Keiichi Inoue

High Mobility Group Protein 1 (HMGB1) Quantified by ELISA with a Monoclonal Antibody That Does Not Cross-React with HMGB2

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)(2)(3)(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).nnRecently, 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.nnTo 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 thymus-derived HMGB1, which was 98% homologous to human HMGB1. Each protein sample used for the analysis was prepared as follows.nnThe peptides were purified and separated by HPLC. The peptide synthesized was added to maleimidobenzoyl- N -hydroxysuccinimide ester (Pierce Chemical Co.)-labeled keyhole limpet hemocyanin (Calbiochem) or maleimidobenzoyl- …

A new Lp(a) assay that is unaffected by apo(a) size polymorphism

We developed sandwich ELISA methods in which anti-apo(a) kringle 4 type 5 through protease (K4 x 5-Pro) domain monoclonal antibody (clone: 203E2) is employed in each instance as the capture antibody and one of the three species of monoclonal antibody [Mab] (clones: 108B8, 202A9, 2B3) is used as the labeled antibody. Using serum containing apo(a) with 34 repeats of kringle 4 as the calibrator, a commercial kit using anti-Lp(a) polyclonal antibody (Pab) or anti-apo(a) Mab overestimated the Lp(a) concentration in samples containing apo(a) with more than 34 repeats of kringle 4 and underestimated the Lp(a) concentration in samples containing apo(a) with fewer than 34 repeats of kringle 4. Moreover, it was demonstrated that the ratios of commercial kit values to anti-apo(a) K4 x 5-Pro Mab-based method values increased as the size of apo(a) increased. The ratios of apo(a) K5 x Pro Mab-based method values to anti-apo(a) K4 x 5-Pro Mab-based method values, however, remained almost constant regardless of the size polymorphism. Thus, we suggest that apo(a) size heterogeneity can significantly affect Lp(a) measurement in the Lp(a) assay using anti-Lp(a) Pab. The novel Lp(a) assay method, using only anti-apo(a) K4 x 5-Pro Mab, is not subject to this phenomenon.