WO2016095273A1

WO2016095273A1 - Antigen stimulant for detecting mycobacterium tuberculosis infection, kit, and applications of antigen stimulant

Info

Publication number: WO2016095273A1
Application number: PCT/CN2014/095543
Authority: WO
Inventors: 曾谷城; 汪华
Original assignee: 广州一代医药科技有限公司
Priority date: 2014-12-17
Filing date: 2014-12-30
Publication date: 2016-06-23
Also published as: CN107064498B; CN107121546B; CN107121546A; CN107064498A; CN104597239A; CN107102137A; CN104597239B

Abstract

Disclosed are an antigen stimulant for detecting mycobacterium tuberculosis infection, and a kit comprising the antigen stimulant. Further disclosed are applications of the antigen stimulant in reagents for detecting mycobacterium tuberculosis infection. The antigen stimulant comprises at least one polypeptide or analogues thereof of polypeptides shown by sequences 1 to 11 in a sequence table, wherein the polypeptides respectively come from tuberculosis specific antigen polypeptides ESAT-6 and tuberculosis specific antigen polypeptides CFP-10. The disclosed antigen stimulant can stimulate peripheral blood T lymphocytes of a tuberculosis infection patient to generate IFN-γ to diagnose the tuberculosis infection, and is not affected by BCG inoculation or other underlying diseases.

Description

Antigen stimulator, kit for detecting Mycobacterium tuberculosis infection and application thereof

Technical field

The invention belongs to the field of biomedical examination, and particularly relates to an antigen stimulator for detecting infection of Mycobacterium tuberculosis and an application thereof, and a kit containing the stimulator of the antigen.

Background technique

Tuberculosis is a type of infectious disease caused by Mycobacterium tuberculosis that is seriously harmful to human health. Although M. tuberculosis initially infects the respiratory system of the human body, it can spread to almost all organs of the human body and cause the corresponding diseases to occur. It is estimated that about one-third of the world's population is infected with M. tuberculosis, and there are 8 million new infections every year, and 2 million people die of tuberculosis each year. China's tuberculosis epidemic and tuberculosis infection are quite serious. It is one of the 22 countries with high burden of tuberculosis in the world, and the number of tuberculosis patients ranks second in the world.

The M. tuberculosis infection generally refers to a population with active tuberculosis or latent tuberculosis infection, or may be a healthy person. After the body infected with M. tuberculosis, a small number of active tuberculosis developed, showing corresponding clinical symptoms, such as fever, cough, cough, chest X-ray abnormalities. Most people do not have the corresponding clinical symptoms after infection, but the body can not clear all the Mycobacterium tuberculosis, which is latent tuberculosis infection. People with latent tuberculosis can develop active tuberculosis in cases of low immunity.

Early diagnosis of tuberculosis is important for the effective control of tuberculosis progression and the spread of M. tuberculosis. At present, there are limited methods for diagnosing tuberculosis infection, including tuberculosis skin test (TST), sputum smear, sputum culture, radioactive X-ray film, serological antibody antigen immunoassay, and PCR and nucleic acid hybridization methods. The interferon-gamma release assay (IGRA) caused by Mycobacterium tuberculosis infection is a new method developed in recent years and can be used to diagnose active tuberculosis and tuberculosis latent infection. The T-SPOT.TB kit (Oxford Immunotec Limited, Aingdon, United Kingdom) and the QuantiFERON-TBGold kit, which have been approved by the US FDA, are based on the IGRA principle, and the tuberculosis antigen-specific protein-6KD encoded by the RD1 region gene is early. Secretory targeting antigen (ESAT-6) and 10KD culture filter protein (CFP-10) are used as stimuli to diagnose tuberculosis infection by detecting T lymphocytes specific for IFN-γ release from peripheral blood. Sensitivity and specificity Both are higher.

The current traditional methods for diagnosing tuberculosis infection have many shortcomings and shortcomings. The Mycobacterium tuberculosis pure protein derivative (PPD) used in the Mycobacterium tuberculosis skin test (TST) contains many mycobacterial species (including pathogenicity). The antigenic molecules shared by mycobacteria, environmental mycobacteria and BCG), therefore, the specificity of PPD for the diagnosis of tuberculosis is poor, and it is impossible to accurately distinguish the positive results of PPD experiments because of BCG vaccination and exposure to various non-tuberculous mycobacteria in the environment. Sensitization is also caused by true M. tuberculosis infection. Although the sputum smear method is simple and easy, but a large part of tuberculosis There is no necessarily M. tuberculosis in the human sputum (the so-called "cash" tuberculosis). Therefore, the sputum smear culture method has the disadvantages of low diagnostic positive rate, easy leak detection, long sputum culture period, and low culture success rate (only about 80%). The detection of nucleic acid components in sputum specimens not only faces the dilemma of diagnosis of sputum sputum tuberculosis, but also has complicated procedures and is prone to false positives. Therefore, immunological techniques have become an important method for the diagnosis of M. tuberculosis infection.

The T-SPOT.TB kit and the QuantiFERON-TB Gold kit are complex, expensive, and costly, severely restricting their use for millions of active tuberculosis patients and hundreds of millions of tuberculosis latent infections. Conduct large-scale tuberculosis screening and diagnosis. In addition, the antigen peptides contained in these kits are extremely complex and are not designed, screened and verified based on the major histocompatibility antigens of Chinese people, so the applicability and use effects need to be further improved. Chinese patent application with the publication number CN 102516356 A screened 8-11 consecutive polypeptide fragments from the antigen Rv3615c as a stimulus source, and diagnosed tuberculosis infection by enzyme-linked immunospot assay (ELISPOT), but its sensitivity was only 64%, far reaching Not required for commercial kits. The antigen stimulator used in Chinese Patent Application Publication No. CN 102297968 A is a combination of 9 polypeptides of Mycobacterium tuberculosis ESAT-6 antigen and CFP-10 antigen, and IFN-γ, TNF-α, IL-2 are used. The magnetic beads coated with five antibodies, MIG and IP-10, simultaneously detect the five cytokines of the cell culture supernatant, which complicates the detection method and the results of the analysis, and requires the use of flow cytometry, which increases The cost of testing limits its promotion. The Chinese patent application with the publication number CN 103604933 A is a kit for detecting active tuberculosis based on TNF-α cytokine ELISA, and the antigen stimulator used in the kit is ESAT-6 and CFP-10 whole protein, however The spatial conformation of the protein may prevent the effective epitope from binding to the recognition molecule on the surface of the T lymphocyte, and its other epitopes may cause negative regulation, affecting the secretion of cytokines, causing the kit to be latently infected. The diagnosis of tuberculosis is poor.

Summary of the invention

An object of the present invention is to provide an antigen stimulating agent for detecting Mycobacterium tuberculosis infection and a kit containing the same. The antigen stimulator provided by the invention can effectively stimulate the peripheral blood T lymphocytes of TB-infected patients to produce IFN-γ, thereby being capable of highly sensitive and highly specific diagnosis of tuberculosis infection, and is not affected by BCG vaccination or other basic diseases.

In order to achieve the object, the technical scheme adopted by the present invention is as follows:

A first aspect of the present invention provides an antigen stimulator for detecting a Mycobacterium tuberculosis infection, which comprises at least one polypeptide or an analogue thereof as shown in Sequences 1 to 11 in the Sequence Listing. The amino acid sequence is as shown in the sequences 1 to 11, and the sequence is as follows:

SEQ ID NO. 1: AGIEAAASAIQGNVTSI

SEQ ID NO. 2: YQGVQQKWDATATELNNALQNL

SEQ ID NO. 3: RTISEAGQAMASTEGNVTGMFA

SEQ ID NO. 4: MAEMKTDAATLAQEAGNF

SEQ ID NO. 5: GAAGTAAQAAVVRFQEAA

SEQ ID NO. 6: VVRFQEAANKQKQELDEI

SEQ ID NO. 7: YQGVQQKWDATATELNNALQ

SEQ ID NO. 8: ISEAGQAMASTEGNVTGMFA

SEQ ID NO.9: MAEMKTDAATLAQEA

SEQ ID NO. 10: AAGTAAQAAVVRFQE

SEQ ID NO. 11: VRFQEAANKQKQELD

Wherein SEQ ID NO. 1 to 3 and SEQ ID NO. 7 to 8 are derived from the tuberculosis specific antigen polypeptide ESAT-6, and SEQ ID NO. 4 to 6 and SEQ ID NO. 9 to 11 are derived from the tuberculosis specific antigen polypeptide. CFP-10.

Preferably, the antigen stimulator consists of the polypeptide represented by SEQ ID NO: 1 and SEQ ID NO: 7 to 8 in the sequence listing, or the analog stimulant thereof, the polypeptide represented by SEQ ID NO: 9 to 11 in the sequence listing or Analog composition.

Most preferably, the antigen stimulator consists of the polypeptide represented by the sequences 1 to 3 in the sequence listing or an analog thereof; or the antigen stimulator is represented by the polypeptides shown in the sequences 4 to 6 of the sequence listing or the like. composition. Using this preferred method, the detection effect is the best, the detection sensitivity is the best, the positive rate of detection for suspected tuberculosis infection specimens is as high as 89%, and the negative coincidence rate is as high as 100%. Using a preferred antigen stimulator to test a wide range of volunteers, it can effectively avoid the interference of other non-tuberculous mycobacterial lung infections, thus effectively identifying M. tuberculosis infection, so it is highly sensitive Sex and specificity. In addition, our kit positive coincidence rate (sensitivity and specificity) is significantly higher than the existing patent technology, the sensitivity of some existing patented technologies is about 60% (such sensitivity is difficult to use clinically). In addition, the kit of this patent can perform tuberculosis screening for large-scale unknown population. As can be seen from Example 4 (Fig. 5 and Fig. 6), one tuberculosis patient can be screened out from 37 unknown persons, and the tumor is excluded. Other lung infections, flu and other diseases have high specificity and sensitivity, which can be applied to large-scale population screening. This feature is particularly important for screening TB patients in China, and its diagnostic sensitivity and specificity. Significantly superior to existing patented technology.

Said analog means that the properties are identical to said polypeptide, including that it can also bind specifically to an antibody, such homologous peptides typically having at least 70% homology, preferably at least 90%, 95% identical Source. The differences typically arise from substitutions, insertions, deletions, and modifications of amino acid residues that can occur at the N-terminus, C-terminus, or any other position of the sequence.

The polypeptide of the present invention can be synthesized by a conventional chemical synthesis method or can be prepared by a genetic engineering technique known to those skilled in the art, and a representative method is to obtain the above polypeptide by solid phase synthesis. The basic principle is: firstly, the hydroxyl group of the hydroxy terminal amino acid of the peptide chain to be synthesized is covalently bonded to the same insoluble high score. The sub-resin is linked, and then the amino acid bound to the solid support is used as an amino component to undergo deamination of the protective group and react with an excess of the activated carboxyl component to lengthen the peptide chain. Repeat (condensation → washing → deprotection → neutralization and washing → next round condensation) operation to achieve the length of the peptide chain to be synthesized, and finally the peptide chain is cleaved from the resin, and after purification and the like, the desired polypeptide is obtained. The α-amino group is protected by BOC (tert-butoxycarbonyl), which is called BOC solid phase synthesis, and the α-amino group is protected by FMOC (9-fluorenylmethoxycarbonyl), which is called FMOC solid phase synthesis.

The antigen stimulator provided by the invention can be applied to the detection of Mycobacterium tuberculosis infection, and the basic detection principle is: stimulating the T cells of the M. tuberculosis host by the antigen stimulator provided by the invention, and then detecting the cells released by the T cells. Factors to determine whether T cells recognize these polypeptides or analogs, thereby indirectly reflecting whether the host is infected with M. tuberculosis.

The antigen stimulators provided by the present invention can be used in the preparation of an agent for detecting M. tuberculosis infection.

The present invention also provides a kit for detecting M. tuberculosis infection, comprising an antigen stimulator as described above, and a capture antibody, a detection antibody, horseradish peroxidase-labeled streptavidin, and 3-amino-9-ethylcarbazole (AEC) chromogenic substrate.

Further, the capture antibody is a monoclonal antibody against human IFN-γ, which is a biotin-labeled antibody against human IFN-γ.

The kit also includes a positive control stimulator and a negative control reagent. The antigen selected for the positive control can respond to most individual T cells, such as commercially available PHA (phytohemagglutinin), and the negative control does not contain antigen. Ingredients, use medium or other buffers.

Another aspect of the present invention provides a method for detecting a Mycobacterium tuberculosis infection in vitro, which comprises contacting an antigen stimulant as described above with a T cell of a Mycobacterium tuberculosis host, and determining a T cell by detecting a cytokine secreted by the T cell. Whether the antigen stimulator is recognized, thereby indirectly reflecting whether the host is infected with M. tuberculosis.

The host refers to a human or other mammal, such as a primate, a rodent such as a cow, a sheep, a pig, a mouse, and a rat.

The T cells are usually presensitized in vivo by antigens from M. tuberculosis, and these antigen-sensitized T cells are usually present in the peripheral blood of the host, and may also be present in alveolar lavage fluid, pleural effusion, cerebrospinal fluid, Lymph nodes or other tissue sites containing T cells. This T cell can be a CD4 ⁺ T cell or a CD8 ⁺ T cell. In the method, T cells can be contacted with a peptide (antigen stimulator) in vitro or in vivo, and it can be determined in vitro or in vivo whether the T cell recognizes the peptide. Typically, it is determined whether a T cell recognizes the peptide by determining a change in the state of the T cell in the presence of the peptide or determining whether the T cell binds to the peptide. The state change of T cells may be that T cells begin to secrete substances or increase secretion, such as cytokines, particularly IFN-γ, IL-2 or TNF-α. Preferably, the secretion of IFN-γ is determined. The substance can usually be detected by binding these substances to a specific binding partner and detecting the presence of a complex of the combination with these substances. Specific binding partners are typically antibodies, such as monoclonal or polyclonal antibodies, which are generally commercially available or can be prepared using standard techniques. The method for measuring cytokines is usually a commonly used method in the field, such as ELISA (enzyme-linked immunosorbent assay), ELISPOT (enzyme-linked immunospot assay), Immunoblotting (immunoblotting), intracellular cytokine staining, T cell proliferation assay, etc. .

In one embodiment of the invention, the ELISPOT method is employed to detect secretion of IFN-[gamma] by T cells. The IFN-γ monoclonal antibody pre-coated on the solid phase carrier first binds to IFN-γ to form a complex, which is then bound to the biotin-labeled second IFN-γ antibody, and then the horseradish peroxidase label The streptavidin specifically binds to biotin and finally forms a spot by the color reaction of the enzyme and the substrate, thereby reflecting the number of activated T cells. The T cells in the method may be isolated in vitro, and of course, may be unprocessed or in vivo. In one embodiment, monocytes are isolated from peripheral blood or other samples, including T cells and APCs (antigen-presenting cells), which are cells capable of presenting peptides to T cells. In one embodiment, the peptide itself is added directly to an experiment comprising T cells and APC. In such assays, APC can present peptides to T cells. APC is not required when using peptides that are recognized by T cells without the need for presentation by APC.

The length of time the peptide is contacted with T cells can vary depending on the method used to determine peptide recognition. Typically, 10 ⁵ to 10 ⁷ peripheral blood mononuclear cells (PBMC) are added to each experiment, preferably 2.5 × 10 ⁵ to 10 ⁶ . When the peptide is directly added to the experiment, its concentration is 0.1 to 100 μg/ml, preferably 1 to 10 μg/ml. A typical time for T cells to be incubated with peptides is 16 to 36 hours.

The antigen stimulator provided by the invention and the kit containing the specific antigen stimulator can effectively detect the Mycobacterium tuberculosis infection in vitro without being affected by the inoculation of BCG (Bacillus Calmette-Guerin), and have high sensitivity and specificity. Sex, both for clinical diagnosis of M. tuberculosis infection and for screening for M. tuberculosis infection in large populations. The kit provided by the invention is designed according to Chinese major histocompatibility complex and screens to verify the dominant antigen polypeptide of Mycobacterium tuberculosis, and has better effect in China, has high specificity and high sensitivity, and the present invention and abroad Compared with the kit, the price is lower and it is more suitable for promotion in China and developing countries.

In the clinical application, the IFN-γ-ELISPOT kit provided by the present invention detects blood samples of 55 patients with highly suspected infection of Mycobacterium tuberculosis, and diagnoses 41 patients with M. tuberculosis infection, and the sputum smear and sputum Bacterial culture, chest X-ray and clinical symptoms were diagnosed as 46 patients with tuberculosis, with a positive coincidence rate of 89.1%; 9 patients with non-tuberculosis diagnosed with the IFN-γ-ELISPOT kit of the present invention, sputum smear The sputum culture, chest X-ray results and clinical symptom diagnosis were all negative results, and the negative coincidence rate reached 100%. The kit provided by the invention has good clinical applicability in the diagnosis of tuberculosis, has good specificity, high sensitivity and high coincidence rate with other diagnostic techniques.

DRAWINGS

Figure 1: Results of the superior peptide verification of the IFN-γ-ELISPOT tuberculosis diagnostic kit of the present invention: (A) IFN-γ-ELISPOT results of the TB-specific polypeptide antigen peptides SEQ ID NO. (The number of spots is 123); (B) IFN-γ-ELISPOT results after stimulating cells of the tuberculosis-specific polypeptide antigen peptides SEQ ID NO. 4 to 6 (the number of spots is 62); (C) negative control (spots) The number is 0); (D) positive (PHA) control (number of spots is 801); (E) is a background control without cells.

Figure 2: The results of the preferred polypeptide verification of the IFN-γ-ELISPOT tuberculosis diagnostic kit of the present invention: In Figure 2, A is the combined stimulation result of the polypeptide antigen SEQ ID NO. 1 and SEQ ID NO. 7-8 (the number of spots is 160), B is the product of the kit tube specific polypeptide peptide SEQ ID NO. 9-11 after stimulating the cells (the number of spots is 146), C is the negative control (the number of spots is 0), D is the positive control (PHA) stimulation results (The number of spots is 783), and E is the background control without cells (the number of spots is 0).

Figure 3: Diagnostic results of this IFN-γ-ELISPOT kit for a suspected tuberculosis patient: (A) negative control (number of spots 1); (B) IFN-γ-induced ESAT-6 non-immunodominative polypeptide after stimulation of cells ELISPOT results (number of spots is 5); (C) IFN-γ-ELISPOT results after stimulating cells of tuberculosis-specific peptide antigen peptides SEQ ID NO. 1 to 3 (number of spots is 22); (D) tuberculosis specificity of the product The IFN-γ-ELISPOT results after stimulating the cells of the polypeptide antigen peptides SEQ ID NOS. 4 to 6 (the number of spots was 19); (E) the IFN-γ-ELISPOT results (the number of spots was 475) after stimulating the cells by the PHA positive control.

Figure 4 is a graph showing the results of diagnosis of the IFN-γ-ELISPOT kit polypeptide sequences 1, 7 to 11 of a suspected tuberculosis patient. In Fig. 4, (A) is the negative control (the number of spots is 1), and Fig. 4 (B) is the IFN-γ-ELISPOT result (the number of spots is 4) after the ESAT-6 non-immunogenic polypeptide stimulating cells, Fig. 4 (C) Is the IFN-γ-ELISPOT result (the number of spots is 38) after stimulating cells of the tuberculosis-specific polypeptide antigen peptides SEQ ID NO. 1 and SEQ ID NO. 7 to 8, and Figure 4 (D) is the tuberculosis-specific polypeptide antigen of the product. SEQ ID NO. 9-11 The IFN-γ-ELISPOT results after stimulating cells (the number of spots was 43), and Fig. 4(E) shows the IFN-γ-ELISPOT results after stimulating cells with PHA positive control (the number of spots was 330) .

Figure 5: Results of tuberculosis screening of 37 volunteers using the IFN-γ-ELISPOT kit of the present invention.

Figure 6: shows the red box in Figure 5 as one of the results of IFN-γ-ELISPOT tuberculosis test indicating tuberculosis positive: (A) negative control (number of spots is 1); (B) ESAT-6 non-immune dominant peptide IFN-γ-ELISPOT results after stimulating cells (number of spots is 6); (C) IFN-γ-ELISPOT results after stimulating cells of tuberculosis-specific polypeptide antigen peptides SEQ ID NOS. 1 to 3 (number of spots is 17); (D) This product tuberculosis specific peptide antigen peptide SEQ ID NO. 4 to 6 The IFN-γ-ELISPOT results after stimulating the cells (the number of spots was 9); (E) the IFN-γ-ELISPOT results (the number of spots was 466) after the PHA positive control stimulated the cells.

detailed description

The technical solution of the present invention will be further described below with reference to the accompanying drawings. The experimental methods in the following examples, unless otherwise specified, are conventional methods in the art. The experimental materials used in the examples are all commercially available conventional biochemical reagents unless otherwise specified, and % of the examples are mass percentages unless otherwise specified.

Example 1: Preparation of tuberculosis-specific T cell dominant epitope polypeptide and IFN-γ-ELISPOT Mycobacterium tuberculosis

Development of dye diagnostic kit

Design and screen T-dominant epitopes by bioinformatics analysis and in vitro detection of T cell immune responses, including the following steps:

(1) Predicting the T cell epitopes of the proteins ESAT-6 and CFP-10 encoded by the RD1 region by bioinformatics technology and corresponding databases such as ProPred, MacVestor, Preotean, etc., and analyzing them with different HLA typing Identification relationship between.

(2) Combining the results of biological analysis, 9 ESAT-6 polypeptides and 11 CFP-10 polypeptides were designed and optimized by Overlap method, and each polypeptide contained 9-22 amino acids.

(3) Collect blood samples from tuberculosis patients, 2 to 5 ml each. The criteria for inclusion of tuberculosis patients were: cough, cough, chest X-ray results, sputum smear or sputum culture, and tuberculosis treatment for less than three weeks, HIV test was negative.

(4) Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll lymphocyte separation solution. PBMC was washed twice with 5-10 ml PBS or PRMI1640, and then resuspended in RPMI1640 medium containing 10% fetal bovine serum (FBS, Life). (Life company) and calculate the number of cells.

(5) ELISPOT assay detects IFN-γ released by T cells after stimulation. The specific procedure is: coating human IFN-γ monoclonal antibody (ebioscience) overnight in 96-well plate (Millipore) with PVDF membrane, RPMI1640 +10% FBS was blocked and 2.5×105 cells and peptides were added to each well. The positive control group was added with phytohemagglutinin (PHA), the negative control group was added to the medium or the lysing reagent for dissolving the polypeptide, and the other group was not added with cells. As a background control. Incubate for 22 hours in the incubator, and then add the biotin-labeled anti-human IFN-γ antibody, horseradish peroxidase-labeled streptavidin, 3-amino-9-ethylcarbazole (AEC). The substrate develops color and forms visible red spots on the PVDF film. The result is read on the ELISPOT reader. As a result, it was judged that the number of spots was ≥10, and the number of spots larger than 2 times of the negative control holes was judged to be a positive result; the number of spots <10 or less than 2 times the number of spots of the negative control holes was judged to be a negative result.

(6) Analysis of results: According to the frequency of positive results of stimulating reaction of peptides to tuberculosis patients, three ESAT-6 dominant epitope polypeptides SEQ ID NO. 1 to 3 were finally screened (the amino acid sequence can be found in sequence 1 in the sequence listing) ～3), 2 ESAT-6 preferred epitope peptides SEQ ID NO. 7-8 (the amino acid sequence can be found in SEQ ID NO: 7-8 of the sequence listing), and 3 CFP-10 dominant epitope polypeptides SEQ ID NO. 4 to 6 (the amino acid sequence can be referred to the sequences 4 to 6 in the sequence listing) 3 CFP-10 preferred epitope polypeptides SEQ ID NOS. 9 to 11 (the amino acid sequences can be referred to the sequences 9 to 11 in the sequence listing). Figure 1 shows a typical IFN-γ-ELISPOT result screened for polypeptides 1-6. Figure 1 (A) shows the IFN-γ-ELISPOT results (the number of spots is 123) after the combination of the tuberculosis-specific polypeptide antigen peptides SEQ ID NO. 1 to 3 (the number of spots is 123), and Figure 1 (B) is the kit. The IFN-γ-ELISPOT results (the number of spots is 62) of the tuberculosis-specific polypeptide antigen peptides SEQ ID NOS. 4 to 6 combined with the stimulating cells, and the negative control (the number of spots is 0) in Fig. 1 (C), Fig. 1 (D) A positive (PHA) control (number of spots is 801) and Figure 1 (E) is a background control without cells. Figure 2 is a typical IFN-[gamma]-ELISPOT result for peptide screening of sequences 1, 7-11. 2A is a result of a combination of a polypeptide antigen peptide SEQ ID NO. 1 and SEQ ID NO. 7 to 8 (the number of spots is 160), and FIG. 2B is a combination of the present invention tube-specific polypeptide antigen peptides SEQ ID NO. 9 to 11. The results after stimulating the cells (the number of spots was 146), Figure 2C is the negative control (the number of spots is 0), Figure 2D is the positive control (PHA) stimulation results (the number of spots is 783), and Figure 2E is the background control without cells ( The number of spots is 0).

Example 2: A diagnostic kit for IFN-γ-ELISPOT Mycobacterium tuberculosis infection developed by the present invention, comprising:

(1) An antigen stimulator, which is an amino acid sequence selected in Example 1, such as a combination of one or more polypeptides of the polypeptides shown in SEQ ID NOs: 1-11 in the Sequence Listing, and may also be an analog of these polypeptides. ;

(2), capture antibody, a monoclonal antibody against human IFN-γ, available from ebioscience;

(3) detecting the antibody, which is a biotin-labeled antibody against human IFN-γ;

(4) horseradish peroxidase-labeled streptavidin;

(5), 3-amino-9-ethylcarbazole (AEC) chromogenic substrate;

(6), positive control stimulator, is phytohemagglutinin (PHA);

(7) A negative control is a medium (eg, RPMI 1640 + 10% FBS) or a lysing reagent (eg, DMSO) for solubilizing the polypeptide.

Example 3: Clinical diagnosis of the IFN-γ-ELISPOT kit of the present invention for suspected patients with Mycobacterium tuberculosis infection

OBJECTIVE: To test the clinical applicability, specificity, sensitivity and compatibility with other diagnostic techniques of the IFN-γ-ELISPOT Mycobacterium tuberculosis infection diagnostic kit of the present invention in the diagnosis of tuberculosis.

(1) Blood samples of 55 patients with highly suspected infection of Mycobacterium tuberculosis were collected, each with 2 to 5 ml.

(2) Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll lymphocyte separation solution. PBMC was washed twice with 5-10 ml PBS or PRMI1640, and then resuspended in RPMI1640 medium containing 10% fetal bovine serum (FBS). The number of cells.

(3) ELISPOT assay detects IFN-γ released by T cells after stimulation. The specific steps are: coating human IFN-γ monoclonal antibody in 96-well plate with PVDF membrane overnight, RPMI1640+10% FBS closed per well Adding 2.5 x 105 cells and polypeptides SEQ ID 1-3 polypeptides (or preferred polypeptide combinations, SEQ ID NO. 1 polypeptides in combination with SEQ ID NOS. 7-8 polypeptides) or polypeptides SEQ ID 4-6 (or preferred polypeptide SEQ ID NO) The combination of .9-11), the positive control group was added with phytohemagglutinin (PHA), the negative control group was added with 10% FBS/1640 medium or the lysing reagent DMSO for dissolving the polypeptide, and the other group was not added with cells as the background control. Incubate for 22 hours in the incubator, and then add the biotin-labeled anti-human IFN-γ antibody, horseradish peroxidase-labeled streptavidin, 3-amino-9-ethylcarbazole (AEC). The substrate develops color and forms visible red spots on the PVDF film. The result is read on the ELISPOT reader. As a result, it was judged that the number of spots was ≥10, and the number of spots larger than 2 times of the negative control holes was judged to be a positive result; the number of spots <10 or less than 2 times the number of spots of the negative control holes was judged to be a negative result.

(4) Analysis of results: Among 55 patients with suspected Mycobacterium tuberculosis infection, 46 patients with tuberculosis were diagnosed by sputum smear, sputum culture, chest X-ray and clinical symptoms, using IFN-γ-ELISPOT of the present invention. The kit was diagnosed as 41 patients with M. tuberculosis infection, so the positive coincidence rate was 89.1%; 9 patients with non-tuberculosis diagnosed with this IFN-γ-ELISPOT kit, sputum smear, sputum culture, chest The results of the film and the diagnosis of the clinical symptoms were all negative, so the negative coincidence rate reached 100%. Figure 3 shows the diagnosis result of the IFN-γ-ELISPOT kit of a suspected tuberculosis patient. The antigen stimulator contained in the kit used is SEQ ID NO. 1 to 3 or SEQ ID NO. 4 ~6). Figure 3 (A) shows the negative control (number of spots is 1), and Figure 3 (B) shows the IFN-γ-ELISPOT results (number of spots 5) after stimulation of cells with ESAT-6 non-immunogenic polypeptide, Figure 3

(C) is the IFN-γ-ELISPOT result (the number of spots is 22) after the combined stimulation of the tuberculosis-specific polypeptide antigen peptides SEQ ID NOS. 1 to 3, and FIG. 3(D) is the tuberculosis-specific polypeptide antigen peptide of the product. The IFN-γ-ELISPOT results (number of spots) of ID NO. 4 to 6 combined with the stimulator cells (Fig. 3 (E) are the IFN-γ-ELISPOT results (the number of spots is 475) after the PHA positive control stimulator cells. Figure 4 is a diagram showing the diagnosis result of a preferred IFN-γ-ELISPOT kit for a suspected tuberculosis patient (the result of the test is that the antigen stimulator contained in the kit is a combination of the polypeptide SEQ ID NO. 1 and SEQ ID NO. 7 to 8. Or a combination of the polypeptides SEQ ID NOS. 9 to 11). Figure 4 (A) shows the negative control (number of spots is 1), and Figure 4 (B) shows the IFN-γ-ELISPOT results (number of spots 4) after stimulation of cells with ESAT-6 non-immunodominant polypeptide, Figure 4 (C) Is the IFN-γ-ELISPOT result (the number of spots is 38) after the combination of the tuberculosis-specific polypeptide SEQ ID NO. 1 and SEQ ID NO. 7 to 8 (Fig. 4(D) is the product knot The IFN-γ-ELISPOT results (the number of spots 43) of the nuclear specific polypeptide antigen peptides SEQ ID NOS. 9 to 11 combined with the stimulating cells, and Fig. 4 (E) are the IFN-γ-ELISPOT results after the PHA positive control stimulator cells. (The number of spots is 330).

Example 4: Applicability of the IFN-γ-ELISPOT kit of the present invention in tuberculosis screening in an unknown population

OBJECTIVE: To test the IFN-γ-ELISPOT Mycobacterium tuberculosis infection diagnostic kit of the present invention (the kit used in this embodiment contains the antigen stimulator as a combination of the polypeptides SEQ ID NOS. 1-3, or the polypeptide SEQ ID NO .4~6 combination) Applicability, specificity and sensitivity in large-scale population tuberculosis screening.

(1) Blood samples from 37 HIV-negative volunteers were collected, 2 to 3 ml each. These people have been vaccinated with BCG.

(3) ELISPOT assay detects IFN-γ released by T cells after stimulation. The specific steps are: coating human IFN-γ monoclonal antibody in 96-well plate with PVDF membrane overnight, RPMI1640+10% FBS closed per well 2.5×105 cells and sequence 1-3 polypeptide or sequence 4-6 polypeptide were added, the positive control group was added with phytohemagglutinin (PHA), the negative control group was added to the medium or the lysing reagent for dissolving the polypeptide, and the other group was not added with cells. As a background control. Incubate for 22 hours in the incubator, and then add the biotin-labeled anti-human IFN-γ antibody, horseradish peroxidase-labeled streptavidin, 3-amino-9-ethylcarbazole (AEC). The substrate develops color and forms visible red spots on the PVDF film. The result is read on the ELISPOT reader. As a result, it was judged that the number of spots was ≥10, and the number of spots larger than 2 times of the negative control holes was judged to be a positive result; the number of spots <10 or less than 2 times the number of spots of the negative control holes was judged to be a negative result.

(4) Analysis of results: Figure 5 shows the results of the IFN-γ-ELISPOT kit applied to the tuberculosis test in unknown population. The red box in Figure 5 shows the result of a positive test for IFN-γ-ELISPOT tuberculosis ( See Fig. 6), in which Figure 6 (A) is the negative control (number of spots is 1), and Figure 6 (B) is the IFN-γ-ELISPOT result after the ESAT-6 non-immunodominant polypeptide stimulates cells (the number of spots is 6) Fig. 6(C) shows the results of IFN-γ-ELISPOT (the number of spots is 17) after the combination of the tuberculosis-specific polypeptide antigen peptides SEQ ID NO. 1 to 3, and Figure 6 (D) is the tuberculosis-specific polypeptide antigen of the product. The IFN-γ-ELISPOT results (number of spots 9) of the peptides SEQ ID NO. 4 to 6 combined with the stimulating cells, and Fig. 6 (E) are the IFN-γ-ELISPOT results after the PHA positive control stimulator cells (the number of spots was 466) The results suggest that the volunteers were positive for IFN-γ-ELISPOT tuberculosis, and that one of the 37 volunteer samples was positive for IFN-γ-ELISPOT tuberculosis, and the volunteers were further tested and confirmed to be active. Mycobacterium tuberculosis infection, indicating that the IFN-γ-ELISPOT kit developed by the present invention can be applied to a large-scale population tuberculosis screening and is not affected by the BCG epidemic Effect of inoculation of other underlying disease or the like. In addition, large-scale tuberculosis screening experiments were performed on similar polypeptide sequences 7 to 11 for similar unknown populations, and it was found that preferred polypeptide sequences 7 to 11 have similarities. The specificity and sensitivity of polypeptide sequence 1 to 6 for tuberculosis screening in large populations will not be described here.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way. Therefore, any of the above embodiments can be made according to the technical essence of the present invention without departing from the technical solution of the present invention. Simple modifications, equivalent changes and modifications are still within the scope of the technical solutions of the present invention.

Claims

An antigen stimulator for detecting a Mycobacterium tuberculosis infection, which comprises at least one polypeptide or an analog thereof as shown in Sequences 1 to 11 in the Sequence Listing.
The antigen stimulator according to claim 1, wherein the antigen stimulator consists of the polypeptide represented by the sequences 1 to 3 in the sequence listing or an analog thereof; or the antigen stimulator is sequenced from the sequence listing. The polypeptide represented by 4-6 or its analog composition.
The antigen stimulator according to claim 1, wherein the antigen stimulator consists of the polypeptide represented by SEQ ID NO: 1 and SEQ ID NO: 7 to 8 in the Sequence Listing; or the antigen stimulant shown by The polypeptides represented by the sequences 9 to 11 in the list or the analogs thereof are composed.
Use of the antigen stimulator according to any one of claims 1 to 3 for the preparation of a reagent for detecting Mycobacterium tuberculosis infection.
A kit for detecting a Mycobacterium tuberculosis infection, comprising the antigen stimulating agent according to any one of claims 1 to 3, and a capture antibody, a detection antibody, and a horseradish peroxidase marker Streptavidin and 3-amino-9-ethylcarbazole chromogenic substrate.
The kit according to claim 5, wherein the capture antibody is a monoclonal antibody against human IFN-γ, and the detection antibody is a biotin-labeled antibody against human IFN-γ.
A method for detecting a Mycobacterium tuberculosis infection in vitro, which comprises contacting the antigen stimulating agent according to any one of claims 1 to 3 with a T cell of a Mycobacterium tuberculosis host, and detecting a cell secreted by the T cell. A factor that determines whether the T cell recognizes the antigen stimulator.
The method of claim 7 wherein said host is a human or other mammal.
The method according to claim 7, wherein the cytokine is IFN-γ, IL-2 or TNF-α, and the T cell is derived from peripheral blood of the host, alveolar lavage fluid, pleural effusion, cerebrospinal fluid, Lymph nodes or other tissue sites containing T cells.
The method according to claim 7, wherein the secretion of T cell cytokines is detected by the following methods: ELISA, ELISPOT, immunoblotting, intracellular cytokine staining, T cell proliferation assay.