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

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 commonly 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-291194 describes a lateral flow assay and a device for detecting an analyte in such an assay. The device comprises a porous carrier provided with a labelled binding reagent which is freely mobile on the carrier upon application of a liquid test sample, the reagent being capable of binding to the analyte, and an unlabeled binding reagent also capable of binding to the analyte and which is permanently immobilised at a detection zone downstream 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 binding 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. 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 on a support and present an epitope for binding to an antibody in free solution which is mobile. 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 antibody. The mobile antibody, which may be labelled or conjugated with an enzyme, suitably 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 "sandwich" 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 unlabeled 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.

Competition assays allow extension of the dynamic range of an assay when employed in conjunction with a sandwich assay by enabling analysis for analyte at levels where the "hook" effect may reduce the usefulness of a sandwich assay. Competition assays are particularly 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 binding.

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.

Apparatus and methods are known for separating and washing reacted analyte-assay species and then detecting them. These methods tend to involve carrying out the assay reaction to react the sample with the binding reagents separately from the subsequent separation and washing step. The sample-assay mixture is then suitably transferred to a zone for separation, washing and detection of the sample. This involves manual or mechanical intervention and sampling handling after the reaction has occurred and apparatus used for the separation and washing process may be complex in its construction and be costly to produce which is especially disadvantageous where analysis of large numbers of samples is required. Furthermore, known methods and devices for instance devices employing centrifugal force to effect separation may be disadvantageous in that they are able to accommodate only a single sample per device increasing costs of operation and complexity further.

The present invention seeks to ameliorate drawbacks of conventional assays by carrying out the sample-assay reaction employing a mobile solid phase and moving the solid phase having a bound analyte through a liquid phase so effecting a separation of labelled analyte and unbound label.

In a first 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 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.

The invention further provides a single step assay method for detecting the presence of an analyte in a sample comprising:

i) carrying out an assay reaction in a first zone comprising 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.

An assay reaction is carried out in the first zone by contacting the sample with the support having the site to which the analyte may bind. Suitably, the first zone and second zone are maintained in fixed relationship and the bound analyte is moved to the second zone by the application of a force which acts on the bound analyte support to effect relative movement from the first zone to the second zone for example gravity, magnetic, electric and especially centrifugal force. The support is moved suitably without external manual or mechanical intervention to move the first zone which contains the sample and the support relative to the second zone.

The invention enables the assay reaction and washing and detection of the sample to be carried out without requiring sample handling, either manual or mechanical, between the assay reaction and washing steps. 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 allows for the detection of the analyte. As compared to known methods, the invention provides a simple and cost effective method to analyse samples, separate unbound labelled species and detect the presence of the analyte without requiring intervention to move the reacted sample assay to different apparatus for the washing and separation step and without using complex separation apparatus. Further, the method enables a plurality of samples to be analysed and processed simultaneously.

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

Advantageously, the user adds the sample to the solid support and binding partners in the first zone and no further intervention to transfer the reacting materials is required. The analyte in the sample reacts with the solid support and is then transferred to the wash zone whereby the process is carried out in a single step.

Suitably, the first zone and second zone are in fixed relation to each other. Movement of the bound analyte from the first zone is suitably effected without manual or mechanical intervention to transfer the reacted sample to the wash zone. Preferably, movement of the bound analyte is effected by moving the apparatus, for example a an assay device comprising an elongate vessel, defining the first and second zone such that motive force is applied to the bound analyte and it passes from the first zone to the second zone. The force may be generated externally for example by rotation of the apparatus to generate a centrifugal force or where the support is susceptible to magnetic or electrical force, by external application of a magnetic or electrical field such that movement from the first zone to the second zone of the solid phase with the bound analyte is effected.

In a preferred embodiment, the first zone comprises a proximal end through which the sample is loaded and a distal end through which the analyte bound to the support passes into the second zone and the proximal end and the distal end of the first zone are aligned and coincident with the direction of the force applied to effect movement of the bound analyte to the second zone. For example, where a centrifugal force is applied by a centrifuge to effect movement of the bound analyte by rotation of the apparatus and generation of the centrifugal force, the proximal opening and distal opening of the first zone both lie on the line of the centrifugal force and sweep through a circular locus in and the proximal opening is radially inward of the distal opening whereby the centrifugal force causes the bound analyte and support to move through the distal opening of the first zone and into 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 preferably to effect an assay reaction;

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 analyte may be detected by detecting the labelling species;

iii) separating unbound labelled binding partner from the analyte-labelled binding partner complex by moving the said complex to a second zone; and

iv) detecting the label to indicate the presence or absence of the analyte.

The invention also provides an assay method for 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 to effect an assay reaction;

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 analyte may be detected by detecting the labelling species; iii) separating unbound labelled binding partner from the analyte-labelled binding partner complex by moving the said complex to a second zone; and

iv) detecting the label to indicate the presence or absence of the analyte. The method is especial suitable for analysing a plurality of samples simultaneously. The support with or without the bound analyte and optionally with bound mobile binding partner 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 binding or by coating, an antibody or other material which is able to present a binding site for the analyte. 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. Suitably the support is not coloured so as to reduce the risk of interference with the detection of the analyte where light emission or colour change is employed as a means of detection.

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 1 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 invention is especially beneficial in detecting the presence of an analyte in a complex biological fluid. In a further 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 to provide 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 _c 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 _w wherein D _w is greater than D _c ;

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.

This method also provides a single step assay method wherein, in step iii), the sample is contacted with a mobile support to effect an assay reaction. The assay reaction and wash step are suitably carried out without manual or mechanical intervention other than application of a force to effect movement of the bound analyte from the contact phase to the wash phase.

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 particle having coated on it a binding ligand, for example an antibody. The support having the bound analyte, if present in the sample, may then be contacted with a mobile binding partner to produce a complex or alternatively, the bound analyte/support may be passed to the wash zone for detection, provided that the bound analyte support is detectable in the wash zone using appropriate detection means. In a preferred embodiment, the mobile binding partner is suitably employed and preferably comprises an antibody which has a detectable 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, preferably at least 10 seconds, more preferably at least 1 minute and especially at least 5 minutes. The incubation time is suitably not longer than 30 minutes, preferably 20 minutes and desirably 15 minutes. 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. 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.

Desirably, the contact phase has a density D _c of greater than 1g/cm ³ , preferably 1.001 to 1.010, for example 1.006, and the wash phase has a density D _w 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.

The invention further provides a single step assay device comprising a vessel which is preferably elongate and preferably has an open end and a closed end, the vessel having 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.

Suitably the single step assay device is adapted for rotation about an axis which is perpendicular to the axis of the elongate vessel such that the vessel sweeps radially about the axis of rotation with the closed end being further from the axis of rotation than the open end whereby the sample is urged centrifugally from the open end of the vessel to the closed end of the vessel as a result of the rotation. 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 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 arrangement of the upper portion, lower portion and sloping floor is such that on adding the liquid phase and solid phase to the upper portion, the mixture rests in the upper portion or on the sloping floor and reaction of the analyte with the solid phase particle occurs and, on application of centrifugal force along the axis of the vessel, travels across the sloping floor to the lower portion and through a wash solution. 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 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. The method of the invention allows a plurality of samples to be analysed simultaneously by employing a plurality of the assay devices according to the invention with a single apparatus for effecting movement of the bound analyte/solid phase from the first zone to the second zone. 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 invention further provides a centrifugal assay device comprising a rotatable support adapted to receive a single step assay device according to the invention and means for rotating the support. Preferably the centrifugal device comprises a plurality of single step assay devices.

The assay device of the invention is illustrated by reference to the accompanying drawings in which: Figure 1 shows a side elevation of a vessel of an assay device according to the invention;

Figure 2 shows a plan view of the vessel in Figure 1.

Figures 3 to 5 show a side elevation of a vessel according to the invention showing successive stages of the single step process of the method of the invention; and

Figure 6 show a perspective view of an array of eight vessels arranged in a card.

The vessel in Figure 1 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 upper portion (4) defines the first zone which comprises the contact zone and the lower portion (5) defines the second zone comprising the wash zone. 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. In other embodiments, the axes may be coincident with the upper portion (4) having a sloping floor (6) which is in the form of an annular ring extending inwardly from the outer wall of upper portion (4) to the edge of lower portion (5). 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 contact phase (8) in the first zone is retained 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 suitably a gap between the contact phase (8) in the first zone and the second zone (9). This is shown clearly in Figure 2.

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 the volume of the wash phase is suitably around 70 microlitres.

The sloping floor (6) 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 ) to apply a centrifugal force in a direction from open end (2) to closed end (3) 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, preferably between 10 and 35 degrees, more preferably from 15 to 30 degrees, 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 which is suitably from 1 to 20 minutes, 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.

The first zone has a proximal end which is the open end (2) through which the sample is added to vessel into the contact zone and also has a distal end which is defined by the point at which the sloping floor meets the top of the lower portion and is the interface between the first zone and the second zone.

In Figure 3, the sample (10) is added to solid support having an assay reagent (11) in the contact zone to provide a contact phase (8) which contains solid phase and mobile binding partner and the sample. The vessel is suitably held at an angle to prevent contact between the assay reagent in the contact phase and the wash reagent in the wash phase (12) in the second zone (9) until required by the user.

Figure 4 shows the single step apparatus during use, that is upon application of a centrifugal force in the direction of the arrow marked F. Figure 4A shows where the force becomes aligned with the length of the lower portion (5) and the contact phase passes along the slope (6) into the lower portion (5) and contacts the wash phase (12) in the second zone. The solid phase (11) passes from the contact phase into the wash phase as it is centrifuged and is progressively washed by the wash reagent in the wash zone (9) as shown in Figure 4B.

Figure 5 shows the single step apparatus after centrifugation. The detectable species, for example an enzyme labelled binding partner attached to the solid phase, now produces a detectable signal, for example light (13), in the wash zone (9), suitably at the bottom (3) of the vessel. Figure 6 shows a perspective representation of an array of vessels arranged in a card (14) with the open end (2) of the vessel on the top of the card. The sample has been added and moved from the contact zone into contact with the wash zone. The solid support is not shown. The vessels are suitably arranged in the card with the open (2) arranged for receipt of a sample into the upper portion (4) of the vessel (1). Suitably the vessels are charged with wash solution (12) in the wash zone (9) and solid support (10) in the first zone.

Multiples of these cards may also be arranged in a block analogous to microplate. The vessels and cards are suitably processed simultaneously. 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- triazole, 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, 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 / 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 1 - '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.

20μΙ of 100 g/ml biotinylated goat anti-human osteoprotegerin (R&D Systems BAF805) in 0.1% BSA, PBS pH 7.4 was added to 0.8ml 0.1M Tris-HCI pH7.4. A suspension of streptavidin-coated silica particles was also prepared by adding 16μΙ of a 25mg/ml suspension of streptavidin silica particles (Kisker, 1.5 micron, density 2.0) to 0.8ml 0.1 M 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 100ng/ml solution. Dilutions of this solution were prepared in 0.1%w/v BSA/PBS pH 7.4.

Peroxidase-conjugated anti - human Fc (Sigma Aldrich) was diluted 1000-fold to 5 pg/ml in 8% by volume skimmed milk in 0.1 M Tris-HCI pH7.4.

Method.

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

2. To the wall of the vessel of the assay device near the top, a 10μΙ volume of osteoprotegerin-Fc solution was dispensed. To this was added a 10μΙ aliquot of peroxidase- conjugated anti-human Fc followed by 10μΙ 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 min.

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

Figure 7 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 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: 10μΙ of 0.5mg/ml suspension.

Biotinylated peroxidase: 5μΙ of 50ng/ml in 0.1%w/v BSA/PBS pH 7.4.

Biotin 10 ^'7 M, 0-5μΙ Method

1. 37μΙ of ELISA Femto reagent was added to each vessel then centrifuged at 200g for 1 minute.

2. 10μΙ 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 8. The light output from the bottom of the vessel is inversely proportional to biotin concentration as depicted in the graph of Figure 8.