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ELISA method for detecting African swine fever CD2v protein antibody, kit and application

A technology for African swine fever and African swine fever virus, which is applied in the field of ELISA for detecting African swine fever CD2v protein antibodies, can solve the problems of inability to distinguish vaccine strains from wild virus strains, etc., and achieves rapid detection, broad application prospects and good specificity. Effect

Inactive Publication Date: 2020-03-27
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the kit can only be used for a large number of screenings of African swine fever virus antibodies, and cannot distinguish vaccine strains from wild strains

Method used

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  • ELISA method for detecting African swine fever CD2v protein antibody, kit and application
  • ELISA method for detecting African swine fever CD2v protein antibody, kit and application
  • ELISA method for detecting African swine fever CD2v protein antibody, kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Preparation of CD2v antigen

[0045]1. Expression of African swine fever CD2v protein

[0046] According to the CD2v gene sequence of African swine fever (ASFV) Chinese epidemic strain Pig / HLJ / 2018 (GenBank: MK333180.1) in GenBank, the whole CD2v gene was artificially synthesized as a template, and a pair of primers CD2v-F / CD2v-R were designed to pass PCR Amplified and cloned into the eukaryotic expression vector pIRESpuro2 (purchased from Clontech, USA), constructed the recombinant plasmid pIRES-CD2v, and transfected CHO-K1 cells (purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) after sequencing verification, After adding puromycin and 2-3 rounds of single cell cloning and identification by indirect immunofluorescence (IFA). The specific identification method is as follows: the cells of the single cell clone are transferred to a 12-well cell culture plate to grow into a dense monolayer and th...

Embodiment 2

[0064] Example 2 Establishment of a double-antigen sandwich ELISA kit for detecting African swine fever virus CD2v antibody

[0065] 1. The detection principle of the kit of the present invention

[0066] The present invention adopts the double antigen sandwich method to coat the recombinant African swine fever virus CD2v protein in the microwell plate, then seal the microplate with 1% (w / v) BSA solution, add the sample to be tested and the standard positive control , negative control. The African swine fever virus CD2v antibody in the sample or standard product can react with the CD2v antigen coated in the microtiter plate. After adding the HRP-labeled CD2v antigen, the enzyme-labeled antigen binds to the CD2v antigen-antibody complex that has completed the reaction in the previous step. Subsequently, horseradish peroxidase (HRP) substrate TMB was added for color development, the stop solution terminated the reaction, and the absorbance value of each hole was measured by a m...

Embodiment 3

[0079] The detection of African swine fever virus CD2v antibody in the sample of embodiment 3

[0080] 1. Use the kit prepared above for detection

[0081] 1) Dilute the serum to be tested, the positive control and the negative control with the serum diluent in proportion, add 100 μL per well to the microtiter plate in Example 2, incubate at 37°C for 1 hour, discard the solution and wash with washing solution for 3-5 Second-rate.

[0082] 2) Add 100 μL (0.2 μg / ml) of HRP-labeled CD2v antigen protein to each well, incubate at 37° C. for 30 minutes, discard the solution and wash with washing solution for 3 to 5 times.

[0083] 3) Add 100 μL of TMB substrate solution to each well, and react for 10 to 15 minutes at room temperature in the dark.

[0084] 4) Add 100 μL 2M H2 SO 4 , to terminate the reaction, and detect the absorbance value at 450 nm with a microplate reader.

[0085] 2. Analysis of test results

[0086] 1) Judgment criteria for negative and positive samples

...

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Abstract

The invention discloses an ELISA (Enzyme Linked Immuno Sorbent Assay) method for detecting an African swine fever CD2v protein antibody, a kit and application. The ELISA method is a double-antigen sandwich method. Through the method, the ELISA kit for detecting the African swine fever CD2v protein antibody is obtained, and the ELISA kit comprises a horse radish peroxidase labeled CD2v antigen anda CD2v protein coated elisa plate. By utilizing the kit designed by the invention, a large number of African swine fever virus antibodies can be screened; the kit can also be used for identifying African swine fever vaccine strains and wild strains in CD2v-deleted immune swine herds, is rapid and sensitive in detection, good in specificity, very suitable for large-area rapid detection and generalinvestigation of African swine fever epidemic situations and purification and eradication of African swine fever in pig farms in the future, and wide in application prospect.

Description

technical field [0001] The invention belongs to the technical field of molecular biology diagnosis of animal epidemic diseases in veterinary medicine, and in particular relates to an ELISA method, a kit and an application for detecting African swine fever CD2v protein antibody. Background technique [0002] African swine fever (ASF) is a highly contagious disease with high lethality caused by African swine fever virus (ASFV) infecting domestic pigs and wild boars. Clinically, high fever, extensive hemorrhage of internal organs, splenomegaly and black color due to hemorrhage are the main features. There are many genotypes of African swine fever virus, and 24 genotypes are currently known. So far, there is no commercial vaccine for the disease. The prevention and control of the epidemic mainly relies on strengthening the biosafety management of farms, restricting the cross-regional flow of live pigs and pig products, and adopting disposal methods such as clearing and culling ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/58G01N33/569
CPCG01N33/6854G01N33/581G01N33/56983G01N2333/01G01N2469/20
Inventor 蒋智勇李春玲蔡汝健郭怡德宋帅李艳楚品品勾红潮徐民生卞志标
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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