Third component of rat complement. Purification from plasma and radioimmunoassay in culture media from cell lines

A double antibody radioimmunoassay for rat C3 has been developed. The assay required the preparation of C3 from plasma. A new purification procedure using ion exchange high performance liquid chromatography is described. The final product was homogeneous on SDS-PAGE analysis. Rat C3 has an apparent molecular weight of 187,000 and is composed of two polypeptide chains with molecular weights of 125,000 and 73,000, respectively. The purified C3 antigen with high hemolytic reactivity, as assessed by its specific functional activity, was used in preparing anti-C3 sera to perform a specific radioimmunoassay for quantifying C3 in the presence of heterologous sera contained in the cell culture media. All the validating criteria, such as precision, recovery and dilution studies, were investigated. The high sensitivity of the method allowed replicate determination of C3 in small aliquots of the cell culture medium.