Limiting dilution analysis of Epstein-Barr virus-induced immunoglobulin production

The major goal of this work is to establish a culture system for the growth of human B lymphocytes at the single-cell level so that the immunoglobulin secreted by the clonal progeny of that cell can be analyzed. A method which involves culturing small numbers (1-1000) of lymphocytes, which have been infected with Epstein-Barr virus (EBV) prior to plating, in round-bottom microtiter plates is described. A feeder layer of irradiated (2500 R) umbilical cord blood lymphocytes to which phytohemagglutinin has been added was found to be optimal. Culture supernatants collected from 3 to 6 weeks postinfection are assayed for the production of IgG and IgM by radioimmunoassay in order to determine the overall cloning efficiency of the system. We have shown that up to 33% of surface Ig-positive cells produce detectable clones in this system. Umbilical cord blood cells are superior to T-cell and macrophage cell lines as feeder layers. Furthermore, culture supernatants from phytohemagglutinin-stimulated umbilical cord lymphocytes do not adequately replace these cells. We also observed that while most IgM-secreting clones continued to produce immunoglobulin during the 7-week time period analyzed, the majority of IgG-secreting clones had a relatively short half-life in vitro. This culture system allows us to examine a significant proportion of the human B-cell population and carry out studies on the frequency of specific antibody- and isotype-producing clones.