A Novel Method for Real-Time, Continuous, Fluorescence-Based Analysis of Anti-DNA Abzyme Activity in Systemic Lupus
Figure 4
Discontinuous measurement of hydrolysis of oligo 18-mer by DNAse I, lupus patient, and normal donor anti-ssDNA antibody by UV spectrophotometry. *5′-6-FAM-ATATAGCGC5T5-DQ1-3′ 18-mer hydrolysis probe was incubated with DNAse I or anti-ssDNA antibody purified from lupus patient or normal healthy donor serum. Analysis by UV spectrophotometry demonstrates a change in A260 reading after 3 hours indicating hydrolysis by lupus anti-poly-(dT) ssDNA antibody. Normal healthy donor-derived anti-poly-(dT) ssDNA antibody does not display hydrolytic activity. No change was seen in replicates containing buffer only and those containing substrate only throughout the trials. Endpoint analysis via one-way ANOVA comparing hydrolytic activity revealed a significant difference between activity of antibody purified from lupus patients and normal donors (F = 5465.06, ). (a) Determination of ability of DNAse I and lupus anti-ssDNA antibody to hydrolyze probe by UV spectrophotometry. The DNAse reaction peaks after approximately 15 minutes and comes to completion in 30 minutes. Activity starts at 0.03 hour, peaks at 0.2 hours, and subsides at 0.5 hours and is much faster than DNA hydrolysis by anti-ssDNA antibody isolated from SLE patient serum. (b) Comparison of ability of purified lupus and normal anti-ssDNA antibodies to hydrolyze probe by UV spectrophotometry. Anti-ssDNA antibodies isolated from normal donors did not demonstrate hydrolytic activity; however, anti-ssDNA antibodies isolated from SLE patients show cleavage ability during the 3 hour incubation time point. These results were consistent across all samples analyzed. * for normal individuals versus SLE patients.
