Development of enantioselective immunoassays for free plasma metanephrines.
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.
Manz
Bernhard
B
Labor Centrum Nordhorn, 48531 Nordhorn, Germany. Manz@labor-nordhorn.de
Kuper
Matthias
M
Booltink
Essy
E
Fischer-Brügge
Ulrich
U
D003429:Cross Reactions; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D008676:Metanephrine / Q000097:blood* / Q000737:chemistry; D011863:Radioimmunoassay / Q000379:methods*; D012680:Sensitivity and Specificity; D013237:Stereoisomerism
D008676:Metanephrine