Detection of cell surface antigens on biopsied human tumor cells using monoclonal antibody‐containing fluorescent microspheres

An immunoassay using fluorescent microspheres was developed for the detection of cell surface antigens on biopsied human cancer cells. A murine IgM monoclonal antibody 202 that was raised against a human melanoma cell line detected monosialogangliosides having sialic acid‐galactose residue, and was used as an antibody source. Purified 202 antibody was coupled with 1‐μm fluorescent micro‐spheres (Immunosphere) by carbondiimide reaction at pH 4.7–5.5, and the assay was standardized using an antigen‐positive human melanoma cell line. Over 90% of cells were positive when 4 μg antibody‐containing immunospheres were used. The specificity of the reactivity was demonstrated by a blocking assay using uncoupled 202 antibody. The binding activity was stable for 2 weeks after preparation. Biopsied specimens that had been viably frozen in a form of single‐cell suspension were thawed and purified by Ficoll density gradient centrifugation. All of the five melanoma specimens tested were positive (>5% fluorescent cells) with a mean percent of positive cells = 35%, whereas only two of the five nonmelanoma tumor specimens were positive (m = 10.8%). None of the ten noncancer tissues, including skin, liver, kidney, and lymphocyte cells, showed greater than 5% positivity. When these tissues were tested after a 3‐day incubation of the cells in a CO 2 incubator, a significant increase in the antibody reactivity was obtained in melanoma tissues (m = 57.8%). In contrast, the antigenic expression of other types of human cancer was changed only slightly (m = 16.2%) within the same period of incubation. Whether the assay is applicable to cell surface antigens of other epitopes of human biopsied tissues or only to ganglioside antigens is currently under investigation.