Establishment And Preliminary Application Of High-risk Human Papillomavirus(HPV)52 Immunochemical Quality Analysis Method

Human papillomavirus(HPV)infection is the main cause of cervical cancer,the second largest gynaecological malignancy in the world.HPV52 is a high-risk HPV with a high prevalence in Asia.HPV52 L1 can self-assemble into a T=7 icosahedral virus-like particle(VLP)in vitro in the absence of viral genes and L2.VLP is highly similar to natural virus and can stimulate the body to produce neutralizing antibodies,but VLP is not contagious.VLP is the best candidate antigen for HPV vaccine.The HPV vaccine is one of the most effective methods to prevent HPV infection.There are currently three VLP-based HPV vaccines that have played an important role in preventing the disease caused by HPV infection.Safety and effectiveness are essential prerequisites for the circulation and use of vaccines in the market,and each treatment in the production of vaccine preparations may have an impact on the final preparation.Therefore,a corresponding quality analysis needs to be established.The method monitors the production process of the vaccine and further monitors the efficacy of the vaccine formulation.The immunochemical quality analysis method can not only classify the virus,identify the conformation of VLP,but also evaluate the immunogenicity of the vaccine preparation.Screening out the good monoclonal antibody(mAb)is essential for constructing an effective immunochemical quality analysis method.In this study,25 HPV52 monoclonal cell lines were obtained by hybridoma technique and clonal screening,and their mAbs were obtained by ascites production and purification.Combined with 17 mAbs in the antibody bank,a total of 42 HPV52 mAbs were obtained.The subtypes,binding activity,binding tendency to the capsomeres,conformational sensitivity and the neutralizing activity of HPV52 mAbs were detected and analyzed,11 outstanding mAbs were screened out.At the same time,we found that chemical treatments(dithiothreitol,hydrogen peroxide,formaldehyde)may affect the structure,thermal stability and sensitivity with wnzyme treated of HPV52 VLP.The binding activity between 42 HPV52 mAbs and antigens with chemical treatments were detected by enzyme-linked immunosorbent assay(ELISA).For the comaparetive analysis of the binding activity,42 mAbs can be divided into three categories respectively.Besides that,the neutralizing activity,binding activity,binding tendency of the capsomeres and serum blocking ability of the mAbs are comprehensively considered,and 5 excellent mAbs sensitive to chemical treatments are screened out.Some mAbs was selected from the above-mentioned to construct three HPV52 vaccine quality anlysis methods:label-free surface plasmon resonance(SPR)method,antigen solution competition method and based-double epitope analysis of sandwi ch ELISA method.The conditions of each experimental method were optimized,and the sensitivity and reproducibility of each experimental method were further investigated.Finally,the SPR detection method based on 7 monoclonal antibodies was successfully constructed,and the antigen solution competition method based on 6 monoclonal antibodies and sandwich ELISA were successfully constructed.In addition,the study also used the sandwich ELISA constructed here to analysis the effect of thermal acceleration on the HPV52 vaccine stock solution,we found that it did not affect the antigenicity of HPV52 vaccine stock solution when it was placed at 37 ? or 45 ? for 14 days.This study identified the physicochemical properties of the panel of the HPV52 mAbs in detail,and further analyzed the difference in antigen binding activity between mAb and HPV52 VLP treated with chemical reagents(DTT,H2O2,HCHO).At the same time,the HPV52 VLP mass analysis method was established by using the suprior mAbs,and the established double-antibody sandwich ELISA method was initially used in the thermal stability analysis of HPV52 VLP stock solution.This study used the superior mAbs sensitive to chemical treatments(dithiothreitol,hydrogen peroxide,f'ormaldehyde)to establish an immunochemical quality analysis method for HPV52 VLP vaccine,which provides an effective tool for the subsequent monitoring of chemical treatments in the vaccine production process.At the same time,it also provides research ideas for the subsequent development of monoclonal antibodies and the construction of quality analysis methods for other types of HPV.In addition,the established HPV52 quality analysis method provides management support for early research,process improvement,product launch and release of HPV52 vaccine.