Procollagen II NTerminal Propeptide (PIINP) is a specific proteolytic fragment generated during the biosynthesis of type II collagen, the major structural collagen in hyaline cartilage, intervertebral discs, and the vitreous body of the eye. During chondrocyte-mediated collagen maturation, the N-terminal propeptide is enzymatically cleaved from procollagen II and released into the extracellular matrix and biological fluids. As a direct byproduct of type II collagen synthesis, PIINP serves as a sensitive and specific biomarker for chondrocyte anabolic activity and cartilage formation. It is widely used in research and clinical assessments to evaluate cartilage turnover, monitor disease progression in osteoarthritis and rheumatoid arthritis, and reflect the integrity of cartilage metabolism. GP6, a platelet receptor, can bind to PIINP and mediate cell-matrix adhesion and downstream signaling events. Briefly, GP6 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μL were then transferred to PIINP-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-GP6 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 human PIINP is lmmobilized at 2 μg/mL(100 μLwell), the concentration of GP6 that produces 50% optimal bindingresponse is found to be approximately 0.378 μg/mL.