Study Of Indirect Enzyme-linked Immunosobent Assay For Detecting Serotype-specific Antibody Of Actinobacillus Pleuropneumoniae

In this study,capsular polysaccharide(CPS) and lipopolysaccharide(LPS) were both extracted from APP serotype 1～10. Indirect ELISA was developed to detect the serotype specific antibody by these two kinds of antigens respectively.The purpose is to find out the most serotype-specific antigen through the cross-reaction test between different APP serotype,and can provide information for identifying the APP serotype.CPS and LPS were both extracted from APP and the specific serotype ELISA were developped by these two antigens respectively.Through the optimization of the reaction condition,The optimal coating concentration of CPS was 13μg/mL,and the LPS was 11μg/mL; PBS contain 1%BSA was determined to be the best blocking reagent, 37℃,60min was the best reaction condition;A screening dilution of 1:200 for sero was confirmed to be the best dilution and the reaction condition was 37℃, 60min;The optimal dillution of HRP-SPA was 1:4000 and reaction time was 30min; The blocking test indicated the CPS and LPS was specificity because CPS and LPS can both block the reaction between antisero and corresponding coating antigens. The cut off value was initially.determined, the P/N≥2.1 were confirmed to be positive and the P/N＜2.1 was confirmed to be negative.The indirect ELISA was confirmed to have good reproducibility by the repeating test.CPS and LPS from all APP serotype were used as coating antigens resipectively in indirect ELISA in order to detect positive sero of other APP serotype. CPS from APP serotype 1 and serotype 4 ,and the LPS from APP serotype 2 and serotype 10 only had reaction with antisero of homologous APP serotype,without cross-reaction with other APP serotype.These antigens were thought to have the best serotype specificity, and could beused as coating antigens for detecting antibody of APP serotype 1,serotype 2,serotype 4 and serotype 10.But the CPS and LPS from other serotype have different level of cross reaction.Indirect ELISA was developed respectively by CPS from APP serotype 1 and 4, LPS from APP serotype 2 and 10. 11 clinic pig sero from APP suspect pig farm were detected by these four ELISA. The result is that 5 samples were showed positive of APP1, 4 samples were showed positive of APP4, 2 samples were showed positive of APP2 and 3 samples were showed positive of APP serotype 10. The result also indicated that there were more than one APP serotype on the same pig.