Multilayer fluorescent immunoassay technique.

Abstract

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.

Automated separation for heterogeneous immunoassays

“…The automation is performed with a conveyor belt moving the slides under different dispensers and the fluorimeter, in a temperature-controlled area. In the Opus system, developed by PB Diagnostics (a venture between Polaroi'd and Behring Diagnostics), two different devices are used: one for a film technology, the other for Elisa processes [7]. In the Elisa device, antibodies are fixed in a glass-fibre support and the specimen is delivered onto it.…”

Clinical evaluation of five therapeutic drugs using dry film multilayer technology on the opus™ immunoassay system

“…However, these technologies have been limited to the use of chromogenic chemical reactions or enzymatic methods (2). The OPUS immunoassay system (PB Diagnostic Systems, Westwood, MA, 02090) utilizes a combination of multilayer film technology and competitive immunoassay methods for the analysis of therapeutic drugs (5)(6)(7).…”

“…The coating above the signal layer contains iron oxide which serves as a screen to block excitation of the label outside the signal layer. The topcoat layer, containing buffer components, surfactants and other reagents, acts as a filter that prevents protein interference with the immunoreaction (5)(6)(7).…”

“…'The fluorescent intensity is inversely proportional to the antigen concentration in the serum sample. Since only 2% of the total volume of applied serum is actually used in the signal layer, boundtfree separation is achieved by diffusion alone and fluorescent interference is minimized since only a fraction of the total amount of fluorescent substances in the sample can be excited (5)(6)(7).…”