Fig. 2

Screening of monoclonal antibodies that recognize desHMGB1 but do not recognize intact HMGB1. A The cleavage site of HMGB1, located between amino acid residues R10 and G11, as well as the amino acid sequence of the P-peptide beginning from G11 of HMGB1 and the N-peptide beginning from K7 of HMGB1, are shown. B, C Of the 3800 hybridoma populations, 95 populations had supernatants that were highly reactive to the desHMGB1 peptide conjugate and underwent the second screening. Microtiter plates were coated with P- or N-peptide and incubated with the supernatants of each hybridoma population (B). Alternatively, polystyrene microtiter plates were coated with the capture antibody in the HMGB1 ELISA, incubated with P- or N-peptide, and then incubated with the supernatants of each hybridoma population (C). After washing, the plates were incubated with anti-mouse IgG peroxidase-conjugated antibodies, washed again, and incubated with the chromogenic substrate. The optical density (OD) of each well was analyzed and is shown as a heat map. In this screening test, three hybridoma populations (#47, #69, and #86) were specifically reactive to P-peptide but unreactive to N-peptide. D, E These three hybridoma populations were then each distributed into 95 wells, and the same screening procedure was again conducted to obtain a single hybridoma clone. The results of the antigen (P- or N-peptide)-coated ELISA and the antibody-coated ELISA are shown in D and E, respectively. F The results of the antibody-coated ELISA with different concentrations of desHMGB1 and different types of detection antibody (47A–C, 69A–C, 86 A–C) are shown. G We selected the hybridoma clone 47C1, which produced antibodies that specifically recognized desHMGB1