EV71 (human enterovirus 71) antigen enzyme-linked reaction detection kit and its preparation method

A technology of EV71 and enzyme-linked reaction, applied in the biological field, can solve the problems such as the preparation method report of the monoclonal antibody enzyme-linked reaction detection kit that has not been found yet

Inactive Publication Date: 2013-01-02
BEIJING HUAWEI BRAVOBIO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0024] Through literature search, etc., so far, no new monoclonal antibody against the conserved region gene in EV71 virus Vp1, the corresponding enzyme-linked reaction detection kit and its preparation method have been found.

Method used

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  • EV71 (human enterovirus 71) antigen enzyme-linked reaction detection kit and its preparation method
  • EV71 (human enterovirus 71) antigen enzyme-linked reaction detection kit and its preparation method
  • EV71 (human enterovirus 71) antigen enzyme-linked reaction detection kit and its preparation method

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Experimental program
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Effect test

preparation example Construction

[0131] (1) Preparation of antigen reference products;

[0132] (2) Preparation of monoclonal antibodies;

[0133] (3) Preparation of anti-EV71 monoclonal antibody-coated plate;

[0134] (4) Preparation of enzyme-labeled antibody;

[0135] (5) Configuration of sample diluent, concentrated washing solution and stop solution;

[0136] (6) Configuration of chromogenic solution;

[0137] Wherein, the EV71 antigen detection method is a double-antibody sandwich enzyme-linked immunosorbent assay;

[0138] (1) The preparation method of the antigen reference product is as follows:

[0139] The purified solution of EV71 virus Vp1 prepared by the genetic engineering method is prepared as a solution containing antigen, and then bovine serum albumin is added to the solution as a protective agent. It is subpackaged to obtain the EV71 antigen reference product;

[0140] (2) The preparation method of the monoclonal antibody is as follows:

[0141] ①Use the prepared EV71 purified liquid ...

Embodiment 1

[0205] Embodiment 1, the preparation of EV71 antigen reference product

[0206] According to the inventor's research on the enterovirus EV71 in different epidemic areas of the country for many years, the gene and protein sequences of different strains were analyzed, the inventor selected the conserved region gene in the EV71 virus structural protein Vp1 to clone, and adopted different expression systems Expression is performed to prepare the antigen. During the expression process, a prokaryotic or eukaryotic expression system is used to make the prepared antigen have more natural components to ensure the specificity of the prepared antibody.

[0207] The purified solution of EV71 virus Vp1 prepared by the genetic engineering method is prepared as a solution with an antigen content of 1000 ng / ml, and then 1% bovine serum albumin is added to the solution as a protective agent. It is divided into 1.0ml / bottle to prepare EV71 antigen reference substance.

Embodiment 2

[0208] Embodiment 2, the preparation of the monoclonal antibody that recognizes EV71 antigen

[0209] BALB / c mice were immunized with the prepared EV71 purified solution. The titer was determined by indirect ELISA method, and spleen cells from the BALB / c mouse with the highest titer were fused with SP2 / 0 myeloma cells in proportion, and the fusion and hybrid tumor cells were cultured on HAT medium. Three times of subcloning were carried out, and each time of subcloning, the indirect ELISA titer of each cell well was detected, and the cells in the wells with an OD value greater than 0.6 were cloned to obtain high-titer monoclonal antibody cell lines targeting different sites . The obtained cell lines were cultured on a large scale, and the cultured cells were injected into the peritoneal cavity of BALB / c mice to prepare anti-EV71 monoclonal antibody ascites. The site specificity of the resulting antibodies was confirmed by ELISA (competition method) in reaction with native vi...

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Abstract

The invention relates to the biotechnical field, concretely relates to a virus antigen content detection kit and its preparation method, and more concretely relates to an enzyme-linked reaction kit for qualitatively and quantitatively detecting the EV71 virus antigen content, and its preparation method. The double-antibody enzyme-linked reaction detection kit adopted in the invention and its preparation method have the characteristics of good specificity, high sensitivity, convenience, fastness, economy and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a virus antigen content detection kit and a preparation method thereof, more specifically to an enzyme-linked reaction immunoassay kit for qualitative and quantitative evaluation of the EV71 virus antigen content and its preparation method. Background technique [0002] (1) Research progress of EV71 virus and its vaccine [0003] 1. Overview and pathogenic mechanism [0004] Enterovirus 71 (English name: Human enterovirus 71), referred to as EV71: EV71 virus belongs to the Picornaviridae family, and the virus particles have a typical icosahedral structure. Due to the wide range of impacts and the severity of the damage caused by EV71, hand-foot-mouth disease (HFMD) has been included in the management of Class C infectious diseases in China. In view of this, scholars and biological companies from all over the world are working on the research of EV71 virus vaccine. [0005] The EV...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/569G01N33/558
Inventor 史晋刘昊智鲍路伟王文灏程超张爱晖
Owner BEIJING HUAWEI BRAVOBIO
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