Abstract
The third component of human complement (C3) was purified to homogeneity by the method of Tack and Prahl (Biochem. 15: 4513, 1976). Sheep and rabbits were made hyperimmune with C3, as well as with activation fragments C3b, C3c, and C3d, as determined by standard double-diffusion assays. Radiolabeling of C3 and each of the fragments was performed by reductive alkylation with tritiated sodium borohydride (10 Ci/mmol) and formaldehyde. This method, which effects the methylation of free amino groups, allowed labeling to specific activities of ∼ 4 × 105 cpm/µg, with minimal change in function as determined by the hemolytic activity assay for C3. Competitive binding studies were conducted by incubation of the labeled and native C3 proteins with a 1/10,000 dilution of the sheep antiserum; free protein was separated from bound with a burro anti-sheep IgG antiserum. This radioimmunoassay indicated an affinity constant of about 5 × 109 liters/mole and with 100-µl samples could be used quantitatively in the range from 2 to 80 ng of C3.
