Monoclonal antibodies against HPV11 virus-like particles: functional characteristics and application on quality assessment

目的分析和鉴定抗HPV11病毒样颗粒(virus-like particle,VLP)鼠源单克隆抗体的性质,筛选性质和生物学活性较优的抗体,并初步应用于抗原或疫苗的质量分析。方法分别利用间接ELISA法和Western blot对HPV11的22株单克隆抗体的亚类、与HPV11 VLP的结合能力和构象敏感性进行检测;采用血凝抑制实验对单克隆抗体的血凝抑制活性进行分析;运用基于假病毒的抗体中和实验对单克隆抗体的中和活性进行鉴定,选出中和活性高的单抗进行两两配对,采用双抗夹心ELISA法捕获单抗并筛选合适的配对双抗。结果对22株单抗的性质进行了详细和完整的鉴定,并根据构象敏感性进行排序,筛选出6株型别特异、结合活性强且中和活性高的单抗(2A2、4A1-3、16G7、14A6、9C12和19C7);成功建立了基于单抗的双抗夹心(14A6∶Ag∶9C12-HRP)ELISA定量分析方法。结论获得了较全面的HPV11 VLP单抗性质信息,建立了重组HPV11抗原质量分析的双抗夹心ELISA法,为HPV11抗原的生命周期管理或尖锐湿疣疫苗的研发、工艺优化、产品放行和稳定性研究等提供了技术支持。Objective To quantitatively analyze the characteristics of a panel of murine anti-human papillomavirus( HPV) 11 L1-derived virus-like particle( VLP) monoclonal antibodies( m Abs) and establish the m Ab-based methods for antigen quality analysis. Methods A panel of 22 murine anti-HPV11 m Abs were characterized in details with their isotype,and binding affinity,conformational sensitivity were examined quantitatively in the direct binding ELISA and Western blot. The hemagglutination inhibition activity of m Abs were identified using the hemagglutination inhibition assay and the pseudovirus( Ps V) neutralization efficiency were examined quantitatively using the Ps V-based neutralization assay. The type-specific, highly conformational sensitive and neutralizing m Abs were selected to be used in the sandwich ELISA assay. Results Based on the quantitative and semi-quantitative results,six type-specific,highly conformational sensitive and neutralizing m Abs( 2A2,4A1-3,16G7,14A6,9C1 and 19C7) were identified. These m Abs,along with 10D6 were screened as the capture m Ab or as the detection m Ab in the sandwich ELISA. Conclusion The binding affinity,conformational sensitivity and neutralization efficiency of anti-HPV11 m Abs were characterized in details. A m Ab-based sandwich ELISA assay( 14A6∶Ag∶9C12-HRP) were developed,which could be used in the in vitro potency analysis of HPV11 VLP-based vaccine.重大新药创制(2015ZX09101034