以合成胜肽ELISA及阻斷性ELISA快速診斷家禽流行性感冒病毒H5及H7亞型抗體

Abstract

家禽流行性感冒 ( avian influenza, AI ) 是由A型流行性感冒病毒所引起之疾病。依據病毒表面的醣蛋白區分，目前在禽鳥類已被證實的有15種HA ( HA1-HA15 ) 及9種NA ( NA1-NA9 ) 亞型，高病原性家禽流行性感冒 ( HPAI ) 主要由H5及H7血清亞型病毒引起。傳統上在血清學監控方面主要採用酵素連結免疫吸附分析試驗 ( enzyme-linked immunosorbent assay, ELISA ) 及血球凝集抑制試驗，ELISA部分目前並無可區分血清亞型之商品化套組可供使用，而血球凝集抑制試驗部分則有培養活毒之風險且可能受N亞型干擾。本研究主題分為兩部分，一部分以合成胜肽建立間接性酵素連結免疫吸附分析試驗 ( indirect ELISA, IELISA )，另一部分利用H7血清亞型之HA重組蛋白製備單源抗體，建立阻斷性酵素連結免疫吸附分析試驗 ( blocking ELISA, BELISA )。在合成胜肽ELISA部分，根據H5之HA1抗原決定區序列合成5組胜肽，結果發現其中peptide H5 265-300具良好之區分H5亞型血清能力。另外針對H7亞型所合成胜肽部分，則是以peptide H7 197-241對H7亞型具良好的區分力。以peptide H5 265-300檢測所得之吸光值扣除以peptide H7 197-241檢測所得之吸光值，可得到H5高免血清為正值，H7高免血清為負值的結果，而其餘HA亞型高免血清皆介於正負0.1之間。依田間HI陰性血清檢測結果將吸光差值大於0.13訂為H5陽性，吸光差值小於 -0.11訂為H7陰性，則在129隻HI陰性血清中只有2隻判定為H5陽性，特異性可達98.45 %。在阻斷性ELISA部分，以西方轉漬法及間接免疫螢光染色法挑選出對於H7血清亞型之病毒HA蛋白較具特異性之單源抗體，再以胜肽矩陣判定此H7單源抗體主要辨識區為HA第285-294胺基酸序列。將此單源抗體配合原核系統表現之H7血清亞型之HA重組蛋白應用於阻斷性ELISA中，可於15種HA血清亞型中區分出H7血清亞型。利用田間HI陰性血清檢測結果訂定此阻斷性ELISA檢測套組之cut-off value為20 %，特異性可達97.71 %。

Avian influenza (AI) is caused by type A influenza virus. According to their surface glycoprotein, fifteen HA subtypes (HA1-HA15) and nine NA subtypes (HA1-NA9) have been identified. Highly pathogenic avian influenza (HPAI) is mainly caused by H5 and H7 viruses. Conventional methods for serological surveillance of AIV are enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition test (HI). There is no commercial ELISA kit available for subtyping of sera. Hemagglutination inhibition test needs the handling of whole viruses, which might have potential risk. Moreover, it might be interfered by NA subtypes. The goal of this study can be divided into two parts, the first part is using synthetic peptides to establish an indirect enzyme-linked immunosorbent assay (indirect ELISA, IELISA), and the second part is using recombinant HA protein of H7 subtype to prepare monoclonal antibody to establish a blocking enzyme-linked immunosorbent assay (blocking ELISA, BELISA). For synthetic peptide ELISA, five peptides according to the HA1 antigenic determinant site of H5 subtype were synthesized, and the result showed that the peptide H5 265-300 could discriminate the H5 antiserum from sera of other subtypes. Moreover, the peptide H7 197-241 could discriminate the H7 antiserum from sera of other subtypes. The absorbance of H7 peptide 197-241 ELISA is subtracted from the absorbance of H5 peptide 265-300 and positive value was obtained for H5 hyperimmuned serum, whereas negative value was obtained for H7 hyperimmuned serum. The values for other hyperimmuned sera were between 0.1 to -0.1. According to values of field sera, it is determined that H5 positive sera have value over 0.13 and H7 positive sera have value under -0.11. By using this method, only 2 out of the 129 negative sera were found positive and the specificity of the peptide ELISA was considered to be 98.45 %. For blocking ELISA, the monoclonal antibody that had specificity to the HA protein of H7 subtypes by Western blot and indirect immunofluorescence assay are chosen. The binding site of this monoclonal antibody was localized at residues 285-294 of HA protein by using the peptide array. The blocking ELISA developed by using recombinant HA protein of H7 subtype and monoclonal antibody could discriminate the H7 antiserum from sera of other subtypes. By testing filed negative sera, it could determine 20 % of inhibition percentage as the cut-off value, and the specificity of the blocking ELISA was found to be 97.71 %.