Development of enzyme-linked immunoassays for human angiotensin I converting enzyme suitable for large-scale studies

Abstract

Objective To develop and validate a simple immunological assay for human 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 immunoglobulin G. In the plate precipitation assay the ACE activity, quantitatively precipitated from human plasma by MoAb 9B9, was measured by enzymatic fluorimetric assay with p-benzyloxycarboxyl-glycyl-L-histidyl-L-leucine or p-benzyloxycarboxyl-L-phenylalanyl-L-histidyl-L-leucine as substrate, directly in the wells. Results These assays are specific for the amino-terminal domain of ACE and recognize differences in the conformations of native and recombinant ACE The sensitivity of the sandwich ELISA was 200pg/ml assay medium; it quantifies the ACE in 10 µl human plasma or less. Intra- and inter-assay variability coefficients were 6.2 and 13.6%, respectively. Both variants of the assay determined the plasma ACE concentration in the presence of ACE inhibitors or EDTA. The ACE concentrations were determined determined by sandwich ELISA in a population of 138 middleaged healthy Caucasian subjects. They were strongly 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) be determined by this ELISA which is suitable for largescale studies of plasma ACE levels.