Preparation Of HMGB1 Monoclonal Antibody And Its Application In Competitive ELISA

Objective To prepare monoclonal antibody against HMGB1 and to establishcompetitive ELISA for the detection of HMGB 1.Methods HMGB1-BSA conjugate (HMGB1-BSA) and HRP-labeled HMGB1(HMGB1-HRP) were prepared by use of bis (sulfosuccinimidyl) suberate; Balb/cmice were immunized with HMGB1-BSA conjugate in the presence of completeFreund's adjuvant; HMGB1 antibody-secreting hybridoma was produced by fusion ofspleen cells from Balb/c mice and Sp2/0 cells with 50% PEG; Hybridoma cells werecloned by limited dilution; HMGB1 monoclonal antibody was purified from ascitesby DE52 ion-exchange chromatography; the competitive ELISA was establishedusing HMGB1 monoclonal antibody and HMGB1-HRP; Animal model of diabetesmellitus was produced by intravenous injection of streptozocin into SD rats.Results From 120 wells, 17 wells were found to contain HMGB1antibody-secreting hybridoma cells at 3 week after hybridization. Form thehybridoma cells in one (designated as A6-â…¢) of the 17 wells, 7 hybridoma clonesspecific for HMGB1 were obtained by limited dilution. HMGB1 monoclonalantibody was produced with one clone called HMGB1-C1 and purified by DE52ion-exchange chromatography. The competitive ELISA established with theHMGB1-BSA conjugate and the HMGB1-HRP can detect HMGB1 at concentrationsranging from 1 to 320ng/mL, its recovery ranged from 97% to 106%, and its CV (%)from 2.6 to 7.3%. The serum levels of HMGB1, measured by the competitive ELISA,from diabetic rats were much higher than that found in normal rats.Conclusion The competitive ELISA based on HMGB1 monoclonal antibodyis potentially developed into a method characterized by good sensitivity,reproducibility, precision, rapid and easy to conduct.