Polio virus III type D antigen pre-enveloped detection method and detection kit and application thereof

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: 2018-07-03
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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  • Polio virus III type D antigen pre-enveloped detection method and detection kit and application thereof
  • Polio virus III type D antigen pre-enveloped detection method and detection kit and application thereof
  • Polio virus III type D antigen pre-enveloped detection method and detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation of antiserum for type III 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 III monovalent inactivated virus D antigen 10-20ml mixed with Freund's complete adjuvant in equal volume, subcutaneous injection of 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 III poliovirus purified antibody (referred to as anti-III 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 the lowrry method to obtain bovine (rabbit, sheep) anti-III 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] Dissolve 10 mg of horseradish peroxidase in 1 ml of 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 0.5ml sodium borohydride, let it stand for 0.1-1 hour, add saturated ammonium sulfate at 1:1, and centrifuge at 15000rpm; take the precipitate and dissolve it and dialyze overnight; obtain bovine (rabbit, sheep) anti-IPV-IgG- HRP, stored at low temperature for later use.

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Abstract

The invention provides a detection method for pre-enveloped polio virus III type D antigen which can be produced in batches. According to the method, a porous plate is enveloped by a cow (rabbit, sheep) III type polio virus resistant D antigen purified antibody, the enveloped plate and the enveloped antibody are protected by a protective agent and then a pre-enveloped plate is prepared, meanwhile,horseradish peroxidase labelled cow (rabbit, sheep) III type polio virus resistant enzyme labelled antibody and other regents are adopted for matching. By adopting the method (reagent), specific qualitative and quantitative detection can be performed on polio virus III type D antigen, achiasmate among polio virus III type D antigen, I, II type C, D antigens and other enterovirus is achieved, andtherefore, the method is a III type polio virus D antigen detection method which is high in specificity, high in sensitivity, convenient to use and wide in application, and thus having relatively highapplication value in IPV vaccine production and inspection.

Description

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

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56983
Inventor 杨蓉罗芳宇谢忠平龙润乡李华杨婷陈洪波谢天宏岳磊谭振国王正鑫
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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