High Mobility Group Protein 1 (HMGB1) is among the most important chromatin proteins. In the nucleus HMGB1 interacts with nucleosomes, transcription factors, and histones. This nuclear protein organizes the DNA and regulates transcription. Besides, Toll Like Receptor 2 (TLR2) has been identified as an interactor of HMGB1, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant rat HMGB1 and recombinant human TLR2. Briefly, HMGB1 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 ul were then transferred to TLR2-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-HMGB1 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution , wells were incubated 15-25 minutes at 37℃. Finally, add 50 µL stop solution to the wells and read at 450/630 nm immediately. The binding activity of recombinant rat HMGB1 and recombinant human TLR2 was shown in Figure 1, the EC50 for this effect is 0.25 ug/mL.