Solubilization and Partial Isolation of Human Melanoma Tumor-Associated Antigens2

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Abstract
Human melanoma cell membrane tumor-associated antigens (TAA's) were solubilized in an active form by pronase digestion of either a fresh melanoma or cells from a melanoma cell line maintained in tissue culture. Upon elution from Sephadex G-200 column, TAA's solubilized from the melanoma cell line were found in four distinct peaks that had apparent molecular weights of approximately 48,000 (partition coefficient Kd, 0.426), 25,000 (Kd;0.567), 17,000 (Kd;0.699), and 13,000 (Kd;0.831) daltons, respectively. Fetal antigen activity was found in all but the 13,000-dalton peak. HLA antigen activity was detected in the 17,000-dalton material. TAA's prepared from the fresh tumor source eluted from Sephadex G-200 column with an apparent molecular weight of 14,000–25,000 (Kd;0.786-0.572) daltons, as did HLA antigens. A partial resolution of the TAA's from the HLA antigens was achieved with the use of DEAE-cellulose chromatography. Results of antigenic stability assays suggested that the TAA structure is stable to prolonged exposure to low pH. Recovery of TAA activity from the strong denaturing agents 5 m urea, 0.5% (wt/vol) sodium dodecyl sulfate, and 4 m guanidine hydrochloride was partially successful. These properties of the TAA's may be useful for further isolation of the TAA's.