Anti-IFN-γ antibodies

Abstract

The present application relates to an isolated antibody that specifically binds to human interferon-γ (IFN-γ) and its applications in diagnosing active tuberculosis infection. The antibody described herein exhibits high specificity and affinity towards IFN-γ, enabling accurate detection of IFN-γ in extrapulmonary fluid samples. The present application also relates to the use of the antibody in in vitro methods of diagnosis of active TB and to an immunoassay device which incorporates the antibody.

Description

[0001] ANTI-IFN-Y ANTIBODIES

[0002] BACKGROUND OF THE INVENTION

[0003] Tuberculosis (TB) is caused by Mycobacterium tuberculosis, which results in a chronic granulomatous infection characterised by paucibacillary disease and foci of caseous necrosis. The main organ affected is the lung (-85% of cases). However, in about 15% to 35% of persons (depending on several factors including genetic background, nationality, HIV status, etc.) TB can involve organs outside the lung, i.e. extrapulmonary TB (EPTB). The most common form of EPTB in many countries is pleural TB (inflammation of the external lung lining causing accumulation of fluid between the lung and the chest wall. Other forms of EPTB include TB pericarditis and TB peritonitis. Together these 3 entities collectively result in TB serositis (inflammation of the serosal membrane; pleural, pericardium, or peritoneum). A different form of EPTB is TB meningitis, which results in the inflammation of the coverings the brain and spinal cord. Infection of all these sites is characterised by pauci-bacillary infection of fluid within these compartments or body cavities. The burden of disease is often below the detectable limit of existing nucleic acid amplification tests (NAATs), thus making immunodiagnosis an attractive proposition.

[0004] While pulmonary TB is conventionally diagnosed by finding evidence of M. tuberculosis in the sputum, diagnosis of these forms of EPTB (as outlined above) is made by detecting the organisms or genomic material in the EP fluid. Thus, conventionally, individuals with a suggestive clinical presentation together with basic radiological findings (e.g. chest x-ray), undergo aspiration of the fluid from the pleural, pericardial, peritoneal cavities, or from the subarachnoid space (cerebrospinal fluid). However, confirm the diagnosis of TB in these fluids is challenging and problematic. Smear microscopy (visualising the organism under a microscope) is generally only possible in <5% of patients, and culture is usually only positive in about 30 to 40% of patients (given the pauci-bacillary nature of the disease). Using nucleic acid amplification testing is also problematic. For example, in pleural TB a commonly used NAAT (e.g. GeneXpert MTB / RIF Ultra; Cepheid) has a sensitivity of only 30 to 40% (Light (2010), Meldau et al. (2014) and Meldau et al. (2019). Whilst more non-specific biomarkers such adenosine deaminase (ADA) have been used, this biomarker has sub-optimal specificity in pleural TB (-80%), in pericardial TB (-70%), and performs even more poorly in cerebrospinal fluid. It also has a poor negative predictive value.

[0005] An alternative biomarker is unstimulated interferon gamma. Upon antigen presentation, CD4+ and CD8+ T-cells produce large amounts of interferon gamma. This biomarker has generally been used for the diagnosis of latent TB infection (LTBI). Indeed, tests like T-SPOT TB and QuantiFERON Gold In-Tube are used to detect interferon gamma in overnight cultures of peripheral blood mononuclear cells stimulated with TB-specific antigens. However, the poor specificity (due to background LTBI and exposure to environmental mycobacteria) means that this test is not useful for the diagnosis of active TB, and the WHO advises against the use of this test for the diagnosis of active TB for these reasons (WHO Policy Statement (2022)). Thus, in summary, IGRAs are useful for the diagnosis of LTBI, but not active TB involving serosal compartments (Metcalfe et al. (2011 )). Thus, when such tests have been evaluated for pleural TB, for example, they performed poorly (Fan et al. (2012).

[0006] An alternative to the IGRAs is to evaluate unprocessed interferon gamma directly in the extrapulmonary fluid. The functional detection of IFN-y in specific human compartments (e.g. blood versus pleural or pericardial space) is influenced by several factors including pH, serosal membrane permeability, inflammatory factors etc. that modulate the folding of proteins, and accumulation of several molecules (antibodies and / or proteins and / or glycolipids) may bind to antibodies resulting in incorrectly blocking (false negative) or enhancing (false positive) the detection of IFN-y (including the heterophile effect).

[0007] These factors described above are related to complex mechanisms that influence the transport of human proteins into and out of biological compartments (e.g. the blood versus pleural space). The effect on membrane permeability includes, but is not limited to, differential cytokine and chemokine-induced permeability of serosal membranes, biomolecules that impact compartment-specific leucocyte trafficking, the mesothelial electrochemical cellular profile, the mesothelial non neuronal cholinergic system, differential activity of families of transporter systems, differential transcellular versus gap junction-mediated transport, release associated with lipid and other vesicles, differential mesothelial membrane-specific signalling cascades, dynamic protein translocons and their associated receptor systems, and differential molecule specific leakage across the serosal-blood barrier.

[0008] Collectively, the aforementioned factors result in topologically distinct classes of proteins, including IFN-y, being ‘trapped’ and differentially detectable in various compartments (e.g. the pleural space verus the blood compartment). Thus, antibodies that detect human IFN-y in one compartment, like blood, may perform differently in another compartment like the pleural space (both sensitivity and specificity of the biomarker of interest may be impacted).

[0009] Interestingly, whilst other diseases such as community-acquired pneumonia, various infections, auto immune illnesses, malignancies, etc. can also drive some interferon gamma production, this is considerably lower in magnitude compared to TB, and also there is also functional ‘trapping’ of interferon gamma in the relevant compartment. Several studies have shown that unstimulated interferon gamma is a good biomarker for the diagnosis of active TB (Meldau et al. (2014), Meldau et al. (2019), Dheda et al. (2009), Pandie et al. (2014) and Patel et al. (2011 )).

[0010] The present inventors have through screening and clinical validation elucidated an antibody pair that enables highly sensitive and specific detection of human IFN-y in EP human compartments compared to the blood compartment. Furthermore, the present inventors have validated a method to detect IFN-y for the diagnosis of EPTB using the fluid from the EPTB compartment.

[0011] The methods of the present invention do not require the use of blood, or overnight stimulation, or use of additional peptides or proteins. The methods rely on a specific antibody pair that mitigates the pitfalls of alternative pairs which tend to be prone to heterophile and other effects that prevent highly sensitive and specific diagnosis of active EPTB. Mitigation strategies against heterophiles include the use of blocking agents in the assay buffers such as inactivated serum, pre-incubation of clinical samples with antibody fragments, removal of the Fc region of the antibody and / or humanising antibodies at development phase (Kricka (1999)). The antibody pair covered employed the humanising strategy through inoculation of mice with human- derived IFN-y and downstream stable cell line development (sequenced regions into a human framework). The use of the antibodies of the present invention may be employed in any type of device or platform for the diagnosis of EPTB. The method does not diagnose LTBI.

[0012] In summary, several local factors (including pH, inflammatory milieu, heterophile molecules, etc.) result in differential sensitivity in detecting human IFN-y in the blood compartment versus EP compartments. Thus, antibodies that perform well for the detection of EPTB (using EP fluids) may perform sub-optimally when detecting IFN-y in the blood compartment (e.g. when used for the diagnosis of LTBI). This, together with the ‘trapping’ effect and the biological nature of the disease results in very high levels of IFN-y in the EP compartments, such that many studies have now shown that unstimulated IFN-y is an excellent biomarker for the diagnosis of active TB. The present invention relates to an antibody pair that may be used to specifically detect IFN-y with high sensitivity in the EP compartments versus the blood compartment.

[0013] SUMMARY OF THE INVENTION

[0014] Embodiments of the present invention relate to isolated antibodies that specifically bind to human interferon-y (IFN-y) and their applications in diagnosing active tuberculosis infections. The antibodies described herein exhibit high specificity and affinity towards IFN-y, enabling accurate detection of IFN-y in extrapulmonary fluid samples.

[0015] According to a first aspect of the present invention there is provided for an isolated antibody that specifically binds to human interferon-y (IFN-y). The antibody comprises or consists of a heavy chain variable region (VH) comprising or consisting of a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11 ; and a light chain variable region (VL) comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 13; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14.

[0016] Specifically, the antibody comprises or consists of a VH of SEQ ID NO: 1 and a VL of SEQ ID NO: 3.

[0017] In a preferred embodiment the antibody is a monoclonal antibody. More preferably, the antibody is humanized or chimeric.

[0018] In one embodiment of the invention the antibody is a full-length antibody. In an alternative embodiment of the invention the antibody is an antibody fragment. Preferably, the antibody fragment is selected from an Fv, single-chain Fv (scFv), Fab, Fab’, or (Fab’)2.

[0019] In a further embodiment the antibody is a detection antibody and may be conjugated either covalently or non-covalently to a detection label. Most preferably the detection label is selected from the group consisting or comprising of a colourimetric label, a fluorescent label, a chemiluminescent label, biotin, a phosphor-based label, a thermal-based label, an enzymatic label, a gold nanoparticle, a silver nanoparticle or a magnetic bead.

[0020] In a second aspect of the invention there is provided for an in vitro method of diagnosing an active tuberculosis infection in a subject comprising or consisting of the steps of firstly, providing an extrapulmonary fluid sample from a subject. In a preferred embodiment the extrapulmonary fluid sample is selected from pleural fluid, pericardial fluid or cerebrospinal fluid. Consequently, contacting the extrapulmonary fluid sample with a capture antibody which specifically binds to an epitope present on an interferon- y (IFN-y) polypeptide. Thereafter, contacting the combined extrapulmonary fluid sample and capture antibody with the detection antibody described above that specifically binds to a different epitope to the epitope bound by the capture antibody, present on the IFN-y polypeptide. Subsequently, detecting binding of the detection antibody to the different epitope of the IFN-y polypeptide. Finally, diagnosing the subject as having active tuberculosis, wherein binding of the IFN-y polypeptide by the capture antibody paired with binding of IFN-y by the detection antibody indicates an active tuberculosis in the subject.

[0021] In one embodiment of the invention the capture antibody comprises or consists of: a heavy chain variable region (VH) having a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 15; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 16; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17; and a light chain variable region (VL) comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 18; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 19; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

[0022] In one embodiment of the invention the capture antibody is a full-length antibody. In an alternative embodiment of the invention the capture antibody is an antibody fragment. Preferably, the antibody fragment is selected from an Fv, singlechain Fv (scFv), Fab, Fab’, or (Fab’)2.

[0023] In a third aspect of the invention there is provided for an immunoassay device, preferably a lateral flow immunoassay device, for detecting IFN-y in an extrapulmonary fluid sample, comprising or consisting of a mobile phase and a stationary phase. The mobile phase comprising or consisting of a detection antibody, as described herein, conjugated to or otherwise associated with a detection label, wherein the detection antibody is capable of binding specifically to an epitope present on an IFN-y polypeptide. The stationary phase comprising or consisting of a capture antibody, wherein the capture antibody is capable of binding specifically to a different epitope present on the IFN-y polypeptide. In one embodiment, the extrapulmonary fluid sample is selected from pleural fluid, pericardial fluid or cerebrospinal fluid. It will be appreciated that the presence of IFN-y in the extrapulmonary fluid sample results in both the detection antibody and capture antibody binding to the IFN-y polypeptide. In one embodiment the capture antibody is an antibody comprising a heavy chain variable region (VH) having a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 15; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 16; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17; and a light chain variable region (VL) comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 18; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 19; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

[0024] In one embodiment of the invention the capture antibody is a full-length antibody. In an alternative embodiment of the invention the capture antibody is an antibody fragment. Preferably, the antibody fragment is selected from an Fv, singlechain Fv (scFv), Fab, Fab’, or (Fab’)2.

[0025] BRIEF DESCRIPTION OF THE FIGURES

[0026] Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures:

[0027] Figure 1 : Detection of IFN-y dilution series from 300 - 0 pg / ml, using our specific monoclonal antibodies. The optimal antibody pair underwent repetitive indirect ELISAs in which IFN-y was plated in triplicate. The inter-variability of the assay was shown to be optimal as dilution series 1 - 4 are all within the same range from 300 - 0 pg / ml.

[0028] Figure 2: Limit of detection of monoclonal antibody pair is 7.4pg / ml and was calculated using average ODs in Table 1 and the following equation = STEYX (Standard error of the predicted y values for each x in a regression) / slope)*3.3. The limit of detection of the optimal antibody pair was determined by indirect ELISA. The calculation showed 7.4pg / ml as the limit of detection, proving a high affinity of the antibodies for IFN-y.

[0029] Figure 3: The ability of the optimal monoclonal antibody pair (Antrum-AB2 and Antrum-AB6) to sensitively and specifically differentiate TB from non-TB in EPTB compartments (pleural fluid). A subset of definite and non-TB pleural fluid samples were applied to indirect ELISAs consisting of the optimal antibody pair (Antrum-AB2 and Antrum-AB6). The interpolated concentration of the optimal antibody pair in each sample is depicted. These scatter plots show that the optimal pair is superior in differentiating definite and non-TB samples as compared to the commercial antibody pair (Figure 6).

[0030] Figure 4: The ability of the optimal monoclonal antibody pair (Antrum-AB2 and Antrum-AB6) to sensitively and specifically differentiate TB from non-TB in EPTB compartments (pericardial fluid). A subset of definite and non-TB pericardial fluid samples were applied to indirect ELISAs consisting of the optimal antibody pair (Antrum-AB2 and Antrum-AB6). The interpolated concentration of the optimal antibody pair in each sample is depicted. These scatter plots show that the optimal pair is superior in differentiating definite and non-TB samples as compared to the commercial antibody pair (Figure 6).

[0031] Figure 5: The ability of the optimal monoclonal antibody pair (Antrum-AB2 and Antrum-AB6) to sensitively and specifically differentiate TB from non-TB in EPTB compartments (cerebrospinal fluid). A subset of definite and non-TB cerebrospinal fluid samples were applied to indirect ELISAs consisting of the optimal antibody pair (Antrum-AB2 and Antrum-AB6). The interpolated concentration of the optimal antibody pair in each sample is depicted. These scatter plots show that the optimal pair is superior in differentiating definite and non-TB samples as compared to the commercial antibody pair (Figure 6).

[0032] Figure 6: Alternative commercial antibody pair (IFN gamma Monoclonal Antibody (NIB42), eBioscience™, Catalog Number 14-7318-85, Invitrogen and IFN gamma Monoclonal Antibody (4S.B3), Biotin, eBioscience™, Catalog Number 13- 7319-81 , Invitrogen), the performance of which is suboptimal in comparison to our optimal monoclonal antibody pair, the antibodies that abrogate heterophile interferences. A subset of definite and non-TB pleural, pericardial and cerebrospinal fluid samples were applied to indirect ELISAs consisting of the commercial antibody pair. The interpolated concentration of each sample is depicted for alternative antibody pair 1 . The scatter plots show that the commercial antibody pair was not as effective in differentiating definite and non-TB samples across as compared to the antibody pair of the present invention.

[0033] SEQUENCE LISTING

[0034] The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and the standard three letter abbreviations for amino acids. It will be understood by those of skill in the art that only one strand of each nucleic acid sequence is shown, but that the complementary strand is included by any reference to the displayed strand. In the accompanying sequence listing:

[0035] SEQ ID NO:1 - Amino acid sequence of the heavy chain of Antrum-AB2.

[0036] SEQ ID NO:2 - Nucleic acid sequence encoding the heavy chain of Antrum-

[0037] AB2. SEQ ID N0:3 - Amino acid sequence of the light chain of Antrum-AB2],

[0038] SEQ ID NO:4 - Nucleic acid sequence encoding the light chain of Antrum-AB2.

[0039] SEQ ID NO:5 - Amino acid sequence of the heavy chain of Antrum-AB6.

[0040] SEQ ID NO:6 - Nucleic acid sequence encoding the heavy chain of Antrum- AB6.

[0041] SEQ ID NO:7 - Amino acid sequence of the light chain of Antrum-AB6.

[0042] SEQ ID NO:8 - Nucleic acid sequence encoding the heavy chain of Antrum- AB6.

[0043] SEQ ID NO:9 - Antrum-AB2 CDR1 region of VH chain.

[0044] SEQ ID NO:10 - Antrum-AB2CDR2 region of VH chain.

[0045] SEQ ID NO:11 - Antrum-AB2 CDR3 region of VH chain.

[0046] SEQ ID NO:12 - Antrum-AB2 CDR1 region of VL chain.

[0047] SEQ ID NO:13 - Antrum-AB2 CDR2 region of VL chain.

[0048] SEQ ID NO:14 - Antrum-AB2 CDR3 region of VL chain.

[0049] SEQ ID NO:15 - Antrum-AB6 CDR1 region of VH chain.

[0050] SEQ ID NO:16 - Antrum-AB6 CDR2 region of VH chain.

[0051] SEQ ID NO:17 - Antrum-AB6 CDR3 region of VH chain.

[0052] SEQ ID NO:18 - Antrum-AB6 CDR1 region of VL chain.

[0053] SEQ ID NO:19 - Antrum-AB6 CDR2 region of VL chain.

[0054] SEQ ID NQ:20 - Antrum-AB6 CDR3 region of VL chain.

[0055] SEQ ID NO:21 - Amino acid sequence of INF-gamma (Uniprot Accession No. P01579).

[0056] SEQ ID NO:22 - Nucleic acid sequence encoding INF-gamma.

[0057] DETAILED DESCRIPTION OF THE INVENTION

[0058] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.

[0059] The invention as described should not be limited to the specific embodiments disclosed and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0060] As used throughout this specification and in the claims which follow, the singular forms “a”, “an” and “the” include the plural form, unless the context clearly indicates otherwise. The terminology and phraseology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms “comprising”, “containing”, “having” and “including” and variations thereof used herein, are meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0061] The following abbreviations are used in the specification and have the corresponding meanings provided:

[0062] ADA - Adenosine Deaminase

[0063] ELISA - Enzyme-Linked Immunosorbent Assay

[0064] EP - Extrapulmonary

[0065] EPTB - Extrapulmonary TB

[0066] HIV - Human Immunodeficiency Virus

[0067] IFN-y - Interferon Gamma

[0068] IGRA - Interferon Gamma Release Assay

[0069] LAM - Lipoarabinomannan

[0070] LTBI - Latent Tuberculosis Infection

[0071] M. tb - Mycobacterium tuberculosis

[0072] NAATs - Nucleic Acid Amplification Tests

[0073] TB - Tuberculosis

[0074] WHO - World Health Organization

[0075] The functional detection of IFN-y in specific human compartments (e.g. blood versus pleural or pericardial space) is influenced by several factors including pH, serosal permeability, inflammatory milieu that modulates protein folding, and accumulation of several molecules (antibodies and / or proteins and / or glycolipids) that may induce false positive or false negative results (including the heterophile effect). These modulatory factors are related to complex mechanisms that influence the transport of human proteins into and out of biological compartments (e.g. the blood versus pleural space etc.) including differential cytokine and chemokine induced permeability of serosal membranes, biomolecules that impact compartment-specific leucocyte trafficking, the mesothelial electrochemical cellular profile, the mesothelial non neuronal cholinergic system, differential activity of families of transporter systems, differential transcellular versus gap junction-mediated transport, release associated with lipid and other vesicles, differential mesothelial membrane-specific signalling cascades, dynamic protein translocons and their associated receptor systems, and differential molecule specific leakage across the serosal-blood barrier. Collectively these factors result in topologically distinct classes of proteins, including IFN-y, being differentially concentrated and differentially detectable in various compartments. Thus, antibodies that detect human IFN-y in one compartment like blood may perform differently in another compartment like the pleural space (both sensitivity and specificity of the biomarker of interest may be impacted). We have through screening and clinical validation elucidated an antibody sequence that enables highly sensitive and specific detection of human IFN-y in EP human compartments compared to the blood compartment. Furthermore, we have validated a method to detect IFN-y for the diagnosis of EPTB using the fluid from the EPTB compartment. Detection of unstimulated IFN-y in blood is too insensitive and nonspecific. This method does not require the use of blood, or overnight stimulation, or use of additional peptides or proteins. The method relies on a specific antibody sequence that mitigates the pitfalls of alternative pairs prone to heterophile and other effects that prevent highly sensitive and specific diagnosis of active EPTB. The use of these antibodies / sequences may be employed in any type of device or platform for the diagnosis of EPTB. The methods of the present invention do not diagnose LTBL

[0076] A high sensitivity (positive test in those that have the disease) is required to minimise false negativity. TB is a serious disease with high morbidity and a 10 to 20% mortality. Thus, it is imperative that cases are not missed. High sensitivity is therefore critical. The high sensitivity is also linked to negative predictive value (or negative likelihood ratio). This speaks to having a very high proportion of truly disease-free persons in those who test negative. Effectively this means that if the test is negative then there is a very high likelihood that the disease is not TB, and will point to other diagnoses resulting in the initiation of alternative diagnostic and / or therapeutic strategies. In a clinical condition like EPTB, a high specificity is also important so that individuals with other conditions or who are healthy are not falsely diagnosed with TB. A false diagnosis of TB will result in exposure of individuals to toxic therapy over a period of 6 months or more, exposure to life threatening adverse events such as drug- induced hepatitis, increased cost to the healthcare system, increased cost to the patient, and severe psychosocial trauma to the individual as TB is a heavily stigmatised disease. This also relates to a high positive predictive value (or positive likelihood ratio) where a positive test is highly predictive of the condition and the clinician can rely on the test result. These concepts are best encapsulated in the following real-world example: In a group of 1 ,000 individuals who are tested because of a suspicion (low or high) of tuberculosis and the disease prevalence is 10%, then there will be 100 individuals with EPTB. A test with 95% sensitivity will detect 95 out of the 100 patients but miss 5 individuals with EPTB. Thus, it is imperative that as few patients as possible be missed (high sensitivity). However, if the specificity is only 85%, for example, then there will be 150 individuals that will be falsely diagnosed with the disease and exposed to toxic therapy with attendant higher patient and health system levels costs. Thus, 245 patients (95+ 150) will be treated for TB. In this example the number of falsely treated patients far outnumber those who are treated for true disease because of the sub-optimal specificity. Thus, both high sensitivity and specificity are required and these metrics are further linked to a high negative predictive value (rule-out value) and high positive predictive value (rule-in value).

[0077] The term "antibody" includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for instance, bispecific antibodies and polyreactive antibodies), and antibody fragments. Accordingly, the term "antibody" as used in this specification includes, but is not limited to, any specific binding member, immunoglobulin class and / or isotype (for instance: lgG1 , lgG2, lgG3, lgG4, IgM, IgA, IgD, IgE and IgM) or an antibody fragment thereof.

[0078] It is understood in the art that an antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains which are inter-connected by disulfide bonds, or an antigen binding portion thereof. A heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH1 , CH2 and CH3). A light chain comprises a light chain variable region (VL) and a light chain constant region (CL). The variable regions of both the heavy and the light chains comprise framework regions (FR’s) and complementarity determining regions (CDR’s). The four FR’s are relatively conserved while the CDR regions (CDR1 , CDR2 and CDR3) comprise hypervariable regions. The FR’s and CDR’s are arranged from the NH2 terminus to the COOH terminus as follows: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. Further, the constant regions may mediate the binding of the immunoglobulin to host tissues or factors.

[0079] The ’’optimal antibody pair” refers to the monoclonal antibodies Antrum-AB2 and Antrum-AB6. Antrum-AB2 is an antibody comprising a heavy chain amino acid sequence of SEQ ID NO:1 and a light chain amino acid sequence of SEQ ID NO:3. Antrum-AB6 is an antibody comprising a heavy chain amino acid sequence of SEQ ID NO:5 and a light chain amino acid sequence of SEQ ID NO:7.

[0080] The “commercial antibody pair” refers to the Invitrogen™ antibodies having catalogue numbers 14-7318-85 and 13-7319-81.

[0081] Also included in the definition of "antibody" are chimeric antibodies, humanized antibodies, recombinant antibodies, human antibodies generated from a transgenic non-human animal and antibodies selected from libraries using enrichment technologies available to those skilled in the art.

[0082] The term "epitope" as used herein means any antigenic determinant on an antigen to which the paratope of an antibody can bind. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0083] An "antibody fragment" comprises a portion of an intact antibody, such as the antigen binding or variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFV fragments; diabodies; or linear antibodies.

[0084] Papain digestion of antibodies produces two identical "Fab" fragments or antigen-binding fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment of antibodies yields an F(ab')2 fragment that has two antigen-combining sites and which retains its ability to cross-link an antigen.

[0085] The term "Fv" refers to the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This fragment contains a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. The folding of these two domains results in the formation of six hypervariable loops (three loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable region (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind an antigen, although at a lower affinity. "Single-chain Fv" ("sFv" or "scFv") are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. The sFv polypeptide can further comprise a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding.

[0086] The “Fab” fragments contain the constant domain of the light chain and the first constant domain (CH1 ) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art. Variant antibodies also are included within the scope of the invention. Thus, variants of the sequences recited in the application also are included within the scope of the invention. Further variants of the antibody sequences having improved affinity can be obtained using methods known in the art and are included within the scope of the invention. Those skilled in the art can modify the amino acid sequences of a polypeptide utilizing recombinant methods and / or synthetic chemistry techniques for the production of variant polypeptides. For example, amino acid substitutions can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of the nucleotide sequence can be used to improve the efficiency of translation in expression systems for the production of the antibody. Such variant antibody sequences will share 70% or more (i.e., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater) sequence identity with the sequences recited in the application. Such sequence identity is calculated with regard to the full length of the sequence recited in the application.

[0087] The term “polypeptide” should be read to include “peptide” and “protein” and vice versa. As used herein, "polypeptide" refers to an amino acid sequence of a recombinant or non-recombinant polypeptide having an amino acid sequence of i) a native polypeptide, ii) a biologically active fragment of an polypeptide, or iii) a biologically active variant of a polypeptide.

[0088] As used herein, the term "isolated" means a nucleic acid or an antibody which has been removed from its natural environment. Nucleic acids, peptides and proteins which have been "isolated" thus include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and / or polypeptides.

[0089] The terms "subject" and "patient" are used interchangeably herein to mean any animal that may have a need for diagnosis with the antibodies described herein. Subjects and patients thus include, without limitation, primates (including humans), canines, felines, murines and other mammalian subjects. Preferably, the subjects are humans. As will be evidence from the context in which the term is used, subject and patient refer to a subject or patient which has IFN-y, which is detectable using the antibodies of the invention in the extrapulmonary compartments.

[0090] In another embodiment, the invention provides for isolated nucleic acids encoding the isolated anti-IFN-y antibodies, vectors and host cells containing the nucleic acids, and recombinant techniques for the production of the antibodies. The invention also provides for polynucleotide variants that encode the peptide sequences of the heavy and light chains of the anti-IFN-y antibodies. These polynucleotide variants may have at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or greater, sequence identity compared to a polynucleotide sequence of this invention, as determined using the methods described herein. Such contiguous sequences may encode a CDR sequence, or may encode a complete variable region. As is known in the art, a variable region sequence may be fused to any appropriate constant region sequence. One skilled in this art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

[0091] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to single-stranded or double-stranded RNA, DNA, or mixed polymers.

[0092] For recombinant production of the antibody, the nucleic acid encoding it is inserted into a vector for further cloning (amplification of the DNA) or for expression. DNA encoding the antibodies of the invention was isolated according to the methods set out in the Examples. Those of skill in the art will appreciate that many vectors are available for use in the recombinant production of antibodies. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0093] The anti-IFN-y antibodies of this invention may also be produced recombinantly, for instance, as a fusion polypeptide with a heterologous or homologous polypeptide, which include a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide, an immunoglobulin constant region sequence, and the like. A heterologous signal sequence selected preferably may be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native antibody signal sequence, the signal sequence is substituted by a prokaryotic signal sequence selected.

[0094] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0095] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard to the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

[0096] Suitable host cells for cloning or expressing the DNA are prokaryotic, yeast, or higher eukaryotic cells. Host cells transformed with the above-described expression or cloning vectors for anti-IFN-y antibody production are cultured in conventional nutrient media, modified as appropriate, for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Antibody compositions prepared from the cells can be purified using purification techniques known to those of ordinary skill in the art.

[0097] In another embodiment of the invention the antibodies of the invention may be used in a diagnostic composition. A diagnostic composition is a composition containing a compound or antibody, e.g., a labelled compound or antibody, that is used to detect the presence in a sample, such as a biological sample, of an antibody that binds to the compound or an immunogen, antigen or epitope that binds to the antibody; for instance, an anti-IFN-y antibody, antigen or epitope.

[0098] In another embodiment of the invention, an article of manufacture, such as a kit, containing materials useful for the treatment of the disorders described above is provided. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition is one or more antibodies in a formulation of the invention as described above. The label on, or associated with, the container indicates that the composition is used for treating the condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringers solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0099] The following examples are offered by way of illustration and not by way of limitation.

[0100] EXAMPLE 1

[0101] Transient expression of human interferon gamma into HEK cells

[0102] The amino acid sequence for IFN-y (Uniprot Accession No. P01579) (SEQ ID NO:21 ) was synthesised into cDNA (SEQ ID NO:22), codon optimised for human expression, and restriction enzyme sites were included to determine correct insertion into a transient vector. The cDNA underwent ligation and was transformed into E. coli. E. coli strains harbouring the expression vector were cultivated resulting in low- endotoxin plasmid preparation of expression vectors (expression culture). In parallel, HEK-INV cells were grown in serum-free suspension culture, the proprietary INVect transfection reagent (Abzena, Cambridge, UK) was added along with 1 L of the expression culture. The transfected HEK-INV cells were cultured until >60% cell viability was achieved after which the cells were harvested and pelleted by centrifugation.

[0103] Purification was performed by affinity chromatography, using TALON (Clonetech) affinity resin on column. The binding buffer consisted of 50 mM NaH2PO4, 300 mM NaCI pH 7.4 and the resulting IFN-y protein was eluted using Imidazol (250 nm). The eluted fractions underwent SDS-PAGE after which the pooled positive fractions were dialysed into PBS (pH 7.4).

[0104] The resulting isolated human-derived IFN-y underwent filtration (0.22 pm membrane), protein quantification (UV 280 nm), protein purity verification (SDS- PAGE), and endotoxin analysis (Charles River PTS endosafe system).

[0105] EXAMPLE 2

[0106] Antibody generation

[0107] The human-derived IFN-y (5 mgs) was used to immunise and boost 3 Balb / C mice. The mouse serum was applied to a direct ELISA in which biotinylated IFN-y (1 mg) was coated on a streptavidin microtitre plate to determine the mouse with the highest antibody titre.

[0108] Generation of hybridoma primary cultures involved the fusion of the spleen cells from the mouse with the highest antibody titre with the myeloma cell line SP2 / 0. The fusion products were plated into 4 X 96-well plates. The primary cell cultures were screened for specific IgG antibodies, using the same direct ELISA method described above. Hybridoma colonies (positive clones) that showed affinity towards IFN-y were transferred to 24-well plates and then propagated and retested for IFN-y specific antibodies.

[0109] Eight primary cultures with a high level of antibody titre were selected for further cloning by limited dilution. The cloned cultures were screened by direct ELISA as described above. Those identified as the highest positive clones were transferred to 24-well culture plates for cell propagation. The selected hybridoma cultures were retested by direct ELISA to determine antibody titre for final selection of the clones.

[0110] The four final antibody re-cloned cultures were propagated, the Ig class determined (all four antibodies were classified as lgG1 ) and underwent mycoplasma testing. The hybridoma cells were isolated and cryopreserved (3 X 106cells per vial), 3 vials per selected clone.

[0111] EXAMPLE 3

[0112] Stable cell line development

[0113] Hybridoma cell pellets were lysed, mRNA extracted, and the heavy chain and light chain variable region DNA was cloned into sequencing vectors for determination of the heavy and light chain DNA sequences.

[0114] The variable region sequences were codon optimised, synthesised and cloned into the Abzena (Cambridge, UK) expression vector in frame with the relevant heavy and light chain constant regions. The vector was transfected into composite CHO™ cell lines and up to four stable cell line pools were generated.

[0115] The stable pools were assessed for antibody productivity in small scale batch cultures from which 20 mgs of each monoclonal antibody was isolated via affinity chromatography using protein G columns.

[0116] EXAMPLE 4

[0117] Indirect ELISA

[0118] The functionality of each stable cell line-derived antibody was assessed using an indirect ELISA in which the capture antibody (Antrum-AB2 antibody) was coated on a 96-well microtiter plate at a concentration of 1 pg / ml (100 pl per well) in carbonate buffer overnight at 2-8°C. The coating buffer was removed and the plate was blocked with BSA in PBS (200 pl per well) for 1 hour and 30 minutes at room temperature. Following blocking, the plate was washed 4 times with wash buffer (200 pl per well) (Tween-20 in PBS). Human-derived IFN-y was diluted to a starting concentration of 300 pg / ml from which six serial dilutions (150, 75, 38, 19, 9.5, 4.8 pg / ml) were performed in assay buffer (BSA and Tween-20 in PBS). These dilutions, which made up the standard curve, were added in triplicate (100 pl per well) in the microtiter plate along with the final wells containing only assay buffer (100 pl per well) (blank). The standard curve was incubated for 1 hour and 30 minutes at room temperature. Following incubation, the plate was washed 4 times with wash buffer (200 pl per well) (Tween-20 in PBS).

[0119] The detection antibody (Antrum-AB6 antibody) was biotinylated using NHS- Sulfo EZ Link kit and corresponding protocol; Thermoscientific) was added to the plate at a concentration of 4.5 pg / ml (100 pl / well) in detection diluent (BSA in PBS) and incubated in the dark (light sensitive step) at room temperature for 1 hour. Following incubation, the plate was washed 4 times with wash buffer (200 pl per well) (Tween- 20 in PBS). Strep-HRP (1 :200 dilution, Pierce high sensitivity, Thermoscientific) in Strep-HRP diluent (BSA in PBS) was added to the plate (100 pl per well) and incubated in the dark (light sensitive step) at room temperature for 30 minutes. Following incubation, the plate was washed 4 times with wash buffer (200 pl per well) (Tween- 20 in PBS). Chromogen (TMB and Peroxidase at 1 :1 ratio, Thermoscientific) was added to the wells (100 pl per well) and incubated in the dark (light sensitive step) at room temperature for 30 minutes. Following incubation, STOP solution (Sulphuric acid) was added to the plate (100 pl per well) and the plate is read 450 and 595 nm (SmartReader, Accuris, USA).

[0120] The resulting absorbance readings are captured from which a standard curve is determined (Figure 1 ). This was performed in GraphPad Prism (Version 5) and Microsoft Excel.

[0121] Limit of detection

[0122] To determine the limit of detection an indirect ELISA was performed as described above after which the limit of detection was calculated using the average absorbance readings and below mentioned equation = STEYX (Standard error of the predicted y values for each x in a regression) / slope)*3.3 (Figure 2). Table 1 : Absorbance readings of standard curve used to determine the limit of detection of monoclonal antibody pair.

[0123] EXAMPLE 5

[0124] Clinical sample performance

[0125] Pleural, pericardial and cerebrospinal fluid samples were collected over a series of clinical trials by the Centre for Lung Infection and Immunity (CLII) at the University of Cape Town, South Africa. The samples were categorised according to the following definitions below:

[0126] 1. Reference standard for tuberculosis: microbiological confirmation of tuberculosis and / or histological features pathognomonic of tuberculosis together with a compatible baseline clinical picture with improvement on anti-TB treatment.

[0127] 2. Definite-TB: was defined as microbiological confirmation of tuberculosis (at least one positive Mycobacterium tuberculosis culture in liquid broth or Xpert positivity (using either fluid or biopsy)] and / or histological features pathognomonic of tuberculosis (caseating granulomatous inflammation or granulomas with or without AFB) with improvement on anti-TB treatment (positive histology or microbiology (e.g. culture or Xpert) from an alternative site e.g. lymph node, or a positive Alere urine LAM test, with improvement on TB treatment would also count as microbiological confirmation of TB).

[0128] 3. Non-TB: patients for whom no microbiological or histological evidence of M. tuberculosis could not be confirmed or found, and / or for whom an alternative diagnosis is available, and / or there was intervention-free resolution of symptoms and signs. These patients at presentation and on follow-up would not have received anti-TB treatment.

[0129] A selection of these samples were run on the indirect ELISA as described above in which along with the standard curve samples were added in duplicate. The sample concentrations of IFN-y (pg / ml) were interpolated from the resulting standard curve. The performance of each antibody pair around a pre-determined cut-off for each extrapulmonary TB fluid (pleural TB = 20.5 pg / ml, pericardial TB = 10 pg / ml and TB meningitis = 13 pg / ml) was determined using contingency tables (GraphPad Prism, Version 5). The antibody pairs comprised:

[0130] 1 . The optimal antibody pair (Antrum-AB2 antibody and Antrum-AB6 antibody) (Figure 3, 4 and 5), and

[0131] 2. A commercial antibody pair (IFN gamma Monoclonal Antibody (NIB42), eBioscience™, Catalog Number 14-7318-85, Invitrogen and IFN gamma Monoclonal Antibody (4S.B3), Biotin, eBioscience™, Catalog Number 13- 7319-81 , Invitrogen) (Figure 6).

[0132] Table 2: Performance of optimal monoclonal antibody pair in pleural fluid, pericardial fluid and CSF compared to a commercially available antibody pair. The optimal antibody pair showed superior performance in distinguishing active TB from non-TB in comparison to the commercial monoclonal antibody pair. REFERENCES

[0133] Christopher DJ, Esmail A, Scott AJ, et al. Performance of Unstimulated IFN-y (IRISA-TB) for Pleural Tuberculosis: A Prospective Study in South Africa and India, Open Forum Infectious Diseases 2024, Volume 1 1 , Issue 10.

[0134] Dheda K, van Zyl-Smit RN, Sechi L, etal. Utility of quantitative T-cell responses versus unstimulated interferon-gamma for the diagnosis of pleural tuberculosis. Eur Respir J 2009, 34(5):1 118-1126.

[0135] Fan L, Chen Z, Hao XH et al. Interferon-gamma release assays for the diagnosis of extrapulmonary tuberculosis: a systematic review and meta-analysis. FEMS Immunology and Medical Microbiology, 2012, June; 65: 456-466.

[0136] Kricka, L. Human anti-animal antibody interferences in immunological says. 1999. Clin Chem 45:942-956.

[0137] Light RW. Update on tuberculous pleural effusion. Respirology 2010, 15(3):451-458.

[0138] Meldau R, Peter J, Theron G, et al. Comparison of same day diagnostic tools including Gene Xpert and unstimulated IFN-gamma for the evaluation of pleural tuberculosis: a prospective cohort study. BMC Pulm Med 2014; 14:58.

[0139] Meldau R, Randall PJ, Pooran A, Limberis J, Makambwa E, Dhansay M, Esmail A and Dheda K. Same day tools, including Xpert Ultra and unstimulated IFN-y, for the rapid diagnosis of pleural tuberculosis. Journal of Clinical Microbiology, 2019, July; doi:10.1128 / JCM.00614-19.

[0140] Metcalfe JZ, Everett CK, Steingart KR et al. Interferon-c Release Assays for Active Pulmonary Tuberculosis Diagnosis in Adults in Low- and Middle-Income Countries: Systematic Review and Meta-analysis. J I D, 201 1 :204 (Suppl 4).

[0141] Pandie S, Peter JG, Kerbelker ZS, et al. Diagnostic accuracy of quantitative PCR (Xpert MTB / RIF) for tuberculous pericarditis compared to adenosine deaminase and unstimulated interferon-gamma in a high burden setting: a prospective study. BMC Med 2014, 12: 101.

[0142] Patel VB, Singh R, Dheda K, et al. Comparative Utility of Cytokine Levels and Quantitative RD-1 -Specific T Cell Responses for Rapid Immunodiagnosis of Tuberculous Meningitis. Journal of Clinical Microbiology 2011 , 49(11 ): 3971 -3976.

[0143] Randall P, Esmail A, Wilson L, etal. GeneXpert MTB / RIF Ultra vs Unstimulated Interferon y (IRISA-TB) for the Diagnosis of Tuberculous Pericarditis in a Tuberculosis- Endemic Setting. Open Forum Infect Dis. 2024 20 ; 11 (3).

[0144] Randall P, Mutsvangwa J, Nliwasa M, et al. Utility of Cerebrospinal Fluid Unstimulated Interferon-Gamma (IRISA-TB) as a Sarne-Day Test for Tuberculous Meningitis in a Tuberculosis-Endemic, Resource-Poor Setting, Open Forum Infectious Diseases, 2024, Volume 11 , Issue 9.

[0145] WHO Policy Statement. Use of alternative interferon-gamma release assays for the diagnosis of TB infection: WHO policy statement. Geneva: World Health Organization; 2022. Licence: CC BY-NC-SA 3.0 IGO.

Claims

CLAIMS1 . An isolated antibody that specifically binds to human interferon-y (IFN- y), wherein the antibody comprises: a heavy chain variable region (VH) comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11 ; and a light chain variable region (VL) comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14.

2. The antibody of claim 1 , wherein the antibody comprises a VH of SEQ ID NO: 1 and a VL of SEQ ID NO: 3.

3. The antibody of claim 1 or 2, wherein the antibody is a monoclonal antibody.

4. The antibody of any one of claims 1 to 3, wherein the antibody is humanized or chimeric.

5. The antibody of any one of claims 1 to 4, wherein the antibody is a full- length antibody.

6. The antibody of any one of claims 1 to 4, wherein the antibody is an antibody fragment.

7. The antibody of claim 6, wherein the antibody fragment is selected from an Fv, single-chain Fv (scFv), Fab, Fab’, or (Fab’)2.

8. The antibody of any one of claims 1 to 7, wherein the antibody is conjugated covalently or non-covalently to a detection label.

9. The antibody of claim 8, wherein the detection label is selected from the group consisting of colourimetric labels, fluorescent labels, chemiluminescent labels, biotin, phosphor-based labels, thermal-based labels, enzymatic labels, gold nanoparticles, silver nanoparticles and magnetic beads.

10. An in vitro method of diagnosing an active tuberculosis infection in a subject comprising:(i) providing an extrapulmonary fluid sample from the subject, wherein the extrapulmonary fluid sample is selected from pleural fluid, pericardial fluid or cerebrospinal fluid;(ii) contacting the extrapulmonary fluid sample with a capture antibody which specifically binds to an epitope present on an IFN-y polypeptide;(iii) contacting the sample of step (ii) with a detection antibody that specifically binds to a different epitope to the epitope bound by the capture antibody, present on the IFN-y polypeptide, wherein the detection antibody is the antibody of any one of claims 1 to 9;(iv) detecting binding of the detection antibody to the different epitope of the IFN-y polypeptide; and(v) diagnosing the subject as having an active tuberculosis infection, wherein binding of the IFN-y polypeptide by the capture antibody and binding of the IFN-y polypeptide by the detection antibody indicates an active tuberculosis infection in the subject.1 1 . The method of claim 10, wherein the capture antibody comprises: a heavy chain variable region (VH) comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 15; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 16; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17; anda light chain variable region (VL) comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 18; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 19; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

12. The method of claim 10 or 11 , wherein the capture antibody is a full- length antibody.

13. The method of claim 10 or 11 , wherein the capture antibody is an antibody fragment.

14. The method of claim 13, wherein the antibody fragment is selected from an Fv, single-chain Fv (scFv), Fab, Fab’, or (Fab’)2.

15. An immunoassay device for detecting IFN-y in an extrapulmonary fluid sample, comprising:(i) a mobile phase comprising a detection antibody conjugated to or otherwise associated with a detection label, wherein the detection antibody is the antibody of any one of claims 1 to 9, and wherein the detection antibody is capable of binding specifically to an epitope present on an IFN-y polypeptide; and(ii) a stationary phase comprising a capture antibody, wherein the capture antibody is capable of binding specifically to a different epitope present on the IFN-y polypeptide, wherein the extrapulmonary fluid sample is selected from pleural fluid, pericardial fluid or cerebrospinal fluid, and wherein the presence of IFN-y in the extrapulmonary fluid sample results in both the detection antibody and capture antibody binding to the IFN-y polypeptide.

16. The immunoassay device of claim 15, wherein the capture antibody comprises: a heavy chain variable region (VH) comprising:a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 15; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 16; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17; and a light chain variable region (VL) comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 18; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 19; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

17. The immunoassay device of claim 15 or 16, wherein the capture antibody is a full-length antibody.

18. The immunoassay device of claim 15 or 16, wherein the capture antibody is an antibody fragment.

19. The immunoassay device of claim 18, wherein the antibody fragment is selected from an Fv, single-chain Fv (scFv), Fab, Fab’, or (Fab’)2.

Images