A rapid method for determining whether monoclonal antibodies react with the same or different antigens on the cell surface

Abstract

A quick and simple method for determining whether monoclonal antibodies react with the same or different antigens is described which utilises an indirect radioactive binding assay against cells. Six antibodies were selected, BK19.45, BK20.20, GenOx4.17, GenOx4.21, W6-34 and W6-46, which detected antigens present either only on leukocytes (BK19.45 and BK20.20) or on a wider range of cell types including fibroblasts, liver cells and neuroblastoma cells. The saturation binding levels for each antibody, in terms of the amount of 125I-anti-mouse immunoglobulin bound, were determined with respect to a fixed number of cells. The addition of two antibodies with different specities (BK19.45 or BK20.20 to either W6-34 or W6-46) resulted in a higher plateau value of 125I-anti-mouse immunoglobulin bound per fixed number of cells than for either antibody singly. The increase in the amount of antibody bound is equivalent to the sum of the two individual saturation binding levels. In contrast, the addition of BK20.20 to BK19.45 or W6-45 produced no detectable rise in the saturation level. From these data it is concluded that BK20.20 and BK19.45, and W6-34 and W6-46 are bound to either identical epitopes which are in very close spatial relationship. On the other hand 19.45 and W6-34, as expected, detect unrelated antigens. Observations from autoradiograph binding studies on the semi-quantitative distribution on bone marrow cells of the antigens recognised by 19.45 and 20.20 supported the conclusion that the antibodies were recognising identical antigens. A study of the antibodies to HLA (2A1) and β 2 microglobulin (M3) showed an increase in the saturation level, when both antibodies were added together, which was less than the sum of the two individual saturation binding levels. The close association of β 2 microglobulin and HLA on the cell surface may to some extent prevent independent antibody binding. These data suggest that the above approach will differentiate between monoclonal antibodies which detect antigenic determinants that are located on closely associated surface molecules.