PCA-ELISA: A sensitive method to quantify free and masked forms of HMGB1

ObjectiveHMGB1 concentration is currently regarded as an important biological marker in many inflammation-related conditions. Although ELISA has been proposed as a convenient way to quantify HMGB1 in biological fluids, various molecules have been shown to complex with HMGB1 and may interfere with HMGB1 detection by this technique. We describe here a simple technical improvement that dissociates HMGB1 containing complexes and therefore increases ELISA sensitivity. This procedure was validated in sera from patients with septic shock.MethodsWe prepared in vitro complexes containing HMGB1 protein. Recombinant human HMGB1 (rhHMGB1) was incubated in the presence of molecules that are known to form complexes with HMGB1 such as LPS, IL-1β, or a rabbit antiserum directed against HMGB1. Then we tested the capacity of perchloric acid (PCA) to dissociate these complexes by quantifying rhHMGB1 by ELISA immediately or following PCA treatment.ResultsWe demonstrated for the first time that incubation of rhHMGB1 with, IL-1β, LPS or specific antibodies significantly reduce the amount of protein detected by conventional ELISA (p < 0.05). Treating the samples with PCA prior ELISA efficiently reversed this inhibition. As expected, PCA-modified ELISA detected significantly higher amounts of HMGB1 in plasma samples from 40 patients with septic shock compared to conventional ELISA (p = 0.0006).ConclusionsWe designed a performing assay that allows the detection of masked and unmasked forms of HMGB1 with a high sensitivity and practicability.