Generation of murine monoclonal antibodies in serum-free medium.

Abstract

Traditional hybridoma fusion technology requires complete medium with serum supplements to support the growth of hybridoma cells. Serum is also required for subcloning of hybridoma cells to support low density cell growth. IL-6 has been shown to enhance the growth of hybridomas and stimulate antibody production by B cells. We found that the serum requirement in media used for generation of hybridomas can be totally eliminated by substituting with 300 units/ml of IL-6. Stable hybridoma cell lines were generated to peptide and protein antigens using serum-free adapted P3.653 myelomas as the fusion partner and medium containing IL-6. Our results indicate that, in general, the fusion efficiencies of serum-free IL-6 supplemented fusions are lower than the fusions employing serum containing media (40%-60% vs. 80%-100%). However, in spite of the lower fusion efficiency, the number of antigen-specific clones generated using IL-6 was equal to or greater than fusions using serum supplements. The use of IL-6 instead of serum in the generation of monoclonal antibodies (MAbs) has several advantages. We are able to eliminate the costly need for serum in media by using IL-6 that is prepared in house. In addition, we eliminate the need for time-consuming serum-free adaptation of hybridoma cell lines prior to transfer to hollow fiber bioreactors.