H5亞型家禽流行性感冒病毒抗原捕捉型酵素連結免疫吸附法及阻斷型酵素連結免疫吸附法之開發

Abstract

本研究之目的為利用對抗H5N2 亞型家禽流行性感冒病毒 (avian influenza viruses，AIV) HA1區域之單株抗體 (monoclonal antibodies，mAbs) 分別發展抗原捕捉型酵素聯結免疫吸附法 (antigen-capture enzyme-linked-immunosorbent assay，AC-ELISA) 以及阻斷型酵素聯結免疫吸附法 (blocking enzyme-linked-immunosorbent assay，B-ELISA)，以期能早期檢測出H5亞型AIV及抗H5亞型AIV抗體，另外亦進一步分析本研究室之前開發塗鍍全病毒的B-ELISA (簡稱Virus-B-ELISA) 敏感性及特異性，並針對此Virus-B-ELISA塗鍍抗原步驟加以改良以減短操作時間。實驗方法為將mAbs做為捕捉與偵測抗體，以AC-ELISA的架構進行H5亞型病毒檢測，並將mAbs標示過氧化氫酶做為追蹤子 (tracer)，以B-ELISA的架構進行雞隻血清檢測。結果，發展出之AC-ELISA只能偵測到屬於歐亞世系之H5亞型毒株，而無法測得其他世系之AIV (cut-off value=0.1)，其最低辨認限制為4.2×104/0.1 mL EID50；至於塗鍍重組HA1蛋白的B-ELISA (簡稱rHA1-B-ELISA) 則因H5亞型AIV誘發的多株抗體無法與重組蛋白質有效結合，而使H5陰性或陽性血清的測定值無顯著差異；在Virus-B-ELISA方面，與血球凝集抑制試驗相較，其檢測雞隻血清之敏感性為95.76％ (113/118)、特異性為90.78％ (266/293)，且改良前後Virus-B-ELISA之Kappa值為0.9539，代表二者間之結果幾乎完全吻合，故以改良式Virus-B-ELISA能更快速地偵測出H5亞型AIV抗體。以上總結可得，本研究發展的AC-ELISA和Virus-B-ELISA可有效運用於H5亞型歐亞世系AIV抗原及H5亞型AIV抗體檢測。

The purpose of this study is to use monoclonal antibodies (mAbs) against a H5N2 avian influenza virus (AIV) HA1 domain to develop an antigen-capture enzyme-linked-immunosorbent assay (AC-ELISA) and a blocking enzyme-linked-immunosorbent assay (B-ELISA) for early detection of H5 subtype AIV and anti-H5 AIV antibodies, respectively. Besides, we also evaluated the sensitivity and the specificity of a H5-subtype AIV B-ELISA developed in our laboratory by coating with whole virus as the antigen (called Virus-B-ELISA) and improved the virus coated step of the Virus-B-ELISA to save time. These mAbs were used as the capture antibodies and detector antibodies for the detection of H5 AIV by AC-ELISA. These mAbs were also labeled with horseradish peroxidase to become the tracers for the detection of H5 antibodies in chicken serum by B-ELISA. The result showed that the AC-ELISA only detected the H5 subtype Eurasia lineage strain and did not cross react with other lineage AIV (cut-off value=0.1). The detection limit was as little as 4.2×104/0.1 mL EID50. As to the B-ELISA coated with recombinant HA1 protein (rHA1) as the antigen (called rHA1-B-ELISA), the serum polyclonal antibodies induced by H5 AIV couldn’t effective binding with rHA1. The measurements of the H5-positive or negative serum are not significantly different. In Virus-B-ELISA, the sensitivity based on the hemagglutination inhibition test was 95.76％ (113/118) and the specificity was 90.78％ (266/293). The Kappa value between original Virus-B-ELISA and improved Virus-B-ELISA was 0.9539 meant an almost perfect consistency. So the improved Virus-B-ELISA can detect the H5 subtype AIV antibodies more rapidly than the original one. The AC-ELISA and the Virus-B-ELISA are useful to detect H5 subtype Eurasia lineage AIV antigen and H5 subtype AIV antibody, respectively.