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Development Of A Sandwich ELISA For Capture Of Bovine Viral Diarrhea Virus Antigen Based On Monoclonal Antibody And Epidemiological Investigation

Posted on:2020-08-29Degree:MasterType:Thesis
Country:ChinaCandidate:J Y HuFull Text:PDF
GTID:2370330575477624Subject:Veterinary Public Health
Abstract/Summary:PDF Full Text Request
Bovine viral diarrhea is an infectious disease caused by bovine viral diarrhea virus(BVDV),which is characterized by high fever,salivation,diarrhea,mucosal hyperemia,mucosal ulcer,declining of production performance and abortion of pregnant cattle.Calves often exhibit immune tolerance and persistent infection.The disease is widely spread in many countries and regions all over the world,which poses a severe threat to the development of cattle industry.In addition to the cattle,BVDV can also infect the sheep,deer,pig and other wild animals.BVDV is divided into two biotypes based on the cytopathic effect,the cytopathic(CP)and noncytopathic(NCP).According to the nucleotide sequence variation,BVDV is divided to two genotypes,BVDV-I and BVDV-II,where they can be further divided into different subgenotypes.Currently,the BVDV-Ib and BVDV-Im are the dominant subgenotypes,which spread in our country.In recent years,the infection of BVDV is still one of the important infectious diseases that hinder the development of cattle industry in our country.Therefore,the early diagnosis and monitoring of BVDV are particularly important.To establish a specific,sensitive,rapid and simple method for diagnosis of BVDV infection,a sandwich ELISA for capture of BVDV antigen based on monoclonal antibody was established using antibodies raised against the recombinant protein E2 derived from CC13 B strain.E2 gene was amplified and cloned,and recombinant E2 protein was expressed using molecular biology techniques and E.coli expression system,respectively.The monoclonal antibody and polyclonal antibody against protein E2 were then prepared and used to establish the double antibody sandwich ELISA method for detection of BVDV antigen.The optimal concentrations of the antigencapturing monoclonal antibody and polyclonal antibody conjugated-HRP were determined using the square matrix titrimetry as 200 ng/well and 1:1000,respectively.The criterion for sandwich ELISA was determined by assaying the 100 BVDVnegative samples as presented by the average plus three standard deviations.The sample was assessed as positive when its OD490 ?0.1567.The specificity,sensitivity and reproducibility for the ELISA were assayed and the results showed that the established ELISA was specific,sensitive,rapid and reproducible.Moreover,the sandwich ELISA method for capturing BVDV antigen was applied to investigate the infection of BVDV on cattle herds from Shandong Province,Henan Province,Jilin Province and Ningxia Province.It was showed that the rate of BVDV infection was between 0~28.30% in different cattle farms.It indicated that there are different degrees of BVDV infection in cattle herds in different regions.Furthermore,three BVDV isolates were obtained and characterized from the feces samples with positive ELISA results in a cattle farm in Jilin Province,which were named BVDV-JY1,BVDV-JY7 and BVDV-JY33,respectively.Phylogenetic analysis of the nucleotide sequence revealed that the 5'UTR sequences of the isolated BVDV strains have high sequence identity with those of the BVDV-Id subgenotype strains.In summary,the monoclonal antibody and polyclonal antibody against protein E2 were prepared and used to establish the double antibody sandwich ELISA method which is specific,sensitive,rapid and easily used in cattle farm for detection of BVDV antigen.These results provide effective and reliable technical means for early diagnosis and monitoring of BVDV infection in the future.At the same time,the epidemiological investigation of the BVDV pathogen by double antibody sandwich ELISA method provides an epidemiological theoretical basis for the prevention and control of BVD in the future.
Keywords/Search Tags:Bovine viral diarrhea virus, Monoclonal antibody, Polyclonal antibody, Double antibody sandwich ELISA, Epidemiological investigation, Isolation and identification
PDF Full Text Request
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