Development Of ELISA For Detecting IBDV Antibodies And Preparation Of Reference Antigen And Antisera Of IBDV

Infectious bursal disease (IBD) is an acute and highly contagious disease, which caused by infectious bursal disease virus (IBDV). It causes death in young chicken and induced immune suppression by destroying B lymphocytes in bursa of Fabricius, resulted in vaccination failure and secondary/mixed infection. IBD has been regarded as one of the most severe infectious diseases which cause great economic loss in poultry industry. In this study, solubleVP2 and VP3 protein expressed in the E.coli were used to develop ELISA methods to detect antibodies against IBDVAt the same time, reference positive antigen and sera were prepared and characterized. All of these provided effective methods and materials for IBDV diagnosis.1. Establishment of ELISA methods to detect antibodies against IBDVIBDV VP2 and VP3 genes were amplified by RT-PCR and cloned into expression vector pGEX-6p-1. The recombined vectors pGEX-6p-1-VP2 and pGEX-6p-1-VP3 were transformed into E.coli BL21(DE3). The fusion proteins were expressed under IPTG induction. The results of SDS-PAGE showed that the fusion protein of GST-VP2 was expressed highest with IPTG at a concentration of 0.75mM, while the fusion protein of GST-VP3 was expressed best with IPTG at a concentration of 0.25mM. Both GST-VP2 and GST-VP3 could react with IBDV positive sera in Western-blot.GST-VP2 and GST-VP3 recombinant fusion proteins were purified by affinity chromatography and used as coating antigen to establish ELISA methods. The factors in ELISA were optimized. We found that the optimal concentrations of coating antigen were identified as 5μg/ml (GST-VP2) and 0.625μg/ml (GST-VP3), respectively. The sera samples for test were at a dilution of 1:400 before an incubation of 30 minutes.The optimal incubation time of secondary antibodies was also 30 minutes. Substrate developing was stopped by 1%SDS after an incubation of 15min. Total 50 sera samples from SPF chickens were tested to determine critical value. The results showed that positive in VP2-based ELISA was value OD6so>0.12, while positive in VP3-based ELISA was value OD650>0.136. Both VP2-based ELISA and VP3-based ELISA were specific to IBDV-positive sera and showed no cross-reactivity with other sera to other avian viruses. The coefficient of variation within batch or between batches was less than 10%. Two established ELISA were applied to detect 77 chicken sera samples from Jiangsu Province, which compared with two commercial ELISA kit for IBDV antibody detection. As a result, the coincidence rate between VP2-ELISA and commercial ELISA kit were 94.8% and 96.1%, respectively; the coincidence rate between VP3-ELISA and commercial ELISA kit were 58.44% and 59.7%, respectively. This result revealed that VP2-based ELISA was much better for the detection of clinical samples.2. Development of IBDV reference positive antigen and antisera28-days old SPF chickens were challenged with virulent virus of IBDV-JS strain through cloaca and eyes. IBDV antigen was isolated and purified from bursa of Fabricius of dead chicken by centrifugation and sephadex chromatography. Purified virus antigen was identified by AGP test and Western-blot. The contenctration of the virus antigen was measured. All tests showed the purified virus antigen had good qualified immunogenicity and were free from contamination of other avian viruses. The purified virus antigen was sub-packaged and lyophilized. Finally, the reference of IBDV antigen was characterized by loading CV, physical properties, sterility, mycoplasma contamination, uniformity, stability, expiration date.In order to make a reference sera to IBDV,14-days old SPF chickens were immunized by low dose IBDV-JS strain and challenged with virulent virus of IBDV-JS strain through cloaca and eyes after boost immunization twice. At 12-days post challenged, antisera were collected from the whole blood of chickens with high-level antibodies. The titers of antisera in AGP test, IFA and ELISA were 1:128,1:1600, and 1:51200. respectively. Western-blot assay showed antisera specifically recognized IBDV-JS strain and IBDV-Q strain. The antisera had no cross-reactivity with other avian viruses. The reference antisera was sub-packaged, lyophilized and characterized by loading CV, physical properties, sterility, mycoplasma contamination, uniformity, stability, expiration date.