A New Non-Immunoglobulin Producing Human Myeloma Cell Line for Production of Hybridomas

Abstract

We selected an 8-azaguanine resistant mutant of a human myeloma cell line (RPMI 826) by cloning the parental cells on a feeder layer of mouse spleen cells in the presence of increasing concentrations of 8-azaguanine. Culture media and cell-free extracts of both the parental and mutant (8226 AR/NIP4–1) cell lines were assayed for immunoglobulin heavy and light chain by Ouchterlony double immunodiffusion and for lambda chain by radioimmunoassay. Secretion of free lambda chain by the parental cell line was confirmed. In contrast, no immunoglobulin heavy or light chains were detected in culture medium of the mutant cell line by either immunodiffusion or radioimmunoassay. No intracellular lambda chain could be detected in the mutant cells by radioimmunoassay (< 100ng/4×107 cells) of cell free extracts or by immunofluorescence of fixed cells. Hybridomas were produced by fusion of 8226AR/NIP 4–1 cells with lymphocytes from a mesenteric lymph node recovered at surgery from a hyper-transfused (n=8) renal transplant recipient. Twenty hybrid culture media were assayed for immunoglobulin by double immunodiffusion and 15 contained either IgG (lamda) or IgG (kappa). None produced IgM or IgA. One hybridoma producing IgG (kappa) was shown by immunofluorescence not to reexpress lambda chain of the parental myeloma.