Application of specific antibody in preparation of diagnostic reagent for diagnosing mycobacterium tuberculosis infection and diagnostic kit

Abstract

The invention provides application of a specific antibody to preparation of a diagnostic reagent for diagnosing mycobacterium tuberculosis infection and a diagnostic kit, and the specific antibody comprises at least two of a B2M antibody, a TXN antibody and a PRDX5 antibody. The diagnostic reagent provided by the invention can effectively distinguish active tuberculosis (ATB) patients, mycobacterium tuberculosis latent infection (LTBI) patients and / or non-infected (HC) people, and has good sensitivity and specificity.

Description

Technical Field

[0001] The present invention relates to a diagnostic marker and a specific antibody thereof, in particular to the use of the specific antibody in preparing a diagnostic reagent for diagnosing mycobacterium tuberculosis infection and a diagnostic kit comprising the specific antibody. Background Art

[0002] Tuberculosis is a chronic infectious disease caused by infection with Mycobacterium tuberculosis, which mainly affects the lungs and can also invade other organs, seriously endangering human health. It is estimated that about 1 / 4 of the world's population is infected with Mycobacterium tuberculosis, most of whom are in a latent state after infection, and about 10% of 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 being simple, rapid and easy to use, but its positive rate is low and its 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 it takes a long time, which greatly limits the positive detection rate of tuberculosis patients. The application of molecular biology technology is a revolutionary progress in the diagnosis of tuberculosis. It has the advantages of accuracy and efficiency, but it has not fundamentally changed the current situation of difficult diagnosis of tuberculosis. The technology has potential contamination risks and may lead to false positive results. The diagnostic performance in smear-negative specimens is still poor. The high detection cost and relatively complex operation methods make these technologies difficult to promote in countries with low and medium development levels. In addition, most of the above detection methods are based on sputum specimens that are not easy to obtain. Therefore, it is urgent to develop new clinical diagnosis methods for tuberculosis. Summary of the invention

[0004] The purpose of the present invention is to provide a diagnostic marker and / or its specific antibody for use in preparing a diagnostic reagent for diagnosing Mycobacterium tuberculosis infection and a diagnostic kit comprising the diagnostic marker and / or its specific antibody. The diagnostic marker of the present invention can effectively distinguish between active pulmonary tuberculosis (ATB) patients, Mycobacterium tuberculosis latently infected (LTBI) patients and / or non-infected (HC) people, and has good sensitivity and specificity.

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

[0006] According to some embodiments, 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.

[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; or, the specific antibodies include B2M antibody and PRDX5 antibody; or, 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 to prepare a diagnostic chip or a detection device for Mycobacterium tuberculosis infection.

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

[0012] According to some embodiments, the diagnostic reagent is also used to distinguish between people who are latently infected with Mycobacterium tuberculosis and non-infected people.

[0013] An embodiment of the present invention relates to a diagnostic kit for diagnosing Mycobacterium tuberculosis infection, wherein the diagnostic kit comprises specific antibodies.

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

[0015] The above and other objects and features of the present invention will become more apparent through the following description in conjunction with the accompanying drawings which exemplarily illustrate an example, in which: Figure 1A and Figure 1B The results of the expression levels of B2M, TXN, PRDX5, PRTN3, DEFA3, FCGR1A, FTO, GCA, MAD1L1, and CD63 in different blood samples are shown (normalized to total protein); Figure 2 Western blotting results of B2M, TXN, and PRDX5 in different blood samples are shown; Figure 3 Comparison of B2M, TXN, and PRDX5 protein levels in patients with active pulmonary tuberculosis (ATB), those with latent Mycobacterium tuberculosis infection (LTBI), and non-infected (HC) populations is shown; Figure 4 The ROC curve diagram of the diagnostic efficacy evaluation of the protein combination of two selected from B2M, TXN and PRDX5 is shown; Figure 5 A ROC curve graph for the diagnostic efficacy evaluation of the combination of B2M, TXN, and PRDX5 is shown. DETAILED DESCRIPTION

[0016] In the following description, for the purpose of explanation, many specific details are set forth to provide a thorough understanding of various embodiments or implementations of the invention. As used herein, "embodiment" and "implementation" are interchangeable terms and are non-limiting examples of devices or methods that employ 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, the various embodiments may be different, but need not be exclusive.

[0017] It should be understood that when the terms “include” and / or “comprises” are used in the specification, they list the presence of stated materials and / or components, but do not exclude the presence or addition of one or more other materials and / or components.

[0018] 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 is a part. Terms (such as those defined in general dictionaries) should be interpreted as having a meaning consistent with their meaning in the context of the relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0019] The term "diagnostic marker" as used in the present invention refers to a molecule to be used as a target for analyzing a patient's experimental sample. Examples of such molecular targets are proteins or polypeptides. Proteins or polypeptides used as markers in the present invention are expected to include naturally occurring variants of the protein and fragments of the protein or the variant, particularly immunologically detectable fragments. Immunologically detectable fragments preferably contain at least 5, 6, 7, 8, 9, 10, 11, 12, 15 or 20 consecutive amino acids of the marker polypeptide.

[0020] Those skilled in the art will recognize that proteins released by cells or present in the extracellular matrix may be damaged (e.g., during inflammation) and may be degraded or cut into such fragments. Certain markers are synthesized in an inactive form, which may be subsequently activated by proteolysis. As will be appreciated by the skilled artisan, proteins or fragments 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. In addition, or in an alternative, the marker polypeptide or its variant may carry a post-translational modification. Non-limiting examples of post-translational modifications are glycosylation, acylation and / or phosphorylation.

[0021] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

[0022] The present inventors have discovered for the first time that the expression levels of at least one of beta-2 microglobulin (B2M), thioredoxin (TXN) and peroxiredoxin 5 (PRDX5) in peripheral blood neutrophils are significantly different in patients infected with Mycobacterium tuberculosis (patients with active pulmonary tuberculosis, patients latently infected with Mycobacterium tuberculosis) and non-infected people. Specifically, the expression level in patients infected with Mycobacterium tuberculosis is significantly higher than that in non-infected people. Therefore, by detecting the content of at least one of beta-2 microglobulin, thioredoxin and peroxiredoxin 5 in the blood, patients infected with Mycobacterium tuberculosis can be effectively distinguished from non-infected people.

[0023] β-2 microglobulin (B2M) is a polypeptide of approximately 13 kDa that exists in serum and is a component of the class I major histocompatibility complex (MHC) that participates in the presentation of peptide antigens to the immune system. The amino acid sequence of β-2 microglobulin is: MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM.

[0024] Thioredoxin (TXN), also abbreviated as Trx1, belongs to the thioredoxin family. It is mainly located in the cytoplasm, and can also be translocated to the nucleus or secreted outside the cell. This protein is an essential antioxidant protein released by cells under stress, which can protect cells from oxidative damage caused by microbial invasion and physical and chemical stimulation. The amino acid sequence of thioredoxin (TXN) is: MVKQIESKTAFQEALDAAGDKLVVVDFSATWCGPCKMIKPFFHSLSEKYSNVIFLEVDVDDCQDVASECEVKCMPTFQFFKKGQKVGEFSGANKEKLEATINELV.

[0025] Peroxiredoxin 5 (PRDX5) belongs to the peroxiredoxin group and is an antioxidant enzyme that scavenges cellular peroxide substrates to protect cells from oxidative stress. The amino acid sequence of peroxiredoxin 5 (PRDX5) is: MGLAGVCALRRRSAGYILVGGAGGQSAAAAARRYSEGEWASGGVRSFSRAAAAMAPIKVGDAIPAVEVFEGEPGNKVNLAELFKGKKGVLFGVPGAFTPGCSKTHLPG FVEQAEALKAKGVQVVACLSVNDAFVTGEWGRAHKAEGKVRLLADPTGAFGKETDLLLDDSLVSIFGNRRLKRFSMVVQDGIVKALNVEPDGTGLTCSLAPNIISQL.

[0026] According to a first aspect of the present invention, a diagnostic kit for diagnosing Mycobacterium tuberculosis infection is provided. The diagnostic kit for diagnosing Mycobacterium tuberculosis infection may include specific antibodies corresponding to diagnostic markers of at least two of beta-2 microglobulin (B2M), thioredoxin (TXN), and peroxiredoxin 5 (PRDX5). Here, the specific antibody corresponding to the diagnostic marker of at least one of beta-2 microglobulin, thioredoxin, and peroxiredoxin 5 is an antibody that can specifically bind to at least one of beta-2 microglobulin, thioredoxin, and peroxiredoxin 5. They may also be referred to as beta-2 microglobulin antibody (B2M antibody), thioredoxin antibody (TXN antibody), or peroxiredoxin 5 antibody (PRDX5 antibody), respectively. These specific antibodies can specifically bind to the above proteins, respectively, and are thus used for the diagnosis of Mycobacterium tuberculosis infection. Preferably, the diagnostic kit may include specific antibodies corresponding to B2M, TXN and PRDX5, ie, beta-2 microglobulin antibody (B2M antibody), thioredoxin antibody (TXN antibody) and peroxiredoxin 5 antibody (PRDX5 antibody).

[0027] According to an embodiment of the present invention, the diagnostic kit may further include a quantitative detection agent for a diagnostic marker. According to an embodiment, the quantitative detection agent may be a specific antibody for the above-mentioned diagnostic marker, but the embodiment is not limited thereto. In an 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.

[0028] 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 signal strength.

[0029] According to an embodiment of the present invention, the diagnostic kit may further comprise at least one of a solid phase carrier, a blocking solution, a color developer, a calibrator of the diagnostic marker fusion antigen, and a washing buffer.

[0030] According to an embodiment of the present invention, the above diagnostic reagent is used to prepare a diagnostic chip or a detection device for Mycobacterium tuberculosis infection, but the embodiment is not limited thereto.

[0031] 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, especially 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.

[0032] The diagnostic reagent may include specific antibodies that correspond to specific proteins produced during the infection of Mycobacterium tuberculosis, so that the diagnostic reagent can use the specific antibodies to bind to diagnostic markers in the collected sample to indicate whether the sample is infected with Mycobacterium tuberculosis. B2M antibodies, TXN antibodies, and PRDX5 antibodies may also be referred to as anti-B2M antibodies, anti-TXN antibodies, and anti-PRDX5 antibodies.

[0033] According to the use of the specific antibodies provided in the embodiments of the present application in the preparation of diagnostic reagents for diagnosing Mycobacterium tuberculosis infection, when in use, when a diagnostic marker or antigen (i.e., β-2 microglobulin, thioredoxin or peroxiredoxin 5) corresponding to the specific antibody is present in the collected sample, the diagnostic marker and the specific antibody will specifically bind to form a diagnostic marker-antibody complex, and then the diagnostic marker-antibody complex is converted into the concentration of the diagnostic marker in the sample to be detected, thereby obtaining the concentration of the diagnostic marker in the sample to be detected. The obtained concentration of the diagnostic marker can reflect the actual level of the marker in the body, so the difference in concentration can be used to distinguish people in different states.

[0034] In some embodiments, the specific antibody can correspond to the diagnostic markers in the sample to be detected, and correspondingly, the diagnostic markers include at least two of B2M, TXN and PRDX5.

[0035] In these embodiments, the diagnostic markers in the collected samples can specifically bind to the specific antibodies, so that active tuberculosis patients and non-infected people can be determined based on the content of the markers. Since there is a high degree of specific binding between the specific antibodies and the diagnostic markers, the possibility of misdiagnosis and missed diagnosis can be reduced.

[0036] In some embodiments, the diagnostic marker is derived from peripheral blood neutrophils.

[0037] In these embodiments, on the one hand, diagnostic markers can be obtained through collection, and on the other hand, the blood collected from peripheral blood neutrophils can be purified to obtain at least two of B2M, TXN and PRDX5 to further improve the accuracy of subsequent detection.

[0038] In some embodiments, the specific antibodies include B2M antibody, TXN antibody and PRDX5 antibody.

[0039] In some embodiments, the diagnostic reagent is used to prepare a diagnostic chip or detection device for Mycobacterium tuberculosis infection.

[0040] In some embodiments, the diagnostic reagent is used at least to distinguish patients with active tuberculosis from non-infected people.

[0041] In some embodiments, the diagnostic reagent is also used to distinguish between people who are latently infected with Mycobacterium tuberculosis and non-infected people.

[0042] In these embodiments, there is usually a high level of diagnostic marker in active pulmonary tuberculosis patients. Therefore, the concentration of the diagnostic marker measured can significantly increase. There may be a medium level of diagnostic marker in the latent infection of Mycobacterium tuberculosis, but no obvious inflammatory reaction has been caused. Therefore, the concentration of the diagnostic marker measured may be at a medium level, and here, the medium 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 people. There is no relevant marker in the non-infected people or it is at an extremely low level, so the concentration of the diagnostic marker measured should be at an extremely low level or cannot be detected.

[0043] 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 to be a patient with active pulmonary tuberculosis, and 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, they are determined to be a non-infected population, thereby achieving at least the purpose of distinguishing active pulmonary tuberculosis patients from non-infected populations, and its diagnostic accuracy (AUC = 0.9810), sensitivity (95.77%) and specificity (91.51%) all performed well.

[0044] In 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, the patient is determined to be an active pulmonary tuberculosis patient, and 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, the patient is determined to be a non-infected population, thereby achieving at least the purpose of distinguishing active pulmonary tuberculosis patients from non-infected populations. Its diagnostic accuracy (AUC = 0.9799), sensitivity (91.55%) and specificity (93.40%) also reached a high level.

[0045] In 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, the patient is determined to be an active pulmonary tuberculosis patient, and 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, the patient is determined to be a non-infected population, thereby achieving at least the purpose of distinguishing active pulmonary tuberculosis patients from non-infected populations. Its diagnostic accuracy (AUC = 0.9720), sensitivity (92.96%) and specificity (94.34%) are all good.

[0046] 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, the diagnostic marker is determined to be derived 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 to be a non-infected population, thereby achieving at least the purpose of distinguishing active pulmonary tuberculosis patients from non-infected populations. Through this method, its diagnostic accuracy (AUC = 0.9794), sensitivity (94.37%) and specificity (96.23%) reach the optimal level, which can more effectively cope with individual differences and disease complexity, and significantly improve the reliability of diagnosis.

[0047] The present invention is further described below in conjunction with specific embodiments.

[0048] 1. Determination of molecular markers of host infection with Mycobacterium tuberculosis 1.1. Inclusion and Exclusion Criteria of Research Subjects The diagnostic criteria for active pulmonary tuberculosis (ATB) patients included in this study were based on the "Health Industry Standard of the People's Republic of China (WS 288-2017) Diagnosis of Pulmonary Tuberculosis" and had positive etiological tests for sputum or bronchoalveolar lavage fluid specimens (at least one of the three smear / culture / nucleic acid tests was positive), no previous history of tuberculosis (no old tuberculosis lesions in the interview and X-ray chest film examination), and first anti-tuberculosis treatment for less than 7 days.

[0049] The inventors used the interferon-γ release test to screen people with latent tuberculosis infection (LTBI) and non-infected (HC) controls, and the test was performed according to the instructions of the QuantiFERON-TB Gold Plus kit of QIAGEN, Germany. LTBI refers to no history of tuberculosis and related clinical manifestations, no abnormalities in the chest X-ray, and a positive interferon-γ release test; HC refers to no history of tuberculosis and related clinical manifestations, no abnormalities in the chest X-ray, and a negative interferon-γ release test.

[0050] The above subjects were excluded: those aged 65 years or younger than 18 years, pregnant or lactating women, diabetic patients, patients with malignant tumors, patients with immune system diseases or receiving immunotherapy, and those infected with human immunodeficiency virus or other pathogens. This study was approved by the Ethics Committee of the Institute of Pathogenic Biology, Chinese Academy of Medical Sciences. After obtaining informed consent from the subjects, the inventors drew peripheral blood from them for subsequent experiments. The demographic information of the samples is shown in Table 1.

[0051] Table 1

[0052] 1.2. Peripheral blood neutrophil sorting and enrichment 1.5 mL of peripheral blood was collected from the subjects using EDTA anticoagulant tubes, and neutrophils were sorted out from the whole blood. The specific steps are as follows: 1) Prepare the flow tubes, write the numbers (numbered by group name plus number, for example, ATB-001), and add 3 mL of separation buffer to each flow tube; 2) Mix the blood and pipette 1.5 mL into the corresponding numbered flow tube, and mix by pipetting; 3) Centrifuge at 4°C, 600 g for 10 min; 4) Carefully discard the supernatant, avoiding touching the surface of the cell pellet, leaving about 0.5 mL of liquid; 5) Add separation buffer up to 1.5 mL and mix gently; 6) Vortex to mix CD15 magnetic beads and quickly add 40 µL / sample to the diluted blood; 7) Close the lid tightly, place the flow tube properly on the Hula Mixer, and rotate and incubate at 8 rpm for 20 minutes; 8) Take out the incubated cells, centrifuge them briefly, place them on a magnetic rack (Invitrogen™, product number: 12321D) for 2 minutes, and carefully discard the supernatant; 9) Remove the flow tube, add 1.6 mL of separation buffer, blow gently to mix well and transfer to the corresponding 2 mL protein low adsorption tube; let it stand on the magnetic stand for 2 minutes and discard the supernatant; 10) Remove from the magnetic stand, add 1.6 mL of separation buffer, blow gently to mix, let stand on the magnetic stand for 2 minutes, aspirate the supernatant, and repeat once.

[0053] 11) Add 20 µL of protease inhibitor solution, mix gently, and store at -80°C.

[0054] 1.3 Total protein extraction and mass spectrometry identification 1) Take out the cell sample obtained in step 1.2 and add SDS buffer (4% SDS, 100 mM Tris-HCl, pH 7.6); 2) sonication (30 s per cycle, 10 cycles) and boiling at 95 °C for 15 min; 3) After centrifugation at 14000g for 40 min, the protein content of the supernatant was determined using a BCA protein assay kit; 4) Take 20 µg of protein from each sample, mix with 5× loading buffer, and boil for 5 minutes. Separate the proteins on a 4%~20% SDS-PAGE gel (constant voltage 180V, 45 minutes). Visualize the protein bands with Coomassie brilliant blue; 5) Take an equal amount from each sample in this experiment and mix them into one sample for the generation and quality control of the DDA library (data-dependent acquisition based spectral library).

[0055] 6) Mass spectrometry was performed on the samples to obtain the protein expression profiles of peripheral blood neutrophils in ATB, LTBI and HC populations, and proteins significantly associated with the infection status of Mycobacterium tuberculosis were screened. The number of unique peptides of the three proteins B2M, TXN and PRDX5 was greater than 2, indicating that the identification results were highly reliable.

[0056] 1.4 Evaluation of protein expression content The inventors used the method of western blotting to determine the protein content of B2M, TXN and PRDX5 in different samples, wherein commercially available B2M (brand: Near Shore Protein, catalog number: Recombinant Human B2M (N-6His)(Cat.No.:C512)), TXN (brand: Near Shore Protein, catalog number: Recombinant Human TXN (N-6His)(Cat.No.:CE85)) and PRDX5 (brand: Near Shore Protein, 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). In addition, through proteomic analysis, the inventors screened out 7 proteins that were significantly associated with the infection status of Mycobacterium tuberculosis as comparative examples. The 7 proteins are: myeloblastin (PRTN3), neutrophil defensin 3 (DEFA3), high affinity immunoglobulin gamma Fc receptor I (FCGR1A), α-ketoglutarate-dependent dioxygenase FTO (FTO), granular calcium protein (GCA), mitotic spindle assembly checkpoint protein MAD1 (MAD1L1) and CD63 antigen (CD63). 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).

[0057] 1) Determination of protein expression level Refer to the User Guide for Wes from Proteinsimple https: / / www.bio-techne.com / pdf-download-arena-document / user-manual / 031-108 / 25 and the Separation Module Jess Abby&Wes SM W001 to SM W012 https: / / www.bio-techne.com / pdf-download-arena-document / product-insert / pl3-0005 operation manual for operation.

[0058] 2) Experimental results Figure 1A and Figure 1B (1) to (10) show the results of the expression levels of B2M, TXN, PRDX5, PRTN3, DEFA3, FCGR1A, FTO, GCA, MAD1L1 and CD63 in different blood samples, respectively.

[0059] from Figure 1A and Figure 1B The experimental results in show that compared with the HC population, the expression levels of B2M, TXN and PRDX5 in M. tuberculosis infected people (ATB and LTBI) are significantly increased, while the expression levels of other proteins are basically the same between the two groups. Therefore, by detecting the expression level of at least one of B2M, TXN and PRDX5 in peripheral blood neutrophils, M. tuberculosis infected people (ATB and LTBI) and HC populations can be distinguished.

[0060] 2. Analysis of the diagnostic efficacy of molecular marker detection for Mycobacterium tuberculosis infection 2.1. Inclusion and Exclusion Criteria of Research Subjects In this study, 319 validation populations (including 71 cases in the ATB group, 142 cases in the LTBI group, and 106 cases in the HC group) were selected to quantitatively analyze the expression of the target protein and perform statistical analysis of the differences between the groups.

[0061] Inclusion criteria: The diagnostic criteria for active pulmonary tuberculosis (ATB) patients included in this study were based on the "Health Industry Standard of the People's Republic of China (WS 288-2017) Diagnosis of Pulmonary Tuberculosis" and had positive etiological tests for sputum or bronchoalveolar lavage fluid specimens (at least one of the three smear / culture / nucleic acid tests was positive), no previous history of tuberculosis (no old tuberculosis lesions in the interview and X-ray chest film examination), and first anti-tuberculosis treatment for less than 7 days.

[0062] The inventors used the interferon-γ release test to screen people with latent tuberculosis infection (LTBI) and non-infected (HC) controls, and the test was performed according to the instructions of the QuantiFERON-TB Gold Plus kit of QIAGEN, Germany. LTBI refers to no history of tuberculosis and related clinical manifestations, no abnormalities in the chest X-ray, and a positive interferon-γ release test; HC refers to no history of tuberculosis and related clinical manifestations, no abnormalities in the chest X-ray, and a negative interferon-γ release test.

[0063] The above research subjects were excluded: those aged 65 years or younger than 18 years, 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 Pathogenic Biology, Chinese Academy of Medical Sciences. After obtaining informed consent from the subjects, the inventors drew peripheral blood from them for subsequent experiments.

[0064] Table 2 Sample demographic information

[0065] 2.2. Peripheral blood neutrophil sorting and enrichment 1.5 mL of peripheral blood was collected from the subjects using EDTA anticoagulant tubes, and neutrophils were sorted out from the whole blood. The specific steps are as follows: 1) Prepare the flow tubes, write the numbers (numbered by group name plus number, for example, ATB-001), and add 3 mL of separation buffer to each flow tube; 2) Mix the blood and pipette 1.5 mL into the corresponding numbered flow tube, and mix by pipetting; 3) Centrifuge at 4°C, 600 g for 10 min; 4) Carefully discard the supernatant, avoiding touching the surface of the cell pellet, leaving about 0.5 mL of liquid; 5) Add separation buffer up to 1.5 mL and mix gently; 6) Vortex to mix CD15 beads and quickly add 40 µL / sample to the diluted blood; 7) Close the lid tightly, place the flow tube properly on the Hula Mixer, and rotate and incubate at 8 rpm for 20 minutes; 8) Take out the incubated cells, centrifuge them instantly, place them on a magnetic rack for 2 minutes, and carefully discard the supernatant; 9) Remove the flow tube, add 1.6 mL of separation buffer, blow gently to mix well and transfer to the corresponding 2 mL protein low adsorption tube; let it stand on the magnetic stand for 2 minutes and discard the supernatant; 10) Remove from the magnetic stand, add 1.6 mL of separation buffer, blow gently to mix, let stand on the magnetic stand for 2 minutes, aspirate the supernatant, and repeat once.

[0066] 11) Add 20 µL of protease inhibitor solution, mix gently, and store at -80°C.

[0067] 2.3. Extraction and quantification of total protein 1) Take out the cell samples obtained in step 2.2, add RIPA lysis buffer (Biyuntian, product number: P0013B) with the same volume as the sample to each sample, add appropriate amount of grinding balls, and run in a freezing grinder (Shanghai Jingxin, product number: JXFSTPRP-CL-BSC) according to the following parameters: 65 Hz, 50 times, 20 s running, 10 s stopping, 4°C; 2) Centrifuge the samples at 15000g for 10 minutes at 4°C, collect the supernatant, take a small amount of protein supernatant, and use the BCA protein assay kit to determine the concentration of each sample; 3) Fully automated protein immunoblotting experiment According to WES TM The operation manual of the fully automatic protein expression analysis system was used to perform the protein immunoblotting experiment and process the data. The experimental data are shown in Table 3 below. The integral value of the protein immunoblotting chemiluminescence signal in the target protein band signal area reflects its overall expression level. TM The software's built-in algorithms perform visualization, display as virtual blots or peaks, quantify as area under the curve, and calculate concentrations.

[0068] Table 3

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] It should be noted that the diagnostic accuracy of the scheme of this application reaches 0.9810. Taking the case of using B2M antibody and TXN antibody together as an example, although some experimental examples cannot achieve the expected effect, this is only an individual difference and does not affect the overall reliability and effectiveness of the scheme of this application.

[0081] 2.4 Experimental Results The relative expression of the target proteins was calculated by total protein normalization. Kruskal-Wallis one-way analysis of variance was used for statistics among the three groups, and Dunn's test was used for pairwise comparisons among multiple groups. The positive rates of the three target proteins B2M, TXN, and PRDX5 were high, and their protein immunoblotting results were as follows: Figure 2 Statistical analysis showed that the levels of B2M, TXN and PRDX5 in neutrophils of the ATB, LTBI and HC groups were significantly different, among which the levels in the ATB group were significantly higher than those in the HC group (P value < 0.0001), and the levels in the LTBI group were also significantly higher than those in the HC group (P value < 0.0001). There was no significant difference between the ATB group and the LTBI group (P value > 0.05). Figure 3 Overall, the results of protein immunoblotting are basically consistent with the proteomic data, indicating that the analysis results of protein immunoblotting are accurate and reliable.

[0082] 2.5 Analysis of diagnostic efficacy Western blotting quantitative analysis confirmed the association between the expressed proteins screened by proteomics analysis and the infection status of Mycobacterium tuberculosis. Logistic binary regression analysis was further applied to draw the receiver operating characteristic curve (ROC curve) to comprehensively evaluate the value of the detection of three protein biomarkers, B2M, TXN and PRDX5, in the diagnosis of Mycobacterium tuberculosis infection.

[0083] The specific method is as follows: For the analysis of the diagnostic efficacy of protein combinations, logistic binary regression analysis was performed in SPSS, linear combinations were constructed and converted into probability values ​​through logistic functions, and ROC curves were drawn using probability values. The experimental data are shown in Table 4 below. Studies have shown that the AUC is between 0.93 and 0.99, indicating that the diagnostic accuracy is high. In addition, multiple protein combinations as diagnostic markers can improve the accuracy, specificity, and sensitivity of diagnosis while adapting to individual differences and disease complexity.

[0084] 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%.

[0085] Table 4: Diagnostic efficacy of different protein combinations in distinguishing ATB, LTBI and HC

[0086] Figure 4 The ROC curve diagram for the diagnostic efficacy evaluation of a combination of two proteins selected from B2M, TXN, and PRDX5 is shown.

[0087] Reference Figure 4 The results of the diagnostic efficacy analysis for distinguishing ATB patients from HC population showed that the combination of B2M and TXN proteins 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 distinguishing LTBI patients from HC population showed that the combination of TXN and PRDX5 proteins 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 distinguishing Mycobacterium tuberculosis infection from HC population showed that the combination of B2M and TXN proteins had the best diagnostic efficacy, with an AUC of 0.9849 (95%CI: 0.9748 ~ 0.9950), with a sensitivity of 97.79% and a specificity of 92.45%.

[0088] Figure 5 The ROC curve diagram for the diagnostic efficacy evaluation of the protein combination of B2M, TXN and PRDX5 is shown.

[0089] Reference Figure 5 The results of the diagnostic efficacy analysis for distinguishing LTBI patients from HC populations showed that compared with different protein combinations, the diagnostic efficacy of the three-protein combination of B2M, TXN, and PRDX5 was the best, with an AUC of 0.9866 (95%CI: 0.9724 ~ 1.000), a sensitivity of 97.99%, and a specificity of 95.28%.

[0090] In summary, the results of this study showed that the combined detection of B2M and TXN proteins can effectively distinguish ATB patients from HC population, and the combined detection of B2M and TXN proteins can effectively distinguish Mycobacterium tuberculosis infected patients (including ATB and LTBI) from HC population; the combined detection of B2M, TXN and PRDX5 proteins can effectively distinguish LTBI patients from HC population.

[0091] The advantages of the combination of B2M, TXN and PRDX5 are mainly reflected in the following aspects: (1) Higher specificity The specificity of B2M, TXN, and PRDX5 combined was 96.23%, significantly higher than B2M combined with TXN (91.51%) and B2M combined with PRDX5 (93.40%). High specificity means that non-infected people can be excluded more accurately, reducing the possibility of misdiagnosis.

[0092] (2) Better overall performance Although the AUC value of B2M combined with TXN was slightly higher (0.9810), its specificity was lower (91.51%). In practical applications, high specificity is particularly important for reducing misdiagnosis. The combination of B2M, TXN, and PRDX5 achieved a better balance between sensitivity (94.37%) and specificity (96.23%), with better overall performance.

[0093] (3) Addressing the complexity of the disease The pathological mechanism of active pulmonary tuberculosis is complex, and a single or dual protein combination may not fully cover all cases. The combination of B2M, TXN, and PRDX5 can better address individual differences and disease heterogeneity by integrating multiple biomarkers, providing more comprehensive diagnostic information.

[0094] In addition, compared with single proteins, multi-protein combinations (such as B2M, TXN, and PRDX5) have the following significant advantages: (1) Reduce the limitations of a single protein A single protein 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, acute kidney injury). TXN may change in certain lung diseases (such as asthma, chronic obstructive pulmonary disease), and PRDX5 may change in certain cancers (such as endometrial cancer, oral squamous cell carcinoma). Through multi-protein combination, mutual verification can be achieved, reducing the limitations of a single protein and improving the specificity of diagnosis.

[0095] (2) Improving the comprehensiveness of diagnosis A single protein may not fully reflect the complexity of the disease, while a multi-protein combination can provide diagnostic information from different angles. For example, some patients may not show obvious changes in a certain protein (such as B2M), but have significant changes in other proteins (such as TXN or PRDX5). A multi-protein combination can make up for the shortcomings of a single protein and improve sensitivity.

[0096] (3) Adapting to individual differences The pathological conditions of different patients may vary, and a single protein may not cover all cases. Multi-protein combinations can better adapt to individual differences and improve the universality and reliability of diagnosis.

[0097] According to the present invention, the diagnostic markers including at least two of B2M, TXN and PRDX5 can effectively distinguish active pulmonary tuberculosis (ATB) patients, latent tuberculosis infection (LTBI) patients and / or non-infected (HC) people with good sensitivity and specificity. However, the present disclosure is not limited to these aspects and features.

[0098] The above content is an illustration of some embodiments of the present disclosure and will not be interpreted as limiting the present disclosure. Although some embodiments have been described, it will be readily understood by those skilled in the art that various modifications can be made in 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.

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. The use according to any one of claims 1 to 6, wherein: The diagnostic reagent is at least used to distinguish active pulmonary tuberculosis patients from non-infected people.

8. The use according to claim 7, wherein: The diagnostic reagent is also used to distinguish between people who are latently infected with Mycobacterium tuberculosis and non-infected people.

9. A diagnostic kit for diagnosing Mycobacterium tuberculosis infection, wherein: The diagnostic kit comprises the specific antibody according to any one of claims 1 to 8.

10. The diagnostic kit according to claim 9, wherein The specific antibody is a monoclonal antibody.

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