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Antigen and kit for detecting tuberculosis-infected T cells and application of kit

A kit and antigen technology, applied in the direction of biological testing, measuring devices, material inspection products, etc., to achieve good diagnostic coincidence rate, convenient operation, and rapid diagnosis

Active Publication Date: 2017-09-08
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 and kit for detecting tuberculosis-infected T cells and application of kit
  • Antigen and kit for detecting tuberculosis-infected T cells and application of kit
  • Antigen and kit for detecting tuberculosis-infected T cells and application of kit

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1 Polypeptide Stimulation Effectiveness Screening

[0029] In order to achieve the purpose of the present invention, the polypeptides screened in the present invention are the specific proteins of pathogenic Mycobacterium tuberculosis from: Ag85B, Mpt64, RV1985C, RV1989c, Rv3425, and among these proteins, the polypeptides that can target the binding site of lymphocytes are screened, All the screened polypeptides 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 sequence...

Embodiment 2

[0059] Embodiment 2 detection kit preparation

[0060] 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:

[0061] An in vitro release enzyme-linked immunosorbent assay detects whether the body is infected with Mycobacterium tuberculosis detection kit, the kit uses ESAT-6 and CFP-10 or ESAT-6-CFP-10 in embodiment 1 and SEQ ID NO respectively Any eight polypeptides in 1~SEQ ID NO.26 or the combination of more than eight are Mycobacterium tuberculosis specific stimulating antigens, (ie ESAT-6 and CFP-10 and SEQ ID NO.1~SEQ ID in embodiment 1 The specific polypeptide in NO.26, or the specific polypeptide in ESAT-6-CFP-10 and SEQ ID NO.1~SEQ ID...

Embodiment 3

[0064] Embodiment 3 uses kit of the present invention to the detection method of in vitro whole blood detection

[0065] 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.

[0066] 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.

[0067] 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-infected T cells. The amino acid sequence of the antigen is shown as SEQ ID NO.1-SEQ ID NO.26; eight polypeptides are randomly selected from the SEQ ID NO.1-SEQ ID NO.26 and are collaborated with ESAT-6 and CFP-10 polypeptides and derivatives thereof, and specifically-stimulated mycobacterium tuberculosis-infected T cells can specifically secrete IFN-gamma, so that the detection sensitivity is increased. The invention provides a novel kit applicable to detection of the tuberculosis-infected T cells. The kit is used for directly performing antigenic stimulation on peripheral blood, without needing separating mononuclear cells from the peripheral blood; the kit is relatively high in detection sensitivity and detection specificity, easy and convenient to operate, low in detection cost and relatively high in 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): G01N33/569G01N33/577G01N33/535G01N33/68G01N21/64G01N21/76G01N33/52
CPCG01N21/6428G01N21/763G01N33/52G01N33/535G01N33/5695G01N33/577G01N33/6866G01N2021/6439G01N2333/35G01N2333/57G01N2800/12
Inventor 张诗冉丁晓莉路春桃黄俊赵平锋
Owner 武汉海吉力生物科技有限公司
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