Development of Enzyme-Linked Immunosorbent Assays for Detecting the H6 Subtype Avian Influenza Virus and Antibody

• Main Authors: Yi-Tung Chen, 陳怡彤
• Other Authors: 王金和
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/95167584976416639982

碩士 === 國立臺灣大學 === 獸醫學研究所 === 97 === Low pathegenic avian influenza virus of H6N1 has circulated frequently in domestic chickens in Taiwan. Nowadays, enzyme-linked immunosorbent assays (ELISAs) for detecting antibody and antigen can’t differentiate H6 subtype. The purposes of this study are to develop ELISAs for detecting avian influenza virus H6 subtype antibody and antigen. For H6 subtype antibody detection, the monoclonal antibody specifically against H6N1 virus was used as the tracer, whole H6N1 virus and HA1 recombinant protein were used as coating antigens to develop virus blocking ELISA (virus-bELISA) and recombinant HA1 blocking ELISA (rHA1-bELISA), respectively. Hemagglutination inhibition test (HI test) was taken as a gold standard for detection of H6 antibody in sera. One hundred and thirty-eight HI test negative sera were used to evaluate the cut-off values of the virus-bELISA and rHA1-bELISA. The cut-off value of the virus-bELISA was 30%. The sensitivity and specificity were 100% (184/184) and 97% (210/216), respectively. The virus-bELISA detected H6 antibody earlier than HI test in the field. On the other hand, the cut-off value of the rHA1-bELISA was 22%. The sensitivity and specificity were 98% (180/184) and 94% (203/216), respectively. However, HI test was more sensitive than rHA1-bELISA for monitoring H6 antibody in the field. For H6 antigen detection, two monoclonal antibodies specifically against H6N1 virus were used as capture antibody and detector antibody respectively for development of the antigen-capture enzyme-linked immunosorbent assays (AC-ELISA) Poultry respiratory viruses including H5 subtype avian influenza virus, infectious bronchitis virus and Newcastle disease virus were tested by the AC-ELISA for specificity analysis. The results revealed that the AC-ELISA had good specificity. Ten H6N1 viruses were used to evaluate the detection limits of the AC-ELISA. The results showed the AC-ELISA could detect at least 1.3×105 EID50/0.1 mL of virus.