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

Development of a blocking ELISA based on site 2 monoclonal antibodies and foot-and-mouth disease virus-like particles for seromonitoring vaccinated animals

李恆瑋(Heng-Wei Lee)

指導教授 ： 鄭益謙

國立臺灣大學/獸醫專業學院/獸醫學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201603059

摘要

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。

關鍵字

口蹄疫 ； 單源抗體 ； Site 2中和抗原決定位 ； 類病毒空殼蛋白 ； 阻斷型酵素連結免疫吸附試驗

並列摘要

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.

並列關鍵字

foot-and-mouth disease ； monoclonal antibodies ； antigenic site 2 ； virus-like particles ； blocking enzyme-linked immunosorbent assay

參考文獻

Abbott, W.M., Damschroder, M.M., Lowe, D.C., 2014. Current approaches to fine mapping of antigen-antibody interactions. Immunology 142, 526-535.

Abrams, C.C., King, A.M., Belsham, G.J., 1995. Assembly of foot-and-mouth disease virus empty capsids synthesized by a vaccinia virus expression system. J Gen Virol 76 ( Pt 12), 3089-3098.

Armer, H., Moffat, K., Wileman, T., Belsham, G.J., Jackson, T., Duprex, W.P., Ryan, M., Monaghan, P., 2008. Foot-and-mouth disease virus, but not bovine enterovirus, targets the host cell cytoskeleton via the nonstructural protein 3Cpro. J Virol 82, 10556-10566.

Asfor, A.S., Upadhyaya, S., Knowles, N.J., King, D.P., Paton, D.J., Mahapatra, M., 2014. Novel antibody binding determinants on the capsid surface of serotype O foot-and-mouth disease virus. J Gen Virol 95, 1104-1116.

Barnett, P.V., Samuel, A.R., Pullen, L., Ansell, D., Butcher, R.N., Parkhouse, R.M., 1998. Monoclonal antibodies, against O1 serotype foot-and-mouth disease virus, from a natural bovine host, recognize similar antigenic features to those defined by the mouse. J Gen Virol 79 ( Pt 7), 1687-1697.