Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1997-05-16
2001-10-30
Smith, Lynette R. F. (Department: 1645)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C436S528000, C435S007320, C435S006120
Reexamination Certificate
active
06309893
ABSTRACT:
The present invention relates to an improved assay method and to devices for use in such methods. In particular the invention relates to assay methods, especially immunoassays, in which soluble releasable reagents are used.
The method and devices are, in certain embodiments, intended for use in specific binding assay procedures, in particular immunoassay procedures. Examples of such procedures in which soluble releasable reagents may be employed are cited in EP-A-0171148, WO92/09892, WO93/25892 and WO93/25908.
In the assay procedures disclosed in EP-A-0171148, certain ancillary reagent(s) are employed and can be in the form of a releasable reagent coating e.g. a coating of releasable antigen or antibody, or derivative thereof. In WO92/09892, in which a device is described possessing one or more calibration regions for the purposes of internal referencing of an assay method, the use of a polyvinyl alcohol (PVA) capping layer is disclosed, in order to delay the dissolution of the soluble reagent for a few seconds after the addition of the sample to the device. This delayed-release is to prevent the reagents washing from one zone to another thereby precluding an accurate assay. However, although limited effectiveness has been achieved by use of such a capping layer, problems of poor reproducibility and low sensitivity have still been encountered.
In the assay procedures disclosed in WO92/09892, the success of the method of assay depends on the spatial separation (i.e. non-mixing) of the various soluble reagents released into the sample solution. However, in other assay techniques involving only one soluble reagent it is advantageous to ensure a maximum amount of the released reagent remains in a certain defined area to ensure high assay precision and sensitivity.
Additionally, WO-A-93/025908 (ARS Holdings NV) refers generally to the delayed release properties of a coated patch and suggests PVA as a suitable material for such a patch. However, there is no suggestion that cross-linked PVA may be used as a delayed release agent.
We have now found that by employing alternative reagents for delaying the release of soluble reagents in assays unexpected improvements in the assay precision and sensitivity can be achieved as compared with the existing methods used.
Thus according to a first aspect of the present invention, we provide in assays utilising one or more soluble releasable reagents the use of cross-linked PVA or of copolymers of methacrylic acid or methacrylate esters in order to achieve the delayed-release of said soluble releasable reagents.
According to a further aspect of the present invention, we provide a method of improving assay precision in assays utilising one or more soluble releasable reagents in which the release of said reagents is delayed by means of cross-linked PVA or copolymers of methacrylic acid or methacrylate esters.
The present technique may be used for a wide variety of chemical or biochemical test procedures but is especially useful in connection with clinical test procedures, most especially immunoassays.
According to a further aspect of the present invention, there is provided a sensor device for an assay as defined above which carries on a surface thereof one or more soluble releasable reagents coated with or incorporated in cross-linked PVA or copolymers of methacrylic acid or methacrylate esters.
The present method is applicable to a wide variety of devices including, for example, dip-stick or test-strip sensors, devices using a “sample flow-through” configuration or devices employing sample containment. Sample containment devices are preferred for carrying out the method of the invention, with a more preferred device being a capillary fill device, especially a fluorescence capillary device, for example the type of device described in EP-A-171148 or in WO-90/14590. Such capillary fill devices may be used singly or in a suitable holder such as described in WO-90/1830.
As described in EP-A-171148, a capillary fill device (hereinafter CFD) typically consists of two plates of transparent material, e.g. glass, separated by a narrow gap or cavity. One plate acts as an optical waveguide and carries an immobilised reagent appropriate to the test to be carried out in the device. As described in WO-90/14590, the other transparent plate can carry on its surface remote from the cavity a layer of light-absorbing or opaque material. For use in a competition assay, the immobilised reagent may for example be a specific binding partner to the ligand desired to be detected and one of the plates may carry a dissoluble reagent comprising ligand analogue, labelled with a fluorescent dye (the ancillary reagent). When a sample is presented to one end of the CFD it is drawn into the gap by capillary action and dissolves the ancillary reagent. In a competition assay for an antigen, the fluorescently labelled antigen analogue will compete with sample antigen for the limited number of antibody binding sites immobilised onto 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 depending upon the sample matrix, assay type (e.g. sandwich or competitive immunoassay) and antibody affinity. Thus for a competition assay, the amount of fluorescently labelled antigen which becomes indirectly bound to the waveguide by virtue of complex formation will be inversely proportional to the concentration of antigen in the sample. 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 (the ancillary reagent). In a sandwich immunoassay for an antigen, a sample antigen will form a sandwich complex with a fluorescently labelled antibody and an antibody immobilised on the waveguide. Thus, for a sandwich immunoassay, the amount of fluorescently labelled antibody which becomes indirectly bound to the waveguide by virtue of complex formation will be directly proportional to the concentration of antigen in the sample.
In the above assay techniques, it is important that the soluble releasable fluorescently labelled reagent does not dissolve instantaneously and get washed down to one end of the device away from the region of the capture antibody during filling of the CFD. If this does happen then poor assay signals are obtained with very high imprecision producing a meaningless result. The method of the present invention ensures that the wash-down of the soluble reagent is minimised.
Thus, according to a further aspect of the present invention, we provide a specifically-reactive sample-collecting and testing device possessing a cavity or cavities each having a dimension small enough to enable sample liquid to be drawn into the cavity by capillary action wherein a surface of the cavity carries an immobilised reagent appropriate to the assay to be carried out in the device, and wherein said surface is a surface of a transparent solid plate which in use acts as a light transmissive waveguide and which forms a wall of the cavity, and wherein the cavity surface(s) have one or more zones comprising, in releasable form, ancillary reagent(s) suitable for the desired assay, said ancillary reagent(s) being coated with or incorporated in cross-linked PVA or copolymers of methacrylic acid or methacrylate esters.
To provide a suitable delayed release of the soluble reagent by means of cross-linked PVA, two methods can be employed. In a first method, the soluble reagent is microdosed on the device. The reagent is dissolved in a buffer solution containing PVA. A further layer of PVA is then coated, suitably spray-coated, over the printed conjugate, this PVA layer subsequently being cross-linked, suitably by spray coating with a cross-linking reagent. In a second method, the soluble reagent is microdosed on the device. The reagent is dissolved in a buffer solution containing PVA. A cross-linking reagent is then appli
Deeley George
Fletcher Janys Elizabeth
Applied Research Systems ARS Holding NV
Ostrolenk Faber Gerb & Soffen, LLP
Portner Ginny Allen
Smith Lynette R. F.
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