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Cytokine. 2011 Jul;55(1):4-7. doi: 10.1016/j.cyto.2011.03.011. Epub 2011 Apr 6.

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

Author information

1
Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, UMRS 872, Paris F-75006, France. stephanie.barnay-verdier@upmc.fr

Abstract

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-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.

RESULTS:

We 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).

CONCLUSIONS:

We designed a performing assay that allows the detection of masked and unmasked forms of HMGB1 with a high sensitivity and practicability.

PMID:
21474328
DOI:
10.1016/j.cyto.2011.03.011
[Indexed for MEDLINE]

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