Creatine Kinase B (CK-BB)?is a brain-enriched isoform of creatine kinase that plays a critical role in maintaining cellular energy homeostasis by catalyzing the reversible transfer of phosphate between ATP and phosphocreatine. It is highly expressed in neurons and smooth muscle, and its elevated levels in cerebrospinal fluid or blood serve as a biomarker for brain injury and neurodegenerative disorders.?CK-BB interacts with Tau protein (MAPT), potentially influencing Tau phosphorylation and aggregation, which may contribute to impaired energy metabolism in tauopathies like Alzheimer’s disease.Thus a functional ELISA assay was conducted to detect the interaction of recombinant rat CK-BB and recombinant mouse MAPT. Briefly, CK-BB was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to MAPT-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-CK-BB 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 rat CK-BB and recombinant mouse MAPT was shown in Figure 1, the EC50?for this effect is 0.08 μg/mL.