Electrochemical enzyme-immunoassay

Abstract

Immunoassay defines the body of techniques which use the antibody (Ab) macromolecule, usually of the IgG class, for the detection and quantitation of an enormous range of simple and complex antigen (Ag) molecules. The success of the methods relies on both the specificity and formation constant of the Ab used, and the ability to detect the interaction between Ab and Ag. Sensitive assays in complex matrices require some kind of label to be present to provide this ability. The development of immunoassay methods has in large part been driven by the available technology. Thus, although one of the first immunoassays using a label was electrochemically based [1], the state of that art in 1951 was relatively primitive, and could not meet the analytical demands of the Ag. Some years later, Yalow and Berson [2] developed immunoassays based on the radioisotopic label (RIA). For the first time, Ab selectivity was married to very low detection limit technology, and the result was an enormous growth rate in the use of RIA in both the clinical and research laboratory. RIA has the significant advantage that the label used is not a normal constituent of physiological samples, and interferences of this type are therefore absent. RIA, however, has the disadvantages that accompany radioisotope handling, together with an inability to distinguish label which is bound from that which is not. There has therefore been an enormous effort to find suitable replacements for the radiolabel. Where the low detection limit of RIA was not required for a successful assay, labels detectable by spectroscopic methods have become important. Where very low detection limits are demanded, then the concept of amplification of, or by, the label has been developed. The most successful of these have been enzyme linked using an immunosorbent phase for Ag extraction (ELISA), although assays based on the lysis of label-containing liposomes are also exhibiting very low detection limits. In our hands, electrochemically based immunoassays at very low detection limits have also used enzyme amplification and ELISA, with either NAD/NADH and glucose-6-phosphate dehydrogenase [3], or phenyl phosphate/phenol and alkaline phosphatase [4]. The greatest sensitivity thus far has been obtained with ELISA coupled with liquid chromatography with electrochemical detection (LCED) [5]. The power of this approach can be readily illustrated by examining the evolution of its use in an assay for IgG, itself an important analyte. This evolution covers three principal stages of development and a reduction in detection limit of five orders of magnitude. The basic methodology is shown in figure 1. The assay is essentially a sandwich ELISA, using an alkaline phosphatase labelled second Ab for amplification. Oxidative flow amperometry at +875 mV