Stability of DNA/ANTI-DNA complexes. IV. Complement fixation

Abstract

We describe an in vitro assay to study complement fixation of antibody/dsDNA immune complexes formed from SLE sera and radiolabeled dsDNA. The method measures the amount of radiolabeled dsDNA which is part of an immune complex bound to red blood cells via the C3b complement component receptor. The assay is dependent upon active complement, red blood cells, and anti-dsDNA antibodies, but it is independent of the red blood cell donor (type O) and the age of the red blood cells (up to 10 days). The method has been compared in some detail with the Farr assay with respect to the antibody/dsDNA ratio in the immune complexes and the relative stability of the complexes as measured by their resistance to dissociation by excess unlabeled dsDNA. Our results indicate that multiple binding of antibodies to dsDNA is required for complement fixation, and that a significant percentage of those antibodies which fix complement are of high avidity. Finally, a double label assay using both [3H]- and [14C]-dsDNA indicates that the complement fixing potential of the anti-dsDNA antibodies in an SLE serum is strongly influenced by the order of mixing of the isotopes with the serum. The DNA isotope which is added to the serum first is considerably more effective at fixing complement than the isotope which is added 1 h later. The implications of these results with respect to the pathogenesis of SLE are discussed.