Factors masking HMGB1 in human serum and plasma

Abstract

High mobility group box 1 protein (HMGB1) is a ubiquitously expressed architectural chromosomal protein. Recently, it has become obvious that HMGB1 can also act as a proinflammatory mediator when actively secreted during cell activation or passively released from necrotic cells. HMGB1 appears to play an important role in the pathogenesis of diseases, including sepsis and rheumatoid arthritis. However, easy, sensitive, and reliable detection systems are required to investigate the clinical significance of HMGB1 in clinical samples for diagnosis and prognosis of diseases. Here, we describe sensitive ELISAs for the detection of HMGB1 in cell culture medium and cell lysates. However, these assays failed to reliably quantitate HMGB1 in serum and plasma when compared with immunoblot analysis. We found that serum/plasma components bind to HMGB1 and interfere with its detection by ELISA systems. In most serum/plasma samples investigated, including those from healthy individuals, we detected IgG antibodies binding to HMGB1. The titers of these antibodies correlated with the capacity of sera to interfere with the detection of recombinant HMGB1 by ELISA. Furthermore, HMGB1 coimmunoprecipitated with several proteins including IgG1, as identified by mass spectrometry. These HMGB1 interacting proteins are currently characterized and may contribute to complex formation, masking, and possibly, modulation of cytokine activity of HMGB1.

Serum Levels of HMGB1 in Postmenopausal Patients with Rheumatoid Arthritis: Associations with Proinflammatory Cytokines, Acute-phase Reactants, and Clinical Disease Characteristics

“…We observed a modest correlation between HMGB1 levels obtained by ELISA and those from Western blots, in accord with results described by Urbonaviciute, et al 7 . Considering the potential human serum factors including HMGB1-specific antibodies masking HMGB1 7 , it is plausible that the total circulating HMGB1 levels detected by Western blotting are higher than those detectable by ELISA.…”

“…Amounts of HMGB1 were also determined in parallel with Western blots in 20 patients 7 . For statistical analysis nonparametric tests were employed due to unequal data distribution.…”

High-Mobility Group Box-1 Protein (HMGB1) Is Increased in Antineutrophilic Cytoplasmatic Antibody (ANCA)-Associated Vasculitis with Renal Manifestations

“…We analyzed our samples with a commonly used Western blot method, whereas Wibisono used a commercial ELISA. However, Urbonaviciute et al (36) found a discrepancy between Western blot and ELISA results, suggesting that serum/plasma components bind to HMGB1 and interfere with its detection by ELISA systems (36).…”

Immunogene Zelltodmarker HMGB1 und sRAGE als neue prädiktive und prognostische Serum Biomarker bei Tumorerkrankungen/Immunogenic cell death markers HMGB1 and sRAGE as new predictive and prognostic serum biomarkers in cancer disease

“…Wahrscheinlich sind daf ü r maskierende Faktoren wie HMGB1-Antik ö rper, Lipoplysaccharide oder IL-1-β verantwortlich, die an HMGB1 binden und so die Erkennung des Molek ü ls im ELISA verhindern [144] . Eine Vorbehandlung mit Perchlors ä ure f ü hrt zur Abl ö sung dieser Faktoren [137] .…”

“…Au ß erdem k ö nnen beim Western Blot auch Background-Proteine dargestellt werden, was R ü ckschl ü sse auf die Spezifit ä t des Testes zul ä sst [137,144,145] .…”