Detection of tumour associated antigen in eluates from protein A columns used for ex vivo immunoadsorption of plasma from melanoma patients by radioimmunoassay

Tumour associated antigen (TAA) of defined specificity and anti-TAA antibodies were isolated by elution with 0.1 M glycine-HCl buffer (pH 3.5) and 2.5 M MgCl₂ from non-viable Staphylococcus aureus used in ex vivo immunoadsorption of plasma from four melanoma patients. The anti- TAA antibody activity in the MgCl₂ eluate was determined by its ability to bind a melanoma ¹²⁵I-TAA. The melanoma ¹²⁵I-TAA was isolated and purified from the spent culture medium of a human melanoma cell line. The activity and specificity of TAA in the glycine-HCl eluates were determined by competitive inhibition in a radioimmunoassay in which melanoma ¹²⁵I-TAA and an allogeneic antiserum obtained from a melanoma patient were used as the reagents. Results indicated that 0.04-0.81% of the total protein contained in the glycine-HCl eluates was TAA. The proportion of TAA to total protein in these eluates varied from patient to patient and treatment to treatment. Inhibition by the glycine-HCl eluates in the competitive radioimmunoassay was dose-dependent. Similarly, binding of melanoma ¹²⁵I-TAA in a direct radioimmunoassay decreased with decreasing amounts of the anti-TAA antibody fraction. Quantitative analysis revealed that the MgCl₂ eluates contained anti-TAA protein at levels ranging from 0.15 to 5.78% of total protein. Because both TAA and anti-TAA activities were found in eluates from S. aureus (protein A positive) used for immunoadsorption of plasma from melanoma patients, and because melanoma ¹²⁵I-TAA isolated and purified from a human melanoma cell line did not bind to protein A directly, the results indicated that TAAs immunologically similar to the melanoma TAA were circulating in the form of immune complexes in plasma of four patients with melanoma and that these complexes could be removed from plasma by ex vivo immunoadsorption.