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Reagent and method for detecting Mycobacterium tuberculosis infection in vitro

A technology for in vitro detection of Mycobacterium tuberculosis, applied in the field of biomedical testing, can solve the problems of high price, unsatisfactory effect, and difficulty in popularization, and achieve high sensitivity, unsatisfactory overcoming effect, and good specificity

Inactive Publication Date: 2012-01-04
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, studies have shown that the effect of T-SPOT.TB reagents in some countries is not satisfactory, and it is speculated that the major histocompatibility antigens of the populations of these countries may be different, and the peptides used are mainly screened out for the populations of European countries, and The high prevalence of non-tuberculous mycobacteria infection in these countries is related to
In addition, the T-SPOT.TB reagent is expensive, about $80 per person, and it is difficult to promote in developing countries

Method used

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  • Reagent and method for detecting Mycobacterium tuberculosis infection in vitro
  • Reagent and method for detecting Mycobacterium tuberculosis infection in vitro
  • Reagent and method for detecting Mycobacterium tuberculosis infection in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1M232

[0041] The preparation of embodiment 1M232 polypeptide

[0042] The lymphocytes of PPD-positive tuberculosis patients were detected by ELISPOT, and 21 polypeptides (each polypeptide containing 15 amino acids) of early secretory antigen target-6 (ESAT-6) designed by the Overlap (overlap) method were screened, and 1 A specific T cell reactive Mtb polypeptide, comprising the following steps:

[0043] (1) Design polypeptides by the Overlap method, each polypeptide contains 15 amino acids (aa), and 21 polypeptide sequences are obtained, which are synthesized by a solid-phase formylation synthesizer according to the experimental conditions of the synthesizer manual. Peptides were dissolved in DMSO and prepared as a 10 mg / ml stock solution. Before use, dilute with RPMI1640 culture medium (Note: RPMI is the abbreviation of "Roswell Park Memorial Institute," "Roswell Park Memorial Institute", 1640 is the medium code) to a concentration of 10 μg / ml.

[0044] (2) Collect the blood of P...

Embodiment 2

[0048] Embodiment 2 detects lymphocyte of 310 routine PPD positive tuberculosis patients with ELISPOT

[0049] 1 Materials and methods

[0050] 1.1 Experimental materials The blood of 310 PPD-positive tuberculosis patients was collected from Guangzhou Chest Hospital, about 2-5ml each.

[0051] 1.2 Separation of peripheral blood lymphocytes (PBMCs) from tuberculosis patients Ficoll lymphocyte separation medium was used to separate PBMCs, and PBMCs were resuspended in R10 culture medium (10% calf serum in RPMI1640 culture medium) for later use.

[0052] 1.3 ELISPOT analysis Select a 96-well plate with PVDF membrane as the reaction plate, coat it with anti-human γ-interferon (IFNγ) monoclonal antibody (mAb) overnight, add 5 × 10 5 For PBMC and polypeptide M232, 3 wells were set up for each assay and incubated for 18 hours. The following control wells were set up: PMA and Ionomycin were used as positive controls, no polypeptide was used as negative control, no cells were used as...

Embodiment 3

[0055] Example 3 ELISPOT is used to detect the effect of mixed polypeptide M232+M233 (1:1) on lymphocytes of 529 cases of PPD positive tuberculosis patients

[0056] The M233 polypeptide is a polypeptide disclosed in the 200810220523.1 patent application filed by the applicant, and its amino acid sequence is: SEQ ID No.2.

[0057] 1 Materials and methods

[0058] 1.1 Experimental materials The blood of 529 PPD-positive tuberculosis patients was collected from Guangzhou Chest Hospital, about 2-5ml each.

[0059] 1.2 Separation of peripheral blood lymphocytes (PBMCs) from tuberculosis patients Ficoll lymphocyte separation medium was used to separate PBMCs, and PBMCs were resuspended in R10 culture medium (10% calf serum in RPMI1640 culture medium) for later use.

[0060] 1.3 After the synthesis of M232 and M233 polypeptides, they were dissolved in DMSO and prepared as 10 mg / ml stock solutions. Before use, appropriate amounts of M232 and M233 were diluted with RPMI1640 culture s...

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Abstract

The invention discloses a reagent and a method for detecting Mycobacterium tuberculosis infection in vitro. The reagent comprises a segment of specific T cell reactive polypeptide, namely M232 polypeptide shown as SEQ ID No.1 and screened from early secreting antigen target-6 (ESAT-6) polypeptide; and the M232 polypeptide is contacted with a T cell of a Mycobacterium tuberculosis host to detect cell factors released by the T cell and determine whether the T cell identifies the M232 polypeptide. The invention has the advantages of high sensitivity, no influence of Bacillus Calmette-Guerin (BCG) vaccine and nontuberculosis mycobacterial vaccine, high specificity, capacity of detecting patients with active pulmonary tuberculosis and patients with potential infection, and capacity of detecting healthy Mycobacterium tuberculosis contacts. The invention is particular suitable for detecting tuberculosis and / or potential infection of the tuberculosis for Chinese people. The invention also relates to a kit for detecting Mycobacterium tuberculosis infection in vitro, which comprises two mixed polypeptides, namely M232 and M233, and a tool for detecting the identification of the T cell on protein, or polypeptide or analogs of the polypeptide.

Description

【Technical field】 [0001] The invention belongs to the field of biomedical testing, and relates to a cell immunology testing method, in particular to a reagent and a method for in vitro testing mycobacterium tuberculosis infection. 【Background technique】 [0002] Tuberculosis caused by Mycobacterium tuberculosis (Mtb) is an infectious disease that seriously endangers human health. In the past 20 to 30 years, due to population movement, the spread of drug-resistant tuberculosis, AIDS and other factors, the tuberculosis epidemic has rebounded globally. It has now surpassed rabies as the largest cause of death among all infectious diseases, and has become the number one killer of young people. 1 / 3 of the world's population (about 2 billion) is infected with tuberculosis, 95% of which occur in developing countries. Among them, 20 million are patients with active tuberculosis, and 8 to 10 million new tuberculosis patients are added every year, and 75% of them are aged between 15...

Claims

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Application Information

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IPC IPC(8): G01N33/569C12N15/31C12N15/63C07K7/08C07K19/00C07K16/12
Inventor 赖小敏方毅敏
Owner SUN YAT SEN UNIV
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