Photobleaching recovery studies of T-independent antigen mobility on antibody-bearing liposomes.
A physicochemical model for the antigen-specific B cell membrane was prepared by incorporating palmitate-conjugated MOPC 315, an anti-DNP IgA, into 8-micron diameter liposomes prepared from phosphatidylcholine, cholesterol, and cardiolipin (2:2:1). With this model system, we examined how the purely passive cross-linking of membrane-bound immunoglobulin molecules by antigen affects the lateral mobility of antigen-receptor complexes. At 37 degrees C, liposome-bound tetramethylrhodamine isothiocyanate (TRITC)-labeled anti-DNP antibody diffuses at 1.4 X 10(-8) cm2 sec-1, a rate comparable to that observed for the phospholipid analog dil-C18-(3). Both substances exhibit complete fluorescence recovery after bleaching. The binding of TRITC-conjugates of the antigens DNP-polymerized flagellin (DNP-POL) and DNP-dextran (DNP-DEX) to liposomes bearing nonfluorescent palmitoyl-MOPC 315 was then examined. The diffusion coefficients D observed for bound antigens decrease monotonically with increased antigen dose and epitope density. For low epitope density antigens, the DNP-DEX and DNP-POL complexes are almost completely mobile. At higher epitope densities, a fraction of bound antigen appears immobile on the time scale of the experiment. This fraction is dependent on antigen concentration and epitope density and on the amount of palmitoyl MOPC 315 incorporated. The immobile fraction is 31.5% for DNP4.0-POL at 30 micrograms/ml on liposomes bearing 150,000 immunoglobulin molecules. Under these conditions D for mobile antigen is 5.0 X 10(-10) cm2 sec-1. The observed immobile fractions may represent formation of a two-dimensional gel phase of antigen-immunoglobulin aggregates. The results obtained in this study are compared with those obtained in previous work on antigen-specific mouse B lymphocytes.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000955:Antigens, T-Independent / Q000032:analysis*; D003911:Dextrans / Q000276:immunology; D004136:Dinitrobenzenes / Q000276:immunology; D000939:Epitopes / Q000276:immunology; D005408:Flagellin / Q000276:immunology; D005455:Fluorescent Antibody Technique; D007070:Immunoglobulin A / Q000378:metabolism; D008081:Liposomes / Q000276:immunology*; D051379:Mice; D008956:Models, Chemical; D011947:Receptors, Antigen, B-Cell
D000911:Antibodies, Monoclonal; D000955:Antigens, T-Independent; D003911:Dextrans; D004136:Dinitrobenzenes; D000939:Epitopes; D007070:Immunoglobulin A; D008081:Liposomes; D011947:Receptors, Antigen, B-Cell; D005408:Flagellin