Development And Application Of Indirect ELISA For Detecting RA Antibody

Riemerella anatipestifer (RA) is the pathogen of duck Infectious serositis and is one of the most hazardous pathogen affecting ducks raising. The present indirect—ELISA can't discriminate antibody generated by natural infection or immunization. We established Indirect ELISA using UW schizolysised RA bacteria suspension, lipid carbohydrate and CAM protein as coating antigen. We detected convalescent serum from ducks which had been artificial infected with RA and serum from ducks which had been immunized with inactivated vaccine. Experiments above provided methods for vaccine development and serological epidemiology research.The research results as follows :1 . prokaryotic expression of CAM fusion proteinWe choose pET as expression vector and E.coli BL21 (DE3 ) as expression host bacterium. E.coli BL21 expressed maximum recombinant protein through the conditions as follows: the induction temperature is 30℃, the IPTG concentration is 0.4mmol and the induction duration is 4 hours. The expressed protein can be identified by Western Blot. We obtained pure CAM fusion protein about 37 KD through His-Bind affinity purification.2 . Indirect ELISA development(1) This experiment established Indirect ELISA using UW schizolysised AF serotype RA suspension which had 91ug/ml protein concentration as coating antigen, I.e. PRO-ELISA. the optimal ELISA reaction condition had been screened as follow : the optimal working concentration of first antibody is 1:800 and second antibody is 1:800. we used this ELISA method to detect seraum such as pasto-bacillus antibody positive serum from duck, type I viral hepatitis antibody positive serum from ducks, ducks origin E.coli antibody positive serum from ducks, ducks origin enteritidis Bacillus antibody positive serum from ducks, ducks plague antibody positive serum from ducks, all the results were negative, the result of blocking test was negative. The coefficient of variation in one plate and the coefficient of variation among plates were 3.86%-16.8% and 6.17%- 9.46% respectively. (2) This experiment established Indirect ELISA using lipid carbohydrate antigen of AF serotype RA which had 26ug/ml lipid carbohydrate concentration as coating antigen, I.e. LPS-ELISA. The optimal ELISA reaction condition had been screened as follow : the optimal working concentration of first antibody is 1:200 and second antibody is 1:400. we used this ELISA method to detect sera such as pasto-bacillus antibody positive serum from duck, type I viral hepatitis antibody positive serum from ducks, ducks origin E.coli antibody positive serum from ducks, ducks origin enteritidis Bacillus antibody positive serum from ducks, ducks plague antibody positive serum from ducks, all the results were negative, the result of blocking test was negative, the coefficient of variation in one plate and the coefficient of variation among plates were 4.73%-6.90% and 5.30%-11.9% respectively.(3) This experiment established Indirect ELISA using pure CAM fusion protein which had 22.51ug/ml protein concentration as coating antigen, I.e. CAM-ELISA. The optimal ELISA reaction condition had been screened as follow: the optimal working concentration of first antibody is 1:400 and second antibody is 1:200. we used this ELISA method to detect sera such as pasto-bacillus antibody positive serum from ducks, type I viral hepatitis antibody positive serum from ducks, ducks origin E.coli antibody positive serum from ducks, ducks origin enteritidis Bacillus antibody positive serum from ducks, ducks plague antibody positive serum from ducks, all the results were negative; The result of blocking test was negative; The coefficient of variation in one plate and the coefficient of variation among plates were 5.85%-16.3% and 4.22%- 9.37% respectively.3. Using indirect ELISA methods established above to detect convalescent sera from duck which had been artificial infected with RA and sera from duck which had been immunized with inactivated vaccine.The antibody of ducks immunized with inactivated vaccine can be detected on the 5th day after the first immunization through the PRO-ELISA method (P/N=7.11). Using the same indirect ELISA method, We can detected antibody on the third day post infection from ducks which had been artificially infected with RA(P/N=6.12), then the antibody level gradually raise till the 21st day post infection, the maximum was detected (P/N= 17.26), then it began to decrease.The antibody of ducks immunized with inactivated vaccine can't be detected on before the 20th day after the first immunization through the LPS-ELISA method (P/N<3). And aftrer second immunization,the antibody level increased fastly,using the same indirect ELISA method, We can detected antibody on the 5^th day and the 20^th day, the antibody level were 4.25 and 9.34 espectively (P/N) .It can be detected from ducks which had been artificially infected with RA (P/N=4.87) on the 3^th day,on the 21th day it reached the maximum (P/N=17.04) , then it began to decrease.Using the CAM-ELISA method to detect the antibody from serum from ducks immunized with inactivated vaccine, the results were negative neither after first vaccination from 5-35^th day and the second vaccination from 5-25^th day; after artificially infected with RA, ,the antibody level was 4.98 (P/N ) On the 3th day,the antibody level reached the maximum on the 21^st day (P/N=13.83) ,then it began to decrease.