Generation Of Monoclonal Antibody Against TcdA And TcdB By DNA Immunization And Cloning And Analysis Of Ig Gene Sequences From Hybridoma Cell Strains

Part ? Generation and characterization of Tcd A-specific monoclonal antibodies by DNA immunizationObjective: To generate monoclonal antibodies(m Abs)against Tcd A by DNA immunization and study their specificities and protective immunity against Tcd A.Eventually,we can utilize these m Abs to develop the potential diagnostic reagents and antibody-based therapeutics against Clostridium difficile toxin A,which can be useful for Clostridium difficile diagnosis and treatment.Methods: Monoclonal antibodies against Tcd A were generated by Tcd A DNA prime and protein boost immunization.Splenocytes from immunized BALB/c mice were fused with myeloma cells SP2/0.The hybridoma cells secreting Tcd A-specific m Abs were screened with capture ELISA and limiting dilution methods.The antibody specificities were further analyzed by ELISA and their protective functions were determined by Tcd A challenged with CHO cell strain in vitro and BALB/c mice in vivo.Results: We generated six hybridoma cell lines secreting Tcd A-specific high affinity monoclonal antibodies.The Ig subclasses of m Ab 1G3F10 was Ig G1,the other 5 m Abs were Ig G2 a.Three m Ab(4A4F2?2C7B6 and 5D8D5)were further demonstrated partial neutralization activity against Tcd A challenge of CHO cells and BALB/C mice.Two m Abs 1G3F10 and 5D8D5 could recognize the same epitope,m Abs 2D4D3?4A4F2?2C7B6 and 1B5F5 could recognize another epitope.Using 5D8D5 as capture antibody and HRP-2C7B6 as detection antibody,we developed a novel diagnostic kit that can quantatively detect Tcd A from Clostridium difficile clinical isolates.Conclusions: Tcd A-specific m Abs were successfully generated by DNA immunization of mice.Tcd A-specific m Abs could be partially protective against toxin challenges both in vitro and in vivo.A very promising Tcd A diagnostic kit was developed using two paired high affinity m Abs.Part ? Generation and characterization of Tcd B-specific monoclonal antibodies by DNA immunizationObjective: To generate monoclonal antibodies(m Abs)against Tcd B by DNA immunization and study their specificities and protective immunity against Tcd B.Eventually,we can utilize these m Abs to develop the potential diagnostic reagents and antibody-based therapeutics against Clostridium difficile toxin B,which can be useful for Clostridium difficile diagnosis and treatment.Methods: Monoclonal antibodies against Tcd B were generated by DNA prime and protein boost immunization of mice.Splenocytes from immunized BALB/c mice were fused with myeloma cells SP2/0.The hybridoma cells secreting Tcd B-specific m Abs were screened with capture ELISA and limiting dilution methods.The antibody specificities were further analyzed by ELISA.Results: We generated twelve hybridoma cell lines secreting Tcd B-specific high affinity monoclonal antibodies.Three m Abs were specific for Tcd B-N and nine specific for Tcd B-C.The Ig subclass of m Ab 7G1F2?4A9E3 and 1D5B3 was Ig G2 a,4B4B1 was Ig2 b,and the other 8 m Abs were Ig G1.More importantly,using 7E2G3 as capture antibody and HRP-4A9E3 as detection antibody,we developed a novel diagnostic kit that could quantatively detect the toxin B from Clostridium difficile clinical isolatesConclusions: Tcd B-specific m Abs were successfully generated by DNA immunization of mice.A very novel and promising Tcd B diagnostic kit was developed using two paired high affinity m Abs.Part III Cloning and analysis of Ig gene sequences from hybridoma cell strains against various pathogens by DNA immunizationObjective: This study was to generate monoclonal antibodies against various pathogens by DNA immunization and characterize their specificities and functions.Cloning and analysis of m Ab genes explanatorily study the mouse B cell development and evolution after DNA immunizations.Methods: Monoclonal antibodies against various pathogens were generated by DNA prime and protein or cells boosting strategy.DNA vaccines expressing influenza H5-VN HA?influenza H7-ZJU01 HA and HIV-1 Gag-C8 were separately used as immunogens for mice immunization.Splenocytes from immunized BALB/c mice were fused with myeloma cells SP2/0.The hybridoma cells secreting antibodies specific for H5-HA,H7-HA or Gag were screened by capture ELISA and limiting dilution methods.Full length genes of Ig gamma and kappa chain sequences were isolated from hybridoma cell RNA by RLM-RACE.Results: Hyridoma cell lines from thirteen representative m Abs against influenza H5-HA,H7-HA or HIV-1 Gag were produced.The Ig gene of gamma and kappa chains were cloned and analyzed by RLM-RACE technique.Somatic mutations and CDR3 length of gamma chains showed great variability when compared with kappa chain.Conclusions: Many monoclonal antibodies against influenza and HIV-1 antigens were successfully produced by DNA immunizations.The m Ab Ig gene sequences were cloned and analyzed,which can be very useful to study B cell development and maturation in DNA immunized mice.