BVDV E2蛋白抗体阻断ELISA检测方法的建立OA

To establish a blocking enzyme-linked immunosorbent assay(ELISA)for the detection of antibodies against the envelope protein E2 of bovine viral diarrhea virus(BVDV),this study utilized a baculovirus expression system to express the E2 protein.The expression and purity of the recombinant protein were verified by indirect immunofluorescence assay(IFA),Coomassie brilliant blue staining,and Western blot(WB).BALB/c mice were immunized with an inactivated BVDV-1b subtype strain(XZ02)to produce monoclonal antibodies,and the obtained monoclonal antibody was conjugated with horseradish peroxidase(HRP).The reactivity of HRP-labeled monoclonal antibody was confirmed by Coomassie brilliant blue staining and WB analysis.Purified E2 protein was used as the coating antigen,and HRP-labeled monoclonal antibody as the detection antibody.Optimal dilutions of the antigen and antibody were determined by checkerboard titration.Through optimization of serum dilution,blocking buffer,serum incubation time,and cut-off value determination,a blocking ELISA for BVDV E2 protein antibody detection was established.The sensitivity,specificity,and concordance of the method were evaluated,followed by application to clinical sample testing.The results showed that the E2 protein was successfully expressed,and monoclonal antibody B6 exhibited strong reactivity with E2.HRP conjugation of B6 was successful.The optimal coating antigen dilution was 1∶1 000,and the optimal HRP-B6 dilution was 1∶10 000.The best serum dilution was 1∶4,with 5%chicken serum as the optimal blocking buffer,and a serum incubation time of 2.0 hours.The cut-off value was determined to be 0.509.The sensitivity of the assay reached 1∶64.When tested with sera positive for five different bovine pathogens,the assay demonstrated high specificity,with inhibition rates≥51%.The coincidence rate between this blocking ELISA and a commercial BVDV blocking ELISA antibody detection kit was 94.35%.Using this established method,317 clinical bovine serum samples were tested,of which 138 were positive and 179 were negative.The developed BVDV E2 protein antibody blocking ELISA provides a reliable and accurate tool for serological diagnosis and evaluation of vaccine-induced immunity against BVDV,supporting more efficient and precise disease control strategies.