RT Journal Article SR Electronic T1 Quantitation of C3 subcomponents on red cells coated with complement in vitro JF Journal of Clinical Pathology JO J Clin Pathol FD BMJ Publishing Group Ltd and Association of Clinical Pathologists SP 977 OP 983 DO 10.1136/jcp.33.10.977 VO 33 IS 10 A1 J Freedman A1 Anne Massey YR 1980 UL http://jcp.bmj.com/content/33/10/977.abstract AB 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 125I-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. K0 values were lower with anti-C3c than with anti-C3d. Anti-C3d K0 values with the various cells coated with complement in vitro were not statistically different (approximately 107 litres/mol), with the exception of trypsinised alternative pathway-coated cells (approximately 108 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.