人乳头瘤病毒（HPV）58型中和单克隆抗体的制备及初步应用

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2.7×107 mol-1·L和1.9×106 mol-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0.05 μg/mL~0.40 μg/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.

Preparation and Application of Neutralizing Monoclonal Antibodies Against Human Papillomavirus Type 58

Persistent infections with human papillomavirus(HPV) 58 have been proved to be an important etiological factor of cervical cancer.Epidemiological study in Asia shows that HPV58 is a prevalent high-risk type,second only to HPV16/18.Neutralizing monoclonal antibodies(mAbs) against HPV58 can be used in HPV58 vaccine development and mechanism elucidation of virus invasion.Female BALB/c mice were immunized with HPV58 L1 virus-like particles(VLPs).Hybridoma cells were prepared and neutralizing mAb secreting cell strains were identified by VLP-ELISA and pseudovirus neutralization assay.mAbs were purified by rProtein A chromatography from hybridoma cell culture supernatants.Affinities of type-specific neutralizing mAbs were analyzed by ELISA,and properties of mAbs were further identified by ELISA using denatured VLPs and additivity assay.Two type-specific neutralizing mAbs against HPV58,XM-22 and XM-23,were obtained,and affinity constants of them were 2.7×107 mol-1·L and 1.9×106 mol-1·L,respectively.The epitope recognized by XM-22 might be different from that recognized by XM-23.Two cross-neutralizing mAbs,XM-21 and XM-24,which could neutralize both HPV58 and heterogeneous HPVs such as HPV18/6,were also obtained.Concentrations of HPV58 L1 VLPs in samples ranged from 0.05 μg/mL to 0.40 μg/mL could be analyzed accurately by XM-22-based indirect ELISA.The established ELISA could be used in HPV58 L1 VLP vaccine production and four neutralizing mAbs with different characteristics would be helpful for mechanism study on HPV58 invasion.