Development of Enantioselective Immunoassays for Free Plasma Metanephrines

A bstract : The development of an enantioselective radioimmunoassay (RIA) and enzyme immunoassay (EIA) for L‐normetanephrine (NM) and L‐metanephrine (M) were studied. Prior to the immunoassay, the protein matrix of the ethylenediaminetetraacetic acid (EDTA) plasma samples was removed by acid precipitation, followed by derivatization of the L‐metanephrines to N‐acyl‐L‐metanephrines. For the EIA, N‐acyl‐L‐NM and N‐acyl‐L‐M were bound to the surface of microtiter plates. Acylated L‐metanephrines from the sample and solid‐phase‐bound N‐acyl‐L‐NM or N‐acyl‐L‐M competed for a fixed number of rabbit anti‐N‐acyl‐NM or anti‐N‐acyl‐M antibody binding sites. When the system was in equilibrium, free antigens and free antigen‐antibody complexes were removed by washing. The antibodies bound to the respective solid‐phase N‐acyl‐L‐NM or N‐acyl‐L‐M were detected by a goat anti‐rabbit IgG‐peroxidase conjugate using tetramethyl benzidine (TMB) as a substrate. The RIAs were conventional double antibody tests using the above rabbit antisera and specific 125 I‐N‐acyl‐L‐metanephrine tracers. Chiral recognition of the L‐enantiomers was observed not only for the native molecules but for all N‐acyl derivatives tested. The cross‐reactivity to the corresponding D‐enantiomers was always <1%. The detection limits were found to be approximately 0.04 nmol/L (7.5 pg/mL) for M and 0.08 nmol/L (15 pg/mL) for NM in RIA and EIA.