Detection Of Four Serotypes Antibodies Against Riemerella Anatipestifer By Indirect Enzyme-Linked Immunosorbent Assay

A indirect enzyme-linked immunosorbent assay(ELISA) was developed for detection of four serotypes(serotype 1,serotype 2,serotype 4 and serotype 5) antibodies against Riemerella Anatipestifer(R.A.) in ducks.The coating antigen were made by ultrasonic method. The optimum R.A. antigen concentrations were found to be 3.8 n g/ml(serotype 1).4.5 u g/ml(serotype 2),2.8 u g/ml(serotype 4) and 3.7 u g/ml(serotype 5),and the optimum dilution of goat anti-duck IgG labelled horseradish peroxidase was 1:4000,coating time at 37 was 2 hours,blocking time at 37 was 30 minutes.The indirect ELISA were confirmed to be reproducible and specific by cross-assay,blocking-assay and reproductive assay. The coefficient of variation within plate were 2.85% to 9.1% (serotype 1),(2) 6.98% to 8.67% (serotype 2),(3) 3.81% to 9.83%(serotype 4) and 2.68% to 7.33%(serotype 5),and the coefficient of variation between plates were 2.82% to 6.3%(serotype 1),4.22% to 9.63%(serotype 2),2.6% to 9.01%(serotype 4) and 2.37% to 6.13%(serotype 5). The results of detection coating with R.A. were negative when 5:A Pasteurella Multocida positive sera,Duck Viral Hepatitis serotype I positive sera,E. Colibacillosis serotype Kgg positive sera in ducks,Enteritis Salmonella positive sera in ducks,E. Colibacollosis serotype K.99 positive sera in ducks and Duck Plague positive sera were tested,whereas,the results of detection to R.A. sera were positive. The sensitivity of indirect ELISA were 50 to 100 times(serotype 1),(2) 25 to 100 times(serotype 2),(3) 12 to 100 times(serotype 4) and 25 to 200 times(serotype 5) than micro-agglutination test. The results of blocking test to four serotypes were positve when relative antigens were coated.20 different sera samples were examined by indirect ELISA,the graph of absorbance measurement obtained from sera samples at 1:100 dilution versus log 10 titer showed a linear correlation over the range in investigation. Standard line and regression equations were derived from the detective results,the equations were log[ELISA Titer(ET) reciprocal]=l. 107+2.819x(serotype l),log[ET reciprocal]=1.019+2.935x(serotype 2),(3) log[ET reciprocal]=0.99+2.709x(serotype 4) and log[ET reciprocal]=1.052+2.953x (serotype 5). The derived regression lines(equations) were used to transfer the optical density value(OD) obtained from a single 1:100 dilution of any unknown serum sample of duck into ELISA titer .The indirect ELISA technique were used to monitor the variety of the mean antibodies titers of vaccinated ducks with the oil-emusified formalin-inactived R.A. vaccine,and to monitor the antibodies level of unvaccinated ducks. The response curves were established when titers were varies along with the time,and the results showed that the indirect ELISA could used to predict the efficiency of vaccination.