DEVELOPMENT OF ENZYME-LINKED IMMUNOASSAYS FOR HUMAN ANGIOTENSIN-I CONVERTING-ENZYME SUITABLE FOR LARGE-SCALE STUDIES

Objective To develop and validate a simple immunological assay for hum an angiotensin converting enzyme (ACE) based on monoclonal antibodies. Methods Microtitre plates were coated with mouse monoclonal antibody (MoAb) to human ACE (9B9) and incubated with diluted samples of human plasma. In the sandwich enzyme-linked immunosorbent assay (ELISA), the plasma ACE, bound to MoAb 9B9, was revealed using polyclonal anti-ACE antibodies and alkaline phosphatase conjugated to goat anti-rabbit im munoglobulin G. In the plate precipitation assay the ACE activity quan titatively precipitated from human plasma by MoAb 9B9, was measured by enzymatic fluorimetric assay with p-benzyloxycarboxyl-glycyl-L-histid yl-L-leucine or zyloxycarboxyl-L-phenylalanyl-L-histidyl-L-leucine as substrate, directly in the wells. Results These assays are specific fo r the amino-terminal domain of ACE and recognize differences in the co nformations of native and recombinant ACE. The sensitivity of the sand wich ELISA was 200 pg/ml assay medium; it quantifies the ACE in 10 mu l human plasma or less. Intra- and inter-assay variability coefficient s were 6.2 and 13.6%, respectively. Both variants of the assay determi ned the plasma ACE concentration in the presence of ACE inhibitors or EDTA The ACE concentrations were determined by sandwich ELISA in a pop ulation of 138 middle-aged healthy Caucasian subjects. They were stron gly correlated with the ACE gene insertion/deletion (I/D) polymorphism , which accounted for 20% of the variance of plasma ACE concentration in this population and 16-24%, of the variance in plasma ACE activity as measured with three different enzymatic assays. Conclusion The ACE concentration (but not inhibition) can be determined by this ELISA whi ch is suitable for large-scale studies of plasma ACE levels.