NL2016913B1

NL2016913B1 - Solid substrate comprising antigens immobilised thereto and use thereof in a method for detecting the presence of mycobacterial material in a sample

Info

Publication number: NL2016913B1
Application number: NL2016913A
Authority: NL
Inventors: Theodorus Castrop Johannes
Original assignee: Kei International Ltd; Tomorrows Ip Ltd
Priority date: 2016-06-08
Filing date: 2016-06-08
Publication date: 2017-12-18

Abstract

The present invention relates to a solid substrate comprising an antigen immobilised to said substrate which antigen is capable of binding to an antibody which is indicative for the presence of mycobacterial material in a human or animal, and a method of detecting the presence of antibodies against mycobacterial material in a sample which applies said solid substrate comprising said immobilised antigen. The invention also relates to a biosensor comprising said solid substrate.

Description

Solid substrate comprising antigens immobilised thereto and use thereof in a method for detecting the presence of mycobacterial material in a sample

Introduction

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. A reliable and fast way of diagnosing tuberculosis is therefore of utmost importance.

Several methods of diagnosing tuberculosis have been developed, but all methods have their disadvantages.

Diagnosing tuberculosis based merely on signs and symptoms is difficult, as is diagnosing the disease in those who are immunosuppressed. The TB skin test (also called the Mantoux tuberculin skin test), TB blood tests (also called interferon-gamma release assays or IGRAs), and chest radiography (X-ray) , and tests on the presence of acid-fast-bacilli (AFB) on a sputum smear, indicate some of the infected individuals in days.

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. 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. In smear negative persons it is usually not clear at first sight that the person suffers from an illness. In addition, the smear test also gives poor results in extra-pulmonary tuberculosis, pediatric tuberculosis and in patients co-infected with HIV and tuberculosis .

Humans or animals infected with M. tuberculosis normally produce antibodies directed against the Mycobacterium. The presence of these antibodies in a sample taken from infected individuals indicates the infection. The most common Mycobacterium specific antigens are mycolic acids or derivatives thereof. For instance, WO 2005/116654 and WO 2013/186679 describe methods of antibodies in a sample against mycolic acids for the diagnosis of active tuberculosis, by detecting binding of antibodies to immobilised mycolic acid antigens.

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, which indicates that samples derived from healthy subjects contain materials which bind to mycolic acid antigens, but which are not indicative for tuberculosis. This leads to a false positive outcome. Thus, in case 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 obscures 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 of detection of tuberculosis.

Summary of the invention

The aim of the invention has been achieved by the provision of immobilised diacyl glycolipid antigens which are capable of binding to antibodies which are indicative for the presence of mycobacterial material with high specificity and a method of detecting the presence of antibodies against mycobacterial material in a sample which applies said immobilised antigen.

In a first aspect the invention relates to a solid substrate, comprising a diacyl glycolipid antigen immobilised to said solid substrate, wherein said immobilised antigen is capable of binding to an antibody which is indicative for the presence of mycobacterial material in a human or animal, wherein the antigen is a compound represented by the following formula (I),

wherein : R2 and R3, identical or different, are independently chosen from H, S03H, SO3- or S03~/M+, wherein M+ is a metal cation, preferably Na+ or K+; R2,and R3’, identical or different, are acyl groups, wherein the antigen is optionally modified with one or more functional groups that enable immobilisation to said solid substrate, and enantiomers, diastereoisomers, and mixtures thereof.

In a second aspect the invention relates to a method of detecting antibodies against mycobacterial material in a sample, a comprising the steps of: (1) providing a sample from a human or animal; (2) exposing at least part of the sample to the solid substrate comprising an immobilised antigen as defined in the previous paragraph; and (3) detecting binding of antibodies to said immobilised antigen.

In a third aspect the invention relates to a biosensor comprising said solid substrate comprising an immobilised antigen as defined in the paragraph before the previous paragraph.

The inventor has surprisingly found that if a solid substrate with the immobilised diacyl glycolipid antigens in accordance with formula I 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 .

Further in this respect it is noted that when the solid substrate with said immobilised diacyl glycolipid antigens in accordance with the invention is 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 .

Short description of the figures

Fig.l shows a diagram with ELISA results using mycolic acid immobilised on ELISA plates.

Fig.2 shows a diagram of ELISA results using a diacyl glycolipid antigen immobilised on ELISA plates.

Detailed description

In accordance with the invention a diacylglycolipid antigen is immobilised on a solid substrate. This antigen is capable of binding to an antibody which is indicative for the presence of mycobacterial material in a human or animal. With "mycobacterial material" in this context is meant material derived from Mycobacterium tuberculosis which leads to generation of antibodies in a human or animal. The presence of this material indicates tuberculosis. The diacyl glycolipid antigen immobilised on the substrate of the invention is a compound represented by the following formula (I),

wherein: R2 and R3, identical or different, are independently chosen from H, SO3-, S03H or S03_/M+, wherein M+ is a cation, preferably an metal cation , preferably Na+ or K+. In formula (I) R2’ and R3’, identical or different, are acyl groups, wherein the antigen is optionally modified with one or more functional groups that enable immobilisation to said solid substrate, and enantiomers, diastereoisomers, and mixtures thereof.

In formula (I) R2’ and R3’, identical or different, may be

wherein X is independently chosen from an unsaturated or saturated linear or branched hydrocarbon chain, optionally substituted with one or more substituents and/or modified with one or more functional groups.

In a preferred embodiment R2 is SO3-, S03H or S03_/M+, wherein M+ is a cation and R3 is H. It is preferred that in case R2 is S03_/M+ that the cation is a metal cation, preferably Na+ or K+.

In another preferred embodiment R2 and R3 are H.

It is preferred that in one of R2 and R3 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 and R3 X is a saturated branched hydrocarbon chain optionally substituted with one or more substituents and/or modified with one or more functional groups. In this respect it is further preferred that one of R2’ and R3’ is a group represented by the following formula (II)

(II) , wherein R4 is a linear saturated hydrocarbon chain with formula CnHn+i, 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+i, wherein n is an integer between 1 and 20, optionally modified with one or more functional groups. It is preferred that the acyl chains represented by R2’ and R3’ 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 substrate/surface or sensor substrate/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. In this respect in antigens with short R2 and R3 acyl chains R4 may be a linear saturated hydrocarbon chain with formula CnHn+i, 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. The other one of R2’ and R3’ in this respect may be a linear saturated hydrocarbon chain with formula CnHn+i, 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 be compound (III) :

(HI) wherein AcS represents a thio acetate group, and M+ is a cation, such as a proton or metal cation, or absent. 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. For silica substrates the substrate suitably can be coated with a silane active group to which the thiol of the modified antigen can bind.

In case the diacyl glycolipid antigen is present in Mycobacterium, tuberculosis it may be isolated from

Mycobacterium tuberculosis. Alternatively, it 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.

It is highly preferred that the antigen is a diacylated sulfoglycolipid (AC2SGL) as found in Mycobacterium tuberculosis, optionally modified with one or more functional groups. In a preferred embodiment said AC2SGL is 2-palmitoyl-3-hydroxyphthioceranoyl-2'-sulfate-οί-α' -D-trehalose or 2-stearoyl-3-hydroxyphthioceranoyl-2' -sulfate-α-α'-D-trehalose, optionally modified with one or more functional groups. AC2SGL 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 (IV)

(IV) wherein R2 is SO3-, S03H or S03_/M+, wherein M+ is a cation, preferably a metal cation, such as Na+ or K+.

These antigens lead to significant improvement with regard to the 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. In addition, these particular antigens perform excellent when applied to samples derived from patients co-infected with HIV and tuberculosis.

Because AC2SGL molecules exist in Mycobacterium tuberculosis they can be isolated from the bacteria. In this case the natural antigen is used and, depending on the substrate to which it is to be immobilised, modified for immobilisation purposes if necessary. The levels of AC2SGL in Mycobacteria are rather low, much lower than for instance the levels of mycolic acid. Therefore is it is more advantageous to synthesize AC2SGL molecules. This saves considerable costs. It is therefore preferred that AC2SGL is synthetic. Synthesis can be performed for instance as described in Geerdink et al, 2013.

The solid substrate to which the diacyl glycolipid antigen of the invention is immobilised may contain other immobilised molecules as well.

For instance, compounds may be immobilised to control the hydrophobicity/hydrophilicity of the solid substrate. In particular when antigens are immobilised that have long acyl chains the substrate's surface may become too hydrophobic for efficient binding of antibodies. In that case it may be preferred to immobilise the antigens to the solid substrate in a less dense packing. This may be obtained by co-immobilisation of hydrophilic molecules distributed between the antigens. 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. For instance a combination of different antigens that fall within the scope of formula I or a combination of one or more antigens that fall within the scope of formula I and other molecules .

In addition to the diacyl glycolipid antigens as described above, other antigens that are capable of binding to an antibody which is indicative for the presence of mycobacterial material in a human or animal may be co-immobilised to the solid substrate. Such antigens may include mycolic acids, cord factors, 1-tuberculosinyladenosine (1-TbAd), β—D-mannosyl phosphomycoketides and derivatives of these antigens in any combination.

In case the diacyl glycolipid antigens are immobilised together with further antigens that are capable of binding to an antibody which is indicative for the presence of mycobacterial material in a human or animal, it is preferred that these further antigens are β— D-mannosyl phosphomycoketides or derivatives thereof, optionally modified with one or more functional groups that enable immobilisation to said solid substrate. If the diacyl glycolipid antigens are immobilised with further antigens, it is preferred that they are co-immobilised with the abovementioned β—D-mannosyl phosphomycoketides or derivatives thereof, because the inventor has found high tuberculosis specific binding of antibodies to these antigens in particular in case of samples derived from patients that were tested smear positive. As the immobilised diacyl glycolipid antigens of the invention perform particularly well in case of smear negative subjects, the combination of these mannosyl phosphoketide antigens with the diacyl glycolipid antigens immobilised on a substrate thus opens the possibility to provide one detection or test substrate, which is very suitable for reliable diagnosis TB in both smear negative and smear positive patients. The abovementioned β—D-mannosyl phosphomycoketides or derivatives thereof can be defined as a compound represented by the following formula V:

wherein Y is an integer between 1 and 10 and R is a hydrocarbon group, and wherein X+ is a proton, a cation (such as a proton or metal cation) or absent, wherein the antigen is optionally modified with one or more functional groups that enable immobilisation to said solid substrate, and enantiomers, diastereoisomers, and mixtures thereof. In formula V, Y preferably is an integer between 1 and 5, preferably between 1 and 4, more preferably between 1 and 3, most preferably wherein Y is 2. Further, in formula V, R preferably is an alkyl group, more preferably a C1-C15 alkyl group, such as a C5 alkyl or a C7 alkyl, preferably wherein R is an n-C5-alkyl or a n-C7-alkyl. It is preferred that when the compound of formula V is modified, that R is modified with one or more functional groups that enable immobilisation to said solid substrate. It is particularly preferred that the compound of formula V is as follows:

wherein X is H, a cation or absent, and R is n-C7Hi5 or n-C5H11, optionally wherein R is modified with one or more functional groups that enable immobilisation to said solid substrate, and enantiomers, diastereoisomers, and mixtures thereof. It is highly preferred that R is n-C7Hi5 as this is the form that commonly occurs in Mycobacterium tuberculosis. These further antigens may be isolated from Mycobacterium tuberculosis or be synthetic.

The way antigens (either the diacyl glycolipid antigens or optional further antigens) are immobilised to the 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. For these substrates, the most convenient immobilisation method is direct physical absorption of the antigen to the assay membrane .

Other 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, i.e. the acyl chains of the diacyl glycolipid antigens. Such a functional group can for instance be a thio group, an amine group, an aldehyde group or any other suitable group.

Some coupling methods require that apart from the antigen also the solid substrate to which the antigens are coupled is modified. Examples of 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; • Aldehyde-activated support materials, wherein coupling of the antigen takes place by a reaction called reductive amination; • Azlactone-activated support materials, involving copolymerization of acrylamide with azlactone; • 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; • Sulfhydryl-reactive support materials, wherein the thiol group can be used for direct coupling reactions; • Maleimide-activated support materials, wherein maleimide-activated reagents form stable thioether linkages; • Iodoacetyl-activated support materials, which react with sulfhydryl groups resulting in stable thioether linkages; • Pyridyl disulfide support materials, wherein pyridyl disulfides react with sulfhydryl groups; and • Hydrazide-activated support materials.

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. For instance, 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. Also self-assembly immobilisation methods are possible. For instance, 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. In case of a gold solid substrate, 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. Regarding coupling to nitrocellulose membranes, 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 .

For purposes of detection of the presence of antibodies in a sample the antigens (diacyl glycolipid antigens and optional further 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. It is noted that in this application the term "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. In this respect, 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.

The immobilised solid substrate of the invention can be used in the method of the invention which comprises the steps of: (1) providing a sample from a human or animal; (2) exposing at least part of the sample to the solid substrate in accordance with the invention, which comprises the immobilised diacyl glycolipid antigen as defined above; and (3) detecting binding of antibodies to said immobilised antigen.

In this method, 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. For the sake of reliability it is highly preferred that all samples in one analysis undergo the same treatment in accordance with the steps of the method of the invention. The high specificity of the 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. The high specificity of the diacyl glycolipid antigens 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 such as for instance described in WO 2005/116654 or WO 2013/186679, where mycolic acids are used as immobilised antigens. The method of the invention is therefore less difficult to perform and leads to fast and reliable diagnosis.

In the method of the invention a sample from a human or animal is provided. Normally 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.

As the above defined diacyl glycolipid antigens perform particularly well in case samples derived from smear negative persons are tested, it is preferred that the sample is derived from a smear negative person, i.e. derived from a person in which the bacterial load is less than 10.000 mycobacteria/ ml sputum. The sample may be obtained by any regular means of obtaining blood from a subject. In order to be used in the methods of the invention samples may be used that have been collected at an earlier stage, stored until use under suitable conditions and provided at a suitable moment. Alternatively, 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. In addition 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.

In case 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.

About 55% of whole blood consists of plasma/serum. If a whole blood sample is not filtered sufficiently or if the patient's physical situation necessitates it, it may be desired to dilute the whole blood sample or plasma or serum. The words 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. Depending on the viscosity of the sample, such dilution may take place before the step of separating the plasma from the blood step (filter step or separating step). Alternatively 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. Such buffer may for example be a PBS/AE buffer comprising NaCl, KC1, KH2P04, Na2HP04 and EDTA in water at physiological pH. Such buffer may be a PBS based buffer consisting of 8.0 g NaCl, 0.2 g KC1, 0.2 g KH2PO4, and 1.05 g Na2HP04 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.

Optionally a detergent may be added in low concentration to the blood/plasma/serum to avoid sticking of components of the test system used.

For detection 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.

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.

In a real time method, because detection takes place in real time, binding of antibodies to the antigens immobilised on the detection substrates is directly detected during the binding process. For detection in principle all real-time, label free analysis techniques may be used.

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.

In a real time detection method, the solid substrate carrying the antigen may be silica based, such as substrates based on silicium dioxide. In such an embodiment 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. Preferably 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.

It is also well possible that 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. The term "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 endpoint 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.

In a preferred embodiment of an end-point assay detection takes place by means of an immunogold filtration assay. In such an assay the detection substrate is a microporous membrane, preferably a nitrocellulose membrane or a PVDF membrane, to which an antigen is immobilised.

In an end-point assay interaction of antibodies with the antigens may be carried out using 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).

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. By using a microporous membrane as the carrier for the capture probe and employing the capillary action and permeability of the membrane, antigens and antibodies can easily react and may conveniently be subjected to optional washing and/or blocking steps. When the colloidal gold-labelled probe binds to the capture probe the colloidal gold particles aggregate and a red dot appears which is visible with the naked eye.

In case the end-point assay of the present invention is an immunogold filtration assay, the antigen is immobilised on a microporous membrane, preferably a nitrocellulose membrane. After immobilising the antigen onto the microporous membrane, the optionally pre-treated samples can be applied to the membrane. After addition of the sample fractions and reaction of the immobilised antigens with the antibodies contained in the samples on the membrane, colloidal gold-labelled second antibodies can be added onto the membrane to have gold particle aggregation in the antigen-antibody reaction place. In case of 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.

In an immunogold filtration assay the microporous membrane may be for example a nitrocellulose membrane, a cellulose acetate membrane or a PVDF membrane with a suitable pore diameter. Preferably, nitrocellulose is used. A suitable pore diameter is 0,2 to 5 pm.

Between the various steps of an immunogold filtration assay the membrane may be washed with a suitable buffer, for example a PBS based buffer. Such buffer may for example be a PBS/AE buffer comprising NaCl, KC1, KH2P04, Na2HP04 and EDTA in water at physiological pH. Such buffer may be a PBS based buffer consisting of 8.0 g NaCl, 0.2 g KC1, 0.2 g KH2P04, and 1.05 g Na2HP04 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.

In case DIGFA is used, the antigen may be immobilised to the microporous membrane in a dot wise manner. In a DIGFA assay the samples are also applied to the membrane in the form of dots. Also 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, for instance the red staining in case a DIGFA assay is used as a detection method, 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. In a particular preferred embodiment, 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. In case DIGFA is used, 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. For instance, 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. For instance 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. As mentioned above, the high specificity of the diacyl glycolipid antigens of the invention 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 diacyl glycolipid antigens of the invention would be suitable in such a method. In one embodiment the method of the invention therefore may be a real-time method of detecting the presence of antibodies against mycobacterial material in a sample, comprising the steps of: i) providing a sample from a human or animal; ii) obtaining at least two fractions of said sample; iii) exposing the first of said fractions to a solid substrate carrying the immobilised antigen; iv) exposing the second of said fractions to a solid substrate not carrying the immobilised antigen; v) exposing the sample fraction exposed in step iii) to a solid test substrate carrying the immobilised antigen and subjecting the sample fraction exposed in step iv) to a solid control substrate carrying the immobilised antigen; vi) detecting binding of antibodies to the immobilised antigens of step v) in real time; and 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.

In a comparable embodiment, the method of the invention may be an end-point method of detecting antibodies against mycobacterial material in a sample, comprising the steps of: i) providing a sample from a human or animal; ii) obtaining at least two fractions of said sample; iii) exposing the first of said fractions to a solid substrate carrying the immobilised antigen; iv) exposing the second of said fractions to a solid substrate not carrying the immobilised antigen; or storing at least part of the second of said fractions until step v), skipping the step of exposing the second of said fractions to a solid substrate not carrying the immobilised antigen; v) exposing at least part of the sample fraction exposed in step iii) to a solid test substrate carrying the immobilised antigen and exposing at least part of the sample fraction exposed or stored in step iv) to a solid control substrate carrying the immobilised antigen; vi) detecting binding of antibodies to the immobilised antigen of step v) in an end-point assay; and 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.

In the context of these embodiments it should be understood that lesser can be interpreted qualitatively and quantitatively, i.e. lesser binding may be interpreted as having less binding events as well as having weaker bindings. The advantage of these two embodiments is that no sample of a healthy person is required as a reference or control sample, even if there is still background signal. Only one sample from one subject is necessary.

Further, 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. Teflon®), 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 .

The immobilised diacyl glycolipid antigens and optional further antigens can be incorporated in a biosensor for use in the method of the invention. Such a biosensor can be any biosensor which is suitable for use in any of the abovementioned devices or assays. For instance, the biosensor may comprise a silica based substrate with an immobilised diacyl glycolipid antigen and a Si ring resonator. Such a biosensor can be used for ring resonance. It is also well possible that 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 diacyl glycolipid antigens. The invention also encompasses any other biosensor comprising the solid substrate of the invention.

Examples

The following example is meant to illustrate the principle of the invention and should not be interpreted as limiting the scope of the claims. In the example human serum samples were tested for antibodies that are specific for the presence of mycobacterial material in a human or animal in an ELISA assay. As a detection method an ELISA method was chosen because it is less sensitive than for example Surface Plasmon Resonance or electrochemical impedance spectroscopy (EIS) or Ring Resonance (Interferometry) . Therefore it can be concluded that if satisfactory results are obtained with ELISA, these will also be obtained with more sensitive detection methods.

Materials : • Sample (human plasma or serum) • 0.2 micron spinfliters (Whatman) • polyclonal mycolic acid dissolved in hexane • synthetic AC2SGL (2-palmitoyl-3-hydroxyphthio- ceranoyl-2'-sulfate-α-α'-D-trehalose) dissolved in hexane • Polystyrene ELISA plates

• PBS

• Blocking buffer: 0.5% casein in PBS • 1-step ultra TMB-ELISA substrate solution (Thermo Scientific) • 2M sulfuric acid • Secondary antibody: rabbit anti-human IgM HRP (DAKO) Procedures

Coating of ELISA plates

To immobilise mycolic acid to ELISA plates to each well 50 μΐ of hexane with polyclonal mycolic acid in a concentration 100 pg/ml per well of 96-wells ELISA plate was added. To immobilise AC2SGL to ELISA plates to each well of 96-wells ELISA plates 50 μΐ (of hexane with AC2SGL in a concentration of 100 μg/ml was added. Plates were incubated for 24 hrs at 4°C and subsequently washed two times with PBS.

Sample preparation

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 (-) ) . Also samples were used derived from healthy humans (Healthy 1 and 2). Fractions of 0,5 ml were obtained and transferred each to a 0,2 micron spin filter and centrifuged at 10000g. The flow-through was diluted 1:20 in blocking buffer. ELISA-procedure

To block aspecific binding of antibodies with the antigens in the wells of the ELISA plates, 300 μΐ of blocking buffer was added to each well and incubated for 1 hour. Subsequently, the blocking buffer was replaced with 45 μΐ of sample. Blocking buffer was used as a negative control. After incubation, the plates were washed three times with PBS. The washing PBS buffer was replaced with HRP-conjugated secondary antibody in blocking buffer (1:20.000 diluted). After incubation, the plate was washed again three times with PBS. Subsequently 50 μΐ per well of TMB-ELISA substrate solution was added to the wells and incubated for 15-30 min at room temperature. The reaction was stopped by adding 50 μΐ per well of 2 M sulphuric acid. Absorbance at 450 nm was measured to quantify the binding of antibodies to the immobilised antigens of the ELISA plate.

Results

Fig. 1 shows the results of an ELISA test 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. The samples derived from smear positive patients (patients 1-9 ( + ) , black bars) show absorbance signals which even seem lower than the signal of the healthy persons. The high background signal derived from materials that are unrelated to tuberculosis but that do bind to mycolic acid thus obscures the signal derived from the actual antibodies that indicate tuberculosis .

In contrast, when ELISA tests were performed with immobilised Ac2SGL, 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.

The above results show that if the immobilised antigens in accordance with formula I are used in a method for detecting a marker for tuberculosis a very high tuberculosis specific binding of antibodies to these antigens is detected. This effect is most pronounced in samples that are derived from patients that were tested smear negative. The signal derived from the actual markers for tuberculosis appears to be 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 results in a significant improvement with regard to the sensitivity of detection of markers for tuberculosis.

References WO 2005/116654 WO 2013/186679 EP 1 950 218 Al D. Geerdink, B. ter Horst, M. Lepore, L. Mori, G. Puzo, A.K. H. Hirsch, M. Gilleron, G. de Libero and A. J. Minnaard, Chem. Sci., 2013, p.709 -716

Claims

1. Vast substraat, omvattende een aan het vaste substraat geïmmobiliseerd antigeen, waarbij het geïmmobiliseerde antigeen in staat is om te binden aan een antili-chaam dat indicatief is voor de aanwezigheid van mycobac-terieel materiaal in een mens of dier, waarbij het antigeen een verbinding is die wordt voorgesteld door de volgende formule (I) ,A solid substrate comprising an antigen immobilized on the solid substrate, wherein the immobilized antigen is capable of binding to an antibody indicative of the presence of mycobacterial material in a human or animal, wherein the antigen is a is a compound represented by the following formula (I),

waarbij: R2 en R3, gelijk of verschillend, onafhankelijk gekozen zijn uit H, S03“, S03H of S03-/M+, waarbij M+ een kation is, R2 en R3 , gelijk of verschillend, acylgroepen zijn, waarbij het antigeen eventueel gemodificeerd is met één of meer functionele groepen die immobilisatie aan het vaste substraat mogelijk maken, en enantiomeren, diastereoisomeren, en mengsels daarvan .wherein: R 2 and R 3, the same or different, are independently selected from H, SO 3 -, SO 3 H or SO 3 - / M +, wherein M + is a cation, R 2 and R 3, the same or different, are acyl groups, wherein the antigen is optionally modified with one or more functional groups that allow immobilisation to the solid substrate, and enantiomers, diastereoisomers, and mixtures thereof.

2. Vast substraat volgens conclusie 1, waarbij R2’ en R3' van het antigeen, gelijk of verschillend,The solid substrate according to claim 1, wherein R 2 'and R 3' of the antigen, identical or different,

zijn, waarbij X onafhankelijk gekozen is uit een onverzadigde of verzadigde lineaire of vertakte koolwaterstofketen, eventueel gesubstitueerd met één of meer substituen-ten en/of gemodificeerd met één of meer functionele groepen .wherein X is independently selected from an unsaturated or saturated linear or branched hydrocarbon chain, optionally substituted with one or more substituents and / or modified with one or more functional groups.

3. Vast substraat volgens conclusie 1 of 2, waarbij in het geïmmobiliseerde antigeen R2 S03“, S03H of S03_/M+ is, waarbij M+ een kation is; en waarbij R3 H is.A solid substrate according to claim 1 or 2, wherein in the immobilized antigen, R2 is SO3, SO3 H or SO3 - / M +, where M + is a cation; and wherein R 3 is H.

4. Vast substraat volgens conclusie 1 of 2, waarbij in het geïmmobiliseerde antigeen R2 en R3 H zijn.A solid substrate according to claim 1 or 2, wherein in the immobilized antigen R2 and R3 are H.

5. Vast substraat volgens één van de conclusies 2 tot 4, waarbij in het geïmmobiliseerde antigeen in één van R2, en R3' X een verzadigde lineaire koolwaterstof keten is, eventueel gesubstitueerd met één of meer substituenten en/of gemodificeerd met één of meer functionele groepen, en waarbij in de andere van R2, en R3’ X een verzadigde vertakte koolwaterstofketen is, eventueel gesubstitueerd met één of meer substituenten en/of gemodificeerd met één of meer functionele groepen.The solid substrate according to any of claims 2 to 4, wherein in the immobilized antigen in one of R 2, and R 3 'X is a saturated linear hydrocarbon chain, optionally substituted with one or more substituents and / or modified with one or more functional 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.

6. Vast substraat volgens één van de conclusies 1 tot 5, waarbij in het geïmmobiliseerde antigeen één van R2’ en R3' een groep is die wordt voorgesteld door volgende formule (II):The solid substrate of any one of claims 1 to 5, wherein in the immobilized antigen, one of R2 'and R3' is a group represented by the following formula (II):

waarbij R4 een lineaire verzadigde koolwaterstofketen met formule CnHn+i is, waarbij n een geheel getal tussen 1 en 20 is, eventueel gemodificeerd met één of meer functionele groepen, en waarbij Y een geheel getal tussen 1 en 10 is, en waarbij R5 H of OH is, en de andere van R2< en R3’ een lineaire verzadigde koolwaterstof keten met formule CnHn+i is, waarbij n een geheel getal tussen 1 en 20 is, eventueel gemodificeerd met één of meer functionele groepen.wherein R 4 is a linear saturated hydrocarbon chain of formula C n H n + i, 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 is, and the other of R2 <and R3 'is a linear saturated hydrocarbon chain of formula CnHn + i, wherein n is an integer between 1 and 20, optionally modified with one or more functional groups.

7. Vast substraat volgens conclusie 1, waarbij het antigeen een diacyl sulfoglycolipide (AC2SGL) zoals gevonden in Mycobacterium tuberculosis is, eventueel gemodificeerd met één of meer functionele groepen.The solid substrate of claim 1, wherein the antigen is a diacyl sulfoglycolipid (AC 2 SGL) as found in Mycobacterium tuberculosis, optionally modified with one or more functional groups.

8. Vast substraat volgens conclusie 7, waarbij het AC2SGL 2-palmitoyl-3-hydroxyphthioceranoyl-2'-sulfaat-a-a'-D-trehalose of 2-stearoyl-3-hydroxyphthioceranoyl-2'-sulfaat-α-α' -D-trehalose is, eventueel gemodificeerd met één of meer functionele groepen.The solid substrate of claim 7, wherein the AC 2 SGL 2-palmitoyl-3-hydroxyphthioceranoyl-2'-sulfate-α-a'-D-trehalose or 2-stearoyl-3-hydroxyphthioceranoyl-2'-sulfate-α-α ' -D-trehalose, optionally modified with one or more functional groups.

9. Vast substraat volgens conclusie 7 of 8, waarbij het antigeen AC2SGL geïsoleerd is uit Mycobacterium tuberculosis of waarbij het antigeen AC2SGL synthetisch is.The solid substrate of claim 7 or 8, wherein the AC2SGL antigen is isolated from Mycobacterium tuberculosis or wherein the AC2SGL antigen is synthetic.

10. Vast substraat volgens één van conclusies 1 tot 9, waarbij naast het antigeen een verder antigeen geïmmobiliseerd is op het vaste substraat, waarbij het verdere antigeen gedefinieerd is als een verbinding die wordt voorgesteld door de volgende formule (V):The solid substrate according to any of claims 1 to 9, wherein in addition to the antigen, a further antigen is immobilized on the solid substrate, the further antigen being defined as a compound represented by the following formula (V):

waarbij Y een geheel getal tussen 1 en 10 is en R een koolwaterstofgroep, bij voorkeur een alkylgroep is, en waarbij X+ een kation of afwezig is, waarbij het antigeen eventueel gemodificeerd is met één of meer functionele groepen die immobilisatie op het vaste substraat mogelijk maken, en enantiomeren, diastereoisomeren en mengsels daarvan.wherein Y is an integer between 1 and 10 and R is a hydrocarbon group, preferably an alkyl group, and wherein X + is a cation or absent, wherein the antigen is optionally modified with one or more functional groups that allow immobilisation on the solid substrate and enantiomers, diastereoisomers and mixtures thereof.

11. Vast substraat volgens conclusie 10, waarbij het verdere antigeen een β-mannosylfosfomycoketide is, eventueel gemodificeerd met één of meer functionele groepen die immobilisatie op het vaste substraat mogelijk maken.The solid substrate of claim 10, wherein the further antigen is a β-mannosyl phosphomycoketide, optionally modified with one or more functional groups that allow immobilization on the solid substrate.

12. Vast substraat volgens één van de conclusies 1 tot 11, waarbij het substraat een waarnemingsoppervlak van een oppervlakteplasmonresonantie-inrichting, elektrochemische impedantiespectroscopie-inrichting, isotherme titra-tiecalorimetrie-inrichting, biolaaginterferometrie-inrichting, optische raster-inrichting, fotonisch kristal-inrichting, akoestische resonantieprofilering-inrichting, of kwartskristalmicrobalans-inrichting is.The solid substrate of any one of claims 1 to 11, wherein the substrate is an observation surface of a surface plasma resonance device, electrochemical impedance spectroscopy device, isothermal titration calorimetry device, biolayer interferometry device, optical raster device, photonic crystal device, acoustic resonance profiling device, or quartz crystal microbalance device.

13. Vast substraat volgens één van de conclusies 1 tot 11, waarbij het substraat een detectie-oppervlak in een enzymen-verbonden immunosorbenttest (ELISA), Western blot-test, radioactieve labelingstest, fotospectrometri-sche test, immunofluorescentietest, immunoprecipitatie- test, immunocytochemietest, immunohistochemietest, ampero-metrische detectietest, voltametrische detectietest, of elektrochemische impedantiespectroscopie-test is.The solid substrate of any one of claims 1 to 11, wherein the substrate has a detection surface in an enzyme-linked immunosorbent test (ELISA), Western blot test, radioactive labeling test, photospectrometric test, immunofluorescence test, immunoprecipitation test, immunocytochemistry test , immunohistochemistry test, amperometric detection test, voltametric detection test, or electrochemical impedance spectroscopy test.

14. Werkwijze voor het detecteren van de aanwezigheid van antilichamen tegen mycobacterieel materiaal in een monster, omvattende de stappen van: (1) het verschaffen van een monster, afkomstig van een mens of dier; (2) het blootstellen van ten minste een deel van het monster aan een vast substraat dat een antigeen omvat dat aan het vaste substraat geïmmobiliseerd is volgens één van de conclusies 1 tot 13; en (3) het detecteren van binding van antilichamen aan het geïmmobiliseerde antigeen.A method for detecting the presence of antibodies against mycobacterial material in a sample, comprising the steps of: (1) providing a sample from a human or animal; (2) exposing at least a portion of the sample to a solid substrate comprising an antigen immobilized on the solid substrate according to any of claims 1 to 13; and (3) detecting antibody binding to the immobilized antigen.

15. Werkwijze volgens conclusie 14, waarbij het monster afkomstig is van een persoon waarin de bacteriële belasting minder dan 10.000 Mycobacterium tuberculosis/ ml sputum is.The method of claim 14, wherein the sample is from a person in which the bacterial load is less than 10,000 Mycobacterium tuberculosis / ml of sputum.

16. Werkwijze volgens conclusie 14 of 15, waarbij het detecteren van binding van de antilichamen aan de geïmmobiliseerde antigenen het meten van verandering van massa op het substraat, verandering van brekingsindex, verandering van entropie, verandering van enthalpie, vis-cositeitsverandering, temperatuurverandering of kleurverandering omvat.A method according to claim 14 or 15, wherein detecting binding of the antibodies to the immobilized antigens measuring change in mass on the substrate, change in refractive index, change in entropy, change in enthalpy, viscosity change, temperature change or color change includes.

17. Werkwijze volgens één van de conclusies 14 tot 16, waarbij detectie van binding van antilichamen aan het geïmmobiliseerde antigeen in ware tijd (real-time) plaatsvindt .The method of any one of claims 14 to 16, wherein detection of binding of antibodies to the immobilized antigen takes place in real-time (real-time).

18. Werkwijze volgens conclusie 17, omvattende de stappen van: i) het verschaffen van een monster afkomstig van een mens of dier; ii) het verkrijgen van ten minste twee fracties van het monster; iii) het blootstellen van de eerste van de fracties aan een vast substraat dat het geïmmobiliseerde antigeen draagt; iv) het blootstellen van de tweede van de fracties aan een vast substraat dat het geïmmobiliseerde antigeen niet draagt; v) het blootstellen van de in stap iii) blootgestelde monsterf ractie aan een vast testsubstraat dat het geïmmobiliseerde antigeen draagt en het blootstellen van de in stap iv) blootgestelde monsterf ractie aan een vast controlesubstraat dat het geïmmobiliseerde antigeen draagt; vi) het detecteren van de binding van antilichamen aan het antigeen van stap v) in ware tijd; en viii)het vergelijken van de graad of uitgebreidheid van binding tussen de test- en controlesubstraten, waarbij enige waargenomen mindere binding aan het testsubstraat een indicator van de aanwezigheid van antilichamen tegen het geïmmobiliseerde antigeen in het monster is die actieve tuberculose in de mens of het dier waarvan de monsters afkomstig zijn indiceert.The method of claim 17, comprising the steps of: i) providing a sample from a human or animal; ii) obtaining at least two fractions of the sample; iii) exposing the first of the fractions to a solid substrate that carries the immobilized antigen; iv) exposing the second of the fractions to a solid substrate that does not carry the immobilized antigen; v) exposing the sample fraction exposed in step iii) to a solid test substrate that carries the immobilized antigen and exposing the sample fraction exposed in step iv) to a solid control substrate that carries the immobilized antigen; vi) detecting the binding of antibodies to the antigen of step v) in real time; and viii) comparing the degree or extent of binding between the test and control substrates, any observed less binding to the test substrate being an indicator of the presence of antibodies to the immobilized antigen in the sample that are active tuberculosis in humans or the human. animal from which the samples originate.

19. Werkwijze volgens conclusie 17 of 18, waarbij detectie uitgevoerd wordt bij gebruik van oppervlakteplas-monresonantie of elektrochemische impedantiespectroscopie, isotherme titratiecalorimetrie, biolaaginterferometrie, optische rasters, fotonisch kristal, akoestische resonan-tieprofilering, kwartskristalmicrobalansen.A method according to claim 17 or 18, wherein detection is carried out using surface plasmon resonance or electrochemical impedance spectroscopy, isothermal titration calorimetry, biolayer interferometry, optical grids, photonic crystal, acoustic resonance profiling, quartz crystal microbalances.

20. Werkwijze volgens één van de conclusies 14 tot 16, waarbij detectie van binding van antilichamen aan het geïmmobiliseerde antigeen plaatsvindt door middel van een eindpunttest.The method of any one of claims 14 to 16, wherein detection of antibody binding to the immobilized antigen is by means of an endpoint test.

21. Werkwijze volgens conclusie 20, omvattende de stappen van: i) het verschaffen van een monster afkomstig van een mens of dier; ii) het verkrijgen van ten minste twee fracties van het monster; iii) het blootstellen van de eerste van de fracties aan een vast substraat dat het geïmmobiliseerde antigeen draagt; iv) het blootstellen van de tweede van de fracties aan een vast substraat dat het geïmmobiliseerde antigeen niet draagt; of het bewaren van ten minste een deel van de tweede van de fracties tot stap v) , waarbij de stap van het blootstellen van de tweede van de fracties aan een vast substraat dat het geïmmobiliseerde antigeen niet draagt overgeslagen wordt; v) het blootstellen van ten minste een deel van de in stap iii) blootgestelde monsterf ractie aan een vast testsubstraat dat het geïmmobiliseerde antigeen draagt en het blootstellen van ten minste een deel van de in stap iv) blootgestelde of bewaarde monsterfractie aan een vast controlesubstraat dat het geïmmobiliseerde antigeen draagt; vi) het detecteren van de binding van antilichamen aan het antigeen van stap v) in een eindpunttest; en vii) het vergelijken van de graad of uitgebreidheid van binding tussen de test- en controlesubstraten, waarbij enige waargenomen mindere binding aan het testsubstraat een indicator van de aanwezigheid van antilichamen tegen het geïmmobiliseerde antigeen in het monster is die actieve tuberculose in de mens of het dier waarvan de monsters afkomstig zijn indiceert.The method of claim 20, comprising the steps of: i) providing a sample from a human or animal; ii) obtaining at least two fractions of the sample; iii) exposing the first of the fractions to a solid substrate that carries the immobilized antigen; iv) exposing the second of the fractions to a solid substrate that does not carry the immobilized antigen; or storing at least a portion of the second of the fractions to step v), wherein the step of exposing the second of the fractions to a solid substrate that does not carry the immobilized antigen is skipped; v) exposing at least a portion of the sample fraction exposed in step iii) to a solid test substrate bearing the immobilized antigen and exposing at least a portion of the sample fraction exposed or saved in step iv) to a solid control substrate that carries the immobilized antigen; vi) detecting the binding of antibodies to the antigen of step v) in an endpoint test; and vii) comparing the degree or extent of binding between the test and control substrates, any observed less binding to the test substrate being an indicator of the presence of antibodies to the immobilized antigen in the sample that are active tuberculosis in humans or the human. animal from which the samples originate.

22. Werkwijze volgens conclusie 20 of 21, waarbij de eindpunttest wordt gekozen uit de groep bestaande uit een enzymen-verbonden immunosorbenttest (ELISA), Western blot-test, radioactieve labelingstest, fotospectrometrische test, immunofluorescentie, immunoprecipitatie, immunocyto-chemie, immunohistochemie, elektrochemische impedantie-spectroscopie.The method of claim 20 or 21, wherein the endpoint test is selected from the group consisting of an enzyme-linked immunosorbent test (ELISA), Western blot test, radioactive labeling test, photospectrometric test, immunofluorescence, immunoprecipitation, immunocytochemistry, immunohistochemistry, electrochemical impedance spectroscopy.

23. Werkwijze volgens conclusie 20 of 21, waarbij de eindpunttest een immunogoudfiltratietest is, bij voorkeur waarbij de immunogoudtest een stip-immunogoudtest is (DIG-FA) .A method according to claim 20 or 21, wherein the endpoint test is an immunogold filtration test, preferably wherein the immunogold test is a spot immunogold test (DIG-FA).

24. Werkwijze volgens één van de conclusies 20-23, waarbij detectie visueel plaatsvindt, bij voorkeur met het blote oog.A method according to any one of claims 20-23, wherein detection takes place visually, preferably with the naked eye.

25. Werkwijze volgens één van de conclusies 19-24, waarbij detectie leidt tot een visueel signaal dat geanalyseerd wordt door middel van een mobiele applicatie die ontworpen is om het bindingssignaal afkomstig van verschillende monsters of monsterfracties te vergelijken en die indiceert of de mens of het dier waarvan het monster afkomstig is actieve tuberculose heeft.The method of any one of claims 19-24, wherein detection results in a visual signal that is analyzed by a mobile application designed to compare the binding signal from different samples or sample fractions and that indicates whether the human or the human animal from which the sample comes has active tuberculosis.

26. Biosensor, omvattende het vaste substraat vol gens één van de conclusies 1 tot 13. -o-o-o-A biosensor comprising the solid substrate of any one of claims 1 to 13. -o-o-o-