Chymotrypsin (CTR),?is a?serine?protease?produced by the pancreas. It belongs to the serine protease family and is known for its specificity towards hydrophobic amino acids. Chymotrypsin cleaves peptide bonds at the carboxyl side of aromatic amino acids, such as phenylalanine, tyrosine, and tryptophan. This enzyme is produced in the pancreas and released into the small intestine to aid in the breakdown of proteins into smaller peptides and amino acids.Colipase is a small protein cofactor,which can bind to Chymotrypsin and may affect the activity or specificity of Chymotrypsin.Thus a functional ELISA assay was conducted to detect the interaction of recombinant bovine CTR and recombinant mouse CLPS. Briefly, CTR was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to CLPS-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-CTR 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 bovine CTR and recombinant mouse CLPS was shown in Figure 1, the EC50?for this effect is 0.005ug/mL .