Analysis of serum blocking factors against leukocyte‐dependent antibody in melanoma patients

Abstract

AbstractPrevious studies have shown that leukocyte‐dependent antibody activity against melanoma cells in the sera of melanoma patients is often completely blocked by factors in their sera. In the present study, characterization of these factors blocking melanoma LDA was carried out by a number of sequential separation procedures involving gel filtration on Sepharose 6B, affinity chromatography on concanavalin A (Con A) and electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulphate (PAGE‐SDS). The blocking factors were identified throughout the separation procedure by their blocking activity against melanoma LDA. It was found that blocking activity against melanoma LDA was located in small (<60,000) and large (approx. 300,000) molecular weight fractions obtained by gel filtration. Both of the latter fractions were found to have affinity for concanavalin A and were therefore glucose‐ and/or mannose‐containing glycoproteins. The blocking activity in both fractions was further resolved into a single fraction of 15,000 d by PAGE‐SDS. This small molecular weight fraction appeared to be melanoma antigen as shown by its removal by affinity chromatography on melanoma antibody bound to sepharose and by its blocking activity against melanoma LDA. A similar fraction was isolated by this separation procedure from supernatants and membranes of cultured melanoma cells. These results appear to provide a basis for the development of in vitro assays to monitor tumour growth in melanoma patients and for further characterization of the biological nature of melanoma antigens.