Improved fusion technique. II. Stability and purity of hybrid clones.

Optimal conditions are defined for hybridoma formation between mouse spleen cells and mouse myeloma cells. The results of using different numbers of spleen cells in the fusion process is reported in 2 parts. Part I deals with the number of spleen cells in relation to hybridoma formation and antibody production. Part II treats of the purity of hybridoma clones and the loss of antibody production following fusion. Part I. Two series of experiments show that when cell fusion is performed properly the total number of antibody producing clones is greater than in non-standard conditions. The yield of hybridomas obtained with a ratio of mouse myeloma to mouse spleen cells of 1:10 did not differ from that reported by De Blas et al. (1981). The number of hybridomas formed seems to depend mainly on the number of mouse spleen cells available. The most satisfactory yield of monoclonal antibodies is obtained under conditions producing growth in approximately 100% of the wells. Part II. Two weeks after fusion a number of antibody producing clones were cultured in limiting dilution. Analysis of the hybridomas indicated that at least 40% of the antibody producing clones disappear during the first 3 weeks. Antibody producing hybridomas were as a rule not outgrown by non-antibody producing clones.