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WO1996042017A1 - Determinations immunologiques - Google Patents

Determinations immunologiques Download PDF

Info

Publication number
WO1996042017A1
WO1996042017A1 PCT/GB1996/001393 GB9601393W WO9642017A1 WO 1996042017 A1 WO1996042017 A1 WO 1996042017A1 GB 9601393 W GB9601393 W GB 9601393W WO 9642017 A1 WO9642017 A1 WO 9642017A1
Authority
WO
WIPO (PCT)
Prior art keywords
binding agent
analyte
support
column
label
Prior art date
Application number
PCT/GB1996/001393
Other languages
English (en)
Inventor
Stephen John Minter
Michael Wraith
Original Assignee
Tepnel Medical 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 Tepnel Medical Limited filed Critical Tepnel Medical Limited
Priority to EP96917588A priority Critical patent/EP0834075A1/fr
Priority to AU60112/96A priority patent/AU6011296A/en
Publication of WO1996042017A1 publication Critical patent/WO1996042017A1/fr

Links

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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/538—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by sorbent column, particles or resin strip, i.e. sorbent materials

Definitions

  • the present invention relates to a method of effecting an immunological determination, e.g. an immunoassay.
  • Immunological techniques are well established for use in analytical procedures to determine, for example, the presence and/or amount of an antigen in a sample.
  • the techniques generally rely on the binding of the antigen to an antibody which is specific therefor.
  • Such techniques may be conducted wholly in the liquid phase but it is also know to immobilise antibody onto a solid support which, after the binding reaction has been effected, may be washed to remove unbound antigen. This then permits the detection of immobilised antigen-antibody complex.
  • the assay could be conducted in the presence of inhibitors which may produce assay misreading.
  • the assay can not be used to enhance the signal level, i.e. the analyte can not be concentrated in the reaction vessel.
  • the immobilisation of the antibody on such solid supports is frequently by passive absorption which does not necessarily provide for stable immobilisation of the antibody.
  • WO-A-91/13354 discloses a competitive immunological assay for detecting a target moiety using a column of a solid support material (e.g. tresyl chloride activated Sepharose) having antibody coupled thereto.
  • a solid support material e.g. tresyl chloride activated Sepharose
  • the binding sites of the immobilised antibody are saturated with a labeled form of the target and then a liquid sample containing the target to be determined is flowed through the support thereby causing the target to displace the antigen (i.e. the labeled target).
  • the displaced antigen may then be detected so that the amount of the target in the liquid sample may be determined.
  • the immobilised first binding agent will be an antibody capable of forming an immunological complex with the antigen.
  • the immobilised first binding agent will be an antigen.
  • the immobilised label may be provided in a number of ways.
  • the label may be associated with the analyte so that formation of the immunological complex between the immobilised binding agent and the analyte results in the label becoming immobilised on the support.
  • the immobilised complex formed between the first binding agent and a non-labeled analyte may be treated with a labeled conjugate which is capable of binding to the complex formed between the first binding agent and the analyte.
  • the label of the conjugate thus becomes immobilised on the support and may be used for determining the immobilised analyte.
  • the method of the invention has a number of advantages.
  • the support onto which the first binding agent is to be immobilised will be provided with chemical groups which will react with chemical groups on the first binding agent to form covalent linkages whereby the first binding agent becomes immobilised on the support.
  • the chemical reaction between the groups on the support and those on the first binding agent will occur preferentially (although not necessarily exclusively) to absorption of the latter on the former.
  • the support may be treated to remove passively absorbed binding agent, to block absorption sites and to "block" any chemical groups of the support which have not reacted with a binding agent.
  • the analyte may only bind to the first binding agent and given that the quantity of chemical groups on the original support was known, it is possible to provide an immobilisation support of known binding capacity.
  • a further advantage is that the label is immobilised on the support. Therefore, once the analyte has become bound to the support and the immobilised label has been provided, the immobilised label may be used for more than one determination of the amount of analyte immobilised on the support. This is an advantage for the purpose of checking the result of determination from the amount of analyte bound to the column without the need to repeat the entire experimental procedure. This is not possible with the procedure of WO-A-91/13354 since the labeled antigen is eluted from the column.
  • the column may, for example, have a bed volume (i.e. the volume occupied by the support material) of 1-100 microlitres.
  • the solid support (to which the first binding agent is covalently bound) is preferably a particulate material, e.g. having a size in the range of 3-200 microns (preferably 100 to 200 microns).
  • the use of a flow through column and the particulate supports (with first binding agent immobilised thereto) have a number of advantages.
  • the particulate supports provide a relatively high surface area (e.g. in excess of lm 2 g "1 ) which allows relatively large amounts of first binding agent available for binding with an analyte in the sample under investigation.
  • a relatively large volume of a sample containing only a low concentration of analyte may be passed through the column to build up readily detectable quantities of immobilised immunological complexes (e.g. antibody-antigen). Therefore the apparatus is capable of handling relatively large sample volumes and/or relatively low analyte concentrations.
  • immobilised immunological complexes e.g. antibody-antigen
  • a further advantage of the invention is that, when desired, the column may be washed to ensure a "clean" column on which procedures may be effected.
  • the column may be washed to remove any unbound analyte species so that these do not act as interferents in a subsequent detection operation. If the label is provided on a conjugate of the complex formed between the analyte and the first binding agent then the column may be washed after treatment with the conjugate to remove non-immobilised label.
  • the first binding agent which will generally be an antigen or an antibody
  • the first binding agent which will generally be an antigen or an antibody
  • the support to which the first binding agent is to be covalently linked comprises epoxy groups.
  • Preferred supports for use in immobilising the first binding agent are of silica and have a siloxane matrix to which the epoxy groups are covalently linked.
  • Suitable supports (having a siloxane matrix to which are linked epoxy groups) are disclosed in WO-A- 93/13220 (Tepnel).
  • Supports provided with epoxy groups are particularly convenient for use in immobilising the first binding agent by means of a condensation reaction between the epoxy groups and functional groups on the first binding agent.
  • the latter groups may react with epoxy groups of the support under relatively mild conditions, e.g. in buffered solution at ambient temperature.
  • a blocking agent to displace any passively absorbed blocking agent and to block any unreacted functional groups of the support.
  • a particularly convenient blocking agent is a partially hydrolysed gelatin, e.g. BycoA as available from Sigma.
  • An alternative blocking agent is BSA.
  • the label which is immobilised to the support is preferably an enzyme label, e.g. Horse Radish Peroxidase. It will however be appreciated that a wide range of other enzymes may be used as well understood by those skilled in the art.
  • an enzyme label e.g. Horse Radish Peroxidase.
  • the amount of immobilised antigen is determined by introducing a substrate for the enzyme onto the column, allowing a detectable change (e.g. a colour change) to develop and then measuring the change.
  • the method of the invention may be used for the determination of a wide range of antibodies.
  • antigens include Gliadin, Thyroid Hormone, Pituitory Hormone, hormonenising Hormone (LTH), and Follicle Stimulating Hormone (FSH). Many other examples will be apparent to those skilled in the art.
  • Fig. 1 schematically illustrates one embodiment of apparatus for effecting the method of the invention
  • Fig. 2 illustrates (to a much enlarged scale) one of the particles incorporated in the column of Fig. 1;
  • Fig. 3 indicates the formation of antigen-antibody complexes on the particle of Fig. 2;
  • Fig. 4 illustrates a detection procedure
  • FIG. 1 there is illustrated a flow through column 1 packed with particles 2.
  • the column has inlet lines 3 along which reagents, for wash solutions and
  • SUBSTTTUTE SHEET (RULE 26) samples may be provided as necessary.
  • An outlet line 4 from the column selectively communicates with a waste line 5 or a detector 6.
  • a heater 7 is provided for heating the column, if necessary.
  • the particles 2 may for example be of solid (non-porous) silica and have a size in the range 100 to 200 microns.
  • the particles 2 are each provided with a siloxane matrix 8 (as depicted by the dashed line) which may be formed by treating the particles with 3-glycidoxypropyl trimethoxy silane as described more fully in WO-A-93/13220.
  • the resultant siloxane matrix has free epoxy groups which may be used for immobilising antibodies 9 onto the particles 2 (see the enlarged schematic view of the particle 2 in Fig. 3).
  • the immobilisation may be effected by reaction of amino, hydroxyl or carboxyl groups on the protein.
  • each particle 2 will be bonded to a large number of antibodies 9 (e.g. there may be nano to micromoles of bound protein per gram). Moreover the particles together provide a surface area of the order of about 1 m 2 /gram so that there are very large numbers of antibodies available for use in the analysis procedure described below.
  • the antibodies 9 will be capable of forming an antibody-antigen complex with a specific antigen, e.g. a virus or a hormone (such as thyroid hormone) and as such the column 1 (containing the particles 2) may be used for detecting the presence of that specific antigen in a sample using the following procedure.
  • a specific antigen e.g. a virus or a hormone (such as thyroid hormone)
  • the column 1 containing the particles 2 may be used for detecting the presence of that specific antigen in a sample using the following procedure.
  • the particles 2 may be washed by introducing a suitable wash solution into the column to ensure that the column is "clean" and does not contain any unwanted interferents.
  • the sample to be investigated may now be prepared using known procedures and introduced onto the column so that antigen 10 (if present in the sample) forms antibody-antigen complexes with the antibodies 9.
  • the antibody-antigen complexes (immobilised on the particles 2) are depicted schematically in Fig. 3.
  • a conjugate is for the antibody- antigen complex described above.
  • the conjugate (which is linked to an enzyme as shown - see Fig. 4) becomes bound to the previously form antibody-antigen complex (to which the conjugate is in effect an antibody).
  • the column may be washed once again to remove any unbound material.
  • a substrate for the enzyme is introduced onto the column.
  • the enzyme substrate combination is such that a detectable change will be produced.
  • liquid present in the column may be passed to a microtitre tray or well, optionally treated with a "stop" solution so as to stabilise the change and subjected to detection by known procedures.
  • the change may, for example, be one which can be detected spectroscopically and may be one allowing a quantitative determination of the amount of antigen present in the original sample.
  • the invention is illustrated by the following non-limiting Example.
  • a solid silica support prepared as disclosed in Example 1 of WO-A-93/ 13220 so as to have a siloxane matrix linked to epoxy groups.
  • Rabbit Anti-Gliadin was diluted with the bicarbonate buffer at a ratio of RAG:buffer of 1 :1000. 50 mg of the support was then mixed with the RAG solution, and reaction between the epoxy groups of the supports and amino groups of the RAG allowed to proceed for 2.5 hours at ambient temperature.
  • Approximately 10 mg of the support was then provided (between two frits) in a column having a volume of 200 ⁇ l.
  • the amount of the support was such that the volume of the support (between the frits) was about 50 ⁇ l.
  • the column was then washed with the bicarbonate buffer at a rate of 15 mis per minute for 15 seconds.
  • the supports on the column were treated overnight at 4°C with 100 ⁇ l of the BycoA solution (i.e. (5) above).
  • the column was then washed with the bicarbonate buffer at a rate of 2.5 ml/minute for 15 seconds.
  • TMPa and TMPb (the substrate for HRP) were then aspirated into the column and incubated for 2 minutes.
  • a volume of 100 ⁇ l was then dispersed from the column into a microtitre well and 100 ⁇ l of 0.1 m sulphuric acid (acting as a stop solution to provide a stable colour), were added to the well.
  • the optical density of the solution in the well was determined at 490 nm and was found to be 0.54 as compared to a volume of 0.24 obtained on a "blank" experiment conducted as set out above save that the Gliadin-HRP conjugate was not added to the column.
  • the Example demonstrates that the RAG had become covalently linked to the support and could be used for the determination of Gliadin.

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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)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Procédé permettant d'effectuer la détermination immunologique d'un élément à analyser placé dans un échantillon de liquide. Ce procédé comprend les étapes suivantes: (i) utilisation d'une colonne en matériau de support solide possédant un premier agent de liaison lié par covalence au matériau de support solide et capable de former un complexe immunologique avec ledit élément à analyser, (ii) introduction de l'échantillon liquide dans la colonne afin de permettre à l'élément à analyser (s'il est présent) de former un complexe immunologique avec ledit premier agent de liaison, (iii) utilisation d'un marqueur lié au complexe immunologique formé en (ii), et (iv) utilisation du marqueur immobilisé in situ sur le support afin de déterminer l'élément à analyser.
PCT/GB1996/001393 1995-06-08 1996-06-10 Determinations immunologiques WO1996042017A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP96917588A EP0834075A1 (fr) 1995-06-08 1996-06-10 Determinations immunologiques
AU60112/96A AU6011296A (en) 1995-06-08 1996-06-10 Immunological determinations

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9511586.1A GB9511586D0 (en) 1995-06-08 1995-06-08 Immunological determinations
GB9511586.1 1995-06-08

Publications (1)

Publication Number Publication Date
WO1996042017A1 true WO1996042017A1 (fr) 1996-12-27

Family

ID=10775705

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1996/001393 WO1996042017A1 (fr) 1995-06-08 1996-06-10 Determinations immunologiques

Country Status (5)

Country Link
EP (1) EP0834075A1 (fr)
AU (1) AU6011296A (fr)
CA (1) CA2224157A1 (fr)
GB (1) GB9511586D0 (fr)
WO (1) WO1996042017A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7410808B1 (en) 2003-12-08 2008-08-12 Charm Sciences, Inc. Method and assay for detection of residues
US9008373B2 (en) 2010-05-06 2015-04-14 Charm Sciences, Inc. Device, system and method for transit testing of samples

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0023989A1 (fr) * 1979-07-28 1981-02-18 Medac Gesellschaft für klinische Spezialpräparate mbH Essai immunoenzymatique pour la détection d'anticorps spécifiques aux pathogènes et trousse de réactifs pour sa mise en oeuvre
GB2109931A (en) * 1981-09-30 1983-06-08 Amano Pharma Co Ltd Enzyme immunoassay
WO1986003589A1 (fr) * 1984-12-07 1986-06-19 Pharmacia Ab Procede, appareil et systeme pour effectuer une analyse d'affinite biospecifique a l'aide d'une colonne munie d'une partie temoin
WO1987007384A1 (fr) * 1986-05-30 1987-12-03 Quidel Dispositif d'analyse immunologique enzymatique
EP0274198A2 (fr) * 1986-11-10 1988-07-13 Unilever Plc Kit d'essai immunologique
WO1992015674A2 (fr) * 1991-03-02 1992-09-17 Tepnel Medical Limited Enzymes modifiant l'adn ou l'arn immobilise sur un support
WO1993013220A1 (fr) * 1991-12-24 1993-07-08 Tepnel Medical Limited Manipulation de sequences d'acides nucleiques____________________

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0023989A1 (fr) * 1979-07-28 1981-02-18 Medac Gesellschaft für klinische Spezialpräparate mbH Essai immunoenzymatique pour la détection d'anticorps spécifiques aux pathogènes et trousse de réactifs pour sa mise en oeuvre
GB2109931A (en) * 1981-09-30 1983-06-08 Amano Pharma Co Ltd Enzyme immunoassay
WO1986003589A1 (fr) * 1984-12-07 1986-06-19 Pharmacia Ab Procede, appareil et systeme pour effectuer une analyse d'affinite biospecifique a l'aide d'une colonne munie d'une partie temoin
WO1987007384A1 (fr) * 1986-05-30 1987-12-03 Quidel Dispositif d'analyse immunologique enzymatique
EP0274198A2 (fr) * 1986-11-10 1988-07-13 Unilever Plc Kit d'essai immunologique
WO1992015674A2 (fr) * 1991-03-02 1992-09-17 Tepnel Medical Limited Enzymes modifiant l'adn ou l'arn immobilise sur un support
WO1993013220A1 (fr) * 1991-12-24 1993-07-08 Tepnel Medical Limited Manipulation de sequences d'acides nucleiques____________________

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7410808B1 (en) 2003-12-08 2008-08-12 Charm Sciences, Inc. Method and assay for detection of residues
US7863057B2 (en) 2003-12-08 2011-01-04 Charm Sciences, Inc. Method and assay for detection of residues
US8592171B1 (en) 2003-12-08 2013-11-26 Charm Sciences, Inc. Method and assay for detection of residues
US9063137B2 (en) 2003-12-08 2015-06-23 Charm Sciences, Inc. Method and assay for detection of residues
US9008373B2 (en) 2010-05-06 2015-04-14 Charm Sciences, Inc. Device, system and method for transit testing of samples

Also Published As

Publication number Publication date
CA2224157A1 (fr) 1996-12-27
EP0834075A1 (fr) 1998-04-08
AU6011296A (en) 1997-01-09
GB9511586D0 (en) 1995-08-02

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