GB2474306A

GB2474306A - Methods and device for detecting an analyte

Info

Publication number: GB2474306A
Application number: GB0917838A
Authority: GB
Inventors: Michael John Marshall
Original assignee: BIOPRODUCTS Ltd
Current assignee: BIOPRODUCTS Ltd
Priority date: 2009-10-12
Filing date: 2009-10-12
Publication date: 2011-04-13
Also published as: GB0917838D0; WO2011045022A1

Abstract

A method of detecting an analyte in a sample comprising contacting the sample with a support having a site to which the analyte can bind in a first zone, contacting the analyte/support with another binding partner/labelling species to form a solid phase complex, moving this complex to a second zone wherein unbound labelling species/binding partner is removed in a wash and detecting the presence of the bound analyte. Movement of the solid phase complex through a contact phase and a wash phase is desirably under gravity in a centrifuge. Alternatively claimed is a single step assay device comprising a vessel having a contact zone for receiving a liquid phase comprising a sample suspected of having an analyte and optionally a mobile binding partner for the analyte and a solid phase support capable of binding to the analyte and a further wash zone that receives a wash phase and is in fluid contact with the contact zone.

Description

ASSAY AND DEVICE THEREFOR

The invention relates to an assay and a device therefor. The invention particularly relates to a method of assaying an analyte which may possibly be present in a fluid, for example a biological fluid.

A wide range of assays and devices for use in assays are known and generally involve the interaction of certain reagents such as antibodies or other biomolecules with an analyte, if present, in the sample undergoing analysis and detection of the presence of a species to provide an indication of the presence or absence of the analyte. Apparatus devised for use in assays include tubes, slides, beads and the like coated with a binding partner for interaction with the analyte. The assay of specific analytes in biological fluids is well-established as a means of analysis, for example in the diagnosis of a particular condition or disease.

Specific binding of an antigen to antibodies immobilised on a solid-phase or the binding of antibody to an antigen on a solid-phase is commonly employed in a wide variety of diagnostic test methods and kits. Such solid-phases are common'y planar or particulate polymeric materials to which the antibody is adsorbed or covalently coupled, examples of which include a "lateral flow" assay and enzyme-linked immunosorbent assay (ELISA).

EP-A-291 194 describes a lateral flow assay and a device for detecting an analyte in such an aay. The device comprises a porous carrier provided with a labelled binding reagent wflMss freely mobile on the carrier upon application of a liquid test sample, the reagent * *** being capable of binding to the analyte, and an unlabelled binding reagent also capable of iId?r to the analyte and which is permanently immobilised at a detection zone ckwream from the labelled reagent. A liquid test sample suspected of containing the analyte is added to the device and, if the analyte is present it interacts with the labelled ***.

bing reagent to form a first analyte-labelled reagent complex. The complex migrates to the detection zone and is captured by the immobilised reagent to provide a complex of the immobilised reagent, the mobilised reagent with the analyte bound to both reagents and provides a visual indication of the presence of the analyte in a so-called "sandwich" assay.

ELISAs may comprise an analyte sandwiched between an immobilised antibody and a labelled antibody.

I

EP-A-291194 describes a separate embodiment in which a mobilisable labelled analyte or a mobilisable labelled reagent complexed with the analyte (or an analyte analogue) is provided on the porous carrier and an immobilised reagent is provided at a detection zone.

The analyte in the sample competes with the labelled analyte or analyte analogue to bind at the immobilised reagent in the detection zone in a so-called "competition" assay.

A "sandwich" assay typically enables a high degree of specificity in detecting the presence of the analyte at low analyte levels due to a binding reaction taking place between the analyte and both a mobile reagent and an immobilised binding reagent at different epitopes on the analyte. The "dose-response" curve for the "sandwich" assay typically shows increasing signal with increasing analyte to the point where the concentration of analyte is such that molecules of the analyte bind to the immobilised reagent without having bound to the mobilised binding reagent thereby preventing formation of the "sandwich" complex.

Accordingly, a lower signal on the dose-response curve is observed at higher levels of analyte in the test fluid and is commonly known as the "hook effect". The "hook effect" limits the dynamic range of assays of this type.

Assays involving a binding reaction between an antibody and analyte in which an enzyme label is conjugated to an antibody and binds to the analyte are known for use in indirect assays and particularly for use in "sandwich" assays. The label typically allows detection and measurement of the level of analyte. In an indirect assay, the analyte may be immobilised c,a.Jpport and present an epitope for binding to an antibody in free solution which is mbil In a sandwich assay an antibody is suitably immobilised on a support and the analyte binds to the immobilised antibody and presents an epitope for binding to a mobile 1t1L5�y. The mobile antibody, which may be labelled or conjugated with an enzyme, suiaI4i binds to the presented epitope on the analyte and the amount of enzyme label bound to the solid-phase via the formation of the analyte/antibody/ enzyme conjugate or the "-wich" complex will be determined by the amount of analyte captured. Generally, a washing step is employed so as to remove excess unbound antibody/enzyme conjugates.

Such assays using enzyme-labels that generate coloured, fluorescent or luminescent signals are widely employed and have achieved considerable sensitivity.

In a sandwich assay, a labelled mobile antibody or reagent may be added to the solid phase after the analyte solution has been passed over an immobilised antibody or reagent to form a complex which is then detectable. Suitably, to achieve high levels of sensitivity and specificity in an immunoassay, excess unbound label is separated from the solid-phase.

This may be achieved through a washing process, typically involving extensive washing of the solid-phase with buffered solutions incorporating detergents, for example Tween 20 and/or irrelevant "blocking" proteins such as bovine serum albumin (BSA), gelatin or casein to block non-specific binding of label. After washing, a solution incorporating a suitable enzyme substrate is typically added to the solid-phase to produce a coloured, fluorescent or luminescent signal to enable detection and quantitation of the enzyme label and hence the analyte bound to the solid-phase. A wide range of labels are known and may be employed in sandwich assays, for example fluorescent or radioactive labels.

In a "competition" assay, higher levels of analyte produce lower levels of signal. Typically, in such assays an analyte in a test sample competes for a binding site on a binding reagent with a labelled analyte or a labelled analyte analogue. At low sample analyte levels, the labelled analyte or analyte analogue is present in a large excess as compared to the analyte in the test sample and binds preferentially so providing a high signal at low sample analyte concentrations. At a higher level of sample analyte, an increasing proportion of the binding reagent reacts with the unlabelled sample analyte so reducing the sites available for binding to the labelled analyte and providing a lower signal. Any unbound binding reagent is suitably removed in a washing step. *S.. * * * * S

QOMtition assays allow extension of the dynamic range of an assay when employed in * SS.

conjunction with a sandwich assay by enabling analysis for analyte at levels where the "hfti14:effect may reduce the usefulness of a sandwich assay. Competition assays are paMiJarly beneficial in enabling the analysis of small analytes which are too small to allow two antibodies to bind in a sandwich assay or which have a single epitope for antibody **SS Idng.

Assays having a combination of a sandwich assay and a competition assay are known and useful in providing a wide dynamic range. The combination of a competition assay and a sandwich assay may be constructed to provide diagnostically useful information at low analyte concentrations via the sandwich assay and, through the competition assay, at higher analyte concentrations at which a sandwich assay may exhibit the hook effect.

Conventional assays generally require excess antibody or excess labelled antibody to be washed away prior to the addition of a suitable substrate solution for generation of a detectable signal and an appropriate level of sensitivity. A separate, sequential washing step is undesirable and the present invention aims to overcome this drawback so as to simplify the assay procedure and to reduce the time required to carry out the assay whilst retaining high levels of sensitivity.

Known assays typically require unbound labelled components to be washed away from the labelled sample analyte which is to be detected to avoid detection of incorrect levels of analyte in the sample. As the liquid phase moves across the stationary solid phase, the required sequential washing adds to complexity and delay in detecting the sample analyte.

The present invention seeks to ameliorate drawbacks of conventional assays by moving a solid phase having a bound analyte through a liquid phase so effecting a separation of labelled analyte and unbound label. In particular the invention provides for the introduction of a specific labelled binding partner to solid-phase particles comprising an immobilised binding partner and an analyte that binds to the immobilised binding partner whereby the labelled binding partner binds to a second binding site on the analyte and separation of the solid phase particles from unbound labelled binding partner. The labelled binding partner 1rci.for the detection of the analyte. * * * *.*

Unlike conventional assays, rapid and efficient separation of unbound label is achieved by mMri the particles to a second zone whereby unbound label is separated from the pks and where a detection signal can be measured away from unbound label. I..

lnirst aspect, the invention provides a method of detecting the presence of an analyte in a sample comprising: i) contacting the sample with a support having a site to which the analyte may bind wherein when the analyte is bound to the said site the presence of the bound analyte is detectable or the bound analyte is contacted with a labelling species such that the presence of the bound analyte may be detected by detecting the labelling species; ii) moving the said bound analyte to a second zone whereby the bound analyte is separated from any unbound labelling species if present; and iii) detecting the presence of the bound analyte in the second zone.

Advantageously, passing the bound analyte from the first zone to the second zone for detection enables the bound analyte to be separated from other materials in the first zone which may otherwise interfere with or mask the detection of the presence of the bound analyte. The movement of the bound analyte on the support relative to a liquid phase in effect provides a wash effect in a simple and convenient manner on passing the bound analyte into the second zone.

The first zone suitably comprises a contact zone in which the analyte and support are contacted and the second zone comprises a wash zone into which the support with bound analyte passes and in which the presence of the bound analyte is detectable.

In a second aspect, the invention provides a method of detecting the presence of an analyte in a sample comprising: I) contacting in a first zone the sample with a support having a site to which the analyte may bind; ii) contacting the support having the bound analyte with a mobile binding partner, for example an antibody, capable of binding to a site on the bound analyte wherein the binding partner has a detectable label or is contacted with a labelling species such that the may be detected by detecting the labelling species; iseparating unbound labelled binding partner from the analyte-labelled binding S.,.

partner complex by moving the said complex to a second zone; and i'v*'* detecting the label to indicate the presence or absence of the analyte.

S..... w

The support with or without the bound analyte and optionally with bound mobile binding pter may be referred to herein as the "solid phase".

Suitably the mobile binding partner is a conjugate in free solution. The conjugate preferably comprises a ligand for example an antibody, specific for one site on the analyte, that desirably is coupled to a label whose detection enables the quantitation of the analyte. The label may be detectable by any means and comprises a species which is detectable by radiation, for example the species is coloured, or may be a fluorescent, magnetic or radioactive moiety. Preferably the label is an enzyme that acts on a substrate whose conversion generates a detectable signal, preferably a coloured, fluorescent or emitted light signal.

The analyte may be bound directly to the support but suitably the support comprises an immobilised binding partner, for example an antibody, which provides a binding site for the analyte and to which the analyte may bind. Reference herein to an analyte is to be taken to include a reference to an analogue of the analyte which is capable of binding to the support or the immobilised binding partner on the support and, where present, the mobile binding partner.

Preferably, the analyte-labelled binding partner complex has a property that allows its selective movement relative to unbound labelled binding partner from the first zone to the second zone. In this process, the said complex suitably passes through a wash medium.

Suitably the support comprises solid-phase particles and they have on their surface a material which presents a binding site for the analyte.

Preferably the binding site is provided by a binding partner, specific to a site on the analyte, coated on the particles and suitably comprises an antibody or polynucleotide. The support suitably comprises any material which is capable of receiving for example by chemical or by coating, an antibody or other material which is able to present a binding site for t1n,plyte. Other than this property, the support is suitably chemically inert and desirably has surfaces that minimise non-specific binding of the components of the assay mixture.

Sty the support is not coloured so as to reduce the risk of interference with the 1on of the analyte where light emission or colour change is employed as a means of detection. *.S.

Examples of suitable materials for the support include organic and inorganic polymeric particles, for example silica, styrenic, acrylic, polyacrylamide and polyethyleneglycol and organic particles, for example erythrocytes and other cells. The material from which the support is made is suitably selected having regard to ionic strength of the environment and the nature of the binding site desired on the particle.

The support preferably has a diameter of 0.05 to 50 and more preferably 0.2 to 20microns.

The support preferably has a density of 1 to 20, more preferably 1 to 10 and desirably greater than I to 3 g/ml. Suitably the solid phase support is of such size and density that it does not rapidly leave the first zone, for example by sedimentation into the second zone so allowing favourable binding kinetics for the analyte.

Suitably the support is essentially non-porous so as to allow efficient washing of the bound analyte or bound analyte/labelled binding partner complex over a short time and short distance in moving from the first to the second zone.. The quantity, size and density of the particles can be optimised by those skilled in the art to provide sufficient surface area for rapid analyte binding, consistent with low non-specific binding of labelled binding partner.

In a preferred embodiment, the binding site on the support is provided by antibodies, lectins or other specific binding proteins or polynucleotides thereon, either chemically or physically bound to the support. A preferred example of a suitable material to provide a binding site on the support is streptavidin and the bound antibody is a biotinylated antibody having an epitope for the suspected analyte.

Advantageously, the invention allows for rapid analysis and provides for a high level of sensitivity. This has a practical benefit of allowing small sample volumes to be assayed at required levels of sensitivity and to detect a wide range of antibodies and antigens. The i4':&tn is especially beneficial in detecting the presence of an analyte in a complex ical fluid. I...

Pftther aspect, the invention provides a method of detecting the presence of an analyte in aample comprising: i) contacting the sample with a support having a site to which the analyte may bind tQpovide a support having the bound analyte, if present in the sample; ii) contacting the support having the bound analyte with a mobile, labelled binding partner in a contact phase in a first zone to produce a complex of the support, analyte and labelled binding partner; iii) effecting movement of the said complex from the contact phase to a wash phase whereby unbound mobile labelled binding partner is left in the first zone such that the said complex is separated from the unbound mobile binding partner; and iv) detecting the complex to indicate the presence or absence of the analyte.

In a further aspect, the invention provides a method of detecting the presence in a sample of an analyte having two binding sites comprising: i) providing a contact phase having a density D comprising a mobile labelled binding partner capable of binding to a free site on the analyte ii) providing a wash phase having a density D wherein D is greater than D; iii) in the contact phase, contacting the sample with a mobile support having a site to which the analyte may bind to provide a mobile solid phase comprising the mobile support having a bound analyte, if present in the sample; iv) passing the mobile solid phase through the contact phase such that the mobile labelled binding partner binds to the mobile solid phase to produce a complex of the support, analyte and labelled binding partner and through the wash phase; and v) detecting the presence or absence of the complex, if present, in the sample.

In the assay of the invention, the sample to be analysed which may or may not contain the analyte to be assayed, is contacted with the support. Suitably the sample is contacted with the support in a contact phase. The analyte, if present in the sample is captured on the support which has a binding site that binds to a specific site on the analyte, desirably a &tle having coated on it a binding ligand, for example an antibody. The support having tJund analyte, if present in the sample, may then be contacted with a mobile binding *1s partner to produce a complex or alternatively, the bound analyte/support may be passed to zone for detection, provided that the bound analyte support is detectable in the waone using appropriate detection means. In a preferred embodiment, the mobile binding partner is suitably employed and preferably comprises an antibody which has a *.* ctable label that binds to a second epitope on the analyte such that the analyte may be detected by detecting the labelling species. Desirably, the mobile binding partner comprises an enzyme-labelled antibody. Preferably the mobile binding partner is present in excess to ensure that all the bound analyte is detected so as to provide, as desired, a quantitative assay. The amount of labelled antibody bound to the particles will thus be determined by the amount of analyte on the particles.

The labelled support is suitably separated from the unbound mobile binding partner, by being transferred into a wash phase and leaving the unbound mobile binding partner in the contact phase. The support having analyte and bound mobile binding partner suitably moves through the contact phase and the wash phase. The relative movement may be achieved by any suitable means including electric, magnetic, surface properties and desirably by sedimentation, especially desirably, by sedimentation under enhanced force, for example by centrifugation.

In a preferred embodiment, the relative movement is based on sedimentation of the support having analyte and bound mobile binding partner through the contact phase and wash phase. Desirably centrifugation is employed to provide or enhance the relative movement through the contact phase and wash phase. Suitably the less dense contact phase containing the unbound mobile binding partner remains upstream of the complex comprising the support, analyte and bound mobile binding partner which suitably resides, for example as a sediment in the wash phase.

In a preferred embodiment, the contact phase and wash phase are not contiguous and preferably are separated by an air gap as the sample is added to the contact phase. The method is suitably carried out in an assay device having a first zone for the contact phase ** : and a second zone for the wash phase where the first zone and second zone are discrete.

The sample is added to the support in the contact phase and resides in the contact phase for a pre-determined period, referred to herein as "incubation" time. The contact phase containing the support and bound analyte is then moved into contact with the wash phase.

The contact phase may be the same or different to the wash phase but is suitably different and of lower density than the wash phase. Suitably, the contact phase and wash phase are not fully miscible such that on contact between the contact phase and the wash phase, an interface between the two phases is formed. Suitably the support passes through the contact phase and into the wash phase such that the mobile labelled binding partner may bind to the support having bound analyte, and unbound mobile labelled binding partner is retained in the contact phase whilst the support with the bound analyte and bound labelled binding partner passes through it and into the wash phase.

Preferably the contact phase comprises a buffered solution, the sample (which may or may not contain analyte), the support and a mobile binding partner in cases where a complex between the analyte and the support and a mobile binding partner is to be formed. The contact phase may desirably also comprise components that minimise non-specific binding of the unbound mobile binding partner to the support.

Preferably the wash phase comprises a buffered solution, dextran or other solute to increase the density of the wash phase. In a preferred embodiment for generation of a detectable signal, the wash phase suitably comprises an enzyme substrate(s) and cofactors to generate a signal in the wash phase, preferably at the bottom of the wash phase, when enzyme label is present. The wash phase may desirably also comprise components that minimise non-specific binding of the unbound mobile binding partner.

By way of example the wash phase may comprise detergent, for example non-denaturing detergent, dextran, buffer, a substrate which interacts with the label on the complex, for example a luminescent substrate for a peroxidise label.

Preferably the complex comprises a label, preferably an enzyme label, on the bound mobile binding partner and the wash phase comprises a substrate on which the label acts to allow detection, for example by providing a coloured, fluorescent or light signal from the conjugate. *SS* * * * ** *

* The wash phase suitably has a greater density than the contact phase. A higher specific density may be obtained by known means, for example by dissolving a solute such as ***** * dextran in the phase. 25

* Desirably, the contact phase has a density D of greater than lg/cm3, preferably 1.001 to 1010, for example 1.006, and the wash phase has a density D of more than 1.01, preferably between 1.01 to 1.10, for example 1.05.

Desirably, the contact phase and wash phase are not miscible or have sufficient difference in density or other physical properties to minimise mixing on contact.

Suitably, the solid phase comprising the support, analyte and bound mobile binding partner has a density and particle size such that it may be suspended, desirably at unit gravity, in the contact phase and has a density greater than that of the wash phase. The solid phase may then in a preferred embodiment be centrifuged through the wash phase to separate it from unbound mobile binding partner, for example enzyme labelled antibody, in the contact phase.

Preferably, detection of an enzyme label in the invention is carried out using a chemiluminescent substrate such as luminol due to the high level of sensitivity achievable.

Low level light signals can be detected and quantified using a highly sensitive CCD camera, photomultiplier tube or an array of photodiodes that are placed to receive light from the wash phase. Image analysis software allows signal quantitation and the detection signals generated may suitably be compared to the signal generated by known concentrations of the analyte, therefore enabling the determination of analyte concentration in test samples by employing calibration.

The assay is especially beneficial in the detection of small analytes for example haptens in a competitive binding format.

The invention allows for multiplexing', whereby different populations of solid support are employed to assay different analytes which may be identified by their different fluorescence characteristics following their deposition in the assay window at the bottom of the wash phase. The emitted light signal or fluorescence signal generated by analyte binding to the *.* particles can be individually quantified and assigned to a particular assay particle population to give an assay of each analyte.

*...S. * *

*: 25 The invention further provides a single step assay device comprising a vessel having i) a contact zone for receiving a liquid phase comprising a sample suspected of containing an *** *..: analyte and optionally a mobile binding partner for the analyte, and a solid phase support capable of binding to the analyte and ii) a wash zone adapted to receive a wash phase and being in fluid communication with the contact zone.

Desirably, the assay device further comprises means for moving the contact zone and wash zone in fixed relation to effect movement of the solid phase from the contact zone to the wash zone, and means of detection of the analyte, if present, on the solid phase in the wash zone. Suitably the assay device comprises an elongate vessel having a closed end and an open end, a first zone for the contact phase disposed at or near the open end and a second zone for the wash phase at or near the closed end of the vessel, the first zone being discrete from the second zone. The open end suitably has a greater width than the closed end.

In a further aspect the invention provides wherein the Suitably, the vessel comprises an upper portion having an open end and a lower portion having a closed end and being of smaller horizontal, cross-sectional surface area than the upper portion and the upper portion and lower portion are in fluid communication and preferably connected by a sloping floor.

Preferably the vessel comprises a generally cylindrical upper portion having an open end and a generally cylindrical lower portion having a closed end and being of smaller radius than the upper portion and radially displaced from the axis of the upper portion. Desirably the contact zone is defined by a part of the floor and a part of the wall of the upper portion and the wash zone is defined by at least a part of the lower portion.

The vessel may comprise means to reduce or prevent contact of the contact phase with the wash phase whilst the complex between the analyte, support and mobile binding partner is being formed. Preferably, the floor slopes gently such that the contact phase rests on the floor and only moves towards the wash phase upon application of external force, for example by centrifugation. **** * * .

** The wash/substrate phase may be dispensed immediately prior to the assay or pre-dispensed into the tube and hermetically sealed for example by a welded sealing film over the top of the tube. In a preferred embodiment, the assay device comprises a vessel 25 containing a wash phase and wherein the open end is hermetically sealed prior to use.

Upon use, the seal may be opened and the contact phase dispensed into the upper portion of the vessel in readiness for receiving the sample to be analysed.

The assay device may comprise an individual vessel but preferably comprises a plurality of vessels according to the invention for use in a method of the invention. Desirably, a plurality of vessels are employed and may be incorporated into a card, disc or plate, for example an 8-well card or disc or 96-well plate comprising 12 cards per plate, as an array to enable a plurality of samples to be assayed simultaneously.

The assay device of the invention is illustrated by reference to the accompanying drawings in which; Figure la shows a side elevation of a vessel of an assay device according to the invention; and Figure lb shows a plan view of the vessel in Figurela.

The vessel in Figure la comprises a vessel (1) having an open end (2) and a closed end (3).

The upper portion (4) of the vessel (1) is generally cylindrical as is the lower portion (5). The axes of the upper portion (4) and the lower portion (5) are parallel but radially offset. The upper portion (4) and lower portion (5) are linked by a sloping floor (6). The sloping floor suitably slopes at around 10 to 15 degrees, for example 12 degrees to a line perpendicular to the longitudinal axis. The closed end (3) is transparent such that light emitted from the complex may be detectable by a suitable detector (7) from beneath the vessel so as to enable an array of vessels to be employed.

The first zone (8) for the contact phase is defined by part of the sloping floor (6) and part of the wall of the upper portion (4). The second zone (9) for the wash phase is defined by the lower portion (5) and there is a gap between the first zone (8) and the second zone (9). This is shown clearly in Figure lb. **** *. In a particularly preferred embodiment, the vessel (1) has a length of approximately 30mm, . the upper portion (4) has a length of approximately 5mm and a diameter of approximately 7mm, the lower portion (5) has a length of approximately 25mm and a diameter of approximately 2mm. The volume of the contact phase is suitably around 20 microlitres and ** 25 the volume of the wash phase is suitably around 70 microlitres. S..

The sloping floor (6) of the first zone (8) suitably facilitates movement of the contents of the contact phase in to the second zone (9) on application of force, for example by centrifuging the vessel (1) so that substantially all the solid phase in the contact phase (8) passes into the wash phase (9) and is urged to the assay window (3) at the base of the second zone (9).

In use, the vessel (1) is suitably held at an angle, for example around 24 degrees to reduce the risk and desirably prevent the contact phase coming into contact with the wash phase to allow any analyte in the sample, support and mobile binding partner to form a complex in the incubation period, for example 10 minutes, prior to passing to the wash phase. Holding the vessel or card or plate containing a plurality of vessels at an angle during the incubation period facilitates sample addition to the upper portion (4) and permits agitation, for example vibrational mixing to ensure efficient analyte capture by the suspended particles should this be required.

Generation of a signal for detection is suitably localised to the solid phase in the wash phase, desirably at the bottom of the lower portion of the vessel and the signal/noise ratio in the assay is suitably maximised by manipulation of the wash phase/substrate solution, composition and the geometry of the vessel tube, its material of construction and the location of the light detectors.

Suitably, only signal generated at the bottom of the wash phase is measured in order to maximise reliability and sensitivity of the assay. This may be affected in a number of ways including: 1) using the geometry of the system. Where light output is detected at an angle to the direction of sedimentation of the solid phase then any signal generated at the interface between the contact phase and the wash phase may be excluded.

2) where an enzyme label is employed, inclusion in the contact phase of a competitive inhibitor of the enzyme label also reduces light output from free enzyme conjugate retained nearer the top of the wash phase (at the interface between the contact phase and the wash * phase). For example, peroxidase stimulated light output from the interface may be reduced by the use of 3-amino-1,2,4-tnazole, for example 25mM; 3) the invention includes further embodiments which employ the generation of a substrate near the solid phase by an enzyme reaction. Where a peroxidise label is employed, *: 25 hydrogen peroxide substrate may be generated locally by employing glucose oxidase (GOX) immobilised on the support. The action of the GOX on glucose in the wash / substrate solution suitably generates hydrogen peroxide only in the vicinity of the solid phase thus localising the signal to that part of the wash phase containing the analyte containing complex; 4) use of a light adsorbing solute in the contact phase and I or the wash phase suitably reduces any signal generated at the interface between the contact phase and the wash phase while having little effect on signal generated close to the detection window; and 5) if a fluorescent label or an enzyme label that generates a fluorescent product is used for detection then the excitation light geometry may be used to exclude any fluorescence from other locations.

The invention is illustrated by the following non-limiting examples.

EXAMPLES

The examples below employed a Starlight Express MX5 camera with appropriate lenses to enable focusing of a card array of tubes at short distance in a light-proof box. Starlight Express software was employed to integrate the light signal and define the area from which it was integrated to optimise the signal/noise ratio. The approximate dimensions of the tube wash phase was 10mm with a diameter of 2mm and the contact phase reservoir above the wash phase was 5mm in diameter with a length of 8mm.

EXAMPLE I -Sandwich' assay of osteoprotegerin-human Fc fusion protein.

Wash Phase/substrate solution: ELISA Femto reagent: Super Signal ELISA Femto (product number 37075, Pierce) incorporating 33% by volume of a 10% w/v solution of dextran (MW 40,000). * * * *

Binding of biotinylated goat anti-human osteoprotegerin to streptavidin silica particles.

* . ..* 2Opl of lOOpgJml biotinylated goat anti-human osteoprotegerin (R&D Systems BAF8O5) in 0.1% BSA, PBS pH 7.4 was added to 0.8m1 0.1M Tris-HCI pH7.4. A suspension of 25 streptavidin-coated silica particles was also prepared by adding 16p1 of a 25mg/mi suspension of streptavidin silica particles (Kisker, 1.5 micron, density 2.0) to 0.8ml 0.IM Tris-HCI pH7.4. This particle suspension was added to the biotinylated goat anti-human osteoprotegerin solution and incubated for 1 hour with mixing. The suspension was centrifuged and supernatant poured off. This wash procedure was repeated four times in 1 ml 0.1M Tris-HCI pH 8.0.

Recombinant human osteoprotegerin-human Fc sample.

Recombinant human osteoprotegerin-Fc (R&D) was diluted in 0.1%w/v BSA/ PBS pH 7.4 to yield a bOng/mi solution. Dilutions of this solution were prepared in 0.1%w/v BSAIPBS pH 7.4.

Peroxidase-conjugated anti -human Fc (Sigma Aldrich) was diluted 1000-fold to 5 pg/mi in 8% by volume skimmed milk in 0.IM Tris-HCI pH7.4.

Method.

1. 37pl of ELISA Femto reagent was added to each vessel as shown in Figure la and centrifuged at 200 g for 1 mm.

2. To the wall of the vessel of the assay device near the top, a lOpI volume of osteoprotegerin-Fc solution was dispensed. To this was added a lOpI aliquot of peroxidase-conjugated anti-human Fc followed by lOp1 of biotinylated anti-human osteoprotegerin support.

3. Other vessels were prepared as controls. This included controls: a) without osteoprotegerin, b) the replacement of the above peroxidase conjugate with an inappropriate peroxidase conjugate, and C) an inappropriate biotinylated goat antibody on the particles.

4. Incubated for 10 minutes.

5. Centrifuged at 200 g for 3 mm.

6. The card holding the vessels was transferred to the box containing the camera and I" photographed with an exposure time of 1 minute. I4**

I

Figure 2 shows the image obtained in a set of vessels with 0 -1000 pg samples of osteoprotegerin-Fc. The upper light signals are due to residual peroxidase at the top of the 25 vessel. This may be blocked by an inhibitor as desired. Specific light signals are generated at the bottom of the vessel indicating the detected complex containing the analyte. The light emission is proportional to the amount of bound OPG-Fc. The relationship between amount of osteoprotegerin-Fc and light signal is plotted in the graph.

EXAMPLE 2

Competitive binding assay for biotin.

Substrate/wash solution: ELISA Femto reagent as in Example 1 Streptavidin coated support particle: lOpI of 0.5mg/mI suspension.

Biotinylated peroxidase: 5p1 of 5Ong/ml in 0.1%w/vBSA/PBS pH 7.4.

Biotin 107M, 0-5pl Method 1. 37p1 of ELISA Femto reagent was added to each vessel then centrifuged at 200g for 1 minute.

2. lOpi of biotinylated-peroxidase solution was dispensed onto the wall of the vessel near the top to provide a contact phase.

3. biotin solution then streptavidin beads were added to the vessel.

4. Incubated for 10 minutes.

5. Centrifuged for 1300 rpm (200g) for 3 minutes.

6. Photograph exposure time was 1 minute.

The results are shown in Figure 3. The light output from the bottom of the vessel is inversely proportional to biotin concentration as depicted in the graph of Figure 3. *..S * * * S. * * .55 * S...

*..S*S * S

S * S

S SS* * ***S

Claims

CLAIMS1. A method of detecting the presence of an analyte in a sample comprising: i) contacting the sample with a support having a site to which the analyte may bind wherein when the analyte is bound to the said site the presence of the bound analyte is detectable or the bound analyte is contacted with a labelling species such that the presence of the bound analyte may be detected by detecting the labelling species; ii) moving the said bound analyte to a second zone in which it is contacted with a wash phase whereby the bound analyte is separated from any unbound labelling species if present; and iii) detecting the presence of the bound analyte in the second zone.
2. A method of detecting the presence of an analyte in a sample comprising: i) contacting in a first zone the sample with a support having a site to which the analyte may bind; ii) contacting the support having the bound analyte with a mobile binding partner, capable of binding to a site on the bound analyte wherein the binding partner *.I.has a detectable label or is contacted with a labelling species such that the analyte may be detected by detecting the labelling species; * 20 iii) separating unbound labelled binding partner from the analyte-labelled binding partner complex by moving the said complex to a second zone in which it is contacted with a wash phase; and *..: iv) detecting the label to indicate the presence or absence of the analyte.
3. A method according to claim 2 wherein: i) the mobile binding partner is labelled and contacted with the support and bound analyte in a contact phase in a first zone to produce a complex of the support, analyte and labelled binding partner; ii) effecting movement of the said complex from the contact phase to a wash phase whereby unbound mobile labelled binding partner is left in the first zone such that the said complex is separated from the unbound mobile binding partner; and iii) detecting the complex to indicate the presence or absence of the analyte.
4. A method according to any one of the preceding claims wherein the analyte to be detected has two binding sites, the method comprising: i) providing a contact phase having a density D comprising a mobile labelled binding partner capable of binding to a free site on the analyte ii) providing a wash phase having a density D wherein D is greater than D; iii) in the contact phase, contacting the sample with a mobile support having a site to which the analyte may bind to provide a mobile solid phase comprising the mobile support having a bound analyte, if present in the sample; iv) passing the mobile solid phase through the contact phase such that the mobile labelled binding partner binds to the mobile solid phase to produce a complex of the support, analyte and labelled binding partner and through the wash phase; and v) detecting the presence or absence of the complex, if present, in the sample.
5. A method according to any one of the preceding claims wherein the support is selected from particles comprising silica or an organic polymer. *
6. A method according to any one of the preceding claims wherein the support comprises particles from O.2pm -2Opm.S 5.5*5

* 20
7. A method according to any one of the preceding claims wherein the support comprises cells. ****

**:
8. A method according to any one of the preceding claims wherein the support comprises particles coated with a binding partner selected from a group consisting of antigen, antibody, avidin, streptavidin, protein ligand, protein receptor, peptide, lectin, DNA, RNA, and a polynucleotide analogue.
9. A method according to any one of the preceding claims wherein the bound analyte support is contacted with a mobile labelled binding partner selected from a group comprising antigen, antibody, hapten, streptavidin, protein ligand, protein receptor, peptide, lectin, DNA, RNA and a polynucleotide analogue.
10. A method according to claim 9 wherein the labelled binding partner comprises an enzyme labelled binding partner.
11. A method according to claim 10 wherein the enzyme label is selected from peroxidase, microperoxidase or alkaline phosphatase, luciferase and beta galactosidase.
12. A method according to any one of the preceding claims wherein the label is fluorescent.
13. A method according to any one of the preceding claims wherein a labelled binding partner comprising an enzyme labelled binding partner is contacted with the analyte bound support and the wash phase comprises a stabilised solution of hydrogen peroxide and luminescence-producing substrate for the enzyme.
14. A method according to claim 13 wherein hydrogen peroxide substrate is generated in the wash phase by an enzyme bound on the support.
15. A method according to any one of the preceding claims comprising assaying a S...*,** plurality of analytes simultaneously in a single vessel wherein the analytes and/or support for each analyte each possesses a unique identifier.

*....S * * 20 *:
16. A method according to any one of the preceding claims wherein the support has a density of 1 to 20g/ml. ****
17. A method according to any one of the preceding claims wherein the support having analyte and optionally bound mobile binding partner passes from the contact phase into the wash phase by sedimentation.
18. A method according to any one of the preceding claims wherein the contact phase and wash phase are not contiguous.
19. A method according to any one of the preceding claims wherein the contact phase is of lower density than the wash phase.
20. A method according to any one of the preceding claims wherein the contact phase and wash phase are not fully miscible such that on contact between the contact phase and the wash phase, an interface between the two phases is formed.
21. A method according to any one of the preceding claims wherein the contact phase has a density D of greater than lg/cm3 and the wash phase has a density D of more than 1.01 and wherein D is less than D
22. A single step assay device comprising a vessel having i) a contact zone for receiving a liquid phase comprising a sample suspected of containing an analyte and optionally a mobile binding partner for the analyte, and a solid phase support capable of binding to the analyte and ii) a wash zone adapted to receive a wash phase and being in fluid communication with the contact zone.
23. A single step assay device according to claim 22 further comprising, means for moving the contact zone and wash zone in fixed relation to effect movement of the b0* : solid phase from the contact zone to the wash zone, and means of detection of the S...analyte, if present, on the solid phase in the wash zone.:: 20
24. A single step assay device according to any one of claim 22 and claim 23 wherein the vessel comprises an elongate vessel having a closed end and an open end, a first zone for the contact phase disposed at or near the open end and a second zone for the wash phase at or near the closed end of the vessel, the first zone being discrete from the second zone and the open end having a greater width than the closed end.
25. A single step assay device according to any one of claims 22 to 24 wherein the vessel comprises an upper portion having an open end and a lower portion having a closed end and being of smaller horizontal, cross-sectional surface area than the upper portion and the upper portion and lower portion being in fluid communication and connected by a sloping floor.
26. A single step assay device according to claim 22 to 24 wherein the vessel comprises a generally cylindrical upper portion having an open end and a generally cylindrical lower portion having a closed end and being of smaller radius than the upper portion and radially coincident with the axis of the upper portion and the upper portion and lower portion being in fluid communication and connected by a sloping floor, the sloping floor defining the contact zone and the lower portion defining the wash zone.
27. A single step assay device according to claim 25 or claim 26 wherein the sloping floor acts to reduce or prevent contact of the contact phase with the wash phase whilst the complex between the analyte, support and optionally mobile binding partner is being formed in the contact phase the said floor sloping gently such that the contact phase rests on the floor and only moves towards the wash phase upon application of external force.
28. A single step assay device according to claim 27 wherein the external force is applied by centrifugation.
29. A single step assay device according to any one of claims 22 to 28 wherein the S..... : vessel comprises a wash phase and the open end is hermetically sealed and _.* adapted to be removed or penetrated to allow a contact phase to be dispensed into the upper portion of the vessel in use. * 2030. A single step assay device according to any one of claims 22 to 29 comprising a plurality of vessels. S...31. A single step assay device according to claim 30 in which the plurality of vessels are located in a card, disc or plate as an array to enable a plurality of samples to be assayed simultaneously.