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Method for detecting pre-coated poliovirus type II D antigen and detection kit and application thereof

A poliomyelitis, quantitative detection method technology, applied in the field of bioengineering, can solve problems such as poor stability of coated microplates, difference in detection results, adverse vaccine quality stability, etc., to improve detection sensitivity and specificity, and reduce detection. and operating errors, reducing the effect of preparation preparation steps

Inactive Publication Date: 2018-07-17
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Since the poliovirus antibody was used in the experiment, it was found that the coated microwell plate had poor stability, so it could not be prepared into a pre-coated plate. Therefore, there is currently no commercially available poliovirus D antigen test kit on the market
The existing D antigen detection methods (reagents) cannot be prepared in batches, and the coating before each use leads to a long detection cycle, which takes at least 3 days to complete a detection. The cycle is too long, which affects the timeliness of vaccine testing and affects the operational skills of the testing personnel. The requirements are high, and changing operators may lead to operational deviations, which is not conducive to the stability of vaccine quality

Method used

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  • Method for detecting pre-coated poliovirus type II D antigen and detection kit and application thereof
  • Method for detecting pre-coated poliovirus type II D antigen and detection kit and application thereof
  • Method for detecting pre-coated poliovirus type II D antigen and detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation of antiserum for type Ⅱ poliovirus D antigen

[0039] Centrifuge the inactivated poliovirus through 10-30% sucrose density gradient at 40,000rpm for 4-8 hours, collect the D antigen layer, and use it for the preparation of immune serum after being confirmed as D antigen by electron microscopy; Yes), first immunization, type II monovalent inactivated virus D antigen 10-20ml mixed with Freund's complete adjuvant in equal volume, subcutaneous injection immunization, and then 3 booster immunizations followed by blood collection, serum separation, and microneutralization test Serum neutralizing antibody titer determination.

Embodiment 2

[0040] Example 2 Preparation of bovine (rabbit, sheep) anti-type II poliovirus purified antibody (referred to as anti-II IPV-IgG)

[0041] 1. Take the protein A / G affinity chromatography column, place it at room temperature for 30 minutes, and equilibrate the column with an equilibration solution 5 times the volume of the column; 2. Mix the antiserum and the equilibration solution in an equal volume ratio before loading the sample. Use 10 times the column volume of the equilibrium solution to elute the impurity protein, leaving the target protein; 3. Use 10 times the column volume of the conventional eluent to elute the target protein from the affinity column, collect the eluted peak, and desalt it for later use 4. Determination of protein content by lowrry method to obtain bovine (rabbit, sheep) anti-II IPV-IgG, which is stored below -20°C for later use; the lotion and balance solution are conventional liquids in the prior art.

Embodiment 3

[0042] Example 3 Preparation of enzyme-labeled antibody

[0043] After dissolving 10 mg of horseradish peroxidase in 1 ml of water for injection, add 0.2 ml of NaIO4, let stand for 0.1-1 hour, add 0.5 ml of ethylene glycol solution, let stand for 0.1-1 hour, then mix with 1 ml of purified antibody obtained in step 2 Mix and dialyze overnight; add 0.5ml sodium borohydride, let it stand for 0.1-1 hour, add saturated ammonium sulfate, centrifuge at 15000rpm for 30-60 minutes; take the precipitate and dissolve it and dialyze overnight; obtain bovine (rabbit, sheep) anti-Ⅱ IPV- IgG-HRP, stored at low temperature for future use.

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Abstract

The invention provides a method for detecting a pre-coated and mass-produced poliovirus type II D antigen. Bovine (rabbit, sheep) anti-type II poliovirus D antigen purified antibodies are used for coating microwell plates, the coated plates and the coated antibodies are protected by using a protective agent to prepare pre-coated plates, and horseradish peroxidase-labeled bovine (rabbit, sheep) anti-type II poliovirus enzyme-labeled antibodies and other reagents are matched. The method (reagent) can conduct specificity qualitative and quantitative detection on the poliovirus type II D antigen,and there is no intersection with a poliovirus type II C antigen, type I, type III C, D antigens and other enteric viruses. The method for detecting the poliovirus type II D antigen has high specificity, high sensitivity, convenient use and wide application, and has high application value in IPV vaccine production verification.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a poliovirus type II D antigen pre-coated detection method, a detection kit and application thereof. Background technique [0002] Poliovirus (polio virus) belongs to the picornaviridae family and the genus Enterovirus, and is divided into three serotypes. It mainly invades motor neurons in the gray matter area of ​​the anterior horn of the spinal cord. Children under the age of 10, especially infants, are also known as polio, which is the main pathogen of the second infectious disease that WHO wants to eliminate after smallpox. There is no specific treatment for poliovirus infection, and it can only be prevented by vaccines. Currently, there are two types of vaccines used to prevent and control polio epidemics: live attenuated polio vaccine (OPV) and inactivated polio vaccine (IPV), OPV was once widely used due to its ease of vaccination and relatively low co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56983G01N2333/105
Inventor 龙润乡谢忠平罗芳宇杨蓉陈洪波岳磊谢天宏李华杨婷王正鑫
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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