T cell-independent B cell response to self-monosialogangliosides: Primary response monoclonal antibodies

Purified GM₁ and GM₂ gangliosides incorporated into liposomes were injected subcutaneously in BALB/c mice every 3-4 days after pretreatment of the animals with low-dose cyclophosphamide. Serum samples were collected at different intervals and tests by ELISA for the presence of anti-ganglioside antibodies. Four doses (50 μg each) were sufficient to raise a measurable primary type of response to GM₁, while nine doses were required to obtain measurable IgM antibody titers to GM₂. Three monoclonal antibodies (MAbs) were generated by fusing splenocytes with mouse myeloma cells. The specificity of MAbs was determined by ELISA and HPTLC-immunostaining using a panel of purified glycolipids. The MAb designated E1 showed a high degree of specificity because it reacted only with N-acetyl GM₂. Monoclonal antibody A3 reacted predominantly with GM₂ and GM₁, but also reacted moderately with the GM₃ ganglioside. The epitope recognized by this MAb is suggested to be the trisaccharide sequence GalNAcβ1-4(NeuAcα2-3)Gal. The third MAb (F6) reacted strongly with GM₁ but a weak reactivity was also observed with GD(1b) as well as with asialo-GM₁, indicating that the terminal tetrasaccharide Galβ1-3GalNAcβ1-4(NeuAcα2-3)Gal- structure is probably involved in antigenic recognition. Formalin-fixed and paraffin-embedded tissue sections were stained with the E1 and A3 MAbs, using the avidin- biotin complex (ABC) technique. Strong immunoreactivity for E1 appeared in the tumor cells of five primary lung carcinomas and in five malignant melanomas. No immunoreactivity was demonstrated in the parenchyma of a lung without malignancy, or in a metastasis from a colon carcinoma.