WO2017211316A1 - Method for detecting the presence of mycobacterial material in a sample using at least two antigens - Google Patents
Method for detecting the presence of mycobacterial material in a sample using at least two antigens Download PDFInfo
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- WO2017211316A1 WO2017211316A1 PCT/CN2017/087547 CN2017087547W WO2017211316A1 WO 2017211316 A1 WO2017211316 A1 WO 2017211316A1 CN 2017087547 W CN2017087547 W CN 2017087547W WO 2017211316 A1 WO2017211316 A1 WO 2017211316A1
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- tuberculosinyl
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- 0 C[C@@]1(*(C=*2)c3c2c(**=C)*(*)c*3)O[C@](COC)C(*)C1* Chemical compound C[C@@]1(*(C=*2)c3c2c(**=C)*(*)c*3)O[C@](COC)C(*)C1* 0.000 description 2
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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
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
Definitions
- the present invention relates to a method of detecting the presence of antibodies against mycobacterial material in a sample comprising detecting binding of antibodies in said sample to at least two types of immobilised antigen, which antigens are capable of binding to an antibody which is indicative for the presence of mycobacterial material in a human or animal.
- the invention also relates to a solid substrate comprising a combination of said at least two antigens immobilised to said substrate, and the invention also relates to a biosensor comprising at least two types of immobilised antigen, which antigens are capable of binding to an antibody which is indicative for the presence of mycobacterial material in a human or animal.
- Mycobacterium tuberculosis is a pathogenic bacterial species in the family Mycobacteriaceae and the causative agent of most cases of tuberculosis (TB) .
- TB is still one of the leading causes of death in many low and middle income countries. In addition, more and more cases are reported of multi-drug resistant TB.
- TB skin test also called the Mantoux tuberculin skin test
- TB blood tests also called interferon-gamma release assays or IGRAs
- chest radiography X-ray
- ARB acid-fast-bacilli
- Sputum smear microscopy (also referred to as smear test) is a common method for diagnosis of pulmonary tuberculosis in low and middle income countries where most TB cases occur. Although it is a simple, rapid and inexpensive technique which is used in areas with a very high prevalence of tuberculosis, there are significant limitations in its performance. For instance, the sensitivity is severely compromised when the bacterial load is less than 10,000 Mycobacterium tuberculosis organisms/ml sputum sample. A person that tests positive in a smear test (smear positive person) thus has a very high bacterial load and will thus be in a very advanced stage of TB.
- smear positive persons In smear positive persons it is usually clear at first sight that the person suffers from an illness. In such advanced stages, often no successful treatment is possible anymore. On the other hand, no conclusion can be drawn if a person tests negative in a smear test (smear negative person) , because that person could suffer from TB in a less advanced stage, which is associated with a lower bacterial load.
- smear negative persons In smear negative persons it is usually not clear at first sight that the person suffers from an illness.
- the smear test also gives poor results in extra-pulmonary tuberculosis, pediatric tuberculosis and in patients co-infected with HIV and tuberculosis.
- the inventor has observed in samples derived from healthy subjects a high degree of binding of materials contained in these samples to immobilised mycolic acid antigens when used as sole type of antigens, which indicates that samples derived from healthy subjects contain materials which bind to mycolic acid antigens, but which are not indicative for tuberculosis. This may lead to a false positive outcome.
- a subject actually suffers from tuberculosis, the problem arises that if materials that are not indicative for tuberculosis bind to immobilised mycolic acid antigens in a detection test, a high background binding signal is produced. This high background signal may obscure the signal derived from the actual markers for tuberculosis. So, there is also the risk of false negative results.
- the inventor therefore considers that there is room for improvement with respect to the sensitivity of detection of tuberculosis.
- the present invention therefore aims to overcome the problems that derive from the binding of materials that are not indicative for tuberculosis to immobilised mycolic acid antigens and to improve the sensitivity and reliability of detection of tuberculosis.
- the aim of the invention has been achieved by the provision of a method of detecting the presence of antibodies against mycobacterial material in a sample comprising the steps of: providing a sample from a human or animal; and detecting binding of antibodies in said sample to at least two types of immobilised antigens capable of binding to an antibody which is indicative for the presence of mycobacterial material in a human or animal, selected from the following group:
- R 2 and R 3 are independently chosen from H, SO 3 H, SO 3- or SO 3- /M + , wherein M + is a cation;
- R 2’ and R 3’ are acyl groups
- antigen represented by formula (I) is optionally modified with one or more functional groups that enable immobilisation to a solid substrate, and enantiomers, diastereoisomers, and mixtures thereof;
- R 1 is H or a group with formula (III)
- R 2 is absent or a group with formula (III) , provided that one of R 1 and R 2 is a group with formula (III) ,
- R 3 and R 4 are selected independently from hydrogen, OH, a carboxylic acid group or an acyl group, in any combination thereof, wherein said tuberculosinyl adenosine antigen is optionally modified with one or more functional groups that enable immobilisation to a solid substrate, and enantiomers, diastereoisomers of the antigens, and mixtures thereof.
- the invention in a second aspect relates to a solid substrate, comprising a combination of 2 or more of the types of tuberculosinyl adenosine antigens, diacyl glycolipid antigens, and mycolic acid derived antigens as defined above in relation to the first aspect of the invention, immobilised to said substrate.
- the invention in a third aspect relates to a biosensor, comprising a combination of 2 or more of the tuberculosinyl adenosine antigens, diacyl glycolipid antigens, and mycolic acid derived antigens as defined above in relation to the first aspect of the invention.
- Fig. 1 shows a diagram with ELISA results using mycolic acid immobilised on ELISA plates (IgM as secondary antibody) .
- Fig. 2 shows a diagram of ELISA results using tuberculosinyl adenosine antigens immobilised on ELISA plates (IgM as secondary antibody) .
- Fig. 3 shows a diagram of ELISA results using diacyl glycolipid antigens immobilised on ELISA plates (IgM as secondary antibody)
- Fig. 4 shows a diagram of ELISA results using diacyl glycolipid antigens according to formula (XIII) immobilised on ELISA plates.
- the present invention is based on the observation that exposing a sample from a human or animal to at least two types of immobilised antigens capable of binding to an antibody which is indicative for the presence of mycobacterial material in a human or animal and selected from the following group:
- tuberculosinyl adenosine antigen as defined above, and detecting binding of antibodies in said sample to said antigens, leads to improved reliability and sensitivity of a tuberculosis test.
- the mycolic acid derived antigens as referred to in the present application may be derived from mycobacteria selected from virulent and pathogenic mycobacteria.
- the term mycolic acid derived antigen is to be understood as a compound comprising a mycolic acid residue which is capable of binding to anti-mycolic acid antibodies.
- the mycolic acid antigen is derived from Mycobacterium tuberculosis.
- Said mycolic acid derived antigen may be at least one selected from the group of mycolic acid, cord factor, chemically modified mycolic acid, chemically modified cord factor, a synthetic mycolic acid derivative, a synthetic cord factor derivative.
- the mycolic acid derived antigen may suitably be selected from one or more of mycolic acids obtained from natural sources, synthetically prepared mycolic acids, sulfur-containing mycolic acids, structural analogues of mycolic acids, and mycolic acid wax esters.
- the mycolic acid derived antigen also includes salts and/or esters of these derivatives.
- Natural sources of mycolic acid derivatives include the cell walls of mycobacteria such as Mycobacterium tuberculosis include mixtures of different classes of compounds and different mycolic acid homologues, often as derivatives in which they are bonded to the wall of the cell.
- Esters of mycolic acid derivatives can also be used such as esters of alcohols (e.g. monohydric alcohols and polyhydric alcohols) and sugar esters.
- Sugar esters are particularly preferred.
- Sugar esters include esters with a monosaccharide, disaccharide or an oligosaccharide. Said saccharides may conveniently include sugar units based on hexoses and/or pentoses. Glucose esters are suitable examples of these esters.
- Further suitable sugar esters are trehalose esters, including trehalose monomycolates and trehalose dimycolates. For instance cord factors, which are trehalose monomycolates or trehalose dimycolates are well known examples of sugar esters that are suitable. These compounds occur in nature as complex mixtures of different classes of mycolic acids and of different homologues within each class.
- semi-synthetic or more preferably synthetic mycolic acid derivatives for the purposes of the invention.
- the structure of the mycolic acid unit affects the biological activity of the cord factor.
- Suitable semi-synthetic derivatives include semi-synthetic cord-factors which may be prepared by attaching mycolic acids to the sugar group. These semi-synthetic factors however still contain mixtures of different homologue. Therefore particular suitable mycolic acid derivatives for use in the context of the present invention are synthetic cord factors, for example the synthetic cord factors described in WO 2010/08667, i.e.
- Salts of mycolic acid derivatives can also be used, for instance ammonium salts, or alkali metal and alkaline earth metal salts.
- Sulfur-containing mycolic acids and/or esters or salts thereof may also be used. These compounds are analogues of natural mycolic acid compounds containing sulfur.
- Mycolic acid wax esters comprise a cyclopropyl group or an alkene and an internal ester group and can be isolated from natural sources or prepared synthetically.
- Suitable compounds for use in the detection method of the invention are described inter alia in WO 2016/024116.
- the mycolic acid antigen may be in a form selected from homogenous and heterogenous compound mixtures.
- the mycolic acid derived antigen may for instance be used in combination with a phospholipid such as phosphatidylcholine.
- mycolic acid involves a risk of false positive/negative signal
- detection of binding of antibodies in the sample to mycolic acid derived antigens in combination with one or two of the other types of antigens as defined above provides an additional indication of whether or not a person is infected with tuberculosis or not. This further enhances the reliability of the method of the invention.
- diacyl glycolipid antigens in this application is meant to refer to diacylated trehalose structures as defined in the following structures (I) and (IV) to (XIII) .
- the inventor has surprisingly found that if these immobilised diacyl glycolipid antigens are used in as one of the at least two types of antigens in accordance with the invention, a very high tuberculosis specific binding of antibodies to these antigens is detected.
- the signal derived from the actual markers for tuberculosis is significantly less obscured by a background signal than when immobilised mycolic antigens are used, so that the signal derived from the actual markers for tuberculosis becomes more pronounced. This way the invention provides a significant improvement with regard to the sensitivity of detection of markers for tuberculosis.
- said immobilised diacyl glycolipid antigens in accordance with the invention are used in a method for detecting a marker for tuberculosis a very high tuberculosis specific binding of antibodies to these antigens is detected in particular in case of samples derived from patients that were tested smear negative. This makes it possible to diagnose TB in an early stage, in which there is a higher chance for successful treatment than in a later stage.
- the use of diacyl glycolipid antigens on the solid substrate of the invention makes it possible to detect low amounts of antibodies against mycobacterial material in a sample.
- the immobilised diacyl glycolipid antigens of the invention perform very well when applied to samples derived from patients co-infected with HIV and tuberculosis.
- diacyl glycolipids which are in fact diacylated trehalose antigens and therefore can also be referred to as diacylated trehalose antigens, or derivatives thereof can be defined as a compound represented by the following formula (I) ,
- R 2 and R 3 are independently chosen from H, SO 3 H, SO 3- or SO 3- /M + , wherein M + is a cation, preferably a metal cation such as Na + or K + ;
- R 2’ and R 3’ are acyl groups, wherein the antigen is optionally modified with one or more functional groups that enable immobilisation to a solid substrate.
- the diacyl glycolipids as described herein also include enantiomers, diastereoisomers, and mixtures the compounds of formula (I) .
- R 2’ and R 3’ identical or different, may be
- X is independently chosen from an unsaturated or saturated linear or branched hydrocarbon chain, suitably an alkyl group, optionally substituted with one or more substituents and/or modified with one or more functional groups.
- R 2 is SO 3- , SO 3 H or SO 3-/ M + , wherein M + is a cation and R 3 is H. It is preferred that in case R 2 is SO 3-/ M + that the cation is Na + or K + .
- R 2 and R 3 are H.
- X is a saturated linear hydrocarbon chain optionally substituted with one or more substituents and/or modified with one or more functional groups and wherein in the other of R 2’ and R 3’ X is a saturated branched hydrocarbon chain optionally substituted with one or more substituents and/or modified with one or more functional groups.
- one of R 2’ and R 3’ is a group represented by the following formula (IV) :
- R 4 is a linear saturated hydrocarbon chain with formula C n H n+1 , wherein n is an integer between 1 and 20, optionally modified with one or more functional groups, and wherein Y is an integer between 1 and 10, and wherein R 5 is H or OH, and the other one of R 2’ and R 3’ is a linear saturated hydrocarbon chain with formula C n H n+1 , wherein n is an integer between 1 and 20, optionally modified with one or more functional groups.
- the acyl chains represented by R 2’ and R 3’ are relatively short. This makes the antigens more soluble in aqueous solutions and thus easier in use.
- the increased hydrophilicity that results from relatively short acyl chains makes the detection surface or sensor surface to which the antigens are immobilised more hydrophilic. Because of this, interactions of antibodies in the antigen occur easier and the speed of the detection will be enhanced. Moreover, it will be easier to synthesize the antigens in case synthetic antigens are used.
- R 4 may be a linear saturated hydrocarbon chain with formula C n H n+1 , wherein n is an integer between 1 and 10, such as between 1 and 9, such as between 1 and 8, such as between 1 and 7, such as between 1 and 6 such as between 1 and 5 such as between 1 and 4 such as between 1 and 3, such as 1, 2, 3, 4, 5, 6, 7, 8 or 9, optionally modified with one or more functional groups, and wherein Y is an integer between 1 and 10, such as between 1 and 5, such as between 1 and 4, such as between 1 and 3, such as 1, 2, 3, 4 or 5, optionally modified with one or more functional groups.
- n is an integer between 1 and 10, such as between 1 and 9, such as between 1 and 8, such as between 1 and 7, such as between 1 and 6 such as between 1 and 5 such as between 1 and 4 such as between 1 and 3, such as 1, 2, 3, 4, 5, 6, 7, 8 or 9, optionally modified with one or more functional groups
- Y is an integer between 1 and 10, such as between 1 and 5, such as between 1 and 4, such as between 1 and 3, such as 1, 2, 3, 4
- R 2’ and R 3’ in this respect may be a linear saturated hydrocarbon chain with formula C n H n+1 , wherein n is an integer between 1 and 15, such as between 1 and 14, such as between 1 and 13, such as between 1 and 12, such as between 1 and 11, such as between 1 and 10, such as between 1 and 9, such as between 1 and 8, such as between 1 and 7, such as between 1 and 6 such as between 1 and 5 such as between 1 and 4 such as between 1 and 3, such as between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 optionally modified with one or more functional groups.
- a suitable antigen in this respect would for example be compound (V) :
- This exemplary molecule is modified with a thio acetate group to enable immobilisation to a solid substrate, for instance a silica substrate or gold substrate.
- An antigen with a thiol group can be directly immobilised to gold.
- the substrate suitably can be coated with a silane active group to which the thiol of the modified antigen can bind.
- the diacyl glycolipid antigen is present in Mycobacterium, tuberculosis it may be isolated from Mycobacterium tuberculosis.
- the antigen may be synthesized, for instance by a method as adapted from what is described in EP 1 950 218 A1. If the antigen is not present in Mycobacterium tuberculosis it may be modified from an antigen isolated from Mycobacterium tuberculosis or synthesized.
- the further antigen is a diacylated sulfoglycolipid (Ac 2 SGL) as found in Mycobacterium tuberculosis, optionally modified with one or more functional groups.
- Ac 2 SGL is 2-palmitoyl-3-hydroxyphthioceranoyl-2’-sulfate- ⁇ - ⁇ ’-D-trehalose or 2-stearoyl-3-hydroxyphthioceranoyl-2’-sulfate- ⁇ - ⁇ ’-D-trehalose, optionally modified with one or more functional groups.
- Ac 2 SGL molecules feature a trehalose 2’-sulfate core, and are diacylated with either a palmitic or stearic residue at the 2-position and hydroxyphthioceranic acid, with varying length and methyl substituents, at the 3-position, for instance compound (VI) :
- R 2 is SO 3- , SO 3 H or SO 3-/ M + , wherein M + is a kation, preferably a metal cation, preferably Na + or K + .
- SO 3-/ Na + moiety may also be SO 3- , SO 3 H and Na+ may also be another kation such as K + .
- diacyl glycolipid antigens as defined above may be represented by formula (X) :
- R2’and R3’ are in formula (X) as defined for formula (I) above. It is preferred that one or both acyl chains herein are modified to enable immobilisation to a solid substrate. It is in particular preferred to use a compound as represented by formula (XI) :
- acyl chains of the compound of formula (XI) may be substituted for any chain as defined in formula (IV) , and the chains may be optionally modified to enable enable immobilisation to a solid substrate.
- Suitable modification include the incorporation of a thio group on one of the acyl chains or the incorporation of a unsaturated bond at the end of one of the acyl chains, e.g. a double bond.
- Suitable examples of such modified molecules include compounds according to formula (XII) , which has an alkene group at the terminus of one of the acyl chains or (XIII) which has a thiol group at the terminus of one of the acyl chains.
- alkyl chains of the compound of formulae (XII) and (XIII) may be substituted for any chain having the formula C n H n+1 or formula (IV) modified with the above shown alkene or thiol group.
- the diacyl glycolipid antigens provide a means for obtaining high sensitivity of detection of markers for tuberculosis. Very high tuberculosis specific binding of antibodies to these antigens is detected in case of samples derived from patients that were tested smear negative. This makes it possible to diagnose TB in an early stage, in which there is a higher chance for successful treatment without complications compared to TB in a late stage.
- these particular antigens perform excellent when applied to samples derived from patients co-infected with HIV and tuberculosis.
- tuberculosinyl adenosine antigens as referred to in this application are compounds represented by the following formula (II) ,
- R 1 is H or a group with formula (III)
- R 2 is absent or a group with formula (III) , provided that one of R 1 and R 2 is a group with formula (III) , R 3 and R 4 are selected independently from hydrogen, OH, an acyl chain, a carboxylic acid group comprising an acyl chain, in any combination thereof, wherein the antigens are optionally modified with one or more functional groups that enable immobilisation to a solid substrate, and enantiomers, diastereoisomers of the antigens, and mixtures thereof.
- R 4 is OH, and even more preferred that R 4 and R 3 are OH.
- R 1 may be a group of formula (III) and R 2 may be absent. In other embodiments R 1 may be H and R 2 may be a group of formula (III) . In case R 2 is a group of formula (III) , the nitrogen to which it is attached carries a positive charge.
- the functional groups that enables immobilisation to a solid substrate is preferably included in the group of formula (III) .
- R 3 or R 4 are or comprise an acyl group it is preferred that only one of R 3 and R 4 is or comprises an acyl group, or in particular a fatty acid group.
- Acyl groups preferably have a suitable length and hydrophobicity to enable immobilisation to a solid substrate.
- the acyl chain may be modified with a functional group to enable immobilisation to a solid substrate.
- An acyl chain may be a mycolic acid chain as described in WO 2014/210327.
- the antigen of formula (II) has an acyl chain that the acyl chain is rather short. This makes the antigens more soluble in aqueous solutions and thus easier in use.
- the increased hydrophilicity that results from relatively short hydrocarbon chains makes the detection surface or sensor surface to which the antigens are immobilised more hydrophilic. Because of this, interactions of antibodies in the antigen occur easier and the speed of the detection will be enhanced. Moreover, it will be easier to synthesize the antigens in case synthetic antigens are used.
- the acyl chain may be a linear or branched C 1 -C 20 chain, such as a C 5 -C 20 chain.
- the antigen of formula (II) is represented by a compound of formula (XIV) or (XV) , optionally modified with one or more functional groups that enable immobilisation to a solid substrate.
- the antigen in accordance with formula (II) is selected from the group of 1-tuberculosinyladenosine (as in formula (XIV)) , 1-tuberculosinyl-2’-deoxyadenosine, 1-tuberculosinyl-O-acetyladenosine or a combination thereof.
- These compounds occur naturally in Mycobacterium tuberculosis bacteria and can be isolated therefrom.
- the natural antigen may be used and, depending on the substrate to which it is to be immobilised, modified for immobilisation purposes if necessary.
- the levels of these compounds in Mycobacteria are rather low, much lower than for instance the levels of mycolic acid. Therefore, it is more advantageous to chemically synthesize these molecules, or to synthesize them in a production host micro-organism followed by isolation and optional further purification steps. This saves considerable costs. It is therefore preferred that these compounds are synthetic.
- Synthetic antigens in accordance with formula (II) may for instance be synthetized in accordance with the method described in Buter et al., 2016.
- tuberculosinyl adenosine antigens as defined above are used in a method for detecting a marker for tuberculosis a very high tuberculosis specific binding of antibodies to these antigens is detected.
- the signal derived from the actual markers for tuberculosis is significantly less obscured by a background signal than when immobilised mycolic antigens are used, so that the signal derived from the actual markers for tuberculosis becomes more pronounced. This way the invention provides a significant improvement with regard to the sensitivity of detection of markers for tuberculosis.
- tuberculosinyl adenosine antigens as defined above are used in a method for detecting a marker for tuberculosis in combination with other types of antigens that are capable of binding to an antibody which is indicative for the presence of mycobacterial material in a human or animal, a more reliable indication is provided that the subject from which the sample is derived has active tuberculosis.
- antigens such as the other antigens described herein, although they may serve as reliable markers of the presence of antibodies against mycobacterial material, may not distinguish between an active tuberculosis infection (such as in smear negative, smear positive, HIV-positive/tuberculosis co-infected, paediatric and extra-pulmonary patients) and an inactive tuberculosis infection (such as in a Bacillus Calmette–Guérin (BCG) vaccinated subject or a subject cured from a tuberculosis infection) .
- BCG Bacillus Calmette–Guérin
- tuberculosinyl adenosine antigens as defined above are not properly detectable in samples derived from subjects vaccinated with a BCG vaccine or from subjects cured from a tuberculosis infection. In other words, if a sample contains a measurable amount of antibodies against tuberculosinyl adenosine antigens as defined above this is an indication of active tuberculosis. This makes it possible to distinguish in a tuberculosis test between subjects with active tuberculosis and subjects wherein the infection is inactive.
- the antigens used in the invention may be isolated from Mycobacterium tuberculosis or be synthetic, i.e. obtained by full chemical synthesis or by synthesis in a production host which is not a Mycobacterium, for instance by transgenic expression, e.g. in E. coli, followed by isolation and optional further purification steps.
- one or more samples from a human or animal may be compared to a sample from a human or animal which is confirmed to be healthy and to a sample from a human or animal which is confirmed to have tuberculosis.
- all samples in one analysis undergo the same treatment in accordance with the steps of the method of the invention.
- the high specificity of at least the tuberculosinyl adenosine antigens and diacyl glycolipid antigens for tuberculosis specific antibodies and the associated lower risk of false positives make it even possible to reliably diagnose whether a person has tuberculosis without the necessity of a reference sample obtained from a healthy subject.
- tuberculosinyl adenosine antigens and diacyl glycolipid antigens for tuberculosis specific antibodies make it possible to reliably diagnose whether a person has tuberculosis without the necessity to divide a sample from a human or animal into two sample fractions of which one is exposed to antigens before the two fractions are exposed to a detection substrate with antigens such as for instance described in WO 2005/116654 or WO 2013/186679, where mycolic acids are used as the sole immobilised antigens.
- the method of the invention is therefore less difficult to perform and leads to fast and reliable diagnosis.
- a sample from a human or animal is provided.
- the sample is derived from a human or animal which is suspected of having active tuberculosis, for instance a human or animal that had contact with someone suffering from tuberculosis or who resides in an area or travelled to an area with high prevalence of tuberculosis.
- samples may be used that have been collected at an earlier stage, stored until use under suitable conditions and provided at a suitable moment.
- a sample may be used in the detection method of the invention on the spot, i.e. as a point of care test.
- the sample is preferably a blood derived sample.
- the sample may be a whole blood sample, a plasma sample or a serum sample.
- Blood serum is blood plasma without clotting factors and is preferred as plasma.
- the word plasma in this application may therefore as well refer to (blood) serum.
- Serum is preferred because it contains less different materials than blood plasma, which may lead to aspecific interactions or unwanted biological activity.
- serum may have a lower viscosity than blood plasma. Using serum therefore may circumvent the need for diluting a sample, which saves time and materials.
- the sample is a whole blood sample
- the sample is preferably pre-filtered or separated to plasma or serum.
- a suitable filter for such a pre-filtering step is a 0, 2 micron filter.
- Plasma or serum in this application may therefore also refer to diluted plasma or serum.
- a dilution of the blood or plasma may therefore be implemented in the method of the invention, such as 5 to 10 x dilution, a 10 to 20 x dilution, a 20 to 50 x dilution, a 50 to 100 x dilution, a 250 to 5000 x dilution, a 750 to 1250 x dilution, such as for instance a 5 x, 10 x, 20 x, 50 x, 100 x, 200 x, 500 x, 4000 x, 2000 x or 1000 x dilution.
- such dilution may take place before the step of separating the plasma from the blood step (filter step or separating step) .
- dilution may take place after the filter step.
- Dilution may be performed with any suitable diluent, for example a PBS based buffer, such as a blocking buffer.
- a PBS based buffer such as a blocking buffer.
- Such buffer may for example be a PBS/AE buffer comprising NaCl, KCl, KH 2 PO 4 , Na 2 HPO 4 and EDTA in water at physiological pH.
- Such buffer may be a PBS based buffer consisting of 8.0 g NaCl, 0.2 g KCl, 0.2 g KH 2 PO 4 , and 1.05 g Na 2 HPO 4 per liter of double distilled, deionized water containing 1 mM EDTA and 0.025% (m/v) sodium azide which is adjusted to pH 7.4.
- the whole blood sample or plasma or serum may be further diluted with agents that prevent blood clotting, such as EDTA, heparine or citrate.
- a detergent may be added in low concentration to the blood/plasma/serum to avoid sticking of components of the test system used.
- At least part of the sample is exposed to a solid substrate carrying the immobilised antigen, i.e. to a detection substrate, and binding of antibodies to the antigen is detected.
- antigens for use in the present invention are (mycolic acid derived antigens, tuberculosinyl adenosine antigens and diacyl glycolipid antigens are as defined above) combinations comprising or consisting of 2 or 3 types of antigens as follows:
- Tuberculosinyl adenosine antigens + mycolic acid derived antigens A) Tuberculosinyl adenosine antigens + mycolic acid derived antigens.
- tuberculosinyl adenosine antigens perform particularly well in providing a reliable indication that the subject from which the sample is derived has active tuberculosis while the diacyl glycolipid antigens perform particularly well in case samples derived from smear negative persons are tested.
- This enables very sensitive detection of tuberculosis, irrespective of the bacterial load in a patient.
- use of these antigens which each result in a specific output upon exposure to a sample from a subject suspected of having tuberculosis enables to provide information about the nature and/or disease state of the subject.
- mycolic acid derived antigens as an additional marker for the presence of antibodies mycolic acid derived antigens is used in combination with the diacyl glycolipid antigens and tuberculosinyl adenosine antigens.
- mycolic acid involves a risk of false positive/negative signal
- detection of binding of antibodies in the sample to mycolic acid derived antigens provides an additional indication of whether or not a person is infected with tuberculosis or not. This further enhances the reliability of the method of the invention.
- This combination of three antigens provides an excellent way of detecting tuberculosis and determining whether the tuberculosis is active or not.
- antigens mycolic acid derived type antigens, tuberculosinyl adenosine type antigens and diacyl glycolipid type antigens as defined in this application, no other types of antigens are used as immobilised antigens in the method, solid substrate and biosensor.
- tuberculosis can be detected in an early stage because of the high specificity for smear negative patients, and patients with active tuberculosis can be distinguished from a person with inactive tuberculosis infection, such as in a BCG vaccinated person or a person cured from a tuberculosis infection) .
- a person with inactive tuberculosis infection such as in a BCG vaccinated person or a person cured from a tuberculosis infection
- multiple antigens which each result in a specific output upon exposure to a sample from a subject suspected of having tuberculosis enables to provide information about the nature and/or disease state of the subject.
- the tuberculosinyl adenosine antigen may be 1-tuberculosinyl adenosine
- the mycolic acid derived antigen may be mycolic acid
- the diacyl glycolipid antigen may be the molecule according to formula (XIII) , wherein the hydrophobic tails of these molecules may be modified for immobilisation.
- a sample is exposed to multiple antigens binding of antibodies to the different types of antigens may be detected simultaneously, which is preferred.
- a sample may be divided in subsamples before exposure, each subsample dedicated to be exposed for a particular type of antigen.
- Simultaneous detection may be performed with one or more substrate carrying multiple types of antigens or with multiple substrates each carrying a particular type of antigen, for instance in a bioreactor comprising different compartments each containing a solid substrate with one type of antigen.
- the first type of antigens and one or more further type of antigens may be immobilised on a solid substrate.
- the invention also relates to a solid substrate which comprises a combination of immobilised antigens according to any of the combinations A-D above.
- different solid substrates may be used for performing one detection, each having a particular type of antigen immobilised thereto.
- the sample is exposed to the solid substrate with the immobilised antigen or multiple types of immobilised antigens as described above; followed by detecting binding of antibodies to the antigens immobilised to said substrate.
- Binding of the antibodies to the immobilised antigens can be detected by means of any assay that involves measurement of change of mass on the substrate, change of refractive index, change of entropy, change of enthalpy, viscosity change, temperature change, colour change etc.
- Detection of binding of antibodies to the antigen on the detection substrate may take place with any suitable detection method, including simple visual detection or methods that include voltametrical, amperometrical or any electrochemical detection.
- Detection of binding of antibodies to the antigen on the detection substrate may take place in real time or by means of an end-point assay.
- Suitable real time detection assays include surface plasmon resonance or electrochemical impedance spectroscopy, isothermal titration calorimetry, bio-layer interferometry, optical gratings, photonic crystal, acoustic resonant profiling, quartz crystal microbalances.
- the solid substrate carrying the antigen may be silica based, such as substrates based on silicium dioxide.
- the antigens are preferably modified at one or both of the acyl chains with a functional group that enables immobilisation.
- Silica based substrates are particularly useful when ring resonance technology is used to detect binding of antibodies to the immobilised antigens.
- the detection is carried out using a biosensor chip using a Si-based ring resonator. This enables the method of the invention to be carried out with a very compact device.
- the solid substrate is gold based.
- Gold based substrates are particularly useful when surface plasmon resonance or electrochemical impedance spectroscopy are used to detect binding of antibodies to the immobilised antigens.
- the detection of binding of antibodies and/or other material to the antigen on the detection substrate may be carried out in an automated device.
- Various automated devices will be known to the person skilled in the art and the skilled person will be able to select suitable software means to determine the degree or extent of binding to the detection substrate.
- Detection of binding of antibodies to the antigen on the detection substrate may also take place by means of an end-point assay.
- end-point assay is to be understood as an assay wherein the outcome of interest is the end result after a fixed assay incubation period, in contrast to the aforementioned real-time assay.
- An end-point detection assay may for instance detect changes to levels of color, fluorescence, absorbance or luminescence at the end of a test.
- Suitable end-point assays include enzyme-linked immunosorbent assay (ELISA) , Western blotting, radioactive labelling assay, photospectrometric assay, immunofluorescence, immunoprecipitation, immunocytochemistry, immunohistochemistry, amperometric or voltametric detection assays, or electrochemical impedance spectroscopy.
- ELISA enzyme-linked immunosorbent assay
- Western blotting radioactive labelling assay
- photospectrometric assay immunofluorescence, immunoprecipitation
- immunocytochemistry immunohistochemistry
- amperometric or voltametric detection assays or electrochemical impedance spectroscopy.
- an end-point assay detection takes place by means of an immunogold filtration assay.
- the detection substrate is a microporous membrane, preferably a nitrocellulose membrane or a PVDF membrane, to which an antigen is immobilised.
- secondary antibodies that bind the heavy chain of the primary antibodies that bind to the immobilised antigens.
- Many suitable secondary antibodies are commercially available.
- the secondary antibody may be coupled to nanoparticles or beads, for instance gold beads, or associated with liposomes.
- Examples of secondary antibodies may be protein A or G, possibly conjugated with an enzyme that enables detection.
- a particular suitable technique or detecting the binding of antibodies to the immobilised antigens on the detection substrate is the so-called immunogold filtration assay (IGFA) , and in particular the dot immunogold filtration assay (DIGFA) .
- IGFA immunogold filtration assay
- DIGFA dot immunogold filtration assay
- Immunogold filtration assays are methods combining ELISA and immunogold technique and are methods in which a sample to be assayed is allowed to filtrate through a microporous membrane, preferably a nitrocellulose membrane, and is captured by a capture probe coated on the membrane.
- a colloidal gold-labelled probe is allowed to filtrate through the microporous membrane in the same manner.
- the antigen is immobilised on a microporous membrane, preferably a nitrocellulose membrane.
- the optionally pre-treated samples can be applied to the membrane.
- colloidal gold-labelled second antibodies can be added onto the membrane to have gold particle aggregation in the antigen-antibody reaction place.
- aggregation visible red or brown spots are formed.
- the intensity of the spot is proportional to the amount of reactions between antigen and antibody, i.e. to the amount of antibodies in the sample. In other words a sample from a person suffering from tuberculosis will result in a more intense spot than a sample from a person which is healthy.
- Immunogold filtration assays are simple and rapid detection methods because no instruments are required except a membrane and the reagents and the results can be observed by the naked eye within a few minutes.
- the microporous membrane may be for example a nitrocellulose membrane, a cellulose acetate membrane or a PVDF membrane with a suitable pore diameter.
- nitrocellulose is used.
- a suitable pore diameter is 0, 2 to 5 ⁇ m.
- the membrane may be washed with a suitable buffer, for example a PBS based buffer.
- a PBS based buffer may for example be a PBS/AE buffer comprising NaCl, KCl, KH 2 PO 4 , Na 2 HPO 4 and EDTA in water at physiological pH.
- Such buffer may be a PBS based buffer consisting of 8.0 g NaCl, 0.2 g KCl, 0.2 g KH 2 PO 4 , and 1.05 g Na 2 HPO 4 per liter of double distilled, deionized water containing 1 mM EDTA and 0.025%(m/v) sodium azide which is adjusted to pH 7.4.
- the antigen may be immobilised to the microporous membrane in a dot wise manner.
- a DIGFA assay the samples are also applied to the membrane in the form of dots.
- the colloidal gold-labelled second antibodies are added in the form of dots.
- a DIGFA assay is particularly preferred because at different spots on several membranes various antigens deriving from various mycobacterial strains may be immobilised. This way it becomes possible to provide information on which mycobacterial strain a patient is infected with.
- Another advantage of using DIGFA is that samples derived from different persons can be compared in one test, because DIGFA enables fast and reliable detection of antibody-antigen interaction in an unlimited amount of spots, depending on the size of the membrane.
- the detection of binding of antibodies and/or other material to the immobilised antigens may be carried out in an automated device.
- Various automated devices will be known to the person skilled in the art and the skilled person will be able to select suitable software means to quantify the degree or extent of binding on the detection substrates.
- the detection of binding of antibodies and/or other material to the immobilised antigen may be performed by a visual detection technique or any other suitable detection technique.
- detection by means of the end-point assay takes place visually, preferably with the naked eye. This has the advantage of easy detection without the need for expensive and complicated detection technology.
- binding of antibody antibodies and/or other material to the immobilised antigens may be assessed by means of the naked eye.
- a visual signal e.g. the red staining in case a DIGFA assay is applied as end-point assay, may also be detected with help of a mobile app, i.e. a computer program designed to run on mobile devices such as tablet computers or smart phones.
- a mobile app i.e. a computer program designed to run on mobile devices such as tablet computers or smart phones.
- an app can be used that is designed to compare the binding signal between different samples or sample fractions and which indicates whether the human or animal from which the sample originated has tuberculosis.
- the method of the invention may comprise additional steps that are advantageous for the sensitivity of the method.
- the method may contain further steps of exposing the sample to molecules that have affinity for molecules in the sample that lead to a binding signal which is not specific for tuberculosis in order to scavenge away these non-specific molecules.
- the method may also contain further steps of dividing the sample.
- the high specificity of in particular the diacyl glycolipid antigens and the tuberculosinyl adenosine for tuberculosis specific antibodies and the associated lower risk of false positives also make it possible to reliably diagnose whether a person has tuberculosis without the necessity to divide a sample from a human or animal into two sample fractions of which one is exposed to antigens before the two fractions are exposed to a detection substrate with antigens. Nevertheless, the antigens mentioned herein would be suitable in such a method.
- the method of the invention therefore may be a real-time method comprising the steps of:
- step iii) exposing the sample fraction exposed in step iii) to a solid test substrate which is a solid substrate as described above for the first aspect of the invention and exposing the sample fraction exposed in step iv) to a solid control substrate which which has the same composition as the solid test substrate;
- step vii) comparing the degree or extent of binding between the test and control substrates, any observed lesser binding to the test substrate being an indicator of the presence of antibodies to the immobilised antigen in the sample that indicates tuberculosis in the human or animal from which the samples originated, wherein the antigens immobilised to the solid substrate of step iii) comprise at least one type, preferably all the types of antigens that are immobilised to the solid test and control substrates.
- the method of the invention may be an end-point method of detecting antibodies against mycobacterial material in a sample
- step iii) exposing at least part of the sample fraction exposed in step iii) to a solid test substrate which is a substrate which is a solid substrate as described above for the first aspect of the invention and exposing the sample fraction exposed or stored in step iv) to a solid control substrate which has the same composition as the solid test substrate;
- step vii) comparing the degree or extent of binding between the test and control substrates, any observed lesser binding to the test substrate being an indicator of the presence of antibodies to the immobilised antigen in the sample that indicates tuberculosis in the human or animal from which the samples originated, wherein the antigens immobilised to the solid substrate of step iii) comprise at least one type, preferably all the types of antigens that are immobilised to the solid test and control substrates.
- the substrate to which the antibody is immobilised in step iii) of these embodiments is in general not made of the same material as the test/control substrate.
- the substrate in step iii) may be made of any material that is inert for non-specific binding of molecules of the sample. Such materials include polytetrafluorethylene (e.g. ) , polypropylene, polyetherketone (PEEK) and polyethylene.
- the test and control substrates in these embodiments are sensing surfaces or substrates of a detection device or detection surfaces or substrates in a detection assay as discussed above.
- compounds may be immobilised to control the hydrophobicity/hydrophilicity of the solid substrate.
- the substrate’s surface may become too hydrophobic for efficient binding of antibodies.
- This may be obtained by co-immobilisation of hydrophilic molecules distributed between the antigens or by immobilization of the antigens onto hydrophylic molecules that are immobilized to the solid substrate.
- Such hydrophilic molecules may for instance be PEG or mPEG.
- the solid substrate may contain any combination of antigens that are capable of binding to antibodies which are indicative for tuberculosis, but at least one of the combinations A-D as specified above.
- the antigens may be co-immobilised to the same solid substrate or be provided on separate solid substrates. The latter enables detection of binding of antibodies to the different types of antigens on separate sections or in separate compartments of a biosensor or in sequence.
- solid substrates may thus comprise:
- antigens capable of binding to an antibody which is indicative for the presence of mycobacterial material in a human or animal such as a solid substrate with 1-tuberculosinyladenosine antigens antigens and Ac 2 SGL antigens immobilised thereto; a solid substrate with 1-tuberculosinyladenosine antigens and mycolic acid antigens immobilised thereto; or a solid substrate with Ac 2 SGL antigens and mycolic acid antigens immobilised thereto;
- -a solid substrate with three types of antigens capable of binding to an antibody which is indicative for the presence of mycobacterial material in a human or animal such as a solid substrate with 1-tuberculosinyladenosine antigens, Ac 2 SGL antigens and mycolic acid antigens immobilised thereto.
- a solid substrate with 1-tuberculosinyladenosine antigens, Ac 2 SGL antigens and mycolic acid antigens immobilised thereto may be used for performing one detection assay, each having a particular type of antigen immobilised thereto in any of the combinations mentioned above.
- antigens are immobilised to a substrate depends on the characteristics of the substrate to which the antigens are immobilised.
- Some substrates allow immobilisation of antigens without modification, for instance by means of hydrophobic interaction of one or both of the hydrophobic carbon chains, such as acyl chains, of the antigen with the substrate. This would for instance be possible if the antigens are immobilised to the surface of an ELISA plate, a nitrocellulose membrane or a PVDF membrane.
- the most convenient immobilisation method is direct physical absorption of the antigen to the assay membrane.
- Suitable substrates may require modification of the antigen with a functional group.
- the term “functional group” in this application is therefore to be understood as a group that enables immobilisation of the antigen to a solid substrate. These functional groups are preferably added to one or both of the hydrophobic carbon chains of the antigens.
- tuberculosinyl adenosine antigens may be added to one or more of the hydrophobic carbon chains of the antigens, e.g. the alkyl chains. Alternatively, functional groups may be added to the nitrogen or amine of the adenosine moiety. Alternatively, functional groups may be added to the tuberculosinyl tail with formula (III) of the antigen.
- the functional groups are preferably added to one or both of the hydrophobic carbon chains of the antigens, i.e. the acyl chains of the diacyl glycolipid antigens.
- a functional group can for instance be a thio group, an amine group, an aldehyde group or any other suitable group.
- Such a functional group can for instance be a thio group, an amine group, an aldehyde group, a double bond (i.e. an alkene group) or any other suitable group.
- modified substrates include:
- NHS ester-activated support materials wherein NHS esters are reactive groups formed by EDC activation of carboxylate molecules to react with primary amines on the antigen;
- CDI-activated support materials wherein carbonyl diimidazole (CDI) activates hydroxyls to form reactive imidazole carbamates that form carbamate linkages with primary amine-containing/modified antigens;
- Antigens can be immobilised or “coupled” directly to solid support material by formation of covalent chemical bonds between particular functional groups on the (modified) antigen (e.g., primary amines, sulfhydryls, carboxylic acids, aldehydes) and reactive groups on the support material. It is also possible that antigens are immobilised non-covalent binding such as hydrophobic interaction or via streptavidin-biotin coupling. Various coupling methods are possible.
- thio coupling, amine coupling and carbonyl-reactive immobilisation methods which involve coupling through carbonyl (sugar) groups where cis-diols can be oxidized with sodium periodate to create aldehydes as sites for covalent immobilisation.
- a silica based substrate may be modified with a silane derivative containing a free amine group, e.g. aminopropyltriethoxysilane or aminomethoxysilane. This free amine group reacts spontaneously with a thiol modified antigen to form a covalent bond. This does not require complicated protocols.
- a thiol cysteamine monolayer may be applied to the gold substrate, the free amine of the thiol cysteamine may than be applied in a spontaneous coupling reaction with a thiol modified antigen. This would not require complicated protocols.
- coupling may be based on various mechanisms. An unmodified antigen may be immobilised to the membrane with its hydrophobic tail (s) as discussed above, but other mechanisms are also possible.
- Such mechanisms include covalent attachment of thiolated antigen to an epoxide-functionalized nitrocellulose membrane, attachment of a biotinylated antigen through a nitrocellulose-binding streptavidin anchor protein, and fusion of an antigen to a novel nitrocellulose-binding anchor protein for direct coupling and covalent attachment through an epoxide thiol linkage using a functionalized nitrocellulose membrane immobilisation.
- the antigens are immobilised to a solid substrate which can be exposed to a sample derived from a human or animal so that binding of antibodies to the immobilised antigens can be determined, either qualitatively or quantitatively or both.
- solid substrate means the same as “solid surface” or ” solid support” .
- the solid substrate may be a sensing surface or substrate of a detection device or a detection surface or substrate in a detection assay.
- the substrate to which the antigens are immobilised may be a sensing surface of a surface plasmon resonance device, electrochemical impedance spectroscopy device, isothermal titration calorimetry device, bio-layer interferometry device, optical gratings device, photonic crystal device, acoustic resonant profiling device, or quartz crystal microbalance device or a detection surface in an enzyme-linked immunosorbent assay (ELISA) , a Western blotting assay, radioactive labelling assay, photospectrometric assay, immunofluorescence, immunoprecipitation assay, immunocytochemistry assay, immunohistochemistry assay, voltametric detection assay, amperometric detection assay or electrochemical impedance spectroscopy assay.
- ELISA enzyme-linked immunosorbent assay
- the immobilised solid substrate of the invention can be used in a biosensor.
- This biosensor comprises a combination of 2 or more of the tuberculosinyl adenosine antigens, diacyl glycolipid antigens, and mycolic acid derived antigens as defined above immobilised to one or more solid substrates.
- the antigens may be immobilised to one and the same substrate mixed or at separate locations or at different substrates in the biosensor. Using separate locations or separate substrates enables analysis of the nature of a possible tuberculosis infection for instance for obtaining an indication whether the tested person has active tuberculosis. In one embodiment therefore multiple types of antigens are immobilized to the same substrate. In another embodiment of the biosensor, each type of antigen is immobilized on a separate substrate.
- the combination of antigens in the biosensor may be any of the combinations A-D as described above.
- the biosensor can be any biosensor which is suitable for use in the method according to the invention.
- the biosensor may comprise a silica based substrate with immobilised antigens and a Si ring resonator. Such a biosensor can be used for ring resonance.
- the substrates of the chambers of the biosensor are gold based. Gold based substrates are particularly useful when surface plasmon resonance or electrochemical impedance spectroscopy are used to detect binding of antibodies to the immobilised antigens.
- the invention also encompasses any other biosensor comprising the solid substrate of the invention.
- Serum derived from humans that were known to be suffering of tuberculosis was derived from both smear positive (Patients 1-9 (+) ) and smear negative patients (Patients 11-18 (-) ) .
- Samples of patients 19-40 tested for anti-TbAd antibodies are a random selection of smear negative and positive patients.
- samples were used derived from healthy humans (Healthy 1, 2 and 3) . Fractions of 0, 5 ml were obtained and trans ferred each to a 0, 2 micron spin filter and centrifuged at 10000 g. The flow-through was diluted 1: 20 in blocking buffer.
- Fig. 1 shows the results of an ELISA test with rabbit anti-human IgM HRP as secondary antibody using immobilised mycolic acid. It is clear that very high background signal is obtained in the healthy persons (healthy 1 and 2, white bars) . As these persons were confirmed to be healthy and samples from these persons thus do not contain antibodies against mycobacterial material, this signal is the result of materials that are unrelated to tuberculosis that do bind to mycolic acid and lead to an ELISA signal. The samples derived from smear negative patients (patients 11-18 (-) , grey bars) show absorbance signals which do not significantly exceed the signal of the healthy persons.
- Fig. 2 shows ELISA tests with rabbit anti-human IgM HRP as secondary antibody that were performed with immobilised 1-tuberculosinyladenosine.
- the background signal obtained in the healthy persons appears to be markedly lower than that is obtained by using mycolic acids as immobilised antigens.
- Fig. 2 show that if these immobilised antigens are used in a method for detecting a marker for tuberculosis a very high tuberculosis specific binding of antibodies to these antigens is detected, regardless of whether the samples are derived from smear positive (patients 1-9 (+) , black bars) or smear negative persons (patients 10-18 (-) , dark grey bars) as the signals of both are similar the signals of the random samples (19-40) .
- This high specificity is clearly not the case when mycolic acids immobilised to a solid substrate are applied.
- the signal derived from antibodies against 1-tuberculosinyladenosine appears to be significantly less obscured by a background signal than when immobilised mycolic antigens are used. This results in a significant improvement with regard to the sensitivity of detection of markers for tuberculosis.
- Fig. 3 shows that when ELISA tests with rabbit anti-human IgM HRP as secondary antibody were performed with immobilised Ac 2 SGL, the background signal obtained in the healthy persons (healthy 1 and 2, white bars) appears to be markedly lower.
- the samples derived from smear positive patients (patients 1-9 (+) , black bars) show absorbance signals which are higher than the signal of the healthy persons, thus confirming the presence of specific antibodies that indicate tuberculosis. This effect is even more pronounced in the samples derived from smear negative patients (patients 11-18 (-) , grey bars) which show absorbance signals which are significantly higher than the signal of the healthy persons.
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Abstract
Description
Claims (32)
- Method of detecting the presence of antibodies against mycobacterial material in a sample comprising the steps of: providing a sample from a human or animal; and detecting binding of antibodies in said sample to at least two types of immobilised antigens capable of binding to an antibody which is indicative for the presence of mycobacterial material in a human or animal, selected from the following group:A) a mycolic acid derived antigen, optionally modified with one or more functional groups that enable immobilisation to a solid substrate ,B) a diacyl glycolipid antigen; represented by the following formula (I)wherein:R2 and R3, identical or different, are independently chosen from H, SO3H, SO3- or SO3-/M+, wherein M+ is a cation; andR2’ and R3’ , identical or different, are acyl groups,wherein the antigen represented by formula (III) is optionally modified with one or more functional groups that enable immobilisation to a solid substrate, and enantiomers, diastereoisomers, and mixtures thereof; andC) a tuberculosinyl adenosine antigen; represented by the following formula (II) :wherein in formula (III) R1 is H or a group with formula (III)R2 is absent or a group with formula (III) , provided that one of R1 and R2 is a group with formula (III) ,R3 and R4 are selected independently from hydrogen, OH, a carboxylic acid group or an acyl group, in any combination thereof, wherein said tuberculosinyl adenosine antigen is optionally modified with one or more functional groups that enable immobilisation to a solid substrate, and enantiomers, diastereoisomers of the antigens, and mixtures thereof.
- Method according to claim 1 or 2, wherein in the diacyl glycolipid antigen according to formula (III) one of R2’ and R3’ is a group represented by the following formula (IV)wherein R4 is a linear saturated hydrocarbon chain with formula CnHn+1, wherein n is an integer between 1 and 20, optionally modified with one or more functional groups, and wherein Y is an integer between 1 and 10, and wherein R5 is H or OH, and the other one of R2’ and R3’ is a linear saturated hydrocarbon chain with formula CnHn+1, wherein n is an integer between 1 and 20, optionally modified with one or more functional groups.
- Method according to claims 1 or 3 wherein in the diacyl glycolipid antigen according to is a diacylated sulfoglycolipid (Ac2SGL) as found in Mycobacterium tuberculosis, optionally modified with one or more functional groups, preferably wherein said Ac2SGL is 2-palmitoyl-3-hydroxyphthioceranoyl-2’ -sulfate-α-α’ -D-trehalose or 2-stearoyl-3-hydroxyphthioceranoyl-2’ -sulfate-α-α’ -D-trehalose, optionally modified with one or more functional groups.
- Method according to any of the claims 1-4, wherein wherein in formula (II) of said tuberculosinyl adenosine antigen R4 is OH, preferably wherein R4 and R3 are OH.
- Method according to any of the claims 1-5, wherein said tuberculosinyl adenosine antigen is represented by a compound of formula (XIV) or (XV) or a combination of tuberculosinyl adenosine antigens in accordance with formula (XIV) comprising a compound of formula (XVI) or (XVII) or a combination thereof,optionally modified with one or more functional groups that enable immobilisation to said solid substrate.
- Method according to any of the claims 1-6, wherein said tuberculosinyl adenosine antigens are selected from the group of 1-tuberculosinyladenosine, 1-tuberculosinyl-2’ -deoxyadenosine, 1-tuberculosinyl-O-acetyladenosine or a combination thereof optionally modified with one or more functional groups that enable immobilisation to said solid substrate.
- Method according to any of the claims 1 to 7, comprising comprising detecting binding of antibodies in said sample toa said C) tuberculosinyl adenosine antigen, andto a said B) diacyl glycolipid antigen.
- Method according to any of the claims 1 to 7, comprising detecting binding of antibodies in said sample toa said C) tuberculosinyl adenosine antigen, andto a said A) mycolic acid derived antigen.
- Method according to any of the claims 1 to 7, comprising detecting binding of antibodies in said sample toa said B) diacyl glycolipid antigen, andto a said A) mycolic acid derived antigen.
- Method according to claim 1 to 10, comprising detecting binding of antibodies in said sample toa said C) tuberculosinyl adenosine antigen,to a said B) diacyl glycolipid antigen, andto a said A) mycolic acid derived antigen.
- Method according to any of the previous claims, wherein said antigens are isolated from Mycobacterium tuberculosis or a production host microorganism other than Mycobacterium or wherein the antigens are chemically synthesized.
- Method according to claim any of the previous claims, wherein the antigens are immobilised to one or more solid substrates.
- Method according to any of the previous claims, wherein detecting the binding of the antibodies to the immobilised antigens involves measurement of change of mass on the substrate, change of refractive index, change of entropy, change of enthalpy, viscosity change, temperature change, or colour change.
- Method according to any of the previous claims, in which detecting of binding of antibodies to the immobilised antigen takes place in real time.
- Method according to claim 15, wherein detection is carried out using surface plasmon resonance or electrochemical impedance spectroscopy, isothermal titration calorimetry, bio-layer interferometry, optical gratings, photonic crystal, acoustic resonant profiling, quartz crystal microbalances.
- Method according to any of the claims 1 to 14 in which detecting of binding of antibodies to the immobilised antigen takes place by means of an end-point assay.
- Method according to claim 17, wherein the end-point assay is selected from the group consisting of enzyme-linked immunosorbent assay (ELISA) , Western blotting, radioactive labelling assay, photospectrometric assay, immunofluorescence, immunoprecipitation, immunocytochemistry, immunohistochemistry, electrochemical impedance spectroscopy.
- Solid substrate, comprising a combination of 2 or more of the tuberculosinyl adenosine antigens, diacyl glycolipid antigens, and mycolic acid derived antigens as defined in any of the claim 1 to 7 immobilised to said substrate.
- Solid substrate according to claim 19, wherein said combination is a combination of said tuberculosinyl adenosine antigens and said diacyl glycolipid antigens.
- Solid substrate according to claim 19, wherein said combination is a combination of said tuberculosinyl adenosine antigens and said mycolic acid derived antigens.
- Solid substrate according to claim 19, wherein said combination is a combination of said tuberculosinyl adenosine antigens and said mycolic acid derived antigens.
- Solid substrate according to claim 19 wherein said combination is a combination of said tuberculosinyl adenosine antigens, said diacyl glycolipid antigens and said mycolic acid derived antigens.
- Solid substrate according to any of the claims 1-19-22, wherein said substrate is a sensing surface of a surface plasmon resonance device, electrochemical impedance spectroscopy device, isothermal titration calorimetry device, bio-layer interferometry device, optical gratings device, photonic crystal device, acoustic resonant profiling device, or quartz crystal microbalance device.
- Solid substrate according to any of the claims 1-9, wherein said substrate is a detection surface in an enzyme-linked immunosorbent assay (ELISA) , a Western blotting assay, radioactive labelling assay, photospectrometric assay, immunofluorescence, immunoprecipitation assay, immunocytochemistry assay, immunohistochemistry assay, amperometric detection assay, voltametric detection assay, or electrochemical impedance spectroscopy assay.
- Biosensor, comprising a combination of 2 or more of the tuberculosinyl adenosine antigens, diacyl glycolipid antigens, and mycolic acid derived antigens as defined in any of the claim 1 to 7, immobilised to one or more solid substrates.
- Biosensor according to claim 25, wherein multiple types of antigens are immobilized to the same substrate.
- Biosensor according to claim 25, wherein each type of antigen is immobilized on a separate substrate.
- Biosensor according to any of the claims 25-27, wherein said combination is a combination of said tuberculosinyl adenosine antigens and said diacyl glycolipid antigens.
- Biosensor according to any of the claims 25-27, wherein said combination is a combination of said tuberculosinyl adenosine antigens and said mycolic acid derived antigens.
- Biosensor according to any of the claims 25-27, wherein said combination is a combination of said diacyl glycolipid antigens and said mycolic acid derived antigens.
- Biosensor according to any of the claims 25-27, wherein said combination is a combination of said tuberculosinyl adenosine antigens, said diacyl glycolipid antigens and said mycolic acid derived antigens.
Priority Applications (5)
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US16/307,422 US11243204B2 (en) | 2016-06-08 | 2017-06-08 | Method for detecting the presence of mycobacterial material in a sample using at least two antigens |
RU2018146528A RU2018146528A (en) | 2016-06-08 | 2017-06-08 | METHOD FOR DETECTING THE PRESENCE OF MYCOBACTERIAL MATERIAL IN SAMPLE USING AT LEAST TWO ANTIGENS |
CN201780047043.7A CN109690313A (en) | 2016-06-08 | 2017-06-08 | Use method existing for mycobacteria substance at least two antigen test samples |
EP17809755.6A EP3469366A4 (en) | 2016-06-08 | 2017-06-08 | Method for detecting the presence of mycobacterial material in a sample using at least two antigens |
BR112018075414-5A BR112018075414A2 (en) | 2016-06-08 | 2017-06-08 | method for detecting the presence of mycobacterial material in a sample using at least two antigens, solid substrate and biosensor |
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NL2016914A NL2016914B1 (en) | 2016-06-08 | 2016-06-08 | Solid substrate comprising antigens immobilised thereto and use thereof in a method for detecting the presence of mycobacterial material in a sample |
NL2016913A NL2016913B1 (en) | 2016-06-08 | 2016-06-08 | Solid substrate comprising antigens immobilised thereto and use thereof in a method for detecting the presence of mycobacterial material in a sample |
NL2017204 | 2016-07-21 | ||
NL2017204A NL2017204B1 (en) | 2016-06-08 | 2016-07-21 | Solid substrate comprising antigens immobilised thereto, biosensor comprising said solid substrate and method for detecting the presence of mycobacterial material in a sample |
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PCT/CN2017/087547 WO2017211316A1 (en) | 2016-06-08 | 2017-06-08 | Method for detecting the presence of mycobacterial material in a sample using at least two antigens |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2020030034A1 (en) * | 2018-08-08 | 2020-02-13 | Kei International Limited | Synthetic antigens for tuberculosis detection |
WO2020119669A1 (en) * | 2018-12-10 | 2020-06-18 | Kei International Limited | Nitrocellulose sheet comprising immobilized immunoglobulins and lipid based antigens and use thereof |
US11243204B2 (en) | 2016-06-08 | 2022-02-08 | Kei International Limited | Method for detecting the presence of mycobacterial material in a sample using at least two antigens |
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CN113412427A (en) * | 2018-12-10 | 2021-09-17 | 凯仪国际有限公司 | Nitrocellulose sheet containing immobilized immunoglobulin and lipid-based antigen and use thereof |
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NL2027721B1 (en) | 2021-03-08 | 2022-09-26 | Tomorrows Ip Ltd | Method of preparing a detection substrate, detection substrate, and uses thereof |
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