Multispot, multianalyte, immunoassay

Abstract

Consideration of the basic principles of immunoassay design reveals that highly sensitive assays can, in principle, be developed using amounts of "sensor" antibody far smaller than are currently conventional in this field. Furthermore, when using such amounts, the fractional occupancy of antibody binding sites by analyte is independent of both sample volume and antibody concentration. Labelling of both the sensor-antibody and a developing antibody (designed to recognize either occupied or unoccupied sensor-antibody binding sites) permits the development of "ratiometric" immunoassays relying on measurement of the ratio of signals emitted by the two labelled antibodies. Furthermore, the sensor-antibody can be located within a "microspot" a few microns 2 in area. By labelling both sensor and developing antibodies with fluorescent labels, and scanning the microspot using a highly focussed laser beam, microspot immunoassays at least comparable in sensitivity with conventional "macroscopic" immunoassays are made possible. This in turn permits the development of immunoassay "arrays" capable in principle of measuring very large numbers of different substances within small samples (such as a drop of blood). The general principles and theory underlying these concepts are discussed, and preliminary experimental data using currently available instrumentation reported.