Dependence of T-cell-replacing factor and immunogenic dose for the production of monoclonal antibodies using the in vitro immunization technique.

Antigen-specific hybridomas can be produced using spleen cells that have been immunized in culture as parental cells in the hybridization step. The in vitro immunization of non-immune mouse spleen cells was supported by a lymphokine preparation that contained T-cell-replacing factor (TRF). The influence of TRF, produced by a mixed-thymocyte reaction, on immunization in culture has been investigated using bacterial cells as the immunogen. The cell vols of stimulated splenocytes were monitored and it was found that the induction of antigen-specific blast cells, which could subsequently be immortalized by fusing them with myeloma cells, was completely abolished in immunizations which were not supported by TRF. If serum-free conditions were used during the in vitro immunization step, the frequency of antigen-specific blast cells increased, which resulted in a higher yield of specific hybridomas. This was due to the reduced background stimulation achieved by omitting serum proteins. The relationship between immunogenic dose and response, measured as the specific efficiency obtained in hybridization experiments with in vitro immunized cells, was recorded using different amounts of sperm whale myoglobin as antigen. An antigen-specific response was recorded with as low as 1 ng antigen/ml.