Five New Epitope-defined Monoclonal Antibodies Reactive with G_M2 and Human Glioma and Medulloblastoma Cell Lines¹

In order to investigate G_M2 expression in gliomas, the G_M2-positive human glioma cell line (HGL) D-54 MG, which contains 0.6 nmol G_M2/mg protein, representing 77% of the total monosialoganglioside fraction, was used as an immunogen for the production of anti-G_M2 monoclonal antibodies. For ganglioside designations, see IUPAC-IUB (Eur. J. Biochem., 79: 11–21, 1977) and Svennerholm (J. Neurochem., 10: 613–623, 1963). Five IgM monoclonal antibodies (DMAb-1 through DMAb-5) specifically recognizing the GalNAcβ1-4(NeuAcα2-3)Gal-terminal epitope common to G_M2 and GalNAc-G_D1a are reported. The antibodies did not react with G_M1, G_M3, G_D2, G_D3, G_D1a, G_D1b, G_T1b, and G_Q1b. Purified anti-G_M2 MAbs were used to define the expression of the “G_M2” terminal epitope by cultured human malignant and normal cells by radioimmunoassay and membrane immunofluorescence. Among neuroectodermal tissue-derived cell lines, DMAb-3, at an optimal concentration of 5 µg/ml, showed high reactivity (radioimmunoassay binding ratios > 20) with 9 of 19 HGLs, 3 of 5 medulloblastoma, 4 of 5 neuroblastoma, and 1 of 3 melanoma lines. Moderate reactivity (binding ratio, 10–20) was exhibited by 3 HGL, 2 medulloblastoma, and 1 neuroblastoma lines and low reactivity (binding ratio, 3–10) by 5 HGL lines; no reactivity was detected with 2 HGL and 2 melanoma lines. Densitometric evaluation of monosi-aloganglioside extracts from human glioma and medulloblastoma cell lines in conjunction with immunostaining on thin-layer chromatograms showed that G_M2 represents the major monosialoganglioside in 8 of 10 HGL and in 3 of 4 Med lines. In these lines the amount of G_M2 ranged from <0.1 to 0.6 nmol/mg protein. These results indicate that G_M2 represents a proportionally increased ganglioside of most glioma, medulloblastoma, and neuroblastoma cells in vitro.