A High-Sensitivity Method for Detection and Measurement of HMGB1 Protein Concentration by High-Affinity Binding to DNA Hemicatenanes

Abstract

BackgroundProtein HMGB1, an abundant nuclear non-histone protein that interacts with DNA and has an architectural function in chromatin, was strikingly shown some years ago to also possess an extracellular function as an alarmin and a mediator of inflammation. This extracellular function has since been actively studied, both from a fundamental point of view and in relation to the involvement of HMGB1 in inflammatory diseases. A prerequisite for such studies is the ability to detect HMGB1 in blood or other biological fluids and to accurately measure its concentration.Methodology/Principal FindingsIn addition to classical techniques (western blot, ELISA) that make use of specific anti-HMGB1 antibodies, we present here a new, extremely sensitive technique that is based on the fact that hemicatenated DNA loops (hcDNA) bind HMGB1 with extremely high affinity, higher than the affinity of specific antibodies, similar in that respect to DNA aptamers. DNA-protein complexes formed between HMGB1 and radiolabeled hcDNA are analyzed by electrophoresis on nondenaturing polyacrylamide gels using the band-shift assay method. In addition, using a simple and fast protocol to purify HMGB1 on the basis of its solubility in perchloric acid allowed us to increase the sensitivity by suppressing any nonspecific background. The technique can reliably detect HMGB1 at a concentration of 1 pg per microliter in complex fluids such as serum, and at much lower concentrations in less complex samples. It compares favorably with ELISA in terms of sensitivity and background, and is less prone to interference from masking proteins in serum.ConclusionThe new technique, which illustrates the potential of DNA nanoobjects and aptamers to form high-affinity complexes with selected proteins, should provide a valuable tool to further investigate the extracellular functions of HMGB1 and its involvement in inflammatory pathologies.

Implications of the release of high-mobility group box 1 protein from dying cells during human immunodeficiency virus type 1 infection in vitro

“…and has been connected with necrosis (Chen et al, 2008;Chu & Ng, 2003;Gaillard et al, 2008). These findings are in line with the initial dogma that only necrosis leads to passive release of HMGB1 (Scaffidi et al, 2002).…”

Redox Modulation of HMGB1-Related Signaling

“…It has been described that results of HMGB1 ELISA in plasma/serum do not correlate with those of immunoblotting (26). The formation of complexes of HMGB1 with IL-1b, LPS or anti-HMGB1 antibodies reduces the sensitivity of the conventional ELISA and causes false-negative results.…”

The Effect of HMGB1, a Damage-Associated Molecular Pattern Molecule, on Polymorphonuclear Neutrophil Migration Depends on Its Concentration

“…Contaminating endotoxin was removed with Triton X-114 [10] and full-length HMGB1, domains A and B were tested for endotoxin contamination using the Limulus amebocyte assay. In order to rule out possible denaturation of HMGB1 during protein production, the activity of HMGB1 was assessed in terms of the capacity of HMGB1 to bind to hemicatenated DNA [11,12] and to stimulate migration of 3T3 mouse fibroblasts [13] . …”