A method for the detection and screening of catalytic anti-DNA antibodies

Abstract

We have developed a microtiter plate assay for the detection and screening of anti-DNA hydrolytic antibodies. The affinity-linked oligonucleotide nuclease assay (ALONA) makes use of substrates with a digoxigenin on the 5′-end of the 3′-biotinylated DNA strands. The substrate binds specifically to the wells of streptavidin-coated microtiter plates where the reaction takes place. Uncleaved substrate retains the digoxigenin label, which is then detected with an enzyme-labeled anti-digoxigenin antibody. We first assessed the efficiency of this assay by measuring S1 nuclease and DNase I activities and the inhibitory effect of EDTA on the reaction. The ALONA procedure was then successfully applied to the screening of a high number of hybridoma clones derived from nonimmunized (NZB×NZW)F1 mice with spontaneous lupus erythematosus. We detected three potential catalytic antibodies and investigated their substrate specificity. Overall, our findings demonstrate the value of the ALONA method for high throughput screening of potential nucleases and catalytic antibodies. Although this assay was designed for the selection of catalysts active in DNA hydrolysis, it can be adapted to detect most types of substrate cleavage reaction.