In vitro B-lymphocyte antigen priming against both non-immunogenic and immunogenic molecules requiring low amounts of antigen and applicable in hybridoma technology.

A method to efficiently antigen-prime B-lymphocytes with low amounts (less than 1 microgram/10(8) cells) of either immunogenic or non-immunogenic molecules is described. Keyhole limpet hemocyanin (KLH) and histone were used as prototypes for strongly immunogenic and for phylogenetically conserved non-immunogenic epitopes, respectively. Several modifications of previously reported methods were applied to the system and resulted in the requirement of antigen amounts sufficiently low to be obtainable by elution of proteins from electrophoretic gels. Antigen priming against highly purified antigen preparations is thereby feasible even when purified material cannot be obtained by conventional biochemical procedures. The amount of T- and B-lymphocytes and interleukin-2 production was estimated under various conditions during the priming procedure, and those optimal for generation of a high number of antigen-specific B-lymphocytes determined. In vitro antigen-primed B-lymphocytes were immortalized by conventional hybridoma technology. By fusion of lymphoid cells with myeloma cells at each day during the antigen-priming period, the optimal day of fusion to generate antigen-specific hybridomas was determined. Further, in 12 experiments with different antigens, 11 monoclonal antibodies to histones H3 and H4, two to the murine glucose transporter, 17 to trinitrophenyl-sheep red blood cells, and 20 to KLH were obtained. All specific hybridomas produced IgMs, as the antigen-priming period could not be extended for more than 9-10 days, whereafter a rapid decay in B-lymphocytes occurred.