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Preparation and Characterization of Three Monoclonal Antibodies against OmpA of Riemerella anatipestifer

HAN Wenlong;ZHANG Xuemei;WANG Xiaolan;YU Shengqing;Shanghai Veterinary Research Institute,CAAS;College of Animal Science and Technology,Anhui Agricultural University;  
Riemerella anatipestifer (RA) infection is one of the major bacterial infectious diseases in poultry.R.anatipestifer consists of complex serotypes and lacks cross-protection among serotypes.Serotypes 1,2 and 10 are the dominant domestic serotypes.Outer membrane protein A (OmpA) is one of the virulence factors of RA and well conserved in different serotypes.Preparation and characterization of monoclonal antibodies (mAbs) to OmpA was performed in the present study.BALB/c mice were immunized with OmpA protein.The spleen cells were fused with murine myeloma cells SP2/O and positive hybridomas were screened by indirect ELISA.The ascitic fluid containing mAbs was prepared by intraperitoneal injection of hybridoma cells into BALB/c mice.The mAb titers were titrated by indirect ELISA and the specificity was determined by Western blot and slide agglutination.In addition,colchicine method was used for chromosome analysis of monoclonal cells.As a result,3 mAbs against RA OmpA were obtained and designated as 2D5,2A6,and 4H8.The subtypes of these mAbs were determined as IgG2a.The titers of 3 mAbs were 1:2 048 000.All 3 mAbs reacted with RA serotype 1 strain CH3,serotype 2 strain NJ3,and serotype 10 strain HXb2 but not with other avian pathogens.The availability of specific mAbs would be beneficial to the research of RA pathogenesis and diagnosis.
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