Quantification of antibodies to the C3d subcomponent of human C3

Abstract

Purified soluble C3d has been employed to measure the concentration of anti-C3d antibodies in immune rabbit sera. Multiple batches of C3d, prepared from C3-C3b substrate by treatment with C3b-inactivator (KAF), after labelling with 125I, retained 80% immunoreactivity, and were stable on storage at -50 degrees and +4 degrees. Concentrations of anti-C3d were determined by Scatchard analysis of equilibrium concentrations of bound and free C3d in a mixture of 125I-labelled C3d and anti-C3d. Separation of bound from free C3d was by G-75 Sephadex filtration. Assuming a 1:1 molar ratio in the antibody-C3d complex, anti-C3d antibody concentrations for four rabbit whole antisera and four IgG preparations fell in the range 288-2433 microgram/ml, with Ko values of 6-2 X 10(8)-2-9 X 10(9) litres/mol. One commercial antiglobulin-serum contained 3-6 microgram anti C3d/ml and had a Ko value of 1-7 X 10(8) litres/mol. Values for anti-C3d concentrations measured independently by an indirect method employing 125I-labelled sheep anti-rabbit IgG averaged 20% lower than those obtained with 125I-labelled C3d. Antibody concentrations were correlated with antiglobulin agglutination titres against C3d-coated red cells; a titre of 1 was given by an anti-C3d concentration of 0-5 microgram/ml.