Interaction of the Third Component of Human Complement (C3) with Erythrocytes and Leukocytes

A direct binding assay has been used to assess the kinetic and equilibrium parameters of the interaction between human C3 and its cell surface receptors on human blood cells. The C3 protein was radiolabeled using Na ¹²⁵I and chloramine-T by standard methods to a specific activity of approximately 1 × 10⁶ cpm/µg. The method of assay in present use is performed in the following manner: a) the labeled protein and test cells are incubated at 37°C in isotonic Tris:HCl buffer (pH 7.4) until equilibrium has been reached, b) an aliquot of the cell suspension is then layered on a column of synthetic oil (ρ = 1.033 gm/cc) contained in a microfuge tube, and (c) a rapid separation of free and bound labeled protein is obtained by pelleting the cells. The free ligand concentration is determined by counting an aliquot of the supernatant and the specific and nonspecific cell associated counts by counting the tip of the microfuge tube.