Detergent solubilization, purification, and separation of specificities of HLA antigens from a cultured human lymphoblastoid line, RPMI 4265.
HLA antigens have been purified to homogeneity after detergent solubilization from RPMI 4265, a human lymphoblastoid line. The inhibition of cytotoxicity assay for HLA antigen was modified, using preincubation with bovine serum albumin of antigen samples containing detergent to prevent lysis of target cells by detergent. Solubilization was tested with many types of detergents. A polyethyleneglycol oleyl ether nonionic detergent mixture, Brij 99:Brij 97 (2:1) was selected for solubilization, since it selectively solubilized HLA antigens, had a low absorbance at 280 nm and was uncharded. HLA antigens were then purified by Lens culinaris lectin affinity chromatography and Bio-Gel A-5m filtration. The antigen specifity HLA-A2 was separated from specificities HLA-B7,12 by isoelectric focusing. Purified HLA antigens contained a subunit of Mr=44,000 with NH2-terminal glycine, and a subunit of Mr=12,000, beta2-microglobulin, with NH2-terminal isoleucine.
D000596:Amino Acids / Q000032:analysis; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology; D002498:Centrifugation; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003601:Cytotoxicity Tests, Immunologic; D003902:Detergents; D000939:Epitopes; D006680:HLA Antigens / Q000302:isolation & purification*; D006649:Histocompatibility Antigens / Q000302:isolation & purification*; D006801:Humans; D007525:Isoelectric Focusing; D008214:Lymphocytes / Q000276:immunology* / Q000648:ultrastructure; D010710:Phosphates / Q000032:analysis; D011506:Proteins / Q000032:analysis; D012995:Solubility
D000596:Amino Acids; D003902:Detergents; D000939:Epitopes; D006680:HLA Antigens; D006649:Histocompatibility Antigens; D010710:Phosphates; D011506:Proteins