Lack of binding of C3 to IgG antibodies during the activation of the classical complement pathway on the red cell

We have studied the interaction between C3 and natural human and rabbit anti-MTX IgM and hyperimmune IgG antibody bound to red cells to which MTX was covalently coupled. IgM Ab molecules bound to the cell surface were measured by their interaction with rabbit anti-human mu-chain IgG Ab: the bound anti-mu-chain or anti-MTX IgG was quantitated with 125I-labelled PA. C3 uptake by EMTX AC142 complexes from purified preparation of C3 was detected by the interaction of bound C3 with rabbit anti-C3 IgG Ab; the bound IgG was then measured with radiolabelled PA. In some experiments the uptake of C3 by the EMTX AC142 complexes was measured by using 125I-labelled C3. To find out if C3 was bound to Ab molecules, anti-MTX Abs were eluted from the cells by excess fluid-phase MTX. After dialysis the eluted anti-MTX IgM or IgG Ab was than reattached to fresh EMTX. All cells were then analyzed for Ab and C3 content by a radioimmunoassay. It was found that when anti-MTX IgM or IgG was eluted from EMTX AC423 complexes no C3 was removed from the cells and that eluted IgM or IgG did not carry with them either C3 antigen or 125I-labelled C3. It was concluded that, during the activation of the classical C-pathway at the red cell surface, no C3 was bound to IgM (rabbit or human) or IgG (rabbit) antibody molecules.