ASSAY METHOD

Abstract: A method of assaying for an analyte by the use of surface plasmon resonance spectrometry (SPRS). The analyte is a member of a non-immune ligand-receptor pair. A metal surface carries the analyte or an analogue immobilised with the other member of the ligand-receptor pair reversibly bound thereto. A fluid containing the analyte is brought into contact with the metal surface, and displacement of the other member of the ligand-receptor pair monitored by SPRS.

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

Inventors: CHARLES STEPHEN ALEXANDER, GARLAND PETER BRYAN, CHARLES, STEPHEN, ALEXANDER, GARLAND, PETER, BRYAN
Applicants: AMERSHAM INT PLC, AMERSHAM INTERNATIONAL PLC
Application year: 1990
Main classification: G01N33
IPC codes: C12Q1, G01N33