Purification of DNA antibodies using cibacron blue F3GA affinity chromatography.
Isolation of DNA antibodies by conventional affinity chromatography has been difficult, often requiring harsh eluting conditions and giving low yields. The triazene dye cibacron blue F3GA is a nucleotide analogue which, when bound to an agarose matrix, has been used to isolate numerous DNA-binding proteins. In this study, we have used cibacron blue chromatography to bind and purify another group of DNA-binding proteins, DNA antibodies. Greater than 90% of both ssDNA and dsDNA antibody activity from 4 SLE plasmas bound to the cibacron blue matrix. Between 30 and 65% of this antibody activity could be eluted from the column with 1.0 M NaCl, with a net 50--60-fold antibody purification from plasma. Studies with 2 monoclonal antibodies showed that a DNA antibody directed against the DNA phosphate backbone bound to cibacron blue, but a monoclonal antibody directed primarily against bases did not bind. Inhibition studies showed that DNA antibodies bound to the cibacron blue matrix at the antigen binding site, suggesting that cibacron blue does act as an antigen analogue. Although cibacron blue chromatography yields only partially purified DNA antibodies, this method should be useful in producing enriched DNA antibody preparations from ascites fluid, tissue culture supernatants, or serum.
D000906:Antibodies / Q000302:isolation & purification*; D000974:Antibodies, Antinuclear / Q000302:isolation & purification*; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D002846:Chromatography, Affinity / Q000379:methods*; D004247:DNA / Q000276:immunology*; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000276:immunology; D014227:Triazines
D000906:Antibodies; D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D014227:Triazines; C012842:Cibacron Blue F 3GA; D004247:DNA