Developments in assay technologies: new ways to measure old

C H Self

Clinical Biochemistry, School of Clinical and Laboratory Sciences, The Medical School, University of Newcastle, Newcastle upon Tyne NE20 9HJ, UK.

Summer School 5-8 July 2005
St Aidan’s College, Durham University, Durham, UK

The provision and indeed the continued development of healthcare is critically dependent on assay systems. Through these, early detection, close monitoring and the discovery of new disease markers is possible for a wide range of conditions. Within this area there has been a remarkable expansion of our immunodiagnostic capability over the past few decades - from high throughput testing systems to point-of-need devices. Our efforts have been directed at increasing the sensitivity, specificity and robustness of such systems.

The initial aim was to replace existing cumbersome systems with the simplicity, reproducibility and speed of immunodiagnostic systems. Sensitivity was a prime concern. This led to our development of the Enzyme Amplification technology that provided a very large colorimetric signal and thus allowed rapid ultra-sensitive systems to be devised and readily measured. These were employed in many areas including virology and endocrinology. The availability of such high-intensity signal systems, as the Enzyme Amplification system, underlined an important fundamental fact with respect to immunodiagnostic systems across the board - their potential is best seen when they are employed in non-competitive-format systems. This is clearly seen when applied to the determination of large molecular weight analytes where reagent excess non-competitive-format sandwich assay systems have been increasingly employed. Unfortunately, however, a great many substances of key clinical importance are simply too small to allow the simultaneous binding of two antibodies necessitated by such classical sandwich assays. Instead, for these small molecular weight analytes, inferior competitive-format systems have had to be used. Competitive-format systems suffer from, for example, relatively low sensitivity, precision and robustness - all problems that can directly affect their potential clinical utility. To overcome these key problems, and allow the development of high performance immunoassays for small molecular analytes to match those of non-competitive systems for large molecular weight analytes, we have developed three non-competitive format systems for analytes of low molecular weight. It is expected that these will find wide clinical use, especially within endocrinology.

The first non-competitive system we have developed is the Anti-Complex System. A secondary antibody is obtained to bind the immune-complex of analyte and a primary antibody against it but not to either single component. As this antibody specifically recognises analyte binding it can therefore be used to determine directly analyte in reagent-excess systems with any of the many immunoassay labels and formats from specialised laboratory systems to point-of-need devices.

The second system we have developed is the Selective Antibody system. In this the secondary antibodies are also against a primary anti-analyte antibody but are specifically selected on their ability to bind primary antibody in the presence of its small analyte but not in its absence when a specific blocking reagent (such as an analyte-analogue or an anti-idiotypic antibody) specifically bind the analyte-binding site and prevent binding of the secondary (selective) antibody. Thus binding of the selective antibody with the primary antibody is again analyte dependent, providing a generic system for direct analyte measurement.

We have now developed the Apposition System as a way of dramatically speeding up the development of non-competitive systems. This removes the need even to raise a secondary antibody, taking advantage of existing antibodies. We synthetically position a bindable reporter moiety close to the analyte binding site of a monoclonal antibody against the analyte, such that a secondary detector antibody may bind the moiety in the presence of analyte while, in the absence of analyte, unbound receptor sites are bound by the blocker, preventing binding of the detector antibody. The system is therefore again analyte dependent. Again, it is directly applicable to all formats from high throughput systems to point-of-need systems such as dipsticks where it has been demonstrated to have particular advantages in applications such as road-side drug testing. There are many clear targets within endocrinology to which we wish to employ this technology.

All of these systems lend themselves to rapid diagnosis and especially when applied to micro-array laboratory-on-a-chip approaches. Further applications, leading to indwelling remote systems, offer a very interesting future at this rapidly developing interface between clinical and diagnostic medicine.

The opinions expressed in this paper are those of the speaker and do not necessarily reflect the views of the Society

Revised: 28-Jul-2005