WO2009144506A1

WO2009144506A1 - Assay device

Info

Publication number: WO2009144506A1
Application number: PCT/GB2009/050589
Authority: WO
Inventors: Ezra Linley; David Darrock; Natalie Beesley
Original assignee: Spd Swiss Precision Diagnostics Gmbh
Priority date: 2008-05-31
Filing date: 2009-05-29
Publication date: 2009-12-03
Also published as: GB0809997D0

Abstract

Disclosed is an assay device for determining the presence and/or extent of an analyte in a liquid sample comprising: a porous carrier (12) having respective first and second surfaces (14, 16) comprising a mobilisable first labelled binding reagent for an analyte provided upstream from a detection zone for immobilising the first binding reagent; a porous sample receiver (11) for receiving a liquid sample having respective first and second surfaces (13, 15); wherein said porous sample receiver is provided upstream from and at least partially overlaps said porous carrier at their respective surfaces (15, 14); wherein the first binding reagent is provided substantially away from the surface (14) of the porous carrier.

Description

Assay device Field of the Invention

The present invention relates to an assay device and method for determining the presence or extent of an analyte.

Background of the Invention

Simple lateral flow immunoassay devices have been developed and commercialised for detection of analytes in fluid samples, see for example EP291194. Such devices typically comprise a porous carrier comprising a dried mobilisable labelled binding reagent capable of binding to the analyte in question, and an immobilised binding reagent also capable of binding to the analyte provided at a detection zone downstream from the labelled binding reagent. Detection of the immobilised labelled binding at the detection zone provides an indication of the presence of analyte in the sample.

Alternatively, when the analyte of interest is a hapten, the immunoassay device may employ a competition reaction wherein a labelled analyte or analyte analogue competes with analyte present in the sample for an immobilised binding reagent at a detection zone. Alternatively the assay device may employ an inhibition reaction whereby an immobilised analyte or analyte analogue is provided at a detection zone, the assay device comprising a mobilisable labelled binding reagent for the analyte.

The assay device may comprise a porous sample receiver for receiving a fluid sample. This is very convenient for testing an analyte such as the pregnancy hormone hCG in urine as it allows for "mid-stream" sampling, namely the assay device is sampled directly in the urine stream. Alternatives to mid-stream sampling are the pipetting of a liquid sample onto the assay device or dipping of the assay device into a collection vessel containing the liquid sample.

Summary of the invention In a first aspect the invention provides an assay device for determining the presence and/or extent of an analyte in a liquid sample comprising: a porous carrier (12) having respective first and second surfaces (14, 16) comprising a mobilisable first labelled binding reagent for an analyte provided upstream from a detection zone for immobilising the first binding reagent; and a porous sample receiver (11) for receiving a liquid sample having respective first and second surfaces (13, 15); wherein said porous sample receiver is provided upstream from and at least partially overlaps said porous carrier at their respective surfaces (15, 14); wherein the first binding reagent is provided at surface (16) and/or within the first porous carrier material but substantially away from the surface (14) of the porous carrier.

According to a second aspect of the invention there is provided a method of manufacturing an assay device for determining the presence and/or extent of an analyte in a liquid sample, comprising: a) applying a first labelled binding reagent to the surface (16) of a porous carrier (12) upstream from a detection zone capable of immobilising the first binding reagent, said porous carrier having first and second surfaces (14, 16); b) overlapping a porous sample receiver (11) having first and second surfaces (13, 15) and provided upstream from the porous carrier at their respective surfaces (15, 16).

Steps (a) and (b) may be carried out in either order.

The labelled binding reagent may be provided in the dry state in the assay device.

The detection zone may comprise an immobilised binding reagent which is capable of immobilising the first binding reagent.

The porous sample receiver may have a porosity greater than that of the porous carrier. According to a particular embodiment, the assay device additionally comprises a second binding reagent for the analyte provided upstream from the detection zone. The second binding reagent may be provided in the vicinity of, or at the same position as, the first binding reagent. The second binding reagent is typically unlabelled.

The labelled binding reagent, and the second binding reagent if present, is provided subtantially away from the surface (14) of the first porous carrier. That is to say that the majority of the binding reagent(s) are present either in the interior of the the first porous carrier and/or at the surface (16). Due to the porous nature of the porous carrier, some of the binding reagent(s) applied to the surface (16) of the porous carrier may be transported to the surface (14).

The second binding reagent may be mobilisable or immobilised on the porous carrier. The second binding reagent may be capable of being immobilised at the detection zone.

The method according to the second aspect may comprise applying a mixture of the first and second binding reagents to the surface of the porous carrier.

The porous carrier may comprise one or more porous carrier materials which may overlap in a linear or stacked arrangement or which are fluidically connected. The assay device may be a lateral flow assay device.

According to an embodiment, the porous carrier comprises a first porous carrier material comprising the binding reagent(s) provided upstream from, and at least partially overlapping, a second porous carrier material comprising the detection zone. The first and second porous carrier materials may be in the form of strips or sheets. The first and second porous carrier materials may be the same or different.

The first porous carrier material may overlap the porous sample receiver at its proximal end region and overlap the second porous carrier material at its distal end region. The binding reagent(s) may be applied and be present anywhere along the surface (16) of the first porous carrier material. According to an embodiment, the binding reagent(s) are present more towards the proximal end region than the distal end region of the first porous carrier material.

According to an embodiment, the first reagent is provided on a macroporous carrier. The macroporous carrier material should be low or non-protein-binding, or should be easily blockable by means of reagents such as BSA or PVA, to minimise non-specific binding and to facilitate free movement of the labelled reagent after the macroporous body has become moistened with the liquid sample. The macroporous carrier material can be pre-treated with a surface active agent or solvent, if necessary, to render it more hydrophilic and to promote rapid uptake of the liquid sample. Suitable materials for a macroporous carrier include plastics materials such as polyethylene and polypropylene, or other materials such as paper or glass-fibre. In the case that the labelled binding reagent is labelled with a detectable particle, the macroporous body may have a pore size at least ten times greater than the maximum particle size of the particle label. Larger pore sizes give better release of the labelled reagent.

Suitable materials that may be employed as a porous carrier for providing the detection zone include nitrocellulose, acetate fibre, cellulose or cellulose derivatives, polyester, polyolefm or glass fibre. The porous carrier may comprise nitrocellulose. This has the advantage that a binding reagent can be immobilised firmly without prior chemical treatment. If the porous solid phase material comprises paper, for example, the immobilisation of the antibody in the second zone needs to be performed by chemical coupling using, for example, CNBr, carbonyldiimidazole, or tresyl chloride.

The porous sample receiving member can be made from any bibulous, porous or fibrous material capable of absorbing liquid rapidly. The porosity of the material can be unidirectional (i.e. with pores or fibres running wholly or predominantly parallel to an axis of the member) or multidirectional (omnidirectional, so that the member has an amorphous sponge-like structure). Porous plastics material, such as polypropylene, polyethylene (preferably of very high molecular weight), polyvinylidene fluoride, ethylene vinylacetate, acrylonitrile and polytetrafluoro-ethylene can be used. Other suitable materials include glass-fibre. In an embodiment, the assay device comprises a glass-fibre macroporous carrier provided upstream from, and overlapping at its distal end, a nitrocellulose porous carrier.

The term "binding reagent" refers to a member of a binding pair, i.e., two different molecules wherein one of the molecules specifically binds with the second molecule through chemical or physical means. The two molecules are related in the sense that their binding with each other is such that they are capable of distinguishing their binding partner from other assay constituents having similar characteristics. The members of the specific binding pair may be referred to as ligand and receptor (antiligand), a binding pair member and binding pair partner, and the like. A molecule may also be a binding pair member for an aggregation of molecules; for example an antibody raised against an immune complex of a second antibody and its corresponding antigen may be considered to be a binding pair member for the immune complex. The binding reagent may comprise an antibody or an antibody fragment, capable of binding to an antigen.

In addition to antigen and antibody binding pair members, other binding pairs include, as examples without limitation, biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, complementary peptide sequences, effector and receptor molecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes, a peptide sequence and an antibody specific for the sequence or the entire protein, polymeric acids and bases, dyes and protein binders, peptides and specific protein binders (e.g., ribonuclease, S-peptide and ribonuclease S-protein), and the like. Furthermore, specific binding pairs can include members that are analogues of the original specific binding member.

"Label" when used in the context of a labelled binding reagent, refers to any substance which is capable of producing a signal that is detectable by visual or instrumental means. Various labels suitable for use in the present invention include labels which produce signals through either chemical or physical means, such as being optically detectable. Such labels include enzymes and substrates, chromogens, catalysts, fluorescent compounds, chemiluminescent compounds, electroactive species, dye molecules, radioactive labels and particle labels. The analyte itself may be inherently capable of producing a detectable signal. The label may be covalently attached to the binding reagent.

The label may comprise a particle such as gold, silver, colloidal non-metallic particles such as selenium or tellurium, dyed or coloured particles such as a polymer particle incorporating a dye, or a dye sol. The dye may be of any suitable colour, for example blue. The dye may be fluorescent. Dye sols may be prepared from commercially- available hydrophobic dyestuffs such as Foron Blue SRP (Sandoz) and Resolin Blue BBLS (Bayer). Suitable polymer labels may be chosen from a range of synthetic polymers, such as polystyrene, polyvinyltoluene, polystyrene-acrylic acid and polyacrolein. The monomers used are normally water-insoluble, and are emulsified in aqueous surfactant so that monomer micelles are formed, which are then induced to polymerise by the addition of initiator to the emulsion. Substantially spherical polymer particles are produced. According to an exemplary embodiment the label is a blue polymeric particle.

The liquid sample can be derived from any source, such as an industrial, environmental, agricultural, or biological source. The sample may be derived from or consist of a physiological source including blood, serum, plasma, interstitial fluid, saliva, sputum, ocular lens liquid, sweat, urine, milk, mucous, synovial liquid, peritoneal liquid, transdermal exudates, pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations, cerebrospinal liquid, semen, cervical mucus, vaginal or urethral secretions and amniotic liquid. In particular the source is human and in particular the sample is urine.

Analytes include, but are not limited to, toxins, organic compounds, proteins, peptides, microorganisms, bacteria, viruses, amino acids, nucleic acids, carbohydrates, hormones, steroids, vitamins, drugs (including those administered for therapeutic purposes as well as those administered for illicit purposes), pollutants, pesticides, and metabolites of or antibodies to any of the above substances. The term analyte also includes any antigenic substances, haptens, antibodies, macromolecules, and combinations thereof. In particular the analyte is hCG.

During the course of their investigations, the inventors surprisingly found that for some types of assay device, the result obtained of the amount of analyte in the liquid sample varies depending upon how the assay device is sampled. In particular the results were shown to differ when dipping the assay device with it porous sample receiver lowermost into a liquid sample and when pipetting a liquid sample onto an assay device.

The reasons for this are unclear but it is thought to be due to the velocity at which liquid sample passes along or through the device. When the device is positioned horizontally, for example when pipetting sample, gravity does not contribute to the flow velocity, however, the velocity at which sample exits the pipette may have a contributing factor. When dipping the assay device into a container containing sample, the sample application region is positioned lowermost such that gravity is inhibiting flow along the assay device. Furthermore there may be no initial velocity of the liquid sample, i.e. it is stationary.

Other means of testing are also possible, such as midstream testing whereby the assay device is held in a urine stream. When testing midstream, the angle of which the assay device may be held by the user may vary. The assay device is held with the sample application region uppermost such that gravity may contribute to the flow velocity of the liquid sample. Furthermore the liquid sample may be travelling at different speeds when it contacts the assay device.

The inventors found that this difference in sampling caused a variation in the amount of labelled binding reagent that was detected at the detection zone. In particular this was found to be the case, when a competition type of assay is employed, wherein a first binding reagent competes with a second binding reagent for analyte.

Depending upon the sampling method, liquid sample may pass along the assay device at different velocities. Furthermore it is possible that the assay device may become flooded, namely instead of flowing through the pore structure of the porous carrier, some of the liquid sample may flow over the surface of the carrier. This can happen when, for example, the assay device is held with the porous sample receiver uppermost after having sampled with liquid sample. When the device "floods", the liquid sample is less impeded by the pore structure of the porous carrier and thus proceeds more quickly along the porous carrier.

The flow speed of liquid sample along the porous carrier in turn affects the time of interaction between the binding reagent and the sample. In particular, when two binding reagents are employed, the flow speed of the sample affects the time of interaction between the two binding reagents.

It is thought that this observed difference may be due to the flow velocity of the liquid sample wherein differences in flow velocity cause a change in the time of interaction between the analyte and the binding reagents, resulting in a change in the ratio of the amount of binding of analyte to the first and second binding reagents. Depending upon the binding characteristics of the binding reagents, for example their relative binding affinities and avidities, the relative amounts of binding of the first and second reagents to the analyte may vary. This change in ratio effects the amount of labelled binding reagent that becomes bound at the detection zone, which in turn effects the assay result.

Typically the porous sample receiver is highly porous such that it is able to take up liquid sample rapidly and typically the sample receiver has a higher porosity than the first porous carrier. Thus if liquid sample is taken up by the sample receiver too rapidly, it cannot be absorbed quickly enough by the first porous carrier and a portion of the liquid sample tends to flow along the surface (14) at a higher velocity than in the bulk of the porous carrier. When labelled reagent is provided at the surface (14) according to assay devices of the prior art, the rate at which labelled reagent is resuspended or is in contact with the liquid sample may vary depending upon the flow velocity of the sample. However, when the binding reagent(s) is positioned on or in the porous carrier away from this surface, only liquid sample which is travelling through the porous carrier will interact with said reagent(s) thus effectively normalising the flow rate of the liquid at the point of contact with the binding reagent(s). This has the effect of reducing the variability seen in assay result depending upon the sampling method.

When detecting analytes using an immunoassay, the assay signal tends to decrease at high concentrations due to the "hook effect". In order to detect analyte at high concentration levels, one solution is to employ a second binding reagent which competes with a first labelled binding reagent for the analyte. This has the effect of decreasing the analyte sensitivity.

It was found that assay devices constructed according to the first aspect showed a decrease in variation with different sampling methods, i.e. the variation in result obtained by assay device when sampling by different methods was substantially reduced. It is thought that this occurs due to the positioning of the labelled first binding reagent, and optionally the second binding reagent, away from the flow stream that occurs due to flooding of the device.

In embodiments of the invention, liquid sample passes from the sample receiver primarily to a proximal surface of the porous carrier, the first labelled binding reagent being predominantly provided on or close to the distal surface of the porous carrier and remote from the proximal surface. In this way sample liquid must pass into the porous carrier and penetrate some way towards the distal surface (e.g. typically at least 50% of the depth of the porous carrier) in order to mobilise the first labelled binding reagent. Similarly, a second labelled binding reagent, if present, is preferably predominantly provided on or close to the distal surface of the porous carrier and remote from the proximal surface.

A way to achieve such an assay device is set out in the first and second aspects of the invention. Providing labelled reagent on the distal surface (16) of the porous carrier is a convenient way to remove the labelled reagent, or at least a substantial amount of it, from the proximal surface (14) directly in the path of the flooding flow stream. Other ways of reducing the variability between sampling method and on the assay result and corresponding assay contructs are contemplated. It may not be necessary for example to provide the labelled reagent in a position away from surface (14) of the first porous carrier. For example, the porous sample receiver may comprise a flow restriction means provided upstream from the binding reagent(s) in order to slow and effectively normalise flow. The flow restriction means may comprise a portion of the flow-path of lower porosity than that of the porous sample receiver.

Alternatively the assay device may comprise a first porous carrier material comprise two or more laminates wherein the binding reagent(s) are applied to an upper surface of a first laminate and a second laminate placed on top of the first laminate such that labelled reagent is effectively provided within the first porous carrier material and away from surface (14) of the first porous carrier material.

In yet a further alternative, the assay device may comprise a first porous carrier having two outer surfaces (14,16) may comprise two laminates wherein a distal end region of the porous sample receiver is effectively sandwiched between the two laminates thus directing flow towards the interior of the first porous carrier. The labelled reagent may be applied to either face of the outer surfaces.

The assay device may be provided within a housing. The porous sample receiver may partially extend out of the housing through an aperture.

The assay device may comprise one or more of the following: a central processing unit (CPU) or microcontroller; one or more LEDs; one or more photodetectors; a power source; and associated electrical circuitry. The power source may be a battery or any other suitable power source (e.g. a photovoltaic cell). Conveniently the CPU or microcontroller will be programmed so as determine, from the output of the photodetectors, the rate or amount of signal accumulation and to compare this to control and measurement thresholds.

The assay device may comprise a timing means by which to measure the time of the assay and by which to determine the time of commencement of measurement of the assay. The timing means may for example comprise a sample presence indication means to detect the time at which liquid sample is added to the device such as a pair of electrodes which are able to detect the presence of liquid sample. Alternatively the timing means may be comprised as part of the optical detection means, wherein timing of the assay measurement is commenced at the time liquid sample is determined by the photodetector as having reached a particular detection or reference zone.

The assay device may further comprise a display means to display the result of the assay. The display means may further display further information such as an error message, personal details, time, date, and a timer to inform the user how long the assay has been measured for. The information displayed by the assay may be indicated in words, numbers or symbols, in any font, alphabet or language, for example, "positive", "negative", "+", "-", "pregnant", "not pregnant", "see your doctor", "repeat the test".

The assay device may comprise a signal detection means to determine the extent and/or amount of labelled species present at the detection and control zones. The signal detection means may comprise an optical detection means such as a photodetector to determine the extent and/or amount of labelled species present. The assay device may comprise one or more light sources such as an LED positioned so as to optically illuminate the zones. Light from the light source illuminates the respective zones and is either transmitted or reflected onto a photodetector which records the amount or intensity of the transmitted or reflected light. The presence of labelled binding reagent at the zones will influence the amount of light that is either transmitted or reflected, thus measurement of light at the photodetector is indicative of the presence or amount of the labelled binding reagent.

The assay device may further comprise a reference zone. The purpose of the reference zone is to provide a signal value against which the signal values obtained at the detection and control zones may be compared. Measurement of the reference zone enables measurement of the background levels of reflected or transmitted light from the flow-path. The background level may be due for example to the optical reflectance of the porous carrier, the presence of liquid sample, or of components of the assay such as a labelled binding reagent. The levels of light measured at the detection zone may therefore be corrected with respect to the levels of background light to provide a compensated signal indicative of the amount of labelled binding reagent present at the detection zone. Measurement at the reference zone may also compensate for any variation between fluid samples applied to assay devices, for example urine samples may vary widely in colour.

A suitable light source is an LED. The colour of the LED will be determined by the colour of the labelled binding reagent. For a blue label, a suitable colour for the LED is red. The LED may be illuminated at a particular frequency or frequencies in order to illuminate a particular zone of the assay device. Light is reflected or transmitted from the zone onto a photodetector which records an electrical signal. The number of electrical signals recorded will depend upon the operating frequency of the LED and thus one or more signals may be recorded over time. The signals will typically be expressed as a % absorbance (%A).

Each measurement zone is typically illuminated by a single LED. A photodetector may detected light from one than one measurement zone and therefore reflected light from one than one LED. This may be achieved by carrying out the illumination process sequentially such that device is able to know which from which zone light is being reflected from onto the photodetector. The sequential illumination process may be repeated with a fixed or varied frequency during the duration of the assay such that the levels of signal over time at each zone may be monitored.

The device may comprise a means to detect the time addition of flow to the assay device. For example, the change in levels of light detected from one or more zones may be monitored to determine whether and when a fluid sample has been applied to the device. The timing of the assay test may be started automatically for example when liquid sample has reached a particular zone. The device may comprise a flow control means wherein the change in levels of light detected from one or more zones may be used to determine whether and when a fluid sample has been applied to the device and to determine the flow-rate of liquid sample along the device by measurement of flow between one or more measured zones. Determination of the flow-rate may be used as a further quality control check, for example the assay may be rejected if the flow-rate is either greater than or less than set levels. The computation circuit may be responsive to the signals to calculate a flow rate for a fluid flowing along the carrier, compare the calculated flow rate to upper and lower limits, and reject the assay result if the calculated flow rate is outside the upper and lower limits.

The typical optical detection system will comprise at least one light source and at least one photodetector (such as a photodiode). Preferred light sources are light emitting diodes or LEDs. Reflected light and/or transmitted light may be measured by the photodetector. For the purposes of this disclosure, reflected light is taken to mean that light from the light source is reflected from the porous carrier or other liquid transport carrier onto the photodetector. In this situation, the detector is typically provided on the same side of the carrier as the light source. Transmitted light refers to light that passes through the carrier and typically the detector is provided on the opposite side of the carrier to the light source. For the purposes of a reflectance measurement, the carrier may be provided with a backing such as a white reflective MYLAR® plastic layer. Thus light from the light source will fall upon the carrier, some will be reflected from its surface and some will penetrate into the carrier and be reflected at any depth up to and including the depth at which the reflective layer is provided. Thus, a reflectance type of measurement may actually involve transmission of light through at least some of the thickness of the porous carrier.

The assay device will typically comprise one or more apertures or windows through which light may shine from the one of more sources of illumination onto a particular zone of the assay or assay strip. The windows serve to define the area of light falling onto a particular zone and to define which part of the assay or assay strip is illuminated. Each zone to be illuminated may have a corresponding window. Thus a device having four measurement zones will have four windows. Light reflected from the windows is collected by the one or more photodetectors. For an assay device comprising a flow path having a plurality of zones the time taken for the liquid sample to travel between the zones may be measured.

Measurements of the light reflected from each window may be taken periodically (for example approximately twice a second) and a low pass digital filter may be used to reject noise and smooth the data. Filtered values may be used for detecting flow and determining the assay result.

For each window, a ratio may be calculated of the measured value when the particular measurement zone in the flow-path is dry ("calibration value"), namely before any liquid sample has reached said zone, divided by the measured value when the measurement zone is wet and a line may have developed. This ratio equals the proportion of light reflected after the change in the reflective properties of the flow- path as a consequence of the liquid sample passing along the flow-path. For example when the flow-path comprises a porous carrier such as nitrocellulose the change in reflective properties can be quite marked.

For each window, the window ratio at the reference, control, and test windows is equal to the measured value when the porous carrier is dry, t=0 (prior to addition of sample), divided by the measured value at time t after addition of sample:

For each time point t the window ratios for each window may be evaluated as follows: filtered reference window value _t

Ref ratio, = 'time=0 filtered reference window value time=t

_, . filtered test window value, __n

Test ratio _t = ≥²^- filtered test window value _time=t

„, , . filtered Ctrl window value _t,_mp__n

Ctrl ratio _t = ^tJ≡^- filtered Ctrl window value _time=t Calculation of filtered %A values

For each time point t, %A values may calculated using these ratios for a test line and a control line using the reference ratio as a baseline for the background that would have occurred in all windows had a line not developed.

„ Test . _t ( ,„%/A * \) = ^{Ref ratiθ}t ^{■ test ratio}t ^L x i 1 n0n0o%/

Ref ratio ',X

Ctrl_t (^o/oA) ^ ^{Ref ratiO}- -^{Ctrl ratiO}- x l0Q^o/o Ref ratio _t

The filtered %A value may be defined as follows:

r/oA) = ^{Ref ratiO}- - ^{teSt ratiO}- x l0Q^o/o Ref ratio '.\

The normalised percentage relative attenuation (%A) is given by the difference of the reference window ratio and the window ratio being considered (control or test windows) divided by the reference window ratio and multiplied by 100%.

Typically the %A values will be those obtained at the full assay development time.

Control and measurement signal values may be presented as %A, namely the signal value with respect to the signal measured at a reference zone.

Alternatively, signal values may be presented as %R, namely an absolute value.

If desired, an absorbent "sink" can be provided at the distal end of the carrier material. The absorbent sink may comprise, for example, Whatman 3MM chromatography paper, and should provide sufficient absorptive capacity to allow any unbound labelled binding reagent to wash out of the detection zone. As an alternative to such a sink it can be sufficient to have a length of porous solid phase material which extends beyond the detection zone.

Following the application of a binding reagent to a detection zone, the remainder of the porous solid phase material may be treated to block any remaining binding sites. Blocking can be achieved by treatment for example with protein (e.g. bovine serum albumin or milk protein), or with polyvinylalcohol or ethanolamine, or combinations thereof. To assist the free mobility of the labelled binding reagent when the porous carrier is moistened with the sample, the porous carrier may further comprise a sugar such as sucrose or lactose and/or other substances, such as polyvinyl alcohol (PVA) or polyvinyl pyrrolidone (PVP). Such material may be deposited for example as an aqueous solution in the region to which the labelled binding reagent is to be applied. Such materials could be applied to the porous carrier as a first application followed by the application of the label, alternatively such materials could be mixed with the label and applied to the porous carrier or combinations of both. Such material may be deposited upstream from or at the labelled binding reagent.

Alternatively, the porous carrier may not be blocked at the point of manufacture; instead the means for blocking the porous carrier are included in a material upstream from the porous carrier. On wetting the test strip, the means for blocking the porous carrier are mobilised and the blocking means flow into and through the porous carrier, blocking as the flow progresses. The blocking means include proteins such as BSA and casein as well as polymers such as PVP, PVA as well as sugars and detergents such as Triton - XlOO. The blocking means could be present in the macroporous carrier material.

The nitrocellulose porous carrier may have a pore size of at least about 1 micron, for example greater than about 5 microns, and for example about 8-12 microns. The nitrocellulose porous carrier may be backed e.g. with a plastics sheet, to increase its handling strength. This can be manufactured easily by forming a thin layer of nitrocellulose on a sheet of backing material such as Mylar™.

According to an embodiment, the assay device may comprise a second porous carrier comprising a labelled binding reagent for an analyte provided upstream from a detection zone. The analyte to be detected by the second porous carrier may be the same analyte as to be detected by the first porous carrier. The first and second porous carriers may respectively define two assays wherein the first assay is capable of detecting analyte in a higher concentration range and the second porous carrier is capable of detecting analyte in a lower concentration range. In particular the analyte may be hCG. The second porous carrier may comprise first and second porous carrier materials and may be prepared, for instance, in accordance with the assay device of Example 3 below. The assay device may comprise a common porous sample receiver which is provided upstream from the first and second porous carriers.

In an embodiment, the second porous carrier comprises first and second porous carrier materials wherein labelled binding reagent is provided at the first porous carrier material and the detection zone is provided at the second porous carrier material. The first porous carrier material has surfaces (21, 22) and overlaps at its proximal end region the porous sample receiver at respective surfaces (22, 15) and at its distal end region the second porous carrier material. Labelled binding reagent may be provided substantially at the surface (21) of the first porous carrier material. The labelled binding reagent may be provided more towards the distal end region of the first porous carrier material than the proximal end region.

For the avoidance of doubt it is hereby expressly stated that any feature described herein as "preferred", "convenient", "advantageous", "desirable" or the like may be present in the invention in isolation, or in combination with any one or more other features so described, unless the context dictates otherwise.

Brief description of the figures Figure 1 is a schematic representation of an assay device according to the prior art.

Figure 2a is a schematic representation of an embodiment of an assay device according to the invention.

Figure 2b is a further schematic representation of an embodiment of an assay device according to the invention.

Figure 3 shows a graph of signal value vs. hCG analyte concentration for pipette vs. dipping methods for assay devices prepared according to Example 1.

Figure 4 shows a graph of signal value vs. hCG analyte concentration for pipette vs. dipping methods for assay devices prepared according to Example 2.

Figure 5 shows a graph of signal value vs. hCG analyte concentration for midstream testing for assay devices prepared according to Example 2.

Fig 6 shows the variation in signal values for pipetting and dipping for assays prepared according to Example 3

As seen in Figure 1, a prior art assay device comprises a porous sample receiver (11) having first and second surfaces (13, 15) provided upstream from a first porous carrier material (12) having first and second surfaces (14, 16) which in turn is provided upstream from and partially overlapping a second porous carrier material (17) comprising a detection zone (18). The porous carrier comprises labelled binding reagent (19) provided on surface (14) at a distal end region of the first porous carrier material.

As shown in Figure 2a an assay device in accordance with the present invention comprises a porous sample receiver (11) having first and second surfaces (13, 15) provided upstream from a first porous carrier material (12) having first and second surfaces (14, 16) which in turn is provided upstream from and partially overlapping a second porous carrier material (17) comprising a detection zone (18). The porous carrier comprises labelled binding reagent (19) provided on surface (16) at a proximal end region of the first porous carrier material.

Figure 2b shows an assay device according to an embodiment of the present invention comprising first and second porous carriers having respectively first porous carrier materials (12) and (20), wherein the first porous carrier comprises a labelled binding reagent and a second binding reagent for the analyte (5) provided on the underside (as illustrated by a shaded area) of the first porous carrier material, namely away from the surface (14) and wherein the second porous carrier comprises a labelled binding reagent for the analyte (6) provided on the surface (21) of the first porous carrier material. Binding reagents (5) are provided at a proximal end region of the first porous carrier material (12) and the labelled binding reagent is provided towards the distal end of the first porous carrier material (20). The second porous carrier is a high sensitivity assay, namely it is capable of detecting analyte in a lower concentration range and the first porous carrier is a low sensitivity assay, namely it is capable of detecting analyte in a higher concentration range.

Figures 3 and 4 shows the signal values (expressed as %A) obtained at the detection zone for assay devices prepared according to Examples 1 and 2. Assay devices were tested with samples of hCG in buffer and tested by pipetting of the sample onto the porous sample receiver wherein the assay device was placed horizontally onto a table and by dipping of the porous sampling receiver into the liquid sample.

Figure 5 shows signal values obtained for assay devices prepared according to Example 1 which were tested midstream wherein the porous sample receiver was held in a stream of hCG in buffer designed to be equivalent to midstream testing. The assay devices were held in the buffer stream for varying times of 3 seconds (triangles), 5 seconds (bold stars) or 10 seconds (light crosses).

Figure 6 shows a graph for signal values obtained vs hCG concentration for assays tested with hCG in buffer by both the pipetting (square symbols) and dipping (lozenge symbols) methods. As can be seen, the variation in signal value is much less than for the assay devices prepared according to Example 1, despite the fact that the labelled reagent was provided in the same region of the respective first porous carrier materials. If the signal values obtained for an assay device are substantially independent upon the testing method, there are advantages in providing the labelled reagent at the distal end of the porous carrier material and on the upper surface as in Figure 1. This allows for example a large proportion of the labelled material to flow onto the second porous carrier material and into the detection zone.

Example 1 (Comparative Example)

Assay devices for the detection of hCG analyte in urine were prepared comprising a first porous carrier material comprising first and second binding reagents provided upstream from and overlapping a second porous carrier material comprising a detection zone. A porous sample receiver was provided upstream from and overlapping the first porous carrier material. The binding reagents were provided on the surface (14) of the first porous carrier material and towards its distal end region.

Preparation of the second porous carrier material comprising the detection zone.

The detection zone was prepared by dispensing a line of anti- β-hCG antibody (in- house clone 3468) at a concentration of 3mg/ml in PBSA buffer, at a rate of lμl/cm on onto bands of nitrocellulose of dimensions 350mm length x 40mm width (Whatman) having a pore-size of 8microns and a thickness between 90-lOOmicrons which had been laminated to a 175micron backing layer. The anti- β-hCG antibody was applied using the Biodot xyz3050 dispensing platform as a line ~1.2mm in width and ~300mm in length at a position of 10mm along the length of the nitrocellulose.

A control zone was prepared plotting goat-anti-rabbit antibody (Lampire), 2mg/ml in PBSA buffer at lμl/cm onto nitrocellulose at the 13mm position, 3mm downstream of the detection zone, using a Biodot XYZ3050 dispensing platform.

The bands of nitrocellulose were dried using Hedinair drying oven serial #17494 set at 55°C and speed 5 (single pass). The nitrocellulose was subsequently blocked using a blocking buffer comprising a mixture of 5% ethanol (BDH Analar 104766P) plus 15OmM Sodium Chloride (BDH Analar 1024 IAP) plus 5OmM trizma base from (Sigma T 1503) plus Tween 20 (Sigma P 1379) and 1% (w/v) polyvinyl alcohol (PVA, Sigma 360627).

The blocking buffer was applied at a rate of 1.75μl/mm to the proximal end of the band. Once the blocking solution had soaked into the membrane a solution of 2% (w/v) sucrose (Sigma S8501 in deionised water) was applied using the same apparatus at a rate of 1.6μl/mm and allowed to soak into the nitrocellulose membrane for ~5 minutes).

The bands of NC were then dried using a Hedinair drying oven serial #17494 set at 75°C and speed 5 (single pass).

Preparation of the mobilisable labelled and unlabelled binding reagents on the first porous carrier material.

Mouse-anti-human α-hCG mAb (clone 3299) conjugated to 400nm blue polystyrene latex (Duke Scientific) was mixed with scavenger antibody mAb mouse anti-human β-hCG (in-house clone 3468) at 3mg/ml to give a final % blue latex of 3%, a final 3468 concentration of 0.075mg/ml and 0.06mg/ml concentration of the free anti-β hCG antibody. The resulting mixture was airbrushed onto Whatman glass fibre (F529 25mm wide reels) using the BIODOT XYZS (serial number 1673) at 90g/hr sprayed at 2.02μg/cm onto F529-09 glass fibre at approximately the 20mm position. The sprayed solution spread out to form a band that was approximately 7mm in length.

Labelled binding reagent for the control zone was also deposited onto the same region of the porous carrier as the labelled binding reagent for the analyte as follows: Rabbit IgG (Dako) was conjugated to 400nm blue latex polystyrene latex (Duke Scientific) in BSA/sucrose to give a final % blue latex of 0.7% solids and sprayed at 65g/hr onto glass fibre. The glass fibre was dried using a Hedinar Conveyor Oven Serial number 17494 set at 65°C and speed 5 (single pass). A second pass of latex was deposited onto the glass fibre by repeating the above however at an offset of - 0.8mm from the original position of spray (further downstream of the glass fibre). The glass fibre as dried as described above.

The glass fibre material with sprayed latex was attached to the nitrocellulose membrane using a clear adhesive coated laminate film (Ferrisgate, 38mm wide) arranged such that the sprayed latex was uppermost and the glass fibre overlapped the surface of the nitrocellulose by approximately 2mm along the length (350mm) of the band of nitrocellulose membrane. The glass fibre was provided upstream from the nitrocellulose membrane and the binding reagents were provided towards the distal end of the glass fibre.

The laminated sheet was subsequently cut into test-strips comprising a glass fibre porous carrier material having a width of 6mm and a length 25mm, with the labelled reagents having been applied 20mm along the length of the glass fibre, provided upstream from and overlapping by 2mm, a nitrocellulose membrane having a width of 6mm and a length of 40mm. A porous sample receiver (Filtrona Fibertec Inc., product code 24464) of 45mm length, 12mm width and a thickness of approximately 2.5mm was provided upstream from and overlapping the first porous carrier material by approximately 3mm.

Example 2

Assay devices for the detection of hCG analyte in urine were prepared comprising a first porous carrier material comprising first and second binding reagents provided upstream from and overlapping a second porous carrier material comprising a detection zone. A porous sample receiver was provided upstream from and overlapping the first porous carrier material. The binding reagents were provided on the surface (16) of the first porous carrier material and towards its proximal end region. The detection and control zones of the second porous carrier material were prepared in accordance with Example 1.

Apart from the different positioning of the labelled and second binding reagents in the assay devices according to Example 2 with that according to Example 1 , the procedure for applying the labelled and second binding reagents for Example 2 was the same as described for Example 1.

It was found that when labelled binding reagent for the control zone was provided at the same position of the porous carrier as the binding reagents, the control signal was observed to decrease at the control zone compared with the control signal observed at the control zone for assay devices prepared according to Example 1. This is thought to be due to the fact that labelled binding reagent for the control zone of Example 2 is further from the control zone than the labelled binding reagent for the control zone of Example 1. It was found that when labelled binding reagent for the control zone was provided downstream of the binding reagents, namely nearer to the control zone, the signal observed at the control zone was boosted.

Labelled binding reagent for the control zone was deposited 5mm downstream on the surface (16) of the first porous carrier material as follows:

Rabbit IgG (Dako) was conjugated to 400nm blue latex polystyrene latex (Duke Scientific) in BSA/sucrose to give a final % blue latex of 0.7% solids and sprayed at 65g/hr onto glass fibre.

The glass fibre was dried using a Hedinar Conveyor Oven Serial number 17494 set at 65°C and speed 5 (single pass). A second pass of latex was deposited onto the glass fibre by repeating the above however at an offset of - 0.8mm from the original position of spray (further downstream of the glass fibre). The glass fibre as dried as described above.

The laminated sheet was subsequently cut into test-strips comprising a glass fibre porous carrier material having a width of 6mm and a length 25mm, with the labelled and second binding reagents having been applied 5mm along the length of the glass fibre, the control labelled reagent having been applied 10mm along the length of the glass fibre, wherein the glass fibre was provided upstream from and overlapping by 2mm, a nitrocellulose membrane having a width of 6mm and a length of 40mm. A porous sample receiver (Filtrona Fibertec Inc., product code 24464) of 45mm length, 12mm width and a thickness of approximately 2.5mm was provided upstream from and overlapping the first porous carrier material by approximately 3mm.

Example 3

Assay devices for the detection of hCG analyte in urine were prepared comprising a first porous carrier material comprising a labeled binding reagent provided upstream from and overlapping a second porous carrier material comprising a detection zone. A porous sample receiver was provided upstream from and overlapping the first porous carrier material. The binding reagents were provided on the surface (14) of the first porous carrier material and towards its distal end.

The detection and control zones were prepared in accordance with that of Example 1.

Preparation of the mobilisable labelled binding reagent on the first porous carrier material.

Labelled binding reagent was prepared according to the following protocol:

Coating latex particles with anti-α hCG 1. Dilute blue latex particles from Duke Scientific (400nm in diameter, DBl 040CB at 10% solids (w/v)) to 2% solids (w/v) with 10OmM di- sodium tetra borate buffer pH 8.5 (BDH AnalaR 102676G) (DTB).

2. Wash the diluted latex by centrifuging a volume of (2mls) of diluted latex in two Eppendorf centrifuge tubes at 17000rpm (25,848 rcf) for 10 minutes on an Heraeus Biofuge 17RS centrifuge. Remove and discard the supernatant and re- suspend the pellets in 10OmM DTB to give 4% solids (w/v) in a total volume of ImI.

3. Prepare a mixture of ethanol and sodium acetate (95% Ethanol BDH AnalaR 104766P with 5% w/v Sodium Acetate Sigma S-2889).

4. Add lOOμls ethanol-sodium acetate solution to the washed latex in step 2 (this is 10% of the volume of latex).

5. Dilute the stock antibody (in-house clone 3299) to give ~ 1200μg/ml antibody in DTB.

6. Heat a volume of ImI of the diluted antibody from step 5 in a water bath set at 41.5°C for ~ 2 minutes. Also heat the washed latex plus ethanol-sodium acetate from step 4 in the same water bath for 2 minutes.

7. Add the diluted antibody to the latex plus ethanol-acetate, mix well and incubate for 1 hour in the water bath set at 41.5⁰C whilst mixing using a magnetic stirrer and a magnetic flea placed in the mixture.

8. Prepare 40mg/ml Bovine Serum Albumin (BSA) Solution (Intergen W22903 in de-ionised water). Block the latex by adding an equal volume of 40mg/ml BSA to the mixture of latex/antibody/ethanol-acetate and incubate in the water bath at 41.5°C for 30 minutes with continued stirring. 9. Centrifuge the mixture at 17000rpm for 10 minutes as in step 2, (split the volume into ImI lots between Eppendorf tubes). Remove and discard the supernatant and re-suspend the pellet in 10OmM DTB. Repeat the centrifugation as in step 2, remove and discard the supernatant and re-suspend in pellet in Air Brushing Buffer (20% (w/v) Sucrose Sigma S8501, 10% BSA (w/v) in 10OmM Trizma Base Sigma T 1503 pH to 9). Add Air Brushing Buffer to give 4% solids (w/v) latex.

The conjugated latex was and sprayed in a mixture of BSA and sucrose onto a glass- fibre porous carrier (F529-09, Whatman) at a rate of 50g/hr and 110mm/s and dried using a Hedinar Conveyor Oven Serial number 17494 set at 65°C and speed 5 (single pass).

Labelled binding reagent for the control zone was also deposited onto the same region of the porous carrier as the labelled binding reagent for the analyte as follows: Rabbit IgG (Dako) was conjugated to 400nm blue latex polystyrene latex (Duke Scientific) in BSA/sucrose to give a final % blue latex of 0.7% solids and sprayed at 65g/hr onto glass fibre.

The glass fibre material with sprayed labelled binding reagent was attached to the nitrocellulose membrane using a clear adhesive coated laminate film (Ferrisgate, 38mm wide) arranged such that the labelled reagent was uppermost and the glass fibre overlapped the surface of the nitrocellulose by ~ 2mm along the length (350mm) of the band of nitrocellulose membrane. The glass fibre was attached to the end of the nitrocellulose such that it was upstream of the detection zone.

The assay devices according to Examples 1-3 were tested with samples of hCG in buffer according to two sampling methods, dipping and pipetting and the corresponding signal values obtained at the detection zone are shown respectively in Figures 3 and 4 and 6. Additionally the assay devices prepared according to Example 2 were tested using buffer samples applied to the assay device in a midstream fashion. Assay devices were held in the buffer flow for varying times and the assay signals were measured. The variation in assay signal with respect to hCG concentration is shown in Figure 5.

From Figure 3 it may be seen that a marked difference in signal values for assay devices prepared according to Example 1 may be observed depending upon the sampling method. Assay devices that were tested using the dipping method gave rise to lower signal values than by pipetting at lower analyte levels (lOOOmIU/ml) and higher signal values than by pipetting at higher analyte levels (10⁴mIU).

From Figure 4 which shows the results obtained for assay devices prepared according to Example 2, a much lesser variability in signal values is observed between sampling by pipette and dipping.

From Figure 5 it may be seen that the assay signal at high hCG levels of 10⁴mIU varies depending upon the time for which the assay device is held in the buffer stream. The signal at 10⁴mIU for buffer stream testing is between that of pipetting and dipping.

Claims

1. An assay device for determining the presence and/or extent of an analyte in a liquid sample comprising: a first porous carrier (12) having respective first and second surfaces (14, 16) comprising a mobilisable first labelled binding reagent for an analyte provided upstream from a detection zone for immobilising the first binding reagent; and a porous sample receiver (11) for receiving a liquid sample having respective first and second surfaces (13, 15); wherein said porous sample receiver is provided upstream from and at least partially overlaps said porous carrier at their respective surfaces (15, 14); wherein the first binding reagent is provided at surface (16) and/or within the first porous carrier material but substantially away from the surface (14) of the porous carrier.

2. The assay device according to claim 1, wherein the porous sample receiver has a higher porosity than the porous carrier.

3. The assay device according to claims 1 and 2, wherein the porous carrier comprises first and second porous carrier materials.

4. The assay device according to claim 3, wherein the first and second porous carrier materials overlap each other.

5. The assay device according to any of claims 1-4, additionally comprising a second binding reagent for the analyte provided in the vicinity of or at the same position as the first labelled binding reagent.

6. The assay device according to claims 3 and 4, wherein the binding reagents are provided on the first porous carrier material and the detection zone is provided on the second porous carrier material.

7. The assay device according to claim 6, wherein the second porous carrier material comprises nitrocellulose.

8. The assay device according to any of the preceding claims, wherein the detection zone comprises a binding reagent for the analyte.

9. The assay device according to any of claims 3-8, wherein the first porous carrier material overlaps the porous sample receiver at its near end and overlaps the second porous carrier material at its distal end region and wherein the binding reagents are provided more towards a proximal end region than the distal end region of the first porous carrier material.

10. The assay device according to any of the preceding claims, further comprising a second porous carrier.

11. The assay device according to claim 10, wherein the first porous carrier is capable of measuring an analyte in a higher concentration range and the second porous carrier is capable of measuring an analyte in a lower concentration range.

12. The assay device according to claim 11, wherein the second porous carrier comprises a labelled binding reagent for the analyte provided upstream from a detection zone.

13. The assay device according to claim 12, comprising a single porous sample receiver common to the first and second porous carriers.

14. The assay device according to any of claims 10-13, wherein the first porous carrier material of the first porous carrier comprises a labelled binding reagent for a control zone provided upstream from a control zone provided on the second porous carrier material of the first porous carrier and provided downstream of the labelled binding reagent, and the second binding reagent if present.

15. The assay device according to any of the preceding claims, wherein the analyte is hCG.

16. The assay device according to any of the preceding claims, wherein the liquid sample is urine.

17. A method of manufacturing an assay device for determining the presence and/or extent of an analyte in a liquid sample, comprising: a) applying a first labelled binding reagent to the surface (16) of a porous carrier (14) upstream from a detection zone capable of immobilising the first binding reagent, said porous carrier having first and second surfaces (14, 16); b) overlapping a porous sample receiver (11) having first and second surfaces (13, 15) and provided upstream from the porous carrier at their respective surfaces (15, 16).

18. An assay device substantially as hereinbefore described and with reference to the accompanying drawings.