Cardiac Troponin I (cTnI), as a cytoplasmic protein, is one of the most important regulatory proteins part of the troponin-tropomyosin complex localized on the actin (thin) myofilaments in cardiac myocytes.It is the inhibitory subunit of troponin, the thin filament regulatory complex which confers calcium-sensitivity to striated muscle actomyosin ATPase activity.Besides,Actin Alpha 1, Cardiac Muscle (ACTC1) has been identified as an interactor of cTnI, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human cTnI and recombinant human ACTC1. Briefly, biotin-linked cTnI were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to ACTC1-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 cTnI and ACTC1 was shown in Figure 1, the EC50?for this effect is 0.066ug/mL.