QUANTIFICATION OF ANTIBODIES TO C3D SUBCOMPONENT OF HUMAN C3

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  • 1 January 1977
    • journal article
    • research article
    • Vol. 32  (6) , 1007-1015
Abstract
Purified soluble C3d [d fragment of complement component 3] was 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 labeling with 125I, retained 80% immunoreactivity, and were stable on storage at -50.degree. and +4.degree.. Concentrations of anti-C3d were determined by scatchard analysis of equilibrium concentrations of bound and free C3d in a mixture of 125I-labeled 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 4 rabbit whole antisera and 4 Ig[immunoglobulin]G preparations fell in the range 288-2433 .mu.g/ml, with Ko values of 6.2 .times. 108-2.9 .times. 109 l/mol. One commercial antiglobulin-serum contained 3.6 .mu.g anti-C3d/ml and had a Ko value of 1.7 .times. 108 l/mol. Values for anti-C3d concentrations measured independently by an indirect method employing 125I-labeled sheep anti-rabbit IgG averaged 20% lower than those obtained with 125I-labeled C3d. Antibody concentrations were correlated with antiglobuliin agglutination titers against C3d-coated red cells; a titer of 1 was given by an anti-C3d concentration of 0.5 .mu.g/ml.