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Mycobacterium tuberculosis TB-SA (total body surface area) antibody colloidal gold method detection kit and preparation method thereof

A technology of Mycobacterium tuberculosis and TB-SA, which is applied in biological tests, measuring devices, analysis materials, etc., can solve the problems of high false positive rate of PPD test, poor specificity of tuberculosis diagnosis, and inability to meet clinical needs, and achieve specificity. Good, high sensitivity, suitable for large-scale production effect

Active Publication Date: 2015-04-01
成都赛普克生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the tuberculosis smear test is simple and accurate, the positive rate is low
Mycobacterium tuberculosis culture detection is highly reliable, but it takes a long time and cannot meet clinical needs
In addition, the antigen used in the tuberculin skin test (PPD) test is a mixture of antigens crudely extracted from Mycobacterium tuberculosis, and many non-tuberculous mycobacteria and Bacillus Calmette-Guerin (BCG) have common antigenic components, which leads to the use of this method in the diagnosis of tuberculosis. poor specificity
China is a highly endemic area of ​​tuberculosis. Due to the widespread vaccination of BCG, the false positive rate of PPD test is also very high. Therefore, clinically, auxiliary diagnosis can only be given according to the strength of skin reaction in PPD test.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: Determination of reaction conditions

[0036] The immunochromatographic colloidal gold detection reaction process mainly involves the combination and matching of three aspects, namely: the preparation of gold-labeled conjugates, the coating of detection lines and quality control lines, and the selection of nitrocellulose membranes.

[0037] 1. Determination of preparation of gold-labeled conjugates

[0038] According to the experimental design, the whole process is divided into colloidal gold preparation, labeling, sealing, centrifugal concentration and spraying, and the conditions of each process are determined as follows:

[0039] Preparation of colloidal gold: It is prepared by reducing chloroauric acid with trisodium citrate, and the specific preparation process is determined as follows:

[0040] Preparation: 1% chloroauric acid solution, 1% trisodium citrate solution.

[0041] Preparation: Add 2.0ml of sodium citrate solution (1%) into a conical fla...

Embodiment 2

[0080] 1. Raw materials:

[0081] (1) Mycobacterium tuberculosis TB-SA antigen: a colorless transparent liquid with a concentration greater than 2 mg / ml, determined by SDS-PAGE;

[0082] (2) Goat anti-human polyclonal antibody: it is a colorless transparent liquid with a concentration greater than 2mg / ml;

[0083] (3) SPA protein: it is a colorless transparent liquid with a concentration greater than 2mg / ml, determined by SDS-PAGE;

[0084] (4) Loading buffer: 0.5 g / ml surfactant TWEEN-20 solution.

[0085] 2. Liquid preparation:

[0086] (1) Preparation of sample loading buffer: Weigh 0.5g of surfactant TWEEN-20, add 20mM PB buffer and stir to fully dissolve, then dilute to 100ml and store at 2-8°C.

[0087] (2) Preparation of chloroauric acid solution: weigh 1.00 g of chloroauric acid, add ultrapure water to dissolve it, dilute to 100 mL, and store at 2-8°C in the dark.

[0088] (3) Preparation of trisodium citrate solution: weigh 1.0 g of trisodium citrate, add ultrapur...

Embodiment 3

[0094] Embodiment 3: the preparation of kit, it comprises the following steps:

[0095] S1. Preparation of test strips:

[0096] S11. Preparation of reaction membrane: Dilute the Mycobacterium tuberculosis TB-SA antigen to a coating concentration of 1.0 mg / ml with phosphate buffer, dilute the goat anti-human polyantibody to a coating concentration of 1.5 mg / ml, and draw Membrane spray gold label machine draws the two solutions onto the nitrocellulose membrane respectively, and saves the membrane after drying. The drying temperature is 35°C and the drying time is 120 minutes;

[0097] S12. Preparation of gold-binding pad: label colloidal gold with SPA protein at a concentration of 0.5 mg / ml, centrifuge at 12,000 r / min for 30 min, discard the supernatant, and add colloidal gold conjugate diluent to 1 / 1 of the original volume 10, then infiltrate the cut polyester film with the mixed solution to prepare a gold bonding pad;

[0098] S13. Assembling and cutting strips: Remove 3cm ...

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Abstract

The invention discloses a Mycobacterium tuberculosis TB-SA (total body surface area) antibody colloidal gold method detection kit and a preparation method of the detection kit. The detection kit is composed of a test strip and loading buffer, wherein the test strip is composed of a detection line coated with TB-SA antigen, a quality control line coated with a goat-anti-human multi-antibody nitrocellulose membrane, a gold-labeled pad with colloidal gold marked SPA (Staphylococcus aureus) protein adsorbed on a polyester film, a sample pad and absorbent paper; and the loading buffer is PB (phosphate buffer) solution containing surface active agent. The preparation method of the detection kit comprises the steps of preparing the test strip and the loading buffer. The Mycobacterium tuberculosis TB-SA antibody colloidal gold method detection kit has the characteristics of rapidness, convenience, accuracy, low cost, good specificity and high sensitivity, and the testing result can be read within 5-15min; and the method for preparing detection kit can be operated simply, can be prepared conveniently and is suitable for industrialized mass production.

Description

technical field [0001] The invention relates to an antibody detection kit and a preparation method thereof, in particular to a mycobacterium tuberculosis TB-SA antibody colloidal gold method detection kit and a preparation method thereof. Background technique [0002] Tuberculosis, AIDS and malaria are regarded as the three major diseases affecting global public health so far. Tuberculosis is one of the major diseases that my country focuses on controlling, and it is also a public health and social problem of global concern. [0003] According to the World Health Organization (WHO) and the fifth national tuberculosis epidemiological survey, nearly 1 / 3 of the world's population is infected with Mycobacterium tuberculosis, and about 8 million new cases occur every year, and nearly 3 million people die from this disease. sick. China is one of the 22 countries with a high burden of tuberculosis in the world, and the number of tuberculosis patients ranks second in the world. At...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/569
CPCG01N33/5695G01N2800/12
Inventor 叶成栋刘军郭沛罗春勤王洪
Owner 成都赛普克生物科技股份有限公司