High Mobility Group Protein 1 (HMG1) 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, Cluster Of Differentiation 24 (CD24) has been identified as an interactor of HMG1, thus a binding ELISA assay was conducted to detect the interaction of recombinant human HMG1 and recombinant human CD24. Briefly, biotin-linked HMG1 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to CD24-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30min, then wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50μl stop solution to the wells and read at 450nm immediately. The binding activity of recombinant human HMG1 and recombinant human CD24 was shown in Figure 1, the EC50?for this effect is 0.16ug/mL.