Development And Application Of The Monoclonal Antibodies Against Genotype â…¦ Newcastle Disease Virus

Since 1997, the goose flocks in some regions of South and East China have experienced severe outbreaks of Newcastle disease virus (NDV) infection. The results of the in vitro tests indicate that the goose isolates have stronger ability to induce syncytium formation, when compared to chicken and pigeon isolates. In order to study the variety of NDV and establish new method to detect NDV, monoclonal antibodies (MAbs) against genotype VII NDV were developed.The 8-week-old female BALB/c mice were immunized with lOOug formaldehyde-inactived NDV ZJ1 strain (isolated from Zhejiang province in 2000) emulsified in Freund's complete adjuvant. After the third immunization, spleen cells from immunized mice were fused with Sp2/0-Ag-14 myeloma cells. By the screening tests with indirect ELISA( enzyme linked immunosorbent assay )and HI (hemagglutinin inhibition) , six positive clones were obtained, which were denominated as 1H6, 2G5, 3A4, 3B5, 6B1 and 8C5 respectively. The specificities of these six MAbs were characterized by HI test, indirect ELISA and indirect immunofluorescent assay (IIFA). All MAbs had high ELISA and IIFA liters, but only five of them were positive to HI test. The immunoglobulin subtypes of the MAbs were Igd, except 3B5.Four antigenic sites were found in the hemagglutinin (HN) protein of NDV in a competitive ELISA performed by these MAbs with M22 and F23 MAbs. The five MAbs with the specificity of HI reacted well with most of the NDV isolates. The HI result was found that the MAbs sharing different antigenic sites had the different reaction map's. The HI map of 8C5 was different from that of 2G5, 3A4, 3B5 and 6B1that shared the same sites. We also found that the five MAbs had the different reactive abilities with different original isolates. These results indicated that the five MAbs reacted strongly with the isolates from goose, but reacted weakly with the pigeon isolates.The method of UFA was established with 6B1 as the first antibody. We could detect the virulent NDVs effectively using this method. In sandwich ELISA, plates were coated with different MAbs and crossing reaction was done with different HRP-conjugated MAbs. As a result, two new systems, 2G5-8C5 and M22-8C5 were successfully established. Using the two new systems along with the system M22-F23 to detect the NDV isolates, we found that one of the systems, 2G5-8C5 could react well with goose original NDV isolates, thus it could be used for detecting NDV from goose. The three systems mentioned above could react well with most of NDV isolates, however, the reaction abilities between them were different in some of the NDV isolates, moreover, the results demonstrated that all NDV isolates could react with M22-8C5 system. Accordingly, this system was suitable for preparing the kit for the detection of NDV.