Development Of Indirect ELISA Antibody Test Kit Of Infectious Bursal Disease

CJ801 strain of IBDV was cultured continuously for four times in chicken embryo, and then cultured for seven times in CEF. We obtained adapted virus of CEF. Then lots of these inactivitied virus was purified with methods of sucrose gradient density centrifugation.Using the inactivated CJ801 strain of IBDV as coating antigen,an indirect ELISA was successfully developed for detecting anti-IBDV antibody in serum and ELISA test kit was primarily prepared.Conditions were optimized for using these preparations for an IBDV-specific ELISA. The result was shown that ELISA plates from Costar was suitable for ELISA kit,the optimal concentration of IBDV for coating of plate was 0.0128 u g/ul, the optimal coating condition of IBDV for ELISA was 4℃ overnight, the dilution of serum sample was 1:200 ,the working concentration of HRP-labeled rabbit anti-chicken IgG was 1:800, serum sample for detecting and HRP-labeled rabbit anti-chicken IgG should be incubated at 37℃ for 1h respectively, the substrate for ELISA was incubated at RT for 20 min before reading the OD655. Following the determination of conditions of ELISA, the threshold value of ELISA was 0.17.Serum samples against BJ836,CJ801,2512 , Cu and Harbin were detected for the antibody to IBDV by using the developed ELISA. It was found that there were cross-reaction between the antibodies and CJ801 strain of IBDV. Meanwhile, it was confirmed that there were no cross-reaction between the antibodies against NDV, EDS'76, IBV, AIV and so on. The results revealed that the indirect ELISA by the purified IBDV had good specificity for the detection of IBDV antibody in serum.60 serum samples from chicken were detected for the antibody to IBDV by using the developed ELISA and the HIPRA IBDV antibody test kit simultaneously. It was found that the coincidence of the developed ELISA were 96%. 44 serum samples from chicken were detected for the antibody to IBDV by using the developed ELISA and the HIPRA IBDV antibody test kit simultaneously. Both kits'result took on linary relation.The assay has met the standards of analogic method abroad.Finally, the storage conditions of the plates coated by antigen of IBDV, the diluted HRP-rabbit anti-chicken conjugate, the control positive/negative serum and the kit were explored. So far the kit could be stored for 3 months at 4'C because of time. The study have developed an assay, which provides a available technique for immune detection and serological survey of IBD.