Development And Application Of HPV18 Monoclonal Antibody

Human papillomaviruses(HPVs)are a group of non-enveloped,double-stranded DNA viruses that mainly infect epithelial cells of their vertebrate hosts.Infections of HPVs can further induce a variety of benign or malignant hyperplasia,for instance,cervical cancer,anal cancers,vaginal cancers,mesopharynx cancers,penis cancer and warts.HPV type 18(HPV18)has been identified as one of the carcinogenic HPV types:HPV18 infection rate is the second highest among cervical cancer samples.Vaccination with HPV vaccines can induce the production of protective antibodies by the immune system,which can clear HPV viruses and thus prevent cancers caused by HPV infections.Detection kits developed using HPV18 monoclonal antibodies as the primary materials are an important tool in the development of HPV vaccines.This thesis mainly focuses on the development of HPV 18 monoclonal antibodies(mAbs)as well as an HPV 18 detection kit.(1)Obtained cell lines expression monoclonal antibodies for L1 protein of HPV18.91 cell lines expressing HPV 18-positive mAbs were obtained at the end of the monoclonal antibody production procedure.These mAbs were subsequently characterized using ELISA to test their specificity for homologous multigenotypic proteins,and cell neutralization test.24 positive clones including 4C2 and 6C11 were identified,among which 12 clones were HPV 18 type-specific within 9 genotypes,24 clones had neutralizing activity,and 12 clones were neutralizing as well as HPV 18 type-specific within 9 genotypes.(2)Successful development of an HPV 18 Detection Kit meeting relavent test standards.By means of cross-pairing,we identified a few pairs of mAbs for HPV 18 L1 protein.These pairs were assessed regarding to their specificity,neutralizing activity,ascites yield,purified antibody activity,enzyme labeled antibody activity etc.The pair "4C2 combining 6C11-HRP" was chosen to be the pair for the assembly of the HPV 18 Detection Kit.Using the checkerboard titration method,the ideal concentrations of coating and enzyme-labeled antibodies were determined as 4ug/ml and 2ug/ml respectively.Optimizations on antigen coating method,formulation of the blocking buffer,sample reaction time,enzyme-labeled secondary antibody reaction time,and substrate reaction time were performed for this sandwich ELISA kit.The final test parameters were set as:coating the plate overnight at 4C,blocking it with 2%BSA,sample and secondary antibody reaction time were both 60 minutes,and developing for 10 minutes.A standard curve was set for this Detection Kit;and methodological validations regarding to the accuracy,precision,specificity,durability,linearity,and range of this Detection Kit were carried out.All results suggested that this HPV18 Detection Kit meets relevant testing standards stated in the pharmacopoeia as well as the actual use needs of our company.