Development And Evaluation Of BVDV ElISA Antibody Detection Kit And Antibody Detection Strip

Bovine viral diarrhea(BVD)is an infectious disease with multiple clinical symptoms caused by Bovine viral diarrhea virus(BVDV).The harm to the cattle industry is huge,first reported in Jilin Province in 1980,and then swept the country,causing huge economic losses to China’s cattle industry.The disease is widely endemic around the world,and both the World Health Organization(OIE)and China have designated it as a statutory three types of epidemic disease,with a high mortality rate and strong contagiousness.Early diagnosis,detection and vaccination are the main links in the prevention and purification of the disease.In the process of BVDV prevention and control,efficient,convenient,sensitive and specific detection methods are also of great significance to the prevention and control of BVDV.This article aims to establish two detection methods by using the E2 protein in BVDV structural protein to detect bovine viral diarrhea virus antibodies.These include:colloidal gold test strips,ELISA test kits.The colloidal gold test strip was carried out by the double antigen sandwich method,and the ELISA test kit was carried out by the indirect method.The presence of BVDV in the herd is diagnosed by antibody testing of unvaccinated herds,and antibody testing in vaccinated herds can evaluate whether the vaccine stimulates the herd to produce antibodies against BVDV.Through the search of relevant information,it is understood that E2 glycoprotein is the most important structural protein,which is required for binding to cell surface receptors,and it also contains major antigenic determinants.It is the main target for virus-induced production of neutralizing antibodies.Therefore,E2 protein was selected as the main raw material for this test.Here’s how:Preparation of ELISA detection kit:E2 protein diluted to a certain concentration is coated on the surface of the solid phase carrier as a detection antigen,non-specific binding is reduced by washing,blocking and other steps,and the horseradish peroxidase(HRP)-labeled goat anti-bovine is used as a capture antigen to form an indirect method to detect BVDV antibodies in the sample.The sensitivity test results showed that the test kit diluted 10~4of BVDV antibody positive serum could still be judged as positive;The specific experimental results showed that the test kit only reacted with BVDV antibodies;The results of the repeatability test showed that the coefficient of variation was less than 10%;The results of the compliance test showed that the positive compliance rate was 95%and the negative compliance rate was 100%.Preparation of colloidal gold test strips:goat anti-cattle and E2 protein were diluted to a certain concentration,coated on nitrocellulose membrane and placed on C line and T line,respectively.The colloidal gold particles were coupled with E2 protein to form colloidal gold-E2 protein complexes,which were placed on the glass fiber membrane to form a double antigen sandwich method to detect BVDV antibodies in the sample.The sensitivity test results showed that 4 kinds of antibody positive serum were detected,and the test strip of this test only reacted to BVDV antibody positive serology;Dilution of serum 10~3can be detected,indicating that the test strip is sensitive;The positive compliance rate is90%and the negative compliance rate is 100%.Conclusion:In this study,E2 protein has been successfully established with two detection methods,and it has good specificity,high sensitivity and can be produced in large quantities.It provides theoretical support and diagnostic methods for the purification and prevention and control of BVDV in China’s cattle industry.