Enhancement of limiting dilution in cloning mouse myeloma-spleen hybridomas by human low density lipoproteins

The limiting dilution technique is a critical step in the cloning of hybridomas for the preparation of monoclonal antibodies. We have found that culture medium supplemented with human plasma low density lipoproteins (LDL) markedly enhanced the yield of hybridoma clones derived from P3 X 63 Ag or FO mouse myeloma cell lines upon limiting dilution. Such enhancement was dependent on the concentration of LDL employed, being optimal at 1-10 micrograms/ml. At LDL concentrations greater than 20 micrograms/ml, the increase in yield of hybrid clones was not significant. The mechanism by which LDL enhances the yield of hybrid clones was partially elucidated by the demonstration that LDL could increase the DNA synthesis of hybridomas as assessed by [3H]thymidine incorporation. The data suggest that LDL play a role in the proliferation of hybridomas. It also indicates that LDL can be utilized for limiting dilution to increase the yield of desired clones. Since LDL is one of the most abundant lipoprotein fractions (approximately 500 micrograms/ml) in human plasma and the isolation procedure is simple, hybridoma culture medium supplemented with human LDL will prove to be a valuable reagent for investigators currently employing monoclonal antibody technology.