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J Immunol Methods. 1985 Dec 27;85(2):335-46.

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


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 antibody 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 X 10(-4) and 6 X 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 (less than 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 (greater than 10(7) 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 frequencies of antibody-negative variants may indicate imminent loss of antibody-producing capacity by a clone growing in suspension culture.

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