A screening method to detect clonal secretion of DNP-specific antibody

Abstract

Spontaneous variants of the IgA immunoglobulin secreting mouse myeloma, S194-2, were isolated by cloning the line on soft agar and screening for the loss of secreted S194 immunoglobulin. Because S194 IgA possesses DNP binding activity, the screening method was designed to test for clonal secretion of antibody which specifically precipitated DNP-ferritin conjugates. Precipitates formed over IgA secreting S194 clones, whereas none were evident over nonsecreting XCl clones nor IgG secreting MOPC 21 clones (MOPC 21 IgG does not bind DNP). In addition the method was sensitive to the amount of immunoglobulin secreted. By continual selection of exceptionally reactive clones with this assay, a S194 culture was obtained which secreted five to six times as much IgA as the original mass culture. Spontaneous variants were isolated from six independent subclones of this parent line with an overall frequency estimated at 2.7 X 10(-5) per cell per generation. Biochemical analysis of these variants showed that all of them secreted reduced or undetectable amounts of IgA. No variants were obtained which secreted IgA molecules altered at the DNP binding site, or which secreted immunoglobulin subunits alone. Variants of the latter class have, however, been obtained in high frequency in other myeloma strains by other investigators.