H6亞型家禽流行性感冒病毒抗原及抗體酵素連結免疫吸附法之開發

Abstract

台灣的雞場常發生H6N1低病原性家禽流行性感冒病毒之感染，目前以酵素連結免疫吸附法 (enzyme-linked immunosorbent assays；ELISA) 的方式檢測抗體與抗原皆無法區分出H6亞型，本研究之目的為發展可檢測H6亞型家禽流行性感冒病毒抗體與抗原之ELISAs。為檢測H6亞型抗體，利用專一性抗H6N1病毒之單株抗體作為追蹤子 (tracer)，分別利用H6N1全病毒及HA1重組蛋白質作為塗鍍抗原發展病毒阻斷型ELISA (virus blocking ELISA；virus-bELISA) 和重組HA1蛋白質阻斷型ELISA (recombinant HA1 blocking ELISA；rHA1-bELISA)。以血球凝集抑制試驗 (hemagglutination inhibition test；HI test) 作為檢測血清中H6亞型抗體之金標準 (gold standard)。兩種bELISA皆以138個HI test陰性血清評估其cut-off值，virus-bELISA之cut-off值為30%，應用於其他血清測得敏感性為100% (184/184)，而特異性為97% (210/216)。此virus-bELISA應用於田間能比HI test能早期監測到H6亞型抗體。另外，rHA1-bELISA之cut-off值為22%，敏感性為98% (180/184)，而特異性為94% (203/216)，應用於田間血清之抗體檢測其敏感性較HI test低。利用兩株專一性抗H6N1之單株抗體分別作為capture antibody及detector antibody發展抗原捕捉型酵素連結免疫吸附法 (antigen-capture enzyme-linked immunosorbent assay；AC-ELISA) 以檢測H6亞型抗原。以家禽呼吸道病毒包括H5亞型家禽流行性感冒病毒、傳染性支氣管炎病毒及新城病病毒測試AC-ELISA之特異性，顯示AC-ELISA只能檢測到H6亞型家禽流行性感冒病毒，具有良好之特異性。另外以10株H6亞型病毒株檢測AC-ELISA之靈敏度，結果顯示最低可檢測到1.3×105 EID50/0.1 mL的病毒量。

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.