Two-step enrichment of HLA-DR antigens from spent medium of human malignant melanoma culture by flotation and density separation procedures.
HLA-DR antigens released by cultured human melanoma cells were harvested from spent medium. Antigenic activity was monitored by quantitative absorption analysis in the mixed hemadsorption assay using anti-HLA-DR rabbit antiserum. Following concentration by amicon filtration, and removal of insoluble components by centrifugation at 136,000 g, the spent medium was subjected to KBr (density, 1.23 g/ml) flotation. The antigenic material was enriched in the upper one-third fraction (lipoprotein-rich), by a factor of 7 with 79% recovery. Further purification of this upper fraction by sucrose (5-30%) density gradient resulted in a marked increase in antigenic activity in the bottom fraction (No. 6), by a factor of 167 with 56% recovery from the spent medium concentrate. Thus, these procedures offer a promising approach towards the isolation of HLA-DR antigens from shed material of cultured melanoma cells for further purification and structural studies.
D002460:Cell Line; D002499:Centrifugation, Density Gradient; D003470:Culture Media; D006684:HLA-DR Antigens; D000949:Histocompatibility Antigens Class II / Q000302:isolation & purification*; D006801:Humans; D008545:Melanoma / Q000276:immunology*
D003470:Culture Media; D006684:HLA-DR Antigens; D000949:Histocompatibility Antigens Class II