Heterogeneous enzyme immunoasssay with electrochemical detection: Competitive and 'sandwich'-type immunoassays

Abstract

In these competitive and 'sandwich'-type heterogeneous enzyme immunoassays, based on liquid chromatography with electrochemical detection, rabbit immunoglobulin G is used as a model compound. Alkaline phosphatase (EC 3.1.3.1), the labeling enzyme, catalyzes conversion of phenyl phosphate to phenol. After separation on an octyldecylsilane column, the enzyme-generated phenol is detected in a thin-layer cell at a carbon-paste working electrode. The detection limit for phenol is 5.0 nmol/L. The electrode response varies linearly with concentrations over a range of three orders of magnitude. For the sandwich-type assay procedure the detection limit is 10 ng/L; the linearity ranges over four orders of magnitude. The detection limit of the competitive immunoassay is 5 μg/L. The dynamic range spans two orders of magnitude.