Purification and immunochemical quantitation of human pancreatic secretory trypsin inhibitor

Abstract

Human pancreatic secretory trypsin inhibitor has been purified from pancreatic juice obtained on connection with surgery in the pancreas. The purification was accomplished by gel filtration on Sephadex G-75 and ion-exchange chromatography on SP-Sephadex C-50 giving 49% yield. Five forms of the inhibitor were demonstrated by combined chromatographic and electrophoretic methods. On gel filtration the inhibitor was shown to have the same molecular weight as the Kunitz inhibitor, i.e. about 6500. Inhibitor purified by affinity chromatography on trypsin-Affi-Gel 10 was used for stimulating antiserum production in rabbits. The antiserum was used for immunochemical quantitation of the inhibitor by means of Mancini's single radial immunodiffusion method alone or in combination with a more sensitive double antibody technique followed by autoradiography. It was possible to measure concentrations of inhibitor as low as 1.5 mg/1 and 0.2 mg/1 using these methods.

Key Words:

• gel diffusion tests
• globulins
• immunoassay
• trypsin
• trypsin inhibitors