Establishment Of Monoclonal Antibodies Panel Against Haemagglutinin Of H1 Subtype Influenza Virus And Analysis Of Its Antigenic Variation

H1N1 influenza virus is an important seasonal human influenza virus, has historically been a global pandemic twice. The global epidemic of influenza A H1N1 virus become the world's attention once again in Spring 2009. Hemagglutinin (HA) which is the most important antigen-related protein, has been related with the spread of the virus, virulence and antigenicity closely. This study chooses HA as a major target to find out variation mechanism of H1N1 influenza virus, with a view to provide useful information for vaccine and treatment research of H1N1 influenza virus.First of all, a common influenza virus isolation and culture system were developed, and the optimal conditions of virus cultivation has been determined. A bank of 62 H1N1 virus which will lay a basis for the follow-up study has been setting up after long-term work of virus isolation.Secondly, the antigenic prediction of hemagglutinin gene of 118 H1N1 strains was made by method of phylogenetic tree analysis. Five major clade of H1N1 was determined, and we select 20 strains for the preparation and identification of monoclonal antibody. A panel of 52 H1 HA specific MAb has been established using Clade 2, 4.2, 4.6, 4.1 and an avian strain for immunization, and characterized by HI assay to the 20 H1N1 viruses. According to their HI reactive spectrums, the 52 MAbs can be divided into six categories (M1-M6), and 14 representive MAbs were further selected as a panel to characterize the antigenecity of H1N1 virus. Five groups, I, II, III, IV and V, were identified based on their cross-reactive spectra with this panel of MAbs in neutralisation assay, and four antigenic groups, A, B, C and D, were identified at the same time. At least 20 antigenecity-related amino acid mutation were found out by analysing differences between amino acid sites. Four sites of these, 203 (RBS), 205 (Sb), 206 ( Sb) and 157 (close to Ca1), may play a key role in antigenic drift of H1N1 viruses. In summary, this study established a useful panel of H1 HA MAbs and will facilitate for the recognition of emerging antigenic variants of H1N1 virus and help in monitoring and evaluating the current H1N1 virus.Finally, diagnostics was established using the 52 H1 HA specific MAbs. Its sensitivity and detection rate were 0.5 HA and 89.5%, respectively. And its specificity is good. It can be used for diagnostic purposes of H1N1 virus.