Synchronous rapid identification and quantitative detection method for Polio virus type I, II and III D antigens, detection kit and application thereof

A quantitative detection method, polio technology, applied in the field of bioengineering, can solve problems such as high operational skill requirements for testing personnel, unfavorable vaccine quality stability, personnel operation deviation, etc., to save production raw material costs, testing costs, and testing time Short, good sensitivity effect

Inactive Publication Date: 2018-08-10
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

Existing D antigen detection methods (reagents) do not take 2 to 3 days to complete a test. The cycle is too long, which affects the timeliness of vaccine testing, and requires high operational skills of testers. Changing operators may lead to operational errors. appear, which is not conducive to the stability of vaccine quality

Method used

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  • Synchronous rapid identification and quantitative detection method for Polio virus type I, II and III D antigens, detection kit and application thereof
  • Synchronous rapid identification and quantitative detection method for Polio virus type I, II and III D antigens, detection kit and application thereof
  • Synchronous rapid identification and quantitative detection method for Polio virus type I, II and III D antigens, detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Preparation of antiserum against poliovirus D antigens of types Ⅰ, Ⅱ, and Ⅲ

[0041] Centrifuge the inactivated poliovirus at 40,000rpm with 10-30% sucrose density gradient for 4-8 hours, collect the D antigen layer, and use it for the preparation of immune serum after being confirmed as the required poliovirus-D antigen by electron microscopy; Experimental animals (cattle, rabbits, and sheep are all acceptable), the first immunization, type I, type II, type III monovalent inactivated virus D antigen 10-20ml mixed with Freund's complete adjuvant in equal amounts, subcutaneous injection immunization, each isotype The antigens were immunized to 1-2 animals respectively to avoid crossover. After 3 booster immunizations, blood was collected, serum was separated, and the serum neutralizing antibody titer was determined by micro-neutralization test.

Embodiment 2

[0042] Example 2 Preparation of Monovalent Purified Anti-Poliomyelitis Antigen Antibody of Bovine, Rabbit or Goat Anti-Poliovirus Type I, Type II and Type III (abbreviated as anti-poliomyelitis virus-IgG)

[0043] 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. Use the lowrry method to measure the protein content, and obtain monovalent bovine, rabbit or sheep anti-type I, type II, and type III poliovirus-IgG, and store it below -20°C for later use; ...

Embodiment 3

[0044] Example 3 Preparation of enzyme-labeled antibody

[0045] After dissolving 10 mg of horseradish peroxidase with 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, and 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; , Type III monovalent poliovirus-IgG-HRP, stored at low temperature for later use, different types of antibodies were labeled separately during the labeling process to avoid confusion and crossover. The solutions used above are conventional solutions of the prior art.

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Abstract

The invention provides a method for specific identification detection of type I, II and III Polio virus D antigens, univalent vaccines, trivalent vaccines and combined vaccines simultaneously. According to the method, bovine, rabbit or sheep anti-type I, II and III Polio virus D antigens are mixed in certain proportion to coat an enzyme-labeled detection plate to prepare a precoated plate, at thesame time, and the precoated plate is matched with horseradish peroxidase labelled bovine, rabbit or sheep anti-type I, II and III Polio virus enzyme-labeled antibodies and other reagents, thus forming the method for rapid identification detection of type I, II and III Polio virus D antigens simultaneously. With no cross with non-target type other Polio virus C, D antigens and target type other Cantigens, the method is a type I, II and III Polio virus D antigen detection method with the advantages of high specificity, high sensitivity, convenient use, cost saving and wide application, and hashigh application value in IPV vaccine production and verification.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a detection method capable of simultaneously rapidly qualitatively or quantitatively identifying and detecting type I, type II and type III poliovirus D antigen, 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 inactivate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56983G01N2333/105G01N2469/10
Inventor 龙润乡谢忠平罗芳宇杨蓉蔡玮陈洪波平玲宋霞
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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