Troponin I Type 1, Slow Skeletal (TNNI1) is a slow-twitch skeletal muscle-specific isoform of troponin I, a key regulatory protein in the troponin complex. It inhibits actin-myosin interaction in the absence of calcium by binding actin and tropomyosin. Upon calcium binding to troponin C, TNNI1 undergoes conformational changes, releasing its inhibitory effect and enabling muscle contraction. TNNI1 is essential for the regulation of slow, sustained muscle contractions in type I muscle fibers.Besides,Troponin T Type 3, Fast Skeletal (TNNT3) has been identified as an interactor of TNNI1, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human TNNI1 and recombinant human TNNT3. Briefly, TNNT3 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to TNNI1-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. Measured by its binding ability in a functional ELISA. When Recombinant TNNI1 is lmmobilized at 2 ug/mL(100 uLwell), the concentration of TNNT3 that produces 50% optimal bindingresponse is found to be approximately 0.107 ug/mL.