ENZYME IMMUNOASSAY WITH ELECTROCHEMICAL DETECTION IN FLOW SYSTEMS

Abstract

The selectivity of an antigen/antibody reaction combined with the chemical am plif ication feature of an enzyme label enables high specificity and low detection levels to be achieved by enzyme immunoassays. Electrochemical enzyme immunoassay is based on antigen labeled with an enzyme that catalyzes the production of an electroactive product. Hydrodynamic electrochemical techniques such as liquid chromatography and flow injection analysis with electrochemical detection have proved to be very effective for the rapid determination of the enzyme-generated product in enzyme immunoassays. Heterogeneous enzyme imm unoassays in which antibody is adsorbed on the walls of polystyrene cuvettes have been developed. Alkaline phosphatase, which catalyzes the conversion of electroinactive phenylphosphate to phenol, is the enzyme label. The rate of phenol production is measured by either liquid chromatography/electrochemistry or flow injection analysis/electrochemistry with a thin-layer carbon paste electrode at +0.87V vs. the Ag/AgCl reference electrode. An example is an assay for digoxin, a cardiac glycoside used to treat chronic heart disease. The assay has a detection limit of 50 pg/mL and exhibits good correlation with radioimmunoassay on patient serum samples over the therapeutic range of 0.8-2.0 ng/mL. Assays have also been developed for orosomucoid and rabbit imm unoglobulin G. Homogeneous enzyme immunoassays are based on a reduction in catalytic activity of enzyme-labeled drug when bound to antibody. The enzyme label glucose-6-phosphate dehydrogenase, which catalyzes the conversion of NAD+ to NADH, has been used. The drug is quantitated by measuring the rate of NADH production by liquid chromatography/electrochemistry. Good results have been obtained for clinical samples of serum from patients on drug maintenance for both phenytoin, an antiepileptic drug, and digoxin over their therapeutic ranges. The heterogeneous enzyme immunoassay with electrochemical detection has several advantages. The detection limit, as demonstrated for digoxin and IgG, is typically in the low picogram-per-milliliter range and is a function of the antigen-antibody binding constant rather than the ability to detect phenol. Consequently, even lower limits should be attainable. Because the sample is rinsed out of the reagent tubes before adding substrate, problems with interferences from electroactive constituents in the sample and possible fouling of the electrode by the adsorption of protein films are eliminated. The main advantage of a homogeneous immunoassay is the absence of a step for separating the antibody-bound antigen from the free antigen. This usually translates into a simpler, faster assay procedure. However, homogeneous assays are usually restricted to higher detection levels than are heterogeneous assays and are more susceptible to interferences from other sample constituents that would otherwise be removed in a separation step.