Characterization of monoclonal antibodies produced by immunization with partially purified IgE receptor complexes.

A series of monoclonal antibodies (mAb) were produced following the immunization of mice with partially purified IgE receptors from the rat basophilic leukemia cell line (RBL-2H3). Twelve hybridoma cell lines were selected that secreted monoclonal antibodies capable of binding RBL-2H3 plasma membranes. These antibodies were all of the IgG1, or IgG2a subclass. All 12 antibodies bound to either intact or glutaraldehyde-fixed RBL-2H3 cells. Only one monoclonal (mAb 2AC3) inhibited 125I-labeled IgE binding (IC50 = 65 micrograms/ml compared to 1.0 microgram/ml for unlabeled IgE). This same mAb weakly precipitated the alpha component of the receptor from 125I-surface-labeled cells and directly triggered histamine secretion when incubated with RBL-2H3 cells. Therefore, this hybridoma most likely represents a low affinity anti-receptor antibody. Among the other 11 monoclonals, two caused direct histamine secretion from RBL-2H3 cells. These same two, as well as four others, released greater than 10% of total cellular histamine when rabbit anti-mouse antibody was added to cross-link mAb bound to the cell surface. One monoclonal (mA 1AD3) did not trigger histamine secretion but did inhibit IgE-mediated histamine release when incubated with pre-sensitized RBL-2H3 cells. Except for mAb 2AC3, none of the other monoclonal antibodies that caused or inhibited histamine secretion immunoprecipitated receptor or other protein components from either 125I-surface-labeled or intrinsically-labeled cells. However, two monoclonal antibodies (mAb 1CC4 and 1CD1) immunoprecipitated 45,000 and 55,000 proteins from 125I-surface-labeled cells. One hybridoma (mAb 2AA2) that failed to immunoprecipitate surface-labeled proteins did precipitate a 20,000 band from intrinsically-labeled cells. This band increased slightly in apparent mol. wt after reduction and, therefore, was not the previously described gamma component of the receptor. Because several mAb were capable of modulating histamine secretion, it appeared that some of the present monoclonal antibodies bound to undefined membrane components that are crucial to the secretory process of rat basophilic leukemia cells.