RESUMO
Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the production of antibodies against a variety of self-antigens including nucleic acids. These antibodies are cytotoxic, catalytic (hydrolyzing DNA, RNA, and protein), and nephritogenic. Current methods for investigating catalytic activities of natural abzymes produced by individuals suffering from autoimmunity are mostly discontinuous and often employ hazardous reagents. Here we demonstrate the utility of dual-labeled, fluorogenic DNA hydrolysis probes in highly specific, sensitive, continuous, fluorescence-based measurement of DNA hydrolytic activity of anti-ssDNA abzymes purified from the serum of patients suffering from SLE. An assay for the presence and levels of antibodies exhibiting hydrolytic activity could facilitate disease diagnosis, prediction of flares, monitoring of disease state, and response to therapy. The assay may allow indirect identification of additional targets of anti-DNA antibodies and the discovery of molecules that inhibit their activity. Combined, these approaches may provide new insights into molecular mechanisms of lupus pathogenesis.
RESUMO
A novel method for isolation and purification of anti-ssDNA antibodies from human sera is developed. The process involves: antibody purification based on their affinity for single-stranded sequence of thymidines and removal of remaining components via protein G coated magnetic beads, with high affinity for only IgG subclass. The high degree of purity and molecular weights of healthy versus lupus anti-ssDNA antibodies were confirmed by SDS-PAGE and silver staining. Western blot confirmed IgG isotype. This novel technique allows for diagnostic purposes, structural and functional analysis of anti-DNA antibodies, and studies of their role in autoimmune diseases.
