title: Quantitation of C3 subcomponents on red cells coated with complement in vitro.
abstract: 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.
mesh_terms: D000818:Animals; D000941:Antigens / Q000032:analysis; D001666:Binding Sites, Antibody; D003176:Complement C3 / Q000032:analysis* / Q000276:immunology; D003179:Complement C3b / Q000032:analysis; D003180:Complement C3b Inactivator Proteins; D004910:Erythrocyte Membrane / Q000276:immunology; D004912:Erythrocytes / Q000276:immunology*; D006041:Goats / Q000276:immunology; D006801:Humans; D007106:Immune Sera; D066298:In Vitro Techniques; D011817:Rabbits / Q000276:immunology; D014357:Trypsin
chemical_list: D000941:Antigens; D003176:Complement C3; D003180:Complement C3b Inactivator Proteins; D007106:Immune Sera; D003179:Complement C3b; D014357:Trypsin