Hllllllllllllllllllllllll||||l||l|l||||||||||llllllllllllllllllllllllll
`USU0562237lA
`
`United States Patent
`
`5,622,871
`[11] Patent Number:
`
`May et al.
`[45] Date of Patent:
`Apr. 22, 1997
`
`[191
`
`[54] CAPILLARY IMMUNOASSAY AND DEVICE
`THEREFOR COMPRISING MOBILIZABLE
`PARTICULATE LABELLED REAGENTS
`
`['35]
`
`Inventors: Keith May, Bedfordsltirc; Michael E.
`Prior, Norlhamptonshire; Ian Richards,
`Bedford, all of England
`
`[73] Assignee: Unilever Patent Holdings B.V.,
`Netherlands
`
`3,744,975
`3,793,004
`
`711973 Mailcn
`3.31974 Zerachia Bi Bl.
`
`23x2s9
`42256
`
`(List continued on next page.)
`
`FOREIGN PATENT DOCUMENTS
`
`6007486
`1135882
`
`7/1985 Australia.
`11;‘19Bl Canada.
`
`(List continued on next page.)
`OTHER PUBLICATIONS
`
`[213 App1.No.: 102,313
`
`[22) Filed:
`
`Jul. 15,1993
`
`Glad ct al, Analytical Biochemistry (B5) 1978, pp. 180-187.
`Gribnau et al, J. Chromatography 376 (1986) pp. 175-189.
`
`Related U.S. Application Data
`
`(List continued on next page.)
`
`Primary Examt‘ner—Carol A. Spiegel
`Attorney, Agent, or Ft'mt—Cushrnan Darby & Cushman,
`LLP
`
`[57]
`
`ABSTRACT
`
`An analytical lest device useful for example in pregnancy
`testing, includes a hollow casing (500) constructed of mois-
`tare—impervious solid material, such as plastics materials,
`containing a dry porous carrier (510) which communicates
`indirectly with the exterior of the casing via a bibulous
`sample receiving member (506) which protrudes from the
`casing such that a liquid test sample can be applied to the
`receiving member and permeate therefrom to the porous
`carrier,
`the carrier containing in a first zone a labelled
`specific binding reagent is freely mobile within the porous
`carrier when in the moist state, and in a second zone spatially
`distinct from the first zone unlabelled specific binding
`reagent for the same analyte which unlabelled reagent is
`permanently immobilised on the carrier material and is
`therefore not mobile in the moist state, the two zones being
`arranged such that liquid sample applied to the porous
`carrier can permeate via the first zone into the second zone,
`and the device incorporating an aperture (508) in the casing.
`enabling the extent (if any) to which the labelled reagent
`becomes bound in the second zone to be observed. Prefer-
`ably the device includes a removable cap for the protruding
`bibulous member.
`
`36 Claims, 5 Drawing Sheets
`
`[60] Continuation of Ser. No. 376.449, Apr. 30, 1992. abandoned,
`which is a division of Ser. No. 795.266, Nov. 19, 1991,
`abandoned, which is a continuation of Ser. No. 294,146,
`filed as PC'1‘.~"GBB3r'(l0322, Apr. 26, 1988, abandoned.
`
`[30]
`
`Foreign Application Priority Data
`
`Apr. 27, 198?
`Oct. 30. 1987
`
`[GB]
`[GB]
`
`United Kingdom
`United Kingdom .
`
`.. 3709873
`
`.. 372545‘?
`
`GOIN 331558
`Int. Cl.“
`[51]
`[52] US. Cl. .............................. 436.-‘S14; 422356; 42258;
`422.360; 4351'? .1; 4351962; 4351970; 435!9'l'l;
`4351973; 4361510; 436518; 4363541; 4361305;
`436.-"310; 4-36l8l'}'; 4363318
`722!56—58;436l'50l,
`[58] Field of Search
`436.1530, 310, 814, 514, 515, 513, 523,
`524, 541, 510, 538. 817, 818; 435.1810,
`7.92-7.95, 970; 422J'60; 4-27f2, 2.11
`
`[56]
`
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`.
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`
`
`
`
`
`:11.
`
`
`
`................... .. 43517.4
`1281632
`43517.93
`...... .. 43517.9
`
`
`
`............................ .. 422156
`4361536
`
`
`
`.
`
`43517.4
`.... .. 43515
`
`.. 4361533
`435nr_9
`
`43517.36
`“ 43514
`.. 4355
`_ 435,r3o5
`43517.25
`
`
`
`4361510
`..................... 4351805
`.
`422156
`. 4361533
`1281897
`
`
`
`............................ .. 43517.9
`424111
`. 4361533
`43517.1
`. 4361512
`.. 4361518
`. 4361506
`............................. 4361500
`
`4361514
`43517.91
`
`.................... .. 4361501
`. 4351805 X
`42411.37
`..
`-‘-1-22158
`43517.91
`4361518
`
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`4361500
`4351805
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`43517.82
`4361518
`.. 4361518
`43517.32
`........................ .. 43517.92
`
`
`
`43517.92
`42.U64
`. 4361524
`4351805
`
`43516
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`4361518
`4351805
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`422156
`
`
`
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`
`2 of 21
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`

`
`5,622,871
`
`Page 3
`
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`122331113 131333 g:$d3- PM OE
`91,952
`1H1984 Eumgfi Pat: OE
`.
`0125118
`ll.-11984 E
`P .011".
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`0212603
`311935 European Fat. 011.
`.
`0154749
`9.11935 European Pat. OE. .
`159745
`1911935
`51-9013931! 931- 011- -
`0164180
`1211985 European Fat. 011.
`1313333
`E“’°1’°““ $1‘ 3:" '
`30174247 M936
`Pa: On.‘ ‘
`
`435(1))
`
`Em-ope-an pat Qfi=__
`311935
`Eui'opt:ii.l1 Pat. O11‘.
`.
`711986
`811986 European Pat. OH. .
`1911935
`E‘-WDPCHI1 Pat 011- -
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`31’
`"
`211933 European Pat. on. .
`8,1938 Eumpean M DE‘ A
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`0135190
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`
`0255342
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`0234232
`033120}
`028637!
`0299428
`0303784
`
`59_m95o
`1509563
`1526703
`2915537
`2099573
`2036041
`2111575
`
`WD85l'01354
`wossrossso
`w0g5,«(}45g3
`W087.-‘(}2'i'74
`
`£3139” -
`,H984 1:9“ -
`oilfield’ Kingdom
`311973
`931973 United Kingdom _
`9,-1979 United Kingdom _
`111932 United Kingdom 4355
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`111933
`11911311 K111399111 -
`W '
`311935 WIPO '..
`711935 wnvo .
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`3 of 21
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`US. Patent
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`Apr. 22, 1997
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`Sheet 1 of 5
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`5,622,871
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`40f21
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`U.S. Patent
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`Apr. 22, 1997
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`Sheet 2 of 5
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`5,622,871
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` Fig. 6.
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`5 of 21
`5of21
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`

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`U.S. Patent
`
`Apr. 22, 1997
`
`Sheet 3 of 5
`
`5,622,871
`
`mm
`
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`6 of 21
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` U.S.Patent
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`Apr. 22, 1997
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`Sheet 4 of 5
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`5,622,871
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`
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`U.S. Patent
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`Apr. 22, 1997
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`Sheet 5 of 5
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`5,622,871
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`1
`CAPILLARY IMMUNOASSAY AND DEVICE
`THEREFOR COMPRISING MOBILIZABLE
`PARTICULATE LABELLED REAGENTS
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`This is a continuation of application Ser. No. 0?;’876,449
`filed on Apr. 30, 1992, which was abandoned upon filing
`hereof, which is a Divisional of Appln. Ser. No. 0Tf?95,266
`filed Nov. 19, 1991, now abandoned which is a continuation
`of Apple. Ser. No. {Y?!294,l-46, filed as PC'I‘lGB88;'00322,
`Apr. 26, 1988, now abandoned.
`
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`
`The present invention relates to assays involving specific
`binding, especially immunoassays.
`In particular, he 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
`which is rapid and which requires the minimum degree of
`skill and involvement from the user.
`
`2. Description of the Related Art The use of test devices
`in the home to test for pregnancy and fertile period (ovula-
`tion) is now commonplace, and a wide variety of test devices
`and kits are available commercially. Without exception, the
`commercially-available devices all require the user to per—
`form a sequence of operations before the test result
`is
`observable. ‘These operations necessarily involve time, and
`introduce the possibility of error.
`
`SUMMARY OF THE INVENTION
`
`It is an object of the present invention to provide a test
`device which is readily usable by an unskilled person and
`which preferably merely requires that some portion of the
`device is contacted with the sample (e.g. a urine stream 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. Ideally the analytical result should 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
`the ship material, usually with the aid of an eluting 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 for an analyte suspected of being in the
`sample is immobilised. Analyte present in the sample can
`therefore become bound within the detection zone. The
`extent to which the analytc becomes bound in that zone can
`be determined with the aid of labelled reagents which can
`also be incorporated in the test strip or applied thereto
`subsequently. Examples of prior proposals utilising these
`principles are given in Thyroid Diagnostics Inc GB
`1589234, Boots-Celltech Diagnostics Lintitcd EP 0225054,
`Syntex (USA) Inc EP 0133442, and Behringwerlte AG BP
`0186799.
`
`The present invention is concerned with adapting and
`improving the known techniques, such as those referred to
`in the above publications, to provide diagnostic test devices
`especially suitable for home use which are quick and con-
`venient to use and which require the user to perform as few
`actions as possible.
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`Atypical embodiment of the invention is an analytical test
`device comprising a hollow casing constructed of moisture
`impervious solid material containing a dry porous carrier
`which communicates directly or indirectly with the exterior
`of the casing such that a liquid test sample can be applied to
`the porous carrier, the device also containing a labelled
`specific binding reagent for an analyte which labelled spe-
`cific binding reagent
`is freely mobile within the porous
`carrier when in the moist state, and unlabelled specific
`binding reagent for the same analyte which unlabelled
`reagent is permanently immobilised in a detection zone on
`the carrier material and is therefore not mobile in the moist
`state, the relative positioning of the labelled reagent and
`detection zone being such that liquid sample applied to the
`device can pick up labelled reagent and thereafter permeate
`into the detection zone, and the device incorporating means
`enabling the extent (if any) to which the labelled reagent
`becomes in the detection zone to be observed.
`Another embodiment of the invention is a device for use
`
`in an assay for an analyte, incorporating a porous solid phase
`material carrying in a first zone a labelled reagent which is
`retained in the first zone while the porous material is in the
`dry state but is free to migrate through the porous material
`when the porous material is moistened, for example by the
`application of an aqueous liquid sample suspected of con-
`taining the analyte, the porous material carrying in a second
`zone, which is spatially distinct from the first zone, an
`unlabelled specific binding reagent having specificity for the
`analyte. and which is capable of participating with the
`labelled reagent in either a "sandwich“ or a “competition“
`reaction, the unlabelled specific binding reagent being firmly
`immobilised on the porous material such that it is not free to
`migrate when the porous material is in the moist state.
`'The invention also provides an analytical method in
`which a device as set forth in the proceeding paragraph is
`contacted with an aqueous liquid sample suspected of con-
`taining the analyte, such that the sample permeates by
`capillary action through the porous solid phase material via
`the first zone into the second zone and the labelled reagent
`migrates therewith from the first zone to the second zone, the
`presence of analyte in the sample being tleter-mined by
`observing the extent (if any) to which the labelled reagent
`becomes bound in the second zone.
`
`In one embodiment of the invention, the labelled reagent
`is a specific binding partner for the analyte. The labelled
`reagent. the analyte {if present) and the immobilised unla-
`belled specific binding reagent cooperate together in a
`“sandwich" reaction. This results in the labelled reagent
`being bound in the second zone if analyte is present in the
`sample. The two binding reagents must have specificities for
`different epitopes on the analyte.
`the labelled
`In anothenembodiment of the invention,
`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 dis-
`persibility 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, or at least
`very closely similar. In this second embodiment, the labelled
`analyle or analyte analogue will migrate through the porous
`solid phase material into the second zone and bind with the
`imrnoboliscd reagent. Any analyze present in the sample will
`compete with the labelled reagent in this binding reaction.
`Such competition will result in a reduction in the amount of
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`5,622,871
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`labelled reagent binding in the second zone, and a conse-
`quent decrease in the intensity of the signal observed in the
`second zone in comparison with the signal that is observed
`in the absence of analyte in the sample.
`An important preferred embodiment of the invention is
`the selection of nitrocellulose as the carrier material. This
`has considerable advantage over conventional strip materi-
`als, such as paper, because it has a natural ability to bind
`proteins without requiring prior sensitisation. Specific bind-
`ing reagents, such as immunoglobulins, can be applied
`directly to nitrocellulose and immobilised thereon. No
`chernica] treatment is required which might 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 polyvinylaleohol. 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.
`Another important preferred embodiment of the invention
`is the use of so called “direct labels", attached to one of the
`specific binding reagents. Direct labels such as gold sols and
`dye sols, are already known per se. They can he used to
`produce an instant analytical 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 a
`analytical device which is stored in the dry state. Their
`release on contact with an aqueous sample can be modu-
`lated, for example by the use of soluble glazes.
`An important aspect of the invention is the selection -of
`technical features which enable a direct labelled specific
`binding reagent
`to be used in a carrier.-based analytical
`device, e. g. one based on a strip format, to give a quick and
`clear result.
`Ideally,
`the result of the assay should be
`discernable by eye and to facilitate this, it is necessary for
`the direct label to become concentrated in the detection
`zone. To achieve this, the direct labelled reagent should be
`transportable easily and rapidly by the developing liquid.
`Furthermore, it is preferable that the whole of the developing
`sample liquid is directed through a comparatively small
`detection zone in order that the probability of an observable
`result being obtained in increased.
`Another important aspect of the invention is the use of .1
`directly labelled specific binding reagent on a carrier mate-
`rial comprising nitroccllulose. Preferably the nitrocellulose
`has a pore size of at least one micron. Preferably the
`nitrocellulose has a pore size not greater than about 20
`microns. In a particularly preferred embodiment, the direct
`label
`is a coloured latex particle of spherical or near-
`spherical shape and having a maximum diameter of not
`greater than about 0.5 micron. An ideal size range for such
`particles is from about 0.05 to about 0.5 microns.
`
`In a further embodiment of the present invention. the
`porous solid phase material is linked to a porous receiving
`member to which the liquid sample can be applied and from
`which the sample can permeate into the porous solid phase
`material. Preferably,
`the porous solid phase material
`is
`contained within a moisture-impermeable easing or housing
`and the porous receiving member, with which the porous
`solid phase material is linked, extends out of the housing and
`can act as a means for permitting a liquid sample to enter the
`housing and permeate the porous solid phase material. The
`housing should be provided with means, eg. appropriately
`placed apertures, which enable the second zone of the
`porous solid phase material (carrying the immobilised unla-
`belled specific binding reagent)
`to be observable from
`outside the housing so that the result of the assay can be
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`observed. If desired, the housing may also be provided with
`further means which enable a further zone of the porous
`solid phase material to be observed from outside the housing
`and which further zone incorporates control reagents which
`enable an indication to be given as to whether the assay
`procedure has been completed. Preferably the 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 appli-
`cation, whiie the assay procedure is being performed.
`Optionally, the labelled reagent can be incorporated else-
`where within the device, e.g. in the bibulous sample collec-
`tion member, but this is not preferred.
`An important embodiment of the invention is a pregnancy
`testing device comprising a hollow elongated casing con-
`taining a dry porous nitrocellulose carrier which communi-
`cates indirectly with the exterior of the casing via a bibulous
`urine receiving member which protrudes from the casing
`and which can act as a reservoir from which urine is released
`into the porous carrier, the carrier containing in a first zone
`a higl1ly—specific anti-hCG antibody bearing a coloured
`“direct“ label.
`the labelled antibody being freely mobile
`within the porous carrier when in the moist state, and in a
`second zone spatially distinct from the first zone an highly-
`spccific 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 anti-
`bodies having specificities for different hCG epitopes, the
`two zones being arranged such that a urine sample applied
`to the porous carrier can permeate via the first zone into the
`second zone, and the casing being constructed of opaque or
`translucent material
`incorporating at
`least one 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 absorb-
`ing liquid rapidly. The porosity of the material can be
`unidirectional (is with pores or fibres running wholly or
`predominantly parallel to an axis of the member) or multi-
`directional (omnidirectional, so that
`the member has an
`amorphous sponge-like structure). Porous plastics material,
`such as polypropylene, polyethylene (preferably of very
`high molecular weight), polyvinylidcne flouride, ethylene
`vinylacetate, acrylonitrilc and polytetraflttoro-ethylene can
`be used. It can be advantageous to pre-treat the member with
`a surface-active agent during manufacture, as this can reduce
`any inherent hydrophobicity in the member and therefore
`enhance its ability to take up and deliver a moist sample
`rapidly and elficiontly. Porous sample receiving members
`can also be made from paper or other cellulosic materials,
`such as nitro-cellulose. Materials that are now used in the
`nibs of so-called fibre tipped pens are particularly suitable
`and such materials can be shaped or extruded in a variety of
`lengths and cross-sections appropriate in the context of the
`invention. Preferably the 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 and similar materials are less
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`preferred in any embodiment wherein the porous receiving
`member-protrudes from a housing. The liquid must there-
`after permeate freely from the porous sample receiving
`member into the porous solid phase material.
`If present, the “conI:r'ol" 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 antibody from the first
`zone. e.g. an “a.nti—mouse" antibody if the labelled body is
`one that has been derived using a murine hybtidoma, to
`confirm that the sample has permeated the test strip. Alter-
`natively, the control zone can contain an anhydrous reagent
`that, when moistened, produces a colour change or colour
`formation, e.g. anhydrous copper sulphate which will turn
`blue when moistened by an aqueous sample. As a further
`alternative, a control zone could contain immobilised ana-
`lyte 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 second zone in
`which the desired test result is recorded. A positive control
`indicator therefore tells the user that the sample has perme-
`ated the required distance through the test device.
`The label can be any entity the presence of which can be
`readily detected. Preferably 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 andfor
`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.
`
`Indirect labels, such as enzymes, e.g. alkaline phosphatase
`and horseradish peroxidase, can be used but these usually
`require the addition of one or more developing reagents such
`as substrates before a visible signal can be detected Hence
`these are less preferred. Such additional reagents can be
`incorporated in the porous solid phase material or in the
`sample receiving member, if present, such that they dissolve
`or disperse in the aqueous liquid sample. Alternatively, the
`developing reagents can be added to the sample before
`Contact with the porous material or the porous material can
`be exposed to the developing reagents after the binding
`reaction has taken place.
`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. In the preferred
`embodiment, where the label is a direct label such as a