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.