GB2366370A

GB2366370A - Reagent for scintillation proximity assays

Info

Publication number: GB2366370A
Application number: GB0020915A
Authority: GB
Inventors: Michael A W Brady; Robert Arnold Jessop; John Morse; Nicholas Thomas
Original assignee: Amersham Pharmacia Biotech UK Ltd
Current assignee: GE Healthcare UK Ltd
Priority date: 2000-08-25
Filing date: 2000-08-25
Publication date: 2002-03-06
Anticipated expiration: 2020-08-25
Also published as: GB0020915D0; GB2366370B

Abstract

The invention relates to scintillation proximity assays, <I>in vitro</I> and in cells in multiwell plates or in other formats. The invention, provides a support in which or upon which there is immobilised a system to be studied and wherein the support comprises a phosphor. In one aspect the system comprises one member of a specific binding pair immobilised in or onto the support. In another aspects the support is treated to enable the attachment and/or the growth of cells. The phosphor is capable of absorbing energy in an integrated way from radioactive emissions from a radiolabelled reagent when it is bound to the surface of the support. Upon activation by means of orange, red or infra red radiation, the stored energy is released as light which may be detected using a charge coupled device.

Description

<Desc/Clms Page number 1> Reagent for Scintillation Proximity Assays The present invention relates to the technique of scintillation proximity assay (SPA). In particular the invention provides a new assay support, and a method for performing SPA. Scintillation proximity assay is now a widely used radioisotopic assay technology used in radioimmunoassays, radio-receptor assays and enzyme assays. The technique of SPA is described in US Patent No.4568649 (Bertoglio-Matte, J.). In SPA, the target of interest is immobilised onto a microsphere incorporating a suitable scintillant material. When a radio-isotopically labelled molecule is brought into close proximity with the scintillant, the energy resulting from radioactive decay is transferred to the scintillant, causing an emission of light. Scintillant beads have been prepared from inorganic scintillators, such as yttrium silicate, and from organic, hydrophobic polymers such as polyvinyltoluene. Binding molecules may be covalently or non-covalently attached to the bead, to allow generic links for assay design. Examples of such binding molecules include specific binding pair members, such as antigens, antibodies, DNA probes, receptors and binding proteins. SPA is thus used widely as a high throughput, homogeneous screening technology for the measurement of a variety of biochemical targets. A high level of radiolabel is required in order to generate a response from the scintillant that can be detected and assayed in a reasonably short period of time. This is overcome by the present invention in which phosphors are used in place of conventional scintillant materials in such assays by providing means for integrated capture of energy from radioactive decay over an extended time period and for rapid readout of assay signal, so minimising analysis time and increasing throughput.

As is well known, certain solids emit energy of a specified wavelength when driven by an external energy source (such as cathodoluminescence in a television screen). An interesting class of such materials do not emit energy immediately, but store electrons in "traps" for long periods of time. The trapped electrons can then be released at a later time, when desired, by exposure of the material to less energetic photons. Under these conditions, it appears that energy has been stored for later use. If the emission of visible light is activated by infrared illumination for example, the material may be called a phosphor, eg. an infrared phosphor. Infrared phosphors have been developed by Quantex Corporation and are believed to comprise alkaline earth metal sulphides or selenides doped with rare earth metal oxides, together with a fusable salt. They are described for example in US Patent Nos.4705952 and 4755324 (Lindmayer, J.).

Imaging systems based on storage phosphor technology are now used for quantitative imaging and as a replacement for X-ray films. Storage phosphor screens are available which are highly sensitive to beta particles, gamma rays and X-rays, thus enabling 32P, '4C, 35S, and '25I detection. Storage phosphor screens can trap and store most of the energy in phosphor particles until the energy is released following scanning with a laser. Thus, imaging systems based on europium-doped barium fluorohalides (eg. BaFBr:Eu2+) are available that may be activated by a helium-neon laser. The resultant blue light may be subsequently collected and detected using a photomultiplier-based detection system. However, europium-doped barium fluorohalides are chemically not stable, and in particular are not suitable for imaging in aqueous systems.

European Patent No. 127866 (Fuji Photo Film Co. Ltd) discloses a method of detecting a radioactive sample in a liquid sample, by applying

the liquid sample and maintaining the sample in contact with a storage phosphor sheet. The sheet is subsequently irradiated with electromagnetic wave radiation-, such as visible light or infrared radiation, in order to recover the stored information. The phosphor can be a divalent europium- activated alkaline earth metal fluorohalide. A disadvantage of this system is that the amount of radiation reaching the phosphor sheet depends critically on the geometry of the equipment, particularly the distance of travel of the radiation through the solution. Another disadvantage is the requirement for a transparent polymer layer to protect the phosphor both chemically and physically. None of the above prior art documents describe the use of storage phosphors in biochemical assays, particularly scintillation proximity based assays in aqueous systems. In one aspect of the present invention, there is provided a support, in which or upon which there is immobilised a system to be studied and wherein the support comprises a phosphor capable of absorbing energy in an integrated way from radioactive emissions from a radiolabelled reagent when it is held in close proximity to the support and of releasing that stored energy in a detectable form upon subsequent activation. Suitably, the system may for example, comprise one member of a specific binding pair immobilised in or onto the support. Alternatively, the system may comprise cells cultured adhering to the support for studying a cellular biochemical process. Suitably, activation is by means of heat or relatively long wavelength radiation, for example orange, red or infrared radiation. Preferably,

the stored energy is released in the form of visible light. Most preferably, the phosphor has an emission maximum in the range of 400 - 700nm. The phosphor is capable of absorbing energy in an integrated way from radioactive emissions from a radiolabelled reagent when it is bound to the support, ie. the amount of energy stored is directly proportional to the extent of radioactive decay of bound radiolabelled reagent. The support may be relatively large, for example the surface of an assay vessel, such as the base and/or wall of the wells of a multiwell plate. Alternatively, the support may form the surface of a probe or dipstick. In a first preferred embodiment, the support takes the form of a particle. In a second preferred embodiment, the support takes the form of a bead in which the phosphor is present as a coating applied onto a preformed surface; or may be dispersed in or constitute or form part of the bead. The support particles employed in the first preferred embodiment of the present invention may be inorganic phosphor particles. In the alternative preferred embodiment, the support particles suitably comprise organic polymeric beads which may be porous or non-porous, for example plastics or polymeric materials, such as polystyrene, polyvinyltoluene, polyacrylamide, agarose, polycarbonate or dextran polymers, and the phosphor may be coated onto the surface, or integrated into the matrix, of the bead. See for example, US Patent No.5522993 (Carlsson et al). There are many suitable phosphors that can be used. Most consist of an inorganic host material doped with one or more activators. Examples of such host materials are yttrium silicate, lithium yttrium silicate, yttrium vanadate, rare earth metal oxides, oxyhalides, oxysulphides, fluorohalides, halosilicates and halo(silicate-germanates), alkaline earth metal phosphates, sulphates, aluminates, haloborates, fluorohalides, alkali metal tantalates,

yttrium niobate and niobate-tantalate, lutetium-gadolinium-yttrium silicate, caesium sodium yttrium fluoride, rubidium bromide and calcium and strontium sulphides. The activators are generally lanthanide or actinide moieties, particularly lanthanum, cerium, samarium, europium, gadolinium, terbium and praseodymium, but may also include niobium, tantalum or zirconium. Preferred supports are particles based on yttrium silicate containing cerium and samarium as activators.

In a second aspect, the present invention provides use in a scintillation proximity assay of a phosphor wherein the phosphor is capable of absorbing energy in an integrated way from radioactive emissions from a radiolabelled reagent when it is bound to a support comprising the phosphor and of releasing that stored energy in a detectable form upon subsequent activation.

In a scintillation proximity assay according to the present invention, a support comprising a phosphor is provided, the support having a specific binding pair member immobilised thereon. The support is contacted with a fluid sample containing a radiolabelled reagent which is capable of specifically binding to the specific binding pair member. At least a proportion of the radiolabelled reagent becomes immobilised adjacent to the support comprising the phosphor. The mean free path of electrons or other particles resulting from radioactive disintegrations of the radiolabelled reagent is small relative to the dimensions of the body of the fluid sample, so only that part of the radioisotope immobilised adjacent to the solid support is capable of exciting the phosphor. Any radiolabelled reagent which remains free, dispersed or dissolved in the fluid medium is generally too far away from the support to be capable of exciting the phosphor. The present invention therefore provides an assay method for the detection of a radiolabelled reagent in a fluid sample, the method producing light energy

at a level which is related to the amount of reagent in the sample. The light energy is produced by the use of a phosphor which is capable of absorbing energy in an integrated way from radioactive emissions from a radiolabelled reagent when it is bound to the support and of releasing that stored energy in a detectable form upon subsequent activation. The support comprising a phosphor is typically coated with one member of a specific binding pair that is capable of specifically binding with the second (and complementary) member of the specific binding pair. Thus, in a third aspect of the present invention, there is provided a method for the detection and/or measurement of an analyte in a fluid sample, the method comprising: a) providing a support in which or upon which there is immobilised one member of a specific binding pair and wherein the support comprises a phosphor; b) bringing into contact with the support a fluid sample containing a radiolabelled reagent capable of specifically binding to said one member of a specific binding pair, wherein the phosphor is capable of absorbing energy in an integrated way from radioactive emissions from the radiolabelled reagent when it is bound to the binding pair member; and c) activating the phosphor and detecting release of energy stored by the phosphor. Assay methods according to the present invention may be conveniently categorised into two types, that is whether they are configured in the form of a direct assay or as a competition assay. Thus, in one embodiment of the third aspect, the assay method of the present

invention is a direct assay and the radiolabelled reagent is the analyte. The support is coated with one member of a specific binding pair. A sample containing a radiolabelled reagent is brought into contact with the support, causing the reagent to specifically bind with the specific binding pair member immobilised onto the support. This causes the radiolabelled reagent to be in close enough proximity to the support comprising the phosphor to allow the radiative energy emitted by the radiolabelled reagent to be absorbed by the phosphor. Subsequent activation of the phosphor causes release of visible light which may be detected and measured. The intensity of light produced is related to the amount of radiolabelled reagent bound to the support and the time over which it is bound. This in turn is a measure of the amount of reagent present in the fluid sample. In a further embodiment of the third aspect, the assay method is a competitive assay for the measurement of an analyte which is a member of a specific binding pair, by the use of a support according to the present invention. The method comprises bringing into contact with the support, a fluid sample containing the analyte and a fluid containing a radiolabelled reagent under conditions to cause the radiolabelled reagent to become bound to the support to an extent related to the concentration of the analyte in the sample, and then activating the phosphor and detecting release of energy stored by the phosphor. Both the analyte and the radiolabelled reagent are capable of specifically binding to a specific binding pair member immobilised onto the support. In this embodiment, the radiolabelled reagent is typically an analogue of the analyte to be measured. The analyte in the sample and the radiolabelled reagent compete for binding to the specific binding pair member immobilised onto the support. The radiolabelled reagent becomes bound to the support to an extent which is inversely related .to the concentration of the analyte in the sample.

In a particular embodiment of the third aspect, the assay is a sandwich assay. This is applicable when the analyte is polyepitopic, ie. having several binding sites for binding to complementary specific binding pair members. In this assay format, one component of a specific binding pair, such as a first antibody, is immobilised onto the support of the present invention. Following binding of an analyte tie. the antigen) to the first antibody, a radiolabelled second antigen-specific antibody is then added to the assay mix, so as to bind with the antigen-first-antibody complex to an extent which is directly related to the concentration of the analyte in the sample. The energy emitted by the radiolabelled antibody is absorbed by the phosphor. Subsequent activation of the phosphor causes the emission of visible light, which may be detected and measured as a measure of the concentration of the analyte. The assay fluid for use in methods according to the present invention is generally an aqueous or other liquid. The assay methods according to the invention may use a radiolabelled reagent in which the radioisotope may be present in free form, such as an atom or an ion. This may be useful when it is desired to monitor the uptake of a radioisotope by cells adhering to a support comprising the phosphor for studying a cellular biochemical process. Alternatively, the radioisotope may be used to label an assay reagent, thereby forming the radiolabelled reagent. This may be useful, for example, when an assay reagent is caused to bind with a specific binding partner immobilised onto the support, or to compete with an unlabelled reagent for binding to a specific binding partner immobilised onto the support. The radioisotope is preferably one which emits 0particles or auger electrons having a mean free path of up to 2000p.m in aqueous media. These include isotopes commonly used in biochemical and

molecular biology applications such as 3H, '4C, '25I, 35S, 33 Pr 5,Cr, 55Fe, $611b, and 45Ca.

The assay is preferably performed in the wells of a multiwell plate, eg. a microtitre plate. The support comprising a phosphor may be provided as beads dispensed into the wells of such a plate. Alternatively, the phosphor may be incorporated into the plate itself either by direct incorporation into the plastic of the plate or by coating, together with a binding agent. Examples of binding agents are calcium sulphate, used in the manufacture of tic plates, and low melting plastics such as polystyrene or copolymers of a-methylstyrene and vinyltoluene. The plates may have 24, 96, 384 or higher densities of wells eg. 864 or 1536 wells.

The third step of the method involves activating the phosphor and detecting the release of energy stored by it. The support, with the radiolabelled reagent bound to it, may be held as long as it is desired to permit the build up of stored energy. The time required is related to the concentration of radiolabel on the support and to the specific activity of the radioisotope used. Upon activation, eg. by infrared radiation, the stored energy is rapidly and completely released, preferably in the form of visible light which is easily detected and measured. The surface may be held to permit build-up of stored energy either in the absence of, or more preferably in the presence of the fluid with which it was incubated. Suitably, the detection step may be performed by non-imaging counting (such as liquid scintillation counters, or luminometers) or more preferably by means of a charge coupled device (CCD) imager (such as a scanning imager or an area imager) to image all of the wells of a multiwell plate.

Such assays, in which one component of a specific binding pair binds non-covalently to a second component of the specific binding pair,

may be applied to high throughput screening assays in which samples containing compounds to be screened are tested for their inhibitory effects, potentiation effects, agonistic, or antagonistic effects on the binding of the first component of the specific binding pair to the second component. An example of such an assay is a radioligand binding assay in which a known or a putative receptor active compound (eg. a receptor antagonist) may be optionally included in the assay. The assay method of the present invention may be used in conjunction with any specific binding pair combination in which either member of the specific binding pair may be radiolabelled without affecting its specificity for the second member of the specific binding pair. Either member of the specific binding pair may be immobilised in or onto the support to enable the detection of the binding of the second member. Examples of specific binding pairs include, but are not restricted to, antibodies/antigens, lectins/glycoproteins, antibodies/cell surface receptors, biotin/streptavidin, biotin/avidin, hormone/receptor, enzyme/substrate or cofactor, DNA/DNA, DNA/RNA, RNA/RNA, DNA/binding protein and RNA/binding protein. As an alternative, assays may be performed on living cells which are cultured on, or which become attached to, the support comprising the phosphor. Thus, in a fourth aspect of the present invention, there is provided a method for the measurement of a cellular process by use of a support comprising a phosphor wherein the phosphor is capable of absorbing energy in an integrated way from radioactive emissions from a radiolabelled reagent when it is bound to the support and of releasing that stored energy in a detectable form upon subsequent activation and wherein the support is

suitable for the attachment or growth of cells. The method comprises providing cells adhering to the support in the presence of a fluid medium, introducing into the fluid medium a reagent labelled with a radioisotope under conditions to cause a portion of the radiolabelled reagent to become associated with or released from the cells adhering to the support and activating the phosphor and detecting release of energy stored by the phosphor so as to study the cellular process.

Preferably, the support comprising the phosphor forms the base and/or wall of the wells of a microwell plate.

Preferably, the cells are cultured adhering to the support within the wells of a microwell plate prior to the introduction of the labelled reagent. Preferably, detection may be performed by use of a charge coupled device (CCD) to image the wells of the multiwell plate. Thus, imaging may be performed either by scanning the wells or by simultaneously imaging all of the wells of the multiwell plate.

For cellular based assays, the phosphors used in the present invention may be incorporated into the base of culture plates, for example multiwell plates. Alternatively, the phosphor may be present as a coating applied onto the wall of the wells of a multiwell plate. The phosphor can be incorporated into the plastic material of the multiwell plate by a variety of methods. For example, the phosphor may be added to a monomer mix prior to polymerisation and moulding of the plate. Alternatively, the phosphor may be added to a solution of a polymer and the solvent removed to leave a phosphor layer coating the inside walls and base of the wells of the plate. The support comprising the phosphor is preferably

optically transparent, in order to image light energy released upon activation of the phosphor. For cellular assays, the support comprising the phosphor will typically require treatment or surface modification so as to be suitable for the attachment and/or the growth of cells. Treatment preferably involves the use of high voltage plasma discharge, a well established method for creating a negatively charged surface (Amstein, C.F. and Hartmann, P.A., J.CIin.Microbiol., 2,1, 46-54, (1975)). A range of additional coatings may be applied to the surface, such as positively or negatively charged chemical coatings such as polylysine, components of the extraceliular matrix such as laminin, collagen and fibronectin, lectins such as wheat germ agglutinin (WGA) which are suitable for the attachment of cell membranes or cell types that normally grow in suspension. Methods for coating plastic cultureware with such agents have been described (Boldt, D.T and Lyons, R.D., J.Immunol., 123, 808, (1979)). Microwell plates containing a scintillant base and the use of such plates in cellular assays are described in European Patent No.650396, the disclosure of which is incorporated herein. Culture of cells on a support according to the present invention involves the use of standard cell culture techniques, eg. cells are cultured in a sterile environment at 37 C in an incubator containing a humidified 95% air/5% COz atmosphere. Various cell culture media may be used including media containing undefined biological fluids such as foetal calf serum, as well as media which is fully defined, such as 293 SFM II serum free media (Life Technologies, Ltd, Paisley, UK). The invention may be used with any cell type that can be cultured on standard tissue culture plastic-ware. Such cell types include all normal

and transformed cells derived from any recognised source with respect to species (eg. human, rodent, simian), tissue source (eg. brain, liver, lung, heart, kidney skin, muscle) and cell type (eg. epithelial, endothelial). In addition, cells that have been transfected with recombinant genes may also be cultured using the invention. There are established protocols available for the culture of diverse cell types. (See for example, Freshney, R.I., Culture of Animal Cells: A Manual of Basic Technique, 2"d Edition, Alan R.Liss Inc. 1987). Such protocols may require the use of specialised coatings and selective media to enable cell growth and the expression of specialist cellular functions. None of such protocols is precluded from use with the methods of this invention. Examples of assays performed on live cells suitable for the method of the invention include, but are not limited to, measurement of binding of radio-labelled ligands to cellular receptors, incorporation of radio-labelled precursors, such as nucleosides and amino acids into cellular macromolecules and uptake or efflux of other radiolabelled molecules into or from cells as a result of cellular metabolism. Additionally, cells may be labelled with a second label for the purposes of identifying different cell types within a culture. Suitably, the second label is non-radioactive, preferably fluorescent. Suitable fluorescent labels for labelling cells in culture include antibodies to cell- surface protein markers, ligands specific for cell-surface receptors and lectins. Nucleic acid (DNA and RNA) probes labelled with fluorescent labels may also be employed to detect nucleic acid components of cells following fixing and permeabilisation. Suitable fluorescent labels which may be used include, but are not limited to, fluoresceins, rhodamines, cyanine dyes, coumarins, and the BODIPYTm groups of fluorescent dyes. Detection may be performed by fluorescence imaging.

For illustration, the following examples of cellular based assays are described in More detail. i) Receptor binding Many biological events are initiated or controlled by binding of soluble ligands to cell surface receptors, for example control of glucose metabolism by insulin. To study such mechanisms, for example to measure the numbers of receptors occurring on the surface of different cell types, it is necessary to perform assays of binding of radiolabelled ligands to cell surface receptors. To perform such assays by the method of the invention the cell types to be studied are cultured in suitable culture vessels furnished with a solid support comprising a phosphor. Cells are grown in standard tissue culture media, under standard tissue culture environmental conditions, typically 37 C, 95% RH and 5% CO2, which allow attachment of cells to the surface of the support. Following establishment of cell growth, a radiolabelled reagent, which may be a receptor-active ligand and typically labelled with 3H or '215I, is added to the cell culture medium and incubation is continued to allow binding of the radiolabelled ligand to receptors on the cell surface. Binding of ligand to the cells causes a fraction of the radiolabelled ligand present in the bulk medium to be associated with the cells, and consequently to become partitioned into a volume of space sufficiently close to the phosphor surface to permit the phosphor to store the energy resulting from radioactive decay in an integrated way. Incubation is continued until such time as it is desired to measure the amount of radiolabelled ligand that has become associated with the cells. This is achieved by removing the culture vessel from the tissue culture environment and activating the

phosphor and detecting the release of energy stored during the incubation period.

ii) Thymidine incorporation DNA synthesis is a key event in cell growth, and studies of the effects of agents or drugs on DNA synthesis is key to many areas of medical research including immune response and carcinogenesis. To study DNA synthesis, cells are incubated with radiolabelled thymidine, typically labelled with 3H or '4C, and the amount of the radiolabel incorporated into DNA is determined to give a measure of the amount of DNA synthesis that has occurred. To perform such assays by the method of the invention, cells are cultured in suitable culture vessels furnished with a support surface comprising the phosphor. Cells are grown in standard tissue culture media, under standard tissue culture environmental conditions, typically 37 C, 95% RH and 5%C02, which allow attachment of cells to the support surface. Following establishment of cell growth radiolabelled thymidine is added to the cell culture medium and incubation is continued to allow uptake of the molecule into the cells and incorporation into cellular DNA. Incorporation into DNA causes a fraction of the radiolabelled molecule present in the bulk medium to be associated with the cells, and consequently to become partitioned into a volume of space sufficiently close to the support surface to permit the phosphor to store the energy resulting from radioactive decay in an integrated way. Incubation is continued until which time as it is desired to measure the amount of radio- labelled ligand that has become associated with the cells. This is achieved by removing the culture vessel from the tissue culture environment, activating the phosphor and detecting the release of energy stored during the incubation period.

Advantages of the assay methods according to the invention over conventional scintillation proximity assay both for in vitro applications and for cellular based assays include one or more of the following: i) The quantity of radioactivity used can be much less than previously, particularly if the support comprising the phosphor can be held for a sufficient length of time to permit the build-up of stored energy. In addition, signal accumulation derives from radioactive decay over the duration of the incubation rather than being limited to the decay which occurs only during the period of detection.

ii) Signal accumulation is performed off-instrument. The stored energy can be rapidly and completely released in a short period of time, thus long counting times are avoided. Instrumentation time required for the detection and measurement of the stored energy is thus minimised.

iii) Quantitation is format-free, since imaging is compatible with any two-dimensional distribution of signal. Thus, assays performed using the phosphors of the present invention may be conducted in current plate formats, ie. 96, 384 or 1536 multiwell plates, or in arrays.

For cellular based assays in particular, application of the method of the invention confers a number of advantages over previously described methods.

iv) Signal accumulation by the storage phosphor is carried out in a tissue culture environment, thereby minimising perturbation of the cellular environment associated with signal measurement in the ambient environment, which is a feature of other methods of detection which use direct measurement of radioactive decay.

v) Data acquired by imaging includes spatial information. This is particularly important for analysis using complex cell cultures which contain more than one cell type and where it is desired to determine in which cells a particular event is occurring. In this respect, the method is compatible with the use of non-radioactive probes or markers, e.g. fluorescent antibodies to cell surface markers which are suitable for identifying specific cell types.

The invention is further illustrated by reference to the following example and figures.

Figures Figure 1 is an image of [3H]biotin binding to streptavidin-coated phosphor particles (yttrium silicate doped with 2% cerium and 0.5% samarium) according to the example.

Figure 2 is a plot showing linearity of response of phosphor support particles according to the example.

Example [3H]Biotin binding assay using streptavidin-coated storage phosphor articles Streptavidin-coated yttrium silicate doped with 2% cerium and 0.5% samarium was suspended in 10mM HEPES buffer pH7.5 at a concentration of 1 Omg/ml. 1 Opl of this suspension (1 OOpg particles) was pipetted into each of the wells of a 384-well multiwell plate. 10p1 of a solution of

[3H]biotin at various concentrations in the same buffer was added to the wells. The amount of [3Hlbiotin ranged from 13,750dpm in doubling dilutions down to 107dpm and eight replicates of each concentration were used. 10w1 of buffer was added to a further eight wells to act as controls. The plate was left to stand in the dark overnight at room temperature to allow complete binding of the biotin to the particles. The plate was then scanned in a Typhoon imager (Molecular Dynamics, Sunnyvale, Ca) using an excitation wavelength of 633nm and an emission wavelength of 390nm. The data obtained is shown in Table 1 and is represented graphically in Figure 2.

Table 1: [3H]Biotin binding assay with streptavidin-coated storage phosphor particles DPM Imager signal Signal to noise ratio* (mean of 8 replicates) 13750 4489.05 539.4 6875 2188.12 262.9 3438 1089.11 130.9 1719 505.03 60.7 859 231.29 27.8 430 105.34 12.7 215 47.49 5.7 107 19.04 2.3 *defined as the ratio of the sample signal to the standard deviation of the background signal (3.9 signal units) The data show a good linearity of response down to low levels of [3H]biotin. The limit of detection, defined as a level of at least three times the background standard deviation above the background, is 215dpm.

Claims

Claims 1. A support, in which or upon which there is immobilised a system to be studied and wherein the support comprises a phosphor capable of absorbing energy in an integrated way from radioactive emissions from a radiolabelled reagent when it is held in close proximity to the support and of releasing that stored energy in a detectable form upon subsequent activation.
2. The support as claimed in claim 1 wherein said system comprises one member of a specific binding pair immobilised in or onto the support.
3. The support as claimed in any of claims 1 or 2 wherein the phosphor is capable upon activation by means of orange, red or infrared radiation, of releasing the stored energy in the form of visible light.
4. The support as claimed in any of claims 1 to 3 wherein the phosphor is an inorganic host material doped with an activator which is a lanthanide or actinide moiety.
5. The support as claimed in claim 4 wherein the host material is selected from the group consisting of yttrium silicate, lithium yttrium silicate, yttrium vanadate, rare earth metal oxides, oxyhalides, oxysulphides, fluorohalides, halosilicates and halo(silicate-germanates), alkaline earth metal phosphates, sulphates, aluminates, haloborates, fluorohalides, alkali metal tantalates, yttrium niobate and hiobate-tantalate, lutetium-gadolinium-yttrium silicate, caesium sodium yttrium fluoride, rubidium bromide and calcium and strontium sulphides.

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6. The support as claimed in claim 4 wherein the activator is selected from lanthanum, cerium, samarium, europium, gadolinium, terbium, praseodymiurri, niobium, tantalum and zirconium.
7. The support .as claimed in any of claims 1 to 6 wherein the phosphor has an emission maximum in the range of 400 - 700nm.
8. The support as claimed in any of claims 1 to 7 wherein the radiolabelled reagent comprises a label selected from 3H, '4C, '25I, 35,S, 33P, 51 Cr, 55Fe, ssRb, and 45 Ca.
9. The support as claimed in any of claims 1 to 8 wherein the support is a particle, preferably yttrium silicate containing cerium and samarium as activators.
10. The support as claimed in any of claims 1 to 8 wherein the support forms the base and/or wall of the wells of a multiwell plate.
11. The support as claimed in any of claims 1 to 8 wherein the support forms a surface of a probe or dipstick.
12. The support according to claims 1 to 11 wherein said specific binding pair is selected from the group consisting of: antibodies/antigens, lectins/glycoproteins, antibodies/cell surface receptors, biotin/streptavidin, biotin/avidin, hormone/receptor, enzyme/substrate or cofactor, DNA/DNA, DNA/RNA, RNA/RNA, DNA/binding protein and RNA/binding protein.
13. The support as claimed in claim 1 wherein said system comprises cells cultured adhering to the support for studying a cellular biochemical process.

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14. Use in a scintillation proximity assay of a phosphor wherein the phosphor is capable of absorbing energy in an integrated way from radioactive emissions from a radiolabelled reagent when it is bound to a support comprising the phosphor and of releasing that stored energy in a detectable form upon subsequent activation.
15. A method for the detection and/or measurement of an analyte in a fluid sample, the method comprising: a) providing a support in which or upon which there is immobilised one member of a specific binding pair and wherein the support comprises a phosphor; b) bringing into contact with the support a fluid sample containing a radiolabelled reagent capable of specifically binding to said one member of a specific binding pair, wherein the phosphor is capable of absorbing energy in an integrated way from radioactive emissions from the radiolabelled reagent when it is bound to the binding pair member; and c) activating the phosphor and detecting release of energy stored by the phosphor.
16. The method as claimed in claim 15 wherein said specific binding,pair is selected from the group consisting of: antibodies/antigens, lectins/glycoproteins, antibodies/cell surface receptors, biotin/streptavidin, biotin/avidin, hormone/receptor, enzyme/substrate or cofactor, DNA/DNA, DNA/RNA, RNA/RNA, DNA/binding protein and RNA/binding protein.

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17. A method of performing an assay for an analyte which is a member of a specific binding pair by the use of a support according to any of claims 1 to 12, the method comprising: a) bringing into contact with the solid support a fluid sample containing the analyte and a fluid containing a radiolabelled reagent under conditions to cause the radiolabelled reagent to become bound to the support to an extent related to the concentration of the analyte in the sample; and b) activating the phosphor and detecting release of energy stored by the phosphor.
18. The method as claimed in claim 17 wherein the assay is performed in the wells of a multiwell plate and a charge coupled device is used to image all of the wells of the multiwell plate.
19. A method for the measurement of a cellular process by use of a support comprising a phosphor wherein the phosphor is capable of absorbing energy in an integrated way from radioactive emissions from a radiolabelled reagent when it is bound to the support and of releasing that stored energy in a detectable form upon subsequent activation and wherein the support is suitable for the attachment or growth of cells, which method comprises: a) providing cells adhering to the support in the presence of a fluid medium: b) introducing into the fluid medium a reagent labelled with a radioisotope under conditions to cause a portion of the radiolabelled

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reagent to become associated with or released from the cells adhering to the support; and c) activating the phosphor and detecting release of energy stored by the phosphor so as to study the cellular process.
20. The method as claimed in claim 19 wherein the support comprising the phosphor forms the base and/or wall of the wells of a multiwell plate.
21. The method as claimed in claim 19 or claim 20 wherein the cells are cultured adhering to the support within the wells of a multiwell plate prior to the introduction of the labelled reagent.
22. A method as claimed in any of claims 19 to 21 wherein a charge coupled device is used to image all of the wells of the multiwell plate.