Complement-Dependent Cytotoxic Antitumor Antibody. I. Immunoglobulin Class Determined by Interaction With Protein A or Concanavalin

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Abstract
The specific interaction of concanavalin A (Con A) with IgM antibodies and Staphylococcus protein A with IgG antibodies was used to study the class of complement-dependent cytotoxic antibodies produced after the immunization of rabbits with the antigenically distinct guinea pig hepatomas designated line-1 and line-10. Con A, either in the fluid phase or bound covalently to Sepharose 4B, inhibited complement-dependent IgM cytotoxic antibody activity but had no effect on IgG activity. In contrast, protein A inhibited IgG but not IgM. On the basis of this specificity, the antisera produced in rabbits to line-1 cells consistently proved to be a mixture of IgG and IgM antibodies, whereas the cytotoxic antibody produced against line-10 cells according to the same immunization schedule was exclusively IgG with the exception of one early bleeding in which some IgM activity was detectable. In addition to the nature of the immunizIng target cell, the route of immunization may be important. A comparison of immunization schedules with line-1 cells demonstrated that, under certain conditions, cytotoxic antibody that behaved essentially like IgM could be produced. In all instances the class of antibody predicted on the basis of inhibition with protein A or Con A was confirmed by the behavior of each antiserum in the fixation of the first component of complement. 125I-labeled protein A was utilized to measure the maximum number of IgG molecules bound to a line-10 cell. This value was of the order of 106 molecules/cell. Similar estimates of the number of cell-bound IgM molecules with the use of (125I]Con A could not be made, inasmuch as unsensitized line-10 and line-1 cells bound approximately 1.1 × 106 and 8×105 molecules Con A/cell, respectively.