Procollagen I N‑Terminal Propeptide (PINP) is a specific proteolytic fragment released during the synthesis of type I collagen, the most abundant collagen in the human body, mainly distributed in bone, skin, tendons, and blood vessels. As type I procollagen is processed by proteinases into mature collagen, PINP is cleaved and released into the circulation in a 1:1 stoichiometric ratio, making it a highly sensitive and specific serum biomarker for osteoblast activity and bone formation rate. Clinically, PINP measurement is widely used to evaluate bone turnover, monitor osteoporosis treatment efficacy, assess fracture risk, and diagnose metabolic bone diseases. Unlike bone resorption markers, PINP directly reflects active bone synthesis and is less affected by diurnal variation. BMP2 promotes osteoblastic differentiation and upregulates collagen synthesis, thereby increasing the production and release of PINP during type I collagen maturation. To detect the activity of recombinant PINP , a functional ELISA assay was performed to evaluate the interaction between recombinant human PINP and recombinant human BMP2. Briefly, BMP2 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to PINP-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-BMP2 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 PINP is lmmobilized at 2 µg/mL(100 µLwell), the concentration of BMP2 that produces 50% optimal bindingresponse is found to be approximately 0.415 µg/mL.