以抗Site 2單源抗體與口蹄疫類病毒空殼蛋白建構阻斷型ELISA對免疫動物進行血清學監控

Abstract

口蹄疫可感染所有偶蹄類動物，且具有高度傳染力，在畜牧產業中是相當重要的病毒性疾病。目前，台灣的防疫政策仍然是全面施打不活化疫苗，並以血清中和試驗（serum neutralizing test, SN test）來評估免疫動物獲得中和抗體保護力的情形。然而，SN test耗時、成本高，且需在嚴格的負壓實驗室內操作。近期的研究指出，大多免疫動物的血清中以抗Site 2抗體為主。因此，我們希望以類病毒空殼蛋白（virus-like particles, VLP）和抗Site 2單源抗體（monoclonal antibody, MAb）建構阻斷型酵素連結免疫吸附試驗（blocking enzyme-linked immunosorbent assay, bELISA），並評估是否可取代SN test。VLP的製備以哺乳類細胞表現系統-transient expression assay搭配共轉染策略製造，並經過蔗醣梯度離心（sucrose gradient centrifugation）及sandwich ELISA等實驗間接確定VLP構型。為了從本實驗室已製備的41株MAbs中挑選出抗Site 2抗體，本研究採用單點突變（knock-out mutagenesis）的方式，以免疫螢光染色（immunofluorescence assay, IFA）和indirect ELISA找出可以辨識VLP但無法辨識Site 2單點突變VLP（mutated VLP, mVLP）之MAbs。配合中和抗體力價檢測結果，有6株抗Site 2抗體被篩選出來。經過sandwich ELISA以及少量樣品測試（實驗豬隻血清和臨床檢體）之bELISA，挑選S11E-9為最佳tracer完成bELISA的建構。

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals worldwide. Serum neutralization test (SN test), a gold standard for evaluating the protection rate against FMDV, is still performed for disease control in Taiwan. However, SN test is laborious, expensive and requires a high-containment biosafety lab. Based on the current study on vaccinated animals, antigenic site 2 of FMDV is the most immuno-dominant neutralizing site. We aim to establish a blocking ELISA (bELISA) based on FMD virus-like particles (VLPs) and site 2 monoclonal antibody (MAb) to detect antibodies against site 2 from vaccinated animals and replace the SN test. VLPs were expressed by eukaryotic transient expression assay with co-transfect strategy and examined by sucrose gradient centrifugation accompanied with sandwich ELISA. For mapping MAb against site 2 from 41 anti-FMDV MAbs prepared previously, we performed knock-out mutagenesis with VLPs and mVLPs (site 2 mutated) by immunofluorescence assay (IFA) and indirect ELISA. Combined with neutralization assay, results indicated that 6 MAbs recognized site 2. Based on the results of sandwich ELISA and bELISA with experimental serum, S11E-9 was regarded as the best tracer to establish bELISA with VLPs.