WO2016012449A1

WO2016012449A1 - Monoclonal antibody, method, kit and use

Info

Publication number: WO2016012449A1
Application number: PCT/EP2015/066648
Authority: WO
Inventors: Beston Hamasur; Stefan Svenson
Original assignee: Tbdiadirect Ab
Priority date: 2014-07-22
Filing date: 2015-07-21
Publication date: 2016-01-28

Abstract

Methods for determining the presence of a surface polysaccharide antigen derivable from a mycobacterium, or a fragment of the antigen, a kit for performing the methods disclosed herein and use of monoclonal antibodies disclosed herein are provided. Furthermore, monoclonal antibody MAb 25 produced by the hybridoma cell line deposited under Accession No. DSM ACC3247 and/or monoclonal antibody MAb 170 produced by the hybridoma cell line deposited under Accession No. DSM ACC3246 are disclosed.

Description

MONOCLONAL ANTIBODY, METHOD, KIT AND USE

Field of the invention

The present invention relates to methods for determining the presence of a surface polysaccharide antigen derivable from a mycobacterium, or a fragment of the antigen, in a biological fluid sample using monoclonal antibodies which specifically bind to the antigen, or to a fragment of the antigen, a kit, and antibodies for use therein.

Background

One third of the world's population is infected with bacteria of the

Mycobacterium tuberculosis (Mtb) complex, and tuberculosis (TB) accounts for 1 .4 million deaths annually and one-fifth of all deaths of adults in poor/low- income countries. WHO reports that, if left untreated, each person with active TB infect about ten to fifteen new individuals annually. Therefore, interrupting disease transmission is of major importance and requires early and accurate detection/diagnosis paired with appropriate treatment. Despite the enormous global burden of TB, present tests for diagnosis of active TB both in humans and animals have severe limitations.

Today^'s diagnostic tests for active TB in humans rely on direct smear microscopy, culture of Mtb bacilli, detection of Mtb nucleic acids, and/or clinical symptoms. In poor countries, the most commonly used diagnostic test is direct sputum smear microscopy. It is a technique that was developed in the 1880s and since then it has remained largely unchanged. It detects only about half of patients and is particularly ineffective for diagnosis of TB in young children and in patients co-infected with human immunodeficiency virus (HIV). Although it is often described as a simple technology, microscopy requires a high level of training and diligence. To effectively combat TB, there is therefore an urgent need for new low cost diagnostic technological platforms that move beyond the century-old direct microscopy method.

There are several nucleic acid amplification (NAAT) tests, but they are all rather high-tech, costly and require expensive instrumentations. The tests are basically not adapted as point-of-care tests. Moreover, all these tests perform poorly in populations affected by the HIV epidemic, in immuno- suppressed individuals, and in small children. The most commonly used NAAT technologies for rapid detection of TB are the reverse-transcription polymerase chain reaction (RT-PCR) and line probe assays. Line-probe assays, although simpler and more affordable, are restricted to use in centralized laboratories.

The GenExpert MTB/RIF assay is a cartridge-based diagnostic NAAT test that can identify Mtb and resistance to rifampicin (RIF). It is a fully automated test, but requires an electrical supply and maintenance of instruments, and the production cost is relatively high.

Furthermore, in vitro tests measuring cellular immune responses, e.g. IFN gamma responses of primed immunocompetent memory cells against more or less specific mycobacterial antigen preparations (e.g.

QuantiFERON®-TB Gold (for humans) and Bovigam® (for cattle)), and similar tests have been developed. One inherent drawback of the methods described above is that they depend on a secondary (historical) immunological response and/or memory of the tested individual. This might vary

considerably, and hence obtained data can be difficult to interpret. Also many other parameters, such as exposure to environmental mycobacteria, age and condition of the human/animal, may strongly influence the outcome of these tests.

Over the years there have also been many efforts to use determination of specific antibody titers (different ELISA, EIA formats) to diagnose TB both in humans and in animals. So far, none of these methods have proven specific or sensitive enough to be of any diagnostic value.

Disclosure of the invention

It is an object of the present disclosure to alleviate at least some of the problems associated with the prior art techniques.

In particular, an object of the present disclosure is to provide at least one monoclonal antibody which specifically binds to a surface polysaccharide antigen derivable from a mycobacterium, or to a fragment of the antigen, with high affinity and/or high avidity. Another object of the present disclosure is to provide a method for determining the presence of a surface polysaccharide antigen derivable from a mycobacterium, or a fragment of the antigen, in a biological fluid sample, wherein at least one monoclonal antibody with high affinity and/or high avidity for the antigen is utilized.

Yet another object of the present disclosure is to provide a kit for determining the presence of a surface polysaccharide antigen derivable from a mycobacterium, or a fragment of the antigen, in a biological fluid sample comprising at least one monoclonal antibody which specially binds to the antigen with high affinity and/or high avidity.

The above mentioned objects, as well as other objects that will be apparent to a person skilled in the art when presented with the present disclosure, are each addressed by the aspects of the present invention.

In an aspect, the present invention provides a monoclonal antibody MAb 25 produced by the hybridoma cell line deposited under Accession No. DSM ACC3247 and/or a monoclonal antibody MAb 170 produced by the hybridoma cell line deposited under Accession No. DSM ACC3246. The cell lines deposited under Accession No. DSM ACC3247 and No. DSM ACC3246 are deposited at the DSMZ (Leibniz-lnstitut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) on 10 July, 2014.

The term "monoclonal antibody" as referred to herein, should generally be understood as a monospecific antibody that is the same as another monospecific antibody of the same kind because they are made by identical immune cells that are all clones of a unique parent cell, in contrast to polyclonal antibodies which are made from several different immune cells. Monoclonal antibodies are homogenous. Furthermore, monoclonal antibodies have monovalent affinity, in that they are selective for one single epitope of the antigen. In contrast, polyclonal antibodies are heterogeneous and contain a mixture of antibodies of different affinities recognizing several epitopes.

In the context of the present disclosure, the term "hybridoma" refers to a technology of forming hybrid cell lines (which are called hybridomas) by fusing a specific antibody-producing B cell with a myeloma (B cell cancer) cell that is selected for its ability to grow in tissue culture and for an absence of antibody chain synthesis. The antibodies produced by the hybridoma are all of a single specificity and are therefore monoclonal antibodies (in contrast to polyclonal antibodies). The production of monoclonal antibodies was invented by Cesar Milstein and Georges J. F. Kohler in 1975.

The monoclonal antibodies (MAbs) disclosed herein are produced using in-house produced immunogens as described in Hamasur et al., Clinical and Experimental Immunology ^, 30 (2004).

According to the present disclosure, the monoclonal antibody MAb 25 and/or a monoclonal antibody MAb 170 can be conjugated to a support. For example, the support may be a magnetic support. The term "magnetic support" means any support that is attracted by a magnet. Preferably, the type of magnetic support may not influence the monoclonal antibody^'s activity. For example, the magnetic support may have a size and form which preferably do not sterically hinder the antibody from binding to its antigen. Furthermore, the magnetic support may preferably not affect the antibody^'s stability.

In some embodiments, the magnetic support is bound to a monoclonal antibody by using e.g. reductive amination or iminothiolation. In some examples, iminothiolation is used in order to obtain a high yield of conjugated antibodies. Furthermore, iminothiolation may create a more stable antibody- magnetic support complex compared to other chemical conjugation techniques.

In some embodiments, a magnetic support is conjugated to

approximately 10-2000 monoclonal antibodies. For example, a magnetic support is conjugated to at least 100 monoclonal antibodies, such as at least

500 monoclonal antibodies, such as at least 1000 monoclonal antibodies, or such as at least 1500 monoclonal antibodies.

In some embodiments, the magnetic support may be a magnetic bead.

The term "bead" as referred to herein means a sphere, such as a particle. The surface of the bead may be smooth or irregular. Preferably, the size and form of the magnetic bead may not influence the monoclonal antibody^'s properties. For example, the magnetic bead can be a magnetic nanobead or a magnetic microbead. In some embodiments, the magnetic support, such as the magnetic bead, may be at least partly coated. The term "at least partly" as referred to herein, should generally be understood as to some extent or in some degree. For example, the bead may be at least partly coated with gold. A surface of gold may reduce the number of unspecific bindings. In other words, gold may reduce the background. Furthermore, it has been found that a surface of gold may facilitate binding of more antibodies to one bead compared with a surface lacking gold. In yet other examples, the beads are at least partly coated with silver.

In some embodiments, the magnetic support, such as the magnetic bead, may be at least partly blocked. Blocking of the surface may reduce unspecific binding. For example, suitable blocking molecules can be selected from bovine serum albumin (BSA) and casein.

In an embodiment of the present disclosure, the monoclonal antibody MAb 25 and/or monoclonal antibody MAb 170 is capable of binding to a surface polysaccharide antigen derivable from a mycobacterium, or to a fragment of the antigen. According to the present disclosure, the term

"antigen", also known as antibody generator, refers to any substance which provokes an adaptive immune response. An antigen is often foreign or toxic to the body (for example, a bacterium) which, once in the body, attracts and is bound to a respective and specific antibody, i.e. an antigen is a molecule that also induces an immune response in the body. Each antibody is specifically designed to deal with certain antigens because of variation in the antibody's complementary determining regions. For example, an antigen may be a protein or a polysaccharide. This includes parts, such as coats, capsules, cell walls, flagella, fimbrae, and toxins, of bacteria, viruses, and other

microorganisms. In the context of the present invention, the term "surface polysaccharide antigen" refers to an antigen in the form of a polysaccharide which is derivable from e.g. the surface of bacteria, such as the cell wall of bacteria. The term "fragment" as referred to herein, should generally be understood as a part of an antigen that is recognized by the immune system, specifically by antibodies. According to the present disclosure, the monoclonal antibody MAb 25 and the monoclonal antibody MAb 170 may bind to different epitopes on the surface polysaccharide antigen, or fragment of the antigen.

The monoclonal antibodies of the present disclosure have been found to have a high affinity and/or high avidity for the surface polysaccharide antigen, or a fragment of the antigen, compared with polyclonal antibodies or other monoclonal antibodies known in the art.

In the context of the present invention, "mycobacterium" may be referred to a genus of Actinobacteria, the Mycobacteriaceae. The genus includes pathogens known to cause serious diseases in mammals. For example, the mycobacterium may be Mycobacterium tuberculosis. Mycobacterium tuberculosis may cause tuberculosis in humans.

According to the present disclosure, the surface polysaccharide antigen may be a lipopolysaccharide. The term "lipopolysaccharide" as referred to herein, also known as lipoglycan and endotoxin, is usually a large molecule consisting of a lipid and a polysaccharide composed of O-antigen, outer core and inner core joined by a covalent bond. For example, the

lipopolysaccharide can be lipoarabinomannan (LAM). "LAM" is a major Mycobacterium tuberculosis (Mtb) surface antigen, a glycolipid surface component of the cell wall of Mtb bacilli and may account for up to 15% of the total bacterial weight. LAM consists of a mannan polysaccharide backbone with branched oligoarabinosyl containing saccharide side chains, the former being covalently linked to a phosphatidyl inositol lipid moiety.

The monoclonal antibodies as disclosed herein have been shown to have high affinity and/or high avidity against the lipoarabinomannan antigen, or a fragment of the antigen.

In the context of the present disclosure, the term "affinity" refers to the strength of interaction between an antigen, or fragment of the antigen, and an antibody's antigen binding site.

In the context of the present disclosure, the term "avidity" refers to a measure of the overall strength of an antibody-antigen complex. It is the accumulated strength of multiple affinities of individual non-covalent binding interactions. Avidity is commonly applied to antibody interactions in which multiple antigen-binding sites simultaneously interact with the

target antigenic epitopes. Individually, each binding interaction may be readily broken, however, when many binding interactions are present at the same time, transient unbinding of a single site does not allow the molecule to diffuse away, and binding of that weak interaction is likely to be restored.

In an aspect of the invention, there is provided a method for determining the presence of a lipoarabinomannan antigen, or a fragment of the antigen, in a biological fluid sample, comprising the steps:

• in a biological fluid sample from a subject,

o adding a first monoclonal antibody conjugated to a magnetic support, o adding a second monoclonal antibody,

• allowing the first monoclonal antibody to bind to any lipoarabinomannan antigen, or to a fragment of the antigen, present in the biological fluid sample,

· allowing the second monoclonal antibody to bind to the lipoarabinomannan antigen, or to a fragment of the antigen,

• wherein the first monoclonal antibody, the lipoarabinomannan antigen, and the second monoclonal antibody form an antibody-antigen-antibody complex,

· optionally, removing any unbound lipoarabinomannan antigen and/or second monoclonal antibody from the biological fluid sample,

• determining the presence of the lipoarabinomannan antigen, or a fragment of the antigen, by detecting the antibody-antigen-antibody complex.

The method for determining the presence of a lipoarabinomannan antigen or a fragment of the antigen, in a biological fluid sample from a subject utilizes at least one monoclonal antibody conjugated to a magnetic support and which antibody specifically binds to a lipoarabinomannan antigen, or to a fragment of the antigen.

The term "antibody-antigen-antibody complex" should generally be understood as that a first antibody has bound to an antigen, or to a fragment of an antigen, and a second antibody has bound to the antigen, or to a fragment of the antigen. The result is an antibody-antigen-antibody complex. According to the present disclosure, the presence of lipoarabinomannan antigen, or a fragment of the antigen, in a biological fluid sample can be indicative for a mycobacterial infection and/or disease in a subject. For example, the mycobacterial infection and/or disease can be tuberculosis. Tuberculosis may be caused by Mycobacterium tuberculosis. For example, the subject can be a mammalian. The mammalian can be a human or an animal. In some examples, the mammalian is a human.

According to the present disclosure, at least one first monoclonal antibody can be conjugated to a magnetic support. For example, the magnetic support can be a magnetic bead, such as a magnetic nanobead or magnetic microbead. In specific embodiments, features of the monoclonal antibody, for example the magnetic support, are as disclosed in the embodiments above.

In some embodiments, at least one second monoclonal antibody can be conjugated to a magnetic support, similar to the first monoclonal antibody as disclosed herein.

In an aspect of the invention, there is provided a method for determining the presence of a surface polysaccharide antigen derivable from a

mycobacterium, or a fragment of the antigen, in a biological fluid sample, comprising the steps:

• in a biological fluid sample from a subject,

o adding a first monoclonal antibody MAb 25 produced by the

hybridoma cell line deposited under Accession No. DSM ACC3247 or a first monoclonal antibody MAb 170 produced by the hybridoma cell line deposited under Accession No. DSM ACC3246, and

o adding a second monoclonal antibody MAb 25 produced by the hybridoma cell line deposited under Accession No. DSM ACC3247 or a second monoclonal antibody MAb 170 produced by the hybridoma cell line deposited under Accession No. DSM ACC3246,

· allowing the first monoclonal antibody to bind to any surface

polysaccharide antigen, or to a fragment of the antigen, present in the biological fluid sample, • allowing the second monoclonal antibody to bind to the surface

polysaccharide antigen, or to a fragment of the antigen,

• wherein the first monoclonal antibody, the surface polysaccharide antigen, and the second monoclonal antibody form an antibody-antigen-antibody complex,

• optionally, removing any unbound surface polysaccharide antigen and/or second monoclonal antibody from the biological fluid sample,

• determining the presence of the surface polysaccharide antigen, or a fragment of the antigen, by detecting the antibody-antigen-antibody complex.

The method for determining the presence of a surface polysaccharide antigen derivable from a mycobacterium, or a fragment of the antigen, in a biological fluid sample from a subject utilizes at least one monoclonal antibody MAb 25 produced by the hybridoma cell line deposited under Accession No. DSM ACC3247 and/or at least one monoclonal antibody MAb 170 produced by the hybridoma cell line deposited under Accession No. DSM ACC3246.

According to the present disclosure, the presence of a surface polysaccharide antigen derivable from a mycobacterium, or a fragment of the antigen, in a biological fluid sample can be indicative for a mycobacterial infection and/or disease in a subject. For example, the mycobacterial infection and/or disease can be tuberculosis. Tuberculosis may be caused by

Mycobacterium tuberculosis. For example, the subject can be a mammalian. The mammalian can be a human or an animal. In some examples, the mammalian is a human.

The monoclonal antibodies bind specifically to the surface

polysaccharide antigen, or to a fragment of the antigen. In some

embodiments, the surface polysaccharide antigen, or a fragment of the antigen, can be a lipopolysaccharide antigen. For example, the

lipopolysaccharide can be lipoarabinomannan.

In some embodiments of the present disclosure, at least one first monoclonal antibody is conjugated to a support. In some examples, the support is a magnetic support, such as a magnetic bead. The magnetic bead can be a magnetic nanobead or a magnetic microbead. In specific

embodiments, features of the monoclonal antibody, for example the magnetic support, are as disclosed in the embodiments above.

In some embodiments, at least one second monoclonal antibody is conjugated to a magnetic support, similar to the first monoclonal antibody as disclosed herein.

According to the present disclosure, a method utilizing magnetic particles can be noted as a "magnetic nanoparticle assay" (MNP assay) regardless of the size of the magnetic particles. The term "magnetic nanoparticle assay" refers to an assay comprising at least one first

monoclonal antibody conjugated to a magnetic support, at least one second monoclonal antibody and at least one antigen to be detected.

According to the present invention, it is obvious for a person skilled in the art that "a first monoclonal antibody" means at least one first monoclonal antibody. In a similar fashion, it is obvious that "a second monoclonal antibody" means at least one second monoclonal antibody.

In the context of the present invention, the term "biological fluid" means body fluid and/or bodily fluid. According to the present disclosure, the biological fluid sample can be selected from the group consisting of serum, sperm, blood, sputum, cerebro-spinal fluid, saliva, and urine. For example, the biological fluid sample can be urine. Urine usually comprises few components which may interact with monoclonal antibodies which specifically interact with a surface polysaccharide antigen. For example, urine usually comprises few components which negatively influence the specificity of the monoclonal antibodies.

According to any aspect described herein, the biological fluid sample may be obtained from a subject prior performing any method described herein. For example, the biological fluid sample can be stored in a refrigerator prior performing any method described herein. In other examples, the biological fluid sample is stored and kept at approximately 37 °C prior performing any method described herein.

According to any aspect, the biological fluid sample can be pre-treated prior performing the steps in the method disclosed herein. For example, the biological fluid sample can be heated. In other examples, the biological fluid sample can be filtered. In other examples, the biological fluid sample can be diluted.

According to any aspect, the biological fluid sample can be pre-cultured under conditions permitting the growth of mycobacteria. For example, the mycobacteria can be Mycobacterium tuberculosis. Pre-cultivation of mycobacteria prior to performing the method for determining the presence of a lipoarabinomannan antigen in the sample as disclosed herein, may provide a lower detection limit of lipoarabinomannan antigen. For example, a urine sample from a subject may be kept during optimal growth conditions in order for any mycobacterium in the sample to grow. While the mycobacteria grow, the amount of lipoarabinomannan, or fragment of the antigen, may increase in the sample. An increased amount of lipoarabinomannan antigen in the sample may facilitate performing the method for determining the presence of a lipoarabinomannan antigen, or a fragment of the antigen, in a biological fluid sample. For example, the detection limit for the antigen may become lower than without performing the pre-cultivation step.

Lipoarabinomannan (LAM) in a soluble form is released both from metabolically active and degrading bacterial cells during TB infection. Hence, in active TB disease LAM occurs in serum and subsequently may be cleared through the kidneys and occur in urine in an antigenically intact form.

Furthermore, since LAM is a carbohydrate antigen and thus inherently heat- stable, LAM may be detectable by sensitive immunological techniques, even after heat treatment of urine samples. The amount of LAM in the urine reflects the bacterial load, metabolic activity and/or rate of degradation of the bacteria. Hence, it permits a semi-quantitative assessment of the infectious status.

In some embodiments, the first monoclonal antibody used in any of the aspects disclosed herein can be a monoclonal antibody as disclosed herein, such as for example MAb 25 and/or MAb 170.

According to any aspect, the first monoclonal antibody is a capture antibody. For example, when performing an assay a capture antibody may be the antibody which binds to (captures) the antigen in the sample. According to the present disclosure, a monoclonal antibody which can be conjugated to a magnetic support is preferably selected as capture antibody.

In some embodiments, the second monoclonal antibody used in any of the aspects disclosed herein can be a monoclonal antibody as disclosed herein, such as for example MAb 25 and/or MAb 170.

In some embodiments of the present disclosure, the second monoclonal antibody is a developer antibody. For example, when performing an assay a developer antibody may be the antibody which binds to an antigen bound to a first (monoclonal) antibody. The second antibody can be labelled and can be detected. Hence, detection of a second antibody may indicate detection of an antigen in a sample.

In some embodiments, the first monoclonal antibody is the same as the second monoclonal antibody, such as for example MAb 25 or MAb 170. In other embodiments, the first monoclonal antibody differs from the second monoclonal antibody. For example, the first monoclonal antibody may be MAb 25 and the second monoclonal antibody may be MAb 170. In another example, the first monoclonal antibody is MAb 170 and the second

monoclonal antibody is MAb 25. According to the present disclosure, the first monoclonal antibody preferably differs from the second monoclonal antibody. It has been shown that by using two different monoclonal antibodies, unspecific binding is reduced. In other words, the background is reduced.

According to the present invention, the monoclonal antibodies MAb 25 and MAb 170 complement each other. Experiments show that the monoclonal antibody MAb 25 has a high avidity to a surface polysaccharide antigen, such as a lipopolysaccharide, such as for example LAM. Experiments have shown that the monoclonal antibody MAb 170 has a weaker affinity to a surface polysaccharide antigen, such as a lipopolysaccharide, such as for example LAM.

According to any aspect disclosed herein, the at least one first monoclonal antibody is allowed to bind to an antigen, or to a fragment of the antigen, in a biological fluid sample after being added to the sample. In a subsequent step, at least one second monoclonal antibody is added to the sample and is allowed to bind to an antigen. In some embodiments, the first monoclonal antibody and the second monoclonal antibody are added simultaneously to a biological fluid sample.

In yet another embodiment, unbound antigen is removed from the biological sample before at least one second monoclonal antibody is added to the biological fluid sample.

In yet another embodiment, unbound second monoclonal antibody is removed from the biological fluid sample before the step of determining the presence of a surface polysaccharide antigen, or a fragment of the antigen, is performed.

According to the present invention, the step of removing any unbound antigen and/or second monoclonal antibody can be performed with the aid of a device, such as a magnetic device. For example, the magnetic device may be a magnet.

The inventors have realized that by conjugating a magnetic support to at least one first monoclonal antibody, a magnet can be used in order to separate at least one first monoclonal antibody which is not attracted by the magnet from the rest of the biological fluid sample. For example, when the first monoclonal antibody has bound to an antigen, the antibody-antigen complex (i.e. an antibody bound to an antigen) is attracted to the magnet. In other examples, when a second monoclonal antibody is bound to an antigen which is bound to a first monoclonal antibody, the antibody-antigen-antibody complex is attracted to the magnet. The magnetic support in combination with a separation step using a magnetic device, such as a magnet, facilitates separation of bound antigen complexes from the rest of the biological fluid sample. For example unbound antigen, unbound second monoclonal antibody and other components in a biological fluid sample can be removed from the antibody-antigen-antibody complex to be detected by using a magnet.

Furthermore, any first monoclonal antibodies being unconjugated to a magnetic support may be separated by using a magnetic device.

According to the present invention, the first monoclonal antibody is allowed to bind to any surface polysaccharide antigen, or a fragment of the antigen, present in the biological fluid sample. For example, the first monoclonal antibody is present in the biological fluid sample during at least 5 minutes, such as at least 10 minutes, such as at least 15 minutes, such as at least 20 minutes, such a at least 30 minutes, such as at least 1 hour before being, optionally, removed from the biological fluid sample.

According to the present invention, the second monoclonal antibody is allowed to bind to any surface polysaccharide antigen, or to a fragment of the antigen, present in the biological fluid sample. For example, the second monoclonal antibody is present in the biological fluid sample during at least 5 minutes, such as at least 10 minutes, such as at least 15 minutes, such as at least 20 minutes, such a at least 30 minutes, such as at least 1 hour before being, optionally, removed from the biological fluid sample.

According to the present invention, the step of determining the presence of a lipoarabinomannan antigen, or a fragment of the antigen, is performed by using an indirect or a direct labeling method. For example, the indirect or direct labeling method can be selected from the group consisting of spectrophotometry, fluorescence, enzyme-linked immunosorbent assay (ELISA), magnetic immunoassay (MIA), and radioimmunoassay.

In the context of the present disclosure, "spectrophotometry" is a quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. Spectrophotometry utilizes a

spectrophotometer which is a photometer that can measure intensity as a function of the light source wavelength. For example, absorbance at e.g. 450 nm can be measured using a spectrophotometer.

The term "fluorescence" as referred to herein, can generally be understood as the emission of light by a substance that has absorbed light or other electromagnetic radiation. Fluorescence can be measured by using a fluorometer.

In the context of the present disclosure, the term "ELISA" means detection of an "analyte" (i.e. the specific substance whose presence is being quantitatively or qualitatively analyzed) in a liquid sample by a method that continues to use liquid reagents during the "analysis" (i.e. controlled sequence of biochemical reactions that will generate a signal which can be easily quantified and interpreted as a measure of the amount of analyte in the sample) that stays liquid and remains inside a reaction chamber or well needed to keep the reactants contained. Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody that is linked to an enzyme

through bioconjugation. Between each step, the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are unspecifically bound. After the final wash step, the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.

The term "magnetic immunoassay" as referred to herein, can generally be understood as a type of diagnostic immunoassay using magnetic beads as labels in lieu of conventional enzymes (ELISA), radioisotopes (RIA) or fluorescent moieties (fluorescent immunoassays). This assay involves the specific binding of an antibody to its antigen, where a magnetic label is conjugated to one element of the pair. The presence of magnetic beads is then detected by a magnetic reader (magnetometer) which measures the magnetic field change induced by the beads. The signal measured by the magnetometer is proportional to the analyte (e.g. antigen) quantity in the initial sample.

The term "radioimmunoassay" as referred to herein, means

an assay technique used to measure concentrations of e.g. antigens by use of antibodies, wherein a known quantity of an antigen is made radioactive. It is frequently labeled with gamma-radioactive isotopes of iodine, such as 125- I, attached to tyrosine. In one example, the radiolabelled antigen is mixed with a known amount of antibody for that antigen, and as a result, the two specifically bind to one another. Then, a sample of serum from a patient containing an unknown quantity of that same antigen is added. This causes the unlabeled (or "cold") antigen from the serum to compete with the radiolabeled antigen ("hot") for antibody binding sites. As the concentration of "cold" antigen is increased, more of it binds to the antibody, displacing the radiolabeled variant, and reducing the ratio of antibody-bound radiolabeled antigen to free radiolabeled antigen. The bound antigens are then separated from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured using a gamma counter. Antibodies conjugated to a magnetic support may be utilized in a radioimmunoassay, e.g. when separating antibody-antigen complex from unbound antigen.

In some embodiments, the step of determining the presence of a surface polysaccharide antigen, or a fragment of the antigen, can be performed using a label chosen from the group consisting of biotin, biocytin, iminobiotin, avidin, and streptavidin. In some examples, the second monoclonal antibody is conjugated to biotin. According to the present disclosure, a monoclonal antibody which can be labeled, e.g. be biotinylated, and still be stable and selective can be selected as a developer antibody.

In some embodiments, horse radish peroxidase (HRP) can be added in the step of determining the presence of a surface polysaccharide antigen, or a fragment of the antigen. For example, horse radish peroxidase can be conjugated to streptavidin. It is known in the art that biotin and streptavidin binds to each other. Hence, HRP conjugated to streptavidin may be utilized in order to detect an antibody-antigen-antibody complex if the second antibody is conjugated to biotin.

In some embodiments of the present disclosure, the step of determining the presence of a surface polysaccharide, or a fragment of the antigen, provides a result which is comparable to an appropriate control. For example, the appropriate control can be selected from a positive control, a negative control, or any combination thereof. The term "positive control" as referred to herein, should generally be understood as a sample comprising the desired antigen to be detected and hence a signal is detected from that sample. The term "negative control" as referred to herein, should generally be understood as a sample lacking the desired antigen to be detected and hence no signal is detected. In the context of the present disclosure, the term "a positive control, a negative control, or any combination thereof means a sample which comprises a certain concentration of the desired antigen to be detected.

According to the present invention, the method further comprises a washing step. Preferably, such washing step can utilize a magnet to be able to separate e.g. antibodies conjugated to a magnetic bead, antibody-antigen complexes, and antibody-antigen-antibody complexes from the rest of the solution present in the biological fluid sample. Second monoclonal antibodies conjugated to a label and which are not bound to an antigen are preferably separated from the assay before detecting the antibody-antigen-antibody complex. Otherwise false positive results may be obtained. Furthermore, the second monoclonal antibodies are preferably all conjugated to a label.

Otherwise, an unlabelled second monoclonal antibody bound to an antigen may create a false negative result.

The inventors have found that the monoclonal antibodies as disclosed herein have a high specificity against a surface polysaccharide antigen derivable from a mycobacterium, or a fragment of the antigen. It has also been shown that the monoclonal antibodies as disclosed herein have a high avidity against a surface polysaccharide antigen derivable from a

mycobacterium, or a fragment of the antigen. Furthermore, it has been shown that the monoclonal antibodies disclosed herein have a high affinity against a surface polysaccharide antigen derivable from a mycobacterium, or a fragment of the antigen. The use of monoclonal antibodies as disclosed herein enables a method for determining the presence of a surface polysaccharide antigen derivable from a mycobacterium, or a fragment of the antigen, in a biological fluid sample with high sensitivity.

Experiments have shown that the surface polysaccharide antigen lipoarabinomannan is secreted in the urine in subjects suffering from tuberculosis. Hence, a biological fluid sample, such as urine, may be analyzed in order to detect the presence of a mycobacterium in the sample.

It has been shown that the surface polysaccharide antigen

lipoarabinomannan is heat-stable so a biological fluid sample may be heat treated prior performing any method disclosed herein. However, the inventors have found that a biological fluid sample can be analyzed without pretreatment. Components in e.g. urine may preferably not influence the results when performing any method disclosed herein.

The inventors have found that by using a new immunoassay, based on a magnetic immunoassay (MIA) utilizing monoclonal antibodies conjugated to a magnetic support, the antigen to be detected can be concentrated in order to lower the detection limit of the antigen. MIA can be used in a variety of formats such as conventional enzyme-linked immunosorbent assay (ELISA) or lateral flow test by replacing gold labels with magnetic labels. In other examples, magnetic nanoparticles (MNP) or magnetic microparticles can be utilized in order to detect antibody-antigen complex signals by for example magnetic sensing of the nano- or microparticles or by optical measurements. Hence, the method for determining the presence of a lipoarabinomannan antigen, or a fragment of the antigen, in a biological fluid sample as disclosed herein, may enable a simple amplification of the antigen to be detected.

According to the present disclosure, magnetic beads exhibit several features very well adapted for e.g. a diagnostic assay. For example, they are not affected by e.g. reagent chemistry or photo-bleaching and are therefore stable over time. Furthermore, the magnetic background in a biomolecular sample is usually insignificant. Furthermore, an advantage is that magnetic beads can be manipulated remotely by magnetism, as described throughout the application.

The inventors have realized that there is a need for increasing the sensitivity of previous surface polysaccharide antigen tests, and also to develop tools to make it reproducible and feasible for large-scale production, providing speed, ease-of-use, low cost and robustness and with accuracy as in advanced laboratory tests. To reduce the time required for target detection, a minimal amount of sample manipulation may be essential. The sensitivity of the detection method as disclosed herein is preferably high enough in order to reduce or even eliminate the need for amplification and complicated enrichment steps of the biological fluid sample, comprising the surface polysaccharide antigen, such as lipoarabinomannan, to be detected, prior to performing the method disclosed herein. Hence, the method for determining the presence of a surface polysaccharide antigen, or a fragment of the antigen, in a biological fluid sample as disclosed herein is suitable for point-of- care tests.

According to the present invention, the methods described herein can be used for diagnosing tuberculosis in a subject where a biological fluid sample from the subject is analyzed.

Usually, a complete medical evaluation for tuberculosis must include a medical history, a physical examination, a chest X-ray and microbiological examination (of sputum or some other appropriate sample). Tuberculosis is typically diagnosed by finding Mycobacterium tuberculosis bacteria in a clinical specimen taken from a subject suspected of suffering from

tuberculosis. While other investigations may strongly suggest tuberculosis as the diagnosis, they cannot confirm it. The inventors have surprisingly found that the method of determining the presence of a surface polysaccharide antigen in a biological fluid sample, as disclosed herein, enables detection of both smear-positive and smear-negative tuberculosis. In the context of the present disclosure, "smear-positive" means presence of at least one acid fast bacilli, such as Mycobacterium tuberculosis in at least one sputum sample. The term "smear-negative" should be understood a sputum sample wherein no acid fast bacilli, such as Mycobacterium tuberculosis is detected.

Furthermore, the inventors have found that the method for determining the presence of a surface polysaccharide antigen, or a fragment of the antigen, is suitable for biological fluid samples obtained from subjects suffering from HIV and also for subjects not suffering from HIV.

In an aspect of the present invention, there is provided a kit for determining the presence of a lipoarabinomannan antigen, or a fragment of the antigen, in a biological fluid from a subject, the kit comprising:

• a first monoclonal antibody conjugated to magnetic support,

• a second monoclonal antibody,

• optionally, means for removing any unbound lipoarabinomannan antigen and/or second monoclonal antibody,

• means for detecting an antibody-antigen-antibody complex.

The presence of a lipoarabinomannan antigen, or a fragment of the antigen, in a biological fluid sample from a subject may indicative for a mycobacterial infection and/or disease in the subject. The mycobacterial infection and/or disease can be tuberculosis. The kit may be utilized in order to analyze a biological fluid sample obtained from a subject, such as a mammalian. The mammalian can be a human or an animal. In some examples, the subject is a human.

According to the present invention, the magnetic support can be a magnetic bead. The magnetic bead can be a magnetic nanobead or a magnetic microbead.

In an aspect of the present invention, there is provided a kit for determining the presence of a surface polysaccharide antigen derivable from a mycobacterium, or a fragment of the antigen, in a biological fluid, the kit comprising:

• a first monoclonal antibody MAb 25 produced by the hybridoma cell line deposited under Accession No. DSM ACC3247 or a first monoclonal antibody MAb 170 produced by the hybridoma cell line deposited under Accession No. DSM ACC3246, and

• a second monoclonal antibody MAb 25 produced by the hybridoma cell line deposited under Accession No. DSM ACC3247 or a second monoclonal antibody MAb 170 produced by the hybridoma cell line deposited under Accession No. DSM ACC3246,

• optionally, means for removing any unbound surface polysaccharide antigen and/or second monoclonal antibody,

• means for detecting an antibody-antigen-antibody complex.

The presence of a surface polysaccharide antigen, or a fragment of the antigen, in a biological fluid sample from a subject may be indicative for a mycobacterial infection and/or disease in the subject. The mycobacterial infection and/or disease can be tuberculosis. The kit may be utilized in order to analyze a biological fluid sample obtained from a subject, such as a mammalian. The mammalian can be a human or an animal. In some examples, the subject is a human.

According to the present invention, the kit may be utilized for

determining the presence of a surface polysaccharide antigen, or a fragment of the antigen, in a biological fluid sample. The surface polysaccharide antigen may be a lipopolysacchahde. For example, the lipopolysaccharide can be lipoarabinomannan.

In some embodiments, the first monoclonal antibody can be conjugated to a support. The support can be a magnetic support. For example, the magnetic support can be a magnetic bead. The magnetic bead can be a magnetic nanobead or a magnetic microbead.

According to the present disclosure, the means for removing any unbound antigen and/or second monoclonal antibody can be a magnetic device. In some embodiments, the magnetic device is a magnet.

According to the present disclosure, the means for detecting an antibody-antigen-antibody complex can be selected from means for performing an indirect of a direct labeling method. The indirect or direct labeling method can be selected from the group consisting of

spectrophotometry, fluorescence, enzyme-linked immunosorbent assay (ELISA), magnetic immunoassay (MIA), and radioimmunoassay. In some embodiments, the indirect or direct labeling method can be performed using a label chosen from the group consisting of biotin, biocytin, iminobiotin, avidin, and streptavidin. For example, the second monoclonal antibody can be conjugated to biotin.

According to the present disclosure, the kit may comprise horse radish peroxidase (HRP). For example, the horse radish peroxidase can be conjugated to streptavidin.

Furthermore, the kit may comprise a device for performing the indirect or direct labelling method, for example at least one eppendorf tube and/or at least one microtiter plate.

According to the present disclosure, the kit may comprise at least one control sample. For example, the control sample can be a negative control sample. In yet another example, the control sample can be positive control sample.

According to the present disclosure, the kit may further comprise instructions for carrying out any of the methods disclosed herein.

In another aspect of the present invention, there is provided a use of a monoclonal antibody conjugated to a magnetic support for determining the presence of a lipoarabinomannan antigen, or a fragment of the antigen, in a biological fluid sample. For example, the magnetic support may be a magnetic bead.

In another aspect of the present invention, there is provided a use of a monoclonal antibody MAb 25 produced by the hybridoma cell line deposited under Accession No. DSM ACC3247 and/or monoclonal antibody MAb 170 produced by the hybridoma cell line deposited under Accession No. DSM ACC3246 for determining the presence of a surface polysaccharide antigen, or a fragment of the antigen, in a biological fluid sample. For example, the surface polysaccharide antigen can be a lipopolysaccharide, such as lipoarabinomannan.

In another aspect of the present invention, there is provided a use of a monoclonal antibody MAb 25 produced by the hybridoma cell line deposited under Accession No. DSM ACC3247 and/or monoclonal antibody MAb 170 produced by the hybridoma cell line deposited under Accession No. DSM ACC3246 for diagnosing tuberculosis in a subject. By detecting the presence of a surface polysaccharide antigen, such as lipoarabinomannan, or a fragment of the antigen, in a biological fluid sample obtained from a subject a diagnosis of tuberculosis may be determined.

Itemized listing of embodiments

The following is a non-limiting and itemized listing of embodiments of the present disclosure, presented for the purpose of describing various features and combinations provided by the invention in certain of its aspects. Items:

1 . Monoclonal antibody MAb 25 produced by the hybridoma cell line

deposited under Accession No. DSM ACC3247 and/or monoclonal antibody MAb 170 produced by the hybridoma cell line deposited under Accession No. DSM ACC3246.

2. Monoclonal antibody MAb 25 and/or MAb 170 according to item 1 ,

wherein said cell line is deposited at the DSMZ on 10 July, 2014. 3. Monoclonal antibody MAb 25 and/or MAb 170 according to any one of items 1 -2, wherein said antibody is conjugated to a support.

4. Monoclonal antibody MAb 25 and/or MAb 170 according to item 3,

wherein said support is a magnetic support.

5. Monoclonal antibody MAb 25 and/or MAb 170 according to item 4,

wherein said magnetic support is a magnetic bead.

6. Monoclonal antibody MAb 25 and/or MAb 170 according to item 5,

wherein said magnetic bead is a magnetic nanobead or a magnetic microbead.

7. Monoclonal antibody MAb 25 and/or MAb 170 according to any one of items 1 -6, wherein said antibody is capable of binding to a surface polysaccharide antigen derivable from a mycobacterium, or to a fragment of said antigen.

8. Monoclonal antibody MAb 25 and/or MAb 170 according to item 7,

wherein said mycobacterium is Mycobacterium tuberculosis.

9. Monoclonal antibody MAb 25 and/or MAb 170 according to any one of items 7-8, wherein said surface polysaccharide antigen is a

lipopolysaccharide.

10. Monoclonal antibody MAb 25 and/or MAb 170 according to item 9,

wherein said lipopolysaccharide is lipoarabinomannan.

1 1 . Method for determining the presence of a lipoarabinomannan antigen, or a fragment of said antigen, in a biological fluid sample, comprising the steps:

• in a biological fluid sample from a subject,

o adding a first monoclonal antibody conjugated to a magnetic support,

o adding a second monoclonal antibody,

• allowing said first monoclonal antibody to bind to any

lipoarabinomannan antigen, or to a fragment of said antigen, present in said biological fluid sample,

• allowing said second monoclonal antibody to bind to said

lipoarabinomannan antigen, or to a fragment of said antigen, • wherein said first monoclonal antibody, said lipoarabinomannan antigen, and said second monoclonal antibody form an antibody- antigen-antibody complex,

• optionally, removing any unbound lipoarabinomannan antigen and/or second monoclonal antibody from said biological fluid sample,

• determining the presence of said lipoarabinomannan antigen, or a fragment of said antigen, by detecting said antibody-antigen-antibody complex.

12. Method according to item 1 1 , wherein the presence of said

lipoarabinomannan antigen in said biological fluid sample is indicative for a mycobacterial infection and/or disease in said subject.

13. Method according to item 12, wherein said mycobacterial infection

and/or disease is tuberculosis.

14. Method according to any one of items 1 1 -13, wherein said subject is a human.

15. Method according to any one of items 1 1 -14, wherein said biological fluid sample is selected from the group consisting of serum, sperm, blood, sputum, cerebro-spinal fluid, saliva, and urine.

16. Method according to item 15, wherein said biological fluid sample is urine.

17. Method according to any one of items 1 1 -16, wherein said biological fluid sample is pre-cultured under conditions permitting the growth of mycobacteria.

18. Method according to item 17, wherein said mycobacteria is

Mycobacterium tuberculosis.

19. Method according to any one of items 1 1 -18, wherein said magnetic support is a magnetic bead.

20. Method according to item 19, wherein said magnetic bead is a magnetic nanobead or a magnetic microbead.

21 . Method according to any one of items 1 1 -20, wherein said first

monoclonal antibody is a monoclonal antibody as defined in any one of items 1 -10. 22. Method according to any one of items 1 1 -21 , wherein said first monoclonal antibody is a capture monoclonal antibody.

23. Method according to any one of items 1 1 -22, wherein said second

monoclonal antibody is a monoclonal antibody as defined in any one of items 1 -10.

24. Method according to any one of items 1 1 -23, wherein said second

monoclonal antibody is a developer monoclonal antibody.

25. Method according to any one of items 1 1 -24, wherein the step of

removing any unbound lipoarabinomannan antigen and/or second monoclonal antibody is performed with the aid of a magnetic device.

26. Method according to item 25, wherein said magnetic device is a magnet.

27. Method according to any one of items 1 1 -26, wherein the step of

determining the presence of said lipoarabinomannan antigen, or a fragment of said antigen, is performed by using an indirect or a direct labeling method.

28. Method according to item 27, wherein said indirect or direct labeling

method is selected from the group consisting of spectrophotometry, fluorescence, enzyme-linked immunosorbent assay (ELISA), magnetic immunoassay (MIA), and radioimmunoassay.

29. Method according to item 28, wherein the step of determining the

presence of said lipoarabinomannan antigen, or a fragment of said antigen, is performed using a label chosen from the group consisting of biotin, biocytin, iminobiotin, avidin, and streptavidin.

30. Method according to item 29, wherein said second monoclonal antibody is conjugated to biotin.

31 . Method according to any one of items 1 1 -30, wherein horse radish

peroxidase (HRP) is added in the step of determining the presence of said lipoarabinomannan antigen, or a fragment of said antigen.

32. Method according to item 31 , wherein said horse radish peroxidase in the step of determining the presence of said lipoarabinomannan antigen, or a fragment of said antigen, is conjugated to streptavidin.

33. Method according to any one of items 1 1 -32, wherein the step of

determining the presence of said lipoarabinomannan antigen, or a fragment of said antigen, provides a result comparable to an appropriate control.

34. Method according to item 33, wherein said appropriate control is

selected from a positive control, a negative control, or any combination thereof.

35. Method according to any one of items 1 1 -34, wherein the method further comprises a washing step.

36. A method for determining the presence of a surface polysaccharide

antigen derivable from a mycobacterium, or a fragment of said antigen, in a biological fluid sample, comprising the steps:

• in a biological fluid sample from a subject,

o adding a first monoclonal antibody according to any one of items 1 -10, and

o adding a second monoclonal antibody according to any one of items 1 -10,

• allowing said first monoclonal antibody to bind to any surface

polysaccharide antigen, or to a fragment of said antigen, present in said biological fluid sample,

• allowing said second monoclonal antibody to bind to said surface polysaccharide antigen, or to a fragment of said antigen,

• wherein said first monoclonal antibody, said surface polysaccharide antigen, and said second monoclonal antibody form an antibody- antigen-antibody complex,

• optionally, removing any unbound surface polysaccharide antigen and/or second monoclonal antibody according to any one of items

1 -10 from said biological fluid sample,

• determining the presence of said surface polysaccharide antigen, or a fragment of said antigen, by detecting said antibody-antigen-antibody complex.

37. Method according to item 36, wherein the presence of said surface

polysaccharide antigen, or a fragment of said antigen, in said biological fluid sample, is indicative for a mycobacterial infection and/or disease in said subject. 38. Method according to item 37, wherein said mycobacterial infection and/or disease is tuberculosis.

39. Method according to any one of items 36-38, wherein said subject is a human.

40. Method according to any one of items 36-39, wherein said surface

polysaccharide antigen is a lipopolysaccharide.

41 . Method according to item 40, wherein said lipopolysaccharide is

lipoarabinomannan.

42. Method according to any one of items 36-41 , wherein said first

monoclonal antibody is conjugated to a support.

43. Method according to item 42, wherein said support is a magnetic

support.

44. Method according to item 43, wherein said magnetic support is a

magnetic bead.

45. Method according to item 44, wherein said magnetic bead is a magnetic nanobead or a magnetic microbead.

46. Method according to any one of items 36-45, wherein said biological fluid sample is selected from the group consisting of serum, blood, sputum, cerebro-spinal fluid, saliva, and urine.

47. Method according to item 46, wherein said biological fluid sample is urine.

48. Method according to any one of items 36-47, wherein said biological fluid sample is pre-cultured under conditions permitting the growth of mycobacteria.

49. Method according to item 48, wherein said mycobacteria is

Mycobacterium tuberculosis.

50. Method according to any one of items 36-49, wherein said first

monoclonal antibody is a capture monoclonal antibody.

51 . Method according to any one of items 36-80, wherein said second

monoclonal antibody is a developer monoclonal antibody.

52. Method according to any one of items 36-51 , wherein the step of

removing any unbound surface polysaccharide antigen and/or second monoclonal antibody is performed with the aid of a magnetic device. 53. Method according to item 52, wherein said magnetic device is a magnet.

54. Method according to any one of items 36-53, wherein the step of

determining the presence of said surface polysaccharide antigen, or a fragment of said antigen, is performed by using an indirect or a direct labeling method.

55. Method according to item 54, wherein said indirect or direct labeling

56. Method according to item 55, wherein the step of determining the

presence of said surface polysaccharide antigen, or a fragment of said antigen, is performed using a label chosen from the group consisting of biotin, biocytin, iminobiotin, avidin, and streptavidin.

57. Method according to item 56, wherein said second monoclonal antibody is conjugated to biotin.

58. Method according to any one of items 36-57, wherein horse radish

peroxidase (HRP) is added in the step of determining the presence of said surface polysaccharide antigen, or a fragment of said antigen.

59. Method according to item 58, wherein said horse radish peroxidase in the step of determining the presence of said surface polysaccharide antigen, or a fragment of said antigen, is conjugated to streptavidin.

60. Method according to any one of items 36-59, wherein the step of

determining the presence of said surface polysaccharide antigen, or a fragment of said antigen, provides a result comparable to an appropriate control.

61 . Method according to item 60, wherein said appropriate control is

62. Method according to any one of items 36-61 , wherein the method further comprises a washing step.

63. Kit for determining the presence of a lipoarabinomannan antigen, or a fragment of said antigen, in a biological fluid from a subject, said kit comprising: a first monoclonal antibody conjugated to a magnetic support, a second monoclonal antibody,

optionally, means for removing any unbound lipoarabinomannan antigen and/or second monoclonal antibody,

means for detecting an antibody-antigen-antibody complex.

64. Kit according to item 63, wherein the presence of said

lipoarabinomannan antigen, or a fragment of said antigen, is indicative for a mycobacterial infection and/or disease in said subject.

65. Kit according to item 64, wherein the mycobacterial infection and/or disease is tuberculosis.

66. Kit according to any one of items 64-65, wherein said subject is a

human.

67. Kit according to any one of items 63-66, wherein said magnetic support is a magnetic bead.

68. Kit according to item 67, wherein said magnetic bead is a magnetic nanobead or a magnetic microbead.

69. Kit according to any one of items 63-68, wherein said means for

removing any unbound lipoarabinomannan antigen and/or second monoclonal antibody is a magnetic device.

70. Kit according to item 69, wherein said magnetic device is a magnet.

71 . Kit according to any one of items 63-70, wherein said means for

detecting said antibody-antigen-antibody complex is selected from means for performing an indirect of a direct labeling method.

72. Kit according to item 71 , wherein said indirect or direct labeling method is selected from the group consisting of spectrophotometry,

fluorescence, enzyme-linked immunosorbent assay (ELISA), magnetic immunoassay (MIA), and radioimmunoassay.

73. Kit according to item 72, wherein said indirect or direct labeling method is performed using a label chosen from the group consisting of biotin, biocytin, iminobiotin, avidin, and streptavidin.

74. Kit according to item 73, wherein the second monoclonal antibody is conjugated to biotin. 75. Kit according to any one of items 63-74, wherein the kit comprises horse radish peroxidase (HRP).

76. Kit according to item 75, wherein said horse radish peroxidase is

conjugated to streptavidin.

77. Kit according to any one of items 63-76, wherein said kit comprises at least one control sample.

78. Kit according to any one of items 63-77, further comprising instructions for carrying out the method as defined in any one of items 1 1 -35.

79. Kit for determining the presence of a surface polysaccharide antigen derivable from a mycobacterium, or a fragment of said antigen, in a biological fluid from a subject, said kit comprising:

• a first monoclonal antibody according to any one of items 1 -10,

• a second monoclonal antibody according to any one of items 1 -10,

• means for detecting an antibody-antigen-antibody complex.

80. Kit according to item 79, wherein the presence of a surface

polysaccharide antigen, or a fragment of said antigen, is indicative for a mycobacterial infection and/or disease in said subject.

81 . Kit according to item 80, wherein the mycobacterial infection and/or disease is tuberculosis.

82. Kit according to any one of items 79-81 , wherein said subject is a

human.

83. Kit according to any one of items 79-82, wherein said surface

polysaccharide is a lipopolysaccharide.

84. Kit according to item 83, wherein said lipopolysaccharide is

lipoarabinomannan.

85. Kit according to any one of items 79-84, wherein said first monoclonal antibody is conjugated to a support.

86. Kit according to item 85, wherein said support is a magnetic support. 87. Kit according to item 86, wherein said magnetic support is a magnetic bead. 88. Kit according to item 87, wherein said magnetic bead is a magnetic nanobead or a magnetic microbead.

89. Kit according to any one of items 79-88, wherein said means for

removing any unbound surface polysaccharide antigen and/or second monoclonal antibody is a magnetic device.

90. Kit according to item 89, wherein said magnetic device is a magnet.

91 . Kit according to any one of items 79-90, wherein said means for

92. Kit according to item 91 , wherein said indirect or direct labeling method is selected from the group consisting of spectrophotometry,

93. Kit according to item 92, wherein said indirect or direct labeling method is performed using a label chosen from the group consisting of biotin, biocytin, iminobiotin, avidin, and streptavidin.

94. Kit according to item 93, wherein the second monoclonal antibody is conjugated to biotin.

95. Kit according to any one of items 79-94, wherein the kit comprises horse radish peroxidase (HRP).

96. Kit according to item 95, wherein said horse radish peroxidase is

conjugated to streptavidin.

97. Kit according to any one of items 79-96, wherein said kit comprises at least one control sample.

98. Kit according to any one of items 79-97, further comprising instructions for carrying out the method as defined in any one of items 36-62.

99. Use of a monoclonal antibody conjugated to a magnetic support for determining the presence of a lipoarabinomannan antigen, or a fragment of the antigen, in a biological fluid sample.

100. Use of a monoclonal antibody MAb 25 produced by the hybridoma cell line deposited under Accession No. DSM ACC3247 and/or monoclonal antibody MAb 170 produced by the hybridoma cell line deposited under Accession No. DSM ACC3246 for determining the presence of a surface polysaccharide antigen, or a fragment of the antigen, in a biological fluid sample.

101 . Use of a monoclonal antibody MAb 25 produced by the hybridoma cell line deposited under Accession No. DSM ACC3247 and/or monoclonal antibody MAb 170 produced by the hybridoma cell line deposited under

Accession No. DSM ACC3246 for diagnosing tuberculosis in a subject.

The use of at least one monoclonal antibody according to the invention will now be described in a non-limiting manner by the following Figures and Examples.

Brief description of the appended figures

Figure 1 shows the kinetics of monoclonal antibody MAb 25 and MAb 170 binding to immobilized lipoarabinomannan (LAM) at various monoclonal antibody concentrations (μΜ) using SPR.

Figure 2 shows avidity of monoclonal antibodies MAb 25 and MAb 170 compared to CSU reference MAbs (CSU-35 and CSU-40) using a

spectrophotometer. Detailed description

In some examples, an ELISA assay can be performed using magnetic beads. The ELISA assay can be performed in e.g. a tube, such as an eppendorf tube. The beads can be utilized as a solid support. At least one antibody (e.g. a capture antibody) can be conjugated to a magnetic particle. A biological fluid sample comprising an antigen can be added to the tube comprising at least one antibody conjugated to a magnetic bead. After having allowed the antigen to bind to an antibody, a magnetic device, such as a magnet, can be utilized in a separation and/or washing step. By attracting the antibodies with bound antigen (antibody-antigen complexes) to one side of the tube, the rest of the biological fluid sample can be removed from the tube by using a pipette. Washing solution can be added to the tube. The magnet can be removed from the tube and antibody-antigen complexes can be dispersed in the washing solution. Once again, the magnet can be put close to the tube and the antibodies conjugated to a magnetic bead will be attracted to the side of the tube. Any unbound antigen is present in the washing solution and the washing solution comprising any unbound antigen can be removed using a pipette. Furthermore, a fluid comprising a second antibody, such as a developer antibody, can be added to the tube. By removing the magnet, the antibody-antigen complexes can disperse in the fluid. Second antibody is allowed to bind to the antigen bound to the first antibody. After a certain time, after having allowed the second antibody to bind to the antigen, a magnet can be put close to the tube. In a similar fashion, as described above, a washing solution can be added to the tube. Any unbound second antibody can be removed from the tube. The second antibody can be conjugated to a label, such as biotin, and that label can be detected or measured utilizing techniques as described herein, for example by adding HRP conjugated to streptavidin to the tube followed by the addition of TMB, spectrophotometry can be utilized, for example by measuring absorbance at e.g. 450 nm.

In some examples, the magnetic device can be formed to be able to surround the tube.

In some examples, first antibodies unconjugated to a magnetic bead will not be attracted to a magnetic device and hence may be separated by using a magnetic device.

Examples

In the following non-limiting Examples, the principle of using specific monoclonal antibodies as described herein for determining the presence of a surface polysaccharide antigen derivable from a mycobacterium, or a fragment of the antigen, in a biological fluid sample is shown.

Materials and Methods

Material and Reagents:

Magnetic particles (MNP) (Micromod Partikeltechnologie GmbH, Germany) with a size in the micrometer range are utilized throughout the experiments. At least one monoclonal antibody, as disclosed herein, is conjugated to a magnetic particle. The monoclonal antibody binds specifically to a surface polysaccharide antigen, such as lipoarabinomannan (LAM). The monoclonal antibodies produced in-house are hereinafter denoted as MAb 25 and MAb 170.

Monoclonal antibodies CSU-35 and CSU-40 (MAbs CSU-35 and CSU- 40), directed at lipoarabinomannan (LAM), produced under the NIH, TBVTRM Contract with Colorado State University (CSU) were used as reference monoclonal antibodies.

Preparation of lipoarabinomannan (LAM)

Cell wall preparations from the bacterial strain Mycobacterium

tuberculosis H37Rv were isolated and lipoarabinomannan (LAM) was purified as previously described in Mazurek et ai, PLoS one, in press (2012).

Arabinomannan (AM) was obtained by mild alkaline hydrolysis of LAM as described in Hamasur et al, Journal of Microbiological Methods 45, 41 (2001 ). Sodium periodate oxidation of delipidated purified LAM (i.e. AM) was done by treatment with 10 mm NalO₄ in acetate buffer pH 6.0 at + 4 °C in the dark for 15 min.

Preparation of anti-LAM monoclonal antibodies

The anti-LAM monoclonal antibodies MAb 25 and MAb 170 were produced in-house as described in Hamasur et ai, Clinical and Experimental Immunology 138, 30 (2004). The isotypes of the monoclonal antibodies were identified by enzyme-linked immunosorbent assay (ELISA), using alkaline phosphatase-conjugated goat antimouse IgG subclass specific antibodies (Sigma Chemical Co, USA) and LAM as coating antigen. The monoclonal antibodies of lgG1 subclass were shown to have high affinity and high avidity towards LAM.

Avidity assessments of the anti-LAM monoclonal antibodies

1 . ELISA

The relative titres of the monoclonal antibodies were determined by ELISA. Wells of polystyrene microplates (Maxisorb, Nunc, Denmark) were coated with 100 μΙ of purified LAM (10 pg/ml) in 0.05 M carbonate buffer, pH 9.6, at room temperature overnight. The plates were washed three times with rinsing buffer (PBS containing 0.05% Tween), and then blocked with 0.5% casein for one hour at 37 °C. After washing, 100 μΙ of serial dilutions of each monoclonal antibody were added to the wells and incubated for one hour at 37 °C. After washing with rinsing buffer, 100 μΙ of alkaline

phosphatase-conjugated goat, antimouse IgG (Sigma Chemical Co., diluted 1/2000 in PBS) was added to each well and the plates were incubated for a further one hour at 37 °C. After subsequent washings, the plates were developed at room temperature using p-nitrophenyl phosphate (Sigma Chemical Co.) as substrate and the color reaction was measured by increase in absorbance at 450 nm using an ELISA reader (Dynatech, MR 5000). 2. Surface Plasmon Resonance (SPR)

The interaction between monoclonal antibodies and LAM was analyzed using Surface Plasmon Resonance (SPR) (Biacore 2000; Biacore, GE

Healthcare, Uppsala, Sweden).

Activation of AM: Six mg of LAM was delipidated by alkaline treatment with 0.1 M NaOH for one hour at 80 °C to form arabinomannan (AM). The pH of the suspension was adjusted to 6.0 with 1 M HCI and the lipid precipitate was removed by centrifugation. The delipidated LAM (dLAM) (i.e. AM) was then treated with 0.05 M periodate for 15 min at 4 °C in the dark with stirring.

After stopping the reaction with 100 ml of ethylene glycol, the activated AM was purified on a PD-10 column equilibrated with 0.1 M sodium bicarbonate buffer at pH 8.3.

Activation of the gold surface: Three different linkers were used for immobilization of the activated AM to the gold surface: i) a 12-amino acid long peptide was designed and employed for coating the gold surface on the chip, ii) cystamine activation of the gold surface introduces a 2-carbon atom long spacer arm and iii) 1 1 -amino dodecanethiol results in creating a surface with a 12-carbon atom spacer-arm. All the three treatments leave free amino groups available for covalent conjugation to the activated AM. Conjugation of AM to the gold chip: The sensor chips were coated with different concentration of activated AM ranging from 0.1 -0.5 mg AM on carbohydrate basis. After incubation over night at ambient temperature, in the presence of 20 mg/ml sodium borohydride, the chips were rinsed twice with PBS and the remaining sites on the chips were blocked with 5 mM

mercaptoethanol for 20 min. The chips were then washed gently with PBS and air-dried for two hours at room temperature. Three chips, one for each conjugation method were blocked with 0.5% casein at room temperature for one hour. After rinsing, 100 μΙ of biotinylated anti-LAM antibody, of a concentration of 2 Mg/ml, was added onto the surface of each chip. The chips were rinsed with PBS-Tween buffer after one hour of incubation. Next, the chips were incubated with 100 μΙ of avidin-HRP at 1 :5000 dilution. After one hour of incubation the chips were rinsed gently with washing buffer and 100 μΙ of TMB substrate was loaded on the surface of the chip followed by 15 min incubation. The blue solution developed on the chip surface was then transferred into the wells of a microtitre plate and the reaction stopped with 50 ml of 16% sulphuric acid. The absorbance was recorded with an ELISA reader at 450 nm.

Avidity assessment of monoclonal antibody MAb 25 and MAB 170: All experiments were performed using Biacore 3000 at 30 °C in PBS at 20 ml/min flow rate, and a total injection of 20 μΙ (data not shown). For

regeneration of the sensor chips different buffers were examined: glycine-HCI buffer at pH 1 .5, 2.0, 2.5 and 3, 3 M guanidine hydrochloride, and 100 mM sodium hydroxide. The best condition for regeneration was 100 mM NaOH, which was then used throughout. The sensorgrams clearly show that the highest AM-Ab binding activity, i.e. with highest resonance unit (rRU), was obtained with the cystamine-AM chip (data not shown). This chip could also be regenerated easily compared to the other surfaces, therefore it was chosen for analysis of other monoclonal antibodies throughout the

experiments.

Synthesis of gold coated MNP/PS particles and MNP-MAb conjugate

MNP or PS (polystyrene) particles, with an amount of about 10¹³, were resuspended in 5 ml toluene and the solution was heated to 85 °C.

HAuCI₄-3H₂O (0.05 g) and oleylamine (1 .25 ml) in toluene (5 ml) were injected to the mixture while maintaining the temperature at 85 °C for one hour. The solution was cooled to room temperature to produce a dark purple solution. The particles (0.1 g) were dissolved in 20 ml phenyl ether and then mixed with 2 ml oleic acid (6 mmol) and 2 ml oleylamine (4 mmol) under N₂ with vigorous stirring. 1 ,2-Hexadecanediol (2.85 g) was added to the solution and heated to 120 °C under reflux for two hours, then cooled to room temperature. The Fe3O₄-Au NPs (magnetic nanoparticles coated with gold) or PS-AU NP (polystyrene nanoparticles coated with gold) were precipitated in ethanol (approximate 15 ml) and separated by either centrifugation or by a permanent magnet. The pellets were washed twice with ethanol and re- dispersed in 3-5 ml water. Tri-sodium citrate (0.04 g) was added and the pH of the resulting solution was adjusted to 6.5. The solution was sonicated for 15 min in ultrasonic bath and the particles were collected by magnet or centrifugation and re-dispersed in water and sonicated again for another 15 min.

UV/visible spectra were obtained using a Molecular Devices Spectromax 384 spectrometer. TEM images were obtained using an FEI Tecnai

G2 120 kV TEM operated at 100 kV and visualized using analysis software. Monoclonal antibodies were immobilized to the surface of the gold coated particles by an in-house patented (US Patent 6165468) method.

Magnetic nanoparticle (MNP) assay

Urine samples of 5 ml were placed in tubes of 10 ml and 10 μΙ of the

MNP-AB-Conjugate (i.e. particle-antibody complex) was added to each urine sample. The tubes were incubated for 20 minutes and then placed in a magnet-stand at room temperature for 3 minutes. The supernatant was then removed and the particles were washed with 4 ml of PBS/Tween solution. This procedure was repeated once. Then 500 μΙ of a 0.5 g/ml biotin-labeled MAb solution was added to each tube, vortexed briefly, and incubated for 15 minutes. After washing, a Strep-Avidin-HRP Conjugate (500 μΙ) (with a dilution of 1 :10 000) was added to each sample and incubated for 15 minutes. After washing, 200 μΙ of SURE Blue TMB reagent per sample tube was added and incubated for 5 minutes in the dark. 100 μΙ of the colored supernatant was placed in a well of a 96-well plate. 50 μΙ of Stop Reagent (producent) was added and absorbance was read at 450 nm using a spectrophotometer.

Urine samples

At the TB clinic, Karolinska Hospital, urines were collected from patients with culture confirmed TB. Samples from TB healthy individuals were collected from hospital staff at Sodersjukhuset, Stockholm, Sweden, and were utilized as negative controls. All samples were immediately frozen at - 20°C until tested.

Results

Binding capacity of monoclonal antibodies

Two murine monoclonal antibodies were raised against LAM. SPR analysis revealed that these monoclonal antibodies (MAb 25 and MAb 170) have high avidity (see Fig. 1 ). These data were in full agreement with the ELISA titration curves of the same monoclonal antibodies (see Fig. 2). The in- house monoclonal antibodies exhibited more than 3 logs higher binding in ELISA as compared to the reference anti-LAM monoclonal antibodies CSU- 35 and CSU-40 (see Fig. 2).

Detection limit of LAM by ELISA utilizing anti-LAM monoclonal antibodies

Using the monoclonal antibodies MAb 25 and MAb 170 and urine samples spiked with LAM at increasing concentrations, the detection limit was estimated to be about 1 ng/ml in ELISA (see Table 1 ) (values are considered positive when OD > 0.35). ELISA and MNP assay results with urine spiked with increasing concentrations of lipoarabinomannan (LAM) using MAb 25 and MAb 170

Numbers marked in cursive are positive (OD > 0.35).

Detection limit of the MNP assay at various urine volumes

By utilizing the MNP, with MAb 25 as first/capture monoclonal antibody and MAb 170 as second/developer monoclonal antibody, the detection limit was increased by 50-fold compared to the ELISA (see Table 1 ), reaching a detection limit of about 0.05 ng/ml when using a volume of 5 ml urine. The starting amount of urine had an important effect on the detection limit of the MNP assay (see Table 2) since a larger volume of urine comprises a larger amount of antigen to be detected.

Table 2. Effect of urine volume on detection limit of MNP assay

^* OD at absorbance 450 nm

Numbers marked in cursive are positive (OD > 0.35).

LAM detection by MNP assay in samples obtained from TB patients and control samples

With a cut-off set at the optical density (OD) 0.35, urine samples from 22 healthy individuals were all negative (mean OD 0.17, range 0.12-0.3).

Twenty-five HIV-negative patients with culture confirmed TB were recruited. From each patient one urine sample was collected before onset of TB treatment. Of eight patients that had yielded morning urine, five were detected as positive for TB by the MNP assay. However, only one of seven patients that had yielded non-morning urine was positive.

Detection limit in known prior art tests

There are tests in the prior art using a sandwich ELISA with polyclonal antibodies. The antibodies utilized in these tests are polyclonal rabbit antibodies of low avidity and specificity. The detection limit of LAM in spiked samples using these known tests is about 1 ng/ml. In a few of these studies, concentration of the urine is applied to increase the sensitivity. The samples are boiled and then centrifuged after which the supernatant is further purified through column chromatography and the eluent is concentrated by a rotavapor. In one test, urine is centrifugated by using a 10 K molecular filter.

Since the LAM concentration in urine of ordinary TB patients is extremely low, in many cases in the range of picogram per ml, known LAM tests (e.g. ELISA tests and strip test) which have a detection limit in the range of nanogram per ml, are not sensitive enough for diagnosis in unselected TB suspects. Actually the disappointment in the present LAM tests all lies in the fact that the detection limit of present LAM urine tests is too high, i.e. the tests are not sensitive enough.

The excretion amount of LAM has been reported to vary between 1 ng/ml and several hundred ng/ml depending on the clinical manifestations. Quantitative LAM detection results increase with bacterial burden. Thus, smear positive patients have been shown to be more often positive in LAM ELISA than smear negative patients and a positive correlation between bacterial density in sputum and urine concentration of LAM has been demonstrated. It has also been shown that the sensitivity of urine LAM testing increases with progressive HIV immune suppression (as reflected by falling CD4 cell counts), probably correlating with an increased total mycobacterial burden.

Detection limit of the MNP assay

The present invention provides for a 50-100-fold increase in urinary LAM detection using the MNP assay compared to previous methods. This increase is mainly due to the combination of the high avidity LAM specific monoclonal antibodies and a concentration step using nanoparticle technology.

Sensitivity of the MNP assay

Using a method disclosed herein, urine samples obtained from HIV- negative patients with verified TB and control samples, provided a specificity of 100% and sensitivity close to 80%. This is to be compared with the results of a range of studies using the commercial tests with polyclonal anti-LAM antibodies where the sensitivity of the tests in HIV-negative TB patients was very low compared to HIV-positive patients. The fact that morning samples were more often positive than non-morning samples further supports the importance of concentration of urine for detection of the small amounts of LAM excreted in urine of most TB patients. Feasibility

The format of the MNP platform makes the test easy to perform. It includes a few pipetting and washing steps. A technician can handle 24-48 samples in about two hours. In this study an ELISA reader was used in order to do a quantitative measurement of the LAM concentration, However, for field purposes the ELISA reader can be replaced by either 1 ) a battery operated portable ELISA reader, or preferably ii) by reading the color change by the naked eye. In that case, no electricity is needed. Further feasibility studies should of course be performed in peripheral clinics once the test has been validated.

The inventors have developed a new format of a test for determining the presence of lipoarabinomannan in a biological fluid sample, using monoclonal antibodies with high avidity. The new test increased the assay sensitivity by using new nanotechnology techniques in the form of nano- and/or

microparticles, such as magnetic particles.

The results show that monoclonal antibodies as disclosed herein show high avidity and/or high affinity against a surface polysaccharide antigen derivable from a mycobacterium, or a fragment of the antigen, such as lipoarabinomannan.

Furthermore, the results show that a method for determining the presence of a surface antigen derivable from a mycobacterium, or a fragment of the antigen, such as lipoarabinomannan, in a biological fluid sample, such as urine, utilizing at least one monoclonal antibody as disclosed herein, which have a low detection limit for the surface polysaccharide antigen is provided and such method is useful in e.g. point-of-care applications.

Indications are Made All designations

FOR RECEIVING OFFICE USE ONLY

0-4 This form was received with the

international application: YES

(yes or no)

0-4-1 Authorized officer

Teloni, Monica FOR INTERNATIONAL BUREAU USE ONLY

0-5 This form was received by the

international Bureau on:

0-5-1 Authorized officer

Claims

Method for deternnining the presence of a lipoarabinonnannan antigen, or a fragment of said antigen, in a biological fluid sample, comprising the steps:

• in a biological fluid sample from a subject,

o adding a first monoclonal antibody conjugated to a magnetic support,

o adding a second monoclonal antibody,

• allowing said first monoclonal antibody to bind to any

• allowing said second monoclonal antibody to bind to said

lipoarabinomannan antigen, or to a fragment of said antigen,

• wherein said first monoclonal antibody, said lipoarabinomannan antigen, and said second monoclonal antibody form an antibody- antigen-antibody complex,

Method according to claim 1 , wherein the presence of said

Method according to claim 2, wherein said mycobacterial infection and/or disease is tuberculosis.

Method according to any one of claims 1 -3, wherein said subject is a human.

5. Method according to any one of claims 1 -4, wherein said biological fluid sample is selected from the group consisting of serum, sperm, blood, sputum, cerebro-spinal fluid, saliva, and urine.

6. Method according to claim 5, wherein said biological fluid is urine.

7. Method according to any one of claims 1 -6, wherein said biological fluid sample is pre-cultured under conditions permitting growth of

mycobacteria.

8. Method according to claim 7, wherein said mycobacteria is

Mycobacterium tuberculosis.

9. Method according to any one of claims 1 -8, wherein said magnetic support is a magnetic bead.

10. Method according to any one of claims 1 -9, wherein said first

monoclonal antibody is monoclonal antibody MAb 25 produced by the hybridoma cell line deposited under Accession No. DSM ACC3247 or monoclonal antibody MAb 170 produced by the hybridoma cell line deposited under Accession No. DSM ACC3246.

1 1 . Method according to any one of claims 1 -10, wherein said second

12. Method according to any one of claims 1 -1 1 , wherein the step of

13. Method according to any one of claims 1 -12, wherein the step of

determining the presence of a said lipoarabinomannan antigen, or a fragment of said antigen, is performed using an indirect or a direct labelling method selected from the group consisting of spectrophotometry, fluorescence, enzyme-linked immunosorbent assay (ELISA), magnetic immunoassay (MIA), and radioimmunoassay.

14. Method according to any one of claims 1 -13, wherein the method further comprises a washing step.

15. Monoclonal antibody MAb 25 produced by the hybridoma cell line

16. Monoclonal antibody MAb 25 and/or MAb 170 according to claim 15, wherein said antibody is conjugated to a magnetic support.

17. Monoclonal antibody MAb 25 and/or MAb 170 according to any one of claims 15-16, wherein said antibody is capable of binding to a

lipoarabinomannan antigen, or to a fragment of said antigen.

18. A method for determining the presence of a surface polysaccharide

• in a biological fluid sample from a subject,

o adding a first monoclonal antibody according to any one of claims 15-17, and

o adding a second monoclonal antibody according to any one of claims 15-17,

• allowing said first monoclonal antibody to bind to any surface

· allowing said second monoclonal antibody to bind to said surface polysaccharide antigen, or to a fragment of said antigen,

• wherein said first monoclonal antibody, said surface polysaccharide antigen, and said second monoclonal antibody form an antibody- antigen-antibody complex, • optionally, removing any unbound surface polysaccharide antigen and/or second monoclonal antibody from said biological fluid sample,

19. Method according to claim 18, wherein the presence of said surface polysaccharide antigen in said biological fluid sample is indicative for tuberculosis in said subject.

20. Method according to any one of claims 18-19, wherein said subject is a human.

21 . Method according to any one of claims 18-20, wherein said

lipopolysaccharide is lipoarabinomannan.

22. Method according to any one of claims 18-21 , wherein said biological fluid sample is selected from the group consisting of serum, blood, sputum, cerebro-spinal fluid, saliva, and urine.

23. Method according to any one of claims 18-22, wherein said first

monoclonal antibody is conjugated to a magnetic support.

24. Method according to any one of claims 18-23, wherein the step of

removing any unbound surface polysaccharide antigen and/or second monoclonal antibody is performed with the aid of a magnetic device.

25. Method according to claim 24, wherein said magnetic device is a

magnet.

26. Method according to any one of claims 18-25, wherein the step of

determining the presence of a surface polysaccharide antigen, or a fragment of said antigen, is performed using an indirect or a direct labelling method selected from the group consisting of

spectrophotometry, fluorescence, enzyme-linked immunosorbent assay (ELISA), magnetic immunoassay (MIA), and radioimmunoassay.

Kit for determining the presence of a lipoarabinomannan antigen, or a fragment of said antigen, in a biological fluid from a subject, said kit comprising:

• a first monoclonal antibody conjugated to a magnetic support,

• a second monoclonal antibody,

means for detecting an antibody-antigen-antibody complex.

28. Kit according to claim 27, wherein said first monoclonal antibody is a monoclonal antibody as defined in any one of claims 15-17.

29. Kit according to any one of claims 27-28, wherein said second

monoclonal antibody is a monoclonal antibody as defined in any one of claims 15-17.

30. Kit according to any one of claims 27-29, wherein said means for

31 . Kit for determining the presence of a surface polysaccharide antigen derivable from a mycobacterium, or a fragment of said antigen, in a biological fluid from a subject, said kit comprising:

· a first monoclonal antibody according to any one of items 1 -10,

• a second monoclonal antibody according to any one of items 1 -10,

• means for detecting an antibody-antigen-antibody complex.

32. Kit according to claim 31 , wherein said surface polysaccharide antigen is lipoarabinomannan.

33. Kit according to any one of claims 31 -32, wherein said first monoclonal antibody is conjugated to a magnetic support. Kit according to any one of claims 31 -33, wherein said means for removing any unbound surface polysaccharide antigen and/or second monoclonal antibody is a magnetic device.

Use of a monoclonal antibody conjugated to a magnetic support for determining the presence of a lipoarabinomannan antigen, or a fragment of the antigen, in a biological fluid sample.

Use of a monoclonal antibody MAb 25 produced by the hybridoma cell line deposited under Accession No. DSM ACC3247 and/or monoclonal antibody MAb 170 produced by the hybridoma cell line deposited under Accession No. DSM ACC3246 for determining the presence of a surface polysaccharide antigen, or a fragment of the antigen, in a biological fluid sample.