大鼠抗肠道病毒71型衣壳蛋白VP1特异性中和单克隆抗体的研制与应用

目的研制大鼠抗肠道病毒71型（EV71）衣壳蛋白VPl特异性中和单克隆抗体（单抗），并用Westem印迹、ELISA和免疫荧光检测（IFA）等方法验证其特异性。方法采用单克隆杂交瘤技术人工合成包含EV71 VP1中和抗原决定簇位点SPT0的多肽偶联载体蛋白KLH，腹腔内注射免疫大鼠数次后，取有预期免疫应答的脾细胞与SP2／0小鼠骨髓瘤细胞融合，用间接ELISA法筛选和有限稀释法亚克隆获得稳定的阳性单克隆杂交瘤细胞株并鉴定其IgG亚型。选取其中1株单克隆杂交瘤细胞系BC6，体外培养后注射于SCID裸鼠腹腔制备腹水以产生大量抗体，用ProteinA层析柱亲合纯化后获得纯化抗体。细胞培养微量中和试验测定该单抗中和EV71的滴度，并将其用于多种方法检测EV71特异性抗原。结果共获得8株稳定的单克隆杂交瘤细胞系，分别有6株属于IgGl亚型，2株为IgG2a亚型。通过制备腹水大量产生抗EV7l大鼠单抗BC6并纯化，其对EV71的中和滴度为1：32。BC6用于Western免疫印迹可特异检测大肠埃希菌表达的EV71VPl重组蛋白和EV71样品中的VPl，用于ELISA可灵敏特异地定性或定量检测EV71抗原含量，用于IFA可特异识别被感染Vero细胞内的EV71颗粒，而对柯萨奇病毒A16型无交叉反应。结论成功制备获得了大鼠抗EV71VPl的特异性中和单抗，可应用于Western免疫印迹、ELISA、IFA等方法，成为灵敏特异地检测EV71VPl或全病毒颗粒的有效工具。

Development and application of rat monoclonal neutralizing antibody against enterovirus 71 capsid protein VP1

Objective To generate rat monoclonal neutralizing antibody against enterovirus 71 （EV71） capsid protein VP1 and apply it for Western blot （WB）, enzyme-linked immunosorbent assay （ELISA） and immunofluorescence assay （IFA）. Methods The traditional hybridoma technology was employed. A peptide containing a neutralizing epitope SP70 that spanning amino acids 208-222 of the capsid protein VP1 sequence of EV71 was synthesized and conjugated to the carrier protein KLH. Then it was used as an immunogen for intraperitoneal injection into rat and boosted with standard immunization protocol. Splenocytes from the rat with expected immune responses were removed and fused with the mouse myeloma cell SP2/O. Stable positive hybridoma clones were obtained through screening their cell culture supernatant by indirect ELISA and subcloning by limiting dilution and then isotyped. One of those hybridoma clones, BC6, was chosen to be cultured in vitro and injected intraperitoneaUy into immunodeficient SCID nude mice to induce ascites. The rat monoclonal antibody （mAb） was then purified by protein A affinity chromatography and its neutralizing titer against EV71 was determined by microneutralization test. The mAb was further explored to detect EV71-specific antigen in multiple assays. Results Total eight stable hybridoma cell lines were obtained. Six of them belonged to IgG1 isotype and the other two belonged to IgG2a isotype. Large scale production of the rat mAb BC6 was achieved through ascites induction in SCID nude mice and its neutralizing titer against EVT1 was 1：32. The mAb was proven to be able to selectively detect E. coli expressed recombinant EV71 VP1 and native EV71 viral VP1 in WB, and sensitively measure antigen content in EV71 sample preparations by ELISA. Moreover, it also specifically recognized EVT1 virion rather than coxsakivirus A16 in infected host Vero cells by IFA. Conclusions Rat monoclonal neutralizing antibody against EV71 VP1 is successfully developed and applied in WB, ELISA and IFA. Thus, it provides a very useful alternative tool to detect EVT1 VP1 or whole virion specifically and sensitively.