WO1991017427A1 - Procede de determination d'un ligand specifique dans un echantillon fluide a l'aide d'un champ evanescent, et composant du materiel de mesure necessaire a cette fin - Google Patents

Procede de determination d'un ligand specifique dans un echantillon fluide a l'aide d'un champ evanescent, et composant du materiel de mesure necessaire a cette fin Download PDF

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Publication number
WO1991017427A1
WO1991017427A1 PCT/NL1991/000073 NL9100073W WO9117427A1 WO 1991017427 A1 WO1991017427 A1 WO 1991017427A1 NL 9100073 W NL9100073 W NL 9100073W WO 9117427 A1 WO9117427 A1 WO 9117427A1
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WO
WIPO (PCT)
Prior art keywords
ligand
optical element
particle form
supports
binding partners
Prior art date
Application number
PCT/NL1991/000073
Other languages
English (en)
Inventor
Richardus Bernardus Maria Schasfoort
Original Assignee
Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno
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 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno filed Critical Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno
Priority to EP91909534A priority Critical patent/EP0600862A1/fr
Priority to JP3509330A priority patent/JPH06502717A/ja
Publication of WO1991017427A1 publication Critical patent/WO1991017427A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons
    • 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
    • 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

  • the invention relates to a method for determining a ligand in a fluid sample by bringing the sample into contact with a surface of an optical element which is capable of generating an evanescent field and which surface is provided with a specific binding partner for the ligand to be determined; irradiating the optical element with light of a wavelength suitable for production of the evanescent field; and analysing the reflected light in order to determine the possibly modified evanescent field characteristics of the optical element as a consequence of the formation of a complex between the ligand and the specific binding partner.
  • SPR surface plasmon resonance
  • a method of this type is disclosed in International Patent
  • Fig. 1 (1) represents a shallow well in a clear plastic material, the flat base (2) of which is provided at the surface with a diffraction grating (3).
  • the upper surface of the grating (3) is covered with a semi-reflecting material or a metal film, such as, for example, of silver, aluminium, copper or gold.
  • the grating is coated with a thin film of specific binding partners for the ligand to be determined.
  • specific binding partners for the ligand for example, anti- gens can be used as ligand and antibodies for the particular antigen as specific binding partners (see Fig. 2) .
  • the determination as such takes place in a housing (4) which is shown in Fig. 1 and which is provided with a laser light source (5) , from which laser light is directed at a suitable angle onto the underside of the base (2) in order to generate the "surface plasmon resonance" (SPR) effect in the grating (3) .
  • the reflected light is captured via a light detector (6), which is also fitted in the housing (4), and the electrical signal originating from detector (6) is processed in a measuring apparatus (7) , which may or may not be fitted in the housing (4).
  • the signal originating from detector (6) changes if the relevant ligand, which binds to the specific binding partners bound to the metal layer, is present in the sample (8) , which is placed in the well (1) .
  • the aim indicated above can be achieved if, in the method described in the preamble, the sample is used in combination with a suspension of supports in particle form, which supports carry the ligand (competition assay; see Fig. 3a and in particular 3*>) •
  • the aim according to the invention can also be achieved if, in the method described in the preamble, an optical element is used of which the surface located at the fluid/solid interface is provided with a multiplicity of supports in particle form, which supports contain binding partners specific for the ligand (see Fig. ).
  • the abovementioned aim can likewise be achieved with the aid of an optical element of which the surface located at the fluid/ solid interface contains a multiplicity of specific binding partners which are provided with a "leaving agent" in the form of particles reversibly bound to said specific binding partners (see Fig. 5) •
  • the term "leaving agent" is defined as a support in particle form which is provided with at least one ligand.
  • the abovementioned aim can also be achieved with the aid of an optical element of which the surface located at the fluid/solid interface contains a multiplicity of binding partners specific for the ligand, some of which are provided with the ligand and some with the "leaving agent", in accordance with an equilibrium setting with a medium, which is advantageously circulated, in which the ligand and the "leaving agent" are present in a constant concentration, this equilibrium setting temporarily shifting after administration of a sample to be investigated and giving a corresponding change in the evanescent field (Fig. 6a) .
  • An important advantage of this last-mentioned solution lies in the fact that in this way "continuous" measurements can be carried out using, for example, SPR measuring equipment (Fig.
  • a membrane which is permeable to the ligand to be measured but impermeable to the "leaving agent” is preferably inserted between the optical element and the location at which the sample to be investigated is supplied (Fig. 7).
  • the above ⁇ mentioned medium, which is advantageously circulated has to contain only the relevant ligand in a constant concentration since the "leaving agent" cannot escape from the space between the membrane and the surface of the optical element.
  • a particular variant of the first-mentioned solution consists in the use of an optical element of which the surface located at the fluid/solid interface is provided with a multiplicity of supports in particle form which contain binding partners specific for the ligand, these specific binding partners also being provided with a "leaving agent" in the form of a particle reversibly bound to said specific binding partners (see Fig. 8) .
  • solu ⁇ tions all relate to increasing the change in mass per unit surface area of the optical element, so that it is a matter of "unity of invention” .
  • the optical element used in the method according to the invention consists of a plastic material or glass having a suitable refractive index, which is preferably coated with a metal layer of silver or gold, the support particles loaded with specific binding partners being applied to the metal layer.
  • the thickness of the metal layer is usually 10-100 nm and can be applied in a known manner, such as by vapour deposition and the like.
  • the optical element is usually in the form of a platelet of a specific thick ⁇ ness.
  • optical fibres and planar light conductors can also be used as optical element.
  • support particles can be used as supports in particle form within the framework of the invention.
  • latex spheres which have a diameter in the range of, for example, 10-500 nm are used as support.
  • suitable materials are, inter alia, (monodisperse) polystyrene latex, poly- methyl methacrylate latex or a silica latex.
  • latices are Unisphere latex particles having a diameter of 0 nm (type 10) or 100 nm (type 11) (Brunschwig Chemie B.V., The Nether ⁇ lands) and "Polybead" polystyrene microspheres having a diameter of 50, 100 or 200 nm (Polysciences Corp., Niles/Illinois, USA).
  • Metal sols sol particles
  • gold sols, silver sols and the like can also be used as support in particle form. It is also possible to use combinations of particles, such as, for example, particles of different size, particles containing different functional groups, particles having different intrinsic refractive indices and also latex with a metal sol.
  • the combination of a latex with a metal sol can provide extra sensitivity and specificity in connection with overlap of two evanescent fields, that is to say the evanescent field generated with the apparatus and the evanescent field generated by the metal particle.
  • the "degree of coverage" of the surface of the optical element, which is provided with the loaded supports in particle form can vary greatly.
  • the degree of coverage depends on the size of the support particles or on the type of specific binding partner, such as antibodies and DNA strands.
  • the degree of coverage is 50-99•5 *•
  • “degree of coverage” of the surface of the optical element by the (“spherical") supports in particle form is understood to mean the ratio of the sum of the sur ⁇ face area of the cross-section of the "spherical" supports multiplied by a factor of 100, divided by the total surface area of the optical element.
  • the “degree of loading” of the specific binding partners on the spherical supports is understood to be the ratio of the loaded surface area, that is to say the surface area covered by the specific binding partners, of the spherical supports multiplied by a factor of 100, divided by the total surface area of the spherical supports in the monolayer or sub-monolayer. In general, this degree of loading is 10-90#.
  • a similar "degree of loading" can be quoted for the ligand on the spherical supports (see definition of "leaving agent”) .
  • the immobilisation of the specific binding partners or ligands on the latex spheres is a phenomenon known per se.
  • a general description of the technique is given in "Micro- particle Immunoassay Techniques" (Seradyn Inc., ed. Galloway and Hicks, Particle Technology Division, P.O. Box 1210, Indianapolis, IN 46206, USA).
  • the supports loaded with specific binding partners can be bound to the metal layer of the optical element by means of absorption or by means of the application of a thin layer of adhesive or sticky layer of another type via a dip technique, spin coating or other generally known deposition techniques.
  • the above procedure can be carried out in reverse by first carrying out the adhesion of the spheres to the metal layer of the optical element and then the immobilisation of the specific binding partners on the spheres acting as supports.
  • the supports in particle form can also be applied to the metal surface via a flat coating, such as a polystyrene coating, that is to say the supports in particle form are immobilised in an indirect manner on the metal surface.
  • the sensitivity of the measuring system can be optimised using combinations of supports in particle form, antibodies and antigens. This is dependent on the application which is developed.
  • the supports in particle form act as an interface between the biological domain and the optical domain.
  • the light sources used can be the light sources known from the prior art, such as the helium-neon laser light source and the infrared diode laser light source.
  • the invention also relates to an optical element, suitable for use in an SPR measuring apparatus and consisting of a transparent platelet or fibre made of plastic or glass, a single surface of which is coated with a metal layer of gold or silver, which metal layer is provided with an amount of support-s in particle form, applied thereon, which contain specific binding partners; details in this regard are given above.
  • FIG. 3b is a diagrammatic representation of the "competition assay" in respect of the binding of a mixture of ligands and supports in particle form provided with a ligand to antibodies which are bound to the metal surface (see curves c and a of Fig. 10 for the order of size in respect of the difference in measured angle) .
  • Fig. 4 diagrammatic representation of the method according to the invention, where the specific binding partner is located on supports in particle form adhering to the surface of the optical element and binds to ligands.
  • Fig. 5 diagrammatic representation of the method according to the invention, in which the "leaving agent" is located on specific binding partners for the ligand adhering to the surface of the optical element and is removed by the ligand via a displacement reaction.
  • Fig. 6 diagrammatic representation of the method according to the invention, in which an equilibrium setting is shown between the specific binding partners adhering to the surface of the optical element (9) and the ligand and "leaving agent" present in the medium.
  • Fig. 6a(0 ⁇ 3) show time points in respect of a measurement
  • Fig. 6a(0) representing the equilibrium phase at time t 0
  • Fig. 6a(l) representing the sampling at time t_
  • Fig. 6a(2) representing the equilibrium phase changed because of the sampling, at time t 2
  • Fig. 6a(3) representing the restored equilibrium phase (as at t 0 ) at time t 3 .
  • Fig. diagrammatic representation of the method according to the invention, in which a membrane (10) which is permeable to the ligand but impermeable to the "leaving agent" is fitted between the surface of the optical element (9) with the specific binding partners adhering thereto and the location at which the sample to be investigated is supplied.
  • Fig. 8 diagrammatic representation of the method according to the invention, in which the specific binding partners for the ligand are present on the spheres adhering to the surface of the optical element and in which the specific binding partners for the ligand itself are provided with a "leaving agent", which "leaving agent” is removed by the ligand via a displacement reaction.
  • Fig. shows an optical system which is used for generating surface plasmon resonance effects, as shown in Fig. 10. In this Fig. 9.
  • (11) represents a prism, (12) an incident ray of polarised light, (13) the reflected light ray, (14) a glass substrate, (15) a gold layer applied to the substrate, (16) a layer of specific binding partners, or latex spheres loaded with specific binding partners according to the invention, applied to the gold layer and
  • Fig. 10 shows a graph of the measurement results obtained using SPR (see example below) , where
  • Fig. 11 represents a graph of a competition assay between a blank hCG-coated latex on the one hand (curve a) and a 250 IU hCG & hCG-coated latex mixture on the other hand (curve b) in a SPR measuring apparatus, in which assay poly- clonal anti-hCG applied to the gold layer acted as anti ⁇ body.
  • Fig. 12 is a graph of a SPR measurement where represents Biocryl spheres represents; represents blank (Biocryl spheres with PCR mixture but without DNA fragments); represents Biocryl spheres with PCR mixture and DNA fragments.
  • the measurement set-up shown in Fig. 9 was used in this example.
  • the gold layer was first provided with a coating.
  • HSA human serum albumin
  • Polybead polystyrene (2.5% solids) carboxylated micro- spheres (100 nm) (Polysciences Corp., Niles/Ill. USA) were then applied to the abovementioned coated gold layer. After 10 min the metal surface was rinsed with PBS.
  • the resulting product was then rinsed with a borate buffer (Polysciences Corp., Niles/Ill., USA).
  • protein HSA; Sigma A 3782
  • a solution containing anti-HSA (Sigma, no. A 1151) was dripped onto the coated gold layer. The product was then incubated for 1 hour. It was then rinsed with PBS. The relevant measurements were carried out with the aid of a measurement apparatus as shown in
  • curve (a) shows the measurement result for HSA applied directly to the gold layer coated with polystyrene; this layer was prepared in the above manner omitting the steps involving the latex spheres. It can be seen from this figure that the sensitivity of the gold layer coated with latex spheres has been appreciably increased (c ⁇ d) compared with the uncoated gold layer (a -> b) .
  • a competition mixture was used for the detection, by mixing a hCG-coated latex solution (0.01% solids concentration) with an hCG solution of variable concentration.
  • the blank contains no free hCG.
  • the latex and hCG were diluted in 0.1% gelatine in
  • PBS/Tween 20 200 ⁇ l of solution were applied to the gold platelet.
  • Biocryl spheres (Toso Haas, size 0.1 um, type Biocryl BPA 1000)
  • DNA material DNA from PVA plasmid
  • the aim of the experiment described in this example is to investigate whether it is possible to measure DNA using Biocryl spheres which, because of their positive charge, display a high affinity for DNA. Since Biocryl spheres in PBS buffer themselves already ensure a large angle shift in SPR experiments and DNA ad ⁇ heres to these spheres with screening of the charge of the Biocryl spheres, this DNA screening should give rise to a smaller shift in angle.
  • the experiment was carried out as follows. 1 ml of PBS was placed in two Eppendorf tubes. A number of Biocryl spheres (in total 1 ⁇ l, 1.5 ⁇ l, 2.0 ⁇ l and 2.5 ⁇ l) diluted 10-fold were then added to each tube. The contents of the tubes were mixed using a vortex stirrer. A PCR mixture containing DNA (35 ⁇ l) was then added to one tube. This tube was mixed well using a vortex stirrer. Finally, the contents of both tubes were transferred to two cuvettes, after which the SPR determinations were carried out. On the basis of photographs of the gel, the DNA concentration was estimated to be 2, 3 ⁇ 4 and 6 ⁇ g/ml. The length of the DNA fragments was in the range of from 400- 2900 bp. A blank PCR mixture, that is to say the PCR mixture, but no DNA, was present, served as blank determination in this example.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
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  • Molecular Biology (AREA)
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Abstract

Procédé de détermination d'un ligand dans un échantillon fluide consistant à mettre l'échantillon en contact avec la surface d'un élément optique apte à générer un champ évanescent, ladite surface étant dotée d'un partenaire de liaison spécifique pour le ligand à déterminer; à irradier l'élément optique avec de la lumière dont la longueur d'onde est adaptée pour la production du champ évanescent; et à analyser la lumière réfléchie afin de déterminer toutes modifications des caractéristiques du champ évanescent de l'élément optique qui seraient dues à la formation d'un complexe entre le ligand et le partenaire de liaison spécifique. Le procédé est caractérisé par l'utilisation d'une pluralité de supports sous forme particulaire qui portent le ligand ou des partenaires de liaison spécifiques pour celui-ci.
PCT/NL1991/000073 1990-05-02 1991-05-01 Procede de determination d'un ligand specifique dans un echantillon fluide a l'aide d'un champ evanescent, et composant du materiel de mesure necessaire a cette fin WO1991017427A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP91909534A EP0600862A1 (fr) 1990-05-02 1991-05-01 Procede de determination d'un ligand specifique dans un echantillon fluide a l'aide d'un champ evanescent, et composant du materiel de mesure necessaire a cette fin
JP3509330A JPH06502717A (ja) 1990-05-02 1991-05-01 無限小場を用いて流体サンプル中の特異的リガンドを測定する方法、およびまたこの目的に適切な必須測定装置構成要素

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NL9001052A NL9001052A (nl) 1990-05-02 1990-05-02 Werkwijze voor het bepalen van een specifiek ligand in een vloeibaar monster met behulp van een evanescent veld, alsook een daarvoor geschikt onderdeel van de benodigde meetinrichting.
NL9001052 1990-05-02

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WO1991017427A1 true WO1991017427A1 (fr) 1991-11-14

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EP (1) EP0600862A1 (fr)
JP (1) JPH06502717A (fr)
AU (1) AU7871491A (fr)
NL (1) NL9001052A (fr)
WO (1) WO1991017427A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0597503A2 (fr) * 1992-11-13 1994-05-18 Amgen Inc. Ligands de récepteurs ECK
DE4307042A1 (de) * 1993-03-05 1994-09-08 S & L Ges Fuer Wissenschaftlic Verfahren zum optischen qualitativen und quantitativen Nachweis von Molekülen, Biomolekülen und Mikroorganismen
WO1996036713A2 (fr) * 1995-05-19 1996-11-21 Amgen Inc. Ligands pour le recepteur eck
USRE44693E1 (en) 1996-11-06 2014-01-07 Sequenom, Inc. Beads bound to a solid support and to nucleic acids

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE1817629A1 (de) * 1968-01-02 1969-08-21 Philips Nv Optisches Element zur Detektion von Aerosolen unter Verwendung von innerer Reflexion
US3979184A (en) * 1975-05-27 1976-09-07 General Electric Company Diagnostic device for visually detecting presence of biological particles
US4054646A (en) * 1973-07-30 1977-10-18 General Electric Method and apparatus for detection of antibodies and antigens
WO1988007202A1 (fr) * 1987-03-10 1988-09-22 Ares-Serono Research & Development Limited Partner Technique amelioree d'analyse et appareil prevu a cet effet
EP0326375A2 (fr) * 1988-01-27 1989-08-02 Ortho Diagnostic Systems Inc. Système d'immunoessai
EP0341928A1 (fr) * 1988-05-10 1989-11-15 AMERSHAM INTERNATIONAL plc Capteurs à résonance de plasmons de surface
EP0353937A1 (fr) * 1988-07-25 1990-02-07 Applied Research Systems Ars Holding N.V. Procédé d'essai

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE1817629A1 (de) * 1968-01-02 1969-08-21 Philips Nv Optisches Element zur Detektion von Aerosolen unter Verwendung von innerer Reflexion
US4054646A (en) * 1973-07-30 1977-10-18 General Electric Method and apparatus for detection of antibodies and antigens
US3979184A (en) * 1975-05-27 1976-09-07 General Electric Company Diagnostic device for visually detecting presence of biological particles
WO1988007202A1 (fr) * 1987-03-10 1988-09-22 Ares-Serono Research & Development Limited Partner Technique amelioree d'analyse et appareil prevu a cet effet
EP0326375A2 (fr) * 1988-01-27 1989-08-02 Ortho Diagnostic Systems Inc. Système d'immunoessai
EP0341928A1 (fr) * 1988-05-10 1989-11-15 AMERSHAM INTERNATIONAL plc Capteurs à résonance de plasmons de surface
EP0353937A1 (fr) * 1988-07-25 1990-02-07 Applied Research Systems Ars Holding N.V. Procédé d'essai

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0597503A2 (fr) * 1992-11-13 1994-05-18 Amgen Inc. Ligands de récepteurs ECK
EP0597503A3 (fr) * 1992-11-13 1995-05-24 Amgen Inc Ligands de récepteurs ECK.
US6087167A (en) * 1992-11-13 2000-07-11 Amgen Inc. Eck receptor ligands
US6479250B1 (en) 1992-11-13 2002-11-12 Amgen Inc. Eck receptor ligands
DE4307042A1 (de) * 1993-03-05 1994-09-08 S & L Ges Fuer Wissenschaftlic Verfahren zum optischen qualitativen und quantitativen Nachweis von Molekülen, Biomolekülen und Mikroorganismen
WO1996036713A2 (fr) * 1995-05-19 1996-11-21 Amgen Inc. Ligands pour le recepteur eck
WO1996036713A3 (fr) * 1995-05-19 1997-05-29 Amgen Inc Ligands pour le recepteur eck
USRE44693E1 (en) 1996-11-06 2014-01-07 Sequenom, Inc. Beads bound to a solid support and to nucleic acids

Also Published As

Publication number Publication date
EP0600862A1 (fr) 1994-06-15
JPH06502717A (ja) 1994-03-24
AU7871491A (en) 1991-11-27
NL9001052A (nl) 1991-12-02

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