WO2016095273A1 - Antigène stimulant pour la détection d'une infection à mycobacterium tuberculosis, kit et applications de l'antigène stimulant - Google Patents

Antigène stimulant pour la détection d'une infection à mycobacterium tuberculosis, kit et applications de l'antigène stimulant Download PDF

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WO2016095273A1
WO2016095273A1 PCT/CN2014/095543 CN2014095543W WO2016095273A1 WO 2016095273 A1 WO2016095273 A1 WO 2016095273A1 CN 2014095543 W CN2014095543 W CN 2014095543W WO 2016095273 A1 WO2016095273 A1 WO 2016095273A1
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antigen
tuberculosis
ifn
cells
seq
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曾谷城
汪华
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广州一代医药科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • 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.
  • Tuberculosis is a type of infectious disease caused by Mycobacterium tuberculosis that is seriously harmful to human health.
  • 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.
  • tuberculosis skin test TST
  • sputum smear sputum culture
  • radioactive X-ray film radioactive X-ray film
  • serological antibody antigen immunoassay PCR and nucleic acid hybridization methods.
  • IGRA interferon-gamma release assay
  • 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
  • 10KD culture filter protein CFP-10 are used as stimuli to diagnose tuberculosis infection by detecting T lymphocytes specific for IFN- ⁇ 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.
  • 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.
  • 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- ⁇ , TNF- ⁇ , 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- ⁇ 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.
  • 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- ⁇ , thereby being capable of highly sensitive and highly specific diagnosis of tuberculosis infection, and is not affected by BCG vaccination or other basic diseases.
  • 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. 4 MAEMKTDAATLAQEAGNF
  • SEQ ID NO. 8 ISEAGQAMASTEGNVTGMFA
  • 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.
  • 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.
  • 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.
  • 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%.
  • 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.
  • kits positive coincidence rate 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).
  • 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.
  • 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.
  • 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.
  • the capture antibody is a monoclonal antibody against human IFN- ⁇ , which is a biotin-labeled antibody against human IFN- ⁇ .
  • 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.
  • 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.
  • 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- ⁇ , IL-2 or TNF- ⁇ .
  • the secretion of IFN- ⁇ 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. .
  • the ELISPOT method is employed to detect secretion of IFN-[gamma] by T cells.
  • the IFN- ⁇ monoclonal antibody pre-coated on the solid phase carrier first binds to IFN- ⁇ to form a complex, which is then bound to the biotin-labeled second IFN- ⁇ 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.
  • 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.
  • APCs antigen-presenting cells
  • the peptide itself is added directly to an experiment comprising T cells and APC.
  • 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 ⁇ g/ml, preferably 1 to 10 ⁇ g/ml. A typical time for T cells to be incubated with peptides is 16 to 36 hours.
  • PBMC peripheral blood mononuclear cells
  • 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.
  • the IFN- ⁇ -ELISPOT kit 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- ⁇ -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.
  • Figure 1 Results of the superior peptide verification of the IFN- ⁇ -ELISPOT tuberculosis diagnostic kit of the present invention: (A) IFN- ⁇ -ELISPOT results of the TB-specific polypeptide antigen peptides SEQ ID NO. (The number of spots is 123); (B) IFN- ⁇ -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.
  • A IFN- ⁇ -ELISPOT results of the TB-specific polypeptide antigen peptides SEQ ID NO. (The number of spots is 123); (B) IFN- ⁇ -ELISPOT results after stimulating cells of the tuberculosis-specific polypeptide antigen peptides SEQ ID NO. 4 to 6 (
  • Figure 2 The results of the preferred polypeptide verification of the IFN- ⁇ -ELISPOT tuberculosis diagnostic kit of the present invention:
  • 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)
  • E is the background control without cells (the number of spots is 0).
  • Figure 3 Diagnostic results of this IFN- ⁇ -ELISPOT kit for a suspected tuberculosis patient: (A) negative control (number of spots 1); (B) IFN- ⁇ -induced ESAT-6 non-immunodominative polypeptide after stimulation of cells ELISPOT results (number of spots is 5); (C) IFN- ⁇ -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- ⁇ -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- ⁇ -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- ⁇ -ELISPOT kit polypeptide sequences 1, 7 to 11 of a suspected tuberculosis patient.
  • (A) is the negative control (the number of spots is 1)
  • Fig. 4 (B) is the IFN- ⁇ -ELISPOT result (the number of spots is 4) after the ESAT-6 non-immunogenic polypeptide stimulating cells
  • Fig. 4 (C) Is the IFN- ⁇ -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.
  • Figure 4 (D) is the tuberculosis-specific polypeptide antigen of the product.
  • SEQ ID NO. 9-11 The IFN- ⁇ -ELISPOT results after stimulating cells (the number of spots was 43), and Fig. 4(E) shows the IFN- ⁇ -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- ⁇ -ELISPOT kit of the present invention.
  • Figure 6 shows the red box in Figure 5 as one of the results of IFN- ⁇ -ELISPOT tuberculosis test indicating tuberculosis positive: (A) negative control (number of spots is 1); (B) ESAT-6 non-immune dominant peptide IFN- ⁇ -ELISPOT results after stimulating cells (number of spots is 6); (C) IFN- ⁇ -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- ⁇ -ELISPOT results after stimulating the cells (the number of spots was 9); (E) the IFN- ⁇ -ELISPOT results (the number of spots was 466) after the PHA positive control stimulated the cells.
  • Example 1 Preparation of tuberculosis-specific T cell dominant epitope polypeptide and IFN- ⁇ -ELISPOT Mycobacterium tuberculosis
  • 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.
  • PBMC Peripheral blood mononuclear cells
  • ELISPOT assay detects IFN- ⁇ released by T cells after stimulation.
  • the specific procedure is: coating human IFN- ⁇ 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.
  • PHA phytohemagglutinin
  • Figure 1 shows a typical IFN- ⁇ -ELISPOT result screened for polypeptides 1-6.
  • Figure 1 (A) shows the IFN- ⁇ -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- ⁇ -ELISPOT results (the number of spots is 62) of the tuberculosis-specific polypeptide antigen peptides SEQ ID NOS.
  • FIG. 1 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.
  • 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)
  • Figure 2E is the background control without cells ( The number of spots is 0).
  • Example 2 A diagnostic kit for IFN- ⁇ -ELISPOT Mycobacterium tuberculosis infection developed by the present invention, comprising:
  • 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. ;
  • PHA phytohemagglutinin
  • a negative control is a medium (eg, RPMI 1640 + 10% FBS) or a lysing reagent (eg, DMSO) for solubilizing the polypeptide.
  • a medium eg, RPMI 1640 + 10% FBS
  • a lysing reagent eg, DMSO
  • Example 3 Clinical diagnosis of the IFN- ⁇ -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- ⁇ -ELISPOT Mycobacterium tuberculosis infection diagnostic kit of the present invention in the diagnosis of tuberculosis.
  • PBMC Peripheral blood mononuclear cells
  • ELISPOT assay detects IFN- ⁇ released by T cells after stimulation.
  • the specific steps are: coating human IFN- ⁇ 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.
  • polypeptides or polypeptides SEQ ID 4-6 (or preferred polypeptide SEQ ID NO)
  • PHA phytohemagglutinin
  • 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.
  • AEC 3-amino-9-ethylcarbazole
  • 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.
  • Figure 3 shows the diagnosis result of the IFN- ⁇ -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)
  • Figure 3 (B) shows the IFN- ⁇ -ELISPOT results (number of spots 5) after stimulation of cells with ESAT-6 non-immunogenic polypeptide
  • FIG. 3 (C) is the IFN- ⁇ -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- ⁇ -ELISPOT results (number of spots) of ID NO. 4 to 6 combined with the stimulator cells Fig. 3 (E) are the IFN- ⁇ -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- ⁇ -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)
  • Figure 4 (B) shows the IFN- ⁇ -ELISPOT results (number of spots 4) after stimulation of cells with ESAT-6 non-immunodominant polypeptide
  • Figure 4 (C) Is the IFN- ⁇ -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- ⁇ -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
  • Fig. 4 (E) are the IFN- ⁇ -ELISPOT results after the PHA positive control stimulator cells. (The number of spots is 330).
  • Example 4 Applicability of the IFN- ⁇ -ELISPOT kit of the present invention in tuberculosis screening in an unknown population
  • OBJECTIVE To test the IFN- ⁇ -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.
  • PBMC Peripheral blood mononuclear cells
  • ELISPOT assay detects IFN- ⁇ released by T cells after stimulation.
  • the specific steps are: coating human IFN- ⁇ 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.
  • PHA phytohemagglutinin
  • Figure 5 shows the results of the IFN- ⁇ -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- ⁇ -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- ⁇ -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- ⁇ -ELISPOT (the number of spots is 17) after the combination of the tuberculosis-specific polypeptide antigen peptides SEQ ID NO.
  • Figure 6 (D) is the tuberculosis-specific polypeptide antigen of the product.
  • the results suggest that the volunteers were positive for IFN- ⁇ -ELISPOT tuberculosis, and that one of the 37 volunteer samples was positive for IFN- ⁇ -ELISPOT tuberculosis, and the volunteers were further tested and confirmed to be active.
  • Mycobacterium tuberculosis infection indicating that the IFN- ⁇ -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.
  • 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.

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Abstract

La présente invention se rapporte à un antigène stimulant pour la détection d'une infection à Mycobacterium tuberculosis et à un kit comprenant l'antigène stimulant. L'invention concerne en outre des applications de l'antigène stimulant dans des réactifs pour la détection d'une infection à Mycobacterium tuberculosis. L'antigène stimulant comprend au moins un polypeptide ou des analogues correspondants de polypeptides représentés par les séquences 1 à 11 dans un tableau de séquences, les polypeptides provenant respectivement de polypeptides d'antigène spécifique de la tuberculose ESAT-6 et de polypeptides d'antigène spécifique de la tuberculose CFP-10. L'antigène stimulant selon l'invention peut stimuler les lymphocytes T du sang périphérique d'un patient atteint d'une infection de type tuberculose pour produire de l'IFN-γ afin de diagnostiquer l'infection de type tuberculose et n'est pas affecté par l'inoculation du BCG ou d'autres maladies sous-jacentes.
PCT/CN2014/095543 2014-12-17 2014-12-30 Antigène stimulant pour la détection d'une infection à mycobacterium tuberculosis, kit et applications de l'antigène stimulant WO2016095273A1 (fr)

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CN201410790793.1A CN104597239B (zh) 2014-12-17 2014-12-17 用于检测结核分枝杆菌感染的抗原刺激物、试剂盒及其应用

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114859062A (zh) * 2022-05-08 2022-08-05 吉林大学 一种基于双酶放大系统的化学发光免疫检测方法
CN116102629A (zh) * 2022-09-03 2023-05-12 武汉中纪生物科技有限公司 一种结核分枝杆菌t细胞抗原表位多肽及其应用
CN117304284A (zh) * 2023-08-07 2023-12-29 梅州市人民医院(梅州市医学科学院) 结核分枝杆菌特异性t细胞hla-a*0201限制性表位肽及其应用
CN117777259A (zh) * 2024-02-23 2024-03-29 上海科新生物技术股份有限公司 检测结核感染的抗原组合物、试剂盒及其应用

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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CN105954521B (zh) * 2016-07-08 2017-04-12 广州华弘生物科技有限公司 一种结核分枝杆菌感染诊断试剂盒
CN106353501A (zh) * 2016-08-19 2017-01-25 南通表源生物技术有限公司 一种结核t细胞检测试剂盒及其检测方法
CN108254553A (zh) * 2016-12-29 2018-07-06 广东希格生物科技有限公司 用于特异性检测结核分枝杆菌感染的蛋白Rv1791
CN110865187A (zh) * 2019-10-16 2020-03-06 华中科技大学同济医学院附属同济医院 基于酶联免疫斑点试验试剂盒计算esat-6抗原孔平均斑点面积的方法
CN115078732A (zh) * 2022-06-09 2022-09-20 广州迪澳医疗科技有限公司 结核分枝杆菌特异性刺激抗原、试剂盒及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004005925A2 (fr) * 2002-07-05 2004-01-15 Isis Innovation Limited Methode diagnostique
CN101419237A (zh) * 2008-09-03 2009-04-29 深圳市东湖医院 结核菌感染的酶联免疫斑点诊断试剂盒及特异性抗原制取方法
CN101493454A (zh) * 2008-01-21 2009-07-29 范雄林 结核病抗原特异的全血IFN-γ诊断试剂盒及其制作方法和应用方法
CN102187224A (zh) * 2008-09-22 2011-09-14 俄勒冈健康科学大学 用于检测结核分枝杆菌感染的方法
CN103604933A (zh) * 2013-11-27 2014-02-26 华中科技大学同济医学院附属同济医院 基于抗原特异性TNF-α-ELISA检测活动性结核病的试剂盒及其应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004099771A1 (fr) * 2003-05-08 2004-11-18 Statens Serum Institut Methode de diagnostic immunologique de la tuberculose basee sur de nouveaux epitopes specifiques
CN102297968B (zh) * 2010-06-28 2013-10-30 程小星 辅助诊断结核病的试剂盒
CN102608333B (zh) * 2012-03-30 2013-06-05 中国科学院微生物研究所 一种结核诊断组合物及其应用
CN103760345B (zh) * 2014-01-17 2016-03-02 北京旷博生物技术股份有限公司 一种利用外周血检测结核分枝杆菌感染的试剂盒及其应用
CN104020297B (zh) * 2014-06-10 2016-12-07 上海交通大学医学院 用于结核分枝杆菌感染检测及临床治疗效果监测的试剂盒及其用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004005925A2 (fr) * 2002-07-05 2004-01-15 Isis Innovation Limited Methode diagnostique
CN101493454A (zh) * 2008-01-21 2009-07-29 范雄林 结核病抗原特异的全血IFN-γ诊断试剂盒及其制作方法和应用方法
CN101419237A (zh) * 2008-09-03 2009-04-29 深圳市东湖医院 结核菌感染的酶联免疫斑点诊断试剂盒及特异性抗原制取方法
CN102187224A (zh) * 2008-09-22 2011-09-14 俄勒冈健康科学大学 用于检测结核分枝杆菌感染的方法
CN103604933A (zh) * 2013-11-27 2014-02-26 华中科技大学同济医学院附属同济医院 基于抗原特异性TNF-α-ELISA检测活动性结核病的试剂盒及其应用

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114859062A (zh) * 2022-05-08 2022-08-05 吉林大学 一种基于双酶放大系统的化学发光免疫检测方法
CN116102629A (zh) * 2022-09-03 2023-05-12 武汉中纪生物科技有限公司 一种结核分枝杆菌t细胞抗原表位多肽及其应用
CN116102629B (zh) * 2022-09-03 2023-09-08 武汉中纪生物科技有限公司 一种结核分枝杆菌t细胞抗原表位多肽及其应用
CN117304284A (zh) * 2023-08-07 2023-12-29 梅州市人民医院(梅州市医学科学院) 结核分枝杆菌特异性t细胞hla-a*0201限制性表位肽及其应用
CN117304284B (zh) * 2023-08-07 2024-03-19 梅州市人民医院(梅州市医学科学院) 结核分枝杆菌特异性t细胞hla-a*0201限制性表位肽及其应用
CN117777259A (zh) * 2024-02-23 2024-03-29 上海科新生物技术股份有限公司 检测结核感染的抗原组合物、试剂盒及其应用
CN117777259B (zh) * 2024-02-23 2024-06-07 上海科新生物技术股份有限公司 检测结核感染的抗原组合物、试剂盒及其应用

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