Use of a specific antibody in the preparation of a diagnostic reagent for diagnosing Mycobacterium tuberculosis infection and a diagnostic kit

Abstract

The present invention provides an application of a specific antibody in the preparation of a diagnostic reagent for diagnosing Mycobacterium tuberculosis infection and a diagnostic kit, wherein the specific antibody comprises at least two of a B2M antibody, a TXN antibody, and a PRDX5 antibody. The diagnostic reagent of the present invention can effectively distinguish active pulmonary tuberculosis (ATB) patients, latent Mycobacterium tuberculosis infection (LTBI) individuals, and / or non-infected (HC) populations, and has good sensitivity and specificity.

Description

Technical Field

[0001] The present invention relates to a diagnostic marker and its specific antibody, and more specifically, to the application of the specific antibody in the preparation of a diagnostic reagent for diagnosing Mycobacterium tuberculosis infection and a diagnostic kit including the specific antibody. Background Art

[0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis infection, mainly affecting the lungs, but can also invade other organs, seriously endangering human health. It is estimated that about one-fourth of the people in the world are infected with Mycobacterium tuberculosis. Most people are in a latent infection state after infection, and about 10% of the infected people may develop active tuberculosis.

[0003] The prevention and control of tuberculosis mainly rely on early detection and treatment. At present, the detection methods of tuberculosis mainly include sputum smear, sputum culture and molecular biology detection methods. The sputum smear method has the advantages of simplicity, rapidity and easy operation, but its positive rate is low and the sensitivity is poor. The sputum culture method has high specificity and is the current gold standard for diagnosing active pulmonary tuberculosis, but its sensitivity is low and the time-consuming is long, greatly limiting the positive detection rate of tuberculosis patients. The application of molecular biology technology is a revolutionary progress in tuberculosis diagnosis, with the advantages of accuracy and high efficiency, but it has not fundamentally changed the current situation of difficult tuberculosis diagnosis. This technology has potential contamination risks, may lead to false positive results, and its diagnostic performance in smear-negative specimens is still not good. The high detection cost and relatively complex operation methods make these technologies difficult to popularize in countries with medium and low development levels. In addition, most of the above detection methods are based on sputum specimens that are not easily obtained. Therefore, there is an urgent need to develop new clinical diagnosis methods for tuberculosis. Summary of the Invention

[0004] The object of the present invention is to provide an application of a diagnostic marker and / or its specific antibody in the preparation of a diagnostic reagent for diagnosing Mycobacterium tuberculosis infection and a diagnostic kit including the diagnostic marker and / or its specific antibody. The diagnostic marker of the present invention can effectively distinguish active pulmonary tuberculosis (ATB) patients, latent Mycobacterium tuberculosis infection (LTBI) patients and / or non-infected (HC) populations, and has good sensitivity and specificity.

[0005] An embodiment of the present invention relates to an application of a specific antibody in the preparation of a diagnostic reagent for diagnosing Mycobacterium tuberculosis infection, wherein the specific antibody includes at least two of B2M antibody, TXN antibody and PRDX5 antibody.

[0006] According to some embodiments, the specific antibody can correspond to the diagnostic marker in the sample to be detected, and the diagnostic marker includes at least two of B2M, TXN and PRDX5.

[0007] According to some embodiments, the diagnostic marker is derived from peripheral blood neutrophils.

[0008] According to some embodiments, the specific antibodies include B2M antibody and TXN antibody; alternatively, the specific antibodies include B2M antibody and PRDX5 antibody; alternatively, the specific antibodies include TXN antibody and PRDX5 antibody.

[0009] According to some embodiments, the specific antibodies include B2M antibody, TXN antibody and PRDX5 antibody.

[0010] According to some embodiments, the diagnostic reagent is used for preparing a diagnostic chip or a detection device for Mycobacterium tuberculosis infection.

[0011] According to some embodiments, the diagnostic reagent is at least used to distinguish active pulmonary tuberculosis patients from non-infected populations.

[0012] According to some embodiments, the diagnostic reagent is also used to distinguish latent Mycobacterium tuberculosis infected individuals from non-infected populations.

[0013] Embodiments of the present invention relate to a diagnostic kit for diagnosing Mycobacterium tuberculosis infection, wherein the diagnostic kit includes specific antibodies.

[0014] According to some embodiments, the specific antibody is a monoclonal antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Through the following description with reference to the drawings which exemplarily show an example, the above and other objects and features of the present invention will become clearer, wherein:

[0016] Figure 1A and Figure 1B shows the results of the expression levels of B2M, TXN, PRDX5, PRTN3, DEFA3, FCGR1A, FTO, GCA, MAD1L1 and CD63 in different blood samples (total protein normalized);

[0017] Figure 2 shows the protein immunoblotting results of B2M, TXN and PRDX5 in different blood samples;

[0018] Figure 3 shows the comparison of the protein levels of B2M, TXN, PRDX5 in active pulmonary tuberculosis (ATB) patients, latent Mycobacterium tuberculosis infection (LTBI) individuals and non-infected (HC) populations;

[0019] Figure 4 shows the ROC curve graph for evaluating the diagnostic efficacy of protein combinations selected from two of B2M, TXN and PRDX5;

[0020] Figure 5 The ROC curve graph showing the diagnostic efficacy evaluation of the combination of B2M, TXN, and PRDX5. Detailed implementation manners

[0021] In the following description, for the purpose of explanation, many specific details are set forth to provide a thorough understanding of various embodiments or implementation manners of the invention. As used herein, "embodiment" and "implementation manner" are interchangeable terms and are non-limiting examples of a device or method that employs one or more of the inventive concepts disclosed herein. However, it is apparent that various embodiments may be practiced without these specific details or with one or more equivalent arrangements. In addition, various embodiments may be different but not necessarily exclusive.

[0022] It should be understood that when the terms "comprising" and / or "including" are used in the specification, the recited materials and / or components are present, but do not exclude the presence or addition of one or more other materials and / or components.

[0023] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Terms (such as those defined in a general dictionary) should be interpreted as having a meaning consistent with their meaning in the context of the relevant field and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0024] The term "diagnostic marker" used in the present invention refers to a molecule to be used as a target for analyzing a patient's test sample. Examples of such molecular targets are proteins or polypeptides. The proteins or polypeptides used as markers in the present invention are expected to include naturally occurring variants of the protein as well as fragments of the protein or the variant, particularly immunologically detectable fragments. The immunologically detectable fragment preferably contains at least 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20 consecutive amino acids of the marker polypeptide.

[0025] Those skilled in the art will recognize that proteins released by cells or present in the extracellular matrix may be damaged (e.g., during an inflammatory process) and may be degraded or cleaved into such fragments. Certain markers are synthesized in an inactive form, which can subsequently be activated by proteolysis. As will be appreciated by those skilled in the art, a protein or a fragment thereof may also exist as part of a complex. Such a complex may also be used as a marker in the sense of the present invention. Additionally, or in the alternative, the marker polypeptide or its variant may carry post-translational modifications. Non-limiting examples of post-translational modifications are glycosylation, acylation, and / or phosphorylation.

[0026] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

[0027] The present inventors have first discovered that the expression levels of at least one of β-2 microglobulin (B2M), thioredoxin (TXN), and peroxiredoxin 5 (PRDX5) in peripheral blood neutrophils are significantly different between patients infected with Mycobacterium tuberculosis (active pulmonary tuberculosis patients, latent Mycobacterium tuberculosis-infected individuals) and non-infected populations. Specifically, the expression levels in patients infected with Mycobacterium tuberculosis are significantly higher than those in non-infected populations. Therefore, by detecting the content of at least one of β-2 microglobulin, thioredoxin, and peroxiredoxin 5 in the blood, patients infected with Mycobacterium tuberculosis can be effectively distinguished from non-infected populations.

[0028] β-2 microglobulin (B2M) is a polypeptide of approximately 13 kDa, present in serum, and is a component of the class I major histocompatibility complex (MHC), participating in the presentation of peptide antigens to the immune system. The amino acid sequence of β-2 microglobulin is:

[0029] MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM.

[0030] Thioredoxin (TXN), also abbreviated as Trx1, belongs to the thioredoxin family, is mainly located in the cytoplasm, and can also translocate to the nucleus or be secreted extracellularly. This protein is an essential antioxidant protein released by cells under stress, protecting cells from oxidative damage caused by microbial invasion and physical and chemical stimuli. The amino acid sequence of thioredoxin (TXN) is:

[0031] MVKQIESKTAFQEALDAAGDKLVVVDFSATWCGPCKMIKPFFHSLSEKYSNVIFLEVDVDDCQDVASECEVKCMPTFQFFKKGQKVGEFSGANKEKLEATINELV.

[0032] Peroxiredoxin 5 (PRDX5) belongs to the peroxiredoxin family and is an antioxidant enzyme that can scavenge cellular peroxide substrates to protect cells from oxidative stress. The amino acid sequence of peroxiredoxin 5 (PRDX5) is:

[0033] MGLAGVCALRRSAGYILVGGAGGQSAAAAARRYSEGEWASGGVRSFSRAAAAMAPIKVGDAIPAVEVFEGEPGNKVNLAELFKGKKGVLFGVPGAFTPGCSKTHLPGFVEQAEALKAKGVQVVACLSVNDAFVTGEWGRAHKAEGKVRLLADPTGAFGKETDLLLDDSLVSIFGNRRLKRFSMVVQDGIVKALNVEPDGTGLTCSLAPNIISQL。

[0034] According to a first aspect of the present invention, there is provided a diagnostic kit for diagnosing Mycobacterium tuberculosis infection. The diagnostic kit for diagnosing Mycobacterium tuberculosis infection may include specific antibodies corresponding to diagnostic markers of at least two of β-2 microglobulin (B2M), thioredoxin (TXN), and peroxiredoxin 5 (PRDX5). Here, the specific antibody corresponding to the diagnostic marker of at least one of β-2 microglobulin, thioredoxin, and peroxiredoxin 5 is an antibody capable of specifically binding to at least one of β-2 microglobulin, thioredoxin, and peroxiredoxin 5. They may also be respectively referred to as β-2 microglobulin antibody (B2M antibody), thioredoxin antibody (TXN antibody), or peroxiredoxin 5 antibody (PRDX5 antibody) in sequence. These specific antibodies can specifically bind to the above-mentioned proteins respectively, and thus are used for the diagnosis of Mycobacterium tuberculosis infection. Preferably, the diagnostic kit may include specific antibodies corresponding to B2M, TXN, and PRDX5, that is, β-2 microglobulin antibody (B2M antibody), thioredoxin antibody (TXN antibody), and peroxiredoxin 5 antibody (PRDX5 antibody).

[0035] According to an embodiment of the present invention, the diagnostic kit may further include a quantitative detection agent for the diagnostic marker. According to the embodiment, the quantitative detection agent may be a specific antibody against the above-mentioned diagnostic marker, but the embodiment is not limited thereto. In the embodiment, the specific antibody can specifically bind to the corresponding marker, and the specific antibody can be used to perform immunoblotting, immunoprecipitation, or enzyme-linked immunosorbent assay to detect the diagnostic marker.

[0036] According to an embodiment of the present invention, the specific antibody may be a monoclonal antibody, but the embodiment is not limited thereto. In some embodiments, the specific antibody may have a label for indicating the signal intensity.

[0037] According to an embodiment of the present invention, the diagnostic kit may further contain at least one of a solid-phase carrier, a blocking solution, a chromogenic agent, a calibrator for the diagnostic marker fusion antigen, and a washing buffer.

[0038] According to an embodiment of the present invention, the above diagnostic reagent is used in the preparation of a diagnostic chip or detection device for Mycobacterium tuberculosis infection, but the embodiment is not limited thereto.

[0039] According to a first aspect of the present invention, there is provided an application of a specific antibody in the preparation of a diagnostic reagent for diagnosing Mycobacterium tuberculosis infection, particularly in the preparation of a diagnostic reagent for diagnosing tuberculosis, wherein the specific antibody comprises at least two of a B2M antibody, a TXN antibody and a PRDX5 antibody.

[0040] The diagnostic reagent may comprise specific antibodies that correspond to specific proteins produced during Mycobacterium tuberculosis infection. Thus, the diagnostic reagent can use the specific antibodies to bind to diagnostic markers in a collected sample to indicate whether there is an infection of Mycobacterium tuberculosis in the sample. The B2M antibody, the TXN antibody and the PRDX5 antibody may also be referred to as an anti-B2M antibody, an anti-TXN antibody and an anti-PRDX5 antibody.

[0041] According to the application of the specific antibody provided by the embodiment of the present application in the preparation of a diagnostic reagent for diagnosing Mycobacterium tuberculosis infection, when in use, when there are diagnostic markers or antigens corresponding to the specific antibodies (i.e., β-2 microglobulin, thioredoxin or peroxiredoxin 5) in the collected sample, the diagnostic markers and the specific antibodies will specifically bind to form a diagnostic marker-antibody complex. Then, the diagnostic marker-antibody complex is converted into the concentration of the diagnostic marker in the sample to be detected, so as to obtain the concentration of the diagnostic marker in the sample to be detected. By the obtained concentration of the diagnostic marker, the actual level of the marker in the body can be reflected. Therefore, the difference in concentration can be used to distinguish people in different states.

[0042] In some embodiments, the specific antibody can correspond to a diagnostic marker in the sample to be detected. Correspondingly, the diagnostic marker comprises at least two of B2M, TXN and PRDX5.

[0043] In these embodiments, the diagnostic markers in the collected sample can specifically bind to the specific antibodies, so that active pulmonary tuberculosis patients and non-infected populations can be determined based on the content of the markers. Since there is a high degree of specific binding between the specific antibody and the diagnostic marker, the possibility of misdiagnosis and missed diagnosis can be reduced.

[0044] In some embodiments, the diagnostic marker is derived from peripheral blood neutrophils.

[0045] In these embodiments, on the one hand, diagnostic markers can be obtained through collection, which is convenient. On the other hand, the blood collected from peripheral blood neutrophils can also be purified to obtain at least two of B2M, TXN, and PRDX5, so as to further improve the accuracy of subsequent detection.

[0046] In some embodiments, the specific antibodies include B2M antibody, TXN antibody, and PRDX5 antibody.

[0047] In some embodiments, the diagnostic reagent is used for preparing a diagnostic chip or detection device for Mycobacterium tuberculosis infection.

[0048] In some embodiments, the diagnostic reagent is at least used to distinguish active pulmonary tuberculosis patients from non-infected populations.

[0049] In some embodiments, the diagnostic reagent is also used to distinguish latent Mycobacterium tuberculosis-infected individuals from non-infected populations.

[0050] In these embodiments, active pulmonary tuberculosis patients usually have high levels of diagnostic markers in their bodies. Therefore, the measured concentration of the diagnostic marker will increase significantly. Latent Mycobacterium tuberculosis-infected individuals may have moderate levels of diagnostic markers in their bodies, but no obvious inflammatory response has been triggered. Therefore, the measured concentration of the diagnostic marker may be at a moderate level. Here, the moderate level means that the concentration of the diagnostic marker is lower than that of active pulmonary tuberculosis patients but higher than that of non-infected populations. Non-infected populations do not have relevant markers or have extremely low levels, so the measured concentration of the diagnostic marker should be at an extremely low level or undetectable.

[0051] In some embodiments, when the concentration of B2M is greater than 13.43 ng / mL and the concentration of TXN is greater than 261.00 ng / mL, the person corresponding to the collected sample is determined as an active pulmonary tuberculosis patient. When the concentration of B2M is not greater than 13.43 ng / mL and the concentration of TXN is not greater than 261.00 ng / mL, it is determined as a non-infected population, so as to achieve the purpose of at least distinguishing active pulmonary tuberculosis patients from non-infected populations. Its diagnostic accuracy (AUC = 0.9810), sensitivity (95.77%), and specificity (91.51%) are all good.

[0052] In some other embodiments, when the concentration of B2M is greater than 13.43 ng / mL and the concentration of PRDX5 is greater than 132.50 ng / mL, it is determined as an active pulmonary tuberculosis patient. When the concentration of B2M is not greater than 13.43 ng / mL and the concentration of PRDX5 is not greater than 132.50 ng / mL, it is determined as a non-infected population, thereby achieving the purpose of at least differentiating active pulmonary tuberculosis patients from non-infected populations. Its diagnostic accuracy (AUC = 0.9799), sensitivity (91.55%), and specificity (93.40%) also reach a relatively high level.

[0053] In still some other embodiments, when the concentration of TXN is greater than 261 ng / mL and the concentration of PRDX5 is greater than 132.50 ng / mL, it is determined as an active pulmonary tuberculosis patient. When the concentration of TXN is not greater than 261 ng / mL and the concentration of PRDX5 is not greater than 132.500 ng / mL, it is determined as a non-infected population, thereby achieving the purpose of at least differentiating active pulmonary tuberculosis patients from non-infected populations. Its diagnostic accuracy (AUC = 0.9720), sensitivity (92.96%), and specificity (94.34%) all perform well.

[0054] More preferably, when the concentration of B2M is greater than 13.43 ng / mL, the concentration of TXN is greater than 261.00 ng / mL, and the concentration of PRDX5 is greater than 132.50 ng / mL, it is determined that the diagnostic marker is from an active pulmonary tuberculosis patient; when the concentration of B2M is not greater than 13.43 ng / mL, the concentration of TXN is not greater than 261.00 ng / mL, and the concentration of PRDX5 is not greater than 132.50 ng / mL, it is determined as a non-infected population, thereby achieving the purpose of at least differentiating active pulmonary tuberculosis patients from non-infected populations. By this method, its diagnostic accuracy (AUC = 0.9794), sensitivity (94.37%), and specificity (96.23%) reach the optimal level, can more effectively cope with individual differences and disease complexity, and significantly improve the reliability of diagnosis.

[0055] The following further elaborates the present invention in conjunction with specific embodiments.

[0056] 1. Determination of Molecular Markers for Host Mycobacterium tuberculosis Infection

[0057] 1.1 Inclusion and Exclusion Criteria for Research Subjects

[0058] The diagnostic criteria for the active tuberculosis (ATB) patients included in this study were based on the "Diagnosis of Tuberculosis, Health Industry Standard of the People's Republic of China (WS 288—2017)", and the pathogen detection in sputum or bronchoalveolar lavage fluid specimens was positive (at least one of smear / culture / nucleic acid detection was positive). There was no previous history of tuberculosis (no old tuberculosis lesions were found by medical history inquiry and X-ray chest radiography), and it was the first anti-tuberculosis treatment with less than 7 days of medication.

[0059] The inventors screened latent tuberculosis infection (LTBI) patients and non-infected (HC) controls using the interferon-γ release assay, and the detection was performed according to the instructions of the QuantiFERON-TB Gold Plus kit from QIAGEN, Germany. LTBI refers to no previous history of tuberculosis and related clinical manifestations, normal X-ray chest radiography, and positive interferon-γ release assay; HC refers to no previous history of tuberculosis and related clinical manifestations, normal X-ray chest radiography, and negative interferon-γ release assay.

[0060] The above study subjects were all excluded: those aged over 65 or under 18, pregnant or lactating women, diabetic patients, patients with malignant tumors, patients with immune system diseases or those receiving immunotherapy, and those infected with human immunodeficiency virus or other pathogens. This study was approved by the Ethics Committee of the Institute of Pathogen Biology, Chinese Academy of Medical Sciences. After obtaining the informed consent of the enrolled subjects, the inventors collected their peripheral blood for subsequent experiments. The sample demographic information is shown in Table 1.

[0061] Table 1

[0062]

[0063] 1.2 Separation and enrichment of peripheral blood neutrophils

[0064] Collect 1.5 mL of peripheral blood from the study subjects using an EDTA anticoagulant tube, and separate neutrophils from the whole blood. The specific operation steps are as follows:

[0065] 1) Prepare flow cytometry tubes, write the numbers (numbered in the way of group name plus number, for example, ATB-001), and add 3 mL of separation buffer to each flow cytometry tube;

[0066] 2) Mix the blood evenly, aspirate 1.5 mL into the corresponding numbered flow cytometry tube, and mix by pipetting;

[0067] 3) Centrifuge at 4°C, 600 g for 10 minutes;

[0068] 4) Carefully aspirate and discard the supernatant, avoiding touching the surface of the cell pellet, and leave about 0.5 mL of liquid;

[0069] 5) Add separation buffer to 1.5 mL, and mix gently;

[0070] 6) Vortex and mix the CD15 magnetic beads, and quickly add 40 µL / sample to the diluted blood;

[0071] 7) Close the lid tightly, place the flow cytometry tube properly on the rotary mixer (Hula Mixer), and incubate with rotation at 8 rpm for 20 minutes;

[0072] 8) Take out the incubated cells, centrifuge them instantaneously, let them stand on the magnetic stand (Invitrogen™, product number: 12321D) for 2 minutes, and carefully aspirate and discard the supernatant;

[0073] 9) Remove the flow cytometry tube, add 1.6 mL of separation buffer, gently blow and mix evenly, and then transfer it to the corresponding 2 mL protein low-binding tube; let it stand on the magnetic stand for 2 minutes, and aspirate and discard the supernatant;

[0074] 10) Remove it from the magnetic stand, add 1.6 mL of separation buffer, gently blow and mix evenly, let it stand on the magnetic stand for 2 minutes, aspirate the supernatant, and repeat once.

[0075] 11) Add 20 µL of protease inhibitor solution, gently mix evenly, and store at -80 °C.

[0076] 1.3 Total protein extraction and mass spectrometry identification

[0077] 1) Take out the cell samples obtained in steps 1.2, and add SDS buffer (4% SDS, 100 mM Tris-HCl, pH 7.6);

[0078] 2) Perform ultrasonic treatment (30 seconds per cycle, 10 cycles), and boil at 95 °C for 15 minutes;

[0079] 3) After centrifuging at 14000 g for 40 minutes, measure the protein content of the supernatant using a BCA protein assay kit;

[0080] 4) Take 20 µg of protein from each sample, mix it with 5× loading buffer respectively, and boil for 5 minutes. Separate the proteins on a 4% - 20% SDS-PAGE gel (constant voltage 180 V, 45 minutes). Visualize through Coomassie protein bands;

[0081] 5) Take equal amounts from each sample in this experiment and mix them into one sample for the generation and quality control of the DDA library (spectrum library based on data-dependent acquisition).

[0082] 6) Perform mass spectrometry identification on the enrolled samples to obtain the peripheral blood neutrophil protein expression profiles of the ATB, LTBI, and HC populations, and screen for proteins significantly related to the Mycobacterium tuberculosis infection status. The number of unique peptides of three proteins, B2M, TXN, and PRDX5, is greater than 2, indicating that the identification results have a relatively high credibility.

[0083] 1.4. Evaluation of protein expression levels

[0084] The inventors measured the protein levels of B2M, TXN, and PRDX5 in different samples by Western blotting. Among them, commercially available B2M (brand: Novoprotein, catalog number: Recombinant Human B2M (N-6His)(Cat.No.:C512)), TXN (brand: Novoprotein, catalog number: Recombinant Human TXN (N-6His)(Cat. No.:CE85)), and PRDX5 (brand: Novoprotein, catalog number: Recombinant Human PRDX5 (N-6His)(Cat. No.:CK91)) were used as controls, and the following commercially available antibodies were used for detection: B2M antibody (brand: Santa Cruz, catalog number: sc-46697), TXN antibody (brand: Santa Cruz, catalog number: sc-166393), PRDX5 antibody (brand: Santa Cruz, catalog number: sc-133072). Additionally, through proteomic analysis, the inventors screened out 7 proteins that were significantly associated with the Mycobacterium tuberculosis infection status as comparative examples. The 7 proteins were: promyelocytic leukemia protein (PRTN3), neutrophil defensin 3 (DEFA3), high-affinity immunoglobulin gamma Fc receptor I (FCGR1A), alpha-ketoglutarate-dependent dioxygenase FTO (FTO), granulocalcin (GCA), mitotic spindle assembly checkpoint protein MAD1 (MAD1L1), and CD63 antigen (CD63). Among them, the following commercially available antibodies were used for detection: PRTN3 (brand: Santa Cruz, catalog number: sc-74534), DEFA3 (brand: Santa Cruz, catalog number: sc-390796), FCGR1A (brand: abcam, catalog number: ab134073), FTO (brand: Santa Cruz, catalog number: sc-271713), GCA (brand: Santa Cruz, catalog number: sc-365808), MAD1L1 (brand: Santa Cruz, catalog number: sc-376613), and CD63 (brand: Santa Cruz, catalog number: sc-5275).

[0085] 1) Determination of protein expression levels

[0086] Refer to the "User Guide for Wes" of ProteinSimple company at https: / / www.bio-techne.com / pdf-download-arena-document / user-manual / 031-108 / 25, as well as the "Separation Module Jess Abby & Wes SM W001 to SM W012" at https: / / www.bio-techne.com / pdf-download-arena-document / product-insert / pl3-0005 for operation.

[0087] 2) Experimental results

[0088] Figure 1A and Figure 1B (1) to (10) in show the results of the expression levels of B2M, TXN, PRDX5, PRTN3, DEFA3, FCGR1A, FTO, GCA, MAD1L1, and CD63 in different blood samples.

[0089] From Figure 1A and Figure 1B the experimental results in, it can be seen that compared with the HC population, the expression levels of B2M, TXN, and PRDX5 are significantly increased in Mycobacterium tuberculosis-infected individuals (ATB and LTBI), while the expression levels of other proteins show no significant difference between the two groups. Therefore, by detecting the expression level of at least one of B2M, TXN, and PRDX5 in peripheral blood neutrophils, Mycobacterium tuberculosis-infected individuals (ATB and LTBI) can be distinguished from the HC population.

[0090] 2. Diagnostic efficacy analysis of Mycobacterium tuberculosis infection molecular marker detection

[0091] 2.1. Inclusion and exclusion criteria for research subjects

[0092] In this study, 319 validation subjects (including 71 cases in the ATB group, 142 cases in the LTBI group, and 106 cases in the HC group) were selected for quantitative analysis of the expression levels of target proteins and statistical analysis of inter-group differences.

[0093] Inclusion criteria: The diagnostic criteria for active pulmonary tuberculosis (ATB) patients included in this study were based on the "Diagnosis of Tuberculosis, Health Industry Standard of the People's Republic of China (WS 288—2017)", and the pathogen detection of sputum or bronchoalveolar lavage fluid specimens was positive (at least one of smear / culture / nucleic acid detection was positive), with no previous history of tuberculosis (no old tuberculosis lesions detected by medical history and X-ray chest radiography), and primary anti-tuberculosis treatment with less than 7 days of medication.

[0094] The inventors screened latent Mycobacterium tuberculosis infection (LTBI) patients and non-infected (HC) controls using the interferon-γ release assay, and performed the detection according to the instructions of the QuantiFERON-TB Gold Plus kit from QIAGEN, Germany. LTBI refers to those without a past history of tuberculosis and related clinical manifestations, normal chest X-ray, and positive interferon-γ release assay; HC refers to those without a past history of tuberculosis and related clinical manifestations, normal chest X-ray, and negative interferon-γ release assay.

[0095] All of the above study subjects were excluded: those aged over 65 or under 18, pregnant or lactating women, diabetic patients, patients with malignant tumors, patients with immune system diseases or those receiving immunotherapy, and those infected with human immunodeficiency virus or other pathogens. This study was approved by the Ethics Committee of the Institute of Pathogen Biology, Chinese Academy of Medical Sciences. After obtaining the informed consent of the enrolled subjects, the inventors collected their peripheral blood for subsequent experiments.

[0096] Table 2 Sample Demographic Information

[0097]

[0098] 2.2 Sorting and Enrichment of Peripheral Blood Neutrophils

[0099] 1.5 mL of peripheral blood from the study subjects was collected in an EDTA anticoagulant tube, and neutrophils were sorted from the whole blood. The specific operation steps are as follows:

[0100] 1) Prepare flow cytometry tubes, write the numbers (numbered in the way of group name plus number, for example, ATB-001), and add 3 mL of separation buffer to each flow cytometry tube;

[0101] 2) Mix the blood evenly, aspirate 1.5 mL and transfer it to the corresponding numbered flow cytometry tube, and mix by pipetting;

[0102] 3) Centrifuge at 600 g for 10 minutes at 4°C;

[0103] 4) Carefully aspirate and discard the supernatant, avoiding touching the surface of the cell pellet, and leave about 0.5 mL of liquid;

[0104] 5) Add separation buffer to 1.5 mL and mix gently;

[0105] 6) Vortex the CD15 magnetic beads evenly and quickly add 40 μL / sample to the diluted blood;

[0106] 7) Tighten the lid, place the flow cytometry tube properly on a rotary mixer (Hula Mixer), and incubate with rotation at 8 rpm for 20 minutes;

[0107] 8) Take out the incubated cells, centrifuge them transiently, let them stand on the magnetic stand for 2 minutes, and carefully aspirate and discard the supernatant;

[0108] 9) Remove the flow cytometry tube, add 1.6 mL of separation buffer, gently blow and mix, and transfer it to the corresponding 2 mL protein low-binding tube; let it stand on the magnetic stand for 2 minutes, and aspirate and discard the supernatant;

[0109] 10) Remove it from the magnetic stand, add 1.6 mL of separation buffer, gently blow and mix, let it stand on the magnetic stand for 2 minutes, aspirate the supernatant, and repeat once.

[0110] 11) Add 20 µL of protease inhibitor solution, gently mix, and store at -80 °C.

[0111] 2.3 Extraction and quantification of total protein

[0112] 1) Take out the cell samples obtained in step 2.2, add RIPA lysis buffer (Beyotime, catalog number: P0013B) with the same volume as the sample volume to each sample, add an appropriate amount of grinding beads, and run in a cryogenic grinder (Shanghai Jingxin, product number: JXFSTPRP-CL-BSC) according to the following parameters: 65 Hz, 50 times, run for 20 s and stop for 10 s, at 4 °C;

[0113] 2) Centrifuge the samples at 15000 g for 10 minutes at 4 °C, collect the supernatant, take a small amount of protein supernatant, and measure the concentration of each sample using a BCA protein assay kit;

[0114] 3) Automatic protein immunoblotting experiment

[0115] Perform protein immunoblotting experiment operation and process the off-machine data according to the operation manual of the WES TM Automatic Protein Expression Analysis System. The experimental data is shown in Table 3 below. The integrated value of the protein immunoblotting chemiluminescence signal within the signal region of the target protein band reflects its overall expression level, and is visualized through the built-in algorithm of the Compass TM software, displayed as a virtual blot or peak, quantified as the area under the curve and the concentration is calculated.

[0116] Table 3

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129] It should be noted that according to the solution of this application, the diagnostic accuracy reaches 0.9810. Taking the case of the combination of B2M antibody and TXN antibody as an example, although some experimental examples do not achieve the expected effect, this is only an individual difference and does not affect the overall reliability and effectiveness of the solution of this application.

[0130] 2.4. Experimental Results

[0131] Through total protein normalization, the relative expression levels of target proteins were calculated. Kruskal-Wallis one-way analysis of variance was used for statistics among three groups, and Dunn's test was used for pairwise comparison among multiple groups. The positive rates of three target proteins, B2M, TXN, and PRDX5, were relatively high, and the results of their Western blot are as Figure 2 shown. Statistical analysis showed that there were statistically significant differences in the levels of B2M, TXN, and PRDX5 in neutrophils of the ATB group, LTBI group, and HC group populations. Among them, the ATB group was significantly higher than the HC group (P value < 0.0001), and the LTBI group was also significantly higher than the HC group (P value < 0.0001). There was no statistically significant difference between the ATB group and the LTBI group (P value > 0.05), as Figure 3 shown. Overall, the results of Western blot were basically consistent with the results of proteomic data, indicating that the analysis results of Western blot were accurate and reliable.

[0132] 2.5. Analysis of Diagnostic Efficacy

[0133] Western blot quantitative analysis confirmed the association between the expressed proteins screened by proteomics analysis and the Mycobacterium tuberculosis infection status. Further, Logistic binary regression analysis was applied to plot the receiver operating characteristic curve (ROC curve) to comprehensively evaluate the diagnostic value of the detection of three protein biomarkers, B2M, TXN, and PRDX5, in Mycobacterium tuberculosis infection.

[0134] The specific method is as follows:

[0135] For the analysis of the combined diagnostic efficacy of proteins, in SPSS, a linear combination was constructed through Logistic binary regression analysis and transformed into a probability value through a logistic function, and the ROC curve was plotted using the probability value. The experimental data are shown in Table 4 below. The study showed that the AUC was between 0.93 and 0.99, indicating a relatively high diagnostic accuracy. In addition, the combination of multiple proteins as diagnostic markers can improve the diagnostic accuracy, specificity, and sensitivity, while adapting to individual differences and disease complexity.

[0136] The cutoff value in the table is the threshold for judging a positive result. For example, when both the B2M concentration > 13.43 ng / mL and the TXN concentration > 261.00 ng / mL are satisfied, the patient can be diagnosed as having active pulmonary tuberculosis. At this time, the diagnostic accuracy reaches 0.9810, the sensitivity is 95.77%, and the specificity is 91.51%.

[0137] Table 4: Diagnostic efficacy of different protein combinations in differentiating ATB, LTBI, and HC

[0138]

[0139] Figure 4 The ROC curve graph showing the evaluation of the diagnostic efficacy of protein combinations selected from two of B2M, TXN, and PRDX5 is shown.

[0140] Refer to Figure 4The results of the diagnostic efficacy analysis for differentiating ATB patients from the HC population showed that the combination of the two proteins, B2M and TXN, had the best diagnostic efficacy, with an AUC of 0.9810 (95% CI: 0.9650 - 0.9970), a sensitivity of 95.77%, and a specificity of 91.51%; the results of the diagnostic efficacy analysis for differentiating LTBI patients from the HC population showed that the combination of the two proteins, TXN and PRDX5, had the best diagnostic efficacy, with an AUC of 0.9865 (95% CI: 0.9722 - 1.000), a sensitivity of 97.99%, and a specificity of 95.28%; the results of the diagnostic efficacy analysis for differentiating Mycobacterium tuberculosis-infected patients from the HC population showed that the combination of the two proteins, B2M and TXN, had the best diagnostic efficacy, with an AUC of 0.9849 (95% CI: 0.9748 - 0.9950), a sensitivity of 97.79%, and a specificity of 92.45%.

[0141] Figure 5 The ROC curve graph showing the evaluation of the diagnostic efficacy of the protein combination of B2M, TXN, and PRDX5 is presented.

[0142] Refer to Figure 5 ,the results of the diagnostic efficacy analysis for differentiating LTBI patients from the HC population showed that, compared with different protein combinations, the combination of the three proteins, B2M, TXN, and PRDX5, had the best diagnostic efficacy, with an AUC of 0.9866 (95% CI: 0.9724 - 1.000), a sensitivity of 97.99%, and a specificity of 95.28%.

[0143] In summary, the results of this study showed that the combined detection of the two proteins, B2M and TXN, could effectively differentiate ATB patients from the HC population, and the combined detection of the two proteins, B2M and TXN, could effectively differentiate Mycobacterium tuberculosis-infected patients (including ATB and LTBI) from the HC population; the combined detection of the three proteins, B2M, TXN, and PRDX5, could effectively differentiate LTBI patients from the HC population.

[0144] The advantages of the combination of B2M, TXN, and PRDX5 are mainly reflected in the following aspects:

[0145] (1) Higher specificity

[0146] The specificity of the combination of B2M, TXN, and PRDX5 was 96.23%, significantly higher than that of B2M combined with TXN (91.51%) and B2M combined with PRDX5 (93.40%). Higher specificity means that it can more accurately exclude the non-infected population and reduce the possibility of misdiagnosis.

[0147] (2) Better comprehensive performance

[0148] Although the AUC value of the combination of B2M and TXN is slightly higher (0.9810), its specificity is relatively low (91.51%). In practical applications, high specificity is particularly important for reducing misdiagnosis. The combination of B2M, TXN, and PRDX5 achieves a better balance between sensitivity (94.37%) and specificity (96.23%), with better comprehensive performance.

[0149] (3) Coping with disease complexity

[0150] The pathological mechanism of active pulmonary tuberculosis is complex, and single or dual protein combinations may not comprehensively cover all cases. By integrating multiple biomarkers, the combination of B2M, TXN, and PRDX5 can better cope with individual differences and disease heterogeneity, providing more comprehensive diagnostic information.

[0151] Additionally, compared with single proteins, multi-protein combinations (such as the combination of B2M, TXN, and PRDX5) have the following significant advantages:

[0152] (1) Reducing the limitations of single proteins

[0153] Single proteins may be affected by other diseases or physiological states, leading to misdiagnosis. For example, B2M may be elevated in other diseases such as chronic lymphocytic leukemia and acute kidney injury. TXN may change in certain lung diseases such as asthma and chronic obstructive pulmonary disease, and PRDX5 may change in certain cancers such as endometrial cancer and oral squamous cell carcinoma. Through multi-protein combinations, mutual verification can be carried out to reduce the limitations of single proteins and improve the specificity of diagnosis.

[0154] (2) Improving the comprehensiveness of diagnosis

[0155] Single proteins may not be able to comprehensively reflect the complexity of diseases, while multi-protein combinations can provide diagnostic information from different perspectives. For example, some patients may not show obvious changes in one protein (such as B2M), but have significant changes in other proteins (such as TXN or PRDX5). Multi-protein combinations can make up for the deficiencies of single proteins and improve sensitivity.

[0156] (3) Adapting to individual differences

[0157] The pathological states of different patients may vary, and single proteins may not cover all cases. Multi-protein combinations can better adapt to individual differences and improve the universality and reliability of diagnosis.

[0158] According to the present invention, a diagnostic marker comprising at least two of B2M, TXN, and PRDX5 can effectively distinguish patients with active tuberculosis (ATB), latent tuberculosis infection (LTBI) individuals, and / or non-infected (HC) populations, and has good sensitivity and specificity. However, the present disclosure is not limited to these aspects and features.

[0159] The above content is an illustration of some embodiments of the present disclosure and will not be construed as a limitation on the present disclosure. Although some embodiments have been described, those skilled in the art will readily understand that various modifications can be made to the embodiments without departing from the spirit and scope of the present disclosure.

Claims

1. Use of a specific antibody in the preparation of a diagnostic reagent for diagnosing Mycobacterium tuberculosis infection, wherein: The specific antibodies include at least two of B2M antibody, TXN antibody and PRDX5 antibody, The diagnostic reagent is used to distinguish active pulmonary tuberculosis patients from non-infected people or to distinguish latently infected Mycobacterium tuberculosis patients from non-infected people.

2. The use according to claim 1, wherein: The specific antibody can correspond to the diagnostic markers in the sample to be detected, and the diagnostic markers include at least two of B2M, TXN and PRDX5.

3. The use according to claim 2, wherein: The diagnostic markers are derived from peripheral blood neutrophils.

4. The use according to claim 1, wherein: The specific antibodies include B2M antibody and TXN antibody; or, the specific antibodies include B2M antibody and PRDX5 antibody; or, the specific antibodies include TXN antibody and PRDX5 antibody.

5. The use according to claim 1, wherein: The specific antibodies include B2M antibody, TXN antibody and PRDX5 antibody.

6. The use according to claim 1, wherein: The diagnostic reagent is used for preparing a diagnostic chip or a detection device for Mycobacterium tuberculosis infection.

7. A diagnostic kit for diagnosing Mycobacterium tuberculosis infection, wherein: The diagnostic kit comprises the specific antibody according to any one of claims 1 to 6.

8. The diagnostic kit according to claim 7, wherein The specific antibody is a monoclonal antibody.

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