IE83806B1 - Solid phase analytical device - Google Patents

Abstract

ABSTRACT An analytical test device incorporating a dry porous carrier to which a liquid sample, eg. urine, suspected of containing an analyte such as HCG or LH can be applied indirectly, the device also incorporating a labelled specific binding reagent which is freely mobile in the porous carrier when in the moist state, and an unlabelled specific binding reagent which is permanently immobilised in a detection zone on the carrier material, the labelled and unlabelled specific binding reagents being capable of participating in either a sandwich reaction or a competition reaction in the presence of the analyte, in which prior to the application to the device of a liquid sample suspected of containing the analyte, the labelled specific binding reagent is retained in the dry state in a macroporous body, eg. of plastics material having a pore size of (10) microns or greater, through which the applied liquid sample must pass en route to the porous carrier material, the labelled specific binding reagent being freely soluble or dispersible in any liquid sample which enters the macroporous body.

Description

PATENTS ACT 1992 980110 SOLID PHASE ANALYTICAL DEVICE UNILEVER PLC This patent application is a divisional of Irish Patent Application No. 592/90, corresponding to European patent application No. 9030l697.0, now granted as EP 383619 B.

The present invention relates to assays involving specific binding, especially immunoassays.

In particular, the invention relates to analytical devices which are suitable for use in the home, clinic or doctor's surgery and which are intended to give an analytical result rapidly and which require the minimum degree of skill and involvement from the user. The use of test devices in the home to test for pregnancy and fertile period (ovulation) is now commonplace.

In the specification of UK patent application GB-A—2204398 we describe test devices which are readily usable even by an unskilled person and which typically merely require that some portion of the device is contacted with a sample (e.g. urine in the case of a pregnancy or ovulation test) and thereafter no further actions are required by the user before an analytical result can be observed. The analytical result can be observable within a matter of minutes following sample application, e.g. ten minutes or less.

The use of reagent—impregnated test strips in specific binding assays, such as immunoassays, has previously been proposed. In such procedures a sample is applied to one portion of the test strip and is allowed to permeate through usually with the aid of an eluting the strip material, solvent such as water. In so doing, the sample progresses into or through a detection zone in the test strip wherein a specific binding reagent is immobilised. Analyte present in the sample can participate in a sandwich or a competition reaction within the detection zone, with a labelled reagent which can also be-incorporated in the test strip or applied thereto. Examples of prior proposals utilising these principles are given in Thyroid Diagnostics Inc GB 1589234, (USA) Boots—Celltech Diagnostics Limited EP 0225054, Syntex Inc EP 0183442, and Behringwerke AG EP 0186799.

Other examples of analytical devices, comprising multiple zones or layers of porous membranes or matrices arranged in series, through which reagents can migrate, are described in EP-A2—299359, W0 86/04683 and WO 89/06799. are especially suited to the use of enzyme labels.

These devices The use of insoluble particulate labels is not contemplated, whereas such particulate labels the described in GB-A-2204398 mentioned above. are preferred in -devices (3) discloses EPC, containing a EP-A-323605, citable under Article assay devices having an application The label can be particulate, and the application pad can be made from labelled reagent. for example a selenium colloid, porous polyethylene frit, i.e. a macroporous body comprising plastics material.

In the patent application now granted as IE 81184 (corresponding to EP 383619 B), of which this present application is a divisional, we claim an analytical test device incorporating a dry porous carrier to which device a liquid sample suspected of containing an the device also incorporating a analyte can be applied, specific binding reagent attached to a particulate label which labelled specific binding reagent is freely mobile in carrier when in the moist state, and an the porous reagent which is permanently unlabelled immobilised in a detection zone on the carrier material, labelled unlabelled capable of participating in either a sandwich reaction or a the the specific binding and specific binding reagents being competition reaction in presence of analyte, characterised in that prior to the application to the device of the liquid sample, the particle-labelled specific binding reagent is retained within the device in the dry state in a separate macroporous body comprising plastics material having a pore size not less than 10 times greater than the maximum particle size of the particulate label, through which macroporous body the applied liquid sample must pass thus facilitating uptake of by the en route to the porous carrier, the particle-labelled specific binding reagent applied liquid sample, and in that the porous carrier is linked via the macroporous body to a porous receiving member to which the liquid sample can be applied and from which the sample can permeate into the porous carrier.

In the present application we provide an analytical test device incorporating a dry’ porous carrier strip to which device an aqueous liquid sample suspected of containing an analyte can be applied, the device also incorporating a specific binding reagent attached to a particulate label which labelled specific binding reagent is freely mobile in the porous carrier strip when in the moist state, and an unlabelled immobilised in a detection zone on the porous carrier strip, the labelled and unlabelled specific binding reagents being specific binding reagent which is permanently capable of participating in either a sandwich reaction or a the of the characterised in that prior to the application to the device the particle-labelled specific binding competition reaction in presence analyte, of the liquid sample, reagent is retained in the dry state in a separate macroporous body comprising plastics material having a pore size not less than 10 times greater than the maximum particle size of the particulate label, through which macroporous body the applied liquid sample must pass en route to the proximal end of the porous carrier strip, and in that there is an absorbent sink at the distal end of the the absorbent sink not being merely a length of which the detection zone and extends beyond the detection zone. porous carrier strip, porous carrier material contains The invention therefore encompasses the use of a macroporous body to facilitate the uptake of a labelled specific binding agent by a liquid sample before such a sample is analysed on a test strip or the like.

Preferably, the dry porous carrier material comprises a chromatographic strip, sudu as a strip of nitrocellulose.

If desired, the nitrocellulose can be backed with moisture impermeable material, such as polyester sheet. Using nitrocellulose as the porous carrier material has considerable advantage over more conventional strip materials, such as paper, because nitrocellulose has a natural ability to bind proteins without requiring prior sensitisation. Specific binding reagents, such as immunoglobulins, can be applied directly to nitrocellulose and immobilised thereon. No chemical treatment is required which xnight interfere with the essential specific binding activity of the reagent. Unused binding sites on the nitrocellulose can thereafter be blocked using simple materials, such as polyvinylalcohol. Moreover, nitrocellulose is readily available in a range of pore sizes and this facilitates the selection of a carrier material to suit particularly requirements such as sample flow rate.

Preferably the nitrocellulose has a pore size of at least one um. Preferably the nitrocellulose has a pore size not greater than about 20pm.

The labelled specific binding reagent comprises a specific attached to a label. Such e.g. non-metallic colloids, binding particulate "direct labels", reagent coloured latex particles, gold sols, and dye sols, are already known per instant analytical se. They can be used to produce an result without the need to add further reagents in order to develop a detectable signal. They are robust and stable and can therefore be used readily in an analytical device which is stored in the dry state. Their release on contact with an aqueous sample can be modulated, for example by the use of soluble glazes. Preferably, the particulate label isea latex particle, such as a coloured latex particle which can be readily visible to the eye if it becomes bound in the If desired, by colour reflectance. detection zone. the assay result can be read instrumentally, e.g. Alternatively, the latex particle can incorporate a fluorescent compound which can respond to applied electromagnetic energy such as ultraviolet light or visible light, to provide an emitted that particularly preferred embodiment, signal can be measured instrumentally. In a the direct label is a coloured latex particle of spherical or near-spherical shape and having a maximum diameter of not greater than about .5pm. An ideal size range for such particles is from about .05 to about O.5um.

We have found that use of a macroporous body as the portion of the device wherein the applied liquid sample encounters the particulate label considerably facilitates the ease with which the label is up by the compared to the situation that usually prevails if particulate taken liquid sample, the particulate label is incorporated as a pre—dosed reagent on the dry porous carrier strip. To enable the particulate label to migrate freely out of the macroporous body with the liquid sample, the macroporous body has a pore size at least times greater than the maximum particle size of the particulate label. More preferably, the macroporous body comprises plastics material having an average pore size of because such not less than 10pm, and ideally about 100nm, larger pore sizes give better release of the labelled reagent. The plastics material should not be protein- binding, or should be easily blockable by means of reagents such as BSA or PVA, to minimise non-specific binding to facilitate free movement of the labelled redagent after the macroporous body has become moistened with the liquid The surface active agent or solvent, more hydrophilic and to promote rapid uptake of the liquid sample. plastics material can be pre—treated with if necessary, to render it sample. Alternatively, if desired, a surface active agent can be incorporated in the solution containing the labelled reagent when this is applied to the macroporous material during manufacture of the device.

The labelled incorporated in macroporous material in bulk, reagent is preferably large sheet, form before e.g. it is subdivided into individual bodies for use in a testing device of the invention.

After a solution containing the labelled reagent has been saturate the material, the allowed to macroporous macroporous material should be dried, e.g. by vacuum or air- drying, or preferably by freeze-drying. Optionally, the solution can also contain a surface active agent, such as a detergent, and/or a glazing material, such as a sugar, e.g.

The presence of the glazing material appears to the labelled specific binding reagents SUCIOSE . enhance release of reagent and promotes stability of delicate such as antibodies. labelled rather than pre-dosed onto the carrier the the By incorporating the reagent in a separate macroporous body, material that also incorporates detection zone, following advantages can be obtained: because a substantial able to sensitivity of the test, of the Enhanced take the enhancing potential reaction quantity liquid sample is up the labelled reagent..before ,migrating through carrier material to the detection zone, time without significantly increasing overall test time.

Also, the liquid which permeates the carrier is of a more uniform and consistent composition. Whereas the test devices as described in our earlier patent application GB—A— are primarily, although not exclusively, suited to qualitative assays, those of the present invention are especially suitable for quantitative assays as well as for qualitative assays.

For example, and the Enhanced perceived performance of the test. the detection zone comprises a line of immobilised reagent, when device incorporates a carrier strip the label is a visible direct label, a positive result shows up more clearly, with much reduced temporary background caused by the visible labelled reagent being progressively conveyed past the detection zone.

Ease of manufacture, because the incorporation of the labelled reagent in the separate macroporous body avoids the need to apply the labelled reagent in a special zone in the as described carrier, which may need careful pre—treatment, in our GB-A—2204398.

If the assay device is intended to identify more than one the body incorporate several labelled specific binding reagents each single macroporous can analyte in a sample, carrying a different label, eg. having different colours or fluorescent properties. This will facilitate the manufacture of a multiple analyte testing device.

Ideally, the macroporous body is in direct moisture- conductive contact with the porous material, and~ the detection zone on the porous carrier material is spaced away from the region of contact between the porous carrier In such an embodiment, the material and the macroporous body. the macroporous body is preferably not less than the quantity of quantity of liquid sample required to saturate liquid sample capable of being absorbed by the mass of porous carrier material linking the macroporous body and the detection zone. In other words, the liquid capacity of the macroporous body is at least equal to the liquid capacity of the working portion of the porous carrier.

The invention also provides an analytical method in which a contacted with an aqueous such that device as set forth above is liquid sample suspected of containing the analyte, the sample permeates by capillary action via the macroporous body through the porous solid carrier into the detection zne and the labelled reagent migrates therewith to the detection the presence of analyte in the sample being determined to which the labelled Zone, by observing the extent (if any) reagent becomes bound in the detection zone.

In one embodiment of the invention, the labelled reagent is The labelled immobilised a specific binding partner for the analyte. reagent, the analyte (if present) and the unlabelled specific binding reagent cooperate together in a "sandwich" reaction. This results in the labelled reagent being bound in the detection zone if analyte is present in the The specificities for different epitopes on the analyte. sample. two binding reagents must have In another embodiment of the invention, the labelled reagent is either the analyte itself which has been conjugated with a label, or is an analyte_analogue, ie. a chemical entity having the identical specific binding characteristics as the analyte, and which similarly has been conjugated with a label. In the latter case, it is preferable that the properties of the analyte analogue which influence its solubility or dispersibility in an aqueous liquid sample and its ability to migrate through the moist porous solid phase material should be identical to those of the analyte itself, similar. In this second or at least very closely embodiment, the labelled analyte or analyte analogue will migrate through the porous carrier into the detection zone and bind with the immobilised reagent. Any analyte present in the sample will compete with the labelled reagent in this Such will reduction in the amount of labelled reagent binding in the binding reaction. competition result in a detection zone, and a consequent decrease in the intensity of the signal observed in the detection zone in comparison with the signal that is observed in the absence of analyte in the sample.

In a further alternative embodiment, an analyte or analyte analogue is immobilised in the detection zone, and the labelled reagent is specific for the ,analyte. If an analyte—containing sample is applied to the device, competition between the immobilised and free analyte reduced the extent to which the labelled reagent may become bound in the detection zone.

In a further embodiment of the present invention, the porous carrier is linked via the macro—porous body to a porous receiving member to which the liquid sample can be applied into the porous and from which the sample can permeate carrier. Preferably, the porous carrier and the macroporous body are contained within a nwisture—impermeable casing or housing and the porous receiving member extends out of the housing and can act as a means for permitting a liquid sample to enter the housing and reach the porous carrier.

The should be with e.g. appropriately placed apertures, which enable the detection provided means, housing zone of the porous solid phase carrier material (carrying the immobilised unlabelled specific binding reagent) to be observable from outside the housing so that the result of the assay can be observed. If desired, be provided with further means which enable a further zone the housing may also of the porous solid phase carrier material to be observed from outside the housing and which further zone incorporates one or more control reagents which enable an indication to be iven as to the Preferably the whether assay procedure has been completed. housing is provided with a removable cap or shroud which can protect the protruding porous receiving member during storage before use. If desired, the cap or shroud can be replaced over the protruding porous receiving member, after sample application, while the assay procedure is being performed.

An important embodiment of the invention is a pregnancy testing device comprising a hollow elongated casing containing a dry porous nitrocellulose carrier which communicates indirectly with the exterior of the casing via a bibulous urine receiving member which protrudes from the casing, the porous nitrocellulose carrier and the sample receiving member being linked via a macroporous body such that any sample reaching the porous carrier must first pass through the macroporous body, the sample receiving member and the macroporous body together acting as a reservoir from which the. carrier, the highly-specific anti-hCG label, the labelled antibody being freely mobile within the macroporous body and urine .is .released. into body antibody bearing a coloured porous macroporous containing a "direct" the porous carrier when in the moist state, and in a detection zone on the carrier spatially distant from the macroporous body an highly-specific unlabelled anti—hCG antibody which is permanently immobilised on the carrier material and is therefore not mobile in the moist state, the labelled and unlabelled antibodies having specificities for different hCG the opaque or translucent material‘ incorporating =at- least one epitopes, casing being constructed of aperture through which the analytical result may be observed, together with a removable and replaceable cover for the protruding bibulous urine receiving member. A fertile period prediction device, essentially as just defined except that the analyte is LH, is an important alternative.

Such devices can be provided as kits suitable for home use, comprising’ a plurality (e.g. two) of devices individually wrapped in moisture impervious wrapping and packaged together with appropriate instructions to the user.

The porous sample receiving member can be made from any bibulous, porous or fibrous material capable of absorbing The of the (ie. with pores or fibres running wholly or liquid rapidly. porosity material can be unidirectional predominantly parallel to an axis of 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 polytetrafluoroaethylene can be used. It can be advantageous to pre—treat the member with a surface—active agent during manufacture, as this can reduce inherent hydrophobicity in the member and therefore enhance its ability to take up and deliver a moist any sample rapidly and efficiently. Porous sample receiving members can also be made from paper or other cellulosic such as nitro-cellulose. Materials that are now used in the particularly sutable and such materials can be shaped or materials, nibs of so—called fibre tipped pens are cross-sections Preferably the extruded in a lengths variety of appropriate in the context of the invention. material comprising the porous receiving member should be chosen such that the porous member can be saturated with aqueous liquid within a matter of seconds. Preferably the material remains robust when moist, and for this reason paper embodiment wherein the porous and similar materials are less preferred in any receiving member protrudes _ 13 _ from a housing. The liquid must thereafter permeate freely from the porous sample receiving member into the macroporous body.

If present, the "control" zone can be designed merely to convey an unrelated signal to the user that the device has worked. For example, the control zone can be loaded with an antibody that will bind to the labelled reagent, e.g. an "anti—mouse" antibody if the labelled reagent is an antibody that has been derived using a murine hybridoma, to confirm that the sample has permeated the test strip.

Alternatively, the control zone can contain an anhydrous when moistened, produces a colour change or e.g. turn blue when mostened by an aqueous sample. reagent that, colour formation, anhydrous copper sulphate which will As a further could immobilised _zone_ contain alternative,"_ai.control analyte which will react with excess labelled reagent from the first zone. As the purpose of the control zone is to indicate to the user that the test has been completed, the control zone should be located downstream from the detection zone in which the desired test result is recorded. A positive control indicator therefore tells the user that the sample has permeated the required distance through the test device.

The label is a direct label, ie. an entity which, in its natural state, is readily visible either to the naked eye, or with the aid of an optical filter and/or applied stimulation, e.g. UV light to promote fluorescence. For example, minute coloured particles, such as dye sols, metallic sols (e.g. gold), and coloured latex particles, are very suitable. Of these options, coloured latex particles are most preferred.

Concentration of the label into a small zone or volume should give rise to a readily detectable signal, e.g. a strongly-coloured area. This can be evaluated by eye, or by instruments if desired.

Coupling of the label to the specific binding reagent can be by covalent bonding, if desired, or by hydrophobic bonding.

Such techniques are commonplace in the art, and form no part of the present invention. Because the label is a direct label such as a coloured latex particle, hydrophobic bonding is preferred.

In all embodiments of the invention, it is essential that the labelled reagent migrates with the liquid sample as this The flow of continues beyond the detection zone and sufficient sample is sample to the detection zone. progresses applied to the porous carrier material in order that this may occur and that any excess labelled reagent which does _15_ not participate in any binding reaction in the detection flushed detection by this away from the zone zone is continuing flow. An absorbent "sink" is provided at 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 conjungate 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 materials which extends beyond but this alternative is not within the this the detection zone, scope of the invention claimed in divisional application.

The presence or intensity of the signal from the label which becomes bound in the detection zone can provide a qualitative or iquantitative measurement of analyte in ‘the A plurality of detection zones arranged in series which the can also be sample. on the porous solid phase material, through aqueous liquid sample can pass progressively, used to provide a quantitative measurement of the analyte, loaded with different binding agents to provide a multi—analyte test. or can be individually specific The immobilised reagent in the detection zone is preferably a highly specific antibody, and more preferably a monoclonal antibody. In the embodiment of the invention involving the sandwich reaction, the labelled reagent is also preferably a highly specific antibody, and more preferably a monoclonal antibody.

Preferably the porous carrier material is in the form of a strip or sheet to which during manufacture of the device, one or more reagents can be applied in spacially distinct _ 16 _ zones. During use, the liquid sample is allowed to permeate through the sheet or strip from one side or end to another.

If desired, a device according to the invention can incorporate two or more discrete bodies of porous solid phase carrier material, e.g. separate strips or sheets, each These discrete bodies can be that a carrying immobilised reagents. arranged in parallel, for example, such single application of liquid sample to the device initiates sample flow ixx the discrete bodies simultaneously. The separate analytical results that can be determined in this way can be used as control results, or if different reagents are used on the different carriers, the simultaneous determination of a plurality of analytes in a single sample can be made.

Alternatively, multiple samples can be applied individually to an array of carriers and analysed simultaneously.

The material comprising the porous solid phase is preferably nitrocellulose. This has the advantage that proteinaceous in the detection zone can be If the reagents, such as an antibody, immobilised firmly without prior chemical treatment. porous solid. phase materiall comprises paper, for example, the immobilisation of an antibody in the second zone needs to be performed by chemical coupling using, for example, CNBr, carbonyldiimidazole, or tresyl chloride.

Following the application of the specific binding reagent to the detection zone, the remainder of the porous solid phase material should be treated to block any remaining binding sites elsewhere. Blocking can be achieved by treatment with protein (e.g. bovine serum albumin or milk protein), or with polyvinylalcohol or ethanolamine, or any combination of these agents, for example. Between these process steps the porous solid phase carrier material should be dried.

Preferably the porous solid phase material is nitrocellulose sheet having a pore size of at least about 1pm, even more preferably of greater than about Sum, and yet more preferably about 8—l2um. Very suitable nitrocellose sheet having a nominal pore size of up to approximately 12pm, is available commercially from Schleicher and Schuell GmbH. the nitrocellulose sheet is "backed", e.g. with This can Preferably, plastics sheet, to increase its handling strength. thin layer of The actual be manufactured easily by forming a nitrocellulose on a sheet of backing material. pore size of the nitrocellulose when backed in this manner will than that of ‘the unbacked material. tend to be lower corresponding Alternatively, a pre-formed sheet of nitrocellulose can be tightly sandwiched between two supporting sheets of solid material, e.g. plastics sheets.

It is preferable that the flow rate of an aqueous sample through the porous solid phase material should be such that in the untreated material, aqueous liquid migrates at a rate of 1cm in not more than 2 minutes, but slower flow rates can be used if desired.

The spatial separation between the macroporous body and the and the flow rate characteristics of the detection zone, porous carrier material, can be selected to allow adequate reaction times during which the necessary specific binding -18.... can occur. Further control over these parameters can be achieved by the incorporation of viscosity modifiers (e.g. sugars and modified celluloses) in the sample to slow down the reagent migration.

Preferably, the immobilised reagent in the detection zone is impregnated throughout the thickness of the carrier in the detection zone throughout the thickness of the sheet (e.g. or strip if the carrier is in this form). Such impregnation can enhance the extent to which the immobilised reagent can capture any analyte or labelled reagent, present in the migrating sample.

Reagents can be applied to the porous carrier material in a variety of ways. Various "printing" techniques have previously been proposed for application of liquid reagents to carriers, e.g. micro-syringes, pens using metered pumps, and ink-jet printing, and any of these direct printing context. To (e.g. sheet) treated with the reagents and then subdivided into smaller the can be used in present techniques facilitate manufacture, the carrier can be small narrow strips each embodying (e.g. required reagent-containing zones) portions to provide a plurality of identical carrier units.

An assay based on the above principles can be used to determine a wide variety of analytes by choice of appropriate specific binding reagents. The analytes can be, for example, proteins, haptens, immunoglobulins, hormones, infectious disease agents Streptoccus, polynucleotides, steroids, drugs, (e.g. of bacterial or viral origin) such as Neisseria and Chlamydia. be performed for analytes such as hCG, LH, and infectious Sandwich assays, for example, disease agents, whereas competition assays, for example, may be carried out for analytes such as E—3—G and P—3—G.

The determination of the presence (if any) of more than one analyte in sample can have significant clinical utility.

For example, the ratio of the levels of apolipoproteins A1 and B can be indicative of susceptibility to coronary heart disease. Similarly, the ratio of the levels of glycated haemoglobin (HbA) to unglycated (HbAo) or total (Hb) haemoglobin can aid in the management of diabetes.

Additionally it is possible to configure tests to measure two steroids simultaneously, e.g. E-3—G and P—3—G.

The determination of the presence of more than two (ie. significant may have the" multiple) analytes in any sample clinical utility. For example, detection of presence of various different sereotypes of one bacterium, or the detection of the presence of soluble serological markers in humans may be useful. By way of example, a multiple analyte test for the detection of the presence of different Streptococcus A, B, C and D. A cocktail of monoclonal antibodies, specific for various _pathologically serotypes of can be prepared for groups , each important group serotypes, or a polyclonal antiserum raised against a Streptococcal group, porous carrier strip as a line extending the width of the particular can be dispensed onto a strip of approximately lmm zone length. Multiple lines can be dispensed in spatially discrete zones, each zone containing immunochemically reactive component(s) capable of binding the analyte of interest. Following the application of the multiple zones, via a suitable application procedure (e.g. ink—jet printing, metered pump and pen, airbrush), the remainder of the porous material should be treated with a _ 20 _ reagent (e.g. bovine serum albumin, polyvinylalcohol, ethanolamine) to block any remaining binding sites elsewhere. some features of the invention will detail with the the The described devices do not include By way of example only, now be described in reference to accompanying drawings. an absorbent sink.

Embodiment 1 Figure 1 of the accompanying drawings represents an isometric view of an assay device, and Figure 2 represents a cross-sectional side elevation of the device shown in Figure Referring to Figure 1, the device comprises a housing or casing 100 of elongate rectangular form having at one end 101 a portion 102 of reduced cross-sectional area. A cap 103 can be fitted onto portion 102 and can abut against the shoulder 104 at end 101 of the housing. Cap 103 is shown Extending beyond end 105 of When cap 103 it covers porous 100 The housing is separated front housing 100. portion 102 is a porous sample collector 106. is fitted onto portion 102 of the housing, 106. face 107 incorporates two apertures 108 and 109. sample collector Upper of housing constructed of an upper half 110 and a lower half 111.

Referring to Figure 2, it can be seen that housing 100 is of hollow construction. Porous sample collector 106 extends The inner end 112 of sample collector 106 body 113 of applied to into housing 100. accommodate a macroporous is recessed to plastics material. Aqueous liquid sample collector 106 can pass freely into macroporous body 113, rapidly saturating it. In turn, macroporous body 113 is in liquid permeable contact with a strip of porous carrier material 114. The housing is constructed of an upper half 110 and a lower half 111 and strip 114 overlap to ensure that there is adequate contact between these two components and that a liquid sample applied to sample collector 106 can permeate via macroporous body 113 and into strip 114. Strip 114 extends further into housing 100. To help ensure that no liquid sample reaches Strip 114 without first passing a gap 115 can be left in the 114 to Strip 114 is through macroporous body 113, strip overlap "backed" by a supporting strip 116 formed of transparent mosture- housing 100 by arranging for the macroporous body 113 only partially. impermeable plastics material. Strip 114 extends beyond apertures.108 and 109.. Means are provided within housing 100 by webbs 117 and 118 to hold strip 114 firmly in place.

In this respect, the internal constructional details of the housing are not a significant aspect of the invention as the held firmly the sample collector 106 is firmly retained in the long as strip is in place within housing, housing, and adequate fluid permeable contact is maintained between sample collector 106, macroporous body 113 and strip 114. and apertures 108 and 109 and can act as a seal against The transparent backing strip 116 lies between strip ingress of mosture from outside the housing 100 via these If desired, the residual space 119 within the apertures. housing can contain moisture-absorbant material, such as silica gel, to help maintain the strip 114 in the dry state The reagent-containing detection zone in but the during storage. strip 114 is containing the immobilised unlabelled reagent will lie in not depicted in Figure 2, zone the region exposed through aperture 108 in order that when ...22_ the device has been used in an assay, the result can be observed through aperture 108. Aperture 109 provides means through which a control zone containing further reagents which may enable the adequate permeation of sample through the strip to be observed.

In operation, the protective cap 103 is removed from the holder and sample e.g. by being placed in a urine stream in the case of sample collector 106 is exposed to a liquid a pregnancy test. After exposing sample collector 106 to the liquid sample for a time sufficient to ensure that the collector 106 is saturated with the sample, the cap 103 can be replaced and the device placed aside by the user for an while two or three minutes) 114 to After the appropriate time, (e.g. test appropriate period time the provide strip sample permeates analytical result. the user can observe the test strip through apertures 108 and 109 and can ascertain whether the assay has been completed by observing the control zone through aperture 109, and can ascertain the result of the assay’ by observing the second zone through aperture 108.

During manufacture, the device can be readily assembled from, for example, plastics material with the housing 100 being moulded in two parts (e.g. upper and lower halves 110 and lll) ultrasonic welding) body and test halves and then sandwiched between the two halves. (e.g. by macroporous which can be securely fastened together after the sample collector, one of the The act strip have been placed within of forming this sandwich construction can be used to "crimp" body and test the collector strip together to ensure adequate contact between them. Cap 103 If desired, sample macroporous can be moulded as a separate complete item. _23_ apertures 108 and 109 can be provided with transparent inserts which may insure greater security against ingress of extraneous moisture from outside the housing. By providing a tight fit between the end 105 of housing 100 and the protruding sample collector 106, the application of sample to the protruding member will not result in sample entering the device directly and by—passing collector 106. Collector therefore provides the sole route of access for the sample to the strip within the housing, and can deliver sample to the strip in a controlled manner. The device as a whole therefore combines the functions of sampler and analyser.

By using the test strip materials and reagents as herein described, a device in accordance with Figures 1 and 2 can for use as a be produced which His .eminently. suitable pregnancy test kit or fertile period test kit for use in the The user 1nerely' needs to apply a urine then (after home or‘ clinic. the and sample to exposed porous member optionally replacing the cap) can observe the test result through aperture 108 within a matter of a few minutes.

Although described .with particular reference to pregnancy tests and fertile period tests, it will be appreciated that the device, as just described, can be used to determine the presence of a very wide variety of analytes if appropriate reagents are incorporated in the test strip. It will be further appreciated that aperture 109 is redundant and may be omitted if the test strip does not contain any control means. Further, the general shape of the housing and cap, both in terms of their length, cross-section and other physical features, can be the subject of considerable variation. -24..

Figure 3 of the accompanying drawings shows an enlarged view of the sample collector, macroporous body and test strip in the device illustrated in Figures 1 and 2. is strip 114, linked to the backed by the collector and test bibulous sample body 113 transparent plastics sheet 116, The macroporous such that liquid can flow in the direction shown by the arrows from the sample collector through the macroporous body and into the porous strip.

Test zone 120 incorporates the immobilised specific binding reagent, and control zone 121 contains a reagent to indicate that the sample has permeated a sufficient distance along the test strip.

An aqueous sample deposited in collector 106 can flow into macroporous body 113 and take up labelled reagent therein.

The sample can permeate from macroporous body 113 along the length of strip 114 and in doing so will carry the labelled reagent along the strip and through zone 120.

If desired, e.g. for ease of manufacture, the collector 106 need not be recessed to accommodate the macroporous body 113. overlapping arrangement, and pressed together during assembly of the complete device.

This will which the liquid path will be essentially as depicted in Instead, these components can simply be placed in an together with the porous strip 114, in practice provide a physical arrangement in Figure 3.

Embodiment 2 ._25_ Figures 4 and 5 illustrate another test device, which is seen in plan view in Figure 4 and in cross-section in Figure , the cross-section being an elevation on the line A seen Figure 4.

Referring to Figure 4, the test device comprises a flat rectangular casing 400 incorporating a centrally disposed rectangular aperture 401, adjacent the left hand end 402, and two further apertures 403 and 404 near the mid point of 403 and 404 the device and arranged such that apertures 401, the device lie on central longitudinal axis of corresponding to line A. Although all three apertures are illustrated as being rectangular, their actual shape is not critical.

Referring to the cross-section seen in Figure 5, the device is hollow and incorporates within it a nacroporous sample receiving member 405 adjacent end 402 of casing 400 and lying directly beneath 401. member 405 is in liquid—conductive contact with one end of a aperture Sample receiving test strip 406 back by a transparent plastics sheet 407 also 400, the extreme other end of the casing. sheet 407 is in firm Contact with the upper inner surface 408 of casing 400, and 404 to prevent ingress of moisture or sample into the Although not shown in the drawings, the porous test contained within casing and which extends to The transparent backing and provides a seal against apertures 403 casing. strip 406 incorporates a test zone and a control zone placed in a appropriately in relation to apertures 403 and 404, manner analagous to that described in Embodiment 1. macroporous sample receiving member incorporates a labelled reagent which is readily soluble or dispensable in an applied liquid sample. _ 25 - In operation, an sample can be applied through aqueous aperture 401, e.g. by means of a syringe, to saturate porous receiving member 405 which contains labelled reagent which can be taken up by the sample. Thereafter, the aqueous sample can permeate the test strip and, after an appropriate time, the test result can be observed through apertures 403 and 404.

Example This example does not include a sink, but illustrates other features of the invention. detergent (1.4 mm thick) of commercially—available, macroporous polyethylene having a pore size of A sheet pre—treated, about lO0um was saturated with an aqueous suspension of blue—coloured latex particles (prepared as described in GB A) or particle size about 0.4um. The latex particles carried an anti—beta LH monoclonal antibody. The solution also contained 3% BSA and 4% sugar. The sheet was then freeze—dried and cut into portions each 6 x 12mm, having a liquid capacity of about 50uL. These were incorporated in test devices as described above under embodiment l, with the test strip comprising backing nitrocellulose with an anti- alph LH monoclonal antibody immobilised in the test zone. of the test The liquid capacity of the "working length" strip between the macroporous body and the detection zone was about 40uL.

When a LH-containing urine sample was applied to the device, a positive result showed up as a very clear blue line, with negligible background bluecolour being visible in the detection window while the assay was being run.

Claims (15)

1. An analytical test device incorporating a dry porous carrier strip (114) to which device an aqueous liquid sample suspected of containing an analyte can be applied, the device also incorporating a specific binding reagent attached to a particulate label which labelled specific binding reagent is freely mobile in the porous carrier strip when in the moist state, and an unlabelled specific binding reagent which is permanently immobilised in a detection zone the labelled capable of (119) on the carrier strip, and unlabelled participating in either a sandwich reaction or a competition porous specific binding reagents being reaction in the presence of the analyte, characterised in that prior to the application to the device of the liquid the particle-labelled sample, specific binding reagent is retained in the dry state ix: a separate nacroporous body (113) less than 10 times greater than the maximum particle size of comprising plastics material having a pore size not the particulate label, through which nmcroporous body the applied liquid sample must pass en route to the proximal end of the porous carrier strip, and in that there is an absorbent sink at the distal end of the porous carrier strip, the absorbent sink not being merely a length of porous carrier" material which contains the detection zone and extends beyond the detection zone.

2. An test device according to claim 1, characterised in that the nacroporous body has an average analytical pore size of not less than 10 um. - 29 _

3. An analytical test device according to claim 1 or claim 2, characterised in that the macroporous body has an average pore size of about 100 pm.

4. An analytical test device according to any one of the preceding claims, characterised in that the plastics material has been pre—treated to render it more hydrophilic.

5.S. An analytical test device according to any one of the preceding claims, characterised in that the plastics material is polyethylene.

6. An analytical test device according to any one of. the preceding claims, characterised in that the particulate label is~ selected from the group consisting of metallic (e.g. gold) sols, non-metallic sols, dye sols, and latex particles having a maximum diameter of not greater than about 0.5 p.

7. An analytical test device according to claim 6, characterised in that the particulate label is a gold sol.

8. An analytical test device according to claim 6, characterised in that the particulate label is coloured latex.

9. An analytical test device according to any one of the preceding claims, characterised in that the porous carrier strip is nitrocellulose, having a pore size of at least lum.

10. An analytical test device according to any one of the preceding claims, characterised in that the macroporous body is in direct moisture-conductive contact with the proximal end of the porous carrier strip, and the detection zone on the porous carrier strip is spaced away from the region of Contact of the porous carrier material with the macroporous body.

11. An analytical test device according to any one of the preceding claims, characterised in that the macroporous body and porous carrier strip are contained within a casing or (400) and having a sample entry port constructed of xnoisture—impermeable material (401) the casing or housing also incorporating housing communicating with the macroporous body, (403) from outside the casing or housing. means to enable the detection zone to be qbservable

12. An analytical test device according to claim 11, characterised in that the casing or housing is provided with means (404, 109) which enable a further zone (120) of the porous carrier strip to be observed from outside the housing and which further zone incorporates one or more control indication to be given as to reagents which enable an whether the assay procedure has been completed.

13. An analytical test device according to any one of the preceding claims, characterised in that the liquid sample is urine. device according to claim 13,

14. An test characterised in that the analyte is hCG. analytical

15. An analytical test device according to claim 1, substantially as hereinbefore described with particular reference to and as illustrated in the accompanying drawings. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS.