N-Terminal Pro Brain Natriuretic Peptide (NT-ProBNP) is a cleavage fragment of proBNP predominantly secreted by ventricular cardiomyocytes upon myocardial stretch, ischemia, or volume overload. Synthesized as a 108-amino acid precursor, proBNP splits into bioactive BNP and stable NT-ProBNP during myocardial secretion. Circulating NT-ProBNP clearance relies on renal filtration without receptor-mediated degradation, rendering it a superior cardiac biomarker with longer half-life and stable plasma concentration. It sensitively reflects ventricular wall stress, widely applied for diagnosing, stratifying risk and monitoring heart failure, myocardial infarction, and pulmonary hypertension. Elevated levels correlate strongly with cardiac dysfunction, adverse cardiovascular outcomes, and all-cause mortality, serving as a core clinical indicator for cardiac load assessment. NT-ProBNP interacts with ANGPT2 (ANG2) to jointly exacerbate myocardial microvascular injury and ventricular remodeling under cardiac stress.Briefly, ANG2 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μL were then transferred to NT-ProBNP-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-ANG2 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 mouse NT-ProBNP is immobilized at 2 μg/mL(100 μL/well), the concentration of ANG2 that produces 50% optimal binding-response is found to be approximately 0.256 μg/mL.