WO2000000826A1 - Analyse immunometrique - Google Patents

Analyse immunometrique Download PDF

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Publication number
WO2000000826A1
WO2000000826A1 PCT/GB1999/002019 GB9902019W WO0000826A1 WO 2000000826 A1 WO2000000826 A1 WO 2000000826A1 GB 9902019 W GB9902019 W GB 9902019W WO 0000826 A1 WO0000826 A1 WO 0000826A1
Authority
WO
WIPO (PCT)
Prior art keywords
analyte
species
immobilised
reagent
labelled
Prior art date
Application number
PCT/GB1999/002019
Other languages
English (en)
Inventor
Lorna Jean Cox
Bernadette Yon-Hin
Original Assignee
Cambridge Sensors Limited
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Cambridge Sensors Limited filed Critical Cambridge Sensors Limited
Publication of WO2000000826A1 publication Critical patent/WO2000000826A1/fr

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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
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow

Definitions

  • This invention relates to an immunometric assay whereby low concentrations of molecules having a single binding site can be rapidly detected and quantitated with minimal instrumentation by an untrained person.
  • haptens small molecules
  • aqueous solution Many immunological methods are available for the determination of haptens (small molecules) in aqueous solution. They suffer from two principal disadvantages, i.e. that as the concentration of hapten increases, the signal decreases; and that the signal is strongly affected by the concentration of the reagents used for its quantitation.
  • US-A-5476770 describes a method wherein the hapten reacts immunologically with the antibody, bringing it into close proximity, so that a covalent bond may be created between the two molecules. The immunological bond is then broken, exposing the epitope on the hapten. Reaction with a labelled version of the same antibody then labels the hapten, the signal increasing for increasing amounts of the hapten. This method is technically demanding and time-consuming.
  • US-A-5236830 describes a method in which the hapten is derivatised before reaction, to introduce a marker; it is then removed from solution by reaction with an antibody attached to a carrier, and dissociated from that antibody; the marker concentration is assayed, giving a higher signal for higher analyte concentrations.
  • the advantage of excess antibody concentration is also achieved, but the derivatisation and the dissociation can be time-consuming and are potential sources of error.
  • EP-A-0161107 describes a method of analysis of small molecules which employs a labelled antibody and an immobilised analogue of the analyte; the extent to which the immobilised analyte becomes labelled is greater for smaller amounts of analyte present in solution in the sample. This is recorded, to generate an inverse-sigmoid calibration curve. While this method is described as an immunometric assay, on the ground of employing a labelled antibody rather than a labelled analogue of the hapten, neither a positive slope to the calibration curve nor the use of excess reagent is achieved.
  • US- A-5219730 describes the use of an idiotype-anti-idiotype immunoassay where analyte is detected by its inhibition of binding of a monoclonal antibody to the unoccupied binding site of a monoclonal antibody raised against the analyte. This requires the production of a pair of high-affinity antibodies for each analyte, which is time- consuming and difficult.
  • US-A-5641690 describes a variation of the foregoing method, whereby a hapten reacts with an antibody directed against the hapten; excess antibody then reacts with a secondary binding partner which may be an analogue of the hapten.
  • a selective antibody is then introduced, which binds only to the antibody which has bound the hapten and not to the antibody which has bound to the secondary binding partner.
  • This method requires the preparation of such a selective antibody, exhibiting a large difference between its affinities for the antibody-hapten complex and for the antibody-secondary binding partner complex, and which must be separately prepared for each first antibody. This imposes a preference for a first antibody of defined composition, for example a monoclonal antibody.
  • an assay for an analyte in a fluid sample comprises: adding to the sample an excess of a labelled reagent which binds to the analyte, thereby forming a conjugate if analyte is present; causing the sample to flow along a path that includes, immobilised at sequential first and second positions, a first species that binds the unconjugated reagent and a second species that binds the conjugate; and determining bound conjugate.
  • the method provided by the present invention requires that the solution which may contain analyte is mixed with an excess of a labelled antibody or other primary binding reagent.
  • the mixture is allowed to react whilst migrating along a membrane or channel upon which firstly an analogue of the analyte and secondly a binder for the antibody or other primary binding reagent have been immobilised.
  • unused binding reagent is removed from the migrating mixture.
  • Binding reagent-analyte conjugate is not so removed, and continues to migrate along the membrane or channel until it contacts the "test site", where it is concentrated for quantitation.
  • the method of quantitation depends on the label employed; a preferred embodiment is visual or absorptiometric quantitation of coloured polystyrene microspheres.
  • the signal at the "test site” is higher for a greater amount of analyte. Excess labelled binding reagent is used over the amount of hapten, and also excess immobilised analogue and test-site binder over the amount of labelled binding reagent. Therefore, the signal obtained is relatively unaffected by the exact amount of reagents present.
  • the binding reagent is preferably an antibody or antibody fragment.
  • the capture reagent can be species-specific and obtained commercially.
  • the method is equally applicable to a monoclonal, polyclonal or recombinant first antibody. Once the antibody has been prepared and labelled by conventional means, the only other reagent required individually for each analyte is a pure sample of the hapten of interest or its analogue, derivatised in such a way that it can be immobilised on the assay surface.
  • a preferred method of achieving this is by coupling it to a protein which can adsorb to, say, nitrocellulose; ideally, this is the protein which was used for immunisation if a polyclonal antiserum containing antibodies against this protein is used.
  • Figs. 1A, IB and 1C are each schematic plan views of a device for use in the invention, respectively before, after (negative result) and after (positive result) use.
  • Fig. 2 shows the dose-response curve obtained by assaying samples containing various concentrations of atrazine, according to Example 2. Description of the Invention
  • a suitable device for use in an assay of the present invention comprises a permeable material, or a channel or channels within an impermeable material, capable of transporting an aqueous solution.
  • a permeable material or a channel or channels within an impermeable material, capable of transporting an aqueous solution.
  • Such a material is shown in Fig. 1, and comprises an application zone 1, a first binding species immobilised on lines 2 transverse to the direction of flow indicated by the arrow, and a second binding species immobilised at a test site 3.
  • Such a device, and other features of the invention described herein, are for illustration only.
  • the device described above together with a first reservoir from which the sample and labelled antibody are introduced and a second reservoir into which the reagents flow when they reach the end of the device, can be encased in an opaque housing.
  • the test site may be viewed through a window in the housing. In the case of a multiple-line test site, only those lines selected by the designer as being suitable for quantitation or quality control are visible to the user through the transparent window.
  • the solution to be analysed is caused to flow through the device by a mechanism such as capillary action, non-bibulous flow or outside pressure.
  • Labelled specific binding partners directed against the binding site on the analyte of interest are introduced at the application zone 1, so as to flow together with the solution carrying the analyte. These may be introduced by various means such as the rupturing of a separate reservoir, by separate addition, or by being included in or on the permeable material or channel(s).
  • Any of the standard labels used for assays are suitable; examples are coloured particles, metal sols, coloured or fluorescent dyes and enzymes. Sufficient excess of the labelled specific binding partner is present to ensure that virtually all the analyte is bound by the specific binding partner, as is normal practice in a "sandwich" assay.
  • the analyte or an analogue is immobilised. This may be bound to and displayed upon the surface of a protein. Sufficient of the immobilised species is used, to capture all of the labelled specific binding partner which has not previously bound the analyte of interest. The excess specific binding partner thereby becomes immobilised. Also in this region, if necessary or desired, there is immobilised a sample of the immunising carrier or such other material which may be required to remove contaminating antibodies with specificities other than for the analyte.
  • Labelled specific binding partner-analyte complexes are left to continue to migrate along the device.
  • the test-site 3 located at a defined distance along the membrane or channel, there is immobilised a second binding reagent with high affinity for the labelled specific binding partner-analyte conjugate, upon which this conjugate becomes concentrated.
  • the immobilised binding partner may be, for example, a species-specific antibody directed against the first antibody, or a bacterial or recombinant protein such as Protein A, Protein G, or Protein L, or a hybrid thereof.
  • the first binding partner may, as well as being labelled as above, be derivatised in such as a way as to become part of a high-affinity binding pair, in which case the other partner of the high-affinity binding pair is used as the "test site".
  • the first binding partner is biotinylated, and streptavidin or avidin is immobilised at the "test site".
  • the amount of label accumulated at the "test site” is controlled by the amount of analyte introduced to the device in the sample; the greater the amount of analyte, the higher the signal.
  • a threshold reaction can be produced in which a positive line is obtained only when the analyte sample is above a certain concentration.
  • the intensity of the test line can be used to quantitate the level of analyte.
  • the intensity of the line may be read visually or abso ⁇ tiometrically in the case of a coloured label, or by whatever means is appropriate to the label if an alternative such as fluorescence is employed.
  • a smaller quantity of a "control" antibody may be used, such as an antibody or other protein which has no affinity for the analyte or the immobilised analyte analogue but which is bound with high affinity by the test site species and which is labelled differently from the assay antibody, or which is bound only by a control species located beyond the test site and which is labelled similarly to or differently from the assay antibody. If the sample does not contain any analyte, so that no signal is obtained, a control signal will be provided at the test site or at the control site to demonstrate the integrity of the reagents and their flow. In a preferred embodiment, this control antibody is labelled with a different colour of polystyrene microspheres whose accumulation may be determined visually.
  • Another preferred embodiment utilises polystyrene microspheres of the same colour as those labelling the assay antibody but utilising a different chemistry for reaction, at the "control line", such as biotinylated polystyrene microspheres and a control line comprising immobilised streptavidin.
  • Another preferred feature of this invention comprises adding to the sample a labelled partner of an independent binding pair which does not interact with the analyte, the reagent, the conjugate or either species, and wherein the flow path comprises, immobilised at a third position, the other partner of the independent binding pair.
  • the affinity of the antibody, or other primary binding partner, for analyte in solution must be high and the affinity of antibody, or other primary binding partner, for the immobilised analyte or its analogue should be slightly less high.
  • the immobilised second binding partner must also have high affinity for the labelled antibody or other primary binding partner and for any antibody used as a control.
  • the invention may be used to assay for any analyte for which there is a specific binding partner, for example a monoclonal, polyclonal or recombinant antibody, a receptor or a specific binding protein.
  • a specific binding partner for example a monoclonal, polyclonal or recombinant antibody, a receptor or a specific binding protein.
  • antibody includes both complete immunoglobulin molecules and immunologically active fragments thereof.
  • Examples of analytes include medicaments, drugs of abuse, agrochemicals, hormones, vitamins, peptides and protein.
  • the analyte preferably has a molecular weight below 1000 Daltons.
  • the invention is particularly suitable for assaying those molecular species for which it is advantageous to perform quantitation rapidly and where little technical expertise or instrumentation is needed or available. The following Examples illustrate the invention.
  • Strips of nitrocellulose (5 mm x 70 mm) were prepared as generally indicated by Fig. 1 A.
  • 10 ⁇ g staphylococcal Protein A was immobilised as a narrow band across the width of the strip and, at a second site, 10 ⁇ g purified human CRP was immobilised as several such narrow bands in close proximity to each other.
  • the strips were blocked with a buffer containing polyvinyl alcohol.
  • Aqueous samples with and without CRP were mixed with an excess of a high-affinity antibody to CRP, coupled to coloured polystyrene microspheres.
  • Strips of nitrocellulose (5 mm x 70 mm), nominal pore size 8 ⁇ m, were prepared as generally indicated by Fig. 1A.
  • the atrazine analogue used was not identical to that used to raise the antiserum.
  • the nitrocellulose strips were blocked by saturation with a 0.025% solution of polyvinyl alcohol.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (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)
  • Chemical Kinetics & Catalysis (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Dans une analyse de détection d'un analyte, par exemple d'un analyte doté d'un site de liaison unique, qui met en oeuvre un partenaire de liaison spécifique, un échantillon s'écoule le long d'un canal ou d'une matière perméable à partir d'un site d'application 1. On introduit une quantité excédentaire d'un partenaire de liaison spécifique, étiqueté, qui est dirigé contre le site de liaison situé sur l'analyte d'intérêt. Au niveau d'un site 2, à une distance définie le long de la membrane ou du canal, une quantité excédentaire suffisante de l'analyte est immobilisée de façon à capturer la totalité du partenaire étiqueté qui ne s'est pas précédemment lié à l'analyte d'intérêt. L'excès de partenaire étiqueté est ainsi immobilisé, ce qui permet au complexe analyte-partenaire étiqueté de continuer à migrer le long de la matière. A une autre distance définie le long de la membrane ou du canal, c'est-à-dire au niveau d'un 'site d'essai' 3, un second réactif de liaison ayant une grande affinité pour le conjugué analyte-partenaire étiqueté est immobilisé, ce qui rend possible l'immobilisation de ce conjugué et par conséquent sa détermination.
PCT/GB1999/002019 1998-06-30 1999-06-28 Analyse immunometrique WO2000000826A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9814008.0A GB9814008D0 (en) 1998-06-30 1998-06-30 Method for the analysis of fluids
GB9814008.0 1998-06-30

Publications (1)

Publication Number Publication Date
WO2000000826A1 true WO2000000826A1 (fr) 2000-01-06

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002004915A2 (fr) * 2000-07-11 2002-01-17 Jacques Toledano Dispositif et procede de detection moleculaire
EP1349915A2 (fr) * 2001-01-09 2003-10-08 Large Scale Proteomics Corporation Technique d'immunosoustraction destinee a la preparation d'un echantillon pour electrophorese en gel en deux dimensions (2-dge)
US7790439B2 (en) 2001-08-09 2010-09-07 Panasonic Corporation Biosensor and measurement method
GB2475579A (en) * 2007-04-23 2011-05-25 Selective Antibodies Ltd Dipstick immunoassay with capture zone comprising immobilised hapten
EP2411808A2 (fr) * 2009-03-24 2012-02-01 Biocept, Inc. Dispositifs et procédés de capture et d'analyse de cellules
EP1325329B1 (fr) * 2000-10-11 2012-09-26 Phadia AB Procédé d'éssai
US8389296B2 (en) 2007-04-23 2013-03-05 Selective Antibodies Limited Assay devices and methods and components for use therein
US9671407B2 (en) 2009-03-24 2017-06-06 Biocept, Inc. Devices and methods of cell capture and analysis

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997034150A1 (fr) * 1996-03-14 1997-09-18 Abbott Laboratories Elements de liaison prenant naissance sur des particules et destines a des dosages immunologiques
CA2198948A1 (fr) * 1996-05-31 1997-12-01 Ronald G. Sommer Detection quantitative des composantes d'un melange dans les bandes immunochromatographiques
WO1998039657A1 (fr) * 1997-03-06 1998-09-11 Quidel Corporation Analyses et dispositifs d'analyse quantitative de flux lateral
EP0895084A2 (fr) * 1997-07-25 1999-02-03 Bayer Corporation Dispositif et méthode pour obtenir des raisons d'analytes avec signification clinique

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997034150A1 (fr) * 1996-03-14 1997-09-18 Abbott Laboratories Elements de liaison prenant naissance sur des particules et destines a des dosages immunologiques
CA2198948A1 (fr) * 1996-05-31 1997-12-01 Ronald G. Sommer Detection quantitative des composantes d'un melange dans les bandes immunochromatographiques
WO1998039657A1 (fr) * 1997-03-06 1998-09-11 Quidel Corporation Analyses et dispositifs d'analyse quantitative de flux lateral
EP0895084A2 (fr) * 1997-07-25 1999-02-03 Bayer Corporation Dispositif et méthode pour obtenir des raisons d'analytes avec signification clinique

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002004915A2 (fr) * 2000-07-11 2002-01-17 Jacques Toledano Dispositif et procede de detection moleculaire
FR2811765A1 (fr) * 2000-07-11 2002-01-18 Jacques Toledano Dispositif et procede de detection moleculaire
WO2002004915A3 (fr) * 2000-07-11 2002-04-11 Jacques Toledano Dispositif et procede de detection moleculaire
EP1325329B1 (fr) * 2000-10-11 2012-09-26 Phadia AB Procédé d'éssai
EP1349915A2 (fr) * 2001-01-09 2003-10-08 Large Scale Proteomics Corporation Technique d'immunosoustraction destinee a la preparation d'un echantillon pour electrophorese en gel en deux dimensions (2-dge)
EP1349915A4 (fr) * 2001-01-09 2008-02-27 Large Scale Proteomics Corp Technique d'immunosoustraction destinee a la preparation d'un echantillon pour electrophorese en gel en deux dimensions (2-dge)
US7642089B2 (en) 2001-01-09 2010-01-05 Agilent Technologies, Inc. Immunosubtraction method
US7691645B2 (en) 2001-01-09 2010-04-06 Agilent Technologies, Inc. Immunosubtraction method
US7790439B2 (en) 2001-08-09 2010-09-07 Panasonic Corporation Biosensor and measurement method
GB2475579A (en) * 2007-04-23 2011-05-25 Selective Antibodies Ltd Dipstick immunoassay with capture zone comprising immobilised hapten
GB2475579B (en) * 2007-04-23 2011-12-21 Selective Antibodies Ltd Assay devices and methods and components for use therein
US8389296B2 (en) 2007-04-23 2013-03-05 Selective Antibodies Limited Assay devices and methods and components for use therein
EP2411808A4 (fr) * 2009-03-24 2012-09-12 Biocept Inc Dispositifs et procédés de capture et d'analyse de cellules
JP2012522217A (ja) * 2009-03-24 2012-09-20 バイオセプト インコーポレイティッド 細胞の捕捉および解析のデバイスおよび方法
CN102414562A (zh) * 2009-03-24 2012-04-11 生物概念股份有限公司 细胞捕获和分析的装置和方法
EP2411808A2 (fr) * 2009-03-24 2012-02-01 Biocept, Inc. Dispositifs et procédés de capture et d'analyse de cellules
US9128082B2 (en) 2009-03-24 2015-09-08 Biocept, Inc. Devices and methods of cell capture and analysis
EP2995953A1 (fr) * 2009-03-24 2016-03-16 Biocept, Inc. Dispositifs et procédés de capture et d'analyse de cellules
AU2010229924B2 (en) * 2009-03-24 2016-07-21 Plus Therapeutics Inc. Devices and methods of cell capture and analysis
US9671407B2 (en) 2009-03-24 2017-06-06 Biocept, Inc. Devices and methods of cell capture and analysis
AU2016222325B2 (en) * 2009-03-24 2017-09-14 Plus Therapeutics Inc. Devices and methods of cell capture and analysis
CN110470835A (zh) * 2009-03-24 2019-11-19 生物概念股份有限公司 细胞捕获和分析的装置和方法
US10527611B2 (en) 2009-03-24 2020-01-07 Biocept, Inc. Devices and methods of cell capture and analysis
US11719692B2 (en) 2009-03-24 2023-08-08 Biocept, Inc. Devices and methods of cell capture and analysis

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