Properties of two monoclonal antibodies directed against the Fc and Fab' regions of rat IgE.
Hybridoma antibodies directed against the Fc and Fab portions of rat IgE were produced by immunizing BALB/c mice with rat IgE and fusing the spleen cells with the nonsecreting plasmacytoma P3/X63Ag8.653. Two of the antibodies, designated as A2 and B5, were extensively characterized. Competitive binding experiments using rat IgE from the IR 162 and IR2 immunocytomas and rat IgG indicated that both A2 and B5 were epsilon-chain specific and not anti-idiotype. A2 also exhibited some cross-reactivity with mouse IgE. When IgE was treated with chymotrypsin so as to produce both F(ab')2 and Fab fragments, the enzyme-treated IgE retained reactivity with B5, but the reactivity to A2 was lost. Heat denaturation of IgE at 56 degrees C resulted in a progressive loss of reactivity of the IgE for both A2 and the Fc receptor on rat basophilic leukemia cells; the reactivity of B5 remained unchanged. A2 does not evidently interact with the same site on the Fc of IgE that is involved in binding to the rat basophilic leukemia cell Fc receptor; A2 exerted little influence on the binding of IgE to rat basophilic leukemia cells. Thus, the data indicate that the antigenic site for B5 is in the Fab region of the IgE molecule and that A2 reacts with the IgE Fc. Use of these antibodies to measure cell-bound IgE was also evaluated in dual label experiments, and potential problems in using divalent antibodies to quantitate cell surface antigens are discussed.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D003429:Cross Reactions; D000939:Epitopes; D007073:Immunoglobulin E / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D007141:Immunoglobulin Fc Fragments / Q000276:immunology*; D008407:Mast Cells / Q000276:immunology; D010323:Passive Cutaneous Anaphylaxis; D051381:Rats; D011919:Rats, Inbred Strains; D011947:Receptors, Antigen, B-Cell / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D011947:Receptors, Antigen, B-Cell; D007073:Immunoglobulin E