Screening And Identification Of Hybridoma Cells Of Monoclonal Antibody Against ORFV For Indirect Immunofluorescence Detection

Orf is an acute,highly contagious disease that affects the healthy breeding of small ruminant economic animals such as sheep and goats.The pathogen causing the disease is Orf virus?ORFV?.Conventional ORFV detection methods,such as indirect ELISA,PCR,cell culture techniques and etc.,play an extremely important role in the study of Orf's epidemiological investigation,pathogenic characteristics and virus identification.However,it is difficult to meet the purpose of localization and visual judgment of ORFV antigens in infected tissues and cells.Virus on tissue and cells can be specifically and visually detected and localized by indirect immunofluorescence assay?IFA?,so that monoclonal antibodies against ORFV,which physicochemical traits are uniform and have strong antigen specificity,are of great value for further studies such as laboratory research and diagnosis.In this study,the clinical samples were tested,ORFV was isolated and identified by using the suspected tissue material of goat's lips and applying PCR and cell culture techniques.The ORFV-infected bovine testicular epithelial cells were used as test samples,and the non-cloned hybridoma cells were used as materials.The hybridoma cells secreting ORFV monoclonal antibody were screened by limiting dilution method and indirect immunofluorescence assay?IFA?,and then antibodies were identified.Frozen tissue sections were prepared from the lip tissue of ORFV-infected goat,and the IFA was performed using the identified monoclonal antibody against ORFV.This study obtained the following results:1.A strain of Orf virus was isolated from clinical suspected ORFV-infected goat's tissue,which can be proliferated in bovine testicular epithelial cells.And the TCID50 of F7ORFV was 10^-6.39/100?L in bovine testicular epithelial cells.2.The conditions for IFA detection of antibodies against ORFV were just as follows,bovine testicular epithelial cells were infected with 10^3.70 TCID50 ORFV for 42 h,and the dilution of fluorescent labeled secondary antibodies was 1:400.3.Two monoclonal hybridoma cell lines were screened by IFA and named as C8 and E8.4 The type of monoclonal antibody secreted by C8 was IgG1,which the recognized epitope was 100-130 Ku;the type secreted by E8 was Ig2b,and the recognized protein was about 100 ku;5.The monoclonal antibodies secreted by C8 and E8 hybridoma cell lines showed specific positive results for IFA of cultured cells infected with ORFV.The monoclonal antibody of C8 showed specific positive results for IFA of the lip tissue section of the goat diagnosed with Orf.Conclusion:1.A local strain of Orf virus was isolated successfully;2.The conditions for IFA detection of antibodies against ORFV were optimized;3.Two hybridoma cell lines,which can secret monoclonal antibodies against ORFV,were obtained.One of the monoclonal antibodies can be used for IFA detection of ORFV in diseased tissue.