Anti-ifn-gamma antibodies

Abstract

The present invention relates to an isolated antibody which specifically binds human interferon-gamma (IFN-gamma) and its use in the diagnosis of active tuberculosis infection. The antibody described herein exhibits high specificity and affinity for IFN-gamma, enabling accurate detection of IFN-gamma in extra-pulmonary liquid samples. The present invention also relates to the use of the antibody in a method for in vitro diagnosis of active TB, and to an immunoassay device comprising the antibody.

Description

Background Technology

[0001] Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis). Mycobacterium tuberculosis Tuberculosis (TB) is caused by a chronic granulomatous infection characterized by oliguria and caseous necrosis. The primary organ affected is the lungs (approximately 85% of cases). However, in approximately 15% to 35% of individuals (depending on several factors including genetic background, nationality, and HIV status), TB can affect organs outside the lungs, a condition known as extrapulmonary TB (EPTB). In many countries, the most common form of EPTB is pleural TB (causing inflammation of the external lung lining, which traps fluid between the lungs and the chest wall). Other forms of EPTB include TB pericarditis and TB peritonitis. The presence of these three conditions collectively leads to TB serositis (inflammation of the serosals; pleura, pericardium, or peritoneum). A different form of EPTB is TB meningitis, which causes inflammation of the meninges of the brain and spinal cord. Infections in all these sites are characterized by oliguria-bacillaryinfection of fluid within these compartments or body cavities. The disease burden is often below the detection limits of existing nucleic acid amplification tests (NAATs), making immunodiagnosis an attractive proposal.

[0002] Although traditionally tuberculosis bacteria were found in sputum ( M. tuberculosis While pulmonary TB is often used as evidence, the diagnosis of these forms of EPTB (as described above) is made by detecting biological or genomic material in the EP fluid. Therefore, traditionally, individuals with indicative clinical presentations and basic radiological findings (e.g., chest X-ray) undergo fluid aspiration from the pleural cavity, pericardium, peritoneum, or subarachnoid space (cerebrospinal fluid). However, confirming the diagnosis of TB in these fluids is challenging and problematic. Smear microscopy (observing the organism under a microscope) is typically feasible only in <5% of patients, and cultures are usually positive only in about 30% to 40% of patients (given the sparsely bacterial nature of the disease). The use of nucleic acid amplification tests is also problematic. For example, in pleural TB, the commonly used NAAT (e.g., GeneXpertMTB / RIF Ultra; Cepheid) has only a sensitivity of 30% to 40% (Light (2010), Meldau et al. (2014), and Meldau et al. (2019)). Although more nonspecific biomarkers, such as adenosine deaminase (ADA), have been used, this biomarker has suboptimal specificity in pleural TB (~80%), pericardial TB (~70%), and even worse in cerebrospinal fluid. Its negative predictive value is also poor.

[0003] An alternative biomarker is unstimulated interferon-gamma. CD4+ and CD8+ T cells produce large amounts of interferon-gamma upon antigen presentation. This biomarker is commonly used to diagnose latent TB infection (LTBI). In fact, tests like T-SPOTTB 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, poor specificity (due to background LTBI and exposure to environmental mycobacteria) means that this test is unsuitable for the diagnosis of active TB, and for these reasons, the WHO recommends against its use in the diagnosis of active TB (WTO Policy Statement (2022)). Therefore, in summary, IGRA is used for the diagnosis of LTBI but not for active TB involving serous compartments (Metcalfe et al. (2011)). Consequently, these types of tests perform poorly when used to assess pleural TB (Fan et al (2012)).

[0004] An alternative to IGRA is the direct assessment of untreated interferon-gamma in extrapulmonary fluid. Functional detection of IFN-γ in specific human compartments (e.g., blood and pleural or pericardial spaces) is affected by several factors, including pH that regulates protein folding, serous membrane permeability, inflammatory factors, etc., and the accumulation of several molecules (antibodies and / or proteins and / or glycolipids) can bind to antibodies, leading to erroneous blocking (false negatives) or enhancement (false positives) of IFN-γ detection (including heterophile effects).

[0005] The factors described above are related to complex mechanisms influencing the transport of proteins in and out of biological compartments (e.g., blood vs. pleural cavity). Effects on membrane permeability include, but are not limited to: different cytokines and chemokines that induce serous membrane permeability; biomolecules that affect compartment-specific leukocyte transport; mesothelial electrochemical cellular profiles; mesothelial nonneuronal cholinergic systems; different activities of transport system families; different transcellular and gap junction-mediated transport; release associated with lipids and other vesicles; different mesothelial membrane-specific signaling cascades; dynamic protein translocitons and their associated receptor systems; and different molecular-specific leakage at the serous membrane-blood barrier.

[0006] In summary, the aforementioned factors lead to the "capture" of different classes of proteins (including IFN-γ) on the topology and their differential detection in various compartments (e.g., the pleural and blood compartments). Therefore, an antibody that can detect human IFN-γ in one compartment (e.g., blood) may behave differently in another compartment (e.g., the pleural cavity) (potentially affecting the sensitivity and specificity of the biomarker of interest).

[0007] Interestingly, while other diseases such as community-acquired pneumonia, various infections, autoimmune diseases, and malignant tumors can also drive the production of some interferon-gamma, the amount produced is much lower compared to TB, and functional "capture" of interferon-gamma exists in the relevant compartments. Several studies have shown that unstimulated interferon-gamma is a good biomarker for diagnosing active tuberculosis (Meldau). et al. (2014), Meldau et al. (2019), Dheda et al. (2009), Pandie et al. (2014) and Patel et al. (2011)).

[0008] The inventors have elucidated, through screening and clinical validation, an antibody pair capable of detecting human IFN-γ with high sensitivity and specificity in the human EP compartment compared to the blood compartment. Furthermore, the inventors have validated a method for diagnosing EPTB using liquid detection of IFN-γ from the EPTB compartment.

[0009] The method of this invention does not require the use of blood, overnight stimulation, or additional peptides or proteins. This method relies on specific antibody pairs that mitigate the deficiencies of alternative antibody pairs, which are susceptible to heterophile and other effects that impair high sensitivity and specificity in the diagnosis of active EPTB. Strategies to mitigate heterophile include: the use of blocking agents (such as inactivated serum) in the assay buffer, pre-incubation of clinical samples with antibody fragments, removal of the Fc region of the antibody during development, and / or humanization of the antibody (Kricka (1999)). The antibody pairs covered employ a humanization strategy, which involves inoculating mice with human-derived IFN-γ and developing downstream stable cell lines (sequencing regions into the human frame). The antibodies of this invention can be used with any type of device or platform for diagnosing EPTB. This method is not intended for the diagnosis of LTBI.

[0010] In summary, several local factors (including pH, inflammatory environment, heterophilic molecules, etc.) lead to differences in the sensitivity of human IFN-γ detection in the blood compartment and the EP compartment. Therefore, antibodies that perform well in detecting EPTB (using EP liquid) may be suboptimal in detecting IFN-γ in the blood compartment (e.g., when used for the diagnosis of LTBI). This, along with the “capture” effect and the biological nature of the disease, results in very high IFN-γ levels in the EP compartment, leading many studies to now demonstrate that unstimulated IFN-γ is an excellent biomarker for diagnosing active TB. This invention relates to an antibody pair, relative to the blood compartment, for the highly sensitive and specific detection of IFN-γ in the EP compartment. Summary of the Invention

[0011] Embodiments of this invention relate to isolated antibodies that specifically bind to human interferon-γ (IFN-γ) and their application in the diagnosis of active tuberculosis infection. The antibodies described herein exhibit high specificity and affinity for IFN-γ, enabling accurate detection of IFN-γ in extrapulmonary fluid samples.

[0012] According to a first aspect of the invention, an isolated antibody that specifically binds to human interferon-γ (IFN-γ) is provided. The antibody comprises or consists of: a heavy chain variable region (VH) comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 15, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 16, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17; and a light chain variable region (VL) comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 18, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

[0013] Specifically, the antibody comprises VH of SEQ ID NO: 5 and VL of SEQ ID NO: 7, or is composed of VH of SEQ ID NO: 5 and SEQ ID NO: 7.

[0014] In a preferred embodiment, the antibody is a monoclonal antibody. More preferably, the antibody is humanized or chimeric.

[0015] 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 Fv, single-chain Fv (scFv), Fab, Fab', or (Fab')2.

[0016] In another embodiment, the antibody is a detection antibody and may be covalently or non-covalently conjugated to a detection label. Most preferably, the detection label is selected from or includes the group consisting of: colorimetric labels, fluorescent labels, chemiluminescent labels, biotin, phosphor-based labels, thermal-based labels, enzyme labels, gold nanoparticles, silver nanoparticles, or magnetic beads.

[0017] In a second aspect of the invention, a method for in vitro diagnosis of active tuberculosis infection in a subject is provided, comprising or consisting of the following steps: First, an extrapulmonary fluid sample from the subject is provided. In a preferred embodiment, the extrapulmonary fluid sample is selected from pleural fluid, pericardial fluid, or cerebrospinal fluid. The extrapulmonary fluid sample is then contacted with a capture antibody that specifically binds to an epitope present on an interferon-γ (IFN-γ) polypeptide. Subsequently, the bound extrapulmonary fluid sample and the capture antibody are contacted with a detection antibody described above, wherein the detection antibody specifically binds to an epitope on the IFN-γ polypeptide that is different from the epitope bound by the capture antibody. Subsequently, the binding of the detection antibody to the different epitope of the IFN-γ polypeptide is detected. Finally, the subject is diagnosed with active tuberculosis, wherein the combination of the binding of the capture antibody to the IFN-γ polypeptide and the binding of the detection antibody to IFN-γ indicates that the subject has active tuberculosis.

[0018] In one embodiment of the present invention, the capture antibody comprises or is composed of the following: The heavy chain variable region (VH) comprises CDR-H1 including the amino acid sequence of SEQ ID NO: 9, CDR-H2 including the amino acid sequence of SEQ ID NO: 10, and CDR-H3 including the amino acid sequence of SEQ ID NO: 11; and the light chain variable region (VL) comprises CDR-L1 including the amino acid sequence of SEQ ID NO: 12, CDR-L2 including the amino acid sequence of SEQ ID NO: 13, and CDR-L3 including the amino acid sequence of SEQ ID NO: 14.

[0019] In one embodiment of the invention, the capturing antibody is a full-length antibody. In an alternative embodiment of the invention, the capturing antibody is an antibody fragment. Preferably, the antibody fragment is selected from Fv, single-chain Fv (scFv), Fab, Fab', or (Fab')2.

[0020] In a third aspect of the invention, an immunoassay apparatus, preferably a lateral flow immunoassay apparatus, is provided for detecting IFN-γ in extrapulmonary fluid samples, comprising a mobile phase and a stationary phase, or consisting of a mobile phase and a stationary phase. The mobile phase comprises or consists of a detection antibody as described herein, wherein the detection antibody is conjugated to or otherwise associated with a detection label, wherein the detection antibody is capable of specifically binding to an epitope present on the IFN-γ polypeptide. The stationary phase comprises or consists of a capture antibody, wherein the capture antibody is capable of specifically binding to different epitopes present on the IFN-γ polypeptide. In one embodiment, the extrapulmonary fluid sample is selected from pleural fluid, pericardial fluid, or cerebrospinal fluid. It should be understood that the presence of IFN-γ in the extrapulmonary fluid sample causes both the detection antibody and the capture antibody to bind to the IFN-γ polypeptide.

[0021] In one embodiment, the capture antibody is an antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) has 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, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; and the light chain variable region (VL) comprises: 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.

[0022] In one embodiment of the invention, the capturing antibody is a full-length antibody. In an alternative embodiment of the invention, the capturing antibody is an antibody fragment. Preferably, the antibody fragment is selected from Fv, single-chain Fv (scFv), Fab, Fab', or (Fab')2. Attached Figure Description

[0023] Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures: Figure 1 We used our specific monoclonal antibody to detect IFN-γ dilutions at 300-0 pg / ml. For the optimal antibody pair, we performed a repeat indirect ELISA, in which IFN-γ was plated in triplicate. When dilutions 1-4 were all within the same range of 300-0 pg / ml, it indicated that the assay had optimal inter-variability.

[0024] Figure 2 The detection limit of the monoclonal antibody pair was 7.4 pg / ml, calculated using the average OD in Table 1 and the following equation: STEYX (standard error of the predicted y value for each x in the regression) / slope) * 3.3. The detection limit of the optimal antibody pair was determined by indirect ELISA. The calculation showed a detection limit of 7.4 pg / ml, demonstrating high affinity of the antibody for IFN-γ.

[0025] Figure 3 The ability of the optimal monoclonal antibody pairs (Antrum-AB2 and Antrum-AB6) to sensitively and specifically distinguish between TB and non-TB in the EPTB compartment (pleural effusion). A subset of pleural effusion samples with confirmed TB and non-TB were applied to an indirect ELISA consisting of the optimal antibody pairs (Antrum-AB2 and Antrum-AB6). The interpolated concentrations of the optimal antibody pairs in each sample were plotted. These scatter plots show that the optimal antibody pairs are superior to commercially available antibody pairs in distinguishing between confirmed TB and non-TB samples. Figure 6 ).

[0026] Figure 4 The ability of the optimal monoclonal antibody pair (Antrum-AB2 and Antrum-AB6) to sensitively and specifically distinguish between TB and non-TB in the EPTB compartment (pericardial fluid). A subset of pericardial fluid samples with confirmed TB and non-TB were applied to an indirect ELISA consisting of the optimal antibody pair (Antrum-AB2 and Antrum-AB6). The interpolated concentrations of the optimal antibody pair in each sample were plotted. These scatter plots show that the optimal antibody pair is superior to commercially available antibody pairs in distinguishing between confirmed TB and non-TB samples. Figure 6 ).

[0027] Figure 5 The ability of the optimal monoclonal antibody pair (Antrum-AB2 and Antrum-AB6) to sensitively and specifically distinguish between TB and non-TB in the EPTB compartment (cerebrospinal fluid). A subset of cerebrospinal fluid samples diagnosed with TB and non-TB were applied to an indirect ELISA consisting of the optimal antibody pair (Antrum-AB2 and Antrum-AB6). The interpolated concentrations of the optimal antibody pair in each sample were plotted. These scatter plots show that the optimal antibody pair is superior to commercially available antibody pairs in distinguishing between samples diagnosed with TB and non-TB. Figure 6 ).

[0028] Figure 6 Alternative commercial antibody pairs (IFN-γ monoclonal antibody (NIB42), eBioscience™, catalog number 14-7318-85, Invitrogen and IFN-γ monoclonal antibody (4S.B3), Biotin, eBioscience™, catalog number 13-7319-81, Invitrogen) are suboptimal in performance compared to our best monoclonal antibody pair (antibodies that eliminate heterophile interference). A subset of pleural effusion, pericardial effusion, and cerebrospinal fluid samples, both confirmed and non-TB, were applied to an indirect ELISA consisting of commercially available antibody pairs. The interpolated concentrations for each sample of alternative antibody pair 1 are plotted. Scatter plots show that the commercially available antibody pairs are less effective at distinguishing between confirmed and non-TB samples compared to the antibody pairs of the present invention.

[0029] sequence list The nucleic acid and amino acid sequences listed in the attached sequence listing are represented using standard letter abbreviations for nucleotide bases and standard three-letter abbreviations for amino acids. Those skilled in the art will understand that only one strand is shown for each nucleic acid sequence, but the complementary strand is included herein by reference to any strand shown. In the attached sequence listing: SEQ ID NO: 1 — Amino acid sequence of the heavy chain of Antrum-AB2.

[0030] SEQ ID NO: 2 — Nucleic acid sequence encoding the heavy chain of Antrum-AB2.

[0031] SEQ ID NO: 3 — Amino acid sequence of the light chain of Antrum-AB2.

[0032] SEQ ID NO: 4 — Nucleic acid sequence encoding the light chain of Antrum-AB2.

[0033] SEQ ID NO: 5 — Amino acid sequence of the heavy chain of Antrum-AB6.

[0034] SEQ ID NO: 6 — Nucleic acid sequence encoding the heavy chain of Antrum-AB6.

[0035] SEQ ID NO: 7 — Amino acid sequence of the light chain of Antrum-AB6.

[0036] SEQ ID NO: 8 — Nucleic acid sequence encoding the heavy chain of Antrum-AB6.

[0037] SEQ ID NO: 9 — Antrum-AB2 CDR1 region of the VH chain.

[0038] SEQ ID NO: 10 — Antrum-AB2 CDR2 region of VH chain.

[0039] SEQ ID NO: 11 — Antrum-AB2 CDR3 region of VH chain.

[0040] SEQ ID NO: 12 — Antrum-AB2 CDR1 region of VL chain.

[0041] SEQ ID NO: 13 — Antrum-AB2 CDR2 region of VL chain.

[0042] SEQ ID NO: 14 — Antrum-AB2 CDR3 region of VL chain.

[0043] SEQ ID NO: 15 — Antrum-AB6 CDR1 region of the VH chain.

[0044] SEQ ID NO: 16 — Antrum-AB6 CDR2 region of VH chain.

[0045] SEQ ID NO: 17 — Antrum-AB6 CDR3 region of VH chain.

[0046] SEQ ID NO: 18 — Antrum-AB6 CDR1 region of VL chain.

[0047] SEQ ID NO: 19 — Antrum-AB6 CDR2 region of VL chain.

[0048] SEQ ID NO: 20 — Antrum-AB6 CDR3 region of VL chain.

[0049] SEQ ID NO: 21 — Amino acid sequence of INF-γ (Uniprot registry number P01579).

[0050] SEQ ID NO: 22 — Nucleic acid sequence encoding INF-γ. Detailed Implementation

[0051] The present invention will now be described more fully with reference to the accompanying drawings, which illustrate some, but not all, embodiments of the invention.

[0052] The invention described herein 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 terminology is used herein, it is used in a general and descriptive sense only and is not intended to be limiting.

[0053] As used throughout this specification and the appended claims, the singular forms “a,” “an,” and “the” include the plural forms, unless the context clearly indicates otherwise.

[0054] The terminology and wording used herein are for descriptive purposes and should not be considered restrictive. The terms “comprising,” “containing,” “having,” and “including,” and their variations, as used herein, are intended to cover the items listed thereafter, their equivalents, and additional items.

[0055] The following abbreviations are used in this instruction manual and have the corresponding meanings provided: ADA-adenosine deaminase ELISA (Enzyme-Linked Immunosorbent Assay) EP-extrapulmonary EPTB - Extrapulmonary TB HIV (Human Immunodeficiency Virus) IFN-γ-interferon γ IGRA-interferon-γ release assay LAM-Lipoarabinomannan LTBI - Latent tuberculosis infection M. tb -Mycobacterium tuberculosis NAAT - Nucleic Acid Amplification Test TB - Tuberculosis WHO (World Health Organization) Functional detection of IFN-γ in specific human compartments (e.g., blood vs. pleural or pericardial space) is influenced by several factors, including pH, serous membrane permeability, the inflammatory environment that regulates protein folding, and the accumulation of several molecules (antibodies and / or proteins and / or glycolipids) that may induce false positive or false negative results (including heterophile effects). These regulatory factors are associated with complex mechanisms affecting the transport of human proteins into and out of biological compartments (e.g., blood vs. pleural space), including: different cytokines and chemokines that induce serous membrane permeability; biomolecules that affect compartment-specific leukocyte transport; mesothelial electrochemical cellular profiles; mesothelial nonneuronal cholinergic systems; different activities of transport system families; different transcellular and gap junction-mediated transport; release associated with lipids and other vesicles; different mesothelial membrane-specific signaling cascades; dynamic protein translocitons and their associated receptor systems; and different molecular-specific leakage at the serous membrane-blood barrier. In summary, these factors lead to the differential concentration and detection of different classes of proteins (including IFN-γ) in different compartments on the topology. Therefore, an antibody that detects human IFN-γ in one compartment (e.g., blood) may behave differently in another compartment (e.g., the pleural cavity) (potentially affecting the sensitivity and specificity of the biomarker of interest). We have elucidated an antibody sequence through screening and clinical validation that can detect human IFN-γ in the human EP compartment with high sensitivity and specificity compared to the blood compartment. Furthermore, we have validated a method for diagnosing EPTB using liquid detection of IFN-γ from the EPTB compartment. Detecting unstimulated IFN-γ in blood is too insensitive and nonspecific. This method does not require the use of blood, overnight stimulation, or additional peptides or proteins. This method relies on a specific antibody sequence that mitigates the deficiencies of alternative antibody pairs, which are susceptible to heterophile and other effects that hinder high sensitivity and specificity in the diagnosis of active EPTB. The use of these antibodies / sequences can be applied to any type of device or platform for diagnosing EPTB. The method of this invention is not intended for the diagnosis of LTBI.

[0056] High sensitivity (positive test in patients with the disease) is required to minimize false negatives. Tuberculosis is a serious disease with a high morbidity and a mortality rate of 10% to 20%. Therefore, it is imperative not to miss any cases. Thus, high sensitivity is crucial. High sensitivity is also associated with negative predictive value (or negative likelihood ratio). This indicates that the proportion of truly disease-free individuals among those who test negative is very high. In practice, this means that if the test is negative, the likelihood that the disease is not TB is very high, and it will point to an alternative diagnosis, prompting the initiation of alternative diagnostic and / or treatment strategies. High specificity is also important in clinical conditions like EPTB, ensuring that individuals with other conditions or who are healthy are not misdiagnosed with TB. A misdiagnosis of TB will result in an individual being exposed to toxic treatments for 6 months or longer, exposure to life-threatening adverse events such as drug-induced hepatitis, increased costs to the healthcare system, increased costs to patients, and, because TB is a severely stigmatized disease, it will also cause severe psychosocial trauma to the individual. This also involves high positive predictive value (or positive likelihood ratio), where a positive test highly predicts the disease, and clinicians can rely on the test results. A real-world example best illustrates this concept: In a cohort of 1000 individuals tested for suspected (low or high) tuberculosis, with a disease prevalence of 10%, 100 individuals will have EPTB. A test with 95% sensitivity will detect 95 of these 100 patients but will miss 5 EPTB patients. Therefore, it is crucial to miss as few patients as possible (high sensitivity). However, if the specificity is only 85%, for example, 150 individuals will be misdiagnosed with the disease and exposed to toxic treatment, accompanied by higher costs at the patient and health system levels. Therefore, 245 patients (95 + 150) will receive treatment for TB. In this example, due to suboptimal specificity, the number of patients mistreated far exceeds the number treated for the true disease. Therefore, both high sensitivity and specificity are essential, and these indicators are further correlated with high negative predictive value (exclusion value) and high positive predictive value (inclusion value).

[0057] The term "antibody" includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies and multireactive antibodies), and antibody fragments. Therefore, as used in this specification, the term "antibody" includes, but is not limited to, any specific binding member, immunoglobulin class and / or isotype (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM) or antibody fragments thereof.

[0058] It should be understood in the art that an antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds or their antigen-binding moieties. The heavy chains include a heavy chain variable region (VH) and a heavy chain constant region (CH1, CH2, and CH3). The light chains include a light chain variable region (VL) and a light chain constant region (CL). Both the heavy and light chain variable regions include a framework region (FR) and a complementarity-determining region (CDR). The four FRs are relatively conserved, while the CDR regions (CDR1, CDR2, and CDR3) include hypervariable regions. The FRs and CDRs are arranged from the NH2 terminus to the COOH terminus as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of both the heavy and light chains contain binding domains that interact with the antigen. Furthermore, the constant regions mediate the binding of the immunoglobulin to host tissues or factors.

[0059] The “best antibody pair” refers to the monoclonal antibodies Antrum-AB2 and Antrum-AB6. Antrum-AB2 is an antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 3. Antrum-AB6 is an antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 5 and the light chain amino acid sequence of SEQ ID NO: 7.

[0060] "Commercially available antibody pairs" refers to the Invitrogen™ antibodies with catalog numbers 14-7318-85 and 13-7319-81.

[0061] The definition of “antibody” also includes chimeric antibodies, humanized antibodies, recombinant antibodies, human antibodies produced from transgenic non-human animals, and antibodies selected from a library using enrichment techniques available to those skilled in the art.

[0062] As used herein, the term "epitope" refers to any antigenic determinant on an antigen that can be bound to the complementary site of an antibody. Epitope determinants typically consist of chemically active surface groups of a molecule, such as amino acid or sugar side chains, and usually possess specific three-dimensional structural features and specific charge characteristics.

[0063] "Antibody fragments" include a portion of a complete antibody, such as the antigen-binding region or variable region of a complete antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFV fragments; biantibodies; or linear antibodies.

[0064] Antibodies digested with papain produce two identical "Fab" fragments, or antigen-binding fragments, each with a single antigen-binding site, and a residual "Fc" fragment, the name reflecting its tendency to crystallize. Antibodies treated with pepsin produce the F(ab')2 fragment, which has two antigen-binding sites and retains its ability to cross-link antigens.

[0065] The term "Fv" refers to the smallest antibody fragment containing both complete antigen recognition and antigen binding sites. This fragment comprises a dimer of a tightly non-covalently associated heavy chain variable region domain and a light chain variable region domain. Folding of these two domains results in the formation of six hypervariable rings (three rings each for the H and L chains), which provide 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 containing only three antigen-specific CDRs) can recognize and bind antigens, albeit with lower affinity. A "single-chain Fv" ("sFv" or "scFv") is an antibody fragment containing VH and VL antibody domains linked together as a single polypeptide chain. sFv polypeptides may further include a polypeptide linker between the VH and VL domains, enabling the sFv to form the desired structure for antigen binding.

[0066] The “Fab” fragment comprises a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab’ fragment differs from the Fab fragment in that it has residues added to the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. Herein, the Fab’ fragment with a free thiol residue on the cysteine ​​residue of the constant domain is designated Fab’-SH. The F(ab’)2 antibody fragment was initially generated as a pair of Fab’ fragments, with a hinge cysteine ​​residue between the pair of Fab’ fragments. Other chemical conjugations of antibody fragments are also known in the art.

[0067] Variant antibodies are also covered within the scope of this invention. Therefore, variants of the sequences listed in this application are also covered within the scope of this invention. Other variants of antibody sequences with improved affinity can be obtained using methods known in the art, and these are also covered within the scope of this invention. Those skilled in the art can modify the amino acid sequence of a polypeptide using recombinant methods and / or synthetic chemistry techniques to generate 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 translation efficiency in the expression system used to generate the antibody. Such variant antibody sequences will share 70% or more (i.e., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) of sequence identity with the sequences listed in this application. This sequence identity is calculated relative to the full length of the sequences listed in this application.

[0068] The term “polypeptide” should be understood to encompass both “peptide” and “protein”, and vice versa. As used herein, “polypeptide” refers to the amino acid sequence of a recombinant or non-recombinant polypeptide that has i) the amino acid sequence of a native polypeptide, ii) a biologically active fragment of a polypeptide, or iii) a biologically active variant of a polypeptide.

[0069] As used herein, the term "isolated" refers to nucleic acids or antibodies that have been removed from their natural environment. Therefore, "isolated" nucleic acids, peptides, and proteins include nucleic acids and proteins purified using standard purification methods. The term also covers nucleic acids, peptides, and proteins prepared in host cells through recombinant expression, as well as chemically synthesized nucleic acids and / or polypeptides.

[0070] The terms “subject” and “patient” are used interchangeably herein and refer to any animal that may require diagnosis using the antibodies described herein. Therefore, subjects and patients include, but are not limited to, primates (including humans), canines, felines, murines, and other mammalian subjects. Preferably, the subject is a human. From the context of the use of the term, it can be understood that a subject and patient refers to a subject or patient who has IFN-γ in an extrapulmonary compartment that can be detected using the antibodies of the present invention.

[0071] In another embodiment, the present invention provides isolated nucleic acid encoding an isolated anti-IFN-γ antibody, a vector containing the nucleic acid and a host cell, and a recombinant technology for producing the antibody.

[0072] This invention also provides polynucleotide variants of the heavy and light chain peptide sequences encoding anti-IFN-γ 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 higher sequence identity compared to the polynucleotide sequences of this invention, as determined using the methods described herein. Such continuous sequences may encode CDR sequences or may encode complete variable regions. As is known in the art, variable region sequences can be fused with any suitable constant region sequence. Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by the two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame localization, etc.

[0073] The terms “nucleic acid” and “polynucleotide” are used interchangeably in this article and refer to single-stranded or double-stranded RNA, DNA, or a mixture of polymers.

[0074] To produce antibodies recombinantly, the nucleic acid encoding the antibody is inserted into a vector for further cloning (DNA amplification) or expression. The DNA encoding the antibody of the present invention is isolated according to the methods shown in the examples. Those skilled in the art will understand that many vectors can be used for the recombinant production of antibodies. Vector components typically include, but are not limited to, one or more of the following: signal sequences, origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences.

[0075] The anti-IFN-γ antibody of the present invention can also be recombinantly produced using heterologous or homologous polypeptides in the form of fusion polypeptides, including signal sequences or other polypeptides having specific cleavage sites at the N-terminus of mature proteins or polypeptides, immunoglobulin constant region sequences, etc. Preferably, the selected heterologous signal sequence can be a signal sequence that is recognized and processed by host cells (i.e., cleaved by signal peptidase). For prokaryotic host cells that do not recognize and process natural antibody signal sequences, the signal sequence is replaced by a selected prokaryotic signal sequence.

[0076] When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, that nucleic acid is "operably linked." For example, if the DNA of a pre-sequence or secretory leader sequence is expressed as a pre-protein involved in polypeptide secretion, then the DNA of the pre-sequence or secretory leader sequence is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence; or, if a ribosome binding site is positioned to facilitate translation, then the ribosome binding site is operably linked to the coding sequence. Generally, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a secretory leader sequence, contiguous and within the reading frame. However, enhancers do not need to be contiguous. Ligation is accomplished by joining at a convenient restriction site. If such a site is not available, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

[0077] As used herein, the terms “cell,” “cell line,” and “cell culture” are used interchangeably, and all such names include progeny. Therefore, the terms “transformation” and “transformed cell” include primary subject cells and cultures derived from them, regardless of the number of passages. It should also be understood that the DNA content of all progeny may not be entirely identical due to intentional or unintentional mutations. This includes mutant progeny with the same function or biological activity as those screened in the initially transformed cells. Where different names are intended to be used, the context will be clear.

[0078] Suitable host cells for cloning or expressing DNA are prokaryotic cells, yeast cells, or higher eukaryotic cells. Host cells transformed with the above-described expression or cloning vectors for the production of anti-IFN-γ antibodies are cultured in standard nutrient media and appropriately modified for inducing promoters, selecting transformants, or amplifying genes encoding desired sequences. Antibody compositions prepared from cells can be purified using purification techniques known to those skilled in the art.

[0079] In another embodiment of the invention, the antibody of the invention can be used in a diagnostic composition. A diagnostic composition is a composition comprising a compound or antibody (e.g., a labeled compound or antibody) for detecting 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 example, an anti-IFN-γ antibody, antigen, or epitope.

[0080] In another embodiment of the invention, an article of manufacture, such as a kit, is provided containing materials that can be used to treat the aforementioned conditions. The article of manufacture includes a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from various materials such as glass or plastic. The container contains a composition for effectively treating the condition and may have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a subcutaneous needle). The active agent in the composition is one or more antibodies as described above in the formulations of the invention. A label on or associated with the container indicates that the composition is intended to treat the selected condition. The article of manufacture may further include a second container containing pharmaceutically acceptable buffer solutions, such as phosphate-buffered saline, Ringer's solution, and glucose solution. It may also include other materials desired from a commercial and user perspective, including other buffer solutions, diluents, filters, needles, syringes, and packaging inserts with instructions for use.

[0081] The following are illustrative and non-limiting examples.

[0082] Example 1 Transient expression of human interferon-γ in HEK cells The amino acid sequence of IFN-γ (Uniprot accession number P01579) (SEQ ID NO: 21) was synthesized into cDNA (SEQ ID NO: 22), and codon optimization suitable for human expression was performed. Restriction enzyme sites were introduced to determine the correct insertion into the transient vector. The cDNA was ligated and transformed into *E. coli*. E. coliA strain of *E. coli* carrying the expression vector was cultured to prepare a low-endotoxin plasmid of the expression vector (expression culture). HEK-INV cells were grown in serum-free suspension culture medium, and 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 precipitated by centrifugation.

[0083] Purification was performed on a column using TALON (Clonetech) affinity chromatography. The binding buffer consisted of 50 mM NaH2PO4, 300 mM NaCl, pH 7.4, and the IFN-γ protein was eluted with imidazole (250 nM). The eluted fraction was subjected to SDS-PAGE, the positive fractions were combined, and then dialyzed into PBS (pH 7.4).

[0084] The obtained isolated human IFN-γ was filtered (0.22µm membrane), quantified (UV 280 nm), validated for protein purity (SDS-PAGE), and analyzed for endotoxin (Charles River PTS endosafe system).

[0085] Example 2 Antibody production Three Balb / C mice were immunized and boosted with human IFN-γ (5 mg). Mouse serum was used in a direct ELISA in which biotinylated IFN-γ (1 mg) was coated on a streptavidin microtiter plate to identify the mouse with the highest antibody titer.

[0086] The generation of primary hybridoma cultures involved the fusion of spleen cells from mice with the highest antibody titers with the myeloma cell line SP2 / 0. The fusion products were plated into 4×96-well plates. Specific IgG antibodies against the primary cell cultures were screened using the same direct ELISA method described above. Hybridoma colonies (positive clones) exhibiting affinity for IFN-γ were transferred to 24-well plates, proliferated, and retested for IFN-γ-specific antibodies.

[0087] Eight primary cultures with high antibody titers were selected for further cloning using limiting dilutions. Cloned cultures were screened by direct ELISA as described above. Clones identified as having the highest positive rates were transferred to 24-well plates for cell proliferation. The selected hybridoma cultures were retested by direct ELISA to determine the antibody titers used for final clone selection.

[0088] Four final antibody recloning cultures were propagated, Ig classes were determined (all four antibodies were classified as IgG1), and mycoplasma testing was performed. Hybridoma cells were isolated and cryopreserved (3 × 10⁶ cells per vial). 6 (3 cells), each selected clone is preserved in 3 vials.

[0089] Example 3 Stable cell line development Hybridoma cell precipitates were lysed, mRNA was extracted, and heavy and light chain variable region DNA was cloned into sequencing vectors to determine the heavy and light chain DNA sequences.

[0090] The variable region sequence was codon-optimized, synthesized, and cloned in frame with the relevant heavy and light chain constant regions into the Abzena (Cambridge, UK) expression vector. The vector was transfected into the Composite CHO™ cell line, resulting in up to four stable cell line libraries.

[0091] The antibody production rate of this stable library was assessed using small-scale batch cultures, from which 20 mg of each monoclonal antibody was isolated by affinity chromatography using a protein G column.

[0092] Example 4 Indirect ELISA The function of each antibody derived from a stable cell line was assessed using an indirect ELISA. The capture antibody (Antrum-AB2 antibody) was coated onto a 96-well microtiter plate at a concentration of 1 μg / ml (100 μL / well) in carbonate buffer and incubated overnight at 2–8°C. After removing the coating buffer, the plate was blocked for 1 hour and 30 minutes at room temperature with BSA (200 μL / well) in PBS. Following blocking, the plate was washed four times with washing buffer (200 μL / well) (Tween-20, in PBS). Human IFN-γ was diluted to an initial concentration of 300 pg / ml and then serially diluted six times (150, 75, 38, 19, 9.5, 4.8 pg / ml) in assay buffer (BSA and Tween-20, in PBS). Add triplicate (100 µL / well) of the diluents used to construct the standard curve to a microtiter plate, and simultaneously set the final wells to contain only assay buffer (100 µL / well) (blank). Incubate the standard curve at room temperature for 1 hour and 30 minutes. After incubation, wash the plate four times with wash buffer (200 µL / well) (Tween-20, in PBS).

[0093] The detection antibody (Antrum-AB6 antibody) was biotinylated using the NHS-Sulfo EZ Link kit and the corresponding protocol (Thermoscientific) to a concentration of 4.5 μg / ml (100 μL / well) in the detection diluent (BSA, in PBS). The plate was incubated at room temperature in the dark for 1 hour (photosensitive step). After incubation, the plate was washed four times with wash buffer (200 μL / well) (Tween-20, in PBS). Strep-HRP (1:200 dilution, Pierce High Sensitivity, Thermoscientific) in the Strep-HRP diluent (BSA, in PBS) was added to the plate (100 μL / well) and incubated at room temperature in the dark for 30 minutes (photosensitive step). After incubation, the plate was washed four times with wash buffer (200 μL / well) (Tween-20, in PBS). Chromogen (TMB and peroxidase in a 1:1 ratio, Thermoscientific) was added to the wells (100 μL / well) and incubated at room temperature in the dark (photosensitive step) for 30 minutes. After incubation, stop solution (sulfuric acid) was added to the plate (100 μL / well), and the plate was read at 450 nm and 595 nm (SmartReader, Accuris, USA).

[0094] Obtain the absorbance readings and use them to determine the standard curve. Figure 1 This was done in GraphPad Prism (version 5) and Microsoft Excel.

[0095] Detection limit To determine the limit of detection, an indirect ELISA was performed as described above, and the limit of detection was then calculated using the average absorbance reading and the equation mentioned below: STEYX(standard error of the predicted y value for each x in the regression / slope) * 3.3. Figure 2 ).

[0096] Table 1: Absorbance readings of the standard curve used to determine the detection limit of monoclonal antibody pairs.

[0097]

[0098] Example 5 Clinical sample status The Centre for Lung Infection and Immunity (CLII) at the University of Cape Town, South Africa, collected pleural fluid, pericardial fluid, and cerebrospinal fluid samples through a series of clinical trials. The samples were classified according to the following definitions: 1. Reference criteria for tuberculosis: microbiological confirmation of tuberculosis and / or histopathological features of tuberculosis, together with compatible baseline clinical images showing improvement with anti-TB treatment.

[0099] 2. Definite-TB: Defined as microbiological confirmation of tuberculosis (at least one positive culture of Mycobacterium tuberculosis in liquid broth, or a positive Xpert test (using liquid or biopsy)) and / or pathological histological features of tuberculosis (caseous granulomatous inflammation, or granulomas with or without AFB), with improvement in anti-TB treatment (positive histology or microbiology (e.g., culture or Xpert) from alternative sites such as lymph nodes; or a positive Alere urine LAM test, with improvement in tuberculosis treatment also counted as microbiological confirmation of TB).

[0100] 3. Non-TB: Patients for whom no microbiological or histological evidence of Mycobacterium tuberculosis can be confirmed or found, and / or for whom an alternative diagnosis can be obtained, and / or whose symptoms and signs resolve without intervention. These patients did not receive anti-TB treatment at presentation and follow-up.

[0101] These samples were selected and run on an indirect ELISA as described above, with duplicates added along with the standard curve samples. The IFN-γ concentration (pg / ml) of the samples was interpolated from the resulting standard curve. The performance of each antibody pair near predetermined cutoff values ​​was determined using a contingency table (GraphPad Prism, version 5) for each extrapulmonary TB fluid (pleural TB = 20.5 pg / ml, pericardial TB = 10 pg / ml, and TB meningitis = 13 pg / ml). The antibody pairs included: 1. Optimal antibody pair (Antrum-AB2 antibody and Antrum-AB6 antibody) Figure 3 , Figure 4 and Figure 5 ),as well as 2. Commercially available antibody pairs (IFN-γ monoclonal antibody (NIB42), eBioscience™, catalog number 14-7318-85, Invitrogen and IFN-γ monoclonal antibody (4S.B3), biotin, eBioscience™, catalog number 13-7319-81, Invitrogen) Figure 6 ).

[0102] Table 2: Performance comparison of the optimal monoclonal antibody pair with commercially available antibody pairs in pleural fluid, pericardial fluid, and CSF. The optimal antibody pair demonstrated superior performance in distinguishing between active TB and non-TB compared to commercially available monoclonal antibody pairs.

[0103]

[0104] References Christopher DJ, Esmail A, Scott AJ, et al. Performance ofUnstimulated IFN-γ (IRISA-TB) for Pleural Tuberculosis: A Prospective Studyin South Africa and India, Open Forum Infectious Diseases 2024, Volume 11,Issue 10. Dheda K, van Zyl-Smit RN, Sechi L, et al. Utility of quantitative T-cell responses versus unstimulated interferon-gamma for the diagnosis ofpleural tuberculosis. Eur Respir J 2009, 34(5):1118–1126. Fan L, Chen Z, Hao XH et al. Interferon-gamma release assays for thediagnosis of extrapulmonary tuberculosis: a systematic review and meta-analysis. FEMS Immunology and Medical Microbiology, 2012, June; 65: 456-466. Kricka, L. Human anti-animal antibody interferences in immunologicalsays. 1999. Clin Chem 45:942-956. Light RW. Update on tuberculous pleural effusion. Respirology 2010,15(3):451–458. Meldau R, Peter J, Theron G, et al. Comparison of same day diagnostictools including Gene Xpert and unstimulated IFN-gamma for the evaluation ofpleural tuberculosis: a prospective cohort study. BMC Pulm Med 2014; 14:58. Meldau R, Randall PJ, Pooran A, Limberis J, Makambwa E, Dhansay M,Esmail A and Dheda K. Same day tools, including Xpert Ultra and unstimulatedIFN-γ, for the rapid diagnosis of pleural tuberculosis. Journal of ClinicalMicrobiology, 2019, July; doi:10.1128 / JCM.00614-19. Metcalfe JZ, Everett CK, Steingart KR et al. Interferon-c ReleaseAssays for Active Pulmonary Tuberculosis Diagnosis in Adults in Low- andMiddle-Income Countries: Systematic Review and Meta-analysis. JID, 2011:204(Suppl 4). Pandie S, Peter JG, Kerbelker ZS, et al. Diagnostic accuracy ofquantitative PCR (Xpert MTB / RIF) for tuberculous pericarditis compared toadenosine deaminase and unstimulated interferon-gamma in a high burdensetting: a prospective study. BMC Med 2014, 12: 101. Patel VB, Singh R, Dheda K, et al. Comparative Utility of CytokineLevels and Quantitative RD-1-Specific T Cell Responses for RapidImmunodiagnosis of Tuberculous Meningitis. Journal of Clinical Microbiology2011, 49(11): 3971–3976. Randall P, Esmail A, Wilson L, et al. GeneXpert MTB / RIF Ultra vsUnstimulated Interferon γ (IRISA-TB) for the Diagnosis of TuberculousPericarditis in a Tuberculosis-Endemic Setting. Open Forum Infect Dis. 202420;11(3). Randall P, Mutsvangwa J, Nliwasa M, et al. Utility of CerebrospinalFluid Unstimulated Interferon-Gamma (IRISA-TB) as a Same-Day Test forTuberculous Meningitis in a Tuberculosis-Endemic, Resource-Poor Setting, OpenForum Infectious Diseases, 2024, Volume 11, Issue 9. WHO Policy Statement. Use of alternative interferon-gamma releaseassays for the diagnosis of TB infection: WHO policy statement. Geneva: WorldHealth Organization; 2022. Licence: CC BY-NC-SA 3.0 IGO.

Claims

1. An isolated antibody, said isolated antibody specifically binding to human interferon-γ (IFN-γ), wherein, The antibodies include: Heavy chain variable region (VH), the heavy chain variable region includes: CDR-H1 includes the amino acid sequence of SEQ ID NO: 15; CDR-H2, including the amino acid sequence of SEQ ID NO: 16; Including the CDR-H3 amino acid sequence of SEQ ID NO: 17; and Light chain variable region (VL), the light chain variable region comprising: CDR-L1 includes the amino acid sequence of SEQ ID NO: 18; Including the amino acid sequence of SEQ ID NO: 19, CDR-L2; and CDR-L3 includes the amino acid sequence of SEQ ID NO:

20.

2. The antibody according to claim 1, wherein, The antibodies include VH of SEQ ID NO: 5 and VL of SEQ ID NO:

7.

3. The antibody according to claim 1 or 2, wherein, The antibody is a monoclonal antibody.

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

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

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

7. The antibody according to claim 6, wherein, The antibody fragment is selected from Fv, single-chain Fv (scFv), Fab, Fab', or (Fab')2.

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

9. The antibody according to claim 8, wherein, The detection marker is selected from the group consisting of colorimetric markers, fluorescent markers, chemiluminescent markers, biotin, phosphor-based markers, thermal markers, enzyme markers, gold nanoparticles, silver nanoparticles, and magnetic beads.

10. A method for in vitro diagnosis of active tuberculosis infection in a subject, comprising: (i) Provide an extrapulmonary fluid sample from the subject, wherein the extrapulmonary fluid sample is selected from pleural fluid, pericardial fluid or cerebrospinal fluid; (ii) Contact the extrapulmonary fluid sample with a capture antibody that specifically binds to an epitope present on the IFN-γ polypeptide; (iii) Contact the sample from step (ii) with a detection antibody that specifically binds to an epitope on the IFN-γ polypeptide that is different from the epitope that the capture antibody binds to, wherein the detection antibody is the antibody according to any one of claims 1 to 9; (iv) Detecting the binding of the detection antibody to different epitopes of the IFN-γ peptide; and (v) Diagnose the subject with active tuberculosis infection. The binding of the capture antibody to the IFN-γ peptide and the binding of the detection antibody to the IFN-γ peptide indicate active tuberculosis infection in the subject.

11. The method according to claim 10, wherein, The capture antibody includes: Heavy chain variable region (VH), the heavy chain variable region includes: CDR-H1, including the amino acid sequence of SEQ ID NO: 9; CDR-H2 including the amino acid sequence of SEQ ID NO: 10; Including the CDR-H3 amino acid sequence of SEQ ID NO: 11; and Light chain variable region (VL), the light chain variable region comprising: CDR-L1 includes the amino acid sequence of SEQ ID NO: 12; Including the CDR-L2 amino acid sequence of SEQ ID NO: 13; and CDR-L3 includes the amino acid sequence of SEQ ID NO:

14.

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

13. The method according to claim 10 or 11, wherein, The captured antibody is an antibody fragment.

14. The method according to claim 13, wherein, The antibody fragment is selected from Fv, single-chain Fv (scFv), Fab, Fab', or (Fab')2.

15. An immunoassay device for detecting IFN-γ in extrapulmonary fluid samples, 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 according to any one of claims 1 to 9, and wherein the detection antibody is capable of specifically binding to an epitope present on the IFN-γ polypeptide; and (ii) A stationary phase comprising a capture antibody, wherein the capture antibody is capable of specifically binding to different epitopes present on the IFN-γ polypeptide. The extrapulmonary fluid sample is selected from pleural fluid, pericardial fluid, or cerebrospinal fluid, and The presence of IFN-γ in the extrapulmonary fluid sample allows both the detection antibody and the capture antibody to bind to the IFN-γ polypeptide.

16. The immunoassay device according to claim 15, wherein, The capture antibody includes: Heavy chain variable region (VH), the heavy chain variable region includes: CDR-H1, including the amino acid sequence of SEQ ID NO: 9; CDR-H2 including the amino acid sequence of SEQ ID NO: 10; Including the CDR-H3 amino acid sequence of SEQ ID NO: 11; and Light chain variable region (VL), the light chain variable region comprising: CDR-L1 includes the amino acid sequence of SEQ ID NO: 12; Including the CDR-L2 amino acid sequence of SEQ ID NO: 13; and CDR-L3 includes the amino acid sequence of SEQ ID NO:

14.

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

18. The immunoassay device according to claim 15 or 16, wherein, The captured antibody is an antibody fragment.

19. The immunoassay device according to claim 18, wherein, The antibody fragment is selected from Fv, single-chain Fv (scFv), Fab, Fab', or (Fab')2.

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