High mobility group protein 1 (HMGB1) has been implicated in diverse cellular functions, including determination of nucleosomal structure and stability and binding of transcription factors to their cognate DNA sequences (1–4). HMGB1 is also present in a membrane-associated form, termed amphoterin, that mediates neurite outgrowth (5). Amphoterin can interact with macrophage cell surface receptors for advanced glycation end products to enhance expression of tissue-type plasminogen activator (6).

Recently, HMGB1 was identified as a late mediator of endotoxin lethality (7). Mice had increased serum HMGB1 concentrations after exposure to endotoxin, and sepsis patients who succumbed to infection also had increased serum HMGB1. It would therefore be useful to develop an easy and highly sensitive method to measure serum HMGB1. However, this study 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 measures only HMGB1 without simultaneous determination of HMGB2. To prepare an anti-peptide monoclonal antibody reacting only with HMGB1, we selected a peptide sequence (peptide 1; GKGDPKKPRGK) with high antigenicity and different from that of HMGB2. The monoclonal anti-calf HMGB1 antibody was prepared against calf thymusderived HMGB1, which was 98% homologous to human HMGB1. Each protein sample used for the analysis was prepared as follows.