A systems approach to fluorescent immunoassay: general principles and representative applications.

Abstract

We have developed an automated system for the immunoassay of subnanogram quantities of clinically interesting compounds by molecular fluorescence. The system includes all the necessary reagents and an automated fluorometer. The microprocessor-based instrument consists of a measurement and data-processing module and an automated sampling unit. With use of 10 pmol/L amounts of fluorescent dyes such as fluorescein, measurements with precision and accuracy of 1--3% are attained. In a competitive-binding fluorescence immunoassay, antigen labeled with a fluorescent dye competes with antigen in the sample or standard for a limited amount of antibody immobilized on a polyacrylamide bead 2--5 micrometers in diameter. After separating antibody-bound from free tracer, we measure the amount of fluorescence bound to the beads. In representative example assays, correlation of fluorescence immunoassay (y) with a reference radioimmunoassay (x) of thyroxine was y = 1.01x + 13 nmol/L, r = 0.98. Correlation of fluorescence immunoassay (y) with a reference radioimmunoassay (x) of triiodothyronine was y = 0.99x + 0.004 nmol/L, r = 0.96.