A quantitative stability analysis of human monoclonal antibody production by heteromyeloma hybridomas, using an immunofluorescent technique

Abstract

We describe a quantitative method of analysis for assessing stability of human monoclonal antibody production by hybridomas. Clones derived from fusion between the SHM-D33 heteromyeloma line and EBV-stimulated human lymphocytes were studied for antbody presence using a fluorescent labelling technique. Frequencies of antibody-negative variants in clonal populations were measured and measurements on parallel clonal populations were subjected to Luria-Delbrück fluctuation analysis to compute rates of generations of antibody-negative cells. Independent hybridoma clones exhibited a range of stabilities and the corresponding rates varied between 5 × 10-4 and 6 × 10-2 cell-1 generation-1. Rates of generation of antibody-negative variants for the more stable heteromyeloma hybridomas compared well with those of 2 established mouse hybridoma lines tested (< 10-3 cell-1 generation-1). There was a positive correlation between frequency of antibody-negative variants measured in clonal populations grown to large numbers of cells ( >107 per culture) and their rate of loss of antibody production. Large variations in frequency of antibody-negative variants were observed in parallel clonal populations, suggesting that loss of ability to produce antibody is due to random, mutation-like events including chromosome loss (Luria and Delbrück, 1943). High frequencues of antibody-negative variants may indicate imminent oss of antibody-producing capacity by a clone growing in suspension culture. © 1985.