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

Objective: HMGB1 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. Methods: We 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-1 beta, 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. Results: We demonstrated for the first time that incubation of rhHMGB1 with. IL-1 beta, 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). Conclusions: We designed a performing assay that allows the detection of masked and unmasked forms of HMGB1 with a high sensitivity and practicability. (C) 2011 Elsevier Ltd. All rights reserved.