"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

A simple and rapid method is presented for determination of the association constants and stoichiometries describing ligand macromolecule interactions. Based on flow injection analysis and electrochemical detection by amperometry, the only requirements for direct measurements are that the ligand have redox properties and that these properties change upon binding to the macromolecule. Bound ligand may then be measured in the presence of free ligand. Detection limits are of the order of 2 pmol of ligand or less, a level that should provide access to previously unmeasurable systems. For the exemplary system, chlorpromazine and human orosomucoid, K0ass was determined as 0.39 X 10(6) M-1 with 0.76 chlorpromazine binding sites of this affinity per orosomucoid molecule.

W. R. Heineman, H. B. Halsall, K. R. Wehmeyer, M. J. Doyle, D. S. Wright, J. Wang, J. Janata, W. Dabuo, P. Jinghao, O. P. Agrawal, S. P. Khatkar, J. F. van Staden, R. N. Goyal, Rajeshwari, N. C. Mathur, M. Otto, Y. Shouzhuo and N. Lihua, Anal. Proc., 1987, 24, 324 DOI: 10.1039/AP9872400324

Enzyme immunoassays based on chromatographic separation and amperometric detection of an enzyme generated product have been investigated. These assays combine the selectivity of the antigen/antibody reaction with the high sensitivity of thin layer amperometry. The feasibility of utilizing LCEC as a detection scheme was demonstrated using the Syva EMIT® kit for phenytoin. NADH production by glucose-6-phosphate dehydrogenase was monitored following a homogeneous procedure. Heterogeneous assays were developed for alkaline phosphatase labeled species which were based upon LCEC determination of phenol. Assays were designed for a common serum glycoprotein (orosomucoid) and a clinically important drug (digoxin). Detection limits approach the pg/mL level and as such may prove fruitful in the quantitation of numerous antigens of clinical interest.