Differential binding of natural monoclonal antibodies to the surface of fixed or living cells

Abstract

A few hundred monoclonal antibodies derived from normal mice were tested for binding to cell surface antigens in one T-cell hybridoma and one I-Ek-transfected fibroblast cell line. The assay, which is suitable for large screenings, used glutaraldehyde-fixed cells followed by immunoenzymatic detection of immunoglobulin. Of the 331 antibodies tested, 75 showed significant binding, not only on these cells, but also on a macrophage, fibroblast, thymoma, and pre-B cell line, and on normal syngeneic and allogeneic thymocytes. If the assay was modified so as to use live cells in a simplified ELISA on living cells, only 10 of 253 antibodies were found to be positive with the T-cell hybridoma line and 7 with the transfected fibroblast cell line. In both sets of conditions, about 75% of the positive antibodies were found to be 'multireactive' after being tested on a panel of antigens. In contrast, conventional 'immune antibodies' to cell surface antigens could be tested by routine methods in either type of assay. We conclude that, while glutaraldehyde fixation does not affect the reactivity of conventional antibodies, this technique is inappropriate for testing the binding of natural antibodies to cell surface antigens.