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`
`USU(}5985675A
`
`United States Patent
`
`[19]
`
`[11] Patent Number:
`
`5,985,675
`
`Charm et al.
`
`[45] Date of Patent:
`
`Nov. 16,1999
`
`[54] TEST DEVICE FOR DETISCTION OF AN
`1.\NA[,Y1‘[<]
`
`[75]
`
`Inventors: Stanley E. Charm. Boston; Richard
`Skifington‘ Nonh Reading; RU})ert J_
`Markovsky, Amcsbury; Eliezer Zomer,
`Newmn’ an 01' Mass
`
`|'.r'3]
`
`/Xssignec: Charm Sciences, Inc., Maiden, Mass.
`
`|21] Appi. P-«Io.: 091tm1,775
`..
`3
`’
`D°°- 31! 1997
`[331 19""
`[5l]
`Int. Cl.“ ................................................. .. G0lN 331533
`[52] U.S. CI.
`............................ .. 4361514; 422155; 422158;
`4221137; 4221188; 4221104; 4221110; 4221278;
`43517.1; 435..1'7.2; 43517.8; 43517.92; 43517.94;
`43517.95; 4351174; 4351287; 4351970; 4351971;
`43517.93; 4361518; 4361524; 4361538; 4361530;
`4361535; 4361533; 4361810; 43(n18(l7
`Field of Search .............................. .. 422156, 58, 187'.
`4321188‘ 104’ 110, 378; 435111’ 72’ 73’
`733, -194‘ -195‘ 174’ 33-;_1‘ 970, 971’
`793; 4351518, 524‘ 538’ 530‘ 535, 533‘
`310 gm’
`5
`
`[58]
`
`[55]
`
`Ref;-renew; Cited
`U.S. l’AI"I_-INT DOCUMI:LN’l'S
`
`4303,1117
`4_.s2o_.'159
`4_.999_.2s5
`5.238.652
`
`vfs§"“s‘_““Z'
`:a_..'t‘.Jl.t"1-1:5
`5_.622_.8'1‘l
`
`.
`lU1'lO8‘1' Cantpheil ct al.
`511989 Guire el al.
`............................... .. 43514
`311991
`S111,-m .
`319993 Sun -31 €l|-
`P3161 Cl Eli.
`”=':l°"3 “"35 9' 3_'« 1
`I,-I9‘)? Rosensleln .
`41'l99‘1' May cl al. .
`
`............................ ..
`
`-
`
`OTHER PUBLICATIONS
`
`"AShor1Guiclc Developing lmmunochromalographicTest
`Strips"; 1996, 1.11. No. TB5U(J. Milliporc Corporation, Berl-
`[URL MA. USA-
`
`Primm'_vE.1'anii11e'r—Chris10pher 1.. (Thin
`_.
`_
`.
`fif:’“““"‘ f’””f,"“";].3_"°'_TI:‘.”3l’1 ["1 ';',gE3'°:l ,
`"”"’y' 39”’ 0*’
`'”"
`"’ 3"
`'
`’°
`"3’
`[57]
`A|;s1‘RA(;1‘
`
`_
`‘
`A test device. system and method, the device composed of
`an clungatcrl, to0thbrush—:-shaped, transparent, plastic housw
`irig and a lateral-flow lest strip for the detection of an
`analylc, such as a beta-lactam in milk, in the housing, the
`housing having an expansion cavity [0 receive expanded,
`liquid-contacted, absorbing material in the test strip, and to
`control lateral flow rate and times in the test strip.
`
`43111-(1,714
`
`It},-‘I98? Fuisz .
`
`20 Claims, 7 Drawing Sheets
`
`ALERE EX. 1008
`ALERE EX. 1008
`
`1 of 15
`1of15
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`U.S. Patent
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`Nov. 16,1999
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`Sheet 1 of 7
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`5,985,675
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`2 of 15
`2of15
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`

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`U.S. Patent
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`Nov. 16,1999
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`Sheet 2 of 7
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`5,985,675
`
`FIG.4
`
`FIG.3
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`FIG.2
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`3 of 15
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`Nov. 16,1999
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`Shcet30l"?
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`5,985,675
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`NEGATIVE
`
`E 3
`
`_.LWHS0P
`
`0 W
`
`FIG.
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`4 of 15
`4 of 15
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`

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`U.S. Patent
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`Nov. 16,1999
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`Sheet 4 of 7
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`5,985,675
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`FIG. 7
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`5 of 15
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`U.S. Patent
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`Nov. 16,1999
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`Sheet 5 of 7
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`5,985,675
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`6 of 15
`6of15
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`

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` U.S.Patent
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`Nov. 16,1999
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`Sheet 6 of 7
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`5,985,675
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`Fl G .
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`1
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`1
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`7 of 15
`7 of 15
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`

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`U.S. Patent
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`Nov. 16,1999
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`Sheet 7 of 7
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`5,985,675
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`8 of 15
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`5,985,675
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`1
`TEST DEVICE FOR DE'l‘EC'l'ION OF AN
`ANALYTE
`
`BACKGROUND OF THE INVENTION
`
`2
`sample absorbent material for the liquid sample and to
`induce capillary [low to the second end. The sample-
`absorbing zone with the compressed material layer is posi-
`tioned adjacent
`the application cavity,
`the compressed-
`material layer and the application cavity designed to provide
`the compressed-material layer to absorb a selected amount
`of liquid sample to be tested and suflicient to carry out the
`test and to expand from a dry, nonexpandable to a wet,
`expanded state, and to provide for the said material layer in
`the wet, expanded state to fill substantially the application
`cavity and to cause suflicient pressure on the housing walls
`ofthe expansion cavity to drive capillary flow of the liquid
`sample toward the disposal zone in said strip in a selected
`time period and to restrict flow of the liquid sample in the
`application cavity to a selected volume, when the open
`application end of the test device is inserted into a liquid to
`obtain the liquid sample.
`is the
`In the invention, one preferred embodiment
`employment of a housing, such as a one-piece, integral,
`injection-molded, all-transparent, plastic material, with the
`plastic material selected or designed to be subject to incu-
`bator temperatures of 50° C. or more for incubation times;
`for example, of 2 to t0—15 minutes, depending on the
`particular test.
`includes a generally
`The preferred embodiment
`toothbrush-type housing shape, with the enlarged, generally
`rectangular, toothbrush-type head at the open application
`end of the housing, with a dry, inert, porous, expandable,
`liquid-permeable, absorbing material in a generally rectan-
`gular layer as an absorbing zone in the test strip;
`for
`example. of cellulose or nitrocellulose, positioned beneath
`the open bottom of the application cavity or chamber. The
`absorbing layer on contact, such as immersion of the appli-
`cation end of the housing of the test device in a liquid, will
`absorb a preselected amount of the liquid sample required
`for the test. The absorbing-layer material will expand; for
`example. in 1 to 30 seconds. to fill or substantially fill the
`expansion cavity and contact the surrounding walls of the
`expansion-cavity housing, to cause suflicient pressure within
`the expansion cavity and in the expanded state of the
`material to drive capillary flow laterally in the underlying
`test strip toward the end of the elongated housing where the
`test strip is positioned. Thus, proper selection and dimen-
`sioning of the expansion cavity and underlying absorbing-
`layer material which generally mimics two dimensions of
`the expansion cavity, permits absorbing and [iltering of the
`selected amount of liquid sample for the test strip, and aids
`in driving the lateral How of the liquid sample in the test strip
`in the housing toward the end of the test strip; for example,
`the disposal zone,
`to receive the liquid sample where
`employed. If the absorbing layer does not expand sulfi-
`ciently to fill or substantially fill the expansion cavity, then
`lateral or capillary flow rates and times are unsatisfactory;
`that is, flow rate too slow and time period too long. Where
`the absorbing layer is used in excess, then excess pressure
`occurs in the expansion cavity, and the expanded absorbing
`layer tends to retard the desired lateral How of the liquid
`sample.
`The housing with the toothbrush-shaped design may
`comprise a separate,
`injection—moldcd housing with an
`optional end cover, to protect the exposed application end
`before sampling and after sampling, and in the incubation
`chamber. to prevent cross—contamination from other sources.
`The test device with the molded housing enables the user to
`handle the handle end of the housing and to obtain a liquid
`sample merely by dipping the ripen application cavity into a
`liquid.
`
`9 of 15
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`ill
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`15
`
`There are numerous patents and publications which
`describe various lateral—flow or immunochromatographic
`test kits and methods for the detection of the presence or
`concentration of chemical residues or analytes or classes
`thereof from liquid samples. One publication includes “A
`SHORT GUIDE-Developing Irnmunochromatographic '1":-st
`Strips" by Millipore Corporation, Bedford, Mass, while
`US. Pat. Nos. would include 4,703,017,
`issued Oct. 27,
`1987; 5,591,645, issued Jan. 7, 1997; 5,266,497, issued Nov.
`30, 1993; 4,999,285,
`issued Mar. 12, 1991; 5,238,652,
`issued Aug. 24, 1993; and 5,622,871, issued Aug. 22, 1997
`(all hereby incorporated by reference).
`US. Pat. No. 5,622,871 describes analytical test devices
`for biological fluids which include elongated, rectangular,
`hollow casings to contain a test strip, and which casings
`have an aperture to permit visual observation of the test
`results on the test strip. The casings include a protective,
`removable cap to protect and enclose the application end of
`the casing. The internal constructional details of the housing
`are not
`a significant
`feature of the invention, but are
`employed to provide a housing for the particular test strip *
`(column 12, lines 20-26}.
`It is desired to provide a new and improved analyte test
`device and method based on test strips in which the housing
`ofthc test strip is designed to promote the absorption, flow
`and efficiency of the test device and the tests carried out.
`SUMMARY OF THE INVENTION
`
`30
`
`The invention relates to an analyte or chemical residue
`test device and method employing a lateral—tlow test strip for
`the detection of the analyte or residue within a housing and
`a method therefor.
`
`The invention comprises an analyte test device for
`detecting, in the general horizontal position, an analyte in a
`liquid sample by capillary lateral llow in an immunochro-
`matographic test strip, which device comprises an elongated
`housing defining an elongated strip cavity having a one open
`application aperture at one end and having another end, the
`cavity adapted to receive and hold a test strip therein, and
`having a transparent top cover section, to permit the obser-
`vation of test results on the test strip, the housing charac-
`terized by an enlarged application cavity extending out-
`wardly from the top cover and having or adapted to have an
`open end at the application end. The test device includes a
`test strip positioned in the strip cavity.
`The test strip comprises a support strip with a plurality of
`sequential, contacting,
`liquid-sample, permeable zones
`extending from the first to the second ends, which zones
`permit the lateral capillary flow of the liquid sample from the
`lirsl
`to the second end. The zones include a sample-
`absorbing and filtering zone composed of an expandable,
`porous, compressed-material layer which moves, on contact
`with the liquid sample, between a nonexpandable state and
`an expandable state on absorption of a preselected amount of
`the liquid sample, and a releasing zone having a mobile-
`phase layer thereon with a receptor for the analyte of the
`liquid sample thereon, typically a visible area, for example,
`of colored beads. The zones include also a reaction zone
`having at least one stationary-layer analyte reference or test
`line, or generally a test and a separate control line thereon for
`observation, to detect the presence of analytes in the liquid
`sample. and optionally a disposal none of a layer of liquid-
`
`35
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`toothbrush-shaped
`The housing also may comprise a
`design, wherein the expansion cavity is formed in a plastic,
`usually transparent, blister-type package which is sealed
`against a flat support, such as a paper strip or another plastic
`strip, and which encompasses within the blister package the
`selected test strip. The blister package includes a removable
`seal strip at the one application end of the enclosed test strip,
`for peeling or removal prior to use and for the introduction
`of a selected volume of the liquid to the application-
`absorbing zone of the test strip while in the blister package.
`The blister package with the liquid sample and test strip may
`be incubated in the incubator and the test results observed or
`read.
`it has been discovered to be
`In a further embodiment,
`desirable to provide one or more apertures in the housing
`which defines the expansion cavity,
`to permit
`the time-
`controlled and more rapid absorbing of the liquid sample
`into the absorbing material for more ellicient absorption and
`to reduce absorption time of the liquid sample. In particular,
`one or more apertures should be placed on the top cover or
`surface of the expansion-cavity housing, particularly of the
`molded housing, rather than on the sides, so that entrapped
`air, after immersion, will be discharged from the expansion
`cavity, as the absorbing layer expands into the wet,
`absorbing, expanded state. While a flat, rectangular strip of
`absorbing material
`is shown with a generally rectangular
`expansion cavity which mimics and provides for
`the
`it
`expanded, rectangular strip of the absorbing zone,
`is
`recognized that the size, material, dimensions and shape of
`the absorbing material and the shape or form of the expan-
`sion cavity may vary in the practice of the invention.
`Typically the open bottom of the expansion cavity is directly
`above the absorbing layer and usually of about the same
`width and length dimensions. to permit expansion without
`restriction of the absorption layer into the expansion cavity.
`While a fully transparent top cover is desirable to enclose
`the test strip and observe or read the test results on the test
`strip, it is recognized that the top cover may be open or have
`an aperture to view the test results, or only a section of the
`top cover be transparent to view the test results, or where
`applicable, the housing may be modified, so that the test
`results may be determined by optical or electronic instru-
`ment means.
`
`The test device may be packaged for use in a bl ister-type
`package or employ a fixed or sljdable protective cap at the
`application end, to protect the test device from contamina-
`tion prior to use and to protect the test device after contact
`with the liquid sample and in the incubator (where required
`in the test},
`to protect against cross-contamination. The
`protective cap can be removable and enclose totally the
`application end of the housing, or merely be slidably
`extended outwardly from the application end between a
`retracted use position and extended, protective, closed posi-
`tion.
`
`The test device employs a test strip selected to detect the
`presence or concentration of selected analytes or residues,
`either a single residue or classes thereof, and visually by
`reference of a reaction reference zone or reference line in the
`test strip which may be observed or measured. Usually, a
`control zone or line is spaced apan slightly downstream
`from the reference zone or lines for control purposes. The
`housing of the test device is applicable to a wide variety of
`presently employed or described test strips which are based
`on lateral [low or capillary flow, regardless of the nature of
`the particular analyte-residue test, provided only that
`the
`application or liquid contact portion 01‘ the test strip requires
`or uses a
`filtering absorbing material which moves by
`
`ll!
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`liquid-sample Contact between a nonexpanded and an
`expanded state at or toward the one application end of the
`test device. Typically,
`the test strip has a support and
`includes, on one surface, a plurality of contacting, liquid-
`permeable. sequential zones or sections with a stationary
`zone, a mobile zone and, optionally, a disposal zone. 'Ihe test
`device is particularly useful in connection with the liquid
`sample comprising a biological lluid; for example, urine;
`blood; or milk; and in the detection of antibiotics, like beta
`lactams or toxins, viruses and the like; however, the test
`device may employ one or more test strips directed to a
`variety of tests.
`Where applicable. the test device is employed in combi-
`nation with an incubator, such as a portable, electrically
`heated incubator with an incubation chamber which may be
`dimensioned to receive the test-device housing snugly
`therein for heating for a selected incubator time;
`for
`example, 55 to 65° C., and for a period of l
`to 10-15
`minutes. The test device and incubator also include a timer,
`so that the incubation period may be timed by a user.
`In operation, the test device with a protective covering or
`cap has the cover or cap removed and the application end
`contacted with a liquid to be tested, such as by immersion or
`a liquid sample pipctted into the application end for 1 to 10
`seconds and then removed. The absorbing material
`is
`allowed to expand within the expansion cavity; for example,
`1 to 15 seconds, then the test device placed in an incubator
`for a time period, then removed and the test results observed
`or measu red.
`The test device and method will be described for the
`purposes of illustration only in connection with certain
`embodiments; however,
`it
`is recognized that various
`changes, additions, improvements and modifications to the
`illustrated embodiments may be made by those persons
`skilled in the art, all falling within the spirit and scope ofthe
`invention.
`
`BRIEF DESCRIPTION OI: TIIE I)RAWINGS
`
`is a perspective, exploded view of a molded-
`I
`FIG.
`housing test device.
`FIGS. 2, 3 and 4 are schematic, illustrative views of the
`use of the test device of FIG. 1.
`
`FIG. 5 is a perspective view of an incubator and the test
`device with a liquid sample.
`FIG. 6 is an enlarged, front-plan view of the test strip of
`FIGS. 1-5, with enlarged, front, sectional views of positive
`and negative test results.
`FIG. 7 is a perspective. exploded view of a blister-pack
`test device.
`
`FIGS. 8, 9 and 10 are schematic, illustrative, side views
`of the use of the test device of FIG. 7.
`
`FIGS. 11 and 12 are perspective views of an incubator and
`the test device of FIG. 7 with a liquid sample.
`FIG. 13 is an enlarged, front-plan view of the test strip of
`FIGS. 7-12, with enlarged, front, sectional views of positive
`and negative test results.
`FIGS. 1-13 refer
`to FIG.
`drawings.
`
`1 and FIGS. 2-13 of the
`
`DI;-ISCl{Il"l"ION OF 'I'I-lli IiMBODIMl_iN'l'S
`
`In the drawings, FIGS. 1-6 show the analyte test device
`10 which includes an elongated, molded housing 12 of a
`one-piece, injection-molded, transparent styrene polymer to
`define an elongated cavity .14 with an open end 16, and
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`having an enlarged, rectangular application cavity 18 at the
`one open application end of the housing 12. The housing
`includes an elongated bottorn cavity formed during the
`injection-molding process. The housing includes an optional
`removable, friction—fitted or snapon protective styrene cap
`22 adapted to fit over the open application end 16 of the
`housing 12 and expansion apertures 19 in the top cover of
`the application housing cavity, to increase the efficiency of
`expansion of the material 32 within the test time.
`The housing cavity 14 includes therein on the bottom
`surface a lateral-llow test strip 28 adapted to detect the
`presence of an analyte in a liquid sample, such as milk. The
`test strip includes an elongated, adhesive, support or backing
`strip 30 with a plurality of sequential layers comprising a
`rectangular pad of dry, compressed, cellnlosic material 32 as
`a liquid-sample absorbent secured to the face su rface of the
`support strip 30 at the one application end. The pad 32 is
`selected to expand in contact with the milk,
`to [ill
`the
`expansion cavity 18 which the pad 32 mimics.
`in two
`dimensions. For example, with milk, the pad would be about
`3-4 mm by 12-14 mm, while the cavity 18 would be about
`5-6 mm by 15-16 mm by 4-6 mm in height. The expansion
`cavity may be dimensioned to be about 60% to 30% less
`than the full expansion of the sponge material.
`The suppon strip 30 includes a treated, mobile—phase, _
`support
`layer 33 with a visible receptor-probe area 34-, a
`stationary-phase layer 36 which includes a reference line 38
`and a control line 60 for the analyte to be detected, and a
`cellulosic absorbent pad 40 at the distal end of the support
`strip 3|] to capture excess liquid sample. The housing 12
`includes a transparent top cover 42 for visual observation of
`the reference line 38 and control line 40. The test strip 28 is
`placed and positioned loosely in the elongated cavity 14-,
`with the pad 32 positioned beneath the expansion cavity 18,
`and the pad extending generally to about or slightly beyond
`the plane of the open application end, and the end covered
`prior to use by the protective cap 22.
`In operation, the cap 22 is removed prior to use and the
`open application end of the housing 12 inserted briefly (1 to
`[0 seconds) in the liquid; for example, milk, to be tested
`employing the elongated housing I2 as a handle (see FIG.
`2). The test device 10 is removed and the pad 32 allowed to
`expand to fill the expansion cavity 18 and to start the lateral
`tlow of the milk sample through the test strip 28 (2 to 6
`minutes) (see FIGS. 3 and 4). Preferably, cap 22 is inserted
`to protect against cross-contamination, and the test device
`then placed in a horizontal position, with the application
`cavity 18 extending downwardly in an electric-heated incu-
`bator 46 with a cavity 47 shaped to receive the test device,
`and irtcubation carried out; for example,
`1
`to 8 rI1iI1utes,the
`incubation temperature observed through the temperature-
`irtdicator scale 48 (see FIG. 5). The incubated test device 10
`is then removed and reversed, and the front view of the test
`device with reference line 38 and control line 40 observed
`(see FIG. 6). The line readings for positive and negative
`controls are illustrated in FIG. 6 adjacent the front view of
`the test device 10. In the sponge material 32, expansion is
`controlled by the expansion cavity 18 volume and size,
`resulting in the sponge material 32 completely filling the
`cavity 18 with a preselected volume of liquid; for example,
`0.1 to [.0 ml,so the amount of liquid sample taken in for the
`test is controlled to the correct amount. The dimensions of
`the expansion cavity 18 prevent the sponge material 32 from
`full expansion, so that pressure is maintained in the
`expanded sponge (see FIG. 4) to aid in forcing capillary-
`lateral llow of the liquid sample through the test strip 28 in
`the housing 12.
`
`30
`
`35
`
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`
`45
`
`50
`
`55
`
`60
`
`0'5
`
`6
`The drawings in FIGS. 7-13 illustrate a further embodi-
`ment of the test device 50 in a transparent blister package
`which includes a transparent-tape plastic seal strip 52 with
`a peel tag 54 at one end, and a transparent blister package 56
`adhesively secured to the strip 52, to enclose a test strip 28
`therein. The blister package 56 includes an elongated cavity
`68 to hold strip 28 and an expansion cavity—housing 58 at the
`one end to form a generally toothbrush-shaped cavity within
`the plastic blister package 56 and strip 52. The selected test
`strip 28 is sealed and enclosed within the transparent blister
`package.
`FIG. 8 shows a side sectional view of the blister-package
`test device 50 prior to use. FIG. 9 shows the blister-package
`test device 50 with one end peeled back by peel tab 54, to
`expose the expansion housing cavity 58 and the dry filter-
`absorbent sponge pad 32 of the test strip 28, so that a defined
`amount of a liquid sample can he added; for example, by
`pipet, as shown. FIG. ll] illustrates the test device 5|] after
`addition of the liquid sample, and with the peel tab resealed
`and with the sponge pad 32 fully expanded by the liquid
`sample within housing cavity 58 and ready to incubate.
`FIG. 11 illustrates the test device 50 upside down and
`placed in one of two cavities 47 in an incubator 46. FlG.l2
`illustrates the technique of adding the liquid sample (see
`FIG. 9) with pipet, while the peel tab 54 is pulled away from
`the end of the test device 50 in the incubator, the test device
`sealed (see FIG. 10) and incubated. The test results of the
`completed test may then he read through the transparent top
`cover of blister package 56, as shown in FIG. 13, to provide
`positive or negative test results.
`The test; for example, the inhibition assay test, strip 28,
`selected for beta lactams in milk, is a quick test for beta
`lactams in comingled raw and pasteurized milk.
`In
`operation, the incubator 46 temperature gauge 48 is checked
`to ensure an incubator temperature of 55° C.; for example.
`temperature indicator 48 may be colored;
`for example,
`green, for use. The test device 50 is placed on one of the
`incubator 46 cavities 47, with the flat side facing up and the
`peel tab 54 peeled back far enough to expose the sponge pad
`32; for example, 12’: of an inch. The milk to be tested is mixed
`thoroughly before testing, and about 0.5 ml added by pipet
`to the exposed sponge pad 32 and the adhesive tape tab 54
`rescaled by hand pressure, and the incubator 46 cover
`closed. The test device 50 is incubated, for example, at least
`6 to 8 minutes and then removed from the incubator 46 and
`held vertically and a comparison made within about 1 hour
`between the test or reference line 38 with the control line 40.
`If no control line 40 appears, the test is invalid. A negative
`testis when the reference line 38 is the same or darker than
`
`the control line 40. Apositive test is indicated when the test
`or reference line 38 is absent or clearly lighter than the
`control line 40.
`
`In more detail, the test device 10 capable of detecting
`analytes in biological lluids comprises the following corn-
`ponents:
`a). A compressed material 32, such as cellulose, that is
`capable of absorbing said biological fluid and acting as
`a pretilter to remove coarse contaminants, such as hair,
`din, etc. Said compressed material, 32 is sized to
`absorb a fixed amount of sample required to complete
`the assay. This compressed material, when expanded
`and contacting the inside wall of housing 12, causes
`suflicient pressure to drive capillary flow along the
`components 32 and 36 in the time required (3 to 8
`minutes) [or a marketable test. Said component (32)
`overlaps said component 32 I
`to 10 mm such that.
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`30
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`35
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`40
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`7
`when an aqueous sample, such as milk, is added to
`component 36, the sample will llow onto said compo-
`nent 32.
`b). A housing 12 for said components 36, (32), 30, 32, 33,
`34, 36, 38, and 40. A housing should be used to allow
`for addition of biological sample, citlicr by dipping,
`pouring or pipctting. Said housing is constructed of
`either a llexible or hard material, such as polystyrene,
`polypropylene, or polyethylene.
`c). A mobile-phase support pad 33 made of polyester,
`polyethylene or glass fiber that acts as a secondary lilter
`for removal oflcss coarse materials (somatic cells}. The
`support has been pretreated with a chemical solution,
`such as 0.01 to 0.2 M sodium citrate pH 6-8. capable
`of neutralizing interferences found in biological
`samples. The mobi1c—phase support pad 33 overlaps
`said component 36 by 1 to 4 mm.
`d). A mobile phase 34 comprising:
`i) highly specific binding proteins or monoclonal anti-
`bodies capable of binding to an analytc and titrated -
`to a known concentration to make unavailable for
`further reactionldctcction a known amount of ana-
`lytc. This unavailability for
`further
`reactiont
`detection allows for the adjustment of a detection
`level of one or more analytes to a specified level of _
`concern. For example,
`in ccftiofur, a beta-lactam
`with a tolerance level of 50 ppb in milk, sensitivity
`can be changed from 5 ppb to between 40-50 ppb by
`the addition of a monoclonal antibody specific for
`ceftiofur. The specific monoclonal antibody ttemoves
`a specific analyte from binding to a receptor or
`antibody which is capable of binding a family of
`related compounds and;
`ii) highly purilied proteins; for example, beta-lactam
`receptor or anti-tet lgG. prepared by affinity purifi-
`cation andlor a combination of hydrophobics’ion-
`exchange IIPLC which are then attached to a
`colored, fluorescent, or infrared probe 34 which can
`be observed by opticalfinstmmental means or both.
`Attachment of proteins to a probe is called binding
`proteinlprobc complex; and
`iii) the mobile phase is sprayed, using a machine by
`lvek. Bio-dot, or Camag, or absorbed onto pad 33 in
`a solution containing 5 to 20% sugar, such as sucrose
`or trehalose, 5 to 20% protein, such as BSA or
`Primatone, 5 to 100 mm of a bulfer solution
`(phosphate or 'l‘rizn:ta base) with a final pl! of
`between 6-8. The mobile phase is sprayed on the
`upper portion of the mobile-phase support pad 33,
`such that component (32) does not overlap portion of
`said component 32, but rather said component 32
`overlaps said component 33 by placing the topmost
`portion of said component 32 1 to 7’ mm before the
`sprayed portion of said component (32). Astationary
`phase membrane 36 consisting of nitrocellulose or
`nylon which has multiple reaction zones present and
`comprising:
`capture zone{s} islare formed by spraying in a test
`line fonn 38 using a spraying instrument, such as
`Ivek. Biodot or Camag. The purpose of said
`capture zone is to capture unreacted binding
`proteinlprobe complex for viewing or measure-
`ment. (Tapturc zone(s) consist of an analytc of
`detection; that is, penicillin G or a member ofsaid
`analyte family; that is, beta-lactams, coupled to a
`carrier protein; that is, BSA, lgG, KLII, suspended
`in a 5 to lfltl mm buffer solution (phosphate or
`
`45
`
`50
`
`55
`
`60
`
`65
`
`8
`Trizma base) at a pH range of 643. Total protein
`concentration of the antibody solution ranges from
`0.2 to 100 mgtml and;
`a control zone is formed by spraying in a line form
`40 using a spraying instrument, such as lvck,
`Biodot or Camag. The purpose of said control
`zone is to capture binding proteinlprobe complex
`that has not bound to said capture mne(s). The
`control zone consists of an antibody specific to the
`binding protein!probe suspended in S to 100 mm
`of a buffer solution (phosphate or Trizma) at a pH
`range of 6 to 8. Total protein concentration of
`antibody solution ranges from 0.2 to 100 rnglml.
`A comparison ofthc control zone 40 to the capture zonc(s)
`38 yields test result. Typically, if the control zone is darker
`than the capture zone('s), analyte is present at detection level
`or greater (see FIG. 6 and FIG. 13).
`A plastic backing 30 with adhesive is employed for the
`mounting of zones 32, 36 and 4-0.
`A disposal pad 60 made of pressed cellulose or other
`absorbent material
`is employed to keep the sample flow
`consistent and to retain the reacted sample. The disposal pad
`overlaps the stationary-phase membrane 36 by 1 to 5 mm.
`An aqueous biological sample is added to component 32
`of a test device 10. Component 32 serves as a sample pad
`which expands as it absorbs the sample. Component 32
`overlaps component 33, and the fluid [lows onto the mobile-
`phase support where the mobile-phase materials dissolve
`into the biological fluid. Analytes present
`in the sample
`begin binding with the specific binding protein(s) 34
`attached to the probe. At the same time, specific unbound
`antibodies or binding proteins will bind with specific ana-
`lytcs to adjust their sensitivity to the test. The mobile—phase
`support pad 33 overlaps the stationary-phase membrane
`E(36), and the biological fluid, along with the mobile-phase
`materials 34, continue to react as all flow up the stationary
`phase. When the binding protein/probe complex reaches the
`capture zone, a portion of the binding proteinlprobe complex
`will bind to the capture zone. In a positive sample, analyte
`in the sample will have bound to the binding proteinfprobe
`complex, reducing the amount of binding proteinlprobe
`complex capable of binding to the capture zone. When the
`material
`reaches the control zone, a portion of binding
`proteinlprobe complex will bind the control zone. Excess
`reagent is then absorbed into the disposal pad.
`the
`In a negative sample, reagents are titrated, so that
`capture zone will have the same or preferably a greater
`amount of the probe binding to it than in the control zone.
`Conversely, in a positive sample, the control zone will have
`a greater amount of the probe binding to it than the capture
`zone.
`
`in one manifestation of this test device 10, a beta—lactam
`test is made to assay for beta-lactams in milk at a safe level.
`Apartially purified beta—lactam receptor from BST (Bncilitts
`stcnr‘o1lteniioplriItr.s'] is bound to a colloidal gold sol to make
`a beta-lactam binding proteinlgold bead probe 34. This is
`sprayed onto the mobile-phase support along with mono-
`clonal antibodies to ccftiofur and cephapirin. to reduce the
`sensitivity of these two 3Illll}lOl.llI.'S to a safe level. To the
`capture line 38 is sprayed a ccforanidc—BSA conjugate, and
`to the control
`line 40 is sprayed an antibody to the HST
`betavlactam receptor. A raw-milk sample (U.5 ml) is applied
`to the sample pad 32 by pipette in test device 50 or dripping
`the open end 16 of housing 12 in test device 10, and the test
`strip 28 is incubated at 55° C. After 8 minutes, the test strip
`is removed from the incubator 46 and analysed. [l the
`capture (test) line 38 is darker or the same color as the
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`control line, the sample 40 is negative, and, if the capture
`line is lighter than the control line, the sample is positive.
`Test results are as follows:
`
`TABLE I
`
`l3eta—|actarn assay in milk using lateral flow test device.
`
`Number of assays
`
`sample
`
`result
`
`3-U
`10
`10
`10
`"ID
`"ID
`"ID
`"ID
`10
`10
`10
`It}
`10
`10
`10
`"ID
`
`ll)
`
`15
`
`zero control
`penicillin G at 5 ppb
`penicillin I3 at 4 ppb
`penicillin I1-Ir at 3 ppb
`nmpicillin at ti pph
`nmpicillin at 41- pph
`ampicillin at 3 ppb
`nmoxicillin at 6 ppb
`amoxiclliin at 4 ppb
`amoxicillin at 3 ppb
`ceftiofur at 30 ppb
`ccftiofur at 40 ppb
`celtiofttr at 50 ppb
`cephapirin at 12 ppb
`cephapirin at 15 ppb
`ccphzlpirilt at. 20 pph
`
`all negative
`all positive
`5 positive_. 5 negative
`3 positive_. 7 ne