Development of avian influenza H5N1 diagnosis

Abstract

Avian influenza virus (AIV) H5N1 has a significant impact to the public health concern as well as socioeconomic aspects. The sporadic outbreak of AIV H5N1 in humans and endemic outbreaks in poultry in Southeast Asia require an increase attention of rapid diagnosis with high efficiency. Therefore, the objectives of this study were to prepare, purify and characterize H5N1 virus, development of an indirect ELISA test and monoclonal antibodies production. The first step was comparing the viral growth in Madin-Darby canine kidney (MDCK) cell lines and chicken embryonic eggs which indicated that the Thai H5N1 virus propagation in the chicken embryonic eggs was better than that of in the MDCK cells. The next step was to concentrate and purify the crude H5N1 virus using sucrose gradient technique. The purified virus was subsequently tested for the nucleic acid detection and protein analysis. An indirect ELISA was established using the purified virus as the whole antigen for detection of antibodies against AIV. The specificity and sensitivity of an indirect ELISA was acceptable, when used in the field and found to be suitable for screening large number of field chicken sera. The purified virus was also used for developing monoclonal antibodies. Although high antibody responses were obtained, the fusions were not successfully done due to many reasons including HAT-resistant myeloma cell and the quality of fusion solution. When the fusion technique was successfully improved, there were other complicated problems arising such as overgrowing of non-secreting clones. It is recommended that recombinant protein should be used for monoclonal antibody production of H5N1.