Kinetic measurement of an alkaline phosphatase enzyme label was performed with amperometric detection using conversion of substrate 4-aminophenyl phosphate to the electroactive phenolic, measured at +125 to +200 mV (vs. Ag/AgCl). Calibration plots for thyroxine-binding globulin (TBG) and cortisol were obtained by excess reagent and limited reagent immunoassays, respectively, which were comparable with those obtained using colorimetric detection. These provided a measurement range of 31-1000 pg/L for TBG and 100-2000 nM for cortisol. An antibody-bound label was monitored in this system, and covering the electrode with a simple dialysis or polycarbonate membrane avoided electrode fouling. The kinetic mode of detection reduced assay time and helped to simplie the assay protocol.
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