Immunoradiometric assay for antibodies recognizing antigenic gangliosides

An immunoradiometric assay for monoclonal antibodies (MoAb) recognizing gangliosides as antigens (Ag) is described and exemplified by the determination of the immunoreactive fraction of 125I-radiolabeled monoclonal A2B5 and 3G5 IgM antibodies. The assay was performed in 96-well microtiter plates equipped with cellulose filter membranes, using the antigens extracted from cloned rat insulinoma cells (RINm5F). The selectivity of the assay was demonstrated by studying the interactions of A2B5 and 3G5 with specific Ag and irrelevant gangliosides. The binding of 125I-A2B5 and 125I-3G5 to the RINm5F extract containing Ag exceeded 35% of the total applied radioactivity and depended on the degree of protein damage caused by radiolabeling, whereas irrelevant glycolipids bound 0.4% (s.d. 0.3%) of total applied 125I-3G5 and 1.2% (s.d. 0.3%) of 125I-A2B5 regardless of the initial amount of the radiolabeled MoAb used. Additional support for the specificity of this method was acquired in experiments with the A1D2 IgG antibody that interacts with a glycoprotein expressed by RINm5F cells but not with the gangliosidic antigens present in the same cell line. 125I-A1D2 bound neither Ag (0.47%; s.d. 0.12%) nor brain extracts containing irrelevant gangliosides (0.32%; s.d. 0.17%). The data for the specific MoAb-Ag systems were analyzed using a modified Lineweaver-Burk graph. By plotting the reciprocal of the fraction of specifically bound MoAb versus the reciprocal of the antigen concentration, the immunoreactive fraction was determined. The present method is rapid, convenient, and independent of variability in antigen expression on the cell surface.