DETECTION AND CHARACTERIZATION OF MONOCLONAL-ANTIBODIES SPECIFIC TO IGE RECEPTORS ON HUMAN-LYMPHOCYTES BY FLOW-CYTOMETRY

  • 1 January 1985
    • journal article
    • research article
    • Vol. 55  (3) , 481-488
Abstract
BALB/c mice were immunized with human lymphoblastoid cells (RPMI 8866 cells) expressing surface receptors for IgE (Fc.epsilon.R). Spleen cells from animals displaying high titers of anti-Fc.epsilon.R antibodies were fused with hypoxanthine-guanine phosphoribosyltransferase-deficient NS1 myeloma cells. Anti-Fc.epsilon.R antibodies were identified by a flow cytometric assay based on their ability to block the binding of IgE-coated fluorescent latex particles to Fc.epsilon.R-positive cells. Fourteen monoclonal hybridoma cell lines secreting antibody of the required specificity were amplified in tissue culture and then grown in the peritoneal cavity of BALB/c mice to obtain ascitic fluids with high antibody titers. The specificity of each monoclonal antibody (Mab) to lymphocyte Fc.epsilon.R was shown by the following observations: the intact Mab molecule or, in some cases, its F(ab'')2 fragments blocked the binding of IgE to several Fc.epsilon.R(+) cell lines different from that employed for the initial immunization; the Mab bound directly to all the Fc.epsilon.R(+) cell lines tested, but not to several Fc.epsilon.R(-) cells as determined by indirect immunofluorescence; the binding of Mab to Fc.epsilon.R(+) cells was selectively blocked by IgE, but not by the other classes of Ig; and Mab had no effect on the binding of IgG to Fc.gamma.R on normal human peripheral blood mononuclear cells (PBMC).