GB2426049A - Rapid flow through immunoassay - Google Patents
Rapid flow through immunoassay Download PDFInfo
- Publication number
- GB2426049A GB2426049A GB0509104A GB0509104A GB2426049A GB 2426049 A GB2426049 A GB 2426049A GB 0509104 A GB0509104 A GB 0509104A GB 0509104 A GB0509104 A GB 0509104A GB 2426049 A GB2426049 A GB 2426049A
- Authority
- GB
- United Kingdom
- Prior art keywords
- analyte
- immunoassay according
- antibody
- immunoassay
- porous material
- 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- Withdrawn
Links
- 238000003018 immunoassay Methods 0.000 title claims abstract description 25
- 239000012491 analyte Substances 0.000 claims abstract description 23
- 102000016943 Muramidase Human genes 0.000 claims abstract description 4
- 108010014251 Muramidase Proteins 0.000 claims abstract description 4
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims abstract description 4
- 239000008280 blood Substances 0.000 claims abstract description 4
- 210000004369 blood Anatomy 0.000 claims abstract description 4
- 229960000274 lysozyme Drugs 0.000 claims abstract description 4
- 235000010335 lysozyme Nutrition 0.000 claims abstract description 4
- 239000004325 lysozyme Substances 0.000 claims abstract description 4
- 239000011324 bead Substances 0.000 claims abstract description 3
- 239000012528 membrane Substances 0.000 claims abstract description 3
- 210000002381 plasma Anatomy 0.000 claims abstract description 3
- 210000002966 serum Anatomy 0.000 claims abstract description 3
- 239000011148 porous material Substances 0.000 claims abstract 8
- 206010040047 Sepsis Diseases 0.000 claims abstract 2
- 239000002981 blocking agent Substances 0.000 claims abstract 2
- 238000001514 detection method Methods 0.000 claims abstract 2
- 239000007788 liquid Substances 0.000 claims abstract 2
- 239000000463 material Substances 0.000 claims description 12
- 230000027455 binding Effects 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 229940088598 enzyme Drugs 0.000 claims description 3
- 238000003556 assay Methods 0.000 description 10
- 230000000903 blocking effect Effects 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 239000011159 matrix material Substances 0.000 description 5
- 238000000034 method Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 108010093096 Immobilized Enzymes Proteins 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229910052747 lanthanoid Inorganic materials 0.000 description 1
- 150000002602 lanthanoids Chemical class 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
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
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
-
- 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
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
An immunoassay in which the analyte is immobilised by passing a liquid sample through a porous material which does not contain an antibody and subsequent detection of the bound analyte by a labelled antibody in the absence of an extrinsic blocking agent. Preferably the analyte to be detected is lysozyme and the sample is of blood, serum or plasma and is indicative of sepsis. The porous material may be charge or uncharged and be a membrane, column or micro particulate conglomerate such as beads.
Description
A Rapid Immunoassay for Lysozyme in Blood Described here is a novel, flow-
through immunoassay, which has been developed for the purpose of rapidity. The assay is primarily intended for lysozyme in blood, plasma or serum although it could be applied to other analytes in other fluids which are compatible with the assay procedure (the fluid which contains the analyte is herein called the matrix). There are three aspects of novelty that, used in combination, comprise this invention - each making a major contribution to the assay rapidity.
1. The analyte is immobilised by passing the matrix through a porous immobilization material.
2. Analyte immobilization does not involve a capture antibody.
3. No blocking step/reagent is required.
The immunoassay consists of passing the matrix through a porous immobilization material which either adsorbs, or filters out, the analyte. The immobilization material may be charged or uncharged, it may take the form of a membrane, a column or a microparticulate conglomerate (for example, a layer, or a bed, of beads). Anti-analyte antibody (the primary antibody) is then passed through the immobilization material such that it binds to the analyte & is itself immobilized. This antibody may be conjugated to an enzyme, for example Horseradish Peroxidase (HRP) or another marker molecule (for example, a fluorescent entity - including lanthanide chelates) the activity of which can be subsequently used to reveal & quantify the presence of immobilised analyte. This procedure can be called a direct immunoassay.
If the primary antibody is not conjugated in this way, a second antibody (the secondary antibody), which binds to the primary antibody, can be conjugated to an enzyme or marker.
This conjugated secondary antibody can be passed through the immobilization material after the analyte & the unconjugated primary antibody such that it too becomes irnmobilised (by binding to the primary antibody). The presence of analyte on the immobilization material is then again revealed by assaying for activity of the immobilized enzyme or marker. This can be called an indirect immunoassay.
The great rapidity of the immunoassay (<40 minutes) is due to the three (numbered) aspects above used in combination. This may be further explained as follows: I. Matrix flow-through (as opposed to flow-over) the immobilization material enhances the rate of the chemistry of analyte immobilization.
2. Unusually, analyte immobilization is not by way of a capture antibody but rather by way of some other adsorbtion chemistry or by size-exclusion filtration, or by a combination of both. The adsorbtion chemistry may be a direct interaction between the analyte and the immobilization material itself or it may be indirect, in that it is by way of an entity which causes, assists or facilitates the binding. This lack of requirement for a capture antibody obviates the incorporation of a capture antibodyimmobilization step into the assay procedure, which significantly decreases overall assay time.
3. Blocking reagents are usually incorporated into immunoassay procedures in order to prevent non-analyte-related binding of marker (non-specific binding). In the present assay, addition of blocking reagents is not necessary, which obviates the incorporation of a blocking step and so decreases overall assay time. The reason for this may be that the matricies for which this assay is specifically designed have an intrinsic blocking function.
The immobilized enzyme or marker may give rise to optical or electrochemical activity which may be detected on the immobilization material itself or at some other site, following removal from the immobilization material.
Passing of the matrix & other assay components through the immobilization material can be continuous or discontinuous, ifl the latter case, the flow may he stopped or reversed if necessary. For this reason, the assay may also be called a stop-flow immunoassay.
Sample results are shown in Fig. 1.
Claims (15)
- Claims We claim: 1. An immunoassay in which the analyte is immobilised bypassing a liquid sample through a porous material which does not contain an antibody and subsequent detection of the of the bound analyte by means of a labelled antibody in the absence of any extrinsic blocking agent.
- 2. An immunoassay according to Claim 1 in which the analyte is lysozyme.
- 3. An immunoassay according to Claim I in which the sample is blood, plasma or serum.
- 4. An immunoassay according to Claim 2 in which the analyte is indicative of sepsis.
- 5. An immunoassay according to Claim 1 in which the analyte is filtered out from or absorbed from the sample.
- 6. An immunoassay according to Claim 1' in which the porous material is charged.
- 7. An immunoassay according to Claim 1' in which the porous material is uncharged.
- 8. An immunoassay according to Claim 5 in which the porous material takes the form of a membrane.
- 9. An immunoassay according to Claim 5 in which the porous material takes the form of a column of material.
- 10. An immunoassay according to Claim 5 in which the porous material takes the form of a microparticulate conglomerate, for example, a layer or a bed of beads.
- 11. An immunoassay according to Claim I in which the presence of bound antibody is quantified by use of a labelled anti-analyte antibody.
- 12. An immunoassay according to Claim 1111 which the presence of bound antibody is quantified by use of an anti-analyte antibody together with a labelled second antibody capable of binding the first antibody.
- 13. An immunoassay according to Claim 11 or Claim 12 in which the label is an enzyme.
- 14. An immunoassay according to Claim 11 or Claim 12 in which the label is detected optically.
- 15. An immunoassay according to Claim 11 or Claim 12 in which the label is detected electrochemically.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0509104A GB2426049A (en) | 2005-05-04 | 2005-05-04 | Rapid flow through immunoassay |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0509104A GB2426049A (en) | 2005-05-04 | 2005-05-04 | Rapid flow through immunoassay |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| GB0509104D0 GB0509104D0 (en) | 2005-06-08 |
| GB2426049A true GB2426049A (en) | 2006-11-15 |
Family
ID=34674329
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB0509104A Withdrawn GB2426049A (en) | 2005-05-04 | 2005-05-04 | Rapid flow through immunoassay |
Country Status (1)
| Country | Link |
|---|---|
| GB (1) | GB2426049A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6015681A (en) * | 1995-07-28 | 2000-01-18 | The United States Of America As Represented By The Secretary Of The Navy | Rapid immunoassay for cariogenic bacteria |
| WO2004048977A1 (en) * | 2002-11-25 | 2004-06-10 | Albert Missbichler | Method for detecting membrane-bound and/or amyloid proteins |
-
2005
- 2005-05-04 GB GB0509104A patent/GB2426049A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6015681A (en) * | 1995-07-28 | 2000-01-18 | The United States Of America As Represented By The Secretary Of The Navy | Rapid immunoassay for cariogenic bacteria |
| WO2004048977A1 (en) * | 2002-11-25 | 2004-06-10 | Albert Missbichler | Method for detecting membrane-bound and/or amyloid proteins |
Also Published As
| Publication number | Publication date |
|---|---|
| GB0509104D0 (en) | 2005-06-08 |
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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) |