DNA antibodies

Two novel methodologies were developed for purification and functional (DNA hydrolytic) assessment of anti-DNA antibodies of IgG isotype from patients with Systemic Lupus Erythematosus (SLE). Earlier protocols for purification and analysis of antibody hydrolytic abilities were lengthy, laborious, and potentially disruptive to antibody function. Purification protocols failed to capture all four IgG subclasses and produced multiple bands outside the range of IgG on electrophoretic separation. Hydrolysis assays were discontinuous increasing the likelihood of introducing error and making them better suited to analysis of endpoint kinetics rather than reaction kinetics.
A two-step, affinity-based purification protocol was developed which utilized magnetic Dynabeads to capture serum components with binding affinity for a thymine 20mer followed by capture of the antibody components of this initial anti-T 20mer serum fraction using Protein G. A fluorescence-based method for real-time, continuous analysis of anti-DNA antibody hydrolytic activity utilizing hydrolysis probes was developed and used to characterize abzyme reaction kinetic parameters. Anti-DNA antibodies demonstrated significantly different Vmax and Km values in the hydrolysis assay (p <0.001) when compared with a DNAse I control.

This study developed a method for positive selection of anti-single stranded (ss)DNA B cells from peripheral blood mononuclear cells (PBMCs) using a twenty nucleotide oligomer of deoxythymidine (oligo(dT) 20mer), coupled to microscopie magnetic beads. Oligo(dT) 20mer specificity for plasma anti-ssDNA antibody (Ab) was established through indirect enzyme linked immunosorbent assay (ELISA) and flow cytometry. A novel method for semi-quantitative detection of ssDNA specific, B cells from PBMC purified peripheral blood that utilized flow cytometric analysis of oligo(dT) 20mer Texas Red labeled cells was developed. Qualitative purification of ssDNA specific, B cells using oligo(dT) coupled magnetic beads was determined through light microscopy and flow cytometric analysis of positive sclected cell populations. Cross-reaetivity of oligo(dT) 20mer with receptors distinct from membrane Ab, resulted in the use of oligo(dC) 20mer as a useful blocking agent. Results show anti-ssDNA Ab titer does not correlate with numbers of peripheral blood ssDNA specific, B cells.

This study evaluated two methods for the isolation and purification of anti-DNA antibodies. A two-step affinity purification with streptavidin (SA) biotinylated oligodeoxythymidine (dT) M-280 and protein G Dynabeadsª was compared to a two step method using Melon(TM) Gel and cellulose DNA. Although Melon gel allowed for faster antibody purification and a higher recovery rate it gave a product of less purity than the magnetic bead method. Further characterization of the antibodies was done by PhastGel(TM) non-reducing SDS-PAGE and isoelectric focusing in order to analyze purity and confirm the polyclonal nature of anti-DNA antibodies. Agilent 2100, with a higher resolution then SDS-PAGE, revealed possible subclasses of different MW not detected by SDS-PAGE. ELISA showed that all four IgG antibody subclasses were present, while Western blot confirmed the presence of human IgGs. Ultraviolet spectroscopy, Agilent, and fluorescence based assays were used to demonstrate DNA hydrolytic activity of purified anti-DNA antibody.