GB2345133A - Polyspecific antibody immunodiagnostic device - Google Patents
Polyspecific antibody immunodiagnostic device Download PDFInfo
- Publication number
- GB2345133A GB2345133A GB9828664A GB9828664A GB2345133A GB 2345133 A GB2345133 A GB 2345133A GB 9828664 A GB9828664 A GB 9828664A GB 9828664 A GB9828664 A GB 9828664A GB 2345133 A GB2345133 A GB 2345133A
- Authority
- GB
- United Kingdom
- Prior art keywords
- antibody
- analyte
- detecting
- antigen
- layer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
Device for detecting the presence of an analyte in an aqueous fluid, comprises an apertured support substrate provided on a top surface with an immobilised polyspecific antibody which will react with the analyte to release a reporter antigen and provided on a bottom surface with means for detecting the presence of the reporter antigen. Substrate may be a fibrous mat or a porous membrane. Reporter antigen may be an enzyme, fluorescent or electrochemically active species. Polyspecific antibody may be conjugated to latex beads.
Description
IMMUNODIAGNOSTIC DEVICE
This invention relates to an immunodiagnostic device for the determination of analytes in aqueous fluids, notably biological fluids.
There is a requirement for test devices which can be used in the home or a doctor's surgery by inexperienced personnel to test for analytes in biological fluids such as blood, plasma, serum and urine. It is desirable that the test should be easy to perform, rapid, accurate and cost effective. Inexperienced personnel for a number of years have performed tests that measure glucose and test for pregnancy. The specificity of such tests has improved as the tests have become easier to perform.
EP 0 291 194 discloses an analytical device in which antibodies are used in both an immobilised and an aqueous mobile form. A liquid test sample can be applied to a porous carrier. The device contains labelled antibody for an analyte which is freely mobile when wet, and unlabelled antibody for the same analyte, although a different epitope, which unlabelled reagent is permanently immobilised in a detection zone on the carrier material. The liquid sample containing analyte binds to the labelled mobile antibody which is then transferred by lateral wicking along the porous carrier to the detection zone where the immobilised antibody binds the analyte-labelled antibody complex. If analyte is not present the mobile labelled antibody is not detected at the detection zone.
EP 0 511 O11 discloses the use of monoclonal bi-or tri-specific antibodies for diagnostic purposes. The binding of an analyte to a first antibody binding site causes release of a reporter antigen from an adjacent second antibody antigen binding site. The term "polyspecific"will be used hereinafter to refer to an antibody of this type. The term includes bi-specific and tri-specific antibodies.
According to an aspect of the present invention there is provided a device for monitoring or detecting the presence of an analyte in an aqueous fluid, comprising an apertured support substrate provided on a top surface with an immobilised polyspecific antibody which will react with the analyte to release a reporter antigen and provided on a bottom surface with means for detecting the presence of the reporter antigen.
Because the support substrate may be a thin sheet of material, the reporter antigen can quickly reach the detecting means, enabling the device to have a fast response time. The sample volume required is also smaller than for a lateral wicking device, where the sample liquid has to travel through a much greater volume of substrate. This makes the device particularly suitable for use in home testing. The support substrate is preferably between 100 and 400 pm thick.
By having the polyspecific antibody on the top surface and the detection means on the reverse surface, these components may readily be applied by well known coating or printing methods.
To provide a particularly quick response, it is preferred that the apertures in the support substrate are sufficiently large that the aqueous liquid flows through the support substrate from the top surface to the bottom surface substantially under gravity, although capillary flow may also be used.
Suitable support substrates include (but are not limited to) glass fibre, glass fibre-cellulosic, polyester, polypropylene and polyamide fibrous mat.
Examples of membranes which may be used as support substrates include cellulose esters, polyethersulphone (PES), polyester, polyamide, and nitrocellulose.
Any detectable reporter antigen may be used. The reporter antigen may for example be an enzyme, a substance which exhibits fluorescence, or an electrochemically active species.
The detecting means may comprise a reagent which reacts with the antigen to produce a colour change. The colour change can either be visually reported or measured by a reflectance meter to give a quantified response.
Alternatively the antigen could be, or give rise to, an electroactive species. For example the reporter antigen could be the enzyme alkaline phosphatase and the reagent could be 4-nitrophenol phosphate which is converted into electrochemically active 4-nitrophenol.
This would in turn make contact with an electrode surface. In this case 4-nitrophenol phosphate would be a component in an electrode formulation. If the antigen is itself an electroactive species, the detecting means could simply comprise an electrode surface, connected to a suitable circuit. Such circuits will be well known to those skilled in the art and will not be further described here.
To reduce possible interaction between the polyspecific antibody and the detecting means in the absence of the analyte, for example as a result of residual enzyme activity associated with the antibody, a layer of spacer material may be provided between the polyspecific antibody layer and the detecting means.
In a preferred embodiment the polyspecific antibody is conjugated onto beads, preferably latex beads, by known techniques. The beads are secured on a carrier substrate, notably a non-woven material with an openpore structure. The latex beads can be either fixed or mobile when wet. A layer of spacer material is optionally provided under the carrier substrate, and a further thin layer comprising a cellulosic material in which a reagent which will react with the reporter antigen is carried. The spacer layer may be a thin layer of paper. In a preferred embodiment, the spacer layer carries a charge opposite to that of the reporter antigen, notably a positive charge. This facilitates passage of the reporter antigen.
To perform a test, an aqueous sample is placed onto a defined area of the top surface of the non-woven material. If analyte is present, it reacts with the detector arm of the polyspecific antibody, causing release of reporter antigen from a second antibody site. It is believed that the bound reporter antigen is released as a result of steric hindrance between the itself and the incoming analyte.
In a further embodiment of the invention polyspecific antibodies can be applied to the air (top) side of a nitrocellulose membrane. The downstream (bottom) side of the membrane has been previously glazed with for example a sucrose solution, to inhibit reagent from penetrating to the top surface during coating of the bottom surface. After drying the air side, the membrane is turned over and the downstream surface is coated with reagent-containing substrate. This has the advantage of reducing the number of layers in the device and hence giving benefits in precision. Methods of either impregnating or coating the membrane are well known but examples include ink-jet printing, flexographic printing, gravure printing, metered pumps and micro-syringes.
In a further embodiment of the invention polyspecific antibodies attached to beads can be applied to the air side of a membrane. The beads may be retained on the top surface of the membrane by virtue of their being larger than the pore size of the membrane. Reagantcarrying substrate can be applied to the downstream surface of a glazed membrane. Membranes that can be used include for example cellulose esters, polyethersulphone, polyester, polyamide.
Examples of suitable materials and their properties are as follows.
Latex beads
-streptavidin coated, 0.7pm, range 0.1 Um.
-Uncoated latex beads bispecific antibody
conjugated using standard procedures. lOw/v %-coat membrane at this
concentration
Polyspecific antibody,
coat beads (eg with biotinylated antibody) at
between 0.1-1 mg/ml
Nitrocellulose membranes
Pore size 1-15 Rm, Schleicher & Scheull or
Millipore
Other membranes
Pore size 1-lOpm To maximise flow rate it is preferred that the membrane is highly hydrophilic, with an open structure.
Membranes from Millipore, Gelman, Pall, Memtek, among others, may be used.
The invention will now be further described, by way of example, with reference to the following drawing in which:
Figure 1 shows a device in accordance with the
present invention;
Figure 2 is a section through a device in
accordance with one embodiment of the present
invention; and
Figure 3 is a section through a device in
accordance with another embodiment of the present
invention.
The device 2 comprises a test strip which has a zone 4 on its top surface where a fluid sample containing a suspected analyte is to be applied.
In the embodiment shown in Figure 2, latex beads 6 (0.7 gm diameter) are carried on a carrier substrate 8 comprising a glass fibre mat. The beads 6 have been coated with streptavidin and biotinylated antibody having a bound reporter antigen of B-galactosidase. A spacer layer 10 of paper is provided on the bottom surface of the carrier substrate 8, and this is coated with a layer 12 of ethyl hydroxyethyl cellulose (EHEC) and a reagent. The reagent is VBzTMgal (Melford
Laboratories, Suffolk) and reacts with ss-galactosidase to produce a change in optical density observable at 570 nm.
The paper spacer layer 10 is about 100 thick and limits migration of the reporter antigen on long term storage, which could produce a colour change without analyte being present.
When a sample of fluid is applied to the sample zone 4, if hCG analyte is present in the fluid, this reacts with the antibody to release ss-galactosidase, a solution of which falls through the carrier substrate 8 under gravity and reacts with the reagent to produce a colour change.
In the embodiment shown in Figure 3, a layer of biotinylated antibody 14 is provided on the top surface of a carrier substrate comprising a porous nitrocellulose membrane 80. The bottom surface has a layer of sucrose glaze 16 and a layer 12 of EHEC and a reagent. The device works in a similar manner to that of the device of Figure 2.
The invention provides a simple and rapid diagnostic test which requires only a small sample volume.
Claims (18)
- CLAIMS 1. A device for monitoring or detecting the presence of an analyte in an aqueous fluid, comprising an apertured support substrate provided on a top surface with an immobilised polyspecific antibody which will react with the analyte to release a reporter antigen and provided on a bottom surface with means for detecting the presence of the reporter antigen.
- 2. A device as claimed in Claim 1, wherein the polyspecific antibody is conjugated to polymeric beads.
- 3. A device as claimed in Claim 2, wherein the polyspecific antibody is conjugated to latex beads.
- 4. A device as claimed in any one of the preceding claims, wherein the apertures in the support structure are of sufficient size that when an aqueous sample is applied to the top surface, it flows to the bottom surface substantially under gravity.
- 5. A device as claimed in any one of the preceding claims, wherein the support substrate comprises a fibrous mat.
- 6. A device as claimed in Claim 5, wherein the fibrous mat is formed from a material selected from the following group: glass fibre, glass fibre-cellulosic, polyester, polypropylene, polyamide..
- 7. A device as claimed in any one of claims 1 to 4, wherein the support substrate comprises a porous membrane.
- 8. A device as claimed in Claim 7, wherein the porous membrane is made from a material selected from the following group: cellulose esters, PES, polyester, polyamide, nitrocellulose.
- 9. A device as claimed in any one of the preceding claims, wherein a layer of spacer material is disposed between the polyspecific antibody and the detecting means.
- 10. A device as claimed in any one of the preceding claims, wherein a glazing layer is provided between the carrier substrate and the detecting means.
- 11. A device as claimed in Claim lof wherein the glazing layer is sucrose.
- 12. A device as claimed in any one of the preceding claims, wherein the antibody is a bi-specific antibody.
- 13. A device as claimed in any one of the preceding claims, wherein the reporter antigen is B-galactosidase and the reagent is VBzTMgal.
- 14. A device as claimed in any one of the preceding claims, wherein the carrier substrate is 100 to 400 jum thick.
- 15. A device as claimed in an Claim 9 wherein the spacer layer is about 100 um thick.
- 16. A device as claimed in Claim 9, wherein the spacer layer is made of paper.
- 17. A device as claimed in Claim 9, wherein the reporter antigen is electrically charged and the spacer is oppositely charged.
- 18. A device for monitoring or detecting the presence of an analyte in an aqueous fluid substantially as herein described with reference to or as shown in the drawing.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9828664A GB2345133A (en) | 1998-12-24 | 1998-12-24 | Polyspecific antibody immunodiagnostic device |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9828664A GB2345133A (en) | 1998-12-24 | 1998-12-24 | Polyspecific antibody immunodiagnostic device |
Publications (2)
Publication Number | Publication Date |
---|---|
GB9828664D0 GB9828664D0 (en) | 1999-02-17 |
GB2345133A true GB2345133A (en) | 2000-06-28 |
Family
ID=10845046
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB9828664A Withdrawn GB2345133A (en) | 1998-12-24 | 1998-12-24 | Polyspecific antibody immunodiagnostic device |
Country Status (1)
Country | Link |
---|---|
GB (1) | GB2345133A (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006047869A1 (en) * | 2004-11-01 | 2006-05-11 | International Bio-Therapeutic Research Inc. | Disposable immunodiagnostic test system |
US8475735B2 (en) | 2004-11-01 | 2013-07-02 | Uma Mahesh Babu | Disposable immunodiagnostic test system |
US8778276B2 (en) | 2008-06-18 | 2014-07-15 | The Secretary Of State For Defence | Detection device |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0511011A1 (en) * | 1991-04-26 | 1992-10-28 | Surface Active Limited | Novel antibodies and methods for their use |
WO1995004931A1 (en) * | 1993-08-06 | 1995-02-16 | Surface Active Limited | Diagnostic method |
WO1996012188A1 (en) * | 1994-10-18 | 1996-04-25 | Surface Active Limited | Diagnostic method and apparatus for performing the method |
-
1998
- 1998-12-24 GB GB9828664A patent/GB2345133A/en not_active Withdrawn
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0511011A1 (en) * | 1991-04-26 | 1992-10-28 | Surface Active Limited | Novel antibodies and methods for their use |
US5573920A (en) * | 1991-04-26 | 1996-11-12 | Surface Active Limited | Antibodies, and methods for their use |
WO1995004931A1 (en) * | 1993-08-06 | 1995-02-16 | Surface Active Limited | Diagnostic method |
WO1996012188A1 (en) * | 1994-10-18 | 1996-04-25 | Surface Active Limited | Diagnostic method and apparatus for performing the method |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006047869A1 (en) * | 2004-11-01 | 2006-05-11 | International Bio-Therapeutic Research Inc. | Disposable immunodiagnostic test system |
US8475735B2 (en) | 2004-11-01 | 2013-07-02 | Uma Mahesh Babu | Disposable immunodiagnostic test system |
US8778276B2 (en) | 2008-06-18 | 2014-07-15 | The Secretary Of State For Defence | Detection device |
Also Published As
Publication number | Publication date |
---|---|
GB9828664D0 (en) | 1999-02-17 |
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Legal Events
Date | Code | Title | Description |
---|---|---|---|
WAP | Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1) |