Studies On The Rapid Detection Of Avian Influenza Virus By Dot Immuno-Gold Filtration Assay (DIGFA)

AIV H9N2 or H5N1 was grown in chicken embryonated eggs and purified using 25000rmp centrifuge. Eight-week Balb/c mice were immunized according to Kazuhiro's procedure.A hybridoma named 2Cg was generated by fusion of NSo myelomas and Splenocytes from immunized mice . The monoclonal antibody( McAb) produced was screened by ELISA and proved to be IgG1 by AGP, Mouse ascitic fluid(ELISA titre:211) was purified (protein concentration:2.113mg/ml) and proved to react with NP protein in western-blotting. The result of cross-reaction in ELISA demonstrated that the McAb was group-specific.The purified rabbit anti-AIV IgG was labeled with colloidal gold particals of lOnm in diameter . A McAb-mediated DIGFA was developed for detection of AIV. When AIV was to be detected, McAb anti-NP (1:320 diluted) was used to coat nitrocellulose membrane(NCM) and react with AIV for 20 minutes.After being washed,the NCM was immersed in the solution of rabbit anti- AIV IgG(l:64 diluted) for 5 minutes. The sample with obvious red dot was judged as positive reaction for AIV,otherwise,negative result.The detection limit of purified AIV antigen and allantioic fluid of AIV were 1.799ng each dot and 103EIDso respectively.AIV titre in inoculated eggs was highest from 66 to 92 hours postinoculation(p.i.).AIV could be 100% detected in cloacal swabs of specific-pathogen-free(SPF) challenged chickens 2 DPI and 80%- 90% detected in trachea of naturally infected or experimentally challenged chickens by DIGFA.We conclude that DIGFA was a rapid ^ sensitive N simple and non-labor-intensive test for61detecting type-A influenza virus in clinical specimens and could be applied to antigen surveillance of avian influenza especially in clinical diagnosis of primary laboratory.