CA2081888A1 - Method of determining type specific antibodies to herpes simplex virus types 1 and 2 - Google Patents
Method of determining type specific antibodies to herpes simplex virus types 1 and 2Info
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- CA2081888A1 CA2081888A1 CA002081888A CA2081888A CA2081888A1 CA 2081888 A1 CA2081888 A1 CA 2081888A1 CA 002081888 A CA002081888 A CA 002081888A CA 2081888 A CA2081888 A CA 2081888A CA 2081888 A1 CA2081888 A1 CA 2081888A1
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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
- G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
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Abstract
This enclosure concerns a method of serotyping antibodies to HSV
2 or 1 or in a sample suspected to contain HSV antibodies which comprises: a) reacting the sample and at least one monoclonal antibody specific for HSV-2 or HSV-1 glycoprotein G with an antigen wherein said monoclonal antibody is not susceptible to heterologous blocking; and b) determining whether antibody binding has occurred. The serotyping method of the disclosure can be done in a variety of formats such as sequential or simultaneous. In another embodiment, this disclosure concerns kits for serotyping antibodies to HSV-1 and HSV-2.
2 or 1 or in a sample suspected to contain HSV antibodies which comprises: a) reacting the sample and at least one monoclonal antibody specific for HSV-2 or HSV-1 glycoprotein G with an antigen wherein said monoclonal antibody is not susceptible to heterologous blocking; and b) determining whether antibody binding has occurred. The serotyping method of the disclosure can be done in a variety of formats such as sequential or simultaneous. In another embodiment, this disclosure concerns kits for serotyping antibodies to HSV-1 and HSV-2.
Description
WO 91/17~3 ~ 8 ~ PCT/US9~/02837 .
~;~
A METHOD OF DETERMINING TYP~ SPECIFIC ~NTIBODIES TO
~ERPES SIMæLEX VIRUS TYPES 1 AND 2 FIELD OF TH~ E~IQ~
This invention concerns a method of serotyping antibodies to herpes simplex virus types 1 and 2, and, in particular, to a method of serotyping which employs monoclonal antibodies to HSV l gG and HSV-2 gG which are not susceptible to heterologous blocking.
B~CK~RO~ OF ~ YEN~
T~.e most striking characteri~tic of herpes simplex virus (HSV) is its propensity ~or persisting in a quiescent or latent state in man, with recurrence of activity at irregular intervals. ~here are two main immunologic variants, types 1 and 2, which crossreact.
One of the major problems in seroepidemiologic studies of HSV infection is the differentiatiôn of antibodies in human serum directed against HSV l and HSV-2.:
~0 Researchers have attempted to overcome these probl~ms using purified HSV glycoproteins and absorption to remove cross reacting antibodies in an ef ort to develop a rapid method of distinguishing ~SY-l and HSV-2 ~ --antibodies. Such methods have important clinical and ; 25 epidemiological significance because it permits diagnosis of persons with asymptomatic HSV infection, identificat~on of candidates for antiviral therapy, as well as identification of pregnant women who may transmit a potentially ~a~al H5V infect~on to the newborn.
U.~. Patent 4,764,459, lssued to Hampar et al. on - August 16, 1988, and Hampar et al., J. Clin. Microbiol., 21: 496--500 ~l985), describe an ELISA to determine antibodies against HSV ~ypes 1 a~d 2 in hum~n sera. The method utalizes heterologous virus-infected cell .
~, :
W0 91~17443 2 ~ s PCr/US91 /02837 extracts to absorb human sera to remove intertypic cross-reactive antibodies. Specifically, the sera were absorbed with heterologous virus-infected cell extracts-to remove cross-reacting antibodies and then were applied to E~ISA plates containing t~e target antigens, immunoaffinity-purified HSV-1 glycoproteins gC and gD
and HSV-2 glycoproteins gD and gF. T~e absor~ance index, defined as the ratio of the absorbance generated by a serum sample absorbed with a heterologo~s virus-infected cell extract ~ersus the absorbance generated bya serum sample absorbed with an u~infected cell extract, was used to determine the pres~nce or absence of antib~diPs to HSV-1 and HSV-2.
U.S. Patent 4,855,224, issued to Berman et al. on August 8, 1989, describes the use of a molecularly cloned polypeptide with antigenic determinan~s capable of specifically binding complementa-y antibody. Using an HSV-1 glycoprotein D as the polypeptide, HSV
antib~di~s could be determined. ~SV-2 antibodies co~ld be detected using HSV-2 glycoprotein C fragmen~.
Arvin et al. J Infection and Immunity, 40: 189-189 ~1983), describe an immunoaffinity purification of HSV-l glycoprotein C with a monoclonal antibody and the use of this purified glycoprotein in a solid phase RIA for type-specific antibodies to HSV-l.
Svenner~olm et al., ~. Clin. Microb~ol., l9:
235-239 (February 1984), describe the use Of h~
~Qm~S~a lectin puri~ied ~SV-1 and HSV-2 antigens to determine type specific a~tibodies in huma~ sera.
Identi~cation cf ~he h~ Qm~i~ lectin purified HSV
antigens ~HSV-l gC and HSV-? gG) was described by Olofsson et al., J. gen. Virol., 67: 737-744 ~1986).
Lee et al., J. Clin. Mirrobtol., 22: 641-644 (1985), describe the ev~ ~tion or an HSV-2 typo-.
... .
WO91/17~3 2 ~ ~ ~ g ~ 8 PCl/VS9l/02837 specific en~yme-linked immunodot serological assay based on af~ini~y-purified gG-2 as antigen.
Lee et al., J. Virol. Methods, 14: 111-118 ~1986), describe immunoaffinity purification of HSV-1 glycoprotein G using a monoclonal antibody. The purified antigen was coated on a solid phase ~n an lmmunodot enzymatic assay to detect HSV-1 antibodies.
Ross et al., J. Virology, 54: 851-855 ~1985), describe a competitive ELISA to test for human antibodies to antigenic site~ on HSV glycoproteins C and D, which are recognized by mouse monoclonal antibodies.
It was shown that there were antibodies i~ human sera that were capable of blocking the binding of monoclonal antibodies. Thus, even though the monoclonal antibodles used were specific for HSV-l or ~SV-2 the assay çould not be used to distinguish HSV-1 and HSV-2 antibodies because blockinq by heterologous human antibodies occurred~ i.e.,-human antibodies to HSV-l blocXed monoclonal antibodies spec~fic for HSV-2 and human anti~odies to HSV-2 bloeked monoclonal antibodies specific for HSV-1.
SU~ARY_Q~ ~HE I~VENTION
This invention concerns a method of serotyping - antibodies to HSV 2 or 1 or in a sample suspected to contain HSV antibodies wh~ch comprises:
a) reacting the sample and at least one monoclonal antibody sp cific for HSV-~ or HSV-l glycopro~ein G with an anti~en wherein said monoclonal antibody ls not susceptible to heter~logous blocking;
and b) determining whether antibody binding has ` occurred.
The serotyping method of the invention can be done in a variety o~ formats such as ~equential or simultaneous.
~ . .
. .
. . . .. . . ...... . . ... . .
- , . . . . . . : - . .:. .~ .
WO 9]/17443 ~ ~ ~ 8 ~ ~ PCT/US91/0~83, 4 .
In another embodiment, this invention concerns kits for serotyping antibodies to ~SV 1 and HSV-2.
BRIEF 12ESCI~IP~I!;?~ OF THE DRA~
- Figure 1 is a yraph depicting the absorbance values for an assay used to determine anti-HSV-l antibodies.
Figure 2 is a graph depicting the absorbance values for an assay to determine HSV-2 anti}:~odies.
DETAILED DEs~IpTI~2L~l~l~3~
The term "antigen" refers to an antigenic substance which can be natural, recombinant, or synt~etic, and which is capable of binding to the monoclonal antibodies and to the antibodies in the sample. The antigen need not be substantially pure as long as the contaminants do not interfere with the assay method.
~5 The term BSV-2 ylycoprotein G ~HSV-2 gG) refers to the HSV-2 glycoprotein- as described by Roizman et al.j Virology 133 242-24~, 1984; Marsden et al., J. Virology ~iQ, 547-554, l9B4; Lee et al., J. Clin. Microbiol. ~, 641-644, 1985; Balachandran et al., J. Virology ~, 825-B32, 1985; and Su et al., J. Yirology .fi2, 3668-3679, 19B8.
The term HSV-1 glycoprotein G (HSV-1 gG) refers to the BSV-l glycoprotein as described by Ackermann et al., Vir;:>logy-~Q, 207-220, 1986; Richmarl et al., J. Virolo~y ~ Z, 647-655, 1986; and Sullivan et al., J. Gen. Virol.
~, 25~7-2S98, 1987.
The assay of tbe present invention is an extsaordinarily simple and speclfic methsd to serotype antibodies to HSV-2 or HSY~l which overcomes the cross-reactivity problems which have plagued researchers in differentiatirlg these antibodies. HSV-1 antibodles are sero~cypecl by using at le~st one monoclonal antibody specific for ~SV-1 glycc~protein G which is not suscep~le to heterologous blocking. ~SV-~ an~ibodies are serot:yped by using at least one monoclonal ant~body ~, '... '. ... ..
.. ~ , . .. .. . . . .
WO91/17~3 2 ~ P~T/US91/02837 S
specific for HSV-2 glyooproteln G which is not susceptible to heterolo~ous blocking. These monoclonal antibodies can be unlabeled or labeled as is discussed below.
The following is one illustration of the method of the invention: HSV antigen, types 1 and 2, is immobilized on a suita~le support. It is then reacted with a sample, usually serum or plasma, which is suspected to contain HSV antibodies whioh, ~f present, will bind to the antigen, and, thus, block binding of the monoclonal antibody. After excess reagents are removed by washing, a labeled monoclonal antibody is incubated with the mixture. Antibodies to HSV-1 are detected by using a monoclonal antibody specific for HSV-l glycoprotein G . Antibodies to HSV-2 are detected by usiny a monoclonal antibody specific ~or HSV-2 glycoprotein G. The mixture is washed again and antibody binding is determined by measuring the amount of reporter bound to the solid support.
The ~ample need not be limited to serum or plasma.
Any biological fluid suspected to con~ain HSV antibodies can be used as the sample.
Techniques for preparing monoclonal antibodies - which can~~e used to practice the invention are well known. The preparation o~ monoclonal antibodies which recognize either RSV-l or HSV-2 glycoprotein ~ have been described in a variety of publications such as Roizman et al., Virology 1~, 242-247, 1984; ~arsden ~t al., J.
Yirology ~Q, 547-554, ~984; Balachandran et al., J.
- 30 ~irology ~, 825-832, l9B5; Lee et al., J. Clin.
Microbio~. 2~, 641-644, 1985; Ackermann e~ alO, Virology l~Q, 20---220, 198Ç; and R~chman et al. r J. Virology ~, 6~7-~55, 19~.
~; T~e antibody can be labeled with a reporter sr wit~
a m~mber of an immune or ^on-immune speeific binding '~' W~1/17~3 2 0 '3 ~ PCT/US91/02837 pair. When the latter is used, detection is effected by reacting the complex with the other member of the binding pair whic~ is labeled.
Members of specific binding pairs suitable for practicing the invention can be of the immune or non-immune type. Examples of immune specific binding pairs include antigen-antibody sy~tems o~ hapten-anti-hapten systems. The antibody member of the specific binding pair can be produced by customary methods familiar to those skilled in the art. Such methods involve immunizing an animal with the antigen member of the specific binding pair. If the antigen member of the specific binding pair is not immunogenic, i.e., a hapten, it can be covalently coupled to a carrier protein to render it immunogenic. The antibody member whether polyclonal, monoclonal, or an immunoreactive fragment thereof can be produced by conventional technlques well known to those skiiled in ~he art. The terms immunoreactive antibody fragment or immunoreactive fragment mean fra~ments which contain the binding region of the antibody. Such fragments can be Fab-type fragments which are defined as fragments devoid o~ the Fc portion, e.g., Fab, Fab' and F(ab'~2 fragments, or may be so-called "half-molecule~' fragments obtained by reductive cleavage of the disulfide bonds connecting t~e heavy chain components of the intact antibody.
Non-immune binding pairs include systems wherein the two components share a natural affinity for each other but are not antibod~es. Exemplary non-lmmune binding pairs are biotin-avidin or b~otin-streptavidin, folic acid-fola~e binding protei~, complementary probe nucleic acids, etc.
A variety of methods are available ~o 20valently ~abel monoclonal an~ibodies with members of spec~f~c blndin~ pairs. ~ethods are selected based upon the : .
., .
~;~
A METHOD OF DETERMINING TYP~ SPECIFIC ~NTIBODIES TO
~ERPES SIMæLEX VIRUS TYPES 1 AND 2 FIELD OF TH~ E~IQ~
This invention concerns a method of serotyping antibodies to herpes simplex virus types 1 and 2, and, in particular, to a method of serotyping which employs monoclonal antibodies to HSV l gG and HSV-2 gG which are not susceptible to heterologous blocking.
B~CK~RO~ OF ~ YEN~
T~.e most striking characteri~tic of herpes simplex virus (HSV) is its propensity ~or persisting in a quiescent or latent state in man, with recurrence of activity at irregular intervals. ~here are two main immunologic variants, types 1 and 2, which crossreact.
One of the major problems in seroepidemiologic studies of HSV infection is the differentiatiôn of antibodies in human serum directed against HSV l and HSV-2.:
~0 Researchers have attempted to overcome these probl~ms using purified HSV glycoproteins and absorption to remove cross reacting antibodies in an ef ort to develop a rapid method of distinguishing ~SY-l and HSV-2 ~ --antibodies. Such methods have important clinical and ; 25 epidemiological significance because it permits diagnosis of persons with asymptomatic HSV infection, identificat~on of candidates for antiviral therapy, as well as identification of pregnant women who may transmit a potentially ~a~al H5V infect~on to the newborn.
U.~. Patent 4,764,459, lssued to Hampar et al. on - August 16, 1988, and Hampar et al., J. Clin. Microbiol., 21: 496--500 ~l985), describe an ELISA to determine antibodies against HSV ~ypes 1 a~d 2 in hum~n sera. The method utalizes heterologous virus-infected cell .
~, :
W0 91~17443 2 ~ s PCr/US91 /02837 extracts to absorb human sera to remove intertypic cross-reactive antibodies. Specifically, the sera were absorbed with heterologous virus-infected cell extracts-to remove cross-reacting antibodies and then were applied to E~ISA plates containing t~e target antigens, immunoaffinity-purified HSV-1 glycoproteins gC and gD
and HSV-2 glycoproteins gD and gF. T~e absor~ance index, defined as the ratio of the absorbance generated by a serum sample absorbed with a heterologo~s virus-infected cell extract ~ersus the absorbance generated bya serum sample absorbed with an u~infected cell extract, was used to determine the pres~nce or absence of antib~diPs to HSV-1 and HSV-2.
U.S. Patent 4,855,224, issued to Berman et al. on August 8, 1989, describes the use of a molecularly cloned polypeptide with antigenic determinan~s capable of specifically binding complementa-y antibody. Using an HSV-1 glycoprotein D as the polypeptide, HSV
antib~di~s could be determined. ~SV-2 antibodies co~ld be detected using HSV-2 glycoprotein C fragmen~.
Arvin et al. J Infection and Immunity, 40: 189-189 ~1983), describe an immunoaffinity purification of HSV-l glycoprotein C with a monoclonal antibody and the use of this purified glycoprotein in a solid phase RIA for type-specific antibodies to HSV-l.
Svenner~olm et al., ~. Clin. Microb~ol., l9:
235-239 (February 1984), describe the use Of h~
~Qm~S~a lectin puri~ied ~SV-1 and HSV-2 antigens to determine type specific a~tibodies in huma~ sera.
Identi~cation cf ~he h~ Qm~i~ lectin purified HSV
antigens ~HSV-l gC and HSV-? gG) was described by Olofsson et al., J. gen. Virol., 67: 737-744 ~1986).
Lee et al., J. Clin. Mirrobtol., 22: 641-644 (1985), describe the ev~ ~tion or an HSV-2 typo-.
... .
WO91/17~3 2 ~ ~ ~ g ~ 8 PCl/VS9l/02837 specific en~yme-linked immunodot serological assay based on af~ini~y-purified gG-2 as antigen.
Lee et al., J. Virol. Methods, 14: 111-118 ~1986), describe immunoaffinity purification of HSV-1 glycoprotein G using a monoclonal antibody. The purified antigen was coated on a solid phase ~n an lmmunodot enzymatic assay to detect HSV-1 antibodies.
Ross et al., J. Virology, 54: 851-855 ~1985), describe a competitive ELISA to test for human antibodies to antigenic site~ on HSV glycoproteins C and D, which are recognized by mouse monoclonal antibodies.
It was shown that there were antibodies i~ human sera that were capable of blocking the binding of monoclonal antibodies. Thus, even though the monoclonal antibodles used were specific for HSV-l or ~SV-2 the assay çould not be used to distinguish HSV-1 and HSV-2 antibodies because blockinq by heterologous human antibodies occurred~ i.e.,-human antibodies to HSV-l blocXed monoclonal antibodies spec~fic for HSV-2 and human anti~odies to HSV-2 bloeked monoclonal antibodies specific for HSV-1.
SU~ARY_Q~ ~HE I~VENTION
This invention concerns a method of serotyping - antibodies to HSV 2 or 1 or in a sample suspected to contain HSV antibodies wh~ch comprises:
a) reacting the sample and at least one monoclonal antibody sp cific for HSV-~ or HSV-l glycopro~ein G with an anti~en wherein said monoclonal antibody ls not susceptible to heter~logous blocking;
and b) determining whether antibody binding has ` occurred.
The serotyping method of the invention can be done in a variety o~ formats such as ~equential or simultaneous.
~ . .
. .
. . . .. . . ...... . . ... . .
- , . . . . . . : - . .:. .~ .
WO 9]/17443 ~ ~ ~ 8 ~ ~ PCT/US91/0~83, 4 .
In another embodiment, this invention concerns kits for serotyping antibodies to ~SV 1 and HSV-2.
BRIEF 12ESCI~IP~I!;?~ OF THE DRA~
- Figure 1 is a yraph depicting the absorbance values for an assay used to determine anti-HSV-l antibodies.
Figure 2 is a graph depicting the absorbance values for an assay to determine HSV-2 anti}:~odies.
DETAILED DEs~IpTI~2L~l~l~3~
The term "antigen" refers to an antigenic substance which can be natural, recombinant, or synt~etic, and which is capable of binding to the monoclonal antibodies and to the antibodies in the sample. The antigen need not be substantially pure as long as the contaminants do not interfere with the assay method.
~5 The term BSV-2 ylycoprotein G ~HSV-2 gG) refers to the HSV-2 glycoprotein- as described by Roizman et al.j Virology 133 242-24~, 1984; Marsden et al., J. Virology ~iQ, 547-554, l9B4; Lee et al., J. Clin. Microbiol. ~, 641-644, 1985; Balachandran et al., J. Virology ~, 825-B32, 1985; and Su et al., J. Yirology .fi2, 3668-3679, 19B8.
The term HSV-1 glycoprotein G (HSV-1 gG) refers to the BSV-l glycoprotein as described by Ackermann et al., Vir;:>logy-~Q, 207-220, 1986; Richmarl et al., J. Virolo~y ~ Z, 647-655, 1986; and Sullivan et al., J. Gen. Virol.
~, 25~7-2S98, 1987.
The assay of tbe present invention is an extsaordinarily simple and speclfic methsd to serotype antibodies to HSV-2 or HSY~l which overcomes the cross-reactivity problems which have plagued researchers in differentiatirlg these antibodies. HSV-1 antibodles are sero~cypecl by using at le~st one monoclonal antibody specific for ~SV-1 glycc~protein G which is not suscep~le to heterologous blocking. ~SV-~ an~ibodies are serot:yped by using at least one monoclonal ant~body ~, '... '. ... ..
.. ~ , . .. .. . . . .
WO91/17~3 2 ~ P~T/US91/02837 S
specific for HSV-2 glyooproteln G which is not susceptible to heterolo~ous blocking. These monoclonal antibodies can be unlabeled or labeled as is discussed below.
The following is one illustration of the method of the invention: HSV antigen, types 1 and 2, is immobilized on a suita~le support. It is then reacted with a sample, usually serum or plasma, which is suspected to contain HSV antibodies whioh, ~f present, will bind to the antigen, and, thus, block binding of the monoclonal antibody. After excess reagents are removed by washing, a labeled monoclonal antibody is incubated with the mixture. Antibodies to HSV-1 are detected by using a monoclonal antibody specific for HSV-l glycoprotein G . Antibodies to HSV-2 are detected by usiny a monoclonal antibody specific ~or HSV-2 glycoprotein G. The mixture is washed again and antibody binding is determined by measuring the amount of reporter bound to the solid support.
The ~ample need not be limited to serum or plasma.
Any biological fluid suspected to con~ain HSV antibodies can be used as the sample.
Techniques for preparing monoclonal antibodies - which can~~e used to practice the invention are well known. The preparation o~ monoclonal antibodies which recognize either RSV-l or HSV-2 glycoprotein ~ have been described in a variety of publications such as Roizman et al., Virology 1~, 242-247, 1984; ~arsden ~t al., J.
Yirology ~Q, 547-554, ~984; Balachandran et al., J.
- 30 ~irology ~, 825-832, l9B5; Lee et al., J. Clin.
Microbio~. 2~, 641-644, 1985; Ackermann e~ alO, Virology l~Q, 20---220, 198Ç; and R~chman et al. r J. Virology ~, 6~7-~55, 19~.
~; T~e antibody can be labeled with a reporter sr wit~
a m~mber of an immune or ^on-immune speeific binding '~' W~1/17~3 2 0 '3 ~ PCT/US91/02837 pair. When the latter is used, detection is effected by reacting the complex with the other member of the binding pair whic~ is labeled.
Members of specific binding pairs suitable for practicing the invention can be of the immune or non-immune type. Examples of immune specific binding pairs include antigen-antibody sy~tems o~ hapten-anti-hapten systems. The antibody member of the specific binding pair can be produced by customary methods familiar to those skilled in the art. Such methods involve immunizing an animal with the antigen member of the specific binding pair. If the antigen member of the specific binding pair is not immunogenic, i.e., a hapten, it can be covalently coupled to a carrier protein to render it immunogenic. The antibody member whether polyclonal, monoclonal, or an immunoreactive fragment thereof can be produced by conventional technlques well known to those skiiled in ~he art. The terms immunoreactive antibody fragment or immunoreactive fragment mean fra~ments which contain the binding region of the antibody. Such fragments can be Fab-type fragments which are defined as fragments devoid o~ the Fc portion, e.g., Fab, Fab' and F(ab'~2 fragments, or may be so-called "half-molecule~' fragments obtained by reductive cleavage of the disulfide bonds connecting t~e heavy chain components of the intact antibody.
Non-immune binding pairs include systems wherein the two components share a natural affinity for each other but are not antibod~es. Exemplary non-lmmune binding pairs are biotin-avidin or b~otin-streptavidin, folic acid-fola~e binding protei~, complementary probe nucleic acids, etc.
A variety of methods are available ~o 20valently ~abel monoclonal an~ibodies with members of spec~f~c blndin~ pairs. ~ethods are selected based upon the : .
., .
3 2~ PCr/us9l/0~837 nature o~ the member of the specific binding pair, the type of linkage desired, and the tolerance of the antibody to ~arlous conjugation chemistries. Biotin can be covalently coupled to monoclonal antibodies by utilizing commercially available active derivatives.
Some of these are biotin-N-hydroxysuccinimide which - binds to amine groups on proteins; biotin hydrazide which binds to carbohydrate moieties, aldehydes and carboxyl groups via a carbodiimide coupling; and biotin maleimide and iodoacetyl biotin which bind to sulfhydryl groups. Fluorescein can be coupled ~o pro~ei~ amine groups using fluorescein isothiocyanate. Dinitrophenyl groups can be coupled ~o protein amine groups using 2,4-dinitrobenzene sulfonate or 2,4-dinitrofluoro-benzene. Other standard methods of conjugate may beemployed ~o couple monoclonal antibodies to a member of a specific binding pair lncluding dialdehyde, carbodiimide coupling, homofunctional crosslinking, and heterobifunctional crosslinking. Carbodiimide coupling is an effective method of coupling carboxyl groups on one substance to amine groups on another. Carbodiimide coupling is facilitated by using the commercially available reagent, l-ethyl-3-(dimethylaminopropyl)-carbodiimide ~EDAC).
- 25 Homobifunctional crossli~kers, including the bifunctional imidoesters and b~functional N-hydroxy-- succinimide esters, are oommercially available and are employed for coupling amine groups on one suhstance to ami~e groups on another. Heterobifunctional crosslinkers are reagents which possess diffPrent functional groups. The most common commercially available heterobifunctionaI crosslinker~ have an amine ~ reacti~e N-hydroxysuccinimide ester as one functlonal - group, a~d a sulfhydryl reactive group as the ~econd ~ 35 func~ional group. The most common sulfhy~ryl reac~ive , .
., .
Wo91/17~3 2a~
,~ PCT/U591/02837 groups are maleimides, pyridyl disulfides and active halogens. One of the funct.ional groups may be a photoactiv~ aryl nitrene, wh~h upon irradiation reacts with a variety of groups.
A reporter can be linked directly or indirectly, covalently or non-covalent:Ly to the monoclonal antibodies or to a member of a specific binding pair.
Reporters can be radioactive isotopes7 enzymes, fluorogenic, chemiluminescent or electrochemlcal materials. Two commonly used radioisotopes are 125I and 3H. Standard radioactive iso~op~c l~beling procedures include the chloramine T, lactoperoxidase and Bolton-Hunter methods for 125I and reductive methylation for 3H~
Enzymes are also used as reporters fo~
immunoassays. These include, but are-~ot limited to, horseradish peroxidase, alkaline phosphatase, ~-galactosidase, glucose oxidase, luciferas~, ~-lactamase, urease and lysozyme. Labeling with enzymes is facilitated by using dialdehyde, carbodi1mide coupling, homobifunctional crosslin~ers and heterobifunctional crosslinkers as described above for labeling monoclonal antibodies with members of specific ` binding pairs. The labeling method chosen depends on .. . . . . .
the functional groups available on the enzyme and the material to be labeled, and the tolerance of both ~o the conjugation conditions. The labeling method used in the presen~ invention may be one ofl bu~ no~ limited to, any conventional methods currently employed including those described by Engvall and Pearlmann, Immunochemistry 8, 871 (1971), Avramea and Ternynck, Immunochemistry ~, -: 1175 (1971~, Ishikawa e~ al., J. Immunoa~say 4(3):209-327 ~1983) and Jablonski, Anal. ~iochem. 1~8:199 (1985).
Labellng may be aceomplished by indirec~ methods such as using spacers or other members of specific binding : 35 pairs. An example of this is the detection of :, .
WO91/17~3 ~ J PCT/US~1/02837 biotinylated antibodies wi~h unlabeled streptavidin and biotinylated enzyme, wit~ the u~labeled streptavidin and biotinylated en~yme being adcled either sequentially or simultaneously. Detection of enzyme acti~lty may be facilita~ed by measuring chromogenic, fluorogenic, chemiluminescent or electrochemical changes by commonly known methods.
Reporters can be fluorogenic ox chemiluminesce~t in nature. In addition, rep~rtl~rs can be detectable by electrochemical means. Some methods of labeling with these reporters are de cribed above. In a preferred embodiment, the enzyme, horseradish peroxidase is used as the reporter.
In another variation, the monoclonal antibody need not be labeled. If it is unlabeled, it can be detected using a labeled anti-species immunoglobulin reagent.
If more than one monoclonal antibody is used, the combination should be specific for ei~her HSV-l gG or HSV-2 gG.
Another embodimen~ involves the s~multaneous, rather than sequential, addition of sample and monoclonal antibody to a support containing immobilized antigen. This is illustrated ln ~he examples described below.
AlternatiYely, at leas~ one monoclonal antibody can be immobilized on a support. It can then be reacted with labeled an~igen and sample or the labeled antigen and sample can be preincubated and then reacted with the - support con~aining immobilized monoclonal antibody.
Suitable supports include synthetic polymer supports, such as polys~yrene, polypropylene, subs~ituted polystyrene, e.g., amina~ed or carboxylated poIystyre~P; polyacrylamides; polyamides; polyvinyl chlorlde, etc.; glass beads; agarose; nitrocellulose, ~ 35 etc.
:` :
't WO~1/17~3 ~ ~ g ~ PCT/U~91/02837 This invention also concerns a kit for serotyplng antibodies to HSV l or BSV-.2 or bo~h HSV-1 and HSV-2.
kit according to the present invention comprises ~a) a suppor~ con~aining immobilized ~SV antigen, ~b) a quantity of at least one monoclonal antibody speci~ic for HSV-1, ~SV-2 or a combination thereof wherein sa~d antibody is not susceptible to heterolog~us blocking and -. further wherein said antibo~y is either (i) unlabeled, lii) labeled with a reporter, or ~iii) labeled with a first member of a specific binding pair, and (c) a quantity of labeled anti-antibody when the monoclonal antibody is unlabeled or a quantity of a labeled second member of the binding pair when the mcnoclonal an~ibody is labeled with the first mem~er of ~he specific binding pair.
The following examples are intended to illustrate the invention.
~
:`. The monoclonal antibodies were prepared according to the methods o~ L. Pereira et al., a~ described in 25 Infection and Immunity, Vol. 29, No. 2, pp. 724~732 ~Aug. 1980) and Oi, et al., as described in Immuno~lobulin Prod~cing ~ybrid Cell Lines, pp. 351~371, ~n B. ~ishell and S. Schitgi ~ed.), Selected Methods in Cellular Immunology, W. H. Freeman Co., San Francisco.
Monoclonal antibodies were purified from ascites fluid by salt fractionation with 40% saturated ammonlum sulfa e and gel filtratio~ on Sephacryl S~300 ~P~armacia, Inc., Piscatzway, NJ). Purified monoclonal antibodies were labeled with horseradish perox$dase ~HRP) accordin~ to the SMCC method described by Ishikawa - . ,. ::, , -WO 91/17443 2 ~ 8 ~
et al., Journal of Immunoassay, Vol. 4, No. 3, pp.
209-327, 1~83.
~SS~
Polystyrene microplate wells were coated with HSV-2 antigen ~Scripps Laboratories, San Diego, CA) dlluted in O.lM carbonate buffer pH 9.6, and blocked with ~% bovine serum albumin (BSA) in 10 mM phosphate buffered saline (PBS), pH 7.4.
For the sequential (Seq.~ format, a serum specimen containing antibodies to HSV-2 ~Pos.) or a specimen without antibodies to HSV (Neg.) were incubated for 1 hr at 37C. Following washing with PBS containing O.OS~
Tween 20 (PBST), HRP labeled anti-HSV-2 gG monoclonal antibodies 55311, 55312, or 55311 and 55312 combined at 1:1, diluted in 1% BSA-PBST were incubated for 1 hr a~
37C. After washing with PBST, a tetramethylbenzidine (TMB) substrate (SOMA Laboratories, Romeo, MI~ was added for 30 min., the reaction stopped with H2SO4, and the absorbance at 450 nm read on a microplate reader (Vmax, Molecular Devices Corp., Menlo Park, CA).
For the simultaneous (Sim.~ format, the monoclonal - antibodies were diluted into the serum specimens, added to the wells, and incubated for 1 hr at 37C. Following washing with PBST, TMB was added and absorbance determined as described for the sequential ~ormat.
The results in Ta~le I below indicate that ~he ~ anti-~SV-2 containing serum-blocked th~ binding of both - anti-HSV-2 gG monoclonal antibodies when used alone, or in combination, whether in a sequential or simultaneous asaay cormat.
..
. .
.
WO91/17~3 ~ ~ ~
2 ~6 ~ PCT/US91/02837 . ~ ~e~
Blocking of Anti-HSV 2 gG Monoclonal Antibodies by Serum Containi~g Anti-HSV 2 Antibodies rmat ~im~ 0.1:)~
~5311 Seq. Neg. 1.441 : P~s. O.lg3 55312 Seq. Neg. 0.999 Pos . O . 010 55311 + 55312 Seq. Neg. 1.510 Pos. 0.098 55311 Sim. Neg. 0.836 Pos . O . 000 55312 Sim. Meg. 1.033 Pos. O.000 55311 + 55312 Sim. Neg. 1.195 P~s . O . 000 Polystyrene microplate wells were coated with a combination of HSV-1 and HSV-2 antigens ~Scripps ; . La~oratories, San Diego, CA) as described in Example 1 above.
For each of the two specificity determinations, twenty serum samples containing antibodies to HSV-1 and twenty serum samples contain~ng~antibodies to HSV-2, diluted 1:1 with 1% BSA-PBS 0.5% triton X-100, were added to the wells, incubated for 1 hr at 37C, ~nd washed with PBST. To determine specificity to anti-HSV-2,-HRP labeled monoclonal antibody 55312 (anti-HSV-2 gG) dilu~ed $n 1% BSA~PBST, was added for 1 hr at 3~C. To determine specificlty to anti-~SV~ RP
labeled monoclonal antibody 5531~ ~anti-HSV-1 gG) dilut~d i.n 1% BS~-P~Sl~, was added for 1 hr ~t 3~C.
Follo~ing wash~ny with PBS~, TMB was added, and the absorbance determined as described in Example 1.
',, ` .
-WO9~/]7~3 2 ~ 3 ~' ~(s~ ~ PCT/US9l/02837 ~ ~u Lt ~
Figure 1 is ~ graph sho~ing the absorbance valuesfor the twenty anti-HSV-1 and twenty anti-HSV-2 antibody containing sera used in an assay to determlne anti HSV-1 antibodies. The sera containlng anti-HSV 1 an~ibodies blocked the binding of t~e anti-HSV-l gG monoelonal antibody. No such blocking was o~served with the sera containing anti-~SV-2 antibodies. Figure 2 is a graph showing the absorbance values obtained for an assay to determine an~i-HSV-2 antibodies. ~he sera containing anti-HSV-~ antibodies blocked the binding of the anti-HSV-~ gG monoclonal antibody. No such blocking was observed with the sera containing anti-~SV-1 antibodies.
~EI~l ~
Polystyrene microplate wells were coa~ed with a combination of ~SV-l and HSV-2 antigens as described in ~-Example l above.
Twenty serum samples containing antibod~es to ~V
and one negative serum sample were added to the wells in duplicate for 1 hr at 37C, and washed with PBST. To one o~ the duplicate wells, HRP labeled 55312 ` (anti-HSV-2 gG) was added, and to the oth~r, HRP label~d `- 55315 (anti-HSV-l gG) was added.--Labeled antibodies were diluted in 1% BSA-PBST, and incubated for 1 hr at 37C. Following washing wi~h PBST, TMB was added, a~d the absorbance determined as described in Fxample l.
The absorbances are shown in Table II. ~ positive r~sponse was chosen as at le~st a 50% reduction in the - absorbance of the negative control specimen.
Determining an~i-HSV types by this method does not require ~edious antlgen purif~cation procedures, heterol~gsus absorption me~ods, or rat~o d~termi~ati~ns as all the previo~sly described methods reyuire.
:`
' .
WO 9]/17443 2 0 ~3 1 ~ ~ ~ PCr/VS91/02837 ~;
Typing Anti-HSV Antibodies in Serum Specimens Absorbar~ce 450 nm De~ectors ~pecimen No. . ~n~i~Z=;~ ~i~!~
Neg . Control 0 . 621 0 . 5S2 Neg .
0 . 001 0 . 006 1 & 2 2 0 . 62 6 0 . 000 3 0 . 000 0 . 001 1 & 2 4 0 . 000 0 . 001 1 & 2 0 . 569 0 . 003 6 0 . 000 0 . 001 1 & 2 7 0.579 0.003 8 0.489 0.000 9 0 . 513 0 . 001 0 . 000 0 . 003 1 & 2 11 0 . 01 6 0 . 002 l ~ 2 12 0 . 002 0 . 002 1 & 2 13 0 . 53~ 0 . 002 14 0 . 000 0 . 001 l & 2 0 . 003 ~ . 000 1 & 2 16 0.003 9.001 1 Ji 2 17 0 . 617 0 . 002 18 0 . ~1 0 . 001 1 &
~9 0.002 9.0a2 1 ~ 2 0.023 0.330 2 '`' . ,
Some of these are biotin-N-hydroxysuccinimide which - binds to amine groups on proteins; biotin hydrazide which binds to carbohydrate moieties, aldehydes and carboxyl groups via a carbodiimide coupling; and biotin maleimide and iodoacetyl biotin which bind to sulfhydryl groups. Fluorescein can be coupled ~o pro~ei~ amine groups using fluorescein isothiocyanate. Dinitrophenyl groups can be coupled ~o protein amine groups using 2,4-dinitrobenzene sulfonate or 2,4-dinitrofluoro-benzene. Other standard methods of conjugate may beemployed ~o couple monoclonal antibodies to a member of a specific binding pair lncluding dialdehyde, carbodiimide coupling, homofunctional crosslinking, and heterobifunctional crosslinking. Carbodiimide coupling is an effective method of coupling carboxyl groups on one substance to amine groups on another. Carbodiimide coupling is facilitated by using the commercially available reagent, l-ethyl-3-(dimethylaminopropyl)-carbodiimide ~EDAC).
- 25 Homobifunctional crossli~kers, including the bifunctional imidoesters and b~functional N-hydroxy-- succinimide esters, are oommercially available and are employed for coupling amine groups on one suhstance to ami~e groups on another. Heterobifunctional crosslinkers are reagents which possess diffPrent functional groups. The most common commercially available heterobifunctionaI crosslinker~ have an amine ~ reacti~e N-hydroxysuccinimide ester as one functlonal - group, a~d a sulfhydryl reactive group as the ~econd ~ 35 func~ional group. The most common sulfhy~ryl reac~ive , .
., .
Wo91/17~3 2a~
,~ PCT/U591/02837 groups are maleimides, pyridyl disulfides and active halogens. One of the funct.ional groups may be a photoactiv~ aryl nitrene, wh~h upon irradiation reacts with a variety of groups.
A reporter can be linked directly or indirectly, covalently or non-covalent:Ly to the monoclonal antibodies or to a member of a specific binding pair.
Reporters can be radioactive isotopes7 enzymes, fluorogenic, chemiluminescent or electrochemlcal materials. Two commonly used radioisotopes are 125I and 3H. Standard radioactive iso~op~c l~beling procedures include the chloramine T, lactoperoxidase and Bolton-Hunter methods for 125I and reductive methylation for 3H~
Enzymes are also used as reporters fo~
immunoassays. These include, but are-~ot limited to, horseradish peroxidase, alkaline phosphatase, ~-galactosidase, glucose oxidase, luciferas~, ~-lactamase, urease and lysozyme. Labeling with enzymes is facilitated by using dialdehyde, carbodi1mide coupling, homobifunctional crosslin~ers and heterobifunctional crosslinkers as described above for labeling monoclonal antibodies with members of specific ` binding pairs. The labeling method chosen depends on .. . . . . .
the functional groups available on the enzyme and the material to be labeled, and the tolerance of both ~o the conjugation conditions. The labeling method used in the presen~ invention may be one ofl bu~ no~ limited to, any conventional methods currently employed including those described by Engvall and Pearlmann, Immunochemistry 8, 871 (1971), Avramea and Ternynck, Immunochemistry ~, -: 1175 (1971~, Ishikawa e~ al., J. Immunoa~say 4(3):209-327 ~1983) and Jablonski, Anal. ~iochem. 1~8:199 (1985).
Labellng may be aceomplished by indirec~ methods such as using spacers or other members of specific binding : 35 pairs. An example of this is the detection of :, .
WO91/17~3 ~ J PCT/US~1/02837 biotinylated antibodies wi~h unlabeled streptavidin and biotinylated enzyme, wit~ the u~labeled streptavidin and biotinylated en~yme being adcled either sequentially or simultaneously. Detection of enzyme acti~lty may be facilita~ed by measuring chromogenic, fluorogenic, chemiluminescent or electrochemical changes by commonly known methods.
Reporters can be fluorogenic ox chemiluminesce~t in nature. In addition, rep~rtl~rs can be detectable by electrochemical means. Some methods of labeling with these reporters are de cribed above. In a preferred embodiment, the enzyme, horseradish peroxidase is used as the reporter.
In another variation, the monoclonal antibody need not be labeled. If it is unlabeled, it can be detected using a labeled anti-species immunoglobulin reagent.
If more than one monoclonal antibody is used, the combination should be specific for ei~her HSV-l gG or HSV-2 gG.
Another embodimen~ involves the s~multaneous, rather than sequential, addition of sample and monoclonal antibody to a support containing immobilized antigen. This is illustrated ln ~he examples described below.
AlternatiYely, at leas~ one monoclonal antibody can be immobilized on a support. It can then be reacted with labeled an~igen and sample or the labeled antigen and sample can be preincubated and then reacted with the - support con~aining immobilized monoclonal antibody.
Suitable supports include synthetic polymer supports, such as polys~yrene, polypropylene, subs~ituted polystyrene, e.g., amina~ed or carboxylated poIystyre~P; polyacrylamides; polyamides; polyvinyl chlorlde, etc.; glass beads; agarose; nitrocellulose, ~ 35 etc.
:` :
't WO~1/17~3 ~ ~ g ~ PCT/U~91/02837 This invention also concerns a kit for serotyplng antibodies to HSV l or BSV-.2 or bo~h HSV-1 and HSV-2.
kit according to the present invention comprises ~a) a suppor~ con~aining immobilized ~SV antigen, ~b) a quantity of at least one monoclonal antibody speci~ic for HSV-1, ~SV-2 or a combination thereof wherein sa~d antibody is not susceptible to heterolog~us blocking and -. further wherein said antibo~y is either (i) unlabeled, lii) labeled with a reporter, or ~iii) labeled with a first member of a specific binding pair, and (c) a quantity of labeled anti-antibody when the monoclonal antibody is unlabeled or a quantity of a labeled second member of the binding pair when the mcnoclonal an~ibody is labeled with the first mem~er of ~he specific binding pair.
The following examples are intended to illustrate the invention.
~
:`. The monoclonal antibodies were prepared according to the methods o~ L. Pereira et al., a~ described in 25 Infection and Immunity, Vol. 29, No. 2, pp. 724~732 ~Aug. 1980) and Oi, et al., as described in Immuno~lobulin Prod~cing ~ybrid Cell Lines, pp. 351~371, ~n B. ~ishell and S. Schitgi ~ed.), Selected Methods in Cellular Immunology, W. H. Freeman Co., San Francisco.
Monoclonal antibodies were purified from ascites fluid by salt fractionation with 40% saturated ammonlum sulfa e and gel filtratio~ on Sephacryl S~300 ~P~armacia, Inc., Piscatzway, NJ). Purified monoclonal antibodies were labeled with horseradish perox$dase ~HRP) accordin~ to the SMCC method described by Ishikawa - . ,. ::, , -WO 91/17443 2 ~ 8 ~
et al., Journal of Immunoassay, Vol. 4, No. 3, pp.
209-327, 1~83.
~SS~
Polystyrene microplate wells were coated with HSV-2 antigen ~Scripps Laboratories, San Diego, CA) dlluted in O.lM carbonate buffer pH 9.6, and blocked with ~% bovine serum albumin (BSA) in 10 mM phosphate buffered saline (PBS), pH 7.4.
For the sequential (Seq.~ format, a serum specimen containing antibodies to HSV-2 ~Pos.) or a specimen without antibodies to HSV (Neg.) were incubated for 1 hr at 37C. Following washing with PBS containing O.OS~
Tween 20 (PBST), HRP labeled anti-HSV-2 gG monoclonal antibodies 55311, 55312, or 55311 and 55312 combined at 1:1, diluted in 1% BSA-PBST were incubated for 1 hr a~
37C. After washing with PBST, a tetramethylbenzidine (TMB) substrate (SOMA Laboratories, Romeo, MI~ was added for 30 min., the reaction stopped with H2SO4, and the absorbance at 450 nm read on a microplate reader (Vmax, Molecular Devices Corp., Menlo Park, CA).
For the simultaneous (Sim.~ format, the monoclonal - antibodies were diluted into the serum specimens, added to the wells, and incubated for 1 hr at 37C. Following washing with PBST, TMB was added and absorbance determined as described for the sequential ~ormat.
The results in Ta~le I below indicate that ~he ~ anti-~SV-2 containing serum-blocked th~ binding of both - anti-HSV-2 gG monoclonal antibodies when used alone, or in combination, whether in a sequential or simultaneous asaay cormat.
..
. .
.
WO91/17~3 ~ ~ ~
2 ~6 ~ PCT/US91/02837 . ~ ~e~
Blocking of Anti-HSV 2 gG Monoclonal Antibodies by Serum Containi~g Anti-HSV 2 Antibodies rmat ~im~ 0.1:)~
~5311 Seq. Neg. 1.441 : P~s. O.lg3 55312 Seq. Neg. 0.999 Pos . O . 010 55311 + 55312 Seq. Neg. 1.510 Pos. 0.098 55311 Sim. Neg. 0.836 Pos . O . 000 55312 Sim. Meg. 1.033 Pos. O.000 55311 + 55312 Sim. Neg. 1.195 P~s . O . 000 Polystyrene microplate wells were coated with a combination of HSV-1 and HSV-2 antigens ~Scripps ; . La~oratories, San Diego, CA) as described in Example 1 above.
For each of the two specificity determinations, twenty serum samples containing antibodies to HSV-1 and twenty serum samples contain~ng~antibodies to HSV-2, diluted 1:1 with 1% BSA-PBS 0.5% triton X-100, were added to the wells, incubated for 1 hr at 37C, ~nd washed with PBST. To determine specificity to anti-HSV-2,-HRP labeled monoclonal antibody 55312 (anti-HSV-2 gG) dilu~ed $n 1% BSA~PBST, was added for 1 hr at 3~C. To determine specificlty to anti-~SV~ RP
labeled monoclonal antibody 5531~ ~anti-HSV-1 gG) dilut~d i.n 1% BS~-P~Sl~, was added for 1 hr ~t 3~C.
Follo~ing wash~ny with PBS~, TMB was added, and the absorbance determined as described in Example 1.
',, ` .
-WO9~/]7~3 2 ~ 3 ~' ~(s~ ~ PCT/US9l/02837 ~ ~u Lt ~
Figure 1 is ~ graph sho~ing the absorbance valuesfor the twenty anti-HSV-1 and twenty anti-HSV-2 antibody containing sera used in an assay to determlne anti HSV-1 antibodies. The sera containlng anti-HSV 1 an~ibodies blocked the binding of t~e anti-HSV-l gG monoelonal antibody. No such blocking was o~served with the sera containing anti-~SV-2 antibodies. Figure 2 is a graph showing the absorbance values obtained for an assay to determine an~i-HSV-2 antibodies. ~he sera containing anti-HSV-~ antibodies blocked the binding of the anti-HSV-~ gG monoclonal antibody. No such blocking was observed with the sera containing anti-~SV-1 antibodies.
~EI~l ~
Polystyrene microplate wells were coa~ed with a combination of ~SV-l and HSV-2 antigens as described in ~-Example l above.
Twenty serum samples containing antibod~es to ~V
and one negative serum sample were added to the wells in duplicate for 1 hr at 37C, and washed with PBST. To one o~ the duplicate wells, HRP labeled 55312 ` (anti-HSV-2 gG) was added, and to the oth~r, HRP label~d `- 55315 (anti-HSV-l gG) was added.--Labeled antibodies were diluted in 1% BSA-PBST, and incubated for 1 hr at 37C. Following washing wi~h PBST, TMB was added, a~d the absorbance determined as described in Fxample l.
The absorbances are shown in Table II. ~ positive r~sponse was chosen as at le~st a 50% reduction in the - absorbance of the negative control specimen.
Determining an~i-HSV types by this method does not require ~edious antlgen purif~cation procedures, heterol~gsus absorption me~ods, or rat~o d~termi~ati~ns as all the previo~sly described methods reyuire.
:`
' .
WO 9]/17443 2 0 ~3 1 ~ ~ ~ PCr/VS91/02837 ~;
Typing Anti-HSV Antibodies in Serum Specimens Absorbar~ce 450 nm De~ectors ~pecimen No. . ~n~i~Z=;~ ~i~!~
Neg . Control 0 . 621 0 . 5S2 Neg .
0 . 001 0 . 006 1 & 2 2 0 . 62 6 0 . 000 3 0 . 000 0 . 001 1 & 2 4 0 . 000 0 . 001 1 & 2 0 . 569 0 . 003 6 0 . 000 0 . 001 1 & 2 7 0.579 0.003 8 0.489 0.000 9 0 . 513 0 . 001 0 . 000 0 . 003 1 & 2 11 0 . 01 6 0 . 002 l ~ 2 12 0 . 002 0 . 002 1 & 2 13 0 . 53~ 0 . 002 14 0 . 000 0 . 001 l & 2 0 . 003 ~ . 000 1 & 2 16 0.003 9.001 1 Ji 2 17 0 . 617 0 . 002 18 0 . ~1 0 . 001 1 &
~9 0.002 9.0a2 1 ~ 2 0.023 0.330 2 '`' . ,
Claims (16)
1. A method of typing antibodies to HSV 2 in a sample suspected to contain HSV antibodies which comprises:
a) reacting the sample and at least one monoclonal antibody specific for HSV-2 glycoprotein G
with an antigen wherein said monoclonal antibody is not susceptible to heterologous blocking; and b) determining whether antibody binding has occurred.
a) reacting the sample and at least one monoclonal antibody specific for HSV-2 glycoprotein G
with an antigen wherein said monoclonal antibody is not susceptible to heterologous blocking; and b) determining whether antibody binding has occurred.
2. A method of typing antibodies to HSV 1 in a sample suspected to contain HSV antibodies which comprises:
a) reacting the sample and at least one monoclonal antibody specific for HSV-1 glycoprotein G
with an antigen wherein said monoclonal antibody is not susceptible to heterologous blocking; and b) determining whether antibody binding has occurred.
a) reacting the sample and at least one monoclonal antibody specific for HSV-1 glycoprotein G
with an antigen wherein said monoclonal antibody is not susceptible to heterologous blocking; and b) determining whether antibody binding has occurred.
3. A method of typing antibodies to HSV-2 in a sample suspected to contain HSV antibodies which comprises:
a) contacting a support containing immobilized antigen with the sample;
b) reacting the product of step (a) with at least one monoclonal antibody specific for HSV-2 glycoprotein G wherein aid antibody is not susceptible to heterologous blocking and further wherein said antibody is either (i) unlabeled, (ii) labeled with a reporter, or (iii) labeled with a first member of a specific binding pair;
c) reacting the product of step (b)(i) with a labeled anti-antibody or reacting the product of step (b)(iii) with a labeled second member of the specific binding pair; and d) detecting the presence or absence of label.
a) contacting a support containing immobilized antigen with the sample;
b) reacting the product of step (a) with at least one monoclonal antibody specific for HSV-2 glycoprotein G wherein aid antibody is not susceptible to heterologous blocking and further wherein said antibody is either (i) unlabeled, (ii) labeled with a reporter, or (iii) labeled with a first member of a specific binding pair;
c) reacting the product of step (b)(i) with a labeled anti-antibody or reacting the product of step (b)(iii) with a labeled second member of the specific binding pair; and d) detecting the presence or absence of label.
4. A method according to claim 3 wherein the antibody is labeled with horseradish peroxidase.
5. A method according to claim 4 wherein the horseradish peroxidase labeled antibody is reacted with tetramethylbenzidene.
6. A method of serotyping antibodies to HSV-1 in a sample suspected to contain HSV antibodies which comprises:
a) contacting a support containing immobilized antigen with the sample;
b) reacting the product of step (a) with at least one monoclonal antibody specific for HSV-1 glycoprotein G wherein said antibody is not susceptible to heterologous blocking and further wherein said antibody is either (i) unlabeled, (ii) labeled with a reporter, or (iii) labeled with a first member of a specific binding pair;
c) reacting the product of step (b)(i) with a labeled anti-antibody or reacting the product of step (b) (iii) with a labeled second member of the specific binding pair; and d) detecting the presence or absence of label.
a) contacting a support containing immobilized antigen with the sample;
b) reacting the product of step (a) with at least one monoclonal antibody specific for HSV-1 glycoprotein G wherein said antibody is not susceptible to heterologous blocking and further wherein said antibody is either (i) unlabeled, (ii) labeled with a reporter, or (iii) labeled with a first member of a specific binding pair;
c) reacting the product of step (b)(i) with a labeled anti-antibody or reacting the product of step (b) (iii) with a labeled second member of the specific binding pair; and d) detecting the presence or absence of label.
7. A method according to claim 6 wherein the antibody is labeled with horseradish peroxidase.
8. A method according to claim 7 wherein the horseradish peroxidase labeled antibody is reacted with tetramethylbenzidene.
9. A method according to claim 3 wherein steps (a) and (b) are performed simultaneously.
10. A method according to claim 6 wherein steps (a) and (b) are performed simultaneously.
11. A method of typing antibodies to HSV-2 in a sample suspected to contain HSV antibodies which comprises:
a) contacting a support containing at least one immobilized monoclonal antibody specific for HSV-2 glycoprotein G with labeled antigen and the sample wherein said monoclonal antibody is not susceptible to heterologous blocking; and b) detecting the presence or absence of label.
a) contacting a support containing at least one immobilized monoclonal antibody specific for HSV-2 glycoprotein G with labeled antigen and the sample wherein said monoclonal antibody is not susceptible to heterologous blocking; and b) detecting the presence or absence of label.
12. A method of typing antibodies to HSV-1 in a sample suspected to contain HSV antibodies which comprises:
a) contacting a support containing at least one immobilized monoclonal antibody specific for HSV-1 glycoprotein G with labeled antigen and the sample wherein said monoclonal antibody is not susceptible to heterologous blocking; and b) detecting the presence or absence of label.
a) contacting a support containing at least one immobilized monoclonal antibody specific for HSV-1 glycoprotein G with labeled antigen and the sample wherein said monoclonal antibody is not susceptible to heterologous blocking; and b) detecting the presence or absence of label.
13. A method of typing antibodies to HSV-2 in a sample suspected to contain HSV antibodies which comprises:
a) preincubating the sample with labeled antigen;
b) reacting the product of step (a) with a support containing at least one immobilized monoclonal antibody specific for HSV-2 glycoprotein G wherein said antibody is not susceptible to heterologous blocking;
and c) detecting the presence or absence of label.
a) preincubating the sample with labeled antigen;
b) reacting the product of step (a) with a support containing at least one immobilized monoclonal antibody specific for HSV-2 glycoprotein G wherein said antibody is not susceptible to heterologous blocking;
and c) detecting the presence or absence of label.
14. A method of serotyping antibodies to HSV-1 in a sample suspected to contain HSV antibodies which comprises:
a) preincubating the sample with labeled antigen;
b) reacting the product of step (a) with a support containing at least one immobilized monoclonal antibody specific for HSV-1 glycoprotein G wherein said antibody is not susceptible to heterologous blocking;
and c) detecting the presence or absence of label.
a) preincubating the sample with labeled antigen;
b) reacting the product of step (a) with a support containing at least one immobilized monoclonal antibody specific for HSV-1 glycoprotein G wherein said antibody is not susceptible to heterologous blocking;
and c) detecting the presence or absence of label.
15. A kit of reagents for serotyping antibodies to HSV-1, HSV-2, or a combination thereof which comprises:
a) a support containing immobilized HSV
antigen;
b) a quantity of at least one monoclonal antibody specific for HSV-1, HSV-2 or a combination thereof wherein said antibody is not susceptible to heterologous blocking and further wherein said antibody is either (i) unlabeled, (ii) labeled with a reporter, or (iii) labeled with a first member of a specific binding pair; and c) a quantity of labeled anti-antibody when the monoclonal antibody is unlabeled or a quantity of a labeled second member of a binding pair when the monoclonal antibody is labeled with the first member of a specific binding pair.
a) a support containing immobilized HSV
antigen;
b) a quantity of at least one monoclonal antibody specific for HSV-1, HSV-2 or a combination thereof wherein said antibody is not susceptible to heterologous blocking and further wherein said antibody is either (i) unlabeled, (ii) labeled with a reporter, or (iii) labeled with a first member of a specific binding pair; and c) a quantity of labeled anti-antibody when the monoclonal antibody is unlabeled or a quantity of a labeled second member of a binding pair when the monoclonal antibody is labeled with the first member of a specific binding pair.
16. A kit according to claim 15 wherein the antibody is labeled with horseradish peroxidase.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US51932990A | 1990-05-04 | 1990-05-04 | |
| US07/519,329 | 1990-05-04 | ||
| PCT/US1991/002837 WO1991017443A1 (en) | 1990-05-04 | 1991-04-30 | A method of determining type specific antibodies to herpes simplex virus types 1 and 2 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2081888A1 true CA2081888A1 (en) | 1991-11-05 |
Family
ID=24067839
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002081888A Abandoned CA2081888A1 (en) | 1990-05-04 | 1991-04-30 | Method of determining type specific antibodies to herpes simplex virus types 1 and 2 |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0527200A1 (en) |
| CA (1) | CA2081888A1 (en) |
| IE (1) | IE911468A1 (en) |
| WO (1) | WO1991017443A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5965354A (en) * | 1995-07-28 | 1999-10-12 | Chiron Corporation | Herpes simplex virus diagnostics |
| GB9615533D0 (en) * | 1996-07-24 | 1996-09-04 | Peptide Therapeutics Ltd | Diagnostic test for herpes simplex virus type-2 |
-
1991
- 1991-04-30 WO PCT/US1991/002837 patent/WO1991017443A1/en not_active Application Discontinuation
- 1991-04-30 EP EP19910909576 patent/EP0527200A1/en not_active Withdrawn
- 1991-04-30 CA CA002081888A patent/CA2081888A1/en not_active Abandoned
- 1991-05-01 IE IE146891A patent/IE911468A1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| WO1991017443A1 (en) | 1991-11-14 |
| IE911468A1 (en) | 1991-11-06 |
| EP0527200A1 (en) | 1993-02-17 |
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