Preparation Of OmpA Monoclonal Antibody Against Riemerella Anatipestifer And Development Of Immunocolloidal Gold Test Strip

Riemerella anatipestifer is one of the major pathogenic bacteria in poultry,with complex serotypes and lack of cross-protection among serotypes.Serotypes 1,2 and 10 are the domestic dominant serotypes.Outer membrane protein A(OmpA)is an immunogenic protein of R.anatipestifer and well conserved in different serotypes.To develop an immunocolloidal gold test strip for detection of R.anatipestifer,in this study,we used the OmpA protein as an antigen to immunize BALB/c mice for generating monoclonal antibodies against R.anatipestifer OmpA.After cell fusion,positive hybridoma cell lines were screened by indirect ELISA,and monoclonal antibodies were prepared by intraperitoneal injection of hybridoma cells.Indirect ELISA was used to detect the monoclonal antibody titer and stability,colchicine method was used for chromosome analysis and Western blot were used to identify the monoclonal antibody specificity.Three monoclonal antibodies against R.anatipestifer OmpA were developed and named as 2D5,2A6,4H8;the subtypes were determined as IgG2a and the titers were 1:2048000 for all three monoclonal antibodies;The three monoclonal antibodies reacted with R.anatipestifer serotype 1 strain CH3,serotype 2 strain NJ3,and serotype 10 strain HXb2,but not with other avian pathogens,including Escherichia coli,Salmonella and Pasteurella multocida,indicatingthree monoclonal antibodies developed in this study have good antibody titers and specificity,could be used to develop an immunocolloidal gold test strip for detection of R.anatipestifer.By screening of the above three monoclonal antibodies,2D5 and 2A6 were selected to develop an immunocolloidal gold test strip for detection of R.anatipestifer with 2D5 as the colloidal gold-labeled protein and 2A6 as the capture protein(T line),respectively.The goat anti-mouse IgG antibody was labeled on nitrocellulose membrane as a control(C line).The labelling pH was optimized as 10.0 and the concentration of 2D5 labeled to colloidal gold particles was optimized as 18?g/m L.The strip specifically detected R anatipestifer strains,showed no cross-reaction with Escherichia coli,Salmonella enterica and Pasteurella multocida strains.The sensitivity of the strip for detecting R anatipestifer was1.0×10~6 colony forming unit.The strips remained stable for up to 8 months at 4°C and the detection can be completed within 15 min.The strip can detect R.anatipestifer in hearts of the ducks experimentally infected with R.anatipestifer,but not infected with Escherichia coli,which were also confirmed with bacterial isolation followed by multiplex polymerase chain reaction.These results suggested that the strips are reliable methods for detection of R anatipestifer in bacterial culture and in infected ducks.