RESUMO
The density of carbohydrate epitopes on the surface of tumor cells is a governing factor for immune recognition and antibody-mediated targeting of tumor-associated carbohydrate antigens in cancer immunotherapy. A sensitive cell-suspension ELISA (cs-ELISA) is developed for quantitation of the functionally exposed carbohydrate epitopes on the cell surface. The factors affecting the measurement of tumor-cell surface glycoconjugates are evaluated using three human melanoma cell lines before and after exposure to various cell preservation treatments. The results of cs-ELISA are compared with the quantitative profile obtained by biochemical and flow cytometry assays. Cs-ELISA measures the density of the functionally exposed specific sugar epitopes on the surface of tumor cells, even in the presence of other similar carbohydrate antigens, provided that the monoclonal antibodies to carbohydrate epitopes are monospecific and sensitive, and that the cells are viable and present in optimal density. Of the three melanoma cell lines, M10-v and M101 expressed disialolactosyl residues of GD3 at concentrations of 5-6 pmol/10(6) cells and 2-3 pmol/10(6) cells, respectively. In both cell lines, the cell-surface GD2 was less than 1.0 pmol/10(6) cells. M24 melanoma cells expressed trace quantities (< 0.1 pmol/10(6) cells) of GD3 and GD2. Trypsinization of M10-v and M101 cells significantly reduced the cell-surface expression of GD3, suggesting GD3 loss, but increased the expression of GD2, suggesting crypticity of membrane-bound GD2. Cs-ELISA results showed that cryopreservation with 10% DMSO and irradiation at 15 krad decreased melanoma cell viability and ganglioside expression for M10-v but not M101 and M24. Formalinization did not affect cs-ELISA measurement of cell-surface carbohydrates. Cs-ELISA was used to monitor the quantity of incorporation of exogenous GD3 onto the surface of GD3-deficient M24 cells. Cs-ELISA for assessment of density of cell surface carbohydrate epitopes may be useful to characterize different types of tumors, to develop carbohydrate-based whole cell vaccines from tumor biopsies, to monitor the effects of cell preservation treatments commonly used in a whole cell vaccine preparation, and to evaluate the incorporation of a particular glycolipid (antigen or adjuvant) into glycolipid-deficient cells that are useful for carbohydrate-based active specific immunotherapy.
