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Pre-coating combined detection method for Polio virus I, II and III D antigens, detection kit and application thereof

A polio and quantitative detection method technology, applied in the field of bioengineering, can solve problems such as differences in detection results, deviations in personnel operations, and high requirements for detection personnel's operational skills, so as to reduce detection and operation errors, improve detection efficiency, and shorten detection time 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

[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 period, at least 3 days to complete a detection, and requires high operating skills of the detection personnel, and changing the operator may lead to Personnel operation deviation occurs, which is not conducive to the stability of vaccine quality

Method used

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  • Pre-coating combined detection method for Polio virus I, II and III D antigens, detection kit and application thereof
  • Pre-coating combined detection method for Polio virus I, II and III D antigens, detection kit and application thereof
  • Pre-coating combined detection method for Polio virus 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 for type Ⅰ, type Ⅱ and type Ⅲ poliovirus D antigen

[0041] The inactivated poliovirus was centrifuged through 10-30% sucrose density gradient for 4-8 hours, and the D antigen layer was collected, which was confirmed by electron microscopy and then used for the preparation of immune serum; test animals (cattle, rabbits, and sheep are acceptable), For the first immunization, 10-20ml of monovalent inactivated virus D antigen of types I, II, and III was mixed with Freund’s complete adjuvant in equal amounts, and then immunized by subcutaneous injection. Each type of antigen was immunized to 1-2 animals, avoiding crossover, and then carried out After 3 booster immunizations, blood was collected, the serum was separated, and the serum neutralizing antibody titer was determined by micro-neutralization test.

Embodiment 2

[0042] Example 2 Preparation of bovine (rabbit, sheep) anti-type I, type II, and type III poliovirus antigen monovalent purified antibody (abbreviated as anti-IPV-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 various types of bovine (rabbit, sheep) anti-type I, type II, and type III monovalent IPV-IgG, and store them below -20°C for later use; different types of serum a...

Embodiment 3

[0044] Example 3 Preparation of enzyme-labeled antibody

[0045] After dissolving 10 mg of horseradish peroxidase in water for injection, add 0.2 ml of NaIO4, let it stand for 0.1-1 hour, add 0.5 ml of ethylene glycol solution, let it stand for 0.1-1 hour, then mix it with 1 ml of the purified antibody obtained in step 2 , dialyzed overnight; add sodium borohydride 0.5ml, let stand, add saturated ammonium sulfate, centrifuge at 15000 for 30-60 minutes; take the precipitate and dissolve it and dialyze overnight; obtain bovine (rabbit, sheep) anti-type I, type II, type III Poliovirus-IgG-HRP, cryopreserved for future use. During the labeling process, different types of antibodies were labeled separately 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 pre-coating Polio virus I, II and III D antigen detection method for batch production. Bovine (rabbit and sheep) anti-I, II and III Polio virus D antigens are employed to purify an antibody-coated microporous plate, also a protective agent is employed for protection of a coated plate and coating antibodies so as to prepare a pre-coated plate, and the pre-coated plate is matched with horseradish peroxidase labeled bovine (rabbit and sheep) anti-I, II and III Polio virus enzyme-labeled antibodies and other reagents at the same time. The method (reagent) can achieve specific qualitative and quantitative detection of Polio virus I, II and III D antigens, has no cross with Polio virus I, II and III C antigens and other enteroviruses, and is a type I, II and III Polio virus D antigen combined detection method with the advantages of high specificity, high sensitivity, convenient use and extensive use, and has high application value in production and verification of IPV vaccines.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a combined detection method for poliovirus type I, type II and type III D antigen pre-coating, 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 o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535G01N33/569
CPCG01N33/535G01N33/56983
Inventor 罗芳宇谢忠平龙润乡杨蓉陈洪波宋霞张云昆谭振国
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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