Detection of IgA binding site by flow cytometry with fluorescent microspheres.
To establish a sensitive method to detect IgA Fc receptors (Fc alpha R) on human and murine lymphoid cells, fluorescent microspheres (FMS) were used in a flow cytometric assay. The following three assays with FMS were tested and compared with a conventional indirect Fc alpha R assay using FITC-labeled anti-IgA: (1) direct cell binding assay with murine myeloma IgA(MOPC315)-coated FMS(IgA-FMS assay), (2) indirect assay with TNP-BSA-coated FMS which bind to cells preincubated with MOPC315 IgA bearing anti-TNP activity (TNP-BSA-FMS assay), and (3) indirect assay with anti-IgA coated FMS after preincubation of the cells with IgA(anti-IgA-FMS assay). In these three assays for Fc alpha R using FMS, the binding of IgA to the cells was not affected by purified IgM or IgG preparations. In both indirect assays using TNP-BSA-FMS and anti-IgA-FMS, sharp and dose dependent IgA binding was obtained at lower IgA concentrations ranging from 4 to 125 micrograms/ml as compared with the conventional indirect assay. The background MFI levels in all these FMS assays remained as low as those in the conventional assay. These findings suggest that FMS coupled with TNP-BSA or anti-IgA antibodies is suitable for the detection of Fc alpha R on both murine and human T cells.
Shimada
T
T
Department of Pathology, Faculty of Medicine, Kyoto University, Japan.
Yoshida
H
H
Tokumoto
H
H
Yodoi
J
J
Sugiyama
T
T
D000818:Animals; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007070:Immunoglobulin A / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D007132:Immunoglobulin Isotypes / Q000378:metabolism; D007075:Immunoglobulin M / Q000378:metabolism; D051379:Mice; D008863:Microspheres; D011961:Receptors, Fc; D011971:Receptors, Immunologic / Q000032:analysis*; D013601:T-Lymphocytes / Q000276:immunology
C045281:IgA receptor; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007132:Immunoglobulin Isotypes; D007075:Immunoglobulin M; D011961:Receptors, Fc; D011971:Receptors, Immunologic