METHOD FOR DETERMINING A SPECIFIC LIGAND IN A FLUID SAMPLE WITH THE AID OF AN EVANESCENT FIELD, AND ALSO A COMPONENT OF THE REQUISITE MEASURING EQUIPMENT SUITABLE FOR THIS PURPOSE

Abstract: 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 in which method the characteristic feature employed is a multiplicity of supports in particle form which carry the ligand or binding partners specific for the ligand.

Description: Method for determining a specific ligand in a fluid sample with the aid of an evanescent field, and also a component of the requisite measuring equipment suitable for this purpose. 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. " The surface plasmon resonance" (SPR) effect, a planar light conductor or an optical fibre can be used to generate an evanescent field. A method of this type is disclosed in International Patent Application W0 88/07202. More particularly, the method described in this Patent Application is carried out using an apparatus shown in Fig. 1. In said 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. On top of this, the grating is coated with a thin film of specific binding partners for the ligand to be determined. 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) . Although the SPR method described in said Interna¬ tional Patent Application WO 88/07202 is apparently not adversely affected by light-scattering particles in the sample (8) applied, it has, however, been found that the reproducibility of this SPR method is undesirably low. In the case of antibodies, for example, this is to be ascribed to a desorption or to a loss of activity during the immobilisation of these antibodies on the metal film. Moreover, the sensitivity (detection limit) of the method described above also leaves something to be desired. An apparatus of this type, as shown in International Patent Application WO 88/07202, is also described in European Patent Appli¬ cation 0,3^6,016; the apparatus described in said European Patent Application also suffers from the above disadvantages. It has been found that the disadvantages of the method mentioned in the preamble, which have been outlined above, can be overcome if a multiplicity of supports in particle form are used, which supports carry the ligand or binding partners specific for the ligand. More particularly, 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. 6a (0)-(3)), since after the measurement has been carried out the surface of the optical element will again assume the initial equi- librium position and therefore will again be available for carrying out a measurement. In the case of this "continuous" measurement method, 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). In this way, 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) . With regard to the abovementioned solutions for overcoming the disadvantages known from the prior art in respect of measurement methods using an evanescent field it is emphasized that said 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" . With the aid of the abovementioned principle of increasing the change in mass per unit surface area of the optical element it has proved possible to increase both the sensitivity and the repro- ducibility of measurement methods based on changes in the evanescent field, such as the SPR method appreciably. 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. In addition to the optical element defined above, optical fibres and planar light conductors can also be used as optical element. Many diverse types of support particles can be used as supports in particle form within the framework of the invention. Advantageously, latex spheres which have a diameter in the range of, for example, 10-500 nm are used as support. Examples of suitable materials are, inter alia, (monodisperse) polystyrene latex, poly- methyl methacrylate latex or a silica latex. Specific examples of 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), such as 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. For example, 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. In general, it can be stated that the degree of coverage is 50-99•5 *• For the sake of completeness it is pointed out that "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") . Many diverse types of compounds can be used as ligands to be determined in a sample, or, respectively, the specific binding partners complementary thereto. In the above, the antigen has already been mentioned as a ligand and the antibody specific for the antigen as specific binding partner. Reference is made to Table A below for a list, which is not complete, of ligands and specific binding partners which can be used. TABLE A Ligand Specific binding partner antigen antibody antibody antigen hormone hormone receptor hormone receptor hormone hapten antibody for hapten antibody for hapten hapten polynucleotide strand complementary polynucleotide strand avidin biotin biotin avidin protein A immunoglobulin immunoglobulin protein A enzyme enzyme cofactor (substrate) or inhibitor enzyme-cofactor(substrate) enzyme or inhibitor lectin specific carbohydrate specific carbohydrate lectin alginate Ca-alginatecomplex Ca-alginate complex alginate The immobilisation of the specific binding partners or ligands on the latex spheres is a phenomenon known per se. For example, 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). Subsequently 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. For the sake of completeness it is pointed out that, if desired, 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. Furthermore, 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. is a diagram of a set-up for the detection of the surface plasmon resonance effect (prior art) . is a diagrammatic representation of the binding of anti¬ bodies bound on a metal surface to a ligand (prior art) . is a diagrammatic representation of the binding of anti¬ bodies bound on a metal surface to supports in particle form provided with a ligand. 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): These figures 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 ; and 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 (17) the fluid sample to be investigated. Fig. 10: shows a graph of the measurement results obtained using SPR (see example below) , where - curve (a) shows the result for the gold layer with HSA (human serum albumin) applied thereon; - curve (b) shows the result for the gold layer with HSA, which has reacted with the anti-HSA, applied thereon; - curve (c) shows the result for the gold layer coated with carboxylated polystyrene microspheres (100 nm) , loaded with HSA; and - curve (d) shows the result for the gold layer, coated with carboxylated polystyrene microspheres (100 nm) , loaded with HSA, which has reacted with the anti-HSA. 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 invention is explained in more detail with the aid of the example below. EXAMPLE I A) Preparation of optical element with gold layer provided with loaded latex spheres The measurement set-up shown in Fig. 9 was used in this example. In this case, the gold layer was first provided with a coating. This coating consisted of human serum albumin (HSA; Sigma A 3782); concentration 1 mg/ml; the surface of the gold layer was com¬ pletely covered with this product, the coated metal surface being rinsed with PBS (phosphate-buffered saline solution; 0.01 M; pH = 7.4) after half an hour. 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. In the subsequent step, 2 ml of a 2% solution of carbodi- imide, prepared by dissolving 4 mg of carbodiimide in 2 ml of PBS, were dripped onto the resulting metal layer provided with spheres, after which an incubation for 6 hours at room temperature (20°C) and at a relative humidity of 100# was carried out. The resulting product was then rinsed with a borate buffer (Polysciences Corp., Niles/Ill., USA). In the subsequent step, protein (HSA; Sigma A 3782) was applied (1 mg/ml; the surface of the gold layer coated with spheres was completely covered) , after which the product was stored over¬ night at 100 relative atmospheric humidity. After this period, the product was rinsed again with PBS. Finally, the unreacted groups originating from carbodiimide were blocked with the aid of an excess of 0.1 M ethanolamine solu- tion for 30 min at room temperature. B) Reaction with anti-HSA 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 Fig. 9- The results are given in Fig. 10. In this Fig. 10, 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) . EXAMPLE II A) Preparation of optical element with gold laver provided with polvclonal antibody against hCG In this example the measurement set-up shown in Fig. 9 was again used and use was made of the following materials: - 0.1% polystyrene solution in toluene (Dynatech) ; - polyclonal antibody against hCG (Human Chorionic Gonadotrophin) , Lot No. 26099. concentration 17 mg/ml, Flemming GmbH; - hCG-coated latex, Lot No. 26070, diameter 0.238 μm, Flemming GmbH; - hCG, Lot No. 128 F-O65I; 5000 IU/vial; sigma. - 0.1% gelatine in PBS/Tween 20. Gelatine Lot No. 073 29282; Oxoid Limited. - PBS/Tween 20 solution; 1 ml of Tween 20 per litre of PBS (0.1 solution) . - PBS - phosphate buffered saline (0.01 M; pH •= 7-4). The gold layer (surface area = approx. 1 cm 2 ) present on the optical element was first provided with a polystyrene layer by applying 300 μl of 0.1% polystyrene solution in toluene. After incubating for 10 seconds, this polystyrene was spun up for 10 seconds at 4400 rpm. A dilution of the polyclonal hCG antibody (0.08 mg/ml) was then applied to the resulting polystyrene substrate and incubated for 20 minutes. The product was then washed three times with PBS. Washing was followed by a blocking step, which was carried out using a 0.1% gelatine solution in PBS/Tween 20. The incubation time for this was 10 minutes. Finally, the detection itself was carried out. 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 μl of solution were applied to the gold platelet. The results of this example are shown in graph form in Fig. 11. It can be seen from this figure that a distinct change in signal occurs within a measurement time of 20 minutes. In this Fig. 11, the time in minutes is shown on the X axis and the change in signal obtained is shown in arbitrary units (in percentage change in reflection) on the Y axis. EXAMPLE III In this example use was made of the SPR apparatus described in Example I and the following materials were used: Biocryl spheres (Toso Haas, size 0.1 um, type Biocryl BPA 1000) DNA material = DNA from PVA plasmid PBS buffer = phosphate-buffered saline (0.01 M, pH = 7.4) - PCR mixture 0.2 M KC1, 1.5 mM MgCl 2 , 0.02 M Tris (pH = 7.7), 0.01% gelatine, 0.2 mM dNTP's (4x) , 50 pmol of primer, 10 ng of DNA and 2.5 μl of W_ . 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 μl, 1.5 μl, 2.0 μl and 2.5 μl) 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 μl) 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 μg/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. The results of the experiment described in this example are shown in Fig. 12. On the basis of the results obtained, it can be stated that in principle it is possible to measure DNA.

Claims: CLAIMS 1. 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, charact¬ erised in that in said method a multiplicity of supports in particle form are used, which supports carry the ligand or binding partners specific for the ligand. 2. Method according to Claim 1, characterised in that a suspension of supports in particle form, which carry the ligand, are used in combination with the sample. 3- Method according to Claim 1, characterised in that 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. 4. Method according to Claim 1 or 3. characterised in that the evanescent field is generated by means of surface plasmon resonance (SPR) effect and the optical element is provided at the fluid/metal interface with the relevant supports in particle form. 5. Method according to Claim 1 or 3. characterised in that an optical element is used, 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 a support in particle form reversibly bound to said specific binding partners. 6. Method according to Claim 1 or 3. characterised in that an optical element is used, 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, in which the ligand and the "leaving agent" are present, this equilibrium setting temporarily shifting after administration of a sample to be investigated and giving a corres- ponding change in the evanescent field. 7. Method according to Claim 6, characterised in that a mem¬ brane which is permeable to the ligand to be measured and impermeable to the "leaving agent" is fitted between the optical element and the location at which the sample is supplied. 8. Method according to Claim 1 or 3. characterised in that the specific binding partners bound to a support in particle form are also provided with a "leaving agent" in the form of a support in particle form which is reversibly bound to said binding partner. 9. Method according to one or more of Claims 4-8, character- ised in that an optical element is used which is coated with a metal layer of gold or silver and to which metal layer the supports in particle form, with thereon the binding partners specific for the ligand or the specific binding partners which are provided with a "leaving agent", are applied. 10. Method according to Claim 9, characterised in that the supports in particle form are applied to the metal surface via a flat coating, such as a polystyrene coating. 11. Method according to one or more of Claims 1-10, character¬ ised in that the supports in particle form which are used are latex spheres. 12. Method according to Claim 11, characterised in that latex spheres having a diameter in a range of from 10-500 nm are used. 13. Method according to Claim 11 or 12, characterised in that a polymer latex such as a polystyrene latex or a polymethyl meth- acrylate latex is used. 14. Method according to Claim 11 or 12, characterised in that a silica latex is used. 15- Method according to Claim 11 or 12, characterised in that a metal latex (= metal sol) is used. 16. Method according to one or more of Claims 11-15, character¬ ised in that a combination of latices of different types are used. 17. Method according to one or more of Claims 3, and 8-l6, characterised in that the "degree of coverage" of the surface of the optical element by the supports in particle form is 50-99-5%• 18. Method according to one or more of Claims 3. ^ and 8-16, characterised in that the "degree of loading" of the surface of the supports in particle form with the specific binding partners is 10-90% 19. Optical element, suitable for use in an SPR measuring apparatus and consisting of a transparent platelet 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 supports in particle form, applied thereon, which carry specific binding partners, as defined in one or more of Claims 3~l8. 20. Optical element, suitable for use in an SPR apparatus and consisting of a transparent platelet 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 binding partners specific for the ligand which are provided with a "leaving agent" in the form of a support in particle form reversibly bound to said specific binding partners. 21. Optical element according to Claim 19, in which the specific binding partners, bound to the support in particle form, are also provided with a "leaving agent" in the form of a support in particle form reversibly bound to said specific binding partner.

Inventors: SCHASFOORT RICHARDUS BERNARDUS, SCHASFOORT, RICHARDUS, BERNARDUS, MARIA
Applicants: TNO, NEDERLANDSE ORGANISATIE VOOR TOEGEPAST-NATUURWETEN
Application year: 1991
Main classification: G01N21
IPC codes: G01N21, G01N33