應用桿狀病毒表現HA以阻斷型ELISA檢測抗H5抗體

Abstract

禽流感病毒可以根據病毒表面上的血球凝集素（Hemagglutinin, HA）與神經氨酸酶（Neuraminidase, NA）分類成不同亞型，HA是一種三聚體的病毒外膜醣蛋白，能與宿主細胞表面的唾液酸（sialic acid）受體結合進入細胞後造成感染。然而，台灣在2003年分離到低病原性（Low pathogenic avian influenza virus, LPAIV）的H5N2禽流感病毒，並首次於2008年分離到具有高病原性（Highly pathogenic avian influenza virus, HPAIV）潛力的H5N2毒株，表示病毒已經由LPAIV演化為HPAIV之傾向。本實驗使用（A/chicken/Taiwan/1209/03(H5N2)）福馬林不活化全病毒免疫BALB/c小鼠，以融合瘤技術製備單源抗體（αHA MAb），並分別使用全病毒與真核表現系統抗原盤透過免疫螢光染色（immunofluorescent assay, IFA）篩選出12株抗HA的單源抗體。除此之外，利用桿狀昆蟲表現系統表現出具有醣基化的重組HA（rHA△TM）蛋白作為抗原，透過條件最優化下建立1209/H5N2 bELISA及rHA△TM/H5N2 bELISA不同抗原之阻斷型酵素免疫吸附法（blocking ELISA, bELISA）。共有265個雞血清樣品使用兩種bELISA檢測，1209/H5N2 bELISA敏感性為88.3%（76/86）、特異性為98.8%（168/170）; rHA△TM/H5N2 bELISA敏感性為93.0%（80/86）、特異性為98.8%（168/170）並以血球凝集抑制法（HI test）當作檢測的金標準。總結上述結果，建立之兩種bELISA可檢測雞隻血清樣品中抗H5的抗體，敏感性雖稍有不足但具有高特異性。

Avian influenza virus (AIV) is classified into subtypes based on the hemagglutinin (HA) and neuraminidase (NA) expressed on viral surfaces. HA is a viral coat glycoprotein as trimetric forms coded from a gene segment, can attach to sialic acid receptors on the host cell, and cause infection. Moreover, Low pathogenic avian influenza virus (LPAIV) H5N2 have been isolated in Taiwan since 2003. Also, the highly pathogenic avian influenza virus (HPAIV) H5N2 was first isolated in 2008. Indicating that the virus has been mutated from LPAIV to HPAIV. In this study, BALB/c mice were immunized with (A/chicken/Taiwan/1209/03(H5N2) AIV for generating anti-HA monoclonal antibodies (αHA MAb). We have obtained 12 MAbs against HA H5 by immunofluorescent assay (IFA) screening with whole-virus and HTK-H5HA antigen plates. Furthermore, rHA△TM/H5N2 was cloned and constructed into pBacPAK8 to prepare glycosylated HA antigen by the baculovirus expression system. After we optimizing the blocking ELISA (bELISA) condition, two bELISA with 1209/H5N2 and rHA△TM/H5N2 were established. The HI test was taken as the golden-standard of detecting αH5 antibodies in chicken sera. 265 chicken sera were tested by these two bELISA. The sensitivity and specificity of 1209/H5N2 bELISA were 88.3%(76/86) and 98.8%(168/170). The sensitivity and specificity of rHA△TM/H5N2 bELISA were 93.0%(80/86) and 98.8%(168/170)。The preliminary results show that 1209/H5N2 or rHA△TM/H5N2 bELISA has relatively low sensitivity but has high specificity for detecting anit-H5 antibodies in chicken sera.