Enzyme-linked immunosorbent assay for angiotensin-converting enzyme in rat testes

Abstract

An enzyme-linked immunosorbent assay (ELISA) was developed to determine the concentration of angiotensin-converting enzyme (ACE) in the testes of rats. ACE was isolated and purified from the testes by affinity chromatography using the specific ACE inhibitor lisinopril bound to Sepharose CL-4B. Polyclonal antibodies to the purified ACE protein were produced in rabbits. A competitive antigen capture ELISA was developed by using antibody-coated polystyrene tubes as the solid phase and alkaline phosphatase covalently bound to purified ACE as the detector. This paper describes in detail the procedures for preparing the affinity gel and column, isolating and purifying ACE protein, producing and isolating the antibodies, production of the alkaline phosphatase-ACE conjugate, and the development of the ELISA. The assay was tested by determining the amount of ACE protein in rat testis as affected by zinc deficiency, a condition known to reduce ACE activity in this tissue. Preliminary results showed a direct correlation between the reduction in ACE activity and ACE protein in the Zn-deficient rats.