Immunopurification and insertion into liposomes of native and mutant H-2Kb: quantification by solid phase radioimmunoassay.
To study the interaction between T cells and isolated H-2Kb, we developed protocols for the immunopurification of the molecule from monoclonal anti-H-2Kb immunoadsorbent columns and for its insertion in lipid vesicles. Patterns of reactivity of two anti-H-2Kb monoclonal antibodies (mAb) (20-8-4 and Y3) on H-2 recombinant and H-2Kb mutant mice indicated that mAb Y3 reacted with all six mutant forms of H-2Kb tested. Binding competition studies indicated that Y3 and 20-8-4 recognized distinct epitopes of H-2Kb. A solid phase radioimmunoassay was established using these two mAb to monitor H-2Kb activity in detergent containing cell lysates, after immunopurification, and after insertion into liposomes. About 70% of H-2Kb activity could be eluted from anti-H-2Kb-immunoadsorbents in the presence of 3 M NH4SCN and octyl glucoside (pH 7.4). A procedure of liposome formation combining gel dilution and dialysis yielded liposomes bearing H-2Kb molecules which could inhibit antibody plus complement cytolysis and could stimulate in vivo primed T cells to generate cytotoxic T lymphocytes in vitro. The present protocol can be extended to immunopurify and obtain H-2Kbm1 bearing liposomes.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D003602:Cytotoxicity, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D006183:H-2 Antigens / Q000235:genetics / Q000302:isolation & purification*; D008081:Liposomes / Q000276:immunology*; D051379:Mice; D008815:Mice, Inbred Strains; D009154:Mutation; D011863:Radioimmunoassay; D013601:T-Lymphocytes / Q000276:immunology
D000911:Antibodies, Monoclonal; D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; D008081:Liposomes