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Precoating detection method and detection kit for polio virus type I D antigen and application of precoating detection method

A technology for poliomyelitis and quantitative detection method, which is applied in the field of bioengineering, can solve the problems of high requirements on operator skills, inability to prepare pre-coated plate finished products, unfavorable quality and stability of vaccines, etc., so as to shorten detection time and reduce preparation Effects of preparative steps, improved assay sensitivity and specificity

Inactive Publication Date: 2018-07-20
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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  • Precoating detection method and detection kit for polio virus type I D antigen and application of precoating detection method
  • Precoating detection method and detection kit for polio virus type I D antigen and application of precoating detection method
  • Precoating detection method and detection kit for polio virus type I D antigen and application of precoating detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Preparation of antiserum against poliovirus type Ⅰ D antigen

[0040] 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), the first immunization, type I monovalent inactivated virus D antigen 10-20 ml mixed with Freund's complete adjuvant in equal volume, subcutaneous injection immunization, and then three booster immunizations followed by blood collection, serum separation, and micro-neutralization test Serum neutralizing antibody titers were determined.

Embodiment 2

[0041] Example 2 Preparation of bovine (rabbit, sheep) anti-type I poliovirus purified antibody (referred to as anti-type I IPV-IgG)

[0042] 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-type I IPV-IgG, which is stored below -20°C for later use; the lotion and balance solution described in the experiment are conventional liquids in the prior art.

Embodiment 3

[0043] Example 3 Preparation of enzyme-labeled antibody

[0044] 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 1:1, centrifuge at 15000rpm for 30-60 minutes; take the precipitate to dissolve and dialyze overnight; obtain bovine (rabbit, sheep) anti-I Type IPV-IgG-HRP, stored at low temperature for future use.

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Abstract

The invention provides a precoating detection method for a polio virus type I D antigen. The method can be used for batch production. A microporous plate is coated with a bovine (rabbit and sheep) anti-type I polio virus D antigen purification antibody, the coated plate and the coating antibody are protected by a protective agent, and then a precoated plate is prepared; meanwhile, horseradish peroxidase labeled bovine (rabbit and sheep) anti-type I polio virus enzyme labeled antibody and other reagents are matched. According to the method (reagent), specific qualitative and quantitative detection can be performed on the polio virus type I D antigen, the polio virus type I C antigen, polio virus type II and III type C and D antigens and other enteroviruses are non-cross, the precoating detection method for the polio virus type I D antigen is high in specificity and sensitivity, convenient to use and wide in application, and has high application value in IPV (inactivated poliovirus vaccine) production and verification.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a poliovirus type I 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 cos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56983
Inventor 谢忠平龙润乡杨蓉罗芳宇陈洪波杨婷李华岳磊谢天宏王正鑫
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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