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Reagent and method for rapidly detecting antibody of mycobacterium tuberculosis in blood serum

A technology for the detection of mycobacterium tuberculosis and antibodies, applied in the field of biochemical detection, can solve the problems of low accuracy of antigen detection and achieve good application prospects

Inactive Publication Date: 2012-06-13
田绿波 +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antigens such as the 38kDa protein of Mycobacterium tuberculosis have been used in ELISA, EIA and other methods to diagnose tuberculosis, but the current detection accuracy of these antigens is still low

Method used

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  • Reagent and method for rapidly detecting antibody of mycobacterium tuberculosis in blood serum
  • Reagent and method for rapidly detecting antibody of mycobacterium tuberculosis in blood serum
  • Reagent and method for rapidly detecting antibody of mycobacterium tuberculosis in blood serum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Screening of Mycobacterium tuberculosis Specific Antigen

[0031] 1 Screening and determination of specific antigen

[0032] Using proteome analysis, Western blot and other techniques, the protein antigen spectrum of Mycobacterium tuberculosis was analyzed, and six specific antigen components were determined: 38KD, Ag85B, MPT64, 16KD, CFP10 and 10KD protein antigens.

[0033] 2 Cloning, expression and purification of genes encoding specific protein antigens

[0034] Using molecular cloning technology, the identified 38KD, Ag85B, MPT64, 16KD, CFP10 and 10KD protein antigen components were cloned and expressed, and the purified antigen was obtained by molecular sieve and ion exchange column chromatography. After the sequence of the obtained product was verified to be correct , which will be used in subsequent experiments.

[0035] Certainly, the above-mentioned antigenic proteins of Mycobacterium tuberculosis obtained in other ways can also be used in the foll...

Embodiment 2

[0036] Example 2 Diagnosis efficiency analysis of tuberculosis antigen and screening of antigen combination mode

[0037] experiment method

[0038] 1. Determine the best ELISA conditions for each antigen

[0039]1) Determination of protein concentration The protein measuring instrument measures the OD values ​​of the recombinant protein at 260nm and 280nm wavelengths, and calculates the protein content according to the formula: protein content=OD280×1.45+OD260×0.74 (mg / ml).

[0040] 2) Determination of optimal antigen coating concentration Dilute the purified protein antigen to different concentrations (10ug / ml, 8ug / ml, 6ug / ml, 4ug / ml, 2ug / ml, ml, 1ug / ml, 0.75ug / ml, 0.5ug / ml, 0.25ug / ml, 0.1ug / ml, 0.075ug / ml, 0.05ug / ml), add 0.1ml to each well of the ELISA plate, overnight at 4°C Take it out, dry it in air, add 0.2ml 30% calf serum to each well, and overnight at 4°C; add diluted tuberculosis positive serum and negative serum respectively, and use it as a blank control, and i...

Embodiment 3 8

[0076] Example 3 Establishment of colloidal gold-labeled double-antigen sandwich method of 38kDa+CFP10+MPT64 antigen combination

[0077] 1 Establishment of colloidal gold-labeled double-antigen sandwich method

[0078] Methods Colloidal gold-labeled immunochromatography

[0079] 1.1 Reagents and equipment

[0080] 1) Colloidal gold labeled tuberculosis antigen, this test uses the antigen combination 38kDa+CFP10+MPT64 determined in the above test, mixed and dissolved in corresponding reagents.

[0081] 2) Preparation of rabbit anti-human IgG antibody

[0082] 3) Goat anti-rabbit IgG antibody

[0083] 4) Nitrocellulose membrane

[0084] 1.2 Preparation method

[0085] Nitrocellulose membrane is immobilized with 1 detection line (A) and 1 quality control line (B line). Line A fixes reagents that react with tuberculosis IgG and is used to detect anti-tuberculosis IgG antibodies. Line B is goat anti-rabbit IgG antibody. The detection equipment was prepared according to the...

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Abstract

The invention aims to solve the technical problem that an antibody of mycobacterium tuberculosis in blood serum is difficult to rapidly and more accurately detect in the field. The technical scheme of the invention is to provide a detection reagent and rapid detection method for detecting an antibody of mycobacterium tuberculosis in blood serum. The detection reagent of the antibody of the mycobacterium tuberculosis in the blood serum comprises three types of mixed mycobacterium tuberculosis antigen proteins, namely 38kDA antigen protein, CFP10 antigen protein and MPT64 antigen protein. The detection method of the antibody of the mycobacterium tuberculosis in the blood serum comprises the following steps of: contacting in-vitro blood serum with the reagent containing the 38kDA antigen protein, the CFP10 antigen protein and the MPT64 antigen protein of the mycobacterium tuberculosis; and judging whether the in-vitro blood serum contains the mycobacterium tuberculosis according to the result of a process for detecting whether an antigen-antibody reaction exists. The reagent provided by the invention has the characteristics of fastness, sensitivity, specialty, simplicity, convenience, and the like; the reagent can be used for rapidly sieving the antibody of the mycobacterium tuberculosis in the in-vitro blood serum, is applicable to each grade of laboratories, and has a very good application prospect.

Description

technical field [0001] The invention belongs to the field of biochemical detection, and in particular relates to a detection reagent and a detection method for rapidly detecting serum mycobacterium tuberculosis antibody. Background technique [0002] Mycobacterium tuberculosis (M.tuberculosis), commonly known as Mycobacterium tuberculosis, is the pathogenic bacteria that causes tuberculosis. It can invade all organs of the body, but tuberculosis is the most common. Tuberculosis is a chronic infectious disease that seriously endangers human health, and its spread and infection are intensifying globally. For countries and regions where tuberculosis is endemic, strengthening the management and treatment of tuberculosis patients, especially infectious tuberculosis patients, is the most important prevention and control measure. [0003] Due to factors such as the increase of multidrug-resistant tuberculosis, the prevalence of HIV / AIDS, immigration, and the neglect of tuberculos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/558
Inventor 田绿波樊学军杨雨陈肖潇石莹
Owner 田绿波
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