Antigen stimulant and kit for detecting mycobacterium tuberculosis infection, and application of antigen stimulant

A technology of mycobacterium tuberculosis and antigen stimulators, applied in the field of biomedical testing, can solve the problems of complex antigen peptides, affecting cytokine secretion, and high price, achieving high sensitivity and specificity, good clinical applicability, and sensitive sex high effect

Active Publication Date: 2015-05-06
SUN YAT SEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] T-SPOT.TB kit and QuantiFERON-TB Gold kit have complex components, high price, and extremely high cost, which seriously restricts their use in millions of active tuberculosis patients and hundreds of millions of latent tuberculosis infected people Large-scale tuberculosis screening and diagnosis
In addition, the antigenic peptides contained in these kits are extremely complex and are not mainly designed, screened and verified based on the major histocompatibility antigens of Chinese people, so the applicability and use effect need to be further improved
The Chinese patent application whose publication number is CN 102516356 A screens out 8-11 continuous polypeptide fragments from the antigen Rv3615c as a stimulus source, and adopts the enzyme-linked immunospot test (ELISPOT) to diagnose tuberculosis infection, but its sensitivity is only 64%, far reaching Less than the requirements of commercial kits
The antigen stimulus used in the Chinese patent application with publication number CN 102297968 A is a combination of 9 polypeptides of Mycobacterium tuberculosis ESAT-6 antigen and CFP-10 antigen, and uses IFN-γ, TNF-α, IL-2 , MIG and IP-10 five kinds of antibody-coated magnetic beads, simultaneously detect five kinds of cytokines in the cell culture supernatant, which causes the complexity of the detection method and result analysis, and requires the use of flow cytometry, increasing the The cost of detection limits its promotion and use
The Chinese patent application with the publication number CN 103604933 A is a kit for detecting active tuberculosis based on TNF-α cytokine ELISA. The antigenic stimulators used in the kit are the whole protein of ESAT-6 and CFP-10. However, due to the whole The spatial conformation of the protein may hinder the binding of effective epitopes to the recognition molecules on the surface of T lymphocytes, and it contains other epitopes that may produce negative regulation and affect the secretion of cytokines, causing the kit to be effective for latent infection Tuberculosis is poorly diagnosed

Method used

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  • Antigen stimulant and kit for detecting mycobacterium tuberculosis infection, and application of antigen stimulant
  • Antigen stimulant and kit for detecting mycobacterium tuberculosis infection, and application of antigen stimulant
  • Antigen stimulant and kit for detecting mycobacterium tuberculosis infection, and application of antigen stimulant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Preparation of tuberculosis-specific T cell dominant antigen epitope polypeptide and development of IFN-γ-ELISPOT Mycobacterium tuberculosis infection diagnostic kit

[0045] Design and screen T cell dominant antigen epitopes through bioinformatics analysis and in vitro detection of T cell immune response, including the following steps:

[0046] (1) Through bioinformatics technology and corresponding databases such as ProPred, MacVestor, Preotean, etc., predict the T cell antigen epitopes of the proteins ESAT-6 and CFP-10 encoded by the RD1 region, and analyze the relationship between them and different HLA types identification relationship between them.

[0047] (2) Combined with the results of biological analysis, 9 ESAT-6 polypeptides and 11 CFP-10 polypeptides were designed and optimized by the Overlap (overlapping sequence) method, and each polypeptide contained 9-22 amino acids.

[0048] (3) Collect blood samples from tuberculosis patients, 2-5ml each....

Embodiment 2

[0052] Embodiment 2: The IFN-γ-ELISPOT Mycobacterium tuberculosis infection diagnostic kit developed by the present invention comprises:

[0053] (1) Antigen stimulant, which is a combination of one or more than two polypeptides in the amino acid sequence screened in Example 1 as shown in Sequence 1-11 in the Sequence Listing, or an analog of these polypeptides ;

[0054] (2), the capture antibody is a monoclonal antibody against human IFN-γ, which can be purchased from ebioscience;

[0055] (3), the detection antibody is a biotin-labeled anti-human IFN-γ antibody;

[0056] (4), horseradish peroxidase-labeled streptavidin;

[0057] (5), 3-amino-9-ethylcarbazole (AEC) chromogenic substrate;

[0058] (6), the positive control stimulus is phytohemagglutinin (PHA);

[0059] (7) Negative control, which is culture medium (such as RPMI1640+10% FBS) or a dissolving reagent (such as DMSO) for dissolving the polypeptide.

Embodiment 3

[0060] Example 3: The IFN-γ-ELISPOT kit of the present invention is applied to the clinical diagnosis of suspected patients with Mycobacterium tuberculosis infection

[0061] Objective: To test the clinical applicability, specificity, sensitivity and coincidence rate with other diagnostic techniques of the IFN-γ-ELISPOT Mycobacterium tuberculosis infection diagnostic kit of the present invention in tuberculosis diagnosis.

[0062] (1) Collect blood samples from 55 cases of highly suspected Mycobacterium tuberculosis infection, 2-5ml each.

[0063] (2) Separate peripheral blood mononuclear cells (PBMC) with Ficoll lymphocyte separation medium, wash PBMC twice with 5-10ml PBS or PRMI1640, resuspend in RPMI1640 medium containing 10% fetal bovine serum (FBS), and calculate number of cells.

[0064] (3) ELISPOT method is used to detect the IFN-γ released by T cells after being stimulated. The specific steps are: coat human IFN-γ monoclonal antibody on a 96-well plate with PVDF mem...

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Abstract

The invention provides an antigen stimulant for detecting mycobacterium tuberculosis infection, and a kit comprising the antigen stimulant. The invention also provides an application of the antigen stimulant in reagents for detecting mycobacterium tuberculosis infection. The antigen stimulant comprises at least one polypeptide or analogues thereof in polypeptides shown as the sequences 1-11 in a sequence table, wherein the polypeptides respectively come from tuberculosis specific antigen polypeptides ESAT-6 and tuberculosis specific antigen polypeptides CFP-10. According to the antigen stimulant provided by the invention, peripheral blood T lymphocytes of tuberculosis infection patients can be effectively stimulated to generate IFN-gamma, so that the tuberculosis infection can be diagnosed at high sensitivity and high specificity, and the influence on BCC inoculation or other underlying diseases can be avoided.

Description

technical field [0001] The invention belongs to the field of biomedical testing, and in particular relates to an antigenic stimulus for detecting mycobacterium tuberculosis infection, an application thereof, and a kit containing the antigenic stimulus. Background technique [0002] Tuberculosis is a kind of infectious disease that seriously endangers human health caused by Mycobacterium tuberculosis. Although Mycobacterium tuberculosis initially infects the human respiratory system, it can spread to almost all organs of the human body and cause corresponding disease. It is estimated that approximately one-third of the world's population is infected with Mycobacterium tuberculosis, with 8 million new infections and 2 million deaths from TB each year. my country's tuberculosis epidemic and Mycobacterium tuberculosis infection are quite serious. It is one of the 22 countries with a high burden of tuberculosis in the world, and the number of tuberculosis patients ranks second i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCC12Q1/68G01N33/564G01N33/5695G01N33/68
Inventor 曾谷城汪华
Owner SUN YAT SEN UNIV
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