應用流感病毒四聚體神經胺酸酶藉由阻斷型ELISA檢測抗H6N1抗體

Abstract

禽流感病毒(Avian influenza virus, AIV)是具有封套之負向單股RNA病毒，並有八段RNA基因片段，病毒封套上包含三種跨膜蛋白: 血液凝集素(Hemagglutinin, HA)、神經胺酸酶(Neuraminidase, NA)與基質蛋白(Matrix, M1)。AIV可以根據病毒表面蛋白HA與NA進行分型，HA可以與宿主細胞上唾液酸接收器(sialic acid receptor)連接造成感染，而NA主要功能為切割病毒與唾液酸之連結，幫助病毒出芽離開宿主細胞。台灣所發生的禽流感案例，在家禽曾發現的低病原性禽流感病毒亞型，有H6N1與H5N2，分別於1972年與2003年首次於養禽場被檢測出，可能造成人畜共通傳染病等公共衛生議題，因此發展具有特異性與敏感性之檢測工具是迫切需要的。本研究使用不活化A/chicken/Taiwan/2838V/00(H6N1) 以及A/chicken/Miaoli/2904/00(H6N1)禽流感病毒對 BALB/c小鼠進行免疫，以融合瘤技術製備辨識NA之單源抗體 (αNA MAb)，並使用免疫螢光染色法(IFA)、西方墨點法(western blotting)以及間接型酵素連結免疫吸附法(indirect ELISA)評估MAb對NA抗原之特異性與敏感性。同時製備重組NA蛋白做為抗原，並使用桿狀病毒-昆蟲細胞表現系統大量製造具有四聚體結構的NA；此外，以蔗糖梯度純化2904/H6N1全病毒，以完整病毒作為抗原。利用所製備之單源抗體與抗原(rN1和2904/H6N1)建立三明治型酵素連結免疫吸附法(sandwich ELISA)，並優化阻斷型酵素連結免疫吸附法(blocking ELISA, bELISA)之條件，建構MAb-based bELISA，並以NI test檢測受測雞血清輔助確認，期應用於能區別N1與N2亞型動物血清之檢測，協助禽流感診斷、分型以及防疫監測。初步測試顯示，以2904/H6N1為抗原，使用MAb組合61YA1-2a & 61YA1-2a-HRP，前驅測試8個分別H6陽性與陰性雞血清樣品，並搭配NI test輔助確認血清中抗體NA亞型別，在bELISA結果中皆有血清OD讀值顯著下降，NI test中有5個血清NA抗體抑制率達到50%，代表血清含有抗體抑制作用，後續有待大量血清樣品測試以顯示所建立MAb-based bELISA之效能。

Avian Influenza virus (AIV) is an enveloped RNA virus with eight RNA- segments. The viral envelope contains three transmembrane proteins: hemagglutinin(HA), neuraminidase(NA), and matrix. AIV subtypes can be classified by HA and NA. The major function of NA is essential to cleave the sialic receptors and viral membranes, and required for virus budding and releasing. Low pathogenic H6N1 AIV has circulated in poultry in Taiwan for 40 years and H5N2 has been isolated in Taiwan since 2003 respectively. These endemic zoonotic pathogens should potentially intimidate the public health. Therefore, to develop the specific and sensitive detection tools is a crucial need. In this study, BALB/c mice were immunized with inactivated A/chicken/Taiwan/2838V/00(H6N1) and A/chicken/Miaoli/2904/00 (H6N1) AIV for generating anti-NA monoclonal antibodies (αNA MAb). These MAbs were screened by IFA, analyzed by western blotting(WB), and indirect ELISA(iELISA) to evaluate the specificity and sensitivity to NA. We also cloned two recombinant NA from H6N1 AIV: full-length N1(rN1) and GCN4N1, and used baculovirus expression system to produce the recombinant and tetrameric NA. Meanwhile, we purified 2904/H6N1 virus by sucrose gradient and used complete virus as the antigen for ELISA. Monoclonal antibody against NA and NA antigens (rN1 and 2904/H6N1) were applied to establish two sandwich ELISAs (sELISA). The MAb-based bELISA was obtained after the condition of sELISA was optimized. Based on the result of analysis and optimization, we choose 2904/H6N1 for the antigen, and 61YA1-2a & 61YA1-2a-HRP as the MAb pair for the MAb-based bELISA. Then eight chicken sera H6 HI positive and negative chosen for primary test respectivly. All H6 positive sera showed low OD value in the bELISA test and five of them have NI activities, which means that these sera may have the blocking antibody revealed by this MAb-based bELISA. Lastly, the aim to establish a bELISA platform with 2904/H6N1 and αNA MAb/H6N1 and the efficacy the bELISA will be compared with the results of the NI test.