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EP0489074A1 - Assay method for determining an analyte using surface plasmon resonance spectrometry (SPRS) - Google Patents

Assay method for determining an analyte using surface plasmon resonance spectrometry (SPRS)

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Publication number
EP0489074A1
EP0489074A1 EP90912817A EP90912817A EP0489074A1 EP 0489074 A1 EP0489074 A1 EP 0489074A1 EP 90912817 A EP90912817 A EP 90912817A EP 90912817 A EP90912817 A EP 90912817A EP 0489074 A1 EP0489074 A1 EP 0489074A1
Authority
EP
European Patent Office
Prior art keywords
analyte
ligand
plasmon resonance
receptor pair
surface plasmon
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Withdrawn
Application number
EP90912817A
Other languages
German (de)
French (fr)
Inventor
Peter Bryan Garland
Stephen Alexander Charles
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GE Healthcare Ltd
Original Assignee
Amersham International PLC
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 Amersham International PLC filed Critical Amersham International PLC
Publication of EP0489074A1 publication Critical patent/EP0489074A1/en
Withdrawn legal-status Critical Current

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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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • 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
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated

Definitions

  • This invention concerns a method of assaying for a macromolecular analyte by use of the technique of surface plasmon resonance spectrometry (SPRS) .
  • SPRS surface plasmon resonance spectrometry
  • the intensity of monochromatic plain-polarised light (conveniently obtained from a laser) reflected from the interface between an optically transparent material, e.g. glass, and a thin metal layer depends on the refractive index of material on the downstream side of the metal. Accordingly, by measuring changes in intensity of the reflected light an indication can be obtained of changes in refractive index of material at a particular point on the down-stream surface of the metal.
  • the intensity of reflected light also varies with the angle of incidence, and reflectivity drops sharply to a minimum at a particular angle which is characteristic of the equipment.
  • WO 89/08260 describes a method of analysing for an analyte in a ⁇ ...nple by bringing the sample into contact with a metal surface, on which an antibodv has previously been reversibly bound to immobilised analyte or analogue, and monitoring displacement of antibody as indicative of the presence or the concentration of the analyte in the sample. That specification is mainly concerned with hapten analytes and describes only hapten-antibody and antigen- antibody binding pairs.
  • EPA 276142 describes competition assays involving an analyte and two other reagents, in which SPRS is used to monitor the formation of a complex on a solid surface.
  • the assay involves the use of at least one other liquid reagent in addition to the sample.
  • the signal resulting from complex formation on the surface may be obscured by noise resulting from non-specific binding of other macromolecules to the surface.
  • This invention provides a method of assaying for an analyte, preferably a macromolecular analyte, which is a member of a ligand-receptor pair other than a hapten-antibody or an antigen-antibody pair, by the use of a metal surface adapted for surface plasmon resonance spectrometry which metal surface carries the analyte or an analogue thereof immobilised thereon with the other member of the ligand-receptor pair reversibly bound thereto, which method comprises bringing a fluid containing the analyte into contact with the metal surface and observing by surface plasmon resonance spectrometry displacement of the other member of the ligand-receptor pair from the surface.
  • the analyte is a member of a ligand- receptor pair. Many example ⁇ of such pairs are known and include the following:
  • protein is used to 0 include peptides.
  • the method involves the use of a metal surface adapted for surface plasmon resonance spectrometry.
  • the metal may comprise silver or gold, conveniently in the form of a layer e.g. deposited by evaporation on a 5 carrier such as a glass slide.
  • the metal surface carries the analyte or an analogue thereof immobilised thereon.
  • An analogue of the analyte is a substance which competes with the analyte for binding to the other member of the ligand-receptor pair. Often the 0 analogue will be arranged to be as near as possible or even completely identical to the analyte.
  • the use in assays of analyte analogues is well known.
  • Binding of the analyte or analogue to the metal surface without loss of binding power is effected by methods which are well known. Particularly when the analyte or analogue is of low molecular weight, the use of a spacer molecule may be required. To prevent non ⁇ specific binding at a later stage in the assay, any surplus area of the metal surface may be coated e.g. ° with an inert protein.
  • this invention provides an assay device comprising a metal surface adapted for 5 surface plasmon resonance spectrometry, which surface' carries immobilised thereon a member of a ligand- receptor pair, other than a hapten-antibody or an antigen-antibody pair, with the other member of the ligand-receptor pair reversibly bound thereto.
  • That other member of the ligand-receptor pair may have been modified to increase the signal generated to increase the SPR signal generated by its removal from the metal surface. Such modification may involve addition of a molecule or group of low refractive index, or more preferably high refractive index such as polystyrene, titanium dioxide or gold colloid.
  • the method of the invention is very simple.
  • a fluid containing the analyte is brought into contact with the pre-coated metal surface.
  • Analyte in the fluid sample competes with immobilised analyte for binding to the other member of the ligand-receptor pair.
  • Displacement of the other member of the ligand-receptor pair into solution, as a complex with analyte in the sample, is monitored by surface plasmon resonance spectrometry.
  • the method has two particular advantages: (a) The only fluid reagent involved is the sample containing the analyte. When this is brought into contact with the coated metal surface, the presence or the concentration of the analyte in the sample can be assayed within minutes or even seconds. the SPRS signal, generated by removal of a reagent from the metal surface, is not significantly contaminated by noise due to non-specific binding of material to the surface.
  • oligonucleotide A (97mer) was hybridised to a complementary oligonucleotide B (17mer). Oligonucleotide B was then covalently linked to the silver surface of a slide. The slide was then blocked with hybridisation buffer. A further oligonucleotide C (50mer) which had the same sequence as part of oligonucleotide B and therefore complementary to A, was then added and displacement from the silver slide of the hybridised oligonucleotide A was measured. Results using 32P end-labelled oligonucleotide A showed that as the amount of oligonucleotide C was increased more oligonucleotide A was released. Such changes will also be measurable by SPR.
  • oli ⁇ onucleotides A sixteen mer probe and a complementary ninety-seven mer target were prepared using phosphoramidite chemistry on the Applied Biosystems Model 380D DNA synthesizer.
  • the DNA probe was modified in order to permit efficient and stable binding to the ver surface. This was prepared by attaching a terminal primary amine at the 5 ' -end of the molecule.
  • the amino-oligonucleotide was mixed with an equal volume of 0.1M sodium hydrogen carbonate solution pH 8.5 and to this mixture a solution of SPDP (0.25 mg for each 1.0 OD of oligo used) in DMF was added.
  • reaction was left to equilibriate for 90 minutes at room temperature and then eluted through a Sephadex G25 PD10 column.
  • Fractions containing the required product were pooled together and purified by preparative HPLC. After purification the appropriate fractions were dried down by vacuum centrifugation to remove HPLC solvents and then reconstituted in a known volume of water.
  • DNA hybrid consisting of the modified 16 mer and a complementary 97 mer was prepared by mixing 16 mer and 97 mer in a 1.5:1 molar ratio ensuring that the
  • the immobilisable probe 16 mer, the immobilisable probe, was in excess.
  • the preannealing was carried out at 65°C in 2x SSPE (300mM NaC1, 20mM NaH.PO.-H ⁇ , 2mM ethylenediaminetetra- acetic acid) and the mixture was allowed to cool down to room temperature for 2hrs.
  • Results demonstrate displacement of the 97 mer sequence away from the silver surface by the complementary 50 base sequence. This does not occur in the absence of this oligonucleotide nor in the presence of the non-complementary sequence which does not hybridise to the 97 mer.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Inorganic Chemistry (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Procédé d'analyse visant à déterminer la présence d'un analyte, mettant en oeuvre la spectrométrie par résonance de plasmon en surface (SRPS). L'analyte est un élément d'une paire ligand-récepteur non immune. Une surface métallique porte l'analyte ou un analogue immobilisé à l'aide de l'autre élément de la paire ligand-récepteur liée de manière réversible audit analyte. On met un fluide contenant l'analyte en contact avec la surface métallique, puis on contrôle le déplacement de l'autre élément de la paire ligand-récepteur par SRPS.Analysis method aimed at determining the presence of an analyte, using surface plasmon resonance spectrometry (SRPS). The analyte is part of a non-immune ligand-receptor pair. A metal surface carries the analyte or analog immobilized using the other element of the ligand-receptor pair linked reversibly to said analyte. A fluid containing the analyte is brought into contact with the metal surface, then the displacement of the other element of the ligand-receptor pair is controlled by SRPS.

Description

ASSAY METHCB
This invention concerns a method of assaying for a macromolecular analyte by use of the technique of surface plasmon resonance spectrometry (SPRS) .
The phenomenon of SPR is well known and will not be described in detail (see EPA 305109 for example) . Briefly, the intensity of monochromatic plain-polarised light (conveniently obtained from a laser) reflected from the interface between an optically transparent material, e.g. glass, and a thin metal layer depends on the refractive index of material on the downstream side of the metal. Accordingly, by measuring changes in intensity of the reflected light an indication can be obtained of changes in refractive index of material at a particular point on the down-stream surface of the metal. The intensity of reflected light also varies with the angle of incidence, and reflectivity drops sharply to a minimum at a particular angle which is characteristic of the equipment.
WO 89/08260 describes a method of analysing for an analyte in a ε...nple by bringing the sample into contact with a metal surface, on which an antibodv has previously been reversibly bound to immobilised analyte or analogue, and monitoring displacement of antibody as indicative of the presence or the concentration of the analyte in the sample. That specification is mainly concerned with hapten analytes and describes only hapten-antibody and antigen- antibody binding pairs.
EPA 276142 describes competition assays involving an analyte and two other reagents, in which SPRS is used to monitor the formation of a complex on a solid surface. The assay involves the use of at least one other liquid reagent in addition to the sample. The signal resulting from complex formation on the surface may be obscured by noise resulting from non-specific binding of other macromolecules to the surface.
This invention provides a method of assaying for an analyte, preferably a macromolecular analyte, which is a member of a ligand-receptor pair other than a hapten-antibody or an antigen-antibody pair, by the use of a metal surface adapted for surface plasmon resonance spectrometry which metal surface carries the analyte or an analogue thereof immobilised thereon with the other member of the ligand-receptor pair reversibly bound thereto, which method comprises bringing a fluid containing the analyte into contact with the metal surface and observing by surface plasmon resonance spectrometry displacement of the other member of the ligand-receptor pair from the surface. The analyte is a member of a ligand- receptor pair. Many example^ of such pairs are known and include the following:
Differentiation factor or analogue Protein receptor Enzyme Cofactor, substrate or inhibitor
Biotin Avidin
Enzyme Prosthetic group
Oligopeptide Protein
Immunoglobulin Protein A Drug Binding protein
In the above, the term protein is used to 0 include peptides.
The method involves the use of a metal surface adapted for surface plasmon resonance spectrometry. The metal may comprise silver or gold, conveniently in the form of a layer e.g. deposited by evaporation on a 5 carrier such as a glass slide. The metal surface carries the analyte or an analogue thereof immobilised thereon. An analogue of the analyte is a substance which competes with the analyte for binding to the other member of the ligand-receptor pair. Often the 0 analogue will be arranged to be as near as possible or even completely identical to the analyte. The use in assays of analyte analogues is well known. Binding of the analyte or analogue to the metal surface without loss of binding power is effected by methods which are well known. Particularly when the analyte or analogue is of low molecular weight, the use of a spacer molecule may be required. To prevent non¬ specific binding at a later stage in the assay, any surplus area of the metal surface may be coated e.g. ° with an inert protein.
Reversibly bound to the immobilised analyte or analogue is the other member of the ligand-receptor pair. In another aspect, this invention provides an assay device comprising a metal surface adapted for 5 surface plasmon resonance spectrometry, which surface' carries immobilised thereon a member of a ligand- receptor pair, other than a hapten-antibody or an antigen-antibody pair, with the other member of the ligand-receptor pair reversibly bound thereto. That other member of the ligand-receptor pair may have been modified to increase the signal generated to increase the SPR signal generated by its removal from the metal surface. Such modification may involve addition of a molecule or group of low refractive index, or more preferably high refractive index such as polystyrene, titanium dioxide or gold colloid.
Using this pre-formed device, the method of the invention is very simple. A fluid containing the analyte is brought into contact with the pre-coated metal surface. Analyte in the fluid sample competes with immobilised analyte for binding to the other member of the ligand-receptor pair. Displacement of the other member of the ligand-receptor pair into solution, as a complex with analyte in the sample, is monitored by surface plasmon resonance spectrometry. The method has two particular advantages: (a) The only fluid reagent involved is the sample containing the analyte. When this is brought into contact with the coated metal surface, the presence or the concentration of the analyte in the sample can be assayed within minutes or even seconds. the SPRS signal, generated by removal of a reagent from the metal surface, is not significantly contaminated by noise due to non-specific binding of material to the surface. Example 1
Displacement Assay using Nucleic Acid Probes
An oligonucleotide A (97mer) was hybridised to a complementary oligonucleotide B (17mer). Oligonucleotide B was then covalently linked to the silver surface of a slide. The slide was then blocked with hybridisation buffer. A further oligonucleotide C (50mer) which had the same sequence as part of oligonucleotide B and therefore complementary to A, was then added and displacement from the silver slide of the hybridised oligonucleotide A was measured. Results using 32P end-labelled oligonucleotide A showed that as the amount of oligonucleotide C was increased more oligonucleotide A was released. Such changes will also be measurable by SPR.
Example 2
P A Strand ispla ement y SPR
Method
Preparation of oliσonucleotides A sixteen mer probe and a complementary ninety-seven mer target were prepared using phosphoramidite chemistry on the Applied Biosystems Model 380D DNA synthesizer. The DNA probe was modified in order to permit efficient and stable binding to the ver surface. This was prepared by attaching a terminal primary amine at the 5'-end of the molecule. The amino-oligonucleotide was mixed with an equal volume of 0.1M sodium hydrogen carbonate solution pH 8.5 and to this mixture a solution of SPDP (0.25 mg for each 1.0 OD of oligo used) in DMF was added. The reaction was left to equilibriate for 90 minutes at room temperature and then eluted through a Sephadex G25 PD10 column. Fractions containing the required product were pooled together and purified by preparative HPLC. After purification the appropriate fractions were dried down by vacuum centrifugation to remove HPLC solvents and then reconstituted in a known volume of water.
Preparation of DNA Hybrid
DNA hybrid consisting of the modified 16 mer and a complementary 97 mer was prepared by mixing 16 mer and 97 mer in a 1.5:1 molar ratio ensuring that the
16 mer, the immobilisable probe, was in excess. The preannealing was carried out at 65°C in 2x SSPE (300mM NaC1, 20mM NaH.PO.-H^, 2mM ethylenediaminetetra- acetic acid) and the mixture was allowed to cool down to room temperature for 2hrs.
SPR Experiment
Using 1.8 x 1θ"10 moles of hybrid/ml, 1ml was pumped across a silver slide at 1μl/s after priming the slide with 2x SSPE. Following a wash step with 1ml of 2x SSPE the slide was blocked with hybridisation buffer followed by a 2x SSPE wash. 3 x 10 moles of a 50 mer, which is complementary in sequence to the 97 mer, in 1ml 2x SSPE was flowed across the slide at a speed of 1μl/s, and the change in reflectivity was monitored with time.
Control reactions involving no DNA and non- complementary DNA were carried out.
Data is presented below with the No DNA control value deducted from the experimental. Table 1
Target DNA change in reflectivity
No DNA control 0
50 mer -1
Non-complementary DNA +1
Results demonstrate displacement of the 97 mer sequence away from the silver surface by the complementary 50 base sequence. This does not occur in the absence of this oligonucleotide nor in the presence of the non-complementary sequence which does not hybridise to the 97 mer.

Claims

8 -CLAIMS
1. A method of assaying for an analyte which is member of a ligand-receptor pair other than a hapten- antibody or an antigen-antibody pair, by the use of a metal surface adapted for surface plasmon resonance
10 spectrometry which metal surface carries the analyte or an analogue thereof immobilised thereon with the other member of the ligand-receptor pair reversibly bound thereto, which method comprises bringing a fluid containing the analyte into contact with the metal
5 surface and observing by surface plasmon resonance spectrometry displacement of the other member of the ligand-receptor pair from the surface.
2. A method as claimed in claim 1 , wherein the analyte is a macromolecule. 0 3. An assay device comprising a metal surface adapted for surface plasmon resonance spectrometry, which surface carries immobilised thereon a member of a ligand-receptor pair, other than a hapten-antibody or an antigen-antibody pair, with the other member of 5 the ligand-receptor pair reversibly bound thereto.
30
35
EP90912817A 1989-08-25 1990-08-24 Assay method for determining an analyte using surface plasmon resonance spectrometry (SPRS) Withdrawn EP0489074A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB8919411 1989-08-25
GB898919411A GB8919411D0 (en) 1989-08-25 1989-08-25 Assay method

Publications (1)

Publication Number Publication Date
EP0489074A1 true EP0489074A1 (en) 1992-06-10

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Country Link
EP (1) EP0489074A1 (en)
JP (1) JPH05500109A (en)
CA (1) CA2063707A1 (en)
GB (1) GB8919411D0 (en)
WO (1) WO1991002981A1 (en)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9119735D0 (en) * 1991-09-16 1991-10-30 Secr Defence Gene probe biosensor method
GB9212416D0 (en) * 1992-06-11 1992-07-22 Medical Res Council Reversible binding substances
JPH11332595A (en) * 1997-07-09 1999-12-07 Masao Karube Method for detecting DNA using probe PNA
DE19927051C2 (en) 1999-06-14 2002-11-07 November Ag Molekulare Medizin Method and device for identifying a nucleotide sequence
DE19950969A1 (en) * 1999-10-22 2001-05-10 Aventis Res & Tech Gmbh & Co Double-stranded nucleic acid probes and their use
NL1014816C2 (en) * 2000-03-31 2001-10-02 Tno Reversibly binding receptor to sensor surface, useful e.g. for identifying or isolating specific ligands, by attaching to coupling agent that binds to antibody on the surface
AU2001246943A1 (en) * 2000-03-31 2001-10-30 Nederlandse Organisatie Voor Toegepast- Natuurwetenschappelijk Onderzoek Tno Method for determination of binding with natural receptors
US20100075347A1 (en) * 2006-08-18 2010-03-25 Dasaratha Sridhar V Methods of detection using acousto-mechanical detection systems

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4626513A (en) * 1983-11-10 1986-12-02 Massachusetts General Hospital Method and apparatus for ligand detection
DK531185A (en) * 1985-11-18 1987-05-19 Radiometer As SENSOR TO DETERMINE THE CONCENTRATION OF A BIOCHEMICAL SPECIES
EP0245206A1 (en) * 1986-05-05 1987-11-11 IntraCel Corporation Analytical method for detecting and measuring specifically sequenced nucleic acid
IL85137A (en) * 1987-01-21 1992-02-16 Ares Serono Res & Dev Ltd Method of assaying for a ligand using surface plasmon resonance effect
GB8804669D0 (en) * 1988-02-27 1988-03-30 Medical Res Council Immobilisation of haptens
GB8807488D0 (en) * 1988-03-29 1988-05-05 Ares Serono Res & Dev Ltd Method of assay

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9102981A1 *

Also Published As

Publication number Publication date
CA2063707A1 (en) 1991-02-26
WO1991002981A1 (en) 1991-03-07
GB8919411D0 (en) 1989-10-11
JPH05500109A (en) 1993-01-14

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