Comparison and Analysis of Different Methods for Purification of Autoimmune Antibody Reactive with Single Stranded DNA: A Pilot Study

Abstract

Purpose: We have compared a two-step affinity purification method, previously developed in house, which utilizes biotinylated oligo-deoxythymidine (dT) bound streptavidin (SA) M-280 magnetic beads and protein G Dynabeads®, with Melon™ Gel, another two step commercial method, for the isolation and purification of human anti-DNA autoantibodies reactive with single stranded DNA (ssDNA), in order to determine which method is more applicable for analysis of antibody subclasses and, potentially subclass functional activities.Results: Although Melon Gel allowed for faster anti-ssDNA autoantibody purification and higher recovery rate, its final product was of lower purity than that of the magnetic bead method, as confirmed with nanogram silver staining method following PhastGelTM non-reducing SDS-PAGE. The polyclonal nature of anti-ssDNA autoantibodies produced by patients with Systemic Lupus Erythematosus (SLE) has been determined and compared with normal human, and B-Chronic Lymphocytic Leucosis-(B-CLL) anti-ssDNA autoantibodies. Characterization of isolated antibodies by isoelectric focusing, western blot analysis and ELISA confirmed them to be human IgGs and detected the presence of all four IgG antibody subclasses with different participation. Additionally, Lab-on chip (Agilent 2100) method, revealed the presence of different MW patterns within lupus IgG subclasses, not detectable by SDS PAGE, which were not consistent with patterns seen in sera of control or in individuals with B-CLL. Conclusions:The results obtained suggest incomparably better purity of antibodies isolated via the magnetic bead method vs. Melon-gel. SDS-PAGE and Lab-on-chip analyses of antibodies revealed the presence of different IgG patterns within human lupus anti-ssDNA autoantibody subclasses than those observed in healthy individuals and B-CLL patients. These patterns may be associated with SLE disease pathogenesis and highlight the importance of further molecular, structural, and functional studies of lupus anti-ssDNA antibodies.