| Summary: | 碩士 === 國立中興大學 === 獸醫微生物學研究所 === 92 === Avian influenza (AI) is caused by type A influenza virus. According to their surface glycoprotein, fifteen HA subtypes (HA1-HA15) and nine NA subtypes (HA1-NA9) have been identified. Highly pathogenic avian influenza (HPAI) is mainly caused by H5 and H7 viruses. Conventional methods for serological surveillance of AIV are enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition test (HI). There is no commercial ELISA kit available for subtyping of sera. Hemagglutination inhibition test needs the handling of whole viruses, which might have potential risk. Moreover, it might be interfered by NA subtypes. The goal of this study can be divided into two parts, the first part is using synthetic peptides to establish an indirect enzyme-linked immunosorbent assay (indirect ELISA, IELISA), and the second part is using recombinant HA protein of H7 subtype to prepare monoclonal antibody to establish a blocking enzyme-linked immunosorbent assay (blocking ELISA, BELISA). For synthetic peptide ELISA, five peptides according to the HA1 antigenic determinant site of H5 subtype were synthesized, and the result showed that the peptide H5 265-300 could discriminate the H5 antiserum from sera of other subtypes. Moreover, the peptide H7 197-241 could discriminate the H7 antiserum from sera of other subtypes. The absorbance of H7 peptide 197-241 ELISA is subtracted from the absorbance of H5 peptide 265-300 and positive value was obtained for H5 hyperimmuned serum, whereas negative value was obtained for H7 hyperimmuned serum. The values for other hyperimmuned sera were between 0.1 to -0.1. According to values of field sera, it is determined that H5 positive sera have value over 0.13 and H7 positive sera have value under -0.11. By using this method, only 2 out of the 129 negative sera were found positive and the specificity of the peptide ELISA was considered to be 98.45 %. For blocking ELISA, the monoclonal antibody that had specificity to the HA protein of H7 subtypes by Western blot and indirect immunofluorescence assay are chosen. The binding site of this monoclonal antibody was localized at residues 285-294 of HA protein by using the peptide array. The blocking ELISA developed by using recombinant HA protein of H7 subtype and monoclonal antibody could discriminate the H7 antiserum from sera of other subtypes. By testing filed negative sera, it could determine 20 % of inhibition percentage as the cut-off value, and the specificity of the blocking ELISA was found to be 97.71 %.
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