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Antigen for detecting tuberculosis infection T cells, kit and application

A kit and antigen technology, applied in the direction of biological testing, measuring devices, material inspection products, etc., to achieve the effects of high sensitivity, high detection rate, and good diagnostic coincidence rate

Active Publication Date: 2017-09-29
武汉海吉力生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In view of the insufficient detection of some special samples by existing detection methods, such as children, immunocompromised and newly infected samples of Mycobacterium tuberculosis, the object of the present invention is to provide an antigenic composition for detecting T cells infected by Mycobacterium tuberculosis , the antigen composition cooperates with ESAT-6 and CFP-10 polypeptides and their derivatives to specifically stimulate fresh whole blood infected by Mycobacterium tuberculosis, specifically secrete IFN-γ, increase detection sensitivity, and effectively improve detection rate, effectively avoid missed detection

Method used

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  • Antigen for detecting tuberculosis infection T cells, kit and application
  • Antigen for detecting tuberculosis infection T cells, kit and application
  • Antigen for detecting tuberculosis infection T cells, kit and application

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1 Polypeptide Stimulation Effectiveness Screening

[0028] In order to realize the purpose of the present invention, the present invention has screened the specific protein of pathogenic Mycobacterium tuberculosis such as: RV3615, RV3873, RV3878, RV3879c, tb7.7, can be aimed at the polypeptide of binding site of lymphocyte in these proteins, screening All the peptides can specifically stimulate the specific secretion of γ-interferon (IFN-γ) in fresh whole blood of patients infected with Mycobacterium tuberculosis. Through repeated clinical verification experiments, the combination of these peptides ESAT-6 and CFP-10 can improve the detection sensitivity. The cytokine IFN-γ released in the blood indirectly reflects whether the body has been infected with Mycobacterium tuberculosis. Through a large number of experimental studies, the inventor finally obtained the specific polypeptide fragments whose nucleotide sequences are shown in SEQ ID NO.1~SEQ ID NO.28. -6-...

Embodiment 2

[0060] Embodiment 2 detection kit preparation

[0061] A test kit for detecting T cells infected with tuberculosis, including the antigen composition as described in Example 1 above and a reagent for detecting interferon gamma, wherein the reagent for detecting interferon gamma includes enzyme-linked immunoassay reagents, chemical Luminescence detection reagents, immunofluorescence detection reagents. These kits are described in detail below:

[0062]An in vitro release enzyme-linked immunoassay to detect whether the body is infected with Mycobacterium tuberculosis detection kit, the kit is respectively ESAT-6 and CFP-10 or ESAT-6-CFP-10 in embodiment 1 and embodiment 1 The specific polypeptide in SEQ ID NO.1~SEQID NO.28 is Mycobacterium tuberculosis specific stimulating antigen, (ie ESAT-6 and CFP-10 and SEQ ID NO.1~SEQ ID NO. 28, or ESAT-6-CFP-10 and the specific polypeptide in SEQ ID NO.1~SEQ ID NO.28 in embodiment 1 as Mycobacterium tuberculosis specific stimulating anti...

Embodiment 3

[0065] Embodiment 3 uses kit of the present invention to the detection method of in vitro whole blood detection

[0066] The test kit prepared in Example 2 is used for detection, first in vitro whole blood stimulation: before detection, ensure that the constant temperature incubator is 37 ° C, and negative control culture tube (N), tuberculosis stimulation culture tube (T), positive control Centrifuge the culture tube (P) at 3000-5000 rpm for 1 minute, mark the three culture tubes respectively, and add the sample number or name. Then turn on the ultraviolet lamp of the biological safety cabinet and irradiate for 15-20 minutes.

[0067] 1. Collection: venipuncture is used to collect whole blood samples using vacuum blood collection tubes anticoagulated with lithium heparin, and the collection volume is not less than 4mL.

[0068] 2. Dispensing: Invert and mix the blood collection tubes for collecting whole blood samples 5-10 times, and dispense them into "N", "T", and "P" tube...

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Abstract

The invention discloses an antigen for detecting tuberculosis infection T cells. The amino acid sequence of the antigen is shown as SEQ ID NO.1 to SEQ ID NO.28; any eight polypeptides in SEQ ID NO.1 to SEQ ID NO.28 are coordinated with ESAT-6 and CFP-10 polypeptides and derivatives to have a common effect to specifically stimulate mycobacterium tuberculosis infected T cells to specifically secrete IFN-gamma, and the detection sensitivity is increased. The invention provides a novel detection kit for the tuberculosis infection T cells; when the kit is used, peripheral blood is directly subjected to antigen stimulation and mononuclear cells of the peripheral blood do not need to be separated; the detection sensitivity and the detection specificity are relatively high; the novel detection kit is simple and convenient to operate, has relatively low detection cost and has relatively high clinical application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to antigens, kits and applications for detecting tuberculosis-infected T cells. Background technique [0002] Tuberculosis is one of the important infectious diseases that are mainly transmitted through the respiratory tract and seriously endanger my country and the world. According to the World Health Organization, there are 1.7-2 billion Mycobacterium tuberculosis infections in the world, and at least 2 million people die from this disease every year (Xie Jianping, Methodology for Mycobacterium tuberculosis functional genome research, Microbiology Bulletin. 2001.28 (5) :92-97). Therefore, methods of accurately screening infected persons play an extremely important role in the prevention and control and even the eventual elimination of tuberculosis. [0003] Existing diagnostic techniques are very difficult to diagnose patients with Mycobacterium tuberculosis infection. In recent de...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/535G01N33/577G01N33/68G01N21/64G01N21/76G01N33/52
CPCG01N21/6428G01N21/763G01N33/52G01N33/535G01N33/5695G01N33/577G01N33/6866G01N2021/6439G01N2333/35G01N2333/57G01N2800/12
Inventor 丁晓莉张诗冉路春桃熊俊黄俊赵平锋
Owner 武汉海吉力生物科技有限公司
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