Preparation And Identification Of Monoclonal Antibodies Against ORF Virus

Orf,also known as contagious ecthyma(CE),The main diagnostic methods for this disease include PCR detection,serum neutralization experiments,and electron microscopy and so on.The preparation of monoclonal antibodies against ORFV protective antigens has important application value in both rapid diagnostic testing of the disease and laboratory immunology of Orf pathogens.In this study,goat primary testis cells were infected with Orf virus(ORFV),and ORFV was propagated to obtain virus cell lysate.Then,sucrose density gradient centrifugation was used to purify the ORFV.BALB/c mice were immunized with the virus.The obtained spleen cells were fused with myeloma cells by traditional monoclonal antibody preparation techniques.The hybridoma cell lines secreting anti-ORFV monoclonal antibodies were prepared,screened and identified.Anti-ORFV monoclonal antibodies were prepared by screening positive hybridomas,and the obtained monoclonal antibodies were identified.Finally,the feasibility of anti-ORFV monoclonal antibody in indirect immunofluorescence detection of antigen was tentatively explored.The results were as follows:1.The purified virus particles were obtained by sucrose density gradient;2.The serum titer of antibody by ELISA was reached to 1:80000 in.mice which were immuned with purified ORFV;3.An ELISA assay for ORFV antibodies was developed and optimized;4.Three hybridoma cell lines were obtained.The three hybridoma cells have the ability to secrete monoclonal antibodies stably and were named E2,H7,and D9 respectively.The antibody titers in supernatants of H7,E2,and D9 cells were 1:2~9,1:2~4,and 1:2~6,and mouse ascites titers were 1:10~5,1:10~3,and 1:10~4 respectively;The type of monoclonal antibody secreted by H7 and D9 was IgG2b and the antibody type of E2 was IgM;5.The H7 cell strain can be used for immunofluorescence detection.Conclusion:Three hybridoma cell lines which can secret anti-ORFV monoclonal antibodies were obtained in this study.The H7 monoclonal antibodies against ORFV can be used for indirect immunofluorescence detection.