| Newcastle disease (ND) is a highly contagious and fatal disease which could cause substantial economic losses in poultry industries. Its causative agent is Newcastle disease virus (NDV) which belongs to the newly defined genus Avulavirus in the family of paramyxovirus. Hemagglutinin-neuraminidase (HN) is an important immune protective glycoprotein on the NDV envelope and closely related to the viral virulence and pathogenicity. Serologically, NDVs possess a single serotype, avian paramyxovirus 1, but they represent different antigenic subtypes. Monoclonal antibodies (MAbs) are considered an useful tool to detect and identify the antigenic variation on a protein, as a given MAb only recognized one epitope.Hence, in this study, to elucidate the variation of epitiopes on HN protein, the MAbs against different epitopes on the HN were developed and reacted with 30 representive NDVs isolated from different hosts, areas and years.1 Purification of NDV and construction of the eukaryotic expression recombinant plasmids containing HN gene fragmentsNDV strain JS/05/5/Go was grown in the allantoic sac of 10-day-old embryonated SPF eggs at 37°C. Following the death of the embryos, allantoic fluid was harvested and purified by centrifugation at 18,000 rpm. Previous studies have shown that most epitopes of HN protein were resided in the globular regions which comprises of six antiparallelβ-sheets. Most of the antigenic epitopes were located inβ1～β3 (175～396 aa). Therefore, this region, in this paper, was divided into two fragments, 175~288 aa and 237~396 aa. After the two fragments were linked to eukaryotic expression vector pcDNA-3.1(-), respectively, two recombinant plasmids pcDNA-HN1 and pcDNA-HN2 were constructed. The transfected cells with the two resultant plasmids, respectively, could be recognized by the NDV-positive serum, which indicated that the target frgments expressed well after trasfection.2 Development of MAbs against NDV HN proteinThe Balb/c mice were immunized with the purified NDV and the eukaryotic expression recombinant plasmids containing HN gene fragments respectively. Then, spleen cells from the immunized mice were fused with SP2/0-Ag-14 myeloma cells. Indirect enzyme linked immunosorbent assay (ELISA) was used to screen hybridoma cells, and the limiting dilution method was performed to subclone the positive hybridoma cells. With purified NDV as immunogen, 10 positive cell-clones were obtained and were designated as 1B5, 1C5, 2G7, 2G8, 3B5, 4C11, 4H6, 5C2, 5G8 and 5G9, respectively. However, only two positive clones 4H11 and 2E8 were obtained with the expression plasmids-based immunogen. All the MAbs exhibited positive reaction with the CEF infected by rFPV-HN, showing that these MAbs were against the NDV HN protein. The development of these MAbs made a foundation for further studying of the antigenic variation on the HN protein.3 Detection of the antigenic variation on HN proteinThe MAbs against the HN protein were labelled by horseradish peroxidase. The competition antibody binding assay showed that these 15 MAbs altogether recognized 10 epitopes. Subsequently, 10 MAbs against different epitopes were chosen to react with 30 NDV strains isolated from different hosts, areas and years. The results demonstrated that the epitopes for MAbs, 5G8, 5G9, 4H11 and M22 to rcognicze were conservative, while the epitopes for the other Mabs were variant in some strains. According to the reaction profile, 30 NDVs were classified into six distinct groups. The different reactive profile of the MAbs with the NDV strains demonstrated the presence of antigenic variation on the HN protein.
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