United States Patent
`
`[19]
`
`May et al.
`
`[54] ASSAYS
`
`[75]
`
`Inventors: Keith May, Bedfordshire; Michael E.
`Prior, Northamptonshire; Ian Richards,
`Bedford, all of England
`
`[73] Assignee: Unilever Patent Holdings B.V.,
`Netherlands
`
`[*] Notice:
`
`The term of this patent shall not extend
`beyond the expiration date of Pat. No.
`5,599,721.
`
`[21] App]. No; 241,675
`
`[22]
`
`Filed:
`
`May 12, 1994
`
`Related U.S. Application Data
`
`[60] Continuation of Ser. No. 876,448, Apr. 30, 1992, abandoned,
`which is a division of Ser. No. 795.266, Nov. 19, I991.
`abandoned, whichis a continuation of Ser. No. 294,146.
`filed as PCI‘.r'GBB8-‘U0322, Apr. 26. 1988, abandoned.
`
`[30]
`
`Foreign Application Priority Data
`
`Apr. 27, 198'?
`Oct. 30, 1937
`
`[GB]
`[GB]
`
`ETOQST3
`England
`United Kingdom ................. .. 8725457
`
`GOIN 33558
`InL Cl.“
`[51]
`436514; 436501; 436513;
`[52] U.S. Cl.
`436523; 4362524; 436525; 436530; 4361541;
`436E810; 436E814; 436;‘8l'.?; 436.3318; 436a"906;
`43S;'962; 435/970; 4351975; 42'.-'l‘2.l3; 422r‘l5i]
`[58] Field of Search .................................. 4-22356-58, 60;
`4361501, 530, 514, 810, 314, 515, 518,
`523, 524, 541, 817, 818. 906; 43St"r'.92—?.95,
`970, 810, 962, 975; 42';'t'2, 2.11, 2.13
`
`I56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`3,410,839 1lt‘l968 De Carvalho
`3,420,205
`H1969 Morisson
`..... ..
`1lt'l971 Morison
`3.620.677
`
`lI6t'1l4
`
`llllll llllllll ||| lllll ||||| |||l| lllll Illll lllll Illll lllll |||||| III III]! llll
`USO0S602040A
`
`[11] Patent Number:
`
`5,602,040
`
`[45] Date of Patent:
`
`*Feb. 11, 1997
`
`FOREIGN PATENT DOCUMENTS
`
`1185882
`63810
`88636
`
`411985 Canada ................................ .. 4361518
`1110982 European Pat. OE. .
`931983 European Pat. Off. .
`
`(List continued on next page.)
`OTHER PUBLICATIONS
`
`Glad et al, Analytical Biochemistry (B5) 1978, pp. 180-187.
`Gribnan et al, J. Chromatography 3T6 (1986) pp. 175-189.
`Kenna, et al., “Methods for Reducing Non—Specific Anti-
`body Binding in Enzyme—Linked lmrnunosotbent Assays",
`Journal of Immunological Methods, 85 (1985) pp. 409-419.
`
`(List continued on next page.) _
`
`Primary Exami:ter—Carol A. Spiegel
`Attorney, Agent, or Fr'rm—Cushman Darby & Cushman,
`LLP
`
`[57]
`
`ABSTRACT
`
`An analytical test device useful for example in pregnancy
`testing, includes a hollow casing (500) constructed of mois-
`ture-impervious solid material, such as plastics materials,
`containing a dry porous carrier (510) which communicates
`indirectly with the exterior of the casing via a hibulous
`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, wherein the mobility is
`facilitated by a material comprising sugar, in 3! amount
`effective to reduce interaction between the test strip and the
`labelled reagent, and in a second zone spatially distinct from
`the first zone unlabeled specific binding reagent for the same
`analyte which unlabelled reagent is permanently immobi-
`lized 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. Preferably the device includes a
`removable cap for the protruding bibulous member.
`
`(List continued on next page.)
`
`34 Claims, 5 Drawing Sheets
`
`S15
`
`S16
`
`
`
`ALERE EX. 1005
`ALERE EX. 1005
`
`1 of 21
`10f21
`
`

`
`5,602,040
` Page 2
`
`..
`
`U.S. PATENT DOCUMENTS
`511972 Price
`3.666.421
`5119'.-'4 Bauer
`3,811,840
`411975 {(0013,
`3.316.504
`3,915,641 10119115 Wright.
`3.954.564
`511915 Memen
`4.022.876
`511977 Anbar
`4,042,335
`8119?“! Clement
`4,094,647
`6119'!!! Deutsch er al.
`4.122.030 1011978 Smith 9131-
`--
`4,166,105
`B11919 Hirschfeld
`4,168,146
`911979 Gruhh clal. ..
`4,169,138
`911979 Jonsson ....... ..
`4,235,601
`1111980 Deutsch ct al.
`4,244,940
`111981
`Jeong el al.
`..
`4,246,339
`1.-‘I981 Cole 01 al.
`4,302,536
`1111981 Longnccker
`4.313.734
`111982 Leuvcring
`4.315.903
`911932 Z6?
`4,366,241
`1211982 Tbrn ctal.
`4,373,932
`211983 Gribnau eta].
`4,374,925
`211983 Litrnanctal.
`4,435,504
`311984 201:
`............. ..
`4,452,901
`511984 Gordon
`4,461,829
`711934 Greenquist
`4.493.793
`111935 cm
`4,51g_555
`511935 Buggy 61 31
`4,552,839
`1111985 Gould
`4.587.102
`511936 Nugatomo et al.
`4.594.321
`E111!
`4,608,246
`ayer ct
`4,659,678
`411937 Forrest at al
`4,673,757
`711937 Rapkin ......
`4,695,554
`911937 O’Connell
`4,703,017
`101198‘? Campbell etal.
`4,'l'0'I,4-50
`1111987 Nason
`4,725,405
`211938 Common et al
`4,740,468
`411988 Wong (:1 al.
`4.742.011
`511933 Blake 6131-
`
`
`
`422156
`422156
`4351192
`43512383
`- 435137
`- 42411
`422156
`..... .. 43514
`252/3131?
`..... .. 42418
`4351792
`4361524
`422156
`. 4241‘!
`. 43515
`43517.25
`4361525
`43517-91
`43517.91
`4361501
`. 435.-'5
`43517.9}
`43517.92
`43517.72
`5301303
`422153
`4551?
`422156
`13355351:
`.
`435513
`4361169
`4361528
`4361501
`422261
`422158
`4361310
`4361513
`
`.
`1011985 European ‘Pat. O11".
`158146
`2.11986 Euro
`anPa1. 011..
`0170746
`711936
`Euroiicean Fat. 00..
`0186100
`711935 Eumpcan pa1_ ()f1‘__
`0135799
`1111986 European Pat. OJ'.I..
`0191640
`1011936
`E]_]_|,'()pea_'|'[ 1=a1_0{1=__
`0199205
`311937 European Fat. 017..
`0212599
`1211937 European Pa.1.0E..
`0250137
`1211937
`E'|_11'[)pea_|1 P31, 011",
`,
`250137
`111933
`E.u_fopea1_-1 pm, 0111 ,
`0253531
`611988 European Fat. 011'.
`0133442
`311933 European pg; 011 _
`0279097
`911933 Eumpaan 931,011”
`023423;;
`111939 European Pa1_()fl'__
`0299423
`211939 European pa1_0f1‘__
`0303734
`911973
`U1-1i[_e1jKjngdg
`,
`1525-703
`911979 United Kingdom _
`2015537
`511932 Unim-1 Kingdom,
`2035041
`711933 United Kingdom.
`2111676
`3102790 1011931 WIPO .................................. .. 4361518
`w()3503339
`711935 w11=o_
`w03.5,:0453-3
`311935 wlpo,
`w03'11027'14
`511937 w11=o_
`
`4361513
`
`OTHER PUBLICATIONS
`_
`,,
`.
`.
`.
`V0gt,_ Jr. ct al., _Quant1tat1ve d1i1"erences‘among various
`proteins as blocking agents for ELISA m1cro111cr plates",
`Journal of Immunological Mcihods, 101 (198?) pp. 43-50.
`Romano et al., “An Amiglobulin Reagent Labelled With
`Colloidal Gold For Use In Electron Microscopy", Immu-
`‘
`_
`fli17w§'aiuSi:4151icisliczil 9316.33‘ “giilrollcd Nuclcalion
`31' R"
`I
`.
`f1l'h P
`.
`I S.’
`. M Dd.
`G Id
`f
`'3 _“3",f‘“°“°
`° 379°“ "91"
`‘"1 “Fem "
`°’
`Eiléspeensjffls
`9-41-_ 131- 1- 1%73fi§'c113_- 1;3- _ al
`-
`-
`-
`Y a
`35»
`013131111-151
`1011 0
`109 311110
`High—Voltagc Electrophoresis", Journal of Chromatography,
`23 (1967). pp. 379-334.
`Hsu, “Immunogold for Detection of Antigcn on Nitrocellu—
`
`l2c;tf_1;;.;5Jer", Analytical Biochemistry, vol. 142, (1984), pp.
`,,
`.
`.
`.
`.,
`.
`,.
`'
`S‘.”°k "1. 31" V“‘“‘3‘'“‘‘“‘’'“’.’‘ of Anngemc '
`'
`' “.1“"h.°d =
`":?°‘1‘g’l“‘§fI1aya'idl13;:1Phg;1‘=§41f§§gmh C°mm”111°3“°“5»
`-
`v
`s
`v
`-
`-
`Geoghcgan at al., “Passive Gold .
`.
`. Hemagglutination“,
`Journal of Immunological Methods, 34, (1980), pp. 11-21.
`R. Brdicka, “Grundla en Der Ph sikalischen C]'[fi[[1_'i¢_=,"’ 351-.
`lin 1953, pp, 775, 78?-1-'.-'87, (an: English Translation].
`'
`--
`'
`'
`§§i§§§9“il;.o§3.°3ZI c.‘T.“.?f£g§o1L€i”s”é'§g.°“
`95311223
`E 1. 11'“
`1 H.
`*
`*1’P-
`1
`“B15
`“"5,” °“):
`.
`.
`,,
`25133113311611} K01lf;;3d‘=1111°1“11(5C11j~%E 15;§11h15§5-
`1
`11 =
`[Ii-"’»an)d
`7 1313-
`- an
`“E 15
`‘
`3953 '3“-
`Zuk. et al. “Enzyme lmmunoch.roma:‘.ography--A Quantita-
`rive Immunoassay Requiring No Ins1;n.1mcntation", Clinical
`Chcrnistry, vol. 31, No. 7, 1985, pp. 1144-1150.
`Van Hell, et a1., “Particle immunoassays”, Chapter 4, Alter-
`
`Bosch, M. G., “Enzym—und Sol Particle Immunoassays for
`,,
`.
`.
`H°““°“°» s Archms 01 GY'“‘=°°1°Bl’ and 095199109 "01-
`242, NO.
`l—4, U937). P13. 509-512 {and English translation).
`Moocrcmans, at 30., “Sensitive Wsualization .
`.
`. Straining“,
`Journal of Immunological Methods, '14 (1984) pp. 353-360.
`Leuvering, et al., “Sol Particle Immunoassay (SPIA)“,
`Abstract, Journal of Immunoassay, 1(1), pp. 77-91 (1980).
`Leuvering at al, “Optimization of a Sandwich Sol Particle
`Immunoassay for Human Chorionic Gonadotrophin", Jour-
`nal of Immunological Methods, Nov. 1, 1932, vol. 2, pp.
`175-184.
`
`2 of 21
`20f21
`
`Et°m“l-‘t‘;':h::"
`4,806.311
`.
`reen uist
`4,806,312
`211939 Greenguist
`.
`4,310,470
`311939 Burkhardt
`..
`4-,83'1',145
`611989 Liotta
`4_g37,163
`611989 do Jagger 6131
`4,859,612
`811989 Cole et al.
`4,861,552
`811939 Mcsuda ct al.
`4.351.711 1:39 FFEBSED -31 31-
`4,868,106
`1 B9
`Ito et 31.
`4-868-108
`911939 B999 =1
`4,906,439
`311990 Grcnner
`4,920,046
`411990 McFarlandc1a1.
`4,954,452
`911990 Yostet al.
`4,956,275
`911990 Zn]: ctal.
`..
`4,956,302
`911990 Gordon ..
`4,959,30'1
`91' 1990 Olson
`4.960.691
`1011990 00111011
`4-931-735
`1" 1991 D“fi“m 3‘ “L
`.......
`313313” at al‘
`,
`,
`ovmgton ct al.
`5,073,434 1211991 Swanson ...... ..
`5‘08g_394
`211992 Chm-I ________ __
`5,120,643
`611992 China 61 31-
`5,141,850
`811992 Cole ct al.
`FOREIGN PATENT DOCUMENTS
`
`42215
`42.2150
`422166
`43517.94
`4361533
`.... .. 4361523
`4361530
`422155
`4361530
`422156
`422156
`43517.9
`4361524
`43517.1
`4361161
`43511.9
`------ 435-'5
`432155
`
`436510
`422156
`. 43517.92
`435,134
`422156
`43615215
`
`
`
`
`
`0125 1 I 3
`0149168
`0212603
`0154749
`
`1111984-
`'1'.-‘I985
`8.11985
`9.11935
`
`European Pat. Off. .
`European Fat. 011'.
`.
`European Fat. 011'.
`.
`European Pat. OE. .
`
`

`
`U.S. Patent
`
`Feb. 11, 1997
`
`Sheet 1 of 5
`
`5,602,040
`
`‘3 Fig.7.
`
`3 of 21
`3of21
`
`

`
`n/_
`
`nu
`
`9..
`
` U.S.Patent
`
`Fig. 6.
`
`Feb. 11, 1997
`
`Sheet 2 of 5
`
`5,602,040
`
`4 of 21
`4 of 21
`
`
`

`
`U.S. Patent
`
`Feb. 11, 1997
`
`Sheet 3 of 5
`
`5,602,040
`
`Em
`
`Sh
`
`im
`
`“Ian
`
`
`
`E fi”.V“r“r“‘..“‘.‘l“.I.'.I_"“.‘.lI..i..r
`
`gm
`
`5 of 21
`5of21
`
`
`
`

`
` U.S.Patent
`
`Feb. 11, 1997
`
`Sheet 4 of 5
`
`5,602,040
`
`ms
`
`6 of 21
`6 of 21
`
`

`
`U.S. Patent
`
`Feb. 11, 1997
`
`Sheet 5 of 5
`
`5,602,040
`
`7 of 21
`70f21
`
`

`
`5 ,60 2,040
`
`1
`ASSAYS
`
`This is a continuation of application Ser. No. 07.-"87fi,4=1-8.
`filed on Apr. 30. 1992, which was abandoned upon the filing
`hereof which was a Divisional of application Ser. No.
`0't'f?95,2ti6 filed Nov. 19, 1991 which was a continuation of
`application Ser. No. 07l'294.l46 filed Feb. 27’, 1989, now
`abandoned.
`
`5
`
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`
`The present invention relates to assays involving specific
`binding, especially immunoassays. This application is based
`on applications filed in Great Britain, having application
`numbers 8709873 (filed Apr. 27, 1987) and 8725457 (filed
`Oct. 30, 1981-’), as well as PCT application GB88t'00322
`(filed Apr. 26, 1988).
`In particular, the invention relates to analytical devices
`which are suitable for use in the home, clinic or doetor’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.
`
`20
`
`2. Description of the Related Art
`The use of test devices in the home to test for pregnancy
`and fertile period (ovulation) is now commonplace, and a
`wide variety of test devices and kits are available comn1er-
`cially. Without
`exception,
`the
`commercially-available
`devices all
`require the user to perform a sequence of
`operations before the test result is observable. These opera-
`tions necessarily involve time, and introduce the possibility
`of error.
`
`35
`
`45
`
`55
`
`SUMMARY OF TI-[E 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 reagenbimpregnated 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 strip material, usually with the aid of an eluting solvent
`such as water. In so doing. the sample progresses into or
`through a detection sons 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 analyte becomes bound in that none cart
`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, Bo-ots~Celltecl't Diagnostics Limited EP 0225054,
`Syntex (U.S.A.} Inc EP 0183442, and Bchringwerke AG EP
`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-
`
`65
`
`2
`venient to use and which require the user to perform as few
`actions as possible.
`A typical embodiment of the invention is an analytical test
`device comprising a hollow casing constructed of moisture-
`impcrvious 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 analytc 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 analytc 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 “sandwic " 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 none, the
`presence of analyte.
`in the sample being determined 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 analytc (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 analytc is present in the
`sample. The two binding reagents must have specificities for
`diiferent epitopes on the analyte.
`the labelled
`In another embodiment of the invention,
`reagent is either the analyte itself which has been conjugated
`with a label, or is an analytc 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 analytc 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
`analytc or analytc analogue will migrate through the porous
`solid phase material into the second zone and bind with the
`
`8 of 21
`8of21
`
`

`
`3
`
`4
`
`5,602,040
`
`immobilised reagent. Any artalyte 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
`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
`chemical 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 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.
`Another imponant 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 be 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
`discernablc 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 a
`directly labelled specific binding reagent on a ca.rricr mate-
`rial comprising nitrocellulose. 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.
`the
`In a further embodiment of the present
`invention,
`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 casing or housing
`and the porous receiving member, with which the porous
`solid phase material is linked. extends out ofthe 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
`
`10
`
`15
`
`20
`
`25
`
`3D
`
`35
`
`40
`
`45
`
`5!}
`
`55
`
`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
`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, while the assay procedure is being performed.
`Optionally, the labelled reagent can be incorporated else-
`where within the deviec, eg. 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 lhe 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 highly—specilic 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-
`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 anti-
`bodies having specificities for dillerent 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 none, 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 Ll-I, 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 (ie 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), polyvinylidene ilouridc. ethylene
`vinylaeetate, acrylonitrile and polytetrailuoroethylene 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 efficiently. Porous sample receiving members
`can also be made from paper or other cellulosic materials,
`such as nitrocellulose. Materials that are new 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
`
`9 of 21
`9of21
`
`

`
`5
`
`5,602,040
`
`6
`
`ID
`
`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
`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 "control" zone can he 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, eg. an “anti-mouse” antibody if the labelled body is
`one that has been derived using a murine hybridoma, 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 front 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 andtor
`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 arcadily detectable signal, e.g.
`a strongly-coloured area. This can be evaluated by eye, or by
`instruments if desired.
`
`30
`
`35
`
`45
`
`55
`
`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
`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
`progresses to the detection zone. Preferably,
`the flow of
`sample continues beyond the detection zone and sulficient
`sample is applied to the porous material in order that this
`may occur and that any excess labelled reagent from the first
`zone which does not participate in any binding reaction in
`the second zone is flushed away from the detection zone by
`this continuing flow. If desired, an absorbent “sirtlt” 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 sufiicient absorp-
`tivc capacity to allow any unbound conjugagc to wash out of
`the detection zone. As an alternative to such a sink it can be
`sufiicient to have a length of porous solid phase material
`which extends beyond the detection zone.
`The presence or intensity of the signal from the label
`which becomes bound in the second zone can provide a
`qualitative or quantitative measurement of analyte in the
`sample. A plurality of detection zones arranged in series on
`the porous solid phase material, through which the aqueous
`liquid sample can pass progressively. can also be used to
`provide a quantitative measurement of the analyte, or can be
`loaded individually with different specific binding agents to
`provide a multi-analyte test.
`The immobilised specific binding reagent in the second
`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 carrier material is in the form of a strip or
`sheet to which the reagents are applied in spacially distinct
`zones, and 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 incor-
`porate two or more discrete bodies of porous solid phase
`material, e.g. separate strips or sheets, each carrying mobile
`and immobilised reagents. These discrete bodies can be
`arranged in parallel, for example, such that a single appli-
`cation of liquid sample to the device initiates sample flow in
`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
`dilferent carriers, the simultaneous determination of a plu-
`rality of analytes in a single sample can be made. Alterna-
`tively, multiple samples can be applied individually to an
`array of carriers and analysed simultaneously.
`The material comprising the porous solid phase is pref-
`erably nitrocellulose. This has the advantage that the anti-
`body in the second zone 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, carbonyldi-
`imidazole, or tresyl chloride.
`
`Following the application of the antibody 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. The labelled reagent for the first
`zone can then be dispensed onto the dry carrier and will
`become mobile in the carrier when in the moist state.
`Between each of these various process steps (sensitisation,
`application of unlabelled reagent, blocking and application
`of the labelled reagent), the porous solid phase material
`should be dried.
`
`65
`
`To assist the free mobility of the labelled reagent when the
`porous carrier is moistened with the sample, it is preferable
`for the labelled reagent to be applied to the carrier as a
`surface layer, rather than being impregnated in the thickness
`of the carrier. This can rrtinirnise interaction between the
`carrier material and the labelled reagent. In a preferred
`embodiment of the invention, the carrier is prc—treated with
`
`10 of 21
`100f21
`
`

`
`5 ,602,040
`
`7
`
`a glazing material in the region to which the labelled reagent
`is to be applied. Glazing can be achieved, for example. by
`depositing an aqueous sugar or cellulose solution, e.g. of
`sucrose or lactose, on the carrier at the relevant portion, and
`drying. The labelled reagent can then be applied to the
`glazed portion. The remainder of the carrier material should
`not be glazed.
`Preferably the porous solid phase material is nitrocellu-
`lose sheet having a pore size of at least about 1 micron, even
`more preferably