Multi-analyte immunoassay.

Abstract

Immunoassays rely on the molecular recognition properties possessed by antibodies to measure substances defined by a particular structure. They can therefore be defined as "structurally specific", as distinct from "functionally specific" assays, e.g. bioassays, which compare the biological effects of substances which are functionally similar, but which may differ in molecular structure. Within the broad class of "immunoassays", two subclasses may be distinguished, differing in their design. These may be described as "competitive" and "non-competitive", respectively, reflecting their dependence on the use of optimal concentrations of antibody which are either very small or very large. It is demonstrable that "non-competitive" assays are those relying on measurement of occupied antibody binding sites following reaction with analyte; conversely "competitive" assays rely on measurement of unoccupied sites. In certain assay designs, it may be shown that fractional antibody binding site occupancy is independent of (a) antibody concentration, and (b) sample volume. Such assays may be termed "ambient analyte immunoassays". This concept has been exploited in the development of free hormone and drug assays, and currently underlines the development of salivary "dip-stick" assays in the Author's Department. The concept is also being exploited in the development of "multi-analyte" immunoassay systems, enabling the simultaneous measurement of tens or even hundreds of substances simultaneously in the same small sample. These systems depend on measurement of fractional antibody occupancy using two different labels: one labeling the "sensing" antibody, the second labeling a "developing antibody", selected to react either with occupied or unoccupied sites on the "sensing" antibody. The ratio of signals emitted by the two labeled antibodies reveals the analyte concentration to which the sensing antibody has been exposed. An array of sensing antibodies, each labeled with the same fluorescent label, is scanned (by a laser), and the fluorescent signal ratio emitted from each discrete antibody couplet in the array measured. Multi-analyte immunoassay systems of this kind are likely to totally transform medical diagnosis in the foreseeable future and are also likely to be of value in the analysis of complex protein mixtures deriving from recombinant DNA technologies.