本试剂盒应用双抗体夹心酶标免疫分析法测定标本中胰蛋白酶水平。用纯化的抗体包被微孔板，制成固相抗体，往包被抗体的微孔中依次加入胰蛋白酶、生物素化的抗人胰蛋白酶抗体、HRP标记的亲和素，经过彻底洗涤后用底物TMB显色。TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的胰蛋白酶呈正相关。用酶标仪在450nm波长下测定吸光度（OD值），计算样品浓度。
Intended use
This immunoassay kit allows for the specific measurement of human trypsin concentrations in cell culture supernates, serum, and plasma.
Introduction
Trypsin (EC 3.4.21.4) is a serine protease found in the digestive system, where it breaks down proteins. It is used for numerous biotechnological processes.Trypsin is secreted into the intestine, where it acts to hydrolyse proteins into smaller peptides or amino acids. This is necessary for the uptake of protein in the food. Trypsin catalyses the hydrolysis of peptide bonds. The enzymatic mechanism is like all other serine proteases: A catalytic triad serves to make the active site serine nucleophilic. This is achieved by modifying the electrostatic environment of the serine. The enzymatic reaction that trypsins catalyze is thermodynamically favorable but requires significant activation energy (it is ‘kinetically unfavorable’). Trypsins have an optimal operating pH of about 8 and optimal operating temperature of about 37°C.
Trypsins are considered endopeptidases, i.e., the cleavage occurs within the polypeptide chain rather than at the terminal amino acids located at the ends of polypeptides.
Trypsin is produced in the pancreas in the form of inactive zymogen, trypsinogen. It is then secreted into the small intestine, where the enzyme enterokinase activates it into trypsin by proteolytic cleavage. The resulting trypsins themselves activate more trypsinogens (autocatalysis), so only a small amount of enterokinase is necessary to start the reaction. This activation mechanism is common for most serine proteases, and serves to prevent autodigestion of the pancreas.The activity of trypsins is not affected by the inhibitor tosyl phenylalanyl chloromethyl ketone TPCK, which deactivates chymotrypsin. This is important because, in some applications, like mass spectrometry, the specificity of cleavage is important.
Test principle
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal
antibody specific for trypsin has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any trypsin present is bound by the immobilized antibody. An enzyme-linked polyclonal antibody specific for trypsin is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of trypsin bound in the initial step. The color development is stopped and the intensity of the color is measured.
