Lectin-mediated binding of liposome-inserted membrane proteins to red blood cells. A method to detect binding of antibodies to purified rat histocompatibility antigen or binding of insulin to the insulin receptor.
Partially purified membrane proteins such as the rat RT-1 histocompatibility antigen or the insulin receptor of porcine liver were inserted into liposomes. These liposomes then bound efficiently to Con A-coated red blood cells. After attachment of the liposomes to the cells, cell-liposome conjugates could be separated from free liposomes by centrifugation. Binding of FITC-labeled specific antibodies or insulin to membrane proteins inserted into the liposomes could then be analyzed with a cell flow cytofluorometer. The amount of RT-1 antigen that became associated with the cells depended on the composition and concentration of phospholipids during liposome formation. No association of RT-1 antigen to the cells was obtained in the presence of 50 mM alpha-methyl mannoside. The technique allows detection of micrograms amounts of the histocompatibility antigen associated with the red blood cells. It was possible to detect binding of insulin to cells to which approximately 9 ng (3 X 10(-14) mol) insulin receptor/10(6) cells had been attached. This amount of membrane protein was close to the detection limit of the method.
D000818:Animals; D000961:Antilymphocyte Serum / Q000032:analysis; D001666:Binding Sites, Antibody; D003208:Concanavalin A / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005434:Flow Cytometry; D006649:Histocompatibility Antigens / Q000032:analysis* / Q000276:immunology; D066298:In Vitro Techniques; D007330:Insulin Antibodies / Q000032:analysis; D008081:Liposomes / Q000378:metabolism*; D008565:Membrane Proteins / Q000097:blood*; D010713:Phosphatidylcholines / Q000378:metabolism; D051381:Rats; D011920:Rats, Inbred WF; D011972:Receptor, Insulin / Q000302:isolation & purification*
D000961:Antilymphocyte Serum; D006649:Histocompatibility Antigens; D007330:Insulin Antibodies; D008081:Liposomes; D008565:Membrane Proteins; D010713:Phosphatidylcholines; C038353:histocompatibility antigens RT, rat; D003208:Concanavalin A; D011972:Receptor, Insulin