Quantitation of C3 subcomponents on red cells coated with complement in vitro.

In order further to characterise and evaluate the reproducibility of human red cells coated with complement in vitro, the number of molecules of C3 subcomponents/red cell were determined by Scatchard analysis of equilibrium concentrations of bound and free antibody using (125)I-labelled goat anti-rabbit IgG. A 1:1 combining ratio was assumed. Red cells coated via the classical pathway had twice as much bound C3b and C3d as alternative pathway-coated cells. Assays using different anti-C3d sera gave different amounts of bound antigen, but results with any one antiserum versus one cell type were reproducible. Anti-C3d sera raised to C3d-tryp and to C3d-KAF detected significantly different amounts of bound C3d on the same cells. Both trypsinisation and serum KAF treatment of classical pathway-coated cells resulted in marked reduction of C3b molecules/cell (over 90% in both cases). Similar reduction in bound C3b was seen after trypsinisation of alternative pathway-coated cells, but serum KAF treatment of such cells had no significant effect. K(0) values were lower with anti-C3c than with anti-C3d. Anti-C3d K(0) values with the various cells coated with complement in vitro were not statistically different (approximately 10(7) litres/mol), with the exception of trypsinised alternative pathway-coated cells (approximately 10(8) litres/mol, the same order of magnitude observed with cells coated with C3d in vivo). A non-linear relationship between antiglobulin titre and antigen strength was observed. The minimal number of C3d molecules/red cell detectable by agglutination with the various anti-C3d sera ranged from 200 to 670 molecules. The minimal number of C3b molecules detectable by agglutination was approximately 9000 molecules/cell.