Immunochemical Quantitation of Cell Bound C4

Abstract

Abstract A method is described for the determination of the absolute amounts of human (Hu) and guinea pig (GP) C4 bound to cell surfaces. The test is based on the inhibition of anti-C4 antibody by C4. The C4 to be measured is added to a standard amount of anti-C4 antibody and the residual activity determined by its capacity to sensitize appropriate indicator cells (EC4h or EC4gp) for lysis by GPC. By comparing the observed lysis with inhibition-of-lysis curves obtained with known C4 antigen, the amount of test antigen can be estimated. The method is simple, rapid and reproducible and can detect nanogram amounts of C4. Guinea pig hepatoma cells harvested from the peritoneal cavity of guinea pigs contained approximately 105 GPC per cell whereas tumor cells sensitized with anti-Forssman antibody or specific antitumor antibody contained approximately 2 to 3 × 106 C4 per cell. The C4 content of a Hu serum and a pool of GP sera was determined by quantitative precipitin analysis and was found to be 942 ± 260 µg and 1106 ± 106 µg/ml, respectively. Based on this Hu serum standard the average C4 content of 12 human sera was found to be 718 ± 61 µg/ml (range 577 to 942 µg/ml) by the inhibition method described in this paper.