Abstract
Tritiated colicin E1 plasmid DNA ([ 3H]DNA) was purified, characterized and developed as a test antigen for study of anti-DNA antibody activity in a modified Farr assay. The homogeneous molecular weight (4.2 × 10 6 daltons), physical state (intact covalently closed circles), and capacity for intrinsic radioactive labeling to a suitable specific activity were all important properties of the plasmid probe. In addition, the ability of the Farr assay to measure primary antigen/antibody interactions made quantitative determinations of anti-double-stranded DNA (dsDNA) antibody activity possible. Sonication of the plasmid resulted in definable fragments which were thermally denatured and used in the measurement of anti-single-stranded DNA (ssDNA) antibody activity. Results from studies in which unlabeled dsDNA, ssDNA, and free 2′-deoxyribonucleotides were employed to inhibit binding of [ 3H]DNA by anti-DNA antibodies indicated the presence of 3 distinct anti-DNA specificities in SLE sera.
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