Troponin T Type 3, Fast Skeletal (TNNT3) is a fast - twitch skeletal muscle - specific isoform of the troponin T (TnT) protein, which is part of the troponin complex that governs calcium - dependent muscle contraction. TNNT3 has three domains: the N - terminal region is responsible for binding tropomyosin (TPM3), the central region interacts with TnI and TnC, and the C - terminal region is crucial for actin binding. Through anchoring the troponin complex to actin filaments and interacting with TPM3, TNNT3 effectively regulates calcium - mediated muscle contraction.Thus a functional ELISA assay was conducted to detect the interaction of recombinant human TNNT3 and recombinant human TPM3. Briefly, TNNT3 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to TPM3-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-TNNT3 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, wells were aspirated and washed 5 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 450/630nm immediately. The binding activity of recombinant human TNNT3 and recombinant human TPM3 was shown in Figure 1, the EC50?for this effect is 0.009ug/mL.