secreting mAbs specific to NSl of DENV were developed by fusion of SP2/0 cells with splenocytes isolated from BALB/c mice immunized with two affinity-purified, detergent-generated monomeric rNSl proteins of DENV-1 and DENV-2 using established hybridoma technology detailed in Milstein et al., Cell Biology International Reports 3, 1-16,1979.
- Dengue-1 NSl (Nucleotide sequence Acc# X69395)
- microtiter plates were washed 3x with PBS + 0.05% Tween-20 (PBS-T) and treated with hybridoma supernatants diluted to 1 : 100, 1 :500, 1 : 1000 and 1 :2000 in a wash buffer and incubated for 1 h at 37°C. All hybridoma supernatant dilutions were tested in duplicate. After 5 washes, goat anti- mouse IgG horseradish peroxidase (HPR)-conjugated (KPL, Gaithersburg, MD) diluted to 1 : 1000 in blocking solution was added and incubated for 1 h at 37°C in humidified chamber.
- HPR horseradish peroxidase
- TMB 3,3',5,5'-Tetramethylbenzidine
- a fixed cell ELISA was used to assess in situ reactivity of mAbs against native cell surface NS1 of DENV-1 to DENV-4. Briefly, Vero cells maintained in Ml 99 medium (Sigma) supplemented with 5% FBS, 1 % Gentamicin (Gibco, Sparks, MD, US), 1% Penicillin-Streptomycin (Gibco), 3% sodium bicarbonate (Gibco) were seeded into 96-well culture plates at a density 2 ⁇ 10 5 cells/well and incubated at 37°C in 10% CO? for 2 days to obtain a desirable 90% confluent monolayer.
- HPR-conjugated goat anti-mouse IgG (KPL) diluted to 1 : 1000 in blocking buffer were added and incubated at 37°C for 1 hour in a humidified chamber.
- ABTS 2,2'-Azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt
- RT room temperature
- OD OD measured at 405 nm. All mAb dilutions were tested in duplicate.
- DENV-2 expressed in E. coli a total of 50 positive clones were identified, of which 37 were derived from mice immunized with rNS l of DENV-2 and the remaining 13 were from mice immunized with rNSl of DENV1. Three of the 50 positive clones were unreactive after successive subcloning, suggesting that these positive clones might have been lost due to an overgrowth of negative clones or genetic instability.
- Six hybridoma cell lines secreted mAbs (2G 1 , 1A6, 1F4, 3D5, 2G2 and 4G3) reactive to both detergent- denatured rNSl and the native NS1 antigen in DENV-infected Vero cells were selected for further characterization as shown in Figure 4. None of these 6 mAbs (1A6, 1F4, 2G2, 3D5, 4G3, 2G1 ) had reactivity to mock-infected fixed Vero cells.
- Hybridoma cells adapted to low IgG FBS medium at a density of 2 ⁇ 10 6 cells/ml were seeded into a 15 ml Bioreactor cell compartment, incubated for fourteen days at 37°C in a 5% C0 2 humidified atmosphere, the mixed cell suspension harvested, centrifuged at 1000 rpm for 5 min to separate the culture supernatant which was filtered with a 0.2 um filter and stored at -20°C until affinity-purified.
- Hybridoma culture supernatant was screened for mAb activity defined by reactivity to NS1 antigen in the iELISA.
- mAb purification [00207] mAbs from hybridoma culture supernatants were purified by using VivaPure Maxiprep Protein G Spin Columns (Satorius Stedim, Bohemia, NY, USA) according to manufacturer's instructions. The affinity-purified mAbs were dialyzed against PBS pH 7.2 fori 6 hours. The concentrations of dialyzed mAbs were determined byNanoDropR- 100 spectrophotometer at 280 nm (NanoDrop Technologies, Willington, DE, USA). The reactivity of purified mAbs to DENV NSl was then confirmed by iELISA. Aliquots of dialyzed mAbs in 15% sterilized glycerol were stored at -20°C until use.
- Affinity purified and dialyzed mAbs were biotinylated with a spacer arm biotin (NHS-PEG4-B iotin) using EZ-LinkTMNHS-PEG4-Biotinylation Kit (Pierce/Thermo Scientific, Rockford, IL, USA). These biotinylated mAbs were then dialyzed against PBS pH 7.2 for 32 hours and their reactivity to DENV rNSl antigen was determined using iELISA. Biotinylated mAbs in 15% of sterile glycerol were aliquoted and stored at -20°C until use.
- mAbs were isotyped using a mouse mAb isotyping kit (Pierce/Thermo Scientific, Rockford, IL, USA) according to manufacturer's instructions. Briefly, ELISA strip wells pre-coated with anti-mouse heavy chain capture antibody (anti-IgGl, IgG2a, IgGb,IgG3, IgA and IgM) or anti-mouse light chain (kappa or lambda)were used for the assay.
- anti-mouse heavy chain capture antibody anti-IgGl, IgG2a, IgGb,IgG3, IgA and IgM
- anti-mouse light chain kappa or lambda
- Diluted hybridoma culture supernatants (1 :50) pre-mixed with goat anti-mouse IgG + IgA + IgM-HRP conjugated were added and incubated at RT for two hours and results measured at 450 nm.
- mAbs, 2G1, 1A6, 1F4, 3D5, 2G2 and 4G3, bind to monomeric, dimeric and hexameric isoforms of DENV NSl
- the proteins were transferred to nitrocellulose membranes (Invitrogen, 0.22 um), blocked overnight at RT in 5% NFDM in PBS with 0.05% Tween20 (NFDM-PBST) and the membranes incubated with one of the following primary anti-NSl mAbs at a 1 :50,000 dilution: 2G1 ( 1.67 mg/ml), 2G2 (1.26 mg/ml), 4G3 (1.26 mg/ml), 1 F4 (1.26 mg/ml). Following incubation with primary antibody a secondary anti-mouse peroxidase detector antibody diluted atl : 10,000 (KPL, Gaithersburg, MD) was used. The substrate Super-Signal West Pico solution (Pierce/Thermo Scientific) was used to detect proteins.
- Figure 4 summarizes the reactivity of the six mAbs against DENV NSl isoforms. mAbs were reactive to monomeric rNS l protein in the iELISA, a finding further corroborated by Western blot analysis in which all mAbs reacted to the monomeric rNS l band under both non-reducing and heat-denatured or non-denatured conditions. mAbs were reactive to dimeric NSl as observed in the fixed cell ELISA, see Figures 4 and 1 1 A; the dimeric isoform being an exclusively cell membrane-associated protein and is predominant in the fixed cell ELISA.
- reactivity to all 4 DENV serotypes was determined using culture supernatants harvested from DENV-1-4 infected Vero cells in the iELISA. Briefly, 96-well microtiter plates were coated overnight at 4°Cwith 100 ul/well of DENV1-4 infected Vero cell culture supernatants diluted twofold starting from 1 : 160 to 1 : 10, 240 in PBS (pH7.4).
- microtiter plates were blocked with PBS containing 5% NFDM and 0.5% Tween-20 for 30 min at RT, washed 3 with wash buffer (PBS + 0.05% Tween-20), and purified mAbs diluted to 1 :200 in PBS was added and incubated for 1 h at 37°C. After 5 washes, goat anti-mouse IgG HPR-conjugated was diluted to 1 : 1000 in blocking solution and incubated for 1 h at 37°C in a humidified chamber.
- the reactivity profile pattern of four serially diluted affinity- purified mAbs and hybridoma culture supernatants(di luted to 1 :500 for two) was tested against four affinity-purified hexameric rNS l proteins of DENV-1 (Nauru/Western Pacific/1974),DENV-2 (Thailand/16681/84) DE V-3 (Sri Lanka D3/H/IMTSSA- SRI/2000/1266) and DENV-4 (Dominica/814669/1981) expressed in a mammalian cell line (NativeAntigen, Oxfordshire, UK).
- 2G2 had relatively strong reactivity (defined by higher OD value) to DENV-1 while the remaining five mAbs appeared serotype cross-reactive, albeit with varied reactivity pattern.
- 2G1 and 4G3 had strong reactivity to DENV-1 , -2 and -3.
- the other three mAbs (1 F6, 1F4 and 3D5) had relatively weak reactivity to all four DENV serotypes except to DENV-1 , see Figure 1 1 A. All six MAbs displayed relatively weaker reactivity to DENV-4 compared to the other three DENV serotypes.
- Reactivity profiles of the mAbs in the iELISA were determined using affinity-purified hexameric rNS l for DENV- 1 to DENV-4. 2G1 and 4G3 appeared to be serotype cross- reactive in the fixed cell ELISA but their reactivity profiles in the iELISA did not perfectly match these results, see Figures 1 1A - F. Furthermore, the three serotype cross- reactive mAbs 1A6, 1 F4, 3D5 that showed weaker reactivity to DENV-1-4 by fixed cell ELISA were more reactive to immunoaffinity-purified hexameric rNS l of DENV-1-4 in the iELISA, see Figure 1 IB.
- NSl mAbs did not cross-react with YFV and WNV NSl
- Binding of mAbs to synthetic peptides was determined by ELISA in streptavidin pre-coated 96-well plates (Pierce, Rockford, IL, USA).Briefly, 96-well plates were pre-coated with streptavidin, blocked with 2% bovine serum albumin (BSA), washed 3 with Tris-buffered saline (TBS) (25 mM Tris, 150 mM NaCl; pH 7.2), 0.1 % BSA, and 0.05% Tween-20, and 100 ul/well of 10 ug/ml biotinylated synthetic peptide diluted in a wash buffer was added and incubated on a shaker at 200 rotations per minute (rpm) for 2 hours at RT.
- BSA bovine serum albumin
- the binding region/epitope for mAb 2G1 was found to be in 25-VHTWTEQY FQADSP-39 (SEQ ID NO: 2) of DEN VI NS l whereas the binding region/epitope for mAbs 1A6, 1F4, 3D5, 2G2 and 4G3 was found to be in 105- GPQPMEHKYSWKSWG-1 19 (SEQ ID NO: 4) of the DENV1 NSl .
- mAbs 1A6, 1 F4, 3D5, 2G2 and 4G3 showed weak reactivity to peptide, 1 13- YSWKSWGKAKIIGAD-127 (SEQ ID NO:9) of DENV NS l .
- This weak reactivity reflects the 7 overlapping amino acid residues YSWKSWG (SEQ ID NO: l) encompassed by both peptide 105-GPQPMEHKYSWKSWG-l 19 (SEQ ID NO: 4) and peptide 1 13-YS WKSWGKAKIIGAD- 127 (SEQ ID NO:9).
- Figure 4 is a table which shows the biological characteristics of the mAbs such as isotype, epitope type, DENV group cross-reactivity, non-reactivity to NSl of YFV and WNV, and affinity constant. Reactivity of mAbs with dimeric DENV NS l was determined by in vitro ELISA in Vero cells seeded in microplate, infected with each DENV serotype at multiplicity of infection 0.1 and fixed on day five post infection.
- YFV Yellow Fever virus
- WNV West Nile Virus
- FIGS. 6 and 7 are tables showing variants of peptide epitopes recognized by mAbs 2G1 , 1A6, 1F4, 3D5, 2G2 and 4G3.
- Figure 8 is a table showing binding affinities of mAbs 2G1, 1 A6, 1 F4, 3D5, 2G2 and 4G3.
- Figure 9 is a graph showing results of competition ELISA analysis for distinct epitope binding of monoclonal antibodies (mAbs). Paired mAbs competition was performed between biotinylated mAbs (at a fixed optimal dose) and unlabeled mAbs (2- fold serially diluted starting from a saturated concentration). Bars represent OD450 values as a measure of binding of biotinylated mAbs to NS1 antigen coated on the microtiter plate across the various doses of unlabeled mAbs.
- NS1 Antigen capture ELISA and testing clinical samples before and after heat denaturation.
- NS 1 Antigen capture ELISA was identical to a standard antigen capture ELISA except for a brief serum heat denaturation step prior adding the specimen to the microtiter plate. Briefly, microtiter plate wells were coated with capture mAb 4G3 (100 ul/well) at concentration 10 ug/ml overnight at 4°C. Following overnight incubation, excess unbound NSl capture mAb was removed and the plate was blocked for 45 min with PBS or TBS containing 2% BSA plus 0.05% Tween-20 at RT.
- Serum samples were diluted 1 :2 in PBST and heat-denatured in 1 .5-ml Eppendorf tubes at 70°C for 10 min. Following blocking step, the microtiter plate was washed 3x and the serum samples - both heat denatured and non-denatured in parallel wells and incubated for 1 h at 37°C. The microtiter plate was then washed 3x, and biotinylated 2G1 mAb (1 :800) was added and incubated for 1 hour at 37°C. The microtiter plate was then washed 3x and streptavidin-conjugated HRP (100 ul/well) was added.
- NSl antigen capture ELISA for detection of immunecomplexed NSl antigen
- artificial NSl antigen-antibody complexes in vitro were generated by mixing purified rNS l protein at a constant concentration of 1 ug/ml while varying the rabbit anti-NS l polyclonal antibodies (Canada Immunoassay Development, Ontario, Canada). After an overnight incubation at 4°C, immunecomplexed specimens were analyzed by NS l capture ELISA with and without heat denaturation step as described above.
- the negative control included rNSl antigen (1 ug/ml) diluted in PBS and incubated overnight at 4°C.
- mAb 2G1 of LD2 epitope was paired with each one of the 5 different NS l mAbs of LXl epitope and evaluated using the sandwich ELISA format. Based on this analysis, 4G3 and 2G1 provided the most robust DENV NSl detection in the described NS l antigen capture ELISA. The optimal concentration of each mAb was then determined by titration ELISA and the limit of detection (LOD) of the assay by serotype was determined from a standard curve using known concentration of serially diluted rNS l protein from of the four DENV serotypes.
- LOD limit of detection
- LOD 2 ng/ml
- the NSl capture ELISA was used to detect NS l antigen in clinical specimens using a total of 15 DENV NS l -positive serum samples. In addition, 20 dengue negative serum samples were used to validate the assay.
- FIG. 1A is a schematic diagram showing DENV NS l capture ELISA format used.
- mAbs 4G3 and 2G1 have high affinity cross-reactivity to all 4 DENV serotypes and distinct non-overlapping binding sites to DENV NS 1.
- Any of mAbs 1 A6, 1F4, 3D5 and 2G2 can be used in the same or similar capture ELISA assay with mAb 2G1.
- Microtiter plates are coated with mAb 4G3.
- the capture monoclonal antibody (mAb), 4G3 is diluted using a coating buffer, bicarbonate buffer pH 9.6, to make the final concentration of ⁇ 0 ⁇ g/mL ⁇ .
- the microtiter plate is then coated by adding ⁇ of the ⁇ g/mL mAb 4G3 per well and the microtiter plate is then incubated overnight (16-18 hours) at 4°C. The excess unbound capture antibody is then removed and the microtiter plate is tapped against dry paper towels.
- the wells of the microtiter plate are blocked to inhibit non-specific binding by adding 300 ⁇ 1 Tris-buffered saline (25mM Tris, 150mM NaCl; pH 7.2) containing 2% bovine serum albumin (BSA) plus 0.1% Tween-20 per well and incubating at room temp for 45 minutes.
- the microtiter plate is then washed three times with washing buffer (Tris-buffered saline plus 0.05% Tween-20).
- Serum samples are diluted and heat treated and transferred into the microtiter plate.
- serum samples are diluted at dilution 1 :2 in Tris-buffered saline plus 0.05% Tween-20.
- duplicate assay prepare 250ul in 1.5ml Eppendorf tubes. Eppendorf tubes containing serum samples are place in a heat block at 70°C for 10 minutes, producing heat-treated serum samples. The heat-treated serum samples are centrifuged at 12,000 rpm for 3 to 5 minutes. An aliquot of ⁇ of each heat treated serum sample is added in duplicate wells of the microtiter plate. Diluent alone is used as a blank control. The microtiter plate is incubated at 20-37°C for 60-90 minutes and then washed three times with washing buffer (Tris-buffered saline plus 0.05% Tween-20).
- Detectably labeled secondary (detection) antibody mAb 2G1
- mAb 2G1 Detectably labeled secondary (detection) antibody, mAb 2G1 .
- An aliquot of ⁇ of the detection monoclonal antibody, biotinylated mAb 2G1 , at dilution 1 :400-1 :2000 in a (Tris-buffered saline plus 0.05% Tween-20) is added to each well and the microtiter plate is incubated at 20-37°C for 60-90 minutes. Diluent alone is added to wells designated as blank.
- microtiter plate is washed five times with washing buffer (Tris-buffered saline plus 0.05% Tween-20).
- washing buffer Tris-buffered saline plus 0.05% Tween-20.
- An aliquot of ⁇ ⁇ Streptavidin- tagged Horseradish Peroxidase (manufactured by Thermo Scientific) at dilution 1 :2000- 1 :4000 in a blocking buffer (Tris-buffered saline containing 2% BSA and 0.1 % Tween- 20) is added to each well and the microtiter plate is then incubated at 37°C for 1 hour. Diluent alone is added to wells designated as blank.
- the microtiter plate is then washed ten times with washing buffer (Tris-buffered saline plus 0.05% Tween-20).
- washing buffer Tris-buffered saline plus 0.05% Tween-20.
- An aliquot of ⁇ 3,3',5,5'-Tetramethylbenzidine (TMB) liquid substrate (manufactured by KPL, USA) is then added to each well and the microtiter plate is incubated for 10 min at room temp in a dark box.
- the reaction is stopped by adding ⁇ TMB Stop Solution (manufactured by KPL, USA) to each well. Signal from wells of the microtiter plate is then read at 450nm within three minutes.
- FIG. 1 B is a graph showing results of DENV NS1 capture ELISA and showing the lower detection limit (LDL) of the assay for each DENV serotype.
- purified rNS l proteins expressed in mammalian cell line were used.
- Serum samples from dengue positive and negative individuals were tested by performing ELISA using the two mAbs, 4G3 and 2G1 with and without prior heat treatment of the serum samples.
- Figure 3A is a graph showing the effect of heat treatment on detection of artificially immune complex DENV NS 1 with 4G3 mAb at different dilutions by an NS 1 capture ELISA according to aspects of the present invention, wherein immune complex formation was performed overnight at 4°C in PBST containing 10% Normal Human Serum (NHS).
- Figure 3B is a graph showing the effect of heat treatment on detection of artificially immune complex DENV NS1 with 4G3 mAb at different dilutions by the DENV NS 1 capture EL1SA, wherein immune complex formation was performed overnight at 4°C in in PBST without NHS.
- HT-ICD refers to heat-mediated immune complex dissociation
- HT-EDTA-ICD refers to Heat-mediated immune complex dissociation in presence of EDTA (anti-coagulant)
- NHT-ICD refers to no heat- mediated immune complex dissociation.
- Immune complex formation at 1 :100 and 1 :500 of 4G3 dilution, respectively, are shown along with results of incubating NS 1 solution in the absence of immune complex. The concentration of NS 1 either as free or immune complex solution was ⁇ g/ml.
- compositions and methods described herein are presently representative of preferred embodiments, exemplary, and not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art. Such changes and other uses can be made without departing from the scope of the invention as set forth in the claims.
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Abstract
Assays for detection of dengue virus non-structural protein 1 (DENV NS 1) in heat-treated or non-heat-treated biological samples of subjects, mosquitoes or in vitro cells infected or suspected of being infected with dengue virus are provided. Antibodies and antigen binding fragments thereof characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof; are provided.
Description
METHODS AND COMPOSITIONS RELATING TO DENGUE VIRUS
REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 62/014,982, filed June 20, 2014, the entire content of which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to methods and compositions for detection of dengue virus infection in a subject. BACKGROUND OF THE INVENTION
[0003] Dengue is a mosquito-borne disease caused by a virus of the Flaviviridae family, genus Flavivirus. Four serotypes of dengue virus are known, DENV-1 , DENV-2, DENV-3 and DENV-4. Dengue virus infection is one of the most serious mosquito- borne diseases with regard to morbidity and mortality.
[0004] Dengue virus infection has several clinically identifiable phases. An incubation phase begins after a bite by a mosquito harboring the dengue virus and lasts from three to fourteen days, typically four to seven days. Following the incubation phase, viremia occurs and the dengue virus enters the bloodstream from the site of infection. During viremia, an acute febrile phase occurs, lasting two to seven days, typically three to five days. The acute febrile phase is followed by the critical phase, also called the afebrile phase, lasting one to three days, typically about two days. The patient will either recover or, occasionally, progress to dengue hemorrhagic
shock syndrome.
[0005] An individual may be infected with the different serotypes at different times. A first infection is referred to as a primary infection, while a second, third or fourth incidence of infection with a second, third or fourth dengue virus serotype is referred to as a secondary infection. Secondary infection has been identified as a risk factor for progression of the disease to dengue hemorrhagic fever.
[0006] Sensitivity of assays to detect dengue virus (DENV) is generally lower for persons with secondary DENV infections than persons with primary infection. A plausible explanation for these findings is not an absence of viral proteins, rather a
significant proportion of DENV proteins is trapped in immunecomplexes produced by IgG anti-DENV NS l from previous infection(s). Heat treatment may not only dissociate the antigen-antibody complexes but may also denature the viral proteins. Since the serum concentration of DENV NS l in acute-dengue patients (primary and secondary DENV infection status) ranges from l Ong/ml to 50 ug/ml, sensitive assays to detect DENV in biological samples are lacking and required.
[0007] Appropriate treatment of dengue virus-infected individuals requires proper identification of the disease. There is a continuing need for methods and compositions for aiding in detecting dengue virus infection in a human subject having or suspected of having a dengue virus infection.
SUMMARY OF THE INVENTION
[0008] Methods for detecting dengue virus infection in a cell or organism are provided according to aspects of the present invention which include: obtaining a biological sample of the cell or organism; providing one or more antibodies or antigen binding fragments characterized by specific binding to an epitope of dengue virus nonstructural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQY FQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSW SWG (SEQ ID NO:4); RPQPMEHKYSW SWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDL YSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof; and assaying dengue virus non-structural protein 1 in the biological sample using the one or more antibodies or antigen binding fragments, wherein detection of dengue virus non-structural protein 1 is indicative of dengue virus infection in the cell or organism.
[0009] Optionally, the organism is a mosquito. In a further option, the cell is in vitro.
[0010] In still a further option, the organism is a human subject.
[0011] Methods for detecting dengue virus infection in a human subject having or suspected of having a dengue virus infection are provided according to aspects of the present invention which include: obtaining a biological sample from the human subject; providing one or more antibodies or antigen binding fragments characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group
consisting of: YSWKSWG (SEQ ID NO: l ); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof; heating the biological sample to denature any DENV NS1 which may be present in the sample, producing a heat-treated biological sample; and assaying dengue virus non-structural protein 1 in the heat-treated biological sample using the one or more antibodies or antigen binding fragments, wherein detection of dengue virus non-structural protein 1 is indicative of dengue virus infection in the cell or organism.
[0012] The biological sample obtained from the human subject can be any of: whole blood, plasma, serum, urine, extracellular fluid, cytosolic fluid, and tissue.
[0013] Methods for detecting dengue virus infection in a human subject having or suspected of having a dengue virus infection are provided according to aspects of the present invention which include: obtaining a biological sample from the human subject; providing one or more antibodies or antigen binding fragments characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NOT ); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7): TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof; heating the biological sample at a temperature in the range of 70°C - 100°C for a time in the range of more than 5 minutes - one hour to denature any DENV NS 1 which may be present in the sample, producing a heat-treated biological sample; and assaying dengue virus nonstructural protein 1 in the biological sample using the one or more antibodies or antigen binding fragments, wherein detection of dengue virus non-structural protein 1 is indicative of dengue virus infection in the cell or organism.
[0014] Methods for detecting dengue virus infection in a human subject having or suspected of having a dengue virus infection are provided according to aspects of the present invention which include: obtaining a biological sample selected from the group consisting of: whole blood, plasma, serum, urine, extracellular fluid, cytosolic fluid, and tissue; providing one or more antibodies or antigen binding fragments characterized by
specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSW SWG (SEQ ID NO: I); VHTWTEQY FQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSW SWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMEL YSW TWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof; heating the biological sample at a temperature in the range of 98°C - 100°C for a time in the range of more than 5 minutes - one hour to denature any DENV NS1 which may be present in the sample, producing a heat-treated biological sample; and assaying dengue virus non-structural protein 1 in the biological sample using the one or more antibodies or antigen binding fragments, wherein detection of dengue virus non-structural protein 1 is indicative of dengue virus infection in the cell or organism.
[0015] Assaying dengue virus non-structural protein 1 in the biological sample using the one or more antibodies or antigen binding fragments is by an ELISA or an antigen capture assay according to aspects of the present invention. Optionally, the antigen capture assay is a lateral flow assay.
[0016] Methods for detecting dengue virus infection in a cell or organism are provided according to aspects of the present invention which include: obtaining a biological sample of the cell or organism; providing a first antibody or antigen binding fragment characterized by specific binding to a first epitope of dengue virus nonstructural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof; providing a second antibody or antigen binding fragment characterized by specific binding to a second epitope of dengue virus nonstructural protein 1 selected from the group consisting of: VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); and a variant of any thereof; and assaying dengue virus non-structural protein 1 in the biological sample using the first antibody or antigen binding fragment and the second antibody or antigen binding fragment to detect dengue virus non-structural protein 1 in the biological sample, wherein detection of dengue virus non-structural protein 1 is indicative of dengue virus infection in the cell or organism.
[0017] Methods for detecting dengue virus infection in a cell or organism are provided according to aspects of the present invention which include: obtaining a biological sample of the cell or organism; providing a first antibody selected from the group consisting of: 4G3, 2G2, 1 A6, 3D5, 1 F4 and an antigen binding fragment of any thereof; providing a second antibody selected from the group consisting of: 2G1 and antigen binding fragment thereof; and assaying dengue virus non-structural protein 1 in the biological sample using the first antibody or antigen binding fragment and the second antibody or antigen binding fragment to detect dengue virus non-structural protein 1 in the biological sample, wherein detection of dengue virus non-structural protein 1 is indicative of dengue virus in ection in the cell or organism.
[0018] Methods for detecting dengue virus infection in a cell or organism are provided according to aspects of the present invention which include: obtaining a biological sample of the cell or organism; providing a first antibody selected from the group consisting of: 4G3, 2G2, 1 A6, 3D5, 1 F4 and an antigen binding fragment of any thereof; providing a second antibody selected from the group consisting of: 2G 1 and antigen binding fragment thereof; heating the biological sample to denature any DENV NS1 which may be present in the sample, producing a heat-treated biological sample; and assaying dengue virus non-structural protein 1 in the heat-treated biological sample using the first antibody or antigen binding fragment and the second antibody or antigen binding fragment to detect dengue virus non-structural protein 1 in the biological sample, wherein detection of dengue virus non-structural protein 1 is indicative of dengue virus infection in the cell or organism.
[0019] Methods for detecting dengue virus infection in a human subject are provided according to aspects of the present invention which include: obtaining a biological sample from the human subject; providing a first antibody selected from the group consisting of: mAb 4G3, mAb 2G2, mAb 1A6, mAb 3D5, mAb 1 F4 and an antigen binding fragment of any thereof; providing a second antibody selected from the group consisting of: mAb 2G 1 and antigen binding fragment thereof; and assaying dengue virus non-structural protein 1 in the biological sample using the first antibody or antigen binding fragment and the second antibody or antigen binding fragment to detect dengue virus non-structural protein 1 in the biological sample, wherein detection of dengue virus non-structural protein 1 is indicative of dengue virus infection in the human subject.
[0020] Methods for detecting dengue virus infection in a human subject are provided according to aspects of the present invention which include: obtaining a biological sample from the human subject; providing a first antibody selected from the group consisting of: mAb 4G3, mAb 2G2, mAb 1A6, mAb 3D5, inAb 1F4 and an antigen binding fragment of any thereof; providing a second antibody selected from the group consisting of: mAb 2G1 and an antigen binding fragment thereof; heating the biological sample to denature any DENV NS 1 which may be present in the sample, producing a heat-treated biological sample; and assaying dengue virus non-structural protein 1 in the heat-treated biological sample using the first antibody or antigen binding fragment and the second antibody or antigen binding fragment to detect dengue virus non-structural protein 1 in the biological sample, wherein detection of dengue virus nonstructural protein 1 is indicative of dengue virus infection in the human subject.
[0021] Dengue virus non-structural protein 1 immunoassay devices are provided according to aspects of the present invention which include a solid porous or non-porous support comprising a first antibody or antigen binding fragment characterized by specific binding to a first epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO: 8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof.
[0022] Dengue virus non-structural protein 1 immunoassay devices are provided according to aspects of the present invention which include a solid porous or non-porous support comprising a first antibody or antigen binding fragment characterized by specific binding to a first epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: I); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof.
[0023] Dengue virus non-structural protein 1 immunoassay devices are provided according to aspects of the present invention which include a solid porous or non-porous support comprising: a first antibody or antigen binding fragment characterized by
specific binding to a first epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSW SWG (SEQ ID NO: l), VHTWTEQYKFQADSP (SEQ ID NO:2), VHTWTEQYKFQPESP (SEQ ID NO:3), GPQPMEHKYSWKSWG (SEQ ID NO:4), RPQPMEHKYSWKSWG (SEQ ID NO:5), RPQPTEL YS WKTWG (SEQ ID NO:6), TPQPMELKYSWKTWG (SEQ ID NO:7), TPPVSDLKYSWKTWG (SEQ ID NO:8), YSW SWGKAKIIGAD (SEQ ID NO:9), and a variant of any thereof; and a conjugate pad comprising a detectably labeled second antibody or antigen binding fragment characterized by specific binding to a second epitope of dengue virus nonstructural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l ); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof, wherein the second antibody or antigen binding fragment thereof specifically binds to a different DENV NS 1 epitope than the first antibody or antigen binding fragment thereof and wherein the second antibody or antigen binding fragment does not interfere with specific binding of the first antibody or antigen binding fragment thereof to DENV NS 1 .
[0024] Dengue virus non-structural protein 1 immunoassay devices are provided according to aspects of the present invention which include a solid porous or non-porous support comprising: a first antibody or antigen binding fragment characterized by specific binding to a first epitope of dengue virus non-structural protein 1 selected from the group consisting of: mAb 4G3, mAb 2G2, mAb 1A6, mAb 3D5, mAb 1F4 and an antigen binding fragment of any thereof; and a conjugate pad comprising a detectably labeled second antibody mAb 2G1 or an antigen binding fragment thereof.
[0025] Optionally, a dengue virus non-structural protein 1 immunoassay device according to aspects of the present invention further includes a conjugate pad including a detectably labeled dengue virus non-structural protein 1 .
[0026] Optionally, a dengue virus non-structural protein 1 immunoassay device according to aspects of the present invention further includes a wicking pad.
[0027] Optionally, a dengue virus non-structural protein 1 immunoassay device according to aspects of the present invention further includes a housing.
[0028] Isolated antibodies and antigen binding fragments thereof characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l ); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof, are provided according to aspects of the present invention. Optionally, the isolated antibody or antigen binding fragment thereof is an isolated monoclonal antibody, an antigen binding fragment of a monoclonal antibody, a polyclonal antibody or an antigen binding fragment of a polyclonal antibody.
[0029] Isolated antibodies or antigen binding fragments thereof characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof, are provided according to aspects of the present invention, wherein the antibody or antigen binding fragment has an attached detectable label.
[0030] Isolated antibodies or antigen binding fragments thereof characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l ); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO: 5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof, are provided according to aspects of the present invention, wherein the antibody or antigen binding fragment is immobilized on a solid or semi-solid support. Optionally, the antibody or antigen binding fragment immobilized on a solid or semi-solid support is detectably labeled.
[0031] Compositions including isolated monoclonal antibodies mAb 4G3, mAb 2G2, mAb 1A6, mAb 3D5, mAb 1 F4 and mAb 2G1 and antigen binding fragments thereof, are provided according to aspects of the present invention.
[0032] Compositions including detectably labeled isolated monoclonal antibodies mAb 4G3, mAb 2G2, mAb 1 A6, mAb 3D5, mAb 1 F4 and mAb 2G 1 and antigen binding fragments thereof, are provided according to aspects of the present invention.
[0033] Compositions including isolated monoclonal antibodies mAb 4G3, mAb 2G2, mAb 1 A6, mAb 3D5, mAb 1F4 and mAb 2G1 and antigen binding fragments thereof, immobilized on a solid or semi-solid support is detectably labeled are provided according to aspects of the present invention.
[0034] Isolated immunogenic peptides having 25 or fewer amino acid residues and comprising an epitope selected from the group consisting of: YSW SWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHK YS WKS WG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO: 8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof, are provided according to aspects of the present invention. Optionally, the isolated immunogenic peptide is immobilized on a solid or semi-solid support and/or is detectably labeled.
[0035] Immunoassay kits for detecting dengue virus NS 1 in a biological sample are provided according to aspects of the present invention which include one or more antibodies or antigen binding fragments characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHK Y S WKS WG (SEQ ID NO:5): RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof.
[0036] Immunoassay kits for detecting dengue virus infection in a human subject having or suspected of having a dengue virus infection, comprising: one or more antibodies or antigen binding fragments characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2);
VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); PQPTELK Y S WKT WG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSW SWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof.
[0037] Immunoassay kits for detecting dengue virus NSl in a biological sample are provided according to aspects of the present invention which include one or more antibodies or antigen binding fragments characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELK Y S W KT WG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof; and a second antibody or antigen binding fragment characterized by specific binding to VHTWTEQYKFQADSP (SEQ ID NO:2); and/or VHTWTEQYKFQPESP(SEQ ID NO:3); or a variant of either thereof.
[0038] Immunoassay kits for detecting dengue virus NS l in a biological sample are provided according to aspects of the present invention which include one or more antibodies selected from the group consisting of: mAb 1 A6, mAb 1 F4, mAb 3D5, mAb 2G2 and mAb 4G3, and an antigen binding fragment of any one or more thereof; and a second antibody mAb 2G1, or an antigen binding fragment thereof.
[0039] Optionally, an antibody or antigen binding fragment included in an immunoassay kit for detecting dengue virus NS l in a biological sample is immobilized on a solid or semi-solid support. In a further option, the antibody or antigen binding fragment included in an immunoassay kit for detecting dengue virus NS l in a biological sample is detectably labeled. One or more controls is optionally included in an immunoassay kit for detecting dengue virus NS l in a biological sample. According to aspects of immunoassay kits of the present invention, an NSl protein or peptide from a non-Dengue virus of family Flaviviridae, genus Flavivirus is included, such as an NSl protein or peptide from yellow fever virus and/or West Nile virus.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] Figure 1A is a schematic diagram showing a DENY NSl capture ELISA according to aspects of the present invention;
[0041] Figure I B is a graph showing results of a DENV NSl capture ELISA according to aspects of the present invention;
[0042] Figure 2 is a showing a comparison of absorbance values between serum samples with heat treatment (HT) and without heat treatment (NHT) prior to testing by the DENV NS 1 capture ELISA assay according to aspects of the present invention;
[0043] Figure 3A is a graph showing the effect of heat treatment on detection of artificial immune complexes of DENV1 NSl with 4G3 mAb at different dilutions by an NSl capture ELISA in the presence of normal human serum (NHS) according to aspects of the present invention;
[0044] Figure 3B is a graph showing the effect of heat treatment on detection of artificial immune complexes of DEN VI NS l with 4G3 mAb at different dilutions by an NS l capture ELISA without normal human serum (NHS) according to aspects of the present invention;
[0045] Figure 4 is a table which shows affinity constants (KA), isotype and results of two different ELISAs in which the indirect ELISA (iELISA) recombinant proteins were used as coating antigens (^g/ml) and in-cell ELISA using dengue virus infected Vero cells were used to show dimeric binding of antibodies. The absorbance was measured at 450nm; +, weak affinity; ++, good affinity; +++, strong affinity; and ++++, highest affinity to NSl antigen;
[0046] Figure 5A is a schematic diagram of an immunochromatography device for detection of NS l according to aspects of the present invention;
[0047] Figure 5B is a schematic diagram of an immunochromatography device for detection of NSl according to aspects of the present invention;
[0048] Figure 6 is a table showing variants of peptide epitopes recognized by mAbs 2G 1 , 1A6, 1 F4, 3D5, 2G2 and 4G3;
[0049] Figure 7 is a table showing variants of peptide epitopes recognized by mAbs 2G1 , 1A6, 1F4, 3D5, 2G2 and 4G3;
[0050] Figure 8 is a table showing binding affinities of mAbs 2G1, 1 A6, 1F4, 3D5, 2G2 and 4G3;
[0051] Figure 9 is a graph showing results of competition ELISA analysis for distinct epitope binding of monoclonal antibodies (mAbs); wherein paired mAb competition was performed between biotinylated mAbs (at a fixed optimal dose) and unlabeled mAbs (2-fold serially diluted starting from a saturated concentration); bars
represent OD450 values as a measure of binding of biotinylated mAbs to NSl antigen coated on the microtiter plate across the various doses of unlabeled mAbs; dose- dependent binding inhibition was observed when unlabeled mAb 1 F4 was made to compete with biotinylated-mAb B-4G3 that had same binding epitope while no inhibition was observed when the competing mAbs, e.g. 1 F4 vs B-2G1 had distinct non- overlapping binding epitopes;
[0052] Figure 10 is an image showing a Western blot of affinity-purified recombinant NS l of DENV probed with monoclonal antibodies 2G1 , 2G2, 4G3, 1F4 to determine reactivity to both dimeric and monomeric NS l isoforms, DENV serotype cross-reactivity and cross-reactivity with other flaviviruses. Reactivity of 2G1 and 2G2 to NS l from all four DENV serotypes is shown in the left column while reactivity of 4G3 and 1F4 is shown in the right column; the superscripts * and φ in DENV NSl indicate that SDS-PGE was performed under heat-denaturing and non-denaturing conditions, respectively; the estimated sizes of monomeric and dimeric NSl forms are about 50 kDA and about 80 kDA. YFV, yellow fever virus; WNV, West Nile virus, rE, recombinant envelop protein;
[0053] Figure 1 1 A is a graph showing the reactivity pattern of mAbs, or hybridoma culture supernatants at dilution of 1 :500, against native dimeric NSl derived from DENVl- infected Vero cells (fixed cell ELISA) at MOI = 0.1 ;
[0054] Figure 1 I B is a graph showing the reactivity pattern of affinity-purified mAb 2G1 (1 ug/ml) against three-fold serially diluted culture supernatants (native hexameric NSl) from DENV serotypes 1-4 infected Vero cells 5 days post-infection;
[0055] Figure 1 1C is a graph showing the reactivity pattern of affinity purified mAb 1 F4 (serial two-fold dilutions from 250 to 0.012 ng) against the four affinity-purified hexameric recombinant NSl (rNSl) proteins coated on wells of microtiter plate at a concentration of 1 ug/mL;
[0056] Figure 1 ID is a graph showing the reactivity pattern of affinity purified mAb 2G1 (serial two-fold dilutions from 250 to 0.012 ng) against the four affinity-purified hexameric recombinant NSl (rNS l) proteins coated on wells of microtiter plate at a concentration of 1 ug/mL;
[0057] Figure 1 1 E is a graph showing the reactivity pattern of affinity purified mAb 2G2 (serial two-fold dilutions from 250 to 0.012 ng) against the four affinity-purified
hexameric recombinant NS1 (rNSl) proteins coated on wells of microtiter plate at a concentration of 1 ug/mL;
[0058] Figure 1 I F is a graph showing the reactivity pattern of affinity purified niAb 4G3 (serial two-fold dilutions from 250 to 0.012 ng) against the four affinity-purified hexameric recombinant NS1 (rNSl ) proteins coated on wells of microtiter plate at a concentration of 1 ug/mL; and
[0059] Figure 12 is a graph showing the effect of heat treatment on detection of artificially immunecomplexed NS1 (1 ug/ml) with rabbit anti-NSl polyclonal antibodies (poly Abs) at different concentrations (2, 1 and 0 ug/ml) by NS1 capture ELISA as described herein; ICF, immunecomplex formation; ICD, immunecomplex dissociation; heat treatment (HT) and no heat treatment (NHT).
DETAILED DESCRIPTION OF THE INVENTION
[0060] Scientific and technical terms used herein are intended to have the meanings commonly understood by those of ordinary skill in the art. Such terms are found defined and used in context in various standard references illustratively including J. Sambrook and D.W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001 ; F.M. Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002; B. Alberts et al., Molecular Biology of the Cell, 4th Ed., Garland, 2002; D.L. Nelson and M.M. Cox, Lehninger Principles of Biochemistry, 4th Ed., W.H. Freeman & Company, 2004; Engelke, D.R., RNA Interference (RNAi): Nuts and Bolts of RNAi Technology, DNA Press LLC, Eagleville, PA, 2003; Herdewijn, P. (Ed.), Oligonucleotide Synthesis: Methods and Applications, Methods in Molecular Biology, Humana Press, 2004; A. Nagy, M. Gertsenstein, K. Vintersten, R. Behringer, Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press; December 15, 2002, ISBN- 10: 0879695919; Kursad Turksen (Ed.), Embryonic stem cells: methods and protocols in Methods Mol Biol. 2002;185, Humana Press; Current Protocols in Stem Cell Biology, ISBN: 9780470151808.
[0061] The singular terms "a," "an," and "the" are not intended to be limiting and include plural referents unless explicitly stated otherwise or the context clearly indicates otherwise.
[0062] Assays
[0063] Assays are provided according to aspects of the present invention which detect DENV NS1 even below the lower serum concentration of 50 ug/ml found in acute-dengue patients.
[0064] Assays are provided according to aspects of the present invention which incorporate a heating step effective to both dissociate immunecomplexes in a patient sample which interfere with detection of DENV NS 1, allow for detection of denatured DENV NS 1 and detect DENV NS 1 even below the lower serum concentration of 50 ug/ml found in acute-dengue patients.
[0065] Methods for aiding in the detection and diagnosis of dengue virus infection in a human subject suspected of having a dengue infection are provided which include collecting a biological sample from the human subject; heating the biological sample for a time sufficient to irreversibly denature any antibodies present in the biological sample, thereby producing a heat-treated sample; and assaying the heat-treated sample to detect dengue virus non-structural protein 1 (NS 1) in the heat-treated biological sample.
[0066] Methods for detecting dengue virus infection in a human subject having or suspected of having a dengue virus infection which include obtaining a biological sample from the human subject; heating the biological sample for a time sufficient to irreversibly denature any antibodies present in the biological sample, producing a heat-treated biological sample; providing one or more antibodies or antigen binding fragments characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof; and assaying dengue virus non-structural protein 1 in the heat- treated biological sample using the one or more antibodies or antigen binding fragments, wherein detection of dengue virus non-structural protein 1 in the heat-treated sample is indicative dengue virus infection in the human subject.
[0067] Detection of DENV NS 1 in the heat-treated sample of a human subject is indicative of dengue virus infection in the subject.
[0068] Optionally, a non-heat-treated biological sample is assayed for DENV NS1 according to aspects of the present invention. Non-heat-treated samples assayed for
DENV NS1 according to aspects of the present invention are samples of whole or fractionated mosquitoes, or in vitro cells, containing or suspected of containing DENV NS 1.
[0069] A biological sample which is heat treated and assayed for DENV NS1 according to processes of the invention may be any biological sample containing or suspected of containing DENV NSI including, whole blood, plasma, serum, urine, extracellular fluid, cytosolic fluid, and tissue. According to aspects of the present invention, a biological sample which is heat treated and assayed for DENV NSI according to processes of the invention is human whole blood, plasma, serum, urine, extracellular fluid, cytosolic fluid, tissue, or a combination of any two or more thereof. According to aspects of the present invention, the biological sample is human urine, whole blood, plasma, serum or a combination of any two or more thereof.
[0070] The terms "subject" and "patient" are used interchangeably herein and refer to a human individual. It is appreciated that aspects of the present invention are applicable to non-human mammalian or avian subjects for dengue virus. Heat-treatment of a biological sample to produce a heat-treated biological sample includes heating the biological sample to a temperature in the range of 38°C - 100°C for one or more periods of time in the range of 10 seconds - 24 hours, sufficient to irreversibly denature the 3-D structure of DENV NS I protein in the biological sample while retaining the 2-D structure of epitopes YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELK Y S WKT WG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof. Thus, a heat- treated biological sample assayed according to aspects of the present invention includes, or is suspected of including DENV NS I protein characterized by irreversibly denatured 3-D structure of DENV NSI protein while retaining the 2-D structure of the DENV NSI protein or at least epitopes YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof.
[0071] According to further aspects, heat-treatment of a biological sample to produce a heat-treated biological sample includes heating the biological sample to a temperature in the range of 55°C - 100°C for one or more periods of time in the range of 10 seconds - 8 hours, sufficient to irreversibly denature the 3-D structure of DENV NS1 protein in the biological sample while retaining the 2-D structure of epitopes YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YS WKS WGKAKIIGAD (SEQ ID NO:9); or variant thereof.
[0072] According to further aspects, heat-treatment of a biological sample to produce a heat-treated biological sample includes heating the biological sample to a temperature in the range of 60°C - 100°C for one or more periods of time in the range of 10 seconds - 4 hours, sufficient to irreversibly denature the 3-D structure of DENV NS1 protein in the biological sample while retaining the 2-D structure of epitopes YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YS WKS WGKAKIIGAD (SEQ ID NO:9); or variant thereof.
[0073] According to further aspects, heat-treatment of a biological sample to produce a heat-treated biological sample includes heating the biological sample to a temperature in the range of 70°C - 100°C for one or more periods of time in the range of 10 seconds - 4 hours, sufficient to irreversibly denature the 3-D structure of DENV NS1 protein in the biological sample while retaining the 2-D structure of epitopes YSWKSWG (SEQ ID NO: l ); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof.
[0074] According to further aspects, heat-treatment of a biological sample to produce a heat-treated biological sample includes heating the biological sample to a temperature in the range of 98°C - 100°C for one or more periods of time in the range of
>5 minutes - 1 hour, sufficient to irreversibly denature the 3-D structure of DENV NS1 protein in the biological sample while retaining the 2-D structure of epitopes YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof.
[0075] According to aspects of the present invention, an assay method for a DENV NS1 selected from: DENV1 NS1 , DENV2 NS 1 , DENV3 NS1 and DENV4 NS1 is performed including heat-treatment of a biological sample to produce a heat-treated biological sample includes heating the biological sample to a temperature in the range of 70°C - 100°C for one or more periods of time in the range of >5 minutes - 1 hour, sufficient to irreversibly denature the 3-D structure of DENV NS1 protein in the biological sample while retaining the 2-D structure of epitopes YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO: 9); or variant thereof.
[0076] According to aspects of the present invention, an assay method for a DENV NS1 selected from: DENV1 NS1 , DENV2 NS1 , DE V3 NS1 and DENV4 NS1 is performed including heat-treatment of a biological sample to produce a heat-treated biological sample includes heating the biological sample to a temperature in the range of 98°C - 100°C for one or more periods of time in the range of >5 minutes - 1 hour, sufficient to irreversibly denature the 3-D structure of DENV NS1 protein in the biological sample while retaining the 2-D structure of epitopes YSWKSWG (SEQ ID NOT); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof.
[0077] According to aspects of the present invention, an assay method for a DENV NS 1 selected from: DENVl NS 1 , DENV2 NS 1 and DENV3 NS 1 but not DENV4 NS l
is performed including heat-treatment of a biological sample to produce a heat-treated biological sample includes heating the biological sample to a temperature in the range of 70°C - 100°C for one or more periods of time in the range of >5 minutes - 1 hour, sufficient to irreversibly denature the 3-D structure of DENV NSl protein in the biological sample while retaining the 2-D structure of epitopes YSWKSWG (SEQ ID NO: l ); VHTWTEQ Y FQAD S P (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELK Y S WKT WG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO: 8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof.
[0078] According to aspects of the present invention, an assay method for a DENV NSl selected from: DEN VI NSl , DENV2 NS l and DENV3 NSl but not DENV4 NSl is performed including heat-treatment of a biological sample to produce a heat-treated biological sample includes heating the biological sample to a temperature in the range of 98°C - 100°C for one or more periods of time in the range of >5 minutes - 1 hour, sufficient to irreversibly denature the 3-D structure of DENV NS l protein in the biological sample while retaining the 2-D structure of epitopes YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO: 8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof.
[0079] A single mosquito, or multiple pooled mosquitoes are assayed for DENV
NS l to detect dengue virus by immunoassay according to aspects of the present invention using an antibody or antigen binding fragment thereof characterized by specific binding to a DENV NS l epitope selected from: YSWKSWG (SEQ ID NO:l);
VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3);
GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5);
RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID
NO:9); or variant thereof.
[0080] In vitro cells are assayed for DENV NS l to detect dengue virus by immunoassay according to aspects of the present invention using an antibody or antigen
binding fragment thereof characterized by specific binding to an NS1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYS WKS WG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof.
[0081] Mosquito biological samples can be prepared by standard methodology, typically by pulverizing mosquitoes to a fine powder in a physiological buffer, followed by an optional centrifugation to remove non-soluble material. Mosquito biological samples can be prepared by digestion of mosquitoes with an exoskeleton specific enzyme, such as but not limited to chitinase, and dissolved in a physiological buffer, followed by an optional centrifugation to remove non-soluble material.
[0082] In vitro cell samples, such as but not limited to mosquito cell lines such as C6/36 and AP61, can be prepared by standard methodology, such as cell lysis in a physiological buffer, followed by an optional centrifugation to remove non-soluble material.
[0083] Biological samples of in vitro cells or mosquitoes can be heat-treated or non- heat-treated for assays according to aspects of the present invention.
[0084] Optionally, a control or standard is included in a method for aiding in the detection and diagnosis of dengue virus infection in a human subject suspected of having a dengue infection according to aspects of the present invention.
[0085] Optionally, a control or standard is included in a method for aiding in the detection and diagnosis in a mosquito or in vitro cell suspected of containing dengue virus according to aspects of the present invention.
[0086] The terms "control" and "standard" are familiar to those of ordinary skill in the art and refer to any control or standard that can be used for comparison. The control or standard may be determined prior to assay for DENV NS 1 , in parallel, simultaneously, in a multiplex assay or other assay format. A control or standard can be a first NS1 level determined by assay in a first sample obtained from a patient. A control or standard can be a negative control and/or a positive control.
[0087] Detection of DENV NS 1 in a heat-treated biological sample according to aspects of the present invention is accomplished by immunoassay using an antibody or antigen binding fragment thereof characterized by specific binding to a DENV NS1
epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSW TWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof.
[0088] The term "epitope" refers to a portion of an antigen that is bound by an antibody.
[0089] Dengue Virus Non-Structural protein (DENV NS 1) is secreted and found in abundance in serum of patients infected with Dengue virus during the acute-phase of Dengue virus infection.
[0090] Isoforms of NS1 are produced by the four Dengue virus (DENV) serotypes, DEN 1, DENV2, DENV3 and DENV4.
[0091] DENV NS 1 includes NS 1 of serotype DENV 1 identified herein as well as homologues and variants thereof. DENV NS 1 of serotype DENV2 identified herein is a serotype homologue of NS 1 of DENV 1 .
[0092] Methods and compositions of the present invention are not limited to particular amino acid sequences identified herein and variants of a reference peptide or protein are encompassed.
[0093] Variants of a peptide or protein described herein are characterized by conserved functional properties compared to the corresponding peptide or protein.
[0094] DENV NS 1 epitopes encompasses variant peptides having at least 95%, 96%, 97%), 98%), 99%, or greater identity to the epitopes having the amino acid sequences identified herein.
[0095] DENV NS1 epitopes encompasses variant peptides including the amino acid sequence YSWKSWG or YSWKTWG and having at least 95%, 96%, 97%, 98%, 99%, or greater identity to the epitopes having the amino acid sequences identified herein.
[0096] Percent identity is determined by comparison of amino acid or nucleic acid sequences, including a reference amino acid or nucleic acid sequence and a putative homologue amino acid or nucleic acid sequence. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino
acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions/total number of positions X 100%). The two sequences compared are generally the same length or nearly the same length.
[0097] The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. Algorithms used for determination of percent identity illustratively include the algorithms of S. Karlin and S. Altshul, PNAS, 90:5873-5877, 1993; T. Smith and M. Waterman, Adv. Appl. Math. 2:482-489, 1981 , S. Needleman and C. Wunsch, J. Mol. Biol., 48:443-453, 1970, W. Pearson and D. Lipman, PNAS, 85:2444-2448, 1988 and others incorporated into computerized implementations such as, but not limited to, GAP, BESTFIT, FASTA, TFASTA; and BLAST, for example incorporated in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.) and publicly available from the National Center for Biotechnology Information.
[0098] A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, PNAS 87:2264-2268, modified as in Karlin and Altschul, 1993, PNAS. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches are performed with the NBLAST nucleotide program parameters set, e.g., for score=100, word length=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present invention. BLAST protein searches are performed with the XBLAST program parameters set, e.g., to score 50, word length=3 to obtain amino acid sequences homologous to a protein molecule of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST are utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI BLAST is used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) are used. Another preferred, non- limiting example of a mathematical algorithm utilized for the comparison of sequences is
the algorithm of Myers and Miller, 1988, CABIOS 4: 1 1 -17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 is used.
[0099] The percent identity between two sequences is determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
[00100] One of skill in the art will recognize that one or more nucleic acid or amino acid mutations can be introduced without altering the functional properties of a given nucleic acid or protein, respectively. Mutations can be introduced using standard molecular biology techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis, to produce variants. For example, one or more amino acid substitutions, additions, or deletions can be made without altering the functional properties of a reference protein. Similarly, one or more nucleic acid substitutions, additions, or deletions can be made without altering the functional properties of a reference nucleic acid sequence.
[00101] When comparing a reference protein to a putative variant, amino acid similarity may be considered in addition to identity of amino acids at corresponding positions in an amino acid sequence. "Amino acid similarity" refers to amino acid identity and conservative amino acid substitutions in a putative variant compared to the corresponding amino acid positions in a reference protein.
[00102] Conservative amino acid substitutions can be made in reference proteins to produce variants.
[00103] Conservative amino acid substitutions are art recognized substitutions of one amino acid for another amino acid having similar characteristics. For example, each amino acid may be described as having one or more of the following characteristics: electropositive, electronegative, aliphatic, aromatic, polar, hydrophobic and hydrophilic. A conservative substitution is a substitution of one amino acid having a specified structural or functional characteristic for another amino acid having the same characteristic. Acidic amino acids include aspartate, glutamate; basic amino acids include histidine, lysine, arginine; aliphatic amino acids include isoleucine, leucine and valine; aromatic amino acids include phenylalanine, glycine, tyrosine and tryptophan; polar
amino acids include aspartate, glutamate, histidine, lysine, asparagine, glutamine, arginine, serine, threonine and tyrosine; and hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine and tryptophan; and conservative substitutions include substitution among amino acids within each group. Amino acids may also be described in terms of relative size; alanine, cysteine, aspartate, glycine, asparagine, proline, threonine, serine, valine are all typically considered to be small.
[00104] A variant can include synthetic amino acid analogs, amino acid derivatives and/or non-standard amino acids, illustratively including, without limitation, alpha- aminobutyric acid, citrulline, canavanine, cyanoalanine, diaminobutyric acid, diaminopimelic acid, dihydroxy-phenylalanine, djenkolic acid, homoarginine, hydroxyproline, norleucine, norvaline, 3-phosphoserine, homoserine, 5- hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, and ornithine.
[00105] The phrase "specific binding" and grammatical equivalents as used herein in reference to binding of an anti-NSl antibody or antigen binding fragment to a specified NSl epitope refers to binding of the anti-NSl antibody or antigen binding fragment to the specified DENV NSl epitope without substantial binding to other non-NS l substances, such as yellow fever virus NS l and/or West Nile virus NSl , present in a sample to be assayed for presence of DENV NSl . It is understood by the ordinarily skilled artisan that specific binding refers to specific binding as determinable by use of appropriate controls to distinguish it from nonspecific binding.
[00106] According to aspects of the present invention, the phrase "specific binding" and grammatical equivalents as used herein in reference to binding of an anti-NSl antibody or antigen binding fragment to a specified NSl epitope refers to binding of the anti-NS l antibody or antigen binding fragment to the specified DENV NSl epitope without substantial binding to other non-NS l substances, such as yellow fever virus NSl and/or West Nile virus NS l , present in a sample to be assayed for presence of DENV NSl, wherein the antibody has an affinity constant (KA) greater than 1 X 106 for an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6);
TPQPM ELK Y S W KT WG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof.
[00107] Antibodies characterized by specific binding to a DENV NS 1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4);
RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6);
TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof are described herein.
[00108] As used herein, the terms "antibody" and "antibodies" relate to monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, camelized antibodies, single domain antibodies, single-chain Fvs
(scFv), single chain antibodies, disulfide-1 inked Fvs (sdFv), and antigen-binding fragments of any of these. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Immunoglobulin molecules are of any type (e.g., IgG,
IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl , IgG2, IgG3, IgG4, IgAl and IgA2), or subclass.
[00109] As used herein, the term "antigen-binding fragment" defines a fragment of an antibody that immunospecifically binds to a target antigen. Antigen-binding fragment may be generated by any technique known to one of skill in the art. For example, Fab and F(ab')2 antigen-binding fragments may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab antigen- binding fragments) or pepsin (to produce F(ab')2 antigen-binding fragments). Antigen- binding fragments are also produced by recombinant DNA technologies. Antigen- binding fragments encompassed by the present compositions and methods retain the ability to bind DENV NS1 as described herein.
[00110] Antibodies, antigen-binding fragments and methods for their generation are known in the art, for instance, as described in Antibody Engineering, Kontermann, R. and Dubel, S. (Eds.), Springer, 2001 ; Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988; Ausubel, F. et al., (Eds.), Short Protocols in Molecular Biology, Wiley, 2002; J. D. Pound (Ed.) Immunochemical Protocols, Methods in Molecular Biology, Humana Press, 2nd ed., 1998; B.K.C. Lo
(Ed.), Antibody Engineering: Methods and Protocols, Methods in Molecular Biology, Humana Press, 2003; and Kohler, G. and Milstein, C, Nature, 256:495-497 (1975).
[00111] Generally described, antibodies contain heavy chain polypeptides and light chain polypeptides. Antigen recognition is mediated by variable regions of the heavy and light chains. Complementarity determining region (CDR) refers to polypeptide regions within the variable region of heavy and light chains. Three CDRs (CDR1, CDR2 and CDR3) are present in each light chain variable region (VL) and each heavy chain variable region (VH). The CDRs are generally responsible for specific antigen recognition properties of the antibody or antigen-binding fragment.
[00112] Antibodies according to aspects of the present invention are those identified as mouse monoclonal antibodies (mAbs) 2G1 , 2G2, 4G3, 1A6, 3D5, and 1 F4.
[00113] Figure 4 describes the biological characteristics of these mAbs including isotype, epitope type, DENV group cross-reactivity, non-reactivity to NS l of Yellow Fever virus (YFV) and NS l of West Nile Virus (WNV), and affinity constant.
[00114] Hybridomas which produce these antibodies have been isolated including a hybridoma producing mAb 2G1 , a hybridoma producing mAb 2G2, a hybridoma producing mAb 4G3, a hybridoma producing mAb 1A6, a hybridoma producing mAb 3D5 and a hybridoma producing mAb 1F4.
[00115] Mouse monoclonal antibody 4G3 is characterized by specific binding to the DENV NS 1 epitope GPQPMEHKYS WKSWG (SEQ ID NO:4).
[00116] Mouse monoclonal antibody 2G2 is characterized by specific binding to the DENV NS l epitope GPQPMEHKYSWKSWG (SEQ ID NO:4).
[00117] Mouse monoclonal antibody 1A6 is characterized by specific binding to the DENV NS l epitope GPQPMEHKYSWKSWG (SEQ ID NO:4).
[00118] Mouse monoclonal antibody 3D5 is characterized by specific binding to the DENV NSl epitope GPQPMEHKYSWKSWG (SEQ ID NO:4).
[00119] Mouse monoclonal antibody 1F4 is characterized by specific binding to the DENV NS l epitope GPQPMEHKYSWKSWG (SEQ ID NO:4).
[00120] Highly related variants of DENV NSl epitope GPQPMEHKYSWKSWG (SEQ ID NO:4) are YSWKSWG (SEQ ID NO: l); RPQPMEHKYS WKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO: 8); and YSWKSWGKAKIIGAD (SEQ ID NO:9).
[00121] Mouse monoclonal antibody 2G1 is characterized by specific binding to the DENV NS 1 epitope VHTWTEQYKFQADSP (SEQ ID NO:2).
[00122] A highly related variant of DENV NS 1 epitope VHTWTEQYKFQADSP (SEQ ID NO:2) is VHTWTEQYKFQPESP (SEQ ID NO:3).
[00123] Substitution at one or more amino acids in a CDR is possible while retaining antigen-binding function, particularly for those amino acid residues that do not contact the antigen. Such substitutions may be made at positions identified and known in the art or may be made empirically.
[00124] Detection of DENV NS1 in a heat-treated or non-heat-treated biological sample according to aspects of the present invention may include detection of a detectable label directly or indirectly attached to NS1 in the heat-treated or non-heat- treated biological sample.
[00125] Methods of detection of DENV NS1 in a heat-treated or non-heat-treated biological sample according to aspects of the present invention includes contacting a sample containing or suspected of containing DENV NS 1 under antigen/antibody binding conditions with a detectably labeled anti-DENV NS 1 antibody wherein the detectable label is directly or indirectly attached to the anti-DENV NS1 antibody. If present, the DENV NS 1 binds to the detectably labeled anti-DENV NS 1 antibody to form a complex such that specific detection of the complex is indicative of DENV NS1 in the sample.
[00126] The term "detectable label" refers to any atom or moiety that can provide a detectable signal and which can be attached to a binding agent, such as a primary or secondary antibody or antigen binding fragment, or analyte, such as DENV NS1 . Examples of such detectable labels include fluorescent moieties, chemiluminescent moieties, bioluminescent moieties, ligands, particles, latex particles, luminescent particles, magnetic particles, fluorescent particles, colloidal gold, enzymes, enzyme substrates, radioisotopes and chromophores. Such particles can be of any shape, size, composition, or physiochemical characteristics compatible with assay conditions. The particles can be microparticles having a diameter of less than one millimeter, for example, a size ranging from about 0.1 to about 1 ,000 micrometers in diameter, inclusive, such as about 3-25 microns in diameter, inclusive, or about 5-10 microns in diameter, inclusive. The particles can be nanoparticles having a diameter from about 1 nanometer (nm) to about 100,000 nm in diameter, inclusive, for example, a size ranging
from about 10-1,000 nm, inclusive, or for example, a size ranging from 200-500 nm, inclusive.
[00127] Any appropriate method, including but not limited to spectroscopic, optical, photochemical, biochemical, enzymatic, electrical and/or immunochemical is used to detect a detectable label in an assay described herein.
[00128] Immunoassays are well-known in the art and include, but are not limited to, enzyme-linked immunosorbent assay (EL1SA) such as but not limited to, antigen capture ELISA, indirect ELISA, fixed cell ELISA; immunochromatography; antigen capture; flow cytometry; immunoblot; immunoprecipitation; immunodiffusion; immunocytochemistry; radioimmunoassay; and combinations of any of these. Generalized details of immunoassays are described in standard references, illustratively including Wild, D., The Immunoassay Handbook, 3rd Ed., Elsevier Science, 2005; Gosling, J. P., Imunoassays: A Practical Approach, Practical Approach Series, Oxford University Press, 2005; E.Harlow and D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988; F. Breitling and S. Diibel, Recombinant Antibodies, John Wiley & Sons, New York, 1999; H. Zola, Monoclonal Antibodies: Preparation and Use of Monoclonal Antibodies and Engineered Antibody Derivatives, Basics: From Background to Bench, BIOS Scientific Publishers, 2000; B.K.C. Lo, Antibody Engineering: Methods and Protocols, Methods in Molecular Biology, Humana Press, 2003; F. M. Ausubel et al., Eds., Short Protocols in Molecular Biology, Current Protocols, Wiley, 2002; Ormerod, M. G., Flow Cytometry: a practical approach, Oxford University Press, 2000; and Givan, A. L., Flow Cytometry: first principles, Wiley, New York, 2001.
[00129] Immunoassay according to aspects of the present invention may include contacting an anti-NS l antibody or antigen binding fragment thereof with a heat-treated or non-heat-treated biological sample, wherein the anti- DENV NS 1 antibody or antigen binding fragment thereof characterized by specific binding to an DENV NS1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2);
VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEH YSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTEL YSW TWG (SEQ ID NO:6);
TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, is immobilized on a
solid support to detect binding of the anti-NSl antibody or antigen binding fragment thereof with DENV NS1 in the heat-treated or non-heat-treated biological sample.
[00130] A dengue virus non-structural protein 1 immunoassay device is provided according to aspects of the present invention which includes a solid or semi-solid porous or non-porous support which includes an antibody or antigen binding fragment characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHK Y S WKS WG (SEQ ID NO:5); RPQPTEL YSWKTWG (SEQ ID NO:6); TPQPMEL YSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or a variant of any thereof.
[00131] The term "solid support" as used herein includes both solid supports and semi-solid supports. The term "solid porous support" as used herein includes both solid porous supports and semi-solid porous supports. The solid support can be in any of various forms or shapes, including planar, such as but not limited to membranes, silicon chips, glass plates and dipsticks; or three dimensional such as but not limited to particles, microtiter plates, microtiter wells, pins and fibers.
[00132] A solid support for attachment of an anti-NSl antibody or antigen binding fragment characterized by specific binding to an NS 1 epitope selected from:
YSWKSWG (SEQ ID NO: l ); VHTWTEQYKFQADSP (SEQ ID NO:2);
VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4);
PvPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6);
TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, can be any of various materials such as glass; plastic, such as polypropylene, polystyrene, nylon; paper; silicon; nitrocellulose; or any other material to which the anti-NSl antibody or antigen binding fragment can be attached for use in an assay.
[00133] In particular aspects, a solid support to which an anti-NS l antibody or antigen binding fragment is attached is a particle.
[00134] Particles to which an anti- DENV NS 1 antibody or antigen binding fragment characterized by specific binding to a DENV NS 1 epitope selected from: YSWKSWG (SEQ ID NOT); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP
(SEQ ID NO:3); GPQPMEHKYSW SWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID N0:7); TPPVSDLKYSWKTWG (SEQ ID N0:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, is bound can be any particles to which an anti- DENV NS 1 antibody or antigen binding fragment can be attached and which are stable and insoluble under assay conditions. The particles can be of any shape, size, composition, or physiochemical characteristics compatible with assay conditions. The particle characteristics can be chosen so that the particle can be separated from fluid, e.g., on a filter with a particular pore size or by some other physical property, e.g., a magnetic property.
[00135] The particles are optionally latex particles, luminescent particles, magnetic particles, or fluorescent particles.
[00136] The particles can be of any shape, size, composition, or physiochemical characteristics compatible with assay conditions. The particles can be microparticles having a diameter of less than one millimeter, for example, a size ranging from about 0.1 to about 1 ,000 micrometers in diameter, inclusive, such as about 3-25 microns in diameter, inclusive, or about 5-10 microns in diameter, inclusive. The particles can be nanoparticles having a diameter from about 1 nanometer (nm) to about 100,000 nm in diameter, inclusive, for example, a size ranging from about 10-1,000 nm, inclusive, or for example, a size ranging from 200-500 nm, inclusive. Particles to which an antibody or antigen binding fragment characterized by specific binding to an NS1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, is bound are illustratively organic or inorganic particles, such as glass or metal and can be particles of a synthetic or naturally occurring polymer, such as polystyrene, polycarbonate, silicon, nylon, cellulose, agarose, dextran, and polyacrylamide. Particles are latex beads according to aspects of the present invention.
[00137] Particles to which an antibody or antigen binding fragment characterized by specific binding to a DENV NS 1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3);
GPQPMEH YSWKSWG (SEQ ID NO:4); RPQPMEHKY S WKS WG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKY S WKT WG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, is bound are optionally encoded and distinguishable from other particles based on a characteristic such as color, reflective index and/or an imprinted or otherwise optically detectable pattern. For example, the particles may be encoded using optical, chemical, physical, or electronic tags. Encoded particles can contain or be attached to, one or more fluorophores which are distinguishable, for instance, by excitation and/or emission wavelength, emission intensity, excited state lifetime or a combination of these or other optical characteristics. Optical bar codes can be used to encode particles.
[00138] According to aspects of the present invention, immunoassay includes assay of NS1 in a heat-treated biological sample by an ELISA technique.
[00139] According to aspects of the present invention, immunoassay includes assay of DENV NS 1 in a heat-treated or non-heat-treated biological sample by an immunochromatography technique. Broadly described, immunochromatography techniques include flowing a sample containing or suspected of containing an analyte of interest along a solid support including an anti-analyte antibody to detect specific binding of the antibody and analyte.
[00140] According to aspects of the present invention, detection of NS 1 in a heat- treated or non-heat-treated biological sample obtained from a subject includes antigen capture, such as by lateral flow assay.
[00141] Optionally, a heterogenous phase sample, is assayed for DENV NS1 according to aspects of the present invention. A heterogenous phase sample is one which contains materials in two or more phases, generally solids or semi-solids and liquids. Whole blood is an example of a heterogenous phase sample that is assayed according to aspects of the present invention. Crude preparations of tissue or cells containing both solids/semi-solids and liquids, such as pulverized human tissue or pulverized or enzymatically digested mosquito tissue, are heterogeneous phase samples assayed according to aspects of the present invention.
[00142] When a heterogeneous phase sample is assayed, solids and semisolids are optionally removed prior to assay, such as by centrifugation, or may remain in the sample. For lateral flow assay, a filtration material, also called a separator, separation
material or separation membrane, is optionally included in the flow path such that non- solubilized solids/semisolids, such as red blood cells, tissue and mosquito exoskeleton, are retained in the filtration material and the liquid component of the sample flows along the flow path, see for example U.S. 4,933,092.
[00143] According to aspects of the present invention, assay of DENV NS 1 in a heat- treated or non-heat-treated biological sample is performed by a lateral flow assay.
[00144] A lateral flow assay according to aspects of the present invention includes flowing a heat-treated or non-heat-treated biological sample along a solid support including an anti-NS l antibody or antigen binding fragment characterized by specific binding to an NS 1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYS WKT WG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, to detect specific binding of the anti-NS l antibody or antigen binding fragment and DENV NS 1 in the heat-treated or non-heat-treated biological sample.
[00145] The heat-treated or non-heat-treated biological sample may be diluted or processed to purify DENV NS 1 prior to analysis.
[00146] A lateral flow assay according to aspects of the present invention includes flowing a heat-treated or non-heat-treated biological sample along a solid support including an anti-NS l antibody or antigen binding fragment characterized by specific binding to an NS 1 epitope selected from: YSWKSWG (SEQ ID NOT); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, in the presence of a competitor to detect competition for binding of the anti- DENV NS1 antibody or antigen binding fragment, with DENV NS1 in the heat-treated or non-heat-treated biological sample.
[00147] According to aspects of the present invention, a lateral flow assay process for detecting DENV NS 1 includes providing: a conjugate pad where detectably labeled anti- NS 1 antibody or antigen binding fragment characterized by specific binding to an NS1
epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSW SWG (SEQ ID NO:5); RPQPTELK YS WKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYS WKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, or detectably labeled DENV NS 1 is diffusibly bound, the conjugate pad adjacent a solid porous support which allows for lateral flow of the fluid heat-treated or non-heat-treated biological sample and which has at least one test detection zone including a non- diffusibly bound detection reagent and at least one control zone including a non- diffusibly bound control reagent, the solid porous support adjacent a wicking pad that promotes the capillary flow of the fluid biological sample along a flow path including the conjugate pad and the solid porous support.
[00148] A non-diffusibly bound detection reagent is an anti- DENV NS 1 antibody or antigen binding fragment characterized by specific binding to a DENV NS1 epitope selected from: YSWKSWG (SEQ ID NO: l ); VHTWTEQYKFQADSP (SEQ ID NO:2);
VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4);
RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYS WKTWG (SEQ ID NO:6);
TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYS WKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof. According to aspects of the present invention in which the conjugate pad contains a detectably labeled anti-
DENV NS 1 antibody or antigen binding fragment characterized by specific binding to a
DENV NS1 epitope selected from: YSWKSWG (SEQ ID NOT );
VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3);
GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7);
TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID
NO:9); or variant thereof, the detection reagent is non-competitive with the detectably labeled anti-DENV NS1 antibody or antigen binding fragment.
[00149] A heat-treated or non-heat-treated biological sample is applied to the conjugate pad. The heat-treated or non-heat-treated biological sample may be diluted or processed to purify DENV NS 1 prior to application to the conjugate pad.
[00150] According to aspects where a detectably labeled anti-DENV NS 1 antibody or antigen binding fragment characterized by specific binding to a DENV NS 1 epitope
selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHK YS WKS WG (SEQ ID NO:5); RPQPTELKY S WKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYS WKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, is included in the conjugate pad, the detectable label is detected in the test zone to detect DENV NS1 in the sample and greater amounts of detected detectable label are indicative of greater amounts of DENV NS 1 in the sample. According to aspects where a detectably labeled DENV NS1 is included in the conjugate pad, the detectable label is detected in the test zone to detect DENV NS 1 in the sample and lower amounts of detected detectable label are indicative of greater amounts of DENV NS 1 in the sample.
[00151] One or more standards may be used to associate an amount of detected detectable label with an amount of DENV NS1 in a sample.
[00152] The conjugate pad is typically blocked to inhibit non-specific binding. A non-limiting example of a blocking solution is lOmM Borate, 3% BSA, 1%, PVP-40, 0.25% Triton x-100, pH 8.
[00153] Any reaction or diluent buffer compatible with the sample, reagents and reaction can be used, including but not limited to phosphate buffered saline, sodium phosphate buffer, potassium phosphate buffer, Tris-HCl buffer, Tricine buffer and other buffers described herein.
[00154] The conjugate pad is disposed adjacent to the solid porous support and the solid porous support is disposed adjacent to the wicking pad. Each component, the conjugate pad, the solid support and the wicking pad has a top surface in substantially the same plane as the top surface of each other component. The conjugate pad, the solid porous support and the wicking pad may be attached together so that they may be moved as one unit. Alternatively or additionally, the conjugate pad, the solid porous support and the wicking pad may all be attached to a structural support, such as a backing material for support and so that they may be moved as one unit.
[00155] According to aspects of the present invention, a lateral flow assay device is provided including 1 ) a conjugate pad where detectably labeled anti-DENV NS 1 antibody or antigen binding fragment characterized by specific binding to an NS1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID
NO:4); RPQPMEHKYSWKSWG (SEQ ID NO: 5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, or detectably labeled NS 1 is diffusibly bound, 2) a solid porous support which allows for lateral flow of the fluid heat-treated or non-heat-treated biological sample and which has at least one test detection zone including a non-diffusibly bound detection reagent and at least one control zone including a non-diffusibly bound control reagent, and 3) a wicking pad that allows for the capillary flow of the fluid biological sample.
[00156] The term "diffusibly bound" refers to reversible attachment or adsorption of a material to the conjugate pad such that the material moves with the lateral flow when contacted with the heat-treated or non -heat-treated biological sample. The term "non- diffusibly bound" refers to attachment of a material to the solid support wherein a non- diffusibly bound material is immobilized and therefore does not move with the lateral flow when contacted with the heat-treated or non-heat-treated biological sample.
[00157] The term "test detection zone" refers to a region of the solid porous support where the detection reagent is non-diffusibly bound. The test detection zone may have any of various shapes and sizes configured to allow for determination of binding of an analyte to the detection reagent. Typically, the test detection zone is a line of non- diffusibly bound detection reagent referred to as a "test line."
[00158] The term "control zone" refers to a region of the solid porous support where the control reagent is non-diffusibly bound. The control zone may have any of various shapes and sizes configured to allow for determination of binding of a control substance to the control reagent. Typically, the control zone is a line of non-diffusibly bound control reagent, referred to as a "control line."
[00159] A control reagent allows a user to confirm that the immunoassay is working properly. For example, a control reagent may be an antibody which specifically binds to the detectably labeled anti-DENV NS 1 antibody or antigen binding fragment characterized by specific binding to a DENV NS 1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof.
[00160] According to aspects of the present invention, a lateral flow assay device includes 1 ) detectably labeled anti-DENV NS1 antibody or antigen binding fragment characterized by specific binding to an NS 1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, diffusibly bound to the conjugate pad, 2) a solid porous support having a test detection zone including non-diffusibly bound anti-DENV NS1 antibody or antigen binding fragment characterized by specific binding to a DENV NS1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, and 3) a wicking pad. According to this aspect, the detectably labeled anti-DENV NS 1 antibody or antigen binding fragment characterized by specific binding to a DENV NS 1 epitope selected from: YSWKSWG (SEQ ID NO: l ); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, diffusibly bound to the conjugate pad and the anti-NS l antibody or antigen binding fragment characterized by specific binding to a DENV NS 1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, non-diffusibly bound to the solid porous support bind specifically to different epitopes of DENV NS 1.
[00161] According to aspects of the present invention, a lateral flow assay device includes 1) a detectably labeled DENV NS 1 epitope diffusibly bound to the conjugate pad, 2) a solid porous support having a test detection zone including non-diffusibly
bound anti-DENV NS1 antibody or antigen binding fragment characterized by specific binding to a DENV NS 1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, and 3) a wicking pad. According to this aspect, the detectably labeled DENV NS 1 epitope diffusibly bound to the conjugate pad binds specifically to the anti-DENV NS1 antibody or antigen binding fragment characterized by specific binding to a DENV NS1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELK Y S WKT WG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, non-diffusibly bound to the solid porous support and therefore competes with DENV NS 1 in a heat-treated or non-heat-treated biological sample.
[00162] The conjugate pad is a material to which a detectably labeled anti-NSl antibody or antigen binding fragment characterized by specific binding to a DENV NS1 epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof, may be diffusibly attached including, but not limited to, glass fiber, bound glass fiber, polyester, cellulose and cellulose derivatives include cellulose acetate and nitrocellulose, nylon, polyvinylidene fluoride, polyethylene, polycarbonate, polypropylene, polyethersulfone and combinations of any of these.
[00163] The solid porous support may be any solid or semi-solid adsorbent porous material suitable for chromatographic applications including, but not limited to, polyvinylidene fluoride, nylon, polyether sulfone, polyester, polypropylene, paper, silica, rayon, cellulose and cellulose derivatives include cellulose acetate and nitrocellulose, woven or non-woven natural or synthetic fibers and porous gels such as agarose, gelatin, dextran and silica gel. The solid porous support may be self-supporting, such as a
membrane, or may be deposited on a structural support, such as an agarose thin layer deposited on a glass slide. According to aspects of the invention, the solid porous support is a nitrocellulose membrane.
[00164] The wicking pad is an absorbent material that facilitates lateral flow by wicking fluid including, but not limited to, an absorbent synthetic or natural polymer, such as cellulose.
[00165] A structural support to which the conjugate pad, solid porous support, and/or wicking pad are attached can be any material which provides support including, but not limited to, a backing card, glass, silica, ceramic and/or plastic membrane. An adhesive may be used to attach the conjugate pad, solid porous support, and/or wicking pad to the structural support.
[00166] A housing may be included to at least partially enclose the conjugate pad, solid porous support, and wicking pad. The housing may be configured to include a well for application of the fluid biological sample to the conjugate pad. The housing optionally allows the user to directly visualize assay results. Alternatively, the housing may include a detection device, such as an optical scanner, for detection of assay results.
[00167] The fluid biological sample flows by capillary action through the wicking pad to a control line and a test line that have anti-DENV NSl antibodies or antigen binding fragments characterized by specific binding to a DENV NSl epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPE8P (SEQ ID NO:3); GPQPMEH YSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTEL YSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSW TWG (SEQ ID NO:8); and YSWKSWG AKIIGAD (SEQ ID NO:9); disposed at a precise concentration determined through validation experiments. The control line is an internal quality control that ensures the sample has migrated appropriately and validates the assay. The test line determines a positive or negative result for the presence of NSl in the heat-treated or non-heat-treated biological sample.
[00168] Figure 5 A is a schematic illustration of a device, 10, for lateral flow assay of NSl according to aspects of the present invention. The conjugate pad 20, solid or semisolid porous support 30, and wicking pad 40, are attached and disposed adjacent to one another. The conjugate pad 20, solid or semi-solid porous support 30, and wicking pad 40 each have at least a top surface 75 substantially the same plane as each other top
surface 75. The direction of lateral flow 50 is shown. An optional housing, 60, is shown which encloses the conjugate pad 20, solid or semi-solid porous support 30, and wicking pad 40. The housing optionally has one or more openings, 70, such as for application of a sample to be assayed for NSl or visualization of test and/or control results. The housing includes an opening 80 for insertion and removal of the conjugate pad 20, solid or semi-solid porous support 30, and wicking pad 40. A test zone 1 14 and a control zone 1 16 are shown.
[00169] Figure 5B shows the conjugate pad 20, solid or semi-solid porous support 30, and wicking pad 40, test zone 1 14 and a control zone 1 16. A first detectably labeled binding agent capable of specific binding to DENV NS1 102 is diffusibly attached to the conjugate pad 20. A test sample is added to the conjugate pad which contains or is suspected of containing DENV NS 1 100. The DENV NS1 and first detectably labeled binding agent capable of specific binding to DENV NS 1 form a complex 104. The complex 104 is moved by lateral flow in the direction of the test zone 1 14 where it is bound to a second binding agent capable of specific binding to DENV NS1 which is non- competing with the first detectably labeled binding agent capable of specific binding to DENV NS1 , forming complex 106. Complex 106 is detected in the test zone by detection of the detectable label, thereby quantitating NS1 in the test sample. Excess detectably labeled binding agent capable of specific binding to NS1 102 moves by lateral flow to the control zone where it binds to a binding agent 1 2 capable of specific binding to the binding agent capable of specific binding to NS1 102, forming a complex 1 10.
[00170] As noted above, a filtration material, also called a separator, separation material or separation membrane, is optionally included in the flow path such that non- solubilized solids/semisolids, such as red blood cells, tissue and mosquito exoskeleton, are retained in the filtration material and the liquid component of the sample flows along the flow path, see for example U.S. 4,933,092. Thus a separator is optionally included in an immunoassay device, such as but not limited to a lateral flow assay device, according to aspects of the present invention.
[00171] DENV NS1 contained in a heat-treated or non-heat-treated biological sample is optionally purified for assay according to a method of the present invention.
[00172] The term "purified" in the context of a heat-treated or non-heat-treated biological sample refers to separation of DENV NS 1 in the biological sample from at least one other component present in the biological sample.
[00173] In particular embodiments, DENV NSl is optionally substantially purified from the heat-treated or non-heat-treated biological sample to produce a substantially purified sample for use in an inventive assay. The term "substantially purified" refers to a desired material separated from other substances naturally present in a sample obtained from the subject so that the desired material makes up at least about 1 - 100% of the mass, by weight, such as about 1 %, 5%, 10%, 25%, 50% 75% or greater than about 75% of the mass, by weight, of the substantially purified sample.
[00174] Sample purification is achieved by techniques illustratively including electrophoretic methods such as gel electrophoresis and 2-D gel electrophoresis; chromatography methods such as HPLC, ion exchange chromatography, affinity chromatography, size exclusion chromatography, thin layer and paper chromatography. It is appreciated that electrophoresis and chromatographic methods can also be used to separate a peptide or peptides from other components in a sample in the course of performing an assay, as in, for example separation of proteins in immunoblot assays.
[00175] According to one aspect of the present invention, DENV NS l is isolated and concentrated by absorption of DENV NS l onto a solid substrate.
[00176] According to one aspect of the present invention, DENV NS l is isolated and concentrated by binding to beads or other particles coupled with an anti-DENV NSl antibody or antigen binding fragment characterized by specific binding to a DENV NS l epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof.
[00177] According to one aspect of the present invention, DENV NS l is isolated and concentrated by binding to magnetic beads coupled with an anti-DENV NSl antibody or antigen binding fragment characterized by specific binding to a DENV NSl epitope selected from: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof.
[00178] Isolated immunogenic peptides having 25 or fewer amino acid residues which include an epitope selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO: 8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof are provided according to aspects of the present invention. Such peptides are useful as immunogens and as controls in immunoassays, for example. One or more amino acid residues extending from the N' and/or C termini of the specified peptides may be those found in DENV NS 1 proteins or may be different. For example, one or more amino acid residues extending from the N' and/or C' termini of the specified peptides may be added to aid in isolation of the peptides.
[00179] Isolated immunogenic DENV NS1 peptides consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof are provided according to aspects of the present invention. Such peptides are useful as immunogens and as controls in immunoassays, for example.
[00180] According to aspects, immunoassay kits for detecting dengue virus infection in a human subject having or suspected of having a dengue virus infection are provided which include one or more antibodies or antigen binding fragments characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); or variant thereof.
[00181] According to aspects, immunoassay kits for detecting dengue virus infection in a human subject having or suspected of having a dengue virus infection are provided which include one or more antibodies or antigen binding fragments including a first antibody or antigen binding fragment characterized by specific binding to an epitope of
dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSW SWG AKIIGAD (SEQ ID NO:9); or variant thereof and a second antibody or antigen binding fragment characterized by specific binding to VHTWTEQYKFQADSP (SEQ ID NO:2) and/or VHTWTEQYKFQPESP (SEQ ID NO:3); or variant thereof.
[00182] One or more auxiliary components are optionally included in such kits, such as a control DENV NS 1 peptide or DENV NS1 protein, a control NS 1 peptide or NS 1 protein from a non-dengue virus such as yellow fever virus or West Nile virus, a buffer, diluent or a reconstituting agent.
[00183] Embodiments of inventive compositions and methods are illustrated in the following examples. These examples are provided for illustrative purposes and are not considered limitations on the scope of inventive compositions and methods.
[00184] Examples
[00185] Viral strains
[00186] Four prototype DENV strains: DENV-1 (Hawaii), DENV-2, (New Guinea- C), DENV-3 (H87) and DENV-4 (H241 ) detailed in Young et al, J. Clin. Microbiol., 38, 1053-1057, 2000, were used for mAb development and characterization.
[00187] Preparation of DENV serotype-specific culture supernatant
[00188] Vero cells (ATCC, Manassas, VA, USA) were first propagated in M199 medium (Gibco/Life Technologies, Grand Island, NY, USA) supplemented with 5% fetal bovine serum (FBS) in T-25 culture flasks. Cells at 80-90% confluency were washed twice with M199 containing 2.5% FBS and cultured in the same medium for two additional days, and cells were then infected for 24 hours with each DENV serotype at a multiplicity of infection (MOI) of 0.1. Following incubation, culture medium was replaced with serum-free M199 medium and cells were cultured for another four days. Mock-infected cells were included as a control. Supernatants were harvested on day 5 post inoculation, clarified, filtered with a 0.2 um filter, concentrated with Amicon UltraDevice Centricon-30K (Millipore, Billerica, MA, USA), treated with SIGMAFAST protease inhibitor cocktail (Sigma, St. Louis, MO, USA) and stored at -80°C until use. The presence of DENV NS 1 protein in each supernatant was confirmed using the
Panbio® Early ELISA, a serotype-cross-reaetive NS1 ELISA (Panbio Diagnostics, Brisbane, Australia).
[00189] Plasmid construction and recombinant DENV NS 1 protein expression
[00190] Baculovirus Expression System with Gateway Technology (Invitrogen, Carlsbad, CA, USA) was used to produce recombinant NS 1 plasmids and proteins. Viral RNA was extracted from DENV-1 - and DENV-2- virus stocks using QIAmp Viral RNA Minikit (QIAGEN, Gaithersburg, MD, USA), and NS1 sequences were amplified with Superscript III One-Step RT-PCR reaction mix(Invitrogen) and primers (5'- CACCGACTCGGGATGTGTAATTAAC-3 ' (SEQ ID NO: 12) and 5'- CTATGCAGAGACC AATGACTTTACT-3 ' (SEQ ID NO: 13) for DENV1 ; 5'- CACCGATAGTGGTTGCGTTGTGAGC-3 ' (SEQ ID NO: 14) and 5'-
CTAGGCTGTGACCAAGGAGTTGACC-3 ' (SEQ ID NO: 15) for DENV-2). Amplified products were gel-purified, sequenced, and cloned into pENTR/D-TOPO-plasmid (Invitrogen) according to the manufacturer's instructions. Clones containing NS1 gene were verified by sequencing and pENTR/D-TOPO-plasmids containing NS 1 were subcloned into the pDESTl O plasmid (Invitrogen) to add an N-terminus His-tag for later protein purification. Plasmids with an NS1 insert were verified by sequencing. To express the rNS l protein in Sf9 insect cell line, extracted recombinant plasmids, pDESTlO with NS l inserts were transformed into DHlObac E. coli (Invitrogen). Six-His-tagged rNSl proteins of DENV-1 and DENV-2 were expressed but as insoluble protein (inclusion bodies). Thus the rNSl proteins were prepared from cell lysates and solubilized in 0.5% sodium dodecylsulfate (SDS) PBS buffer and purified by Precision Antibody (PA), Custom Antibody Generation Services (Columbia, MD, USA). When NSl was solubilized in 0.5% SDS, it resulted in denaturation of the rNS l exclusively into the monomeric isoform. To determine if the isoform of the purified rNSl was exclusively in monomeric form, native SDS-PAGE gel and western blot were performed. Prior to mice intradermal injection, the 0.5% SDS in PBS buffer used to solubilize monomeric rNSl was adjusted to 0.05%SDS.
[00191] Development of hybridoma cell lines secreting anti-DENV NSl mAbs
[00192] Hybridoma cell lines secreting mAbs specific to NSl of DENV were developed by fusion of SP2/0 cells with splenocytes isolated from BALB/c mice immunized with two affinity-purified, detergent-generated monomeric rNSl proteins of DENV-1 and DENV-2 using established hybridoma technology detailed in Milstein et
al., Cell Biology International Reports 3, 1-16,1979. To generate hybridoma cell lines secreting anti-DENV NS l monoclonal antibodies (mAbs), female BALB/c mice were immunized with recombinant NS l antigens of DENV1 (Hawaii) and DENV2 (NGC) obtained from cell lysate of Sfi) insect cells, solubilized in 0.5%SDS.
[00193] The anti-dengue-1 NS l antibodies, 2G2, 4G3, 1A6, 3D5, and 1F4 were produced against the following sequence:
Dengue-1 NSl (Nucleotide sequence Acc# X69395)
DV1 protein sequence SEQ ID NO: 10
DSGCVINWKGRELKCGSGIFVTNEVHTWTEQYKFQADSPKRLSAAIGKAWEEG VCGIRSATRLENIMWKQISNELNHILLENDMKFTVVVGDVSGILTQGRKMIGPQP MEH YSWKSWGKAKIIGADVQNTTFIIDGPNTPECPDDQRAWIWEVEDYGFGIF TTNIWLKLRDSYTQVCDPRLMSAAIKDSKAVHADMGYWIESEKNETWKLARAS FIEVKTCVWPKSHTLWSNGVLESEMIIPKIYGGPISQHNYRPGYSTQTAGPWHLG LELDFDLCEGTTVVVDEHCGNRGPSLRTTTVTGKIIHEWCCRSCTLPPLRFKGE DGCWYGMEIRPVKD EENLVKSLVSA
[00194] The anti-dengue-2 NSl antibody, 2G1 , was produced against the following sequence:
Dengue-2 NS l (Nucleotide sequence Acc# (AF038403)
DV2 protein sequence SEQ ID NO: 1 1
DSGCVVSWKNKELKCGSGIFITDNVHTWTEQYKFQPESPSKLASAIQKAHEEGIC GIRSVTRLENLMWKQITPELNHILSENEVKLTIMTGDIKGIMQAGKRSLQPQPTEL KYSW TWGKAKMLSTESHNQTFLIDGPETAECPNTNRAWNSLEVEDYGFGVFT TNIWLKLREKQDVFCDSKLMSAAIKDNRAVHADMGYWIESALNDTWKIEKASFI EVKSCHWPKSHTLWSNGVLESEMIIPKNFAGPVSQHNYRPGYHTQTAGPWHLG KLEMDFDFCEGTTVVVTEDCGNRGPSLRTTTASGKLITEWCCRSCTLPPLRYRGE DGCWYGMEIRPLKEKEENLVNSLVTA
[00195] Both dengue NS l recombinant proteins were expressed with N-terminal 6 histidine tags in the Invitrogen Gateway Baculovirus expression system.
[00196] Screening of anti-DENV NSl positive hybridomas
[00197] In order to select desired anti-DENV NSl positive hybridoma cell lines, two screening methods, first round hybridoma screening and second round hybridoma screening were employed.
[00198] First round hybridoma screening
[00199] The reactivity of hybridoma culture supernatants was screened for reactivity to affinity-purified detergent-generated monomeric rNS l of DENV-1 and DENV-2 expressed in E. coli (GenScript, NJ, USA) in iELlSA. Briefly, 96-well microtiter ELISA plates were coated overnight at 4°C with monomeric rNS l at a concentration of 1 ug/ml in phosphate buffered saline (PBS). Individual wells of microtiter plates were blocked with PBS (pH 7.4) containing 5% non-fat dry milk (NFDM) and 0.5% Tween-20 for 30 min at room temperature (RT). After incubation, the microtiter plates were washed 3x with PBS + 0.05% Tween-20 (PBS-T) and treated with hybridoma supernatants diluted to 1 : 100, 1 :500, 1 : 1000 and 1 :2000 in a wash buffer and incubated for 1 h at 37°C. All hybridoma supernatant dilutions were tested in duplicate. After 5 washes, goat anti- mouse IgG horseradish peroxidase (HPR)-conjugated (KPL, Gaithersburg, MD) diluted to 1 : 1000 in blocking solution was added and incubated for 1 h at 37°C in humidified chamber. Following 5 washes, 3,3',5,5'-Tetramethylbenzidine (TMB) liquid substrate(KPL) was added and incubated for 15 min at RT in the dark. Color development reaction was stopped by TMB stop solution (KPL) and the colorimetric optical density (OD) was determined with 450 nm filter on ELISA ELx800 microplate reader (BioTechR, VT, USA).
[00200] Second round hybridoma screening
[00201] A fixed cell ELISA was used to assess in situ reactivity of mAbs against native cell surface NS1 of DENV-1 to DENV-4. Briefly, Vero cells maintained in Ml 99 medium (Sigma) supplemented with 5% FBS, 1 % Gentamicin (Gibco, Sparks, MD, US), 1% Penicillin-Streptomycin (Gibco), 3% sodium bicarbonate (Gibco) were seeded into 96-well culture plates at a density 2 χ 105cells/well and incubated at 37°C in 10% CO? for 2 days to obtain a desirable 90% confluent monolayer. Cells were then infected with each of the four DENV serotypes at an MOI of 0.1 in separate 96-well culture plates. On day five post infection, culture medium was removed and cells were fixed for 30 minutes at -20°C with equal volume of ethanol and methanol. The fixed cells were then washed 5x with PBS and blocked for 30 min at RT with PBS containing 5% NFDM and 0.5% Tween-20 (PBST). Following the blocking step, mAbs diluted tol : 100, 1 :500 and 1 : 1000 were added and incubated for 1 hour at 37°C. After 5 washes with PBS, HPR-conjugated goat anti-mouse IgG (KPL) diluted to 1 : 1000 in blocking buffer were added and incubated at 37°C for 1 hour in a humidified chamber. Following 5 washes with PBS, ABTS (2,2'-Azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt)
substrate (KPL) was added for color development and incubated in the dark for 1 hour at room temperature (RT) and OD measured at 405 nm. All mAb dilutions were tested in duplicate. Positive reactivity was scored if the average OD value for duplicate tests was greater than the corresponding average of mock-infected cells under the same conditions. In each plate run, the cross-serotype reactive NS 1 mAb 7E1 1 (Harlan Laboratories, Inc., U.S.) was included as a positive control.
[00202] Hybridoma cell lines secreting anti-DENV NS 1 mAbs
[00203] Based on reactivity of hybridoma clones to monomeric rNS l of DENV-1 and
DENV-2 expressed in E. coli, a total of 50 positive clones were identified, of which 37 were derived from mice immunized with rNS l of DENV-2 and the remaining 13 were from mice immunized with rNSl of DENV1. Three of the 50 positive clones were unreactive after successive subcloning, suggesting that these positive clones might have been lost due to an overgrowth of negative clones or genetic instability. Six hybridoma cell lines secreted mAbs (2G 1 , 1A6, 1F4, 3D5, 2G2 and 4G3) reactive to both detergent- denatured rNSl and the native NS1 antigen in DENV-infected Vero cells were selected for further characterization as shown in Figure 4. None of these 6 mAbs (1A6, 1F4, 2G2, 3D5, 4G3, 2G1 ) had reactivity to mock-infected fixed Vero cells.
[00204] mAb production
[00205] Large scale mAb production from selected hybridoma clones was carried out using a CELLine 1000 bioreactor (BD Biosciences; Sparks, MD, USA). Prior to production, hybridoma cell lines maintained in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% FBS were adapted to antibiotic free BD CellTM MAb Medium (Gibco) supplemented with 10% HycloneSuper Low IgG FBS (Gibco) and 1% Glutamine (Gibco) by reducing gradually the DMEM supplemented with 10% FBS over a number of passages. Hybridoma cells adapted to low IgG FBS medium at a density of 2 χ 106 cells/ml were seeded into a 15 ml Bioreactor cell compartment, incubated for fourteen days at 37°C in a 5% C02 humidified atmosphere, the mixed cell suspension harvested, centrifuged at 1000 rpm for 5 min to separate the culture supernatant which was filtered with a 0.2 um filter and stored at -20°C until affinity-purified. Hybridoma culture supernatant was screened for mAb activity defined by reactivity to NS1 antigen in the iELISA.
[00206] mAb purification
[00207] mAbs from hybridoma culture supernatants were purified by using VivaPure Maxiprep Protein G Spin Columns (Satorius Stedim, Bohemia, NY, USA) according to manufacturer's instructions. The affinity-purified mAbs were dialyzed against PBS pH 7.2 fori 6 hours. The concentrations of dialyzed mAbs were determined byNanoDropR- 100 spectrophotometer at 280 nm (NanoDrop Technologies, Willington, DE, USA). The reactivity of purified mAbs to DENV NSl was then confirmed by iELISA. Aliquots of dialyzed mAbs in 15% sterilized glycerol were stored at -20°C until use.
[00208] Conjugation of mAbs
[00209] Affinity purified and dialyzed mAbs were biotinylated with a spacer arm biotin (NHS-PEG4-B iotin) using EZ-LinkTMNHS-PEG4-Biotinylation Kit (Pierce/Thermo Scientific, Rockford, IL, USA).These biotinylated mAbs were then dialyzed against PBS pH 7.2 for 32 hours and their reactivity to DENV rNSl antigen was determined using iELISA. Biotinylated mAbs in 15% of sterile glycerol were aliquoted and stored at -20°C until use.
[00210] mAb isotyping
[00211] mAbs were isotyped using a mouse mAb isotyping kit (Pierce/Thermo Scientific, Rockford, IL, USA) according to manufacturer's instructions. Briefly, ELISA strip wells pre-coated with anti-mouse heavy chain capture antibody (anti-IgGl, IgG2a, IgGb,IgG3, IgA and IgM) or anti-mouse light chain (kappa or lambda)were used for the assay. Diluted hybridoma culture supernatants (1 :50) pre-mixed with goat anti-mouse IgG + IgA + IgM-HRP conjugated were added and incubated at RT for two hours and results measured at 450 nm.
[00212] mAb isotypes
[00213] Of the six mAbs (1 A6, 1 F4, 2G2, 3D5, 4G3, 2G1), 1 F4 and 2G2 had IgG2b heavy chain whereas the remaining four mAbs had IgGl heavy chain but all of them had a kappa (κ) light chain as shown in Figure 4. There was no mAb with mixed isotypes, indicating that these mAbs were not produced by mixed hybridoma populations.
[00214] mAbs, 2G1 , 1A6, 1F4, 3D5, 2G2 and 4G3, were characterized by indirect
ELISA, western blotting, in-cell ELISA.
[00215] mAbs, 2G1, 1A6, 1F4, 3D5, 2G2 and 4G3, bind to monomeric, dimeric and hexameric isoforms of DENV NSl
[00216] Western blot
[00217] ELISA results were confirmed by Western blot analysis, which detected both NS l monomer and dimer isoforms, to verify DENV specificity of anti-NS l mAbs. One hundred and twenty five nanograms each of purified DENV-1, DENV-2, DENV-3, DENV-4,YFV, and WNV rNS l antigens (Native Antigen) and 10 ug total cell culture supernatant from COS-1 cells secreting DENV-1 envelope (E) protein (CDC) was separated using Novex NuPAGE 4-12% Bis-Tris gels (Life Technologies) under non- reduced, heat-denatured or non-denatured conditions. Protein size discrimination was determined with MagicMark™ XP Protein standards (20-220 kDa, Invitrogen). COS-1 cells secreting DENV-1 envelope (E) protein are described in detail in Purdy and Chang, Virology, 333(2):239-250, 2005. For Western blot analysis the proteins were transferred to nitrocellulose membranes (Invitrogen, 0.22 um), blocked overnight at RT in 5% NFDM in PBS with 0.05% Tween20 (NFDM-PBST) and the membranes incubated with one of the following primary anti-NSl mAbs at a 1 :50,000 dilution: 2G1 ( 1.67 mg/ml), 2G2 (1.26 mg/ml), 4G3 (1.26 mg/ml), 1 F4 (1.26 mg/ml). Following incubation with primary antibody a secondary anti-mouse peroxidase detector antibody diluted atl : 10,000 (KPL, Gaithersburg, MD) was used. The substrate Super-Signal West Pico solution (Pierce/Thermo Scientific) was used to detect proteins.
[00218] Figure 4 summarizes the reactivity of the six mAbs against DENV NSl isoforms. mAbs were reactive to monomeric rNS l protein in the iELISA, a finding further corroborated by Western blot analysis in which all mAbs reacted to the monomeric rNS l band under both non-reducing and heat-denatured or non-denatured conditions. mAbs were reactive to dimeric NSl as observed in the fixed cell ELISA, see Figures 4 and 1 1 A; the dimeric isoform being an exclusively cell membrane-associated protein and is predominant in the fixed cell ELISA. This was further verified by western blot showing reactivity to the about 80 kDa NS l dimer isoform band, see Figure 10. In addition, mAbs were found reactive to native hexameric NSl in cell culture supernatant obtained from each of the four DENV serotypes, and they also reacted to immunoaffinity-purified hexameric rNS l proteins of DENV-1 to DENV-4 by ELISA as shown in Figures 1 1B-F.
[00219] mAbs serotype- cross-reactivity
[00220] In addition to reactivity in the fixed-cell ELISA, reactivity to all 4 DENV serotypes was determined using culture supernatants harvested from DENV-1-4 infected Vero cells in the iELISA. Briefly, 96-well microtiter plates were coated overnight at
4°Cwith 100 ul/well of DENV1-4 infected Vero cell culture supernatants diluted twofold starting from 1 : 160 to 1 : 10, 240 in PBS (pH7.4). Next day, microtiter plates were blocked with PBS containing 5% NFDM and 0.5% Tween-20 for 30 min at RT, washed 3 with wash buffer (PBS + 0.05% Tween-20), and purified mAbs diluted to 1 :200 in PBS was added and incubated for 1 h at 37°C. After 5 washes, goat anti-mouse IgG HPR-conjugated was diluted to 1 : 1000 in blocking solution and incubated for 1 h at 37°C in a humidified chamber. To eliminate the variability due to NSl expression patterns of the four DENV serotypes, the reactivity profile pattern of four serially diluted affinity- purified mAbs and hybridoma culture supernatants(di luted to 1 :500 for two) was tested against four affinity-purified hexameric rNS l proteins of DENV-1 (Nauru/Western Pacific/1974),DENV-2 (Thailand/16681/84) DE V-3 (Sri Lanka D3/H/IMTSSA- SRI/2000/1266) and DENV-4 (Dominica/814669/1981) expressed in a mammalian cell line (NativeAntigen, Oxfordshire, UK). The size (hexameric form) of these affinity- purified rNSl s was demonstrated by size exclusion chromatograph (SEC). Similar iELISA procedures were followed for affinity-purified rNS l protein as previously described starting at rNSl concentration of 1 ug/ml.
[00221] DENV serotype cross-reactive mAbs
[00222] According to the results obtained from the fixed cell ELISA, 2G2 had relatively strong reactivity (defined by higher OD value) to DENV-1 while the remaining five mAbs appeared serotype cross-reactive, albeit with varied reactivity pattern. Of the five serotype-cross-reactive mAbs, 2G1 and 4G3 had strong reactivity to DENV-1 , -2 and -3. The other three mAbs (1 F6, 1F4 and 3D5) had relatively weak reactivity to all four DENV serotypes except to DENV-1 , see Figure 1 1 A. All six MAbs displayed relatively weaker reactivity to DENV-4 compared to the other three DENV serotypes. Reactivity profiles of the mAbs in the iELISA were determined using affinity-purified hexameric rNS l for DENV- 1 to DENV-4. 2G1 and 4G3 appeared to be serotype cross- reactive in the fixed cell ELISA but their reactivity profiles in the iELISA did not perfectly match these results, see Figures 1 1A - F. Furthermore, the three serotype cross- reactive mAbs 1A6, 1 F4, 3D5 that showed weaker reactivity to DENV-1-4 by fixed cell ELISA were more reactive to immunoaffinity-purified hexameric rNS l of DENV-1-4 in the iELISA, see Figure 1 IB. Also, 2G2 that appeared to be DENV-1 specific in the fixed cell ELSA was serotype cross-reactive in the iELISA. Western blot results confirmed the serotype cross-reactivity of all six MAbs, see Figure 10.
[00223] mAbs cross-reactivity to other flaviviruses
[00224] Cross-reactivity of the six MAbs against commercially available affinity- purified recombinant NS l of Yellow Fever virus (YFV) and West Nile virus (WNV) in iELISA following procedures described in the first round screening selection of positive hybridoma clones.
[00225] NSl mAbs did not cross-react with YFV and WNV NSl
[00226] Both iELISA and western blot indicated that none of the mAbs had cross- reactivity to purified recombinant YFV or WNV NSl as shown in Figures 4 and 10). This was further supported by a significant amino acid sequence difference between the NS Ts of all four DENV serotypes and YFV and WNV at the binding epitopes of MAbs, see Figures 6 and 7.
[00227] Mapping of mAb epitope by synthetic peptide ELISA
[00228] As all of the mAbs were derived from immunization of detergent-generated monomeric NS l, linear epitope mapping with 43 peptides consisting of 15-mers (except the last peptide which is 16-mer) with a seven amino acid overlap to cover the full-length NS l protein of DENV-1 (Hawaii) was performed, see Figure 8. These linear peptides were synthesized by the Biotechnology Core Facility Branch, CDC, Atlanta, with >95% purity. Each synthetic peptide was designed and synthesized to contain (i) biotin in the N-terminal, (ii) a SGSG spacer between biotin and 15-mer amino acids and (iii) an amide in the C-terminal. All cysteines in the homologous amino acid sequences of DENV-1 NS l were replaced by serines in the synthetic peptides to avoid peptide dimerization and oligomerization or covalent linkage to thiols in the mAbs. The size (hexameric form) of these affinity-purified rNS l s was demonstrated by size exclusion chromatograph (SEC).
[00229] Binding of mAbs to synthetic peptides was determined by ELISA in streptavidin pre-coated 96-well plates (Pierce, Rockford, IL, USA).Briefly, 96-well plates were pre-coated with streptavidin, blocked with 2% bovine serum albumin (BSA), washed 3 with Tris-buffered saline (TBS) (25 mM Tris, 150 mM NaCl; pH 7.2), 0.1 % BSA, and 0.05% Tween-20, and 100 ul/well of 10 ug/ml biotinylated synthetic peptide diluted in a wash buffer was added and incubated on a shaker at 200 rotations per minute (rpm) for 2 hours at RT. After 3 washes, the 100 ul/well of hybridoma supernatants diluted at 1 : 100 and 1 :500 were added and incubated for 1 hour at RT. Following 3 washes, HRP-conjugated goat anti-mouse IgG antibody diluted at 1 : 1000 was added and
incubated for 1 hour at RT. Finally, TMB substrate was added and OD450 values were obtained.
[00230] Based on this assay, the binding region/epitope for mAb 2G1 was found to be in 25-VHTWTEQY FQADSP-39 (SEQ ID NO: 2) of DEN VI NS l whereas the binding region/epitope for mAbs 1A6, 1F4, 3D5, 2G2 and 4G3 was found to be in 105- GPQPMEHKYSWKSWG-1 19 (SEQ ID NO: 4) of the DENV1 NSl . In addition, mAbs 1A6, 1 F4, 3D5, 2G2 and 4G3 showed weak reactivity to peptide, 1 13- YSWKSWGKAKIIGAD-127 (SEQ ID NO:9) of DENV NS l . This weak reactivity reflects the 7 overlapping amino acid residues YSWKSWG (SEQ ID NO: l) encompassed by both peptide 105-GPQPMEHKYSWKSWG-l 19 (SEQ ID NO: 4) and peptide 1 13-YS WKSWGKAKIIGAD- 127 (SEQ ID NO:9).
[00231] Epitope for 2G1 and its multiple alignments the sequence of rNSl from all 4 DENV serotypes used to determine the analytical sensitivity of the assay. P4 represents 15-mer synthetic peptides (amino acids 25 to 39) where mAb 2G 1 mapped to bind.
P4 25-VHT TEQYKFQADSP-39 SEQ ID NO 2
DENV1_NAC 25-VHTWTEQYKFQADSP-39 SEQ ID NO 2
DENV3_NAC 25-VHTWTEQYKFQADSP-39 SEQ ID NO 2
DENV2_NAC 25-VHTWTEQYKFQPESP-39 SEQ ID NO 3
DENV4 NAC 25-VHTWTEQYKFQPESP-39 SEQ ID NO 3
[00232] Epitope 4G3 and its multiple alignments the sequence of rNSl from all 4
DENV serotypes used to determine the analytical sensitivity of the assay. PI 4 represents
15-mer synthetic peptides (amino acids 105 to 1 19) where mAb 4G3 mapped to bind. p!4 105-GPQPMEHKYSWKS G-119 SEQ ID NO: 4
DENV1_NAC 105-RPQPMEHKYSWKS G-119 SEQ ID NO: 5
DENV2_NAC 105-RPQPTELKYSWKTWG-119 SEQ ID NO: 6
DENV3_NAC 105-TPQPMELKYSWKTWG-119 SEQ ID NO: 7
DENV4 NAC 105-TPPVSDLKYSWKT G-119 SEQ ID NO: 8
[00233] Figure 4 is a table which shows the biological characteristics of the mAbs such as isotype, epitope type, DENV group cross-reactivity, non-reactivity to NSl of YFV and WNV, and affinity constant. Reactivity of mAbs with dimeric DENV NS l was determined by in vitro ELISA in Vero cells seeded in microplate, infected with each DENV serotype at multiplicity of infection 0.1 and fixed on day five post infection. Recombinant proteins were used as coating antigens (^g/ml) in indirect ELISA
(iELISA) and the absorbance was measured at 450nm; +, OD = 0.3 to 0.8; ++, OD= 1 to 2, +++, OD = 2 to 3 and ++++, OD >3. All mAbs showed no cross-reactivity to NSl of Yellow Fever virus (YFV) and West Nile Virus (WNV). The binding region/epitope for 5 mAbs (1A6, 1 F4, 3D5, 2G2 and 4G3) appeared to be in the same region 105- GPQPMEHKYSWKSWG-1 19 (SEQ ID NO: 4) whereas for mAb 2G1 was in 25- VHTWTEQYKFQADSP-39 (SEQ ID NO: 2) region of the DENV NSl .
[00234] The identified epitopes for mAbs are conserved among all the four DENV serotypes
[00235] Multiple amino acid sequence alignments between the mAbs epitopes and the homologous NS l amino acid sequences of DENV and YFV, WNV, Japanese Encephalitis virus (JEV) and Murray Valley virus (MEV) using strains in the NCBI database were performed using CIustalW2 to test (a) whether the epitopes of mAbs are specific to DENV and conserved among DENV strains, and (b) whether amino acid sequence variation in the prototype DENV strains could explain the differences in observed mAb reactivity pattern between fixed cell ELISA and iELISA. A few amino acid sequence differences were identified at peptide position 105-119 among the DENV strains. In contrast, the amino acid sequence was highly conserved at position 25-39 which was recognized by 2G1, although there was variation at position 36-37 in which 36AD37 was specific to DENV-1 and DENV-3 strains and 36PE37 was specific to DENV-2 and DENV-4 strains. Overall, the epitopes of the mAbs appeared to be highly conserved among DENV strains but were distinct from other flaviviruses. Figures 6 and 7 are tables showing variants of peptide epitopes recognized by mAbs 2G1 , 1A6, 1F4, 3D5, 2G2 and 4G3.
[00236] Figure 8 is a table showing binding affinities of mAbs 2G1, 1 A6, 1 F4, 3D5, 2G2 and 4G3.
[00237] mAb binding competition ELISA
[00238] Competitive binding of the labeled and unlabeled mAbs was tested by ELISA. Briefly, microtiter plates were coated overnight at 4°C with rNS l antigen and then blocked with PBST containing 5% non-fat dry milk (NFDM) for 30 min at RT. Two-fold serial dilution of unlabeled mAbs (starting at a saturating concentration of 40 ug/ml) was performed in low-binding Eppendorf tubes (Fisher Thermo Scientific, IL, USA) and 50 ul of each mAb dilution was added to individual wells and incubated for 90 min at 37°C. Following a pre-incubation step with unlabeled "competitor" mAb, 50 ul of
biotinylated mAb at a fixed optimal dilution was added to each well. After 90 min incubation at 37°C, the plate was washed 3x and incubated for 30 min at 37°C with streptavidin conjugated HRP (Fisher Thermo Scientific,IL, USA). The plate was then washed 5x and incubated for 10 minutes with TMB liquid substrate at RT. The substrate reaction was stopped and the plate was read at OD450.
[00239] The binding of biotinylated 2G 1 (epitope group LD2) to rNS l was not inhibited by pre-incubation with any one of the five unlabeled "competitor" mAbs of the same epitope group (LXl), indicating an absence of competition for binding sites. In contrast, the binding of biotinylated 4G3 (LXl ) to rNS l was inhibited in dose-dependent manner by pre-incubation with any one of the unlabeled competitor mAbs of the same epitope group (LXl, e.g., 1 F4) indicating competition for the binding sites, see Figure 9.
[00240] Figure 9 is a graph showing results of competition ELISA analysis for distinct epitope binding of monoclonal antibodies (mAbs). Paired mAbs competition was performed between biotinylated mAbs (at a fixed optimal dose) and unlabeled mAbs (2- fold serially diluted starting from a saturated concentration). Bars represent OD450 values as a measure of binding of biotinylated mAbs to NS1 antigen coated on the microtiter plate across the various doses of unlabeled mAbs. Dose-dependent binding inhibition was observed when unlabeled mAb (1 F4) was made to compete with biotinylated-mAb (B-4G3) that had same binding epitope while no inhibition was observed when the competing mAbs (e.g. 1F4 vs B-2G1) had distinct non-overlapping biding epitopes.
[00241] NS1 Antigen capture ELISA and testing clinical samples before and after heat denaturation.
[00242] Clinical samples
[00243] An NS1 capture ELISA was developed which incorporates a heat-mediated
ICD step prior to testing for detection of NS 1 antigen in clinical dengue samples. The clinical specimens (n = 15 NS 1 -positive by Panbio® Early ELISA and n = 20 dengue negative serum samples) for this study were submitted for diagnostic testing as part of a dengue surveillance programs - the island-wide, passive dengue surveillance system (PDSS) and tested according to the routine diagnostic algorithm as described in Rigau-
Perez, Puerto Rico Health Sciences Journal, 18:337-345, 1999; and Sharp et al., PLoS
Neglected Tropical Diseases, 7:e2159, 2013.
[00244] NS 1 Antigen capture ELISA
[00245] The NS 1 antigen capture ELISA was identical to a standard antigen capture ELISA except for a brief serum heat denaturation step prior adding the specimen to the microtiter plate. Briefly, microtiter plate wells were coated with capture mAb 4G3 (100 ul/well) at concentration 10 ug/ml overnight at 4°C. Following overnight incubation, excess unbound NSl capture mAb was removed and the plate was blocked for 45 min with PBS or TBS containing 2% BSA plus 0.05% Tween-20 at RT. Serum samples were diluted 1 :2 in PBST and heat-denatured in 1 .5-ml Eppendorf tubes at 70°C for 10 min. Following blocking step, the microtiter plate was washed 3x and the serum samples - both heat denatured and non-denatured in parallel wells and incubated for 1 h at 37°C. The microtiter plate was then washed 3x, and biotinylated 2G1 mAb (1 :800) was added and incubated for 1 hour at 37°C. The microtiter plate was then washed 3x and streptavidin-conjugated HRP (100 ul/well) was added. Following a final 5* washes, the plate was incubated for 1 hour at 37°C, color development and absorbance measurement were performed as described above for iELISA. Paired OD450 values comparison before and after heat-denaturation for each serum specimen was calculated using Wilcoxon Signed-Rank Test.
[00246] Artificial NS l -antibody immune complex formation and heat-mediated dissociation
[00247] To further demonstrate the utility of the NSl antigen capture ELISA for detection of immunecomplexed NSl antigen, artificial NSl antigen-antibody complexes in vitro were generated by mixing purified rNS l protein at a constant concentration of 1 ug/ml while varying the rabbit anti-NS l polyclonal antibodies (Canada Immunoassay Development, Ontario, Canada). After an overnight incubation at 4°C, immunecomplexed specimens were analyzed by NS l capture ELISA with and without heat denaturation step as described above. The negative control included rNSl antigen (1 ug/ml) diluted in PBS and incubated overnight at 4°C.
[00248] Performance of NSl capture ELISA with heat-denatured and non-denatured specimens
[00249] mAb 2G1 of LD2 epitope was paired with each one of the 5 different NS l mAbs of LXl epitope and evaluated using the sandwich ELISA format. Based on this analysis, 4G3 and 2G1 provided the most robust DENV NSl detection in the described NS l antigen capture ELISA. The optimal concentration of each mAb was then determined by titration ELISA and the limit of detection (LOD) of the assay by serotype
was determined from a standard curve using known concentration of serially diluted rNS l protein from of the four DENV serotypes. As shown in Figure IB, the ELISA had the highest analytical sensitivity for DENV-1 (LOD = 2 ng/ml) followed by DENV-2 and DENV-3 (LOD = 4 ng/ml) but had relatively lower analytical sensitivity to DENV-4 (LOD = 8 ng/ml). This data corroborated the cross-reactivity patterns of mAbs 4G3 and 2G1 determined by iELISA, see Figures 11A-F. The NSl capture ELISA was used to detect NS l antigen in clinical specimens using a total of 15 DENV NS l -positive serum samples. In addition, 20 dengue negative serum samples were used to validate the assay. Heat denaturation of DENV-positive serum samples resulted in approximately three-fold increase in OD450 in the capture ELISA. The average value for paired OD450 differences for dengue positive serum samples before and after heat denaturation was 0.9 (range 0.2-2.0, p value 0.007). In contrast to positive sera, however, the average OD450 values for negative control sera was 0.12 with a standard deviation 0.03, and with no significant OD450 value differences between heat-denatured and non-denatured serum samples (Figure 2).The observed increased detection in DENV-positive sera following heat treatment could be due to the mAbs high affinity to heat-denatured NS l , heat- mediated release of NSl from immunecomplexes, or both. Artificial immune complexes were generated that could mimic secondary DENV infections, and these samples were tested with and without heat denaturation by the NS l capture ELISA. Without heat- mediated ICD step, the OD values were inversely proportional to the concentration of polyclonal antibodies used to create the immunecomplexed rNS l protein, indicating the rNSl was trapped in the immunecomplex. Similar to DENV NS l -positive sera, the OD values were significantly enhanced after heat treatment regardless of the concentration of anti-NS l polyclonal antibodies used to immunecomplex rNS l antigen, see Figure 12, evidence for the release of NS l from the immunecomplexes as consequence of heat- mediated ICD. Taken together these results indicated that an NSl capture ELISA according to aspects of the present invention is useful for early detection of DENV secondary infections.
[00250] An antigen capture ELISA for detection of DENV NS l protein was developed using a pair of mAbs, mAb 4G3 and mAb 2G1 . Figure 1A is a schematic diagram showing DENV NS l capture ELISA format used. mAbs 4G3 and 2G1 have high affinity cross-reactivity to all 4 DENV serotypes and distinct non-overlapping
binding sites to DENV NS 1. Any of mAbs 1 A6, 1F4, 3D5 and 2G2 can be used in the same or similar capture ELISA assay with mAb 2G1.
[00251] Microtiter plates are coated with mAb 4G3. For this, the capture monoclonal antibody (mAb), 4G3, is diluted using a coating buffer, bicarbonate buffer pH 9.6, to make the final concentration of \ 0μg/mL·. The microtiter plate is then coated by adding ΙΟΟμΙ of the ^g/mL mAb 4G3 per well and the microtiter plate is then incubated overnight (16-18 hours) at 4°C. The excess unbound capture antibody is then removed and the microtiter plate is tapped against dry paper towels.
[00252] The wells of the microtiter plate are blocked to inhibit non-specific binding by adding 300μ1 Tris-buffered saline (25mM Tris, 150mM NaCl; pH 7.2) containing 2% bovine serum albumin (BSA) plus 0.1% Tween-20 per well and incubating at room temp for 45 minutes. The microtiter plate is then washed three times with washing buffer (Tris-buffered saline plus 0.05% Tween-20).
[00253] Serum samples are diluted and heat treated and transferred into the microtiter plate. For this, serum samples are diluted at dilution 1 :2 in Tris-buffered saline plus 0.05% Tween-20. In order to test serum samples in duplicate assay prepare 250ul in 1.5ml Eppendorf tubes. Eppendorf tubes containing serum samples are place in a heat block at 70°C for 10 minutes, producing heat-treated serum samples. The heat-treated serum samples are centrifuged at 12,000 rpm for 3 to 5 minutes. An aliquot of ΙΟΟμΙ of each heat treated serum sample is added in duplicate wells of the microtiter plate. Diluent alone is used as a blank control. The microtiter plate is incubated at 20-37°C for 60-90 minutes and then washed three times with washing buffer (Tris-buffered saline plus 0.05% Tween-20).
[00254] Detectably labeled secondary (detection) antibody, mAb 2G1 , is added to each well of the microtiter plate following this incubation. An aliquot of ΙΟΟμΙ of the detection monoclonal antibody, biotinylated mAb 2G1 , at dilution 1 :400-1 :2000 in a (Tris-buffered saline plus 0.05% Tween-20) is added to each well and the microtiter plate is incubated at 20-37°C for 60-90 minutes. Diluent alone is added to wells designated as blank. Following this incubation, the microtiter plate is washed five times with washing buffer (Tris-buffered saline plus 0.05% Tween-20). An aliquot of Ι ΟΟμΙ Streptavidin- tagged Horseradish Peroxidase (manufactured by Thermo Scientific) at dilution 1 :2000- 1 :4000 in a blocking buffer (Tris-buffered saline containing 2% BSA and 0.1 % Tween- 20) is added to each well and the microtiter plate is then incubated at 37°C for 1 hour.
Diluent alone is added to wells designated as blank. The microtiter plate is then washed ten times with washing buffer (Tris-buffered saline plus 0.05% Tween-20). An aliquot of ΙΟΟμΙ 3,3',5,5'-Tetramethylbenzidine (TMB) liquid substrate (manufactured by KPL, USA) is then added to each well and the microtiter plate is incubated for 10 min at room temp in a dark box. The reaction is stopped by adding ΙΟΟμΙ TMB Stop Solution (manufactured by KPL, USA) to each well. Signal from wells of the microtiter plate is then read at 450nm within three minutes.
[00255] The ELISA showed good analytical sensitivity for heat treated rNSl proteins from all 4 DENV serotypes except for DENV4. Figure I B is a graph showing results of DENV NS1 capture ELISA and showing the lower detection limit (LDL) of the assay for each DENV serotype. The analytical sensitivity of assay in order: DENVl > DENV3, DENV2 > DENV4. This is consistent with the reactivity patterns of these two mAbs presented in Figure 4. For all 4 DENV serotypes, purified rNS l proteins expressed in mammalian cell line were used.
[00256] Serum samples from dengue positive and negative individuals were tested by performing ELISA using the two mAbs, 4G3 and 2G1 with and without prior heat treatment of the serum samples.
[00257] Figure 2 is a graph showing a comparison of absorbance values between serum samples with and without heat treatment prior to testing by the DENV NS1 capture ELISA. Both dengue positive (n=15) and negative (n=20) serum samples were included in this preliminary evaluation. The average OD value differences for paired heat treated (HT)/non-heat treated (NHT) dengue positive serum samples was 0.9 (rage 0.2 - 2.0) with p value=0.007 (Wilcoxon Signed-Rank Test).The average OD value for negative controls was 0.12 with a SD=0.03. The negative samples are represented by broken lines. As shown in Fig. 2, heat treatment of serum samples resulted in at least three-fold increase in detection signal of the ELISA, indicating the epitopes for the mAbs used were better exposed after heat treatment.
[00258] Figure 3A is a graph showing the effect of heat treatment on detection of artificially immune complex DENV NS 1 with 4G3 mAb at different dilutions by an NS 1 capture ELISA according to aspects of the present invention, wherein immune complex formation was performed overnight at 4°C in PBST containing 10% Normal Human Serum (NHS). Figure 3B is a graph showing the effect of heat treatment on detection of artificially immune complex DENV NS1 with 4G3 mAb at different dilutions by the
DENV NS 1 capture EL1SA, wherein immune complex formation was performed overnight at 4°C in in PBST without NHS.
[00259] In both Figures 3A and 3B, HT-ICD refers to heat-mediated immune complex dissociation; HT-EDTA-ICD refers to Heat-mediated immune complex dissociation in presence of EDTA (anti-coagulant); and NHT-ICD refers to no heat- mediated immune complex dissociation. Immune complex formation at 1 :100 and 1 :500 of 4G3 dilution, respectively, are shown along with results of incubating NS 1 solution in the absence of immune complex. The concentration of NS 1 either as free or immune complex solution was ^g/ml.
[00260] Any patents or publications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication is specifically and individually indicated to be incorporated by reference.
[00261] The compositions and methods described herein are presently representative of preferred embodiments, exemplary, and not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art. Such changes and other uses can be made without departing from the scope of the invention as set forth in the claims.
Claims
1. A method for detecting dengue virus infection in a cell or organism, comprising:
obtaining a biological sample of the cell or organism;
providing one or more antibodies or antigen binding fragments characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQY FQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof; and assaying dengue virus non-structural protein 1 in the biological sample using the one or more antibodies or antigen binding fragments, wherein detection of dengue virus non-structural protein 1 is indicative of dengue virus infection in the cell or organism.
2. The method of claim 1 , wherein the organism is a human subject having or suspected of having a dengue virus infection, and further comprising:
heating the biological sample, producing a heat-treated biological sample prior to assaying dengue virus non-structural protein 1 in the heat-treated biological sample using the one or more antibodies or antigen binding fragments.
3. The method of claim 1 or 2, wherein the biological sample is selected from the group consisting of: whole blood, plasma, serum, urine, extracellular fluid, cytosolic fluid, and tissue.
4. The method of claim 1, wherein the organism is a mosquito.
5. The method of claim 1, wherein the cell is in vitro.
6. The method of any one of claims 1 - 5, wherein the assaying comprises an
ELISA or an antigen capture assay.
7. The method of any one of claims 1- 6, wherein the antigen capture assay is a lateral flow assay.
8. The method of any one of claims 2, 3 or 6-7, wherein the dengue virus infection is a secondary dengue virus infection.
9. A dengue virus non-structural protein 1 immunoassay device, comprising: a solid porous or non-porous support comprising a first antibody or antigen binding fragment characterized by specific binding to a first epitope of dengue virus non- structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l);
VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3);
GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5);
RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7);
TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof.
10. The dengue virus non-structural protein 1 immunoassay device of claim 9, further comprising a conjugate pad comprising a detectably labeled second antibody or antigen binding fragment characterized by specific binding to a second epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof.
1 1. The dengue virus non-structural protein 1 immunoassay device of claim 9 or 10, further comprising a conjugate pad comprising detectably labeled dengue virus non-structural protein 1.
12. The dengue virus non-structural protein 1 immunoassay device of any one of claims 9-1 1, further comprising a wicking pad.
13. The dengue virus non-structural protein 1 immunoassay device of any one of claims 9 - 12, further comprising a housing.
14. An isolated antibody or antigen binding fragment thereof characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof.
15. The isolated antibody or antigen binding fragment thereof of claim 14, wherein the isolated antibody or antigen binding fragment thereof is an isolated monoclonal antibody or antigen binding fragment thereof.
16. The isolated antibody or antigen binding fragment thereof of claim 14, wherein the antibody or antigen binding fragment thereof is an isolated polyclonal antibody or antigen binding fragment thereof.
17. The isolated antibody or antigen binding fragment thereof of any one of claims 14 - 16, comprising an attached detectable label.
18. The isolated antibody or antigen binding fragment thereof of any one of claims 14 - 17, immobilized on a solid or semi-solid support.
19. An isolated immunogenic peptide having 25 or fewer amino acid residues and comprising an epitope selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof.
20. An immunoassay kit for detecting dengue virus infection in a human subject having or suspected of having a dengue virus infection, comprising: one or more antibodies or antigen binding fragments characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHT WTEQ YKFQPE SP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof.
21 . The immunoassay kit for detecting dengue virus infection in a human subject having or suspected of having a dengue virus infection of claim 20, wherein the one or more antibodies or antigen binding fragments comprises a first antibody or antigen binding fragment characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG(SEQ ID NO: l); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO: 8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof; and a second antibody or antigen binding fragment characterized by specific binding to VHTWTEQYKFQADSP (SEQ ID NO:2) and/or VHTWTEQYKFQPESP(SEQ ID NO:3); or a variant of either thereof.
22. The method of any one of claims 1-8, the device of any one of claims 9- 13, the isolated antibody or antigen binding fragment of any one of claims 14 -15 or 17- 18 or the immunoassay kit of claim 20 or 21, wherein the antibody characterized by specific binding to an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: l); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYSWKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof is selected from the group consisting of: 2G1, 1A6, 1F4, 3D5, 2G2 and 4G3; and wherein
the antigen binding fragment is an antigen binding fragment of an antibody selected from the group consisting of: 2G1, 1A6, 1F4, 3D5, 2G2 and 4G3.
23. The method of any one of claims 1-8 and 22, the device of any one of claims 9-13 and 22, the isolated antibody or antigen binding fragment of any one of claims 14 -15, 17-18 and 22 or the immunoassay kit of any of claims 20-22, wherein the antibody is further characterized by lack of specific binding to yellow fever virus NS1 and West Nile virus NS1.
24. The method of any one of claims 1 -8 and 22-23, the device of any one of claims 9-13 and 22-23, the isolated antibody or antigen binding fragment of any one of claims 14 -15, 17-18 and 22-23 or the immunoassay kit of any of claims 20-23, wherein the antibody has an affinity constant (KA) greater than 1 X 106 for an epitope of dengue virus non-structural protein 1 selected from the group consisting of: YSWKSWG (SEQ ID NO: 1); VHTWTEQYKFQADSP (SEQ ID NO:2); VHTWTEQYKFQPESP (SEQ ID NO:3); GPQPMEHKYSWKSWG (SEQ ID NO:4); RPQPMEHKYSWKSWG (SEQ ID NO:5); RPQPTELKYS WKTWG (SEQ ID NO:6); TPQPMELKYSWKTWG (SEQ ID NO:7); TPPVSDLKYSWKTWG (SEQ ID NO:8); and YSWKSWGKAKIIGAD (SEQ ID NO:9); and a variant of any thereof.
25. A method for detecting and/or assessing dengue virus infection in a human subject substantially as described herein.
26. An immunoassay device for assaying dengue virus non-structural protein 1 in a heat-treated biological sample substantially as described herein.
27. An isolated antibody or antigen binding fragment thereof characterized by binding to dengue virus non-structural protein 1 , substantially as described herein.
28. An isolated immunogenic peptide substantially as described herein.
29. A method for detecting and/or assessing dengue virus infection in a mosquito or in vitro cell substantially as described herein.
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WO2017223286A1 (en) * | 2016-06-23 | 2017-12-28 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Dengue virus non-structural protein 1 specific binding polypeptides and methods of using the same |
WO2018208741A1 (en) * | 2017-05-08 | 2018-11-15 | University Of Pittsburgh-Of The Commonwealth System Of Higher Education | Methods and compositions for the detection of flavivirus infections |
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WO2020043069A1 (en) * | 2018-08-28 | 2020-03-05 | 东莞市朋志生物科技有限公司 | Ns1-binding protein |
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US20130011427A1 (en) * | 2009-12-16 | 2013-01-10 | The Johns Hopkins University | Flavivirus species-specific peptide tags for vaccine and diagnostic use |
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US11407818B2 (en) | 2016-06-23 | 2022-08-09 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Antibodies that bind to dengue virus serotype 4 (DENV4) and methods of making and using the same |
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