Alanine Aminotransferase (ALT), also known as glutamate-pyruvate transaminase (GPT), is a cytoplasmic enzyme primarily found in hepatocytes. It plays a critical role in amino acid metabolism by catalyzing the transfer of an amino group between alanine and α-ketoglutarate, generating pyruvate and glutamate. ALT is widely recognized as a key clinical biomarker for liver health; its serum activity increases significantly during hepatocellular damage, such as in viral hepatitis, drug-induced liver injury, or fatty liver disease. Measurement of ALT levels in blood is a standard diagnostic tool to detect and monitor liver dysfunction.Besides,Albumin (ALB) has been identified as an interactor of ALT, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human ALT and recombinant human ALB. Briefly, ALT was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to ALB-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-ALT 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 human ALT and recombinant human ALB was shown in Figure 1, the EC50 for this effect is 1.99 µg/mL.