- Monoclonal antibodies are highly valuable tools in biomedicine but the generation by hybridoma technology is very time-consuming and elaborate. In order to circumvent the consisting drawbacks an in vitro immunization approach was established by which murine as well as human monoclonal antibodies against a viral coat protein could be developed. The in vitro immunization process was performed by isolation of murine hematopoietic stem cells or human monocytes and an in vitro differentiation into immature dendritic cells. After antigen loading the cells were co-cultivated with naive T and B lymphocytes for three days in order to obtain antigen-specific B lymphocytes in culture, followed by fusion with murine myeloma cells or human/murine heteromyeloma cells. Antigen-specific hybridomas were selected and the generated antibodies were purified and characterized in this study by ELISA, western blot, gene sequencing, affinity measurements. Further the characteristics were compared to a monoclonal antibody against the same target generated byMonoclonal antibodies are highly valuable tools in biomedicine but the generation by hybridoma technology is very time-consuming and elaborate. In order to circumvent the consisting drawbacks an in vitro immunization approach was established by which murine as well as human monoclonal antibodies against a viral coat protein could be developed. The in vitro immunization process was performed by isolation of murine hematopoietic stem cells or human monocytes and an in vitro differentiation into immature dendritic cells. After antigen loading the cells were co-cultivated with naive T and B lymphocytes for three days in order to obtain antigen-specific B lymphocytes in culture, followed by fusion with murine myeloma cells or human/murine heteromyeloma cells. Antigen-specific hybridomas were selected and the generated antibodies were purified and characterized in this study by ELISA, western blot, gene sequencing, affinity measurements. Further the characteristics were compared to a monoclonal antibody against the same target generated by conventional hybridoma technology. Isotype detection revealed a murine IgM and a human IgG4 antibody in comparison to an IgG1 for the conventionally generated antibody. The antibodies derived from in vitro immunization showed indeed a lower affinity for the antigen as compared to the conventionally generated one, which is probably based on the significantly shorter B cell maturation (3 days) during the immunization process. Nevertheless, they were suitable for building up a sandwich based detection system. Therefore, the in vitro immunization approach seems to be a good and particularly fast alternative to conventional hybridoma technology.…
