Radiant energy – Photocells; circuits and apparatus – Optical or pre-photocell system
Patent
1990-12-19
1992-11-24
Nelms, David C.
Radiant energy
Photocells; circuits and apparatus
Optical or pre-photocell system
2504581, 356246, H01J 516
Patent
active
051665158
DESCRIPTION:
BRIEF SUMMARY
This invention relates to methods of optical analysis of evanescent signals
and to apparatus for use in such methods.
More particularly, the invention relates to methods of, and apparatus for, detecting evanescent signals produced during ligand/specific binding partner assay procedures, particularly immunoassays.
The prior art contains various disclosures of analytical devices for handling and metering small volumes of test samples, and for the use of such devices in methods of optical analysis.
For example, U.S. Pat. No. 3,939,350 describes optical measurement of fluorescent material bound to the surface of a solid transparent prism by a method involving single total internal reflection and interaction of the evanescent wave at the surface of the prism with the bound material.
EP-A-75353 makes reference to the exponentially decaying (evanescent) external radiation due to light which is propagated longitudinally in a fibre, and its interaction with coatings.
EP-A-170376 describes, inter alia, methods of optical analysis of fluorescence emerging from a waveguide which is part of a chemical test device, whilst EP-A-171148 describes in detail certain chemical test devices, namely fluorescence capillary fill devices, and methods for their manufacture.
As described in EP-A-171148, a fluorescence capillary fill device (hereinafter FCFD) typically consists of two pieces of transparent material, e.g. glass, separated by a narrow gap. One plate acts as an optical waveguide and carries an immobilized reagent appropriate to the test to be carried out in the device. For use in an immunoassay, the immobilized reagent may for example be an antibody to an antigen desired to be detected and one of the plates may carry a dissoluble reagent comprising antigen labelled with a fluorescent dye. When a sample is presented to one end of the FCFD it is drawn into the gap by capillary action and dissolves the reagent. In a competition assay, the fluorescently labelled antigen will compete with sample antigen for the limited number of antibody binding sites immobilized on the waveguide. Because the capillary gap is narrow (typically about 100 microns) the reaction will generally go to completion in a short time, possibly less than 5 minutes. In a sandwich assay, the waveguide will carry a specific binding partner for the ligand desired to be detected and one of the plates will carry a dissoluble reagent comprising a further specific binding partner labelled with a fluorescent dye. In a sandwich immunoassay for an antigen, a sample antigen will form a sandwich complex with a fluorescently labelled antibody and an antibody immobilized on the waveguide. Thus the amount of fluorescently labelled antibody which becomes indirectly bound to the waveguide by virtue of complex formation will be a function of the concentration of antigen in the sample.
The term "antigen" as used herein will be understood to include both antigenic species (for example, proteins, bacteria, bacterial fragments, cells, cell fragments and viruses) and haptens which may be rendered antigenic under suitable conditions.
When using an FCFD in an assay, evanescent wave coupling avoids the need for a separation or washing step. When the labelled reagent is optically excited by a light source of suitable wavelength, fluorescence emission occurs both from fluorophores in the liquid phase and from fluorophores bound, directly or indirectly, to the waveguide. The optical properties of the media involved are such that most of the fluorescence arising from surface-bound fluorophores emerges from the transverse edge of said waveguide at angles to the normal whose magnitude is less than the magnitude of a certain angle .alpha., whilst substantially all of the fluorescence arising from fluorophores in the liquid phase emerges from the edge of said waveguide at angles to the normal whose magnitude is greater than the magnitude of .alpha. (.alpha. being such that -1/2.pi.<.alpha.<1/2.pi.). The value of .alpha. depends on the refractive indices of the liquid solution in the
REFERENCES:
patent: 4668636 (1987-05-01), Ringrose et al.
patent: 4810658 (1989-03-01), Shanks et al.
patent: 4844869 (1989-07-01), Glass
patent: 4880752 (1989-11-01), Keck et al.
Applied Research Systems ARS Holding N.V.
Nelms David C.
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