Development Of Antigen Capture ELISA For Detecting H5 Avian Influenza Virus

In this study, hyper-immunized goat serum was prepared by immunizing for three times with inactive purified H5N1 AIV (DZJ/1100) mixed with Freund's adjuvant or incomplete Freund's adjuvant. The antigen capture ELISA was developed using polyclonal anti-sera (goat serum against AIV) as capture antibody and H5 specificity monoclonal antibody as detecting antibody for detecting H5 AIV. The optimal conditions were determined: capture anti-sera was diluted for 1:1600, H5 AIV specificity McAb was diluted for 1:10000 and the enzyme-labeled antibody was diluted for 1:5000. The optimal reactive time of the detecting antibody and conjugate were one and half an hour separately in the assay.NDV,EDS76,IBV,ILTV,MDV,APV and H1-H15 AIV were detected by the sandwich ELISA and the result proved that there was no crossing-reaction between the antigen capture ELISA and other avian viruses and others fourteen AIV except H5. It proved that the H5 AIV antigen capture ELISA had excellent specificity.Comparing with HA test and egg inoculation test, the sensitivity of the antigen capture ELISA was twice better than that of HA test, but it was less than that of egg inoculation test.Many experimently infected clinical samples were detected by the sandwich ELISA under the most optimal conditions. Finally, spleen, lung, heart and liver were defined as target organ when clinical samples were detected by the ELISA and the cut-off value of spleen was 0.15 and others were 0.1.The ELISA kit which consisted of coated microplate and reactive solutions were kept in room temperature, 4℃and -20℃seperately. The detection results showed that kits had bad stability in room temperature and the period of validity was only one month. At the same time, the period of validity in 4℃was 4 months and over 8 months in -20℃.