AffinityImmuno Products

Introduction

HMGB1 is a nonhistone chromatin protein that, inside the nucleus, helps bend and organize DNA to support replication, transcription, chromatin remodeling, and repair processes such as V(D)J recombination. When cells are stressed, dying, or activated, HMGB1 can be released passively from necrotic or pyroptotic cells or actively secreted by immune cells, and outside the cell it behaves as a damage-associated molecular pattern that signals tissue injury to the immune system. Depending on its oxidation state, extracellular HMGB1 can act more like a chemokine that recruits inflammatory cells, a cytokine that triggers NF-kB-driven release of factors such as TNF and IL-6 through receptors including RAGE and Toll-like receptors, or a tolerogenic signal associated with apoptotic clearance. Because of this dual nuclear and extracellular role, HMGB1 has been linked to processes ranging from normal genome maintenance to tumor cell migration and the broader inflammatory response seen in sepsis, trauma, and other acute injury states.

As one of the best-characterized damage-associated molecular patterns, extracellular HMGB1 is widely measured in sepsis, trauma, and acute injury research as an indicator of tissue damage severity, complementing its distinct role inside the nucleus in normal chromatin organization and DNA repair.

Principle of the Assay

This kit uses a sandwich enzyme immunoassay format in which the microtiter plate wells are pre-coated with a capture antibody specific to Human HMGB1. During incubation, HMGB1 present in standards or samples is captured by this immobilized antibody, followed by addition of a biotin-conjugated detection antibody that recognizes a distinct epitope on the bound analyte. Avidin-conjugated horseradish peroxidase (HRP) is then added to each well, where it binds the biotin label. Upon addition of TMB substrate solution, a colorimetric signal develops exclusively in wells containing Human HMGB1, biotin-conjugated antibody, and Avidin-HRP conjugate. The enzymatic reaction is stopped by addition of a sulphuric acid solution, and absorbance is measured at 450 nm (± 10 nm). Sample concentrations are determined by comparing the optical density of each sample against a standard curve.