以抗HA單源抗體建立阻斷型ELISA區別抗禽流感病毒H5或H6亞型之抗體

Abstract

H6N1及H5N2禽流感病毒 (avian influenza virus, AIV) 分別於1972及2003年出現於台灣並且流行至今，H6N1 AIV的分離株為低病原性，而H5N2 AIV在近幾年已經從感染的雞隻分離到高病原性的病毒，必須要發展出可以區分雞隻血清中帶有抗H5或H6亞型之抗體的檢測工具，以幫助AIV的監測與防疫。本研究以不活化的H5N2或H6N1 AIVs免疫BALB/c小鼠，待小鼠血清力價上升後，以融合瘤技術將脾臟細胞 (splenocytes) 與骨髓瘤細胞 (myelomas) 進行融合，並利用間接螢光抗體染色法 (indirect immunofluorescent assay, IFA)、血球凝集抑制試驗(hemagglutination inhibition test, HI test)、中和試驗 (Neutralization test, NT)、西方墨點法 (Western blot, WB) 及間接型酵素連結免疫吸附法 (indirect ELISA) 篩選出抗血球凝集素 (hemagglutinin, HA) 之單源抗體 (αHA MAbs)，同時鑑定αHA MAbs對H5與H6 AIV抗原之間沒有交叉反應；另一方面，我們使用福馬林不活化的A/chicken/Taiwan/1209/2003(H5N2) 全病毒，與桿狀病毒表現系統所表現的A/chicken/Taiwan/2838V/00(H6N1) HA蛋白 (rHAΔTM/H6N1) 作為抗原，搭配經過篩選的αHA MAbs，挑選出最佳的抗體組合，建立以1209/H5N2或rHAΔTM/H6N1為抗原的sandwich blocking ELISA (1209/H5N2 bELISA與rHAΔTM/H6N1 bELISA)。以HI test作為雞血清樣本中H5、H6亞型抗體存在與否的黃金標準 (golden standard)，以702個雞血清測試兩種bELISA，1209/H5N2 bELISA的臨界值為27%，敏感性為93.31% (265/284)，特異性為90.91% (380/418)；rHAΔTM/H6N1 bELISA的臨界值為28%，敏感性為95.11% (214/225)，特異性為96.44% (460/477)。此兩種bELISA相對於HI test具有較高的敏感性，可檢測到低HI力價的血清抗體，有助於提升國內禽流感血清檢測與防疫監測之成效。

H6N1 and H5N2 AIV have been circulated in Taiwan since 1972 and 2003 respectively. H6N1 strains are low pathogenic avian influenza viruses (LPAIV). However, some of the H5N2 strains isolated currently become highly pathogenic avian influenza viruses (HPAIV). Developing an effective detection tool to differentiate whether the serum antibodies are against H5 or H6 subtypes is essential for AIV surveillance and management. In this study, BALB/c mice were immunized with inactivated H5N2 or H6N1 AIVs. After evaluated the antibody titer of the mice sera by immunofluorescent assay (IFA), we fused mice splenocytes with myelomas to generate hybridomas. Anti-HA Monoclonal antibodies (αHA MAbs) were screened by IFA, hemagglutination inhibition (HI) assay, western blotting (WB) and indirect ELISA (iELISA) to confirm the specificity to HA and no interaction between H5 and H6 AIVs. On the other hand, we established two sandwich blocking ELISAs (1209/H5N2 bELISA；rHAΔTM/H6N1 bELISA) with αHA MAbs and formalin-inactivated 1209/H5N2 viruses or recombinant HA protein of A/chicken/Taiwan/2838V/00 (H6N1) by baculovirus-insect cell expression system. Hemagglutination inhibition test was taken as the golden standard method of detecting anti-H5 or anti-H6 antibodies in the chicken sera. 702 chicken sera were tested by 1209/H5N2 bELISA and rHAΔTM/H6N1 bELISA. The cut-off value of 1209/H5N2 bELISA was 27%. The sensitivity and specificity were 93.31% (265/284) and 90.91% (380/418). In addition, the cut-off value of rHAΔTM/H6N1 bELISA was 28%. The sensitivity and specificity were 95.11% (214/225) and 96.44% (460/477). Also, both of the two bELISAs can detect the antibodies in chicken sera with low HI titer. The results show that these bELISAs could be used to differentiate H5 and H6 infection in the field.