Preparation Of Monoclonal Antibody And Development Of The Colloidal Gold Immunochromatographic Strip For Rapid Detection Of Riemerella Anatipestifer

Riemerella anatipestifer is reported worldwide as the cause of epizootic infectious polyserositis of domestic ducks,and it is also pathogenic for gooses,turkeys and other waterflow.Currently,serotypes 1,2 and 10 strains are responsible for most outbreaks in China,and there is no effective cross-protection between different serotypes.On the other hand,the struture is different between the different serotypes of R.anatipestifer.In this study,the monoclonal antibodies against R.anatipestifer serotype 1,2 and 10 strains.Based on the rabbit anti-OmpA/motb anatibody and monoclonal antibodies,colloidal gold immunochromatographic strip for detecting R.anatipestifer was established.1.Prokaryotic expression and immunological characterization of R.anatipestifer OmpA/motb proteinRecombinant OmpA/motb(rOmpA/motb)was efficiently expressed in E.coli BL21(DE3),as shown by the presence of 55-kDa band in a Coomassie blue-stained SDS-PAGE gel.rOmpA/motb was blotted with duck antisera against R.anatipestifer CH3,NJ3 and HXb2,suggesting it is a cross-immunogenic protein among the strains with different serotypes.Rabbit anti-OmpA/motb antibodies were generated by immunization of rabbit with purified rOmpA/motb.The titer of antiserum was assessed over 1:256,000 by enzyme linked immune-sorbent assay(ELISA).Western blot showed that rabbit anti-OmpA/motb serum reacted well with R.anatipestifer CH3,NJ3 and HXb2,but did not react with avian pathogenic E.coli,S.enterica and P.multocida strains.2.Preparation of monolonal antibodies(McAbs)against R.anatipestiferThree stable hybridoma cell clones that producing anti-R.anatipestifer McAbs were established,and named as 2C2,2D9,and 3E3.The isotype of all McAbs was identified as IgM with?light chain.Ascites fluid titers of 2C2?2D9 and 3E3 were 1:64 000?1:128 000and1:64000,respectively.Western blot analysis showed that three McAbs specifically reacted with R.anatipestifer CH3,NJ3 and HXb2,but did not react with avian pathogenic E.coli,S.enterica and P.multocida strains.These results indicating that three McAbs had good cross-reactivity and specificity.The specifical anti-R.anatipestifer McAbs obtained in this study can be used for development of rapid detection of R.anatipestifer.3 Development of the colloidal gold immunochromatographic strip for rapid detection of R.anatipestiferThe colloidal gold of 22 nm in diameter was prepared by chemical synthesis.McAb3E3 was conjugated to colloidal gold particles and the combination of antibody with colloidal gold particles was characterized by UV-visible(UV-vis)light absorption spectra.The detection line and quality control line in nitrocellulose membrane was sprayed with McAb 2C2 and goat anti-mouse IgG,respectively.Immunochromatographic strips were assembled in regular sequence through different accessories sticked on PVC plate.The strips could specifically detect R.anatipestifer strains,but did not detect E.coli,S.eterica and P.multocida.The minimum detection limit for R.anatipestifer of the strips was2.5×10~6 colony forming units.The immunochromatographic strips prepared in this study offer a specific,sensitive,and rapid detection method for R.anatipestifer,which will have a favorable prospect after further improvement with package of the strips together.