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 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 200 pg/ml assay medium; it quantifies the ACE in 10 microliters 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 by sandwich ELISA in a population of 138 middle-aged 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) can be determined by this ELISA which is suitable for large-scale studies of plasma ACE levels.