| Summary: | 碩士 === 國立中興大學 === 獸醫學系 === 91 === An outbreak of highly pathogenic H5N1 avian influenza occurred in Hong Kong in 1997 and caused the death of 6 people. Nucleotide sequence analysis showed that this virus was transmitted directly from avian species to humans. In 1999 and 2001, the H5N1 virus circulated again in chickens and ducks at Hong Kong and China, and caused a severe economic loss. Because of the rapid and frequent communication between Taiwan and China, the H5N1 virus might invade Taiwan and impose a potential risk to the poultry industry of Taiwan. To develop a rapid method for the survey of this virus infection, we have developed a monoclonal antibody (MAb-H5) against the HA (hemagglutinin) protein of H5 virus, and developed a blocking ELISA for the detection of antibody against the H5 virus. To locate the binding site of MAb-H5, we divided the HA protein (residues 108-299) of the H5 subtype into 17 fragments, and expressed these fragments in E. coli. By Western blot, we were able to localize the binding site of MAb-H5 at residues 200-231 of the HA protein. We also synthesized a gene fragment encoding the residues 200-231 of the HA protein of the H5N1 virus isolated in Hong Kong in 1997. By E. coli expression and Western blot analysis we showed that MAb-H5 could recognized the HA protein of the H5N1 virus isolated in 1997 in Hong Kong. H1-H15 reference sera (1:3 dilution) was tested against this blocking ELISA, and the result showed that the H5 antiserum gave an absorbance 0.208 whereas sera of other subtypes gave absorbance higher than 2.076. The sensitivity of the blocking ELISA was 93.3 when tested against the positive sera, and the specificity was 93.7 when tested against the negative field sera (30% competition was used as the cut-off value). The further work of this study will include the test of more field sera, and modification of the test condition to increase the sensitivity and specificity of this test. We believe that the blocking ELISA could provide a rapid tool for the detection of antibody of the H5 virus, and will contribute to the prevention of the invasion and spreading of H5 virus.
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