PCA-ELISA: a sensitive method to quantify free and masked forms of HMGB1.
Cytokine
; 55(1): 4-7, 2011 Jul.
Article
em En
| MEDLINE
| ID: mdl-21474328
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-1ß, 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-1ß, 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Ensaio de Imunoadsorção Enzimática
/
Percloratos
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Proteína HMGB1
Tipo de estudo:
Diagnostic_studies
Limite:
Female
/
Humans
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Male
/
Middle aged
Idioma:
En
Ano de publicação:
2011
Tipo de documento:
Article