Multilayer fluorescent immunoassay technique

We describe a new multilayer immunoassay element for the determination of haptens in undiluted serum and plasma. Polysaccharide layers are coated onto a plastic base. The signal layer contains an immobilized antibody and a fluorescent-labeled hapten. A second layer, containing a pigment, acts as an optical screen. Sample spreading is achieved by a molded grid in contact with the upper layer of the immunoassay element. After sample is added to the element, endogenous analyte competes with the labeled hapten for the binding sites of the immobilized antibody; equilibrium is reached in 4-12 min. Because of the relative liquid-holding capacities of the layers and the grid, only a small amount of the free components remains in the signal layer. The signal is measured by front-surface fluorimetry. This technology has been applied to theophylline and thyroxin assays. Within- and between-run CVs range from 3% to 6%. Comparisons with fluorescent polarization immunoassays (Abbott TDx) showed excellent correlation (theophylline: r = 0.98, slope = 1.07, intercept = 0.3; thyroxin: r = 0.97, slope = 0.91, intercept = 0.8). The new method requires only one pipetting step (sample delivery) and is potentially applicable to a wide range of analytes.