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High mobility group box 1 (HMGB1) quantified by ELISA that dose not cross-react with HMGB2

Introduction

Recently, high mobility group box 1 (HMGB1) was identified as a late mediator of endotoxin lethality. Patients with sepsis who succumbed to infection had increased serum HMGB1. However, it revealed that HMGB1 and HMGB2, with extremely high homology (81%) to HMGB1, coexist in the serum. We report an ELISA method we have developed that measured only HMGB1 without simultaneous determination of HMGB2.

Methods

We developed anti-HMGB1 polyclonal and anti-HMGB1, 2 monoclonal antibody. We checked that developed antibodies both cross-reacted with porcine, bovine, rabbit, rat and mouse HMGB1 by western blotting because of more than 98% identity in amino acid sequence between these kinds and human. Using these antibodies, we developed a sandwich ELISA method to measure HMGB1 specifically but be insensitive to HMGB2 in human serum. Using this method, we measured HMGB1 in samples obtained from 44 patients with septic shock and examined the following: (1) the participation of HMGB1 in septic shock, (2) the correlation of cytokines and HMGB1 in septic shock, and (3) the correlation of HMGB1 level and the therapeutic effect of endotoxin adsorption therapy (PMX-DHP).

Results

With this method, the detection limit was 1 and 0.3 ng/ml, respectively, using the chromogenic substrate TMBZ and the chemoluminescent substrate PS-atto. This assay system showed 85–108% recovery by external addition recovery test. (1) The HMGB1 concentration in serum was significantly higher in the septic shock death group than the survival group. In addition, there appeared to be a positive correlation between serum HMGB1 concentration and SOFA score. (2) A negative correlation was observed between serum HMGB1 and cytokine concentration. (3) The HMGB1 concentration in serum decreased after PMX-DHP in all cases. In particular, the serum concentration decreased significantly after PMX-DHP in the survival group.

Discussion

Using our assay method, HMGB1 was detected in the blood of patients with septic shock. The results suggested that HMGB1 plays an important role in aggravation of the condition in septic multiple organ failure.

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Yakabe, K., Yamada, S., Kobayashi, M. et al. High mobility group box 1 (HMGB1) quantified by ELISA that dose not cross-react with HMGB2. Crit Care 9, P177 (2005). https://doi.org/10.1186/cc3240

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Keywords

  • Septic Shock
  • Multiple Organ Failure
  • Survival Group
  • ELISA Method
  • Sofa Score