Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1997-05-22
2001-09-25
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C310S311000, C310S31300R
Reexamination Certificate
active
06294391
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method of detecting the presence of an analyte of interest, and an assay device for performing the method.
BACKGROUND OF THE INVENTION
Numerous assays have been described which make use of the specific binding properties of certain molecules to detect the presence of an analyte of interest in a sample. Typically such assays involve the specific binding between immunoglobulins (such as antibodies or functional binding fragments thereof) and haptens or antigens to which the immunoglobulins bind. Examples of such assays include enzyme-linked immunosorbent assays (ELISAs) and radio-immunoassay (RIA).
Conventionally, in order to detect binding between the analyte of interest and a binding partner having specific binding affinity therefor, it is necessary for the binding partner to be labelled. Known labels include enzymes, radio-labels, fluorescent or chemiluminescent labels, electroactive labels (such as redox labels) and coloured particles (e.g. latex beads).
A refinement of assays of the general nature outlined above relates to “displacement” assays. In such assays, the presence of an analyte of interest in a sample causes the displacement either of a labelled binding partner or a labelled ligand from a pre-existing binding partner/ligand complex. Generally speaking the amount of displaced labelled substance will be proportional to the concentration of the analyte of interest in the sample. Alternatively, one may employ “competition” assays, in which there is competition between the analyte of interest and a labelled competitor (such as labelled analyte or analogue) for binding to available binding sites.
Several assay methods relying on competition and/or displacement are described in the prior art. For example, EP 0,324,540 discloses assays designed to measure the amount of free ligand (rather than complexed ligand, which complexed ligand is typically protein-bound) in biological samples such as plasma or serum. The assay method requires the use of a “signal reagent”, which is a labelled monoclonal antibody. The monoclonal binds to free ligand, which is in competition with a ligand analogue (which analogue does not bind to the natural ligand complexing proteins present in the sample). Typically the analogue is immobilised (e.g. on particles or beads). The analogue is selected to have a lower affinity than the ligand for the anti-ligand monoclonal antibody. The assay thus works on the principle of immuno-competition, the presence of free ligand in the sample serving to decrease the amount of labelled antibody which becomes associated with the ligand analogue.
WO 91/05262 discloses a device and method for detecting the presence of molecular analytes in a fluid (especially e.g. steroids, and other low molecular weight analytes). Typically, aqueous biological samples are drawn along a test strip by capillary action. As the sample advances, it carries a labelled analyte from an area of storage at one end of the strip to a first binding means, which is an anti-analyte antibody. In the absence of free analyte in the sample, the labelled analyte (e.g. analyte/enzyme conjugate) will remain bound to the first binding means. However, if free analyte is present in the sample it will tend to displace the labelled analyte (or at least, compete therewith for binding sites on the first binding means) such that some labelled analyte will be bound to the second binding means, which is an anti-enzyme antibody. Colour is developed by placing the strip in an appropriate substrate solution.
EP 0,383,313 discloses a composition and assay method “for measuring haptens, antigens or antibodies by means of a competitive binding method”. The invention disclosed therein requires that either the antibody or its ligand is labelled.
However, useful as such assays are, the requirement for labelling is disadvantageous. Radio-labels represent obvious hazards in handling and disposal. Enzyme or other active labels may deteriorate during storage, affecting the sensitivity of the assay. Use of coloured particles causes problems in that the relatively large surface area of the particles introduces non-specific binding sites which can affect the accuracy of the assay.
The present invention seeks to reduce these difficulties by providing an assay method and device which do not require the use of conventionally-labelled reagents.
SUMMARY OF THE INVENTION
In a first aspect the invention provides a method of detecting the presence of an analyte of interest in a sample, the method comprising the steps of:
providing a binding partner reversibly immobilised on a solid support, said binding partner having binding specificity for the analyte;
contacting the sample with the solid support;
specifically displacing the binding partner from the solid support in response to the presence of the analyte of interest in the sample, said displacement causing a reduction in the mass of material immobilised on the solid support, thereby generating a detectable change in a mass-dependent property of the solid support; and
detecting said change.
In a second aspect the invention provides a method of detecting the presence of an analyte of interest in a sample, the method comprising the steps of:
providing an analogue of the analyte of interest reversibly immobilised on a solid support;
contacting the sample with the solid support;
specifically displacing the analogue from the solid support in response to the presence of the analyte of interest in the sample, said displacement causing a reduction in the mass of material immobilised on the solid support, thereby generating a detectable change in a mass-dependent property of the solid support; and
detecting said change.
Changes in the mass of material immobilised on the solid support can cause detectable changes in a number of mass-dependent properties which can be detected, for example, by acoustic wave or evanescent wave type sensors, or by surface plasmon resonance (SPR) detectors, all of which are known in the art (see, for example, those disclosed in EP 0 341 927, EP 0 416 730 and EP 0 453 224). A particularly suitable mass-dependent property for detection of changes therein is the refractive index of the surface of the solid support to which material is immobilised.
In a third aspect the invention provides a method of detecting the presence of an analyte of interest in a sample, the method comprising the steps of:
providing a binding partner reversibly immobilised on a solid support, said binding partner having binding specificity for the analyte;
contacting the sample with the solid support;
specifically displacing the binding partner from the solid support in response to the presence of the analyte of interest in the sample, said displacement generating a detectable signal; and
detecting said signal;
characterised in that said binding partner does not comprise a conventional label.
In a fourth aspect the invention provides a method of detecting the presence of an analyte of interest in a sample, the method comprising the steps of:
providing an analogue of the analyte of interest reversibly immobilised on a solid support;
contacting the sample with the solid support;
displacing the analogue from the solid support in response to the presence of the analyte of interest in the sample, said displacement generating a detectable signal; and
detecting said signal;
characterised in that said analogue of the analyte does not comprise a conventional label.
The invention also provides an assay device for detecting the presence of an analyte of interest in a sample, the device comprising:
a solid support; a binding partner reversibly immobilised on said solid support, said binding partner having binding specificity for the analyte of interest; and change detection means for detecting a change in a mass-dependent property of the solid support caused by specific displacement of the binding partner from the solid support in response to the presence of the analyte of interest.
The invention additionally provides an assay device for detecting the prese
Badley Robert A.
Berry Mark J.
Porter Philip
Wattam Trevor A.
Chin Christopher L.
Pillsbury & Winthrop LLP
Unilever Patent Holdings B.V.
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