Reagent for detecting tubercle bacillus infection in vitro and method thereof

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

Active Publication Date: 2009-06-03
GUANGZHOU RHFAY BIOTECH CO LTD
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  • 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 ou

Method used

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  • Reagent for detecting tubercle bacillus infection in vitro and method thereof
  • Reagent for detecting tubercle bacillus infection in vitro and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Preparation of M233 polypeptide

[0040] ELISPOT was used to detect the lymphocytes of PPD-positive tuberculosis patients, and 21 polypeptides (each polypeptide containing 14 or 15 amino acids) of early secretory antigen target-6 (ESAT-6) designed by the Overlap method were screened. A specific T cell reactive Mtb polypeptide is produced, comprising the following steps:

[0041] (1) Design polypeptides by the Overlap method, each polypeptide contains 14 or 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 it with RPMI1640 culture medium to a concentration of 10ug / ml.

[0042] (2) Collect the blood of PPD-positive tuberculosis patients from Guangzhou Chest Hospital, about 2-5ml each.

[0043] (3) Peripheral blood mononuclea...

Embodiment 2

[0046] Example 2 Detection of lymphocytes in 47 cases of PPD-positive tuberculosis patients by ELISPOT

[0047] 1 Materials and methods

[0048] 1.1 Experimental materials The blood of 47 PPD-positive tuberculosis patients was collected from Guangzhou Chest Hospital, about 2-5ml each.

[0049] 1.2 Isolation of 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.

[0050] 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 M233, 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 background control, and a group o...

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Abstract

The invention discloses a reagent for detecting tubercle bacillus infection in vitro and a method thereof. The reagent comprises M233 polypeptide represented by SEQ ID No.1; and cytokine released from T cells is detected by contacting the M233 polypeptide or an analog thereof with the T cells of a tubercle bacillus host to determine whether the T cells identify the M233 polypeptide or the analog thereof. The reagent has the advantages of high sensitivity, good specificity, being free from interference of BCG vaccine and non-tuberculosis mycobacteria vaccine, being capable of detecting active pulmonary tuberculosis patients, the patients with dormant infection and healthy persons who contact with mycobacterium nontuberculosis. The reagent and the method are especially applicable to detecting tuberculosis and/or dormant infection thereof for Chinese people.

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 detecting 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. Now it has become the largest cause of death among all infectious diseases (has surpassed rabies), and has become the number one killer of young people. 1 / 3 of the world's population (about 2 billion) has been infected with tuberculosis, 95% of which occurred 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 age...

Claims

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Application Information

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IPC IPC(8): G01N33/53C12N15/31C12N15/63C07K7/08C07K16/12
Inventor 赖小敏董涛方毅敏
Owner GUANGZHOU RHFAY BIOTECH CO LTD
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