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人乳头瘤病毒(human papillomavirus,HPV)58型是宫颈癌的主要诱因之一.HPV58在亚洲地区宫颈癌组织中的检出率仅次于HPV16/18.HPV58中和单克隆抗体可用于HPV病毒样颗粒(virus-like particle,VLP)疫苗的研究,并为病毒感染入侵机制的研究提供实验材料.本研究采用HPV58 L1 VLP免疫BALB/c小鼠,取其脾细胞进行杂交瘤细胞的制备,通过VLP-ELISA和假病毒中和实验筛选杂交瘤细胞株;经rProtein A纯化阳性杂交瘤细胞培养上清获得单抗;采用ELISA测定型别特异性中和单抗的亲和力,采用相加实验及变性VLP-ELISA分析单抗识别表位的性质;选取高亲和力单抗建立定量分析HPV58 L1 VLP的ELISA方法.获得了2株HPV58特异性中和单抗XM-22和XM-23,亲和常数分别为2.7×107mol-1.L和1.9×106mol-1.L,二者识别表位可能不同.同时获得2株具有交叉中和活性的单抗XM-21和XM-24,除可较高水平中和HPV58外,还可分别交叉中和亲缘关系较远的HPV18和HPV6.以XM-22建立的ELISA方法定量分析HPV58 L1 VLP的检测范围为0.05μg/mL～0.40μg/mL.本研究建立的ELISA方法可用于HPV58 L1 VLP疫苗生产的质量控制研究,获得的4株具有不同特点的中和单抗可用于HPV58感染入侵机制的研究.
Human papillomavirus (HPV) type 58 is one of the major causes of cervical cancer.HPV58 is the second most detectable HPV16 / HPV18 in cervical cancer in Asia, and HPV58 neutralizing monoclonal antibody can be used in HPV virus Like particle (VLP) vaccine and provide experimental materials for studying the mechanism of viral infection.In this study, BALB / c mice were immunized with HPV58 L1 VLP and their spleen cells were selected for hybridoma cell preparation, Hybridoma cell lines were screened by VLP-ELISA and pseudovirion neutralization; monoclonal antibodies were obtained by culturing the supernatant of rProtein A-positive hybridoma cells; affinity of type-specific neutralizing mAbs was determined by ELISA; The properties of the monoclonal antibody recognition epitopes were analyzed by denaturing VLP-ELISA, and the high-affinity monoclonal antibody was selected to establish an ELISA method for quantitative analysis of HPV58 L1 VLPs. Two HPV58-specific neutralizing monoclonal antibodies XM-22 and XM-23 were obtained. The affinity constants Respectively 2.7 × 107mol-1.L and 1.9 × 106mol-1.L, the two identified epitopes may be different at the same time obtained two cross-neutralizing activity of the monoclonal antibody XM-21 and XM-24, Horizontal and HPV58, but also can be crossed and kinship distant HPV18 and HPV6.The quantitative detection of HPV58 L1 VLP by ELISA established by XM-22 ranged from 0.05μg / mL to 0.40μg / mL.The ELISA method established in this study can be used for quality control of HPV58 L1 VLP vaccine production, Four different neutralizing McAbs with different characteristics could be used in the study of invasion mechanism of HPV58 infection.