Determination of the Immunoglobulin Class of Complement‐dependent Cytotoxic Antibodies in Serum of D23 Hepatoma‐bearing Rats

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Abstract
The immunoglobulin [Ig] class of the complement-dependent cytotoxic antibodies in serum from D23 hepatoma-bearing rats (D23 TBS) for D23 hepatoma cells was analyzed. When studied by affinity chromatography with concanavalin A, protamine, or staphylococcal protein A conjugated to Sepharose, the cytotoxic activity bound to the former 2 but not to protein A. The binding fractions were further characterized by column chromatography on Sepharose CL-4B. The cytotoxic activity was recovered exclusively in the high MW fractions corresponding to human IgM. Monitoring with IgG- or IgM-specific rabbit antibodies indicated that these high MW cytotoxic fractions contained IgG and IgM. Fractionation of D23 TBS at low pH suggested that cytotoxicity was due to IgM antibodies rather than to immune-complexed IgG antibodies. This was supported by the findings that rabbit anti-rat IgM antibodies inhibited the cytotoxicity of TBS completely when added at high dilutions.