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A kind of enterovirus type 71 inactivated vaccine antigen ELISA detection kit

An enzyme-linked immunoassay and enterovirus technology, applied in the biological field, can solve problems such as the inability to correctly detect the antigen content of inactivated vaccines, the impact of test results, and the inability to monitor vaccines, achieving good specificity, high sensitivity, and good specificity sexual effect

Active Publication Date: 2016-03-23
ZHEJIANG PUKANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has a long cycle and often takes several days to get the results, and because CPE can only detect infectious viruses, it cannot correctly detect the antigen content in inactivated vaccines, such as vacuolar viruses, inactivated viruses etc., so it is impossible to carry out effective, real-time and correct monitoring during the production process of the vaccine
However, the detection of EV71 antigen by enzyme-linked immunosorbent assay often has a certain impact on the detection results due to the source of the antibody, especially the non-specific adsorption of impurities in the vaccine greatly interferes with the detection results.

Method used

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  • A kind of enterovirus type 71 inactivated vaccine antigen ELISA detection kit
  • A kind of enterovirus type 71 inactivated vaccine antigen ELISA detection kit
  • A kind of enterovirus type 71 inactivated vaccine antigen ELISA detection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0028] The preparation of embodiment 1 anti-enterovirus type 71 virus polyclonal antibody

[0029] 1) The expression gene of enterovirus type 71 capsid structural protein (VP1) was cloned to obtain a recombinant expression bacterium expressing enterovirus type 71 capsid structural protein;

[0030] 2) Express enterovirus 71 capsid structural protein (VP1) recombinant antigen with the obtained recombinant bacteria expression bacteria, and obtain relatively pure recombinant antigen through column chromatography;

[0031] 3) Rabbits were immunized with the obtained antigen, immunized four times at intervals of one week, each immunization was 0.5 mg. Serum was collected four weeks after the last immunization;

[0032] 4) The obtained serum was purified by protein A column chromatography to obtain the purified anti-enterovirus type 71 polyclonal antibody. The resulting purified antibody see figure 2 .

Embodiment 2

[0033] Embodiment 2 Preparation of anti-enterovirus 71 virus mouse monoclonal antibody

[0034] 1) The enterovirus type 71 specific polypeptide FGEHKQEKDL synthesized, and coupled with the keyhole limpet hemocyanin carrier protein;

[0035]2) The resulting conjugated protein was immunized with Balb / c mice to obtain antigen-stimulated mouse splenocytes;

[0036] 3) The splenocytes were fused with mouse myeloma cells (SP2 / 0), and screened by indirect ELISA

[0037] Positive clones of hybridoma cells secreting specific antibodies, and then subcloned and identified by limiting dilution

[0038] IgG subtype, obtain a stable monoclonal hybridoma cell line;

[0039] 4) Ascites was prepared by injecting monoclonal hybridoma cells cultured in vitro into the peritoneal cavity of Balb / c mice, and the obtained ascites was subjected to protein A column chromatography to separate and obtain purified anti-enterovirus 71 monoclonal antibodies. The resulting purified antibody see image ...

Embodiment 3

[0040] The preparation of embodiment 3 enterovirus 71 virus antigen standard items

[0041] 1) The enterovirus 71 antigen standard product is obtained by culturing and amplifying the enterovirus 71 vaccine strain on Vero cells, freeze-thawing, centrifugal extraction and column chromatography. It is used for the calibration of the antigen content of enterovirus 71 inactivated vaccine. See the resulting antigen Figure 4 .

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Abstract

The invention relates to an ELISA detection kit for an enterovirus 71 inactivated vaccine antigen, the detection kit comprising: a pre-coated anti-enterovirus 71 polyclonal antibody ELISA plate, a sample diluent, a second Antibodies, Enzyme-labeled Antibodies, Concentrated Wash Solution, Enzyme Substrate Solution, and Stop Solution. The microtiter plate is pre-coated with a polyclonal antibody prepared from recombinant enterovirus type 71 structural protein 1 as an immune source, and the second antibody is a monoclonal antibody prepared from a keyhole limpet blue carrier protein-coupled polypeptide sequence FGEHKQEKDL as an immune source Antibody; enzyme-labeled antibody is horseradish peroxidase-labeled goat anti-mouse immunoglobulin antibody; antigen standard is placed in the box. The invention has better sensitivity when measuring the antigen titer of the EV71 virus inactivated vaccine, better linearity in the range of 5.9-750ng / ml, and the linear correlation coefficient r2 is greater than 0.99. The kit provided by the invention is a simple and convenient detection kit for measuring the antigen titer of EV71 virus inactivated vaccine with good specificity, accurate quantification, high sensitivity, good repeatability.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to an ELISA detection kit for enterovirus 71 (EV71) inactivated vaccine antigen. Background technique [0002] In recent years, hand, foot and mouth disease has ravaged most parts of our country and brought great harm to people. A large number of epidemiological studies have shown that enterovirus 71 is one of the main pathogens causing HFMD. EV71 belongs to the Picornaviridae family, a member of the Enterovirus genus. The infection of this virus mostly occurs in infants under 5 years old, and can cause herpes on the hands, feet, mouth and other parts. Individual patients can cause complications such as myocarditis, pulmonary edema, and aseptic meningoencephalitis. morbidity and mortality rates. Therefore, it is of great significance to develop a safe and effective EV71 vaccine to prevent and control the spread of HFMD. At present, there is no related vaccine in the market, and the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/577
CPCG01N33/577G01N33/68
Inventor 高孟罗永能王平张锋姜云水周康凤唐彩华高丽美朱莲毛子安毛江森
Owner ZHEJIANG PUKANG BIOTECH
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