Development Of Monoclonal Antibodies And Rapid Detection Methods Against Riemerella Anatipestifer

Riemerella anatipestifer(RA) is the causative agent of duck infectious serositis and it causes acute or chronic disease in poultry, especially in ducks. There are various clinical symptoms in infected ducks with lethargy, deflection of neck, limping, greenish diarrhea, serosity excretion from the eyes and nose, and nervous symptoms such as spasm and opisthotonos. The characteristic pathological lesions include fibrinous pericarditis, perihepatitis, airsacculitis, caseous salpingitis and meningitis. The infectious setositis causes a great economic losses due to its high mortality, reduction of reproduction and high cost of treatment A rapid and specific detection method against RA is a key step to prevent and control this disease. In this study, monoclonal antibodies (mAbs) were developed, and double-antibodies sandwich ELISA, immune colloidal gold test strip, PCR method were established. It will be helpful for detection and control of RA infection.1、Preparation of mAbs against RAIn this study, the whole bacterium was used as the immunogen. BALB/c mice were immunized subcutaneously with whole bacterium in complete Freund’s adjuvant, two identical boosters emulsified in incomplete Freund’s were given at2weeks interval. Mice were injected intraperitoneally with the same antigen at6weeks after the first immunization. Three days after last immnunzation, spleen cells of immunized mouse were fused with myeloma cells (SP2/0).4positive hybridomas cell strains were identified and named as2F7,3G9,5G7and2E6, respectively. The2F7was IgG2b isotype and the other three mAbs were IgG3isotype.5G7had cross reaction with serotype1,2,3and11strains, while the other3mAbs with serotype1,2and3strains. There was no cross reaction to Pastuerella multocida, Escherchia coli and Salmonella. The results of western-blot assay showed that mAbs5G7binded with an approximate45-kDa protein.2、Establ ishment of rapid detection methods for RA.Polyclonal antiserum against RA was generated and purified along with mAbs to establish double sandwich ELISA method. Polyclonal antibody was used as capture antibody while mAbs were used as detect antibody, The optimal conditions for ELISA were as following:polyclonal antibodys with184ng per well were added to coat overnight, the plates were blocked by10%BCS for2hours at37℃, the reaction times for detection antigen, polyclonal antibody and enzyme labeled antibody were one hour, the valute of OD450was obtained after20min coloration. This method has no cross-reaction with duck-origin Pasteurella multocida, E. coli and salmonella, and its minimum detectable limit was104CFU. Gold immunochromatographic assay (GICA) was generated by mAb5G7. It could detect all four serotype strains of RA, but not Pasteurella multocida, E. coli and salmonella. The minimum detectable limit for this test strips was6×103CFU. PCR method was established with primers for16srDNA, which produced a PCR product of384bp, and this method could detect all four serotype strains of RA, but not Pasteurella multocida, E. coli and salmonella. The minimum detectable limit for PCR method was200CFU of RA bacteria and20pg of DNA, respectively. The detection results for clinical samples showed that the positive rates for PCR, double sandwich ELISA, GICA and bacterial isolation method were 40.7%(11/27)、55.6%(15/27).51.9%(14/27)、40.7%(11/27) respectively.The coincidence between bacterial isolation method and PCR, double sandwich ELISA, GICA was92.6%,85.2and88.9%, respectively. Therefore, this study provides rapid, specific and sensitive detection methods for the different needs of clinic and practice.