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.