Detection and characterization of monoclonal antibodies specific to IgE receptors on human lymphocytes by flow cytometry.
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 titres of anti-Fc epsilon R antibodies were fused with HGPRT-deficient NSI 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 in order to obtain ascitic fluids with high antibody titres. The specificity of each monoclonal antibody (Mab) to lymphocyte Fc epsilon R was shown by the following observations: (i) the intact monoclonal antibody 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; (ii) 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; (iii) the binding of Mab to Fc epsilon R(+) cells was selectively blocked by IgE, but not by the other classes of Ig; and (iv) Mab had no effect on the binding of IgG to Fc gamma R on normal human peripheral blood mononuclear cells (PBMC).
Rector
E
E
Nakajima
T
T
Rocha
C
C
Duncan
D
D
Lestourgeon
D
D
Mitchell
R S
RS
Fischer
J
J
Sehon
A H
AH
Delespesse
G
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity; D001667:Binding, Competitive; D002460:Cell Line; D005434:Flow Cytometry; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D007152:Immunologic Capping; D008214:Lymphocytes / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D011961:Receptors, Fc / Q000276:immunology*; D017455:Receptors, IgE; D011971:Receptors, Immunologic / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011961:Receptors, Fc; D017455:Receptors, IgE; D011971:Receptors, Immunologic; D007073:Immunoglobulin E