US00579S273A
`5,798,273
`[11] Patent Number:
`Umted States Patent
`Shuler et al.
`[45] Date of Patent:
`Aug. 25, 1998
`
`
`[19]
`
`[54] DIRECT READ LATERAL FLOW ASSAY FOR
`SMALL ANALYTES
`
`4361514. 501.
`[58] Field of Search
`435/507. SIG. 512. 5131435115. 7.93
`
`[75]
`
`Inventors: John K. Shuler. Baltimore: Stephen J.
`Loveli. Towson. both of Md.: Abigail
`5. Fisher. Belmont: Alan J. Weiss.
`Acton. both of Mass.: Robert ‘W.
`Rosens1e'Ln.El]icolt City. Md.
`
`Assignees:
`
`])ic-king)” and Co]nPany_
`Fmnk-un 1_'ak°5- N-1'? Nfiml’°“’-‘
`C°"P°““°"' B'3df°rd* M355’
`
`21
`
`.
`1 App’ ”°
`1
`[22] Filed:
`
`.: '7 9
`1 ’m
`Sep. 25, 1996
`
`151]
`
`6
`[Ill CL --------------------- G01N 331558: GOIN 331566:
`GDIN 33.1564; GOIN 331563
`4361514: 4361501: 4361507:
`[52] U3. CI.
`4-36:'51{}: 436115 12: 436513: 435:’? .5; 435I'.".93
`
`[551
`
`References Cited
`ETENT DOC
`"UM
`
`ENT
`
`S
`
`U'S' P
`435:0‘
`4.11988 Wang et 21.
`4.740.468
`435n.9
`3:199: Ullman et al.
`5.131.303
`422156
`5229.073 W1993 Luo et al.
`.............................. 436514
`l.||'19’9-1
`Prinsary Ebramf.-ur—Lynette F. Smith
`Assistant Examiner—Brett Nelson
`Axioms}: Agent, or F'£rm—Bruee S. Weintraub
`[57]
`ABSTRACT
`
`
`
`_
`_
`_
`The present mventlon relates to a method and assay device
`for detecting small analytes. The results of the assay can be
`directly read from the device. which is a lateral flow device.
`
`32 Claims, 3 Drawing Sheets
`
`ALERE EX. 1010
`ALERE EX. 1010
`
`1 of 8
`1of8
`
`

`
`U.S. Patent
`
`Aug. 25, 1993
`
`Sheet 1 of 3
`
`5,798,273
`
`16
`
`C‘)
`
`::1._..
`
`12
`
`‘T’
`
`(J
`I-:1
`EL:
`
`2 of 8
`2of8
`
`

`
`3U
`
`m
`
`M
`
`3
`
`93:81¢:QTE5,323:
`
`BDIUI
`
`3O00OommMmm
`
`
`
`73WE5424mt,._«z<m7.,E55,:EaomvE5424.5%mv5EaomozqmaE3mD24oAE5424o
`
`3 of 8
`
`

`
`S.U
`
`tnm
`
`A
`
`%w5.,
`
`Mbam
`
`Pwlwi
`
`%
`
`.m
`
`3
`
`5
`
`3M35,
`
`dx_n_mIov
`
`
`
`masE%:oo._8mm,m;_m.oom.ouM7.,o
`
`4 of 8
`
`2:
`
`8
`
`on
`
`m5._§dxa
`
`

`
`5.793.273
`
`1
`DIRECT READ LATERAL FLOW ASSAY FOR
`SMALL ANALYTES
`
`FIELD OF THE INVENTION
`
`The present invention relates to a novel lateral flow assay
`and method for detecting small analytes. Results can be
`directly read from the assay. Small analytes for medical
`diagnostics can be detected by utilization of the device and
`method of the present invention.
`
`BACKGROUND OF THE INVEN1"ION
`
`Although there are many irnmunoassays which exist for
`detection of small analytes. currently existing products
`which are commercially available yield “typical” competi-
`tive inhibition results. meaning. reduction of signal with
`increasing analyte concentration. However.
`the present
`assay. by the method and device to be described herein.
`yields increased signal with increasing analyte concentra-
`tion.
`
`Furthermore. presently existing products which are com-
`mercially available incorporate a read-out zone which
`requires the user to compare the result to a color chart. The
`present
`invention describes an assay which provides a
`multiple read-out: additional line(s) appear at discrete ana-
`lyte concentrations.
`The present invention Pftivides an assay and method
`which is capable of providing a direct reading of the results
`of a competitive inhibition assay for small analytes.
`
`SUMMARY OF TEE INVENTION
`
`The present invention relates to a latuai flow assay and
`method for detecting small analytes. In particular. this assay
`is typically a competitive inhibition assay. The results of this
`assay can be read directly from the assay device. The device
`is contemplated to be used to detect small analytes useful in
`various types of medical diagnostic tests.
`
`35
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`FIG. 1 is a schematic representation of a device of the
`present invention.
`FIG. 2 is a schematic representation of another embodi-
`ment of the device of the present invention as set fonh in
`FIG. 1.
`
`45
`
`FIGS. 3a—3e are a schematic representation of the results
`of a direct read lateral flow assay for PCB with:
`a) {J ppm analyze; 13} >0 and <5 ppm analyte: c) 5 ppm
`analyte; d} >5 and <50 ppm analyte; and e) 50 ppm
`analyte.
`FIG. 4 is a graphic representation of the instrumented
`read-out line 1 in pixel intensity vs. Log IPCBI.
`DI-3'I'Al1.ED DESCRIPTION OF THE
`INVENTION
`
`55
`
`The present invention is directed to a method and device
`for detecting small analytes. In a preferred embodiment of
`the present invention. a sample (i.e.. an extract or solution)
`suspected of containing specific analyte is added to a prepa-
`ration of anti-analyte antibody which. with analyte consti-
`tutes a specific binding pair. To this mixture is added a
`reagent containing a tracer such as colored particles which
`are coated with analyte or analyte analog. Analyte or analyte
`analog may be anachecl to colored particles directly or
`through a carrier molecule. The colored particles will also
`
`2
`contain an additional label which is one member of a second
`specific binding pair. This label may. for example. be biotin.
`The mixture of sample. anti-analyte antibody solution and
`reagent containing colored particles is applied to a lateral
`flow device containing a solid support {such as for exarnple.
`a nitrocellulose membrane) which contains three specific
`areas:
`
`1. A sample addition area:
`2. A capture area containing analyte or analyte analog
`immobilized onto the capture area:
`3. A read-out area which contains one or more zones. each
`zone in the read-out area contairting one or more of the
`following: immobilized anti-analyte antibody. immo-
`bilized complementary binding partner to the label on
`the colored particle (e.g.. antibiotic or avidin). and
`immobilized analyte or analyte analog.
`In the case where a sample does not contain specific
`aualyte. a fraction of the antibody binds to the tracer such as
`colored particles which contain analyte or analyte analog.
`Only a small population of particles migrate past the capture
`area. The minimum number of colored particles would be
`available for travel to and binding to materials in the zonetjs)
`of the read-out area.
`
`In the case where sample contains a specific analyte to be
`deterrnined in the present assay. some antibody would bind
`to analyte and less would be available for binding to the
`analyte or analyte analog containing colored particles. A
`larger population of colored particles migrate past the cap-
`ture area to bind to one or more of the zones in the read-out
`area. As the sample contains increasingly larger amounts of
`analyte. greater amounts of unbound anatyte particles are
`free to bind to more zone(s) in the read-out area resulting in
`a stronger signal or the appearance of additional lines or
`symbols on the assay device. Results can thus be determined
`in both instrumented and most significantly. non-
`instrurnented fashion.
`The assay system of the present invention can detect small
`aualytes for medical diagnostics such as nutrients
`(vitamins). hormones such as estrogen and progesterone.
`drugs of abuse. and peptides. as well as small analytes of
`envitolamental and agricultural interest such as PCB and
`aflatoxin. Other small analytes of interest can include. but
`are not li.1'nited to. trace metals and poisons such as for
`example. household toxins and therapeutic drugs.
`A preferred ernbodirnent of the device of the present
`invention is set forth in FIG. 1. A solid support 1. which can
`be. for example. a nitrocellulose membrane. has a sample
`addition area 8; a capture area 2 having analyse or analyte
`analog immobilized thereon: and a read-out area 3. which
`contains.
`in this embodiment.
`three woes. However.
`it
`should be understood that this read-out area according to the
`present invention. contains at least one or more zones to
`provide the desired results and can contain more than three
`zones or less than three zones if so desired. In the embodi-
`ment set forth in FIG. 1. none 4 is a control zone having an
`irrelevant anti-analyte antibody immobilized thereon
`wherein this is anti—analy'te antibody to a second irrelevant
`analyte which is dilferent
`than the first analyte to be
`determined. and wherein this irrelevant analyte is attached to
`a tracer which can be. for example. a colored particle. The
`irrelevant analyte and tracer are added to the solutionfsample
`to be applied to the present device prior to application of that
`solutionfsaruple mixture to area 8. 'I1:te other zones 5 and 6
`in the read-out area will have immobilized thereon comple-
`mentary binding partner to the label on the colored particle.
`This complementary binding partner may. for example. be
`neutravidin. The area 7 indicates the distal end of the solid
`
`5 of 8
`5of8
`
`

`
`5.798.273
`
`3
`support (or strip of. for example. nitrocelltilose membrane)
`where the assay will come to an end.
`FIG. 2 sets forth another embodiment of the device as
`shown in FIG. 1. In this ernbodirnent. the solid support or
`strip 1 of FIG. 1 is inserted into a housing (such as. for
`example. a plastic housing) 10. The housing 10 has a frame
`having a hole 11 located at the front end of the device. and
`a read—out ‘"window‘' 12 which encompasses a section of the
`read-out area 3 and zones 4. 5 and 6 shown in FIG. 1. The
`frame of the housing 10 also has a rectangular “window” 16
`at the distal end of the plastic housing covering the solid
`support which will be used for viewing the end of the assay.
`A different tracer or marker can be added to the sample to
`enable the user to read the end of the assay by viewing this
`tracer or marker in the window 16. In the embodiment of the
`device set forth in FIG. 2. a section of the sample addition
`area can be seen through the hole 11 and a section of the
`read-out area can be seen through the window 12. and will
`show zones 4. 5 and 6. Howeva: the capture area 2 is not
`visible to the naked eye. It is under the housing 10 between
`the hole 11 and the window 12.
`The solid support which is employed in the assay is
`generally a cellulose ester. with nitrocellulose giving excep-
`tionally good results. It should be understood that the turn
`“nit:rocel.lulose" refers to nitric acid esters of cellulose.
`which may be nitrocellulose alone. or a mixed ester of nitric
`acid and other acids. in particular. aliphatic carboxylic acids
`having from one to seven carbon atoms. with acetic acid
`being preferred. Sud: solid supports which are formed from
`cellulose esterifted with nitric acid alone or a mixture of
`nitric acid and another acid such as acetic acid. are often
`referred to as nitrocellulose papa".
`Although nitrocellulose is a preferred material for pro-
`ducing the solid support. it is to be understood that other
`mattrials. having a surface area suflicient for supptrting a
`binder in a concentration as set forth above may also be
`employed for producing such solid supports including but
`not limited to nylon.
`The solid support employed in the assay is preferably in
`sheet form. with the substrate in drect fonn. generally being
`intheforrnofacard.atestst:ripord.ips1:ick.etic.Itistobe
`understood. however. that other forms are also within the
`spirit and scope of the invention.
`The tracer of the present invention can be. for example. a
`colored particle. A preferred colored particle is a sac. which
`includes a dye or other colored substance as a marlin’.
`whereby the tracer. when used in the assay. is visible without
`destruction of the sac to release the colored substance. ‘The
`sac may be any one of a wide variety of sacs. including. but
`not limited to intact crydttocytes. crylltrocyle ghosts. lipo-
`somes (single walled. sometimes called vesicles. or
`mtdtilainellar). polymer microcapsulcs. for example. those
`made by coacervation. or intra.far:ial polymerization. etc.
`Polymer microcapsules are produced by procedures
`known in the art except that the solution in which the
`microcapsules are forrned also includes a marker whereby
`the interior of me polymer microcapsule includes the
`marker. The preparation of such microcapsules is disclosed.
`for example.
`in Microencapruiation Processes and
`Applications, edited by Jan E. Vandegger (Plenum Press
`1904) which is hereby incorporated by reference.
`As known in the art. liposomes can be prepared from a
`wide variety of lipids. including phosplrolipids. glycolipids.
`steroids. relatively long chain allryl esters. for example.
`allryl phosphates. fatty acid esters. for example. lecithin.
`fatty amines and the like. A mixttne of fatty materials may
`be employed. such as a combination of neutral steroid. a
`
`15
`
`25
`
`4-5
`
`5.5
`
`4
`charged amphiphilc and a phospholipid. The examples of
`phospholipids include lecithin. sphingomyelin. dipalrnitoyl
`phosphatidylcholine. and the like. Steroids may include
`cholesterol. cholestanol. anesterol. and the like. The charged
`amphiphilic compounds may generally contain from twelve
`to thirty carbon atoms and may include mono- or dialkyl
`phosphate esters or an alttylamine. for example. dicetyl
`phosphate. stearyl amine. hexadecyl amine. dilauryl
`phosphate. and the like.
`The liposome sacs are prepared in an aqueous solution
`including the marker whereby the sacs include the marker in
`the interior thereof. The liposome saw are easily prepared by
`vigorous agitation in the solution. followed by removal of
`marker from the exterior of the sac. For the preparation of
`liposomes. see U.S. Pat. No. 4.342.826. PCT International
`Publication No. WO 80101515. U.S. Pat No. 4.539.376 and
`U.S. Pat. No. 4.522.803 which are hereby incorporated by
`reference.
`The tracer including the colored particle may also be
`produced by using an aqueous dispersion of a hydrophobic
`dye or pigment. or of polymer nuclei coated with such a dye
`or pigment. Such tracers are described in more detail in U.S.
`Pat. No. 4.373 .932. which is hereby incorporated by refer-
`ence.
`
`Examples of particles which may be used in the present
`invention include. but are not limited to. fe1'rit:in. phyco-
`ery‘th.rins or other phycobili~proteins. precipitated or
`insoluble metals or alloys. fungal. algal. or bacterial pig-
`ments or derivatives such as bacterial cltlorophylls. plant
`materials or derivatives and the like. The visible colored
`particles may be visible polymer particles. such as colored
`polystyrene particles of spherical shape (i.e.. beads).
`Thus the process of the present
`invention can be as
`follows. for an assay for a specific small analyte: anti-
`analyte antibody is Inixed with tracer such as colored
`particles containing analyte or analyte analog on their
`surface. as well as a second molecule {such as biotin) also
`on the surface and a sample containing analyte. During the
`formation of a complex between anti—analyte antibody and
`analyze or analyte analog on the particles. the more analyte
`present in the sample. the more colored particles are not
`associated with the complex (i.e.. this is the free Eraction).
`The mixture is then applied to a lateral flow device of the
`present invention. consisting of a solid support such as a
`strip of nitrocellulose on which is deposited at line of analyte
`in a capture area. and one or more zones in a read-out area.
`As the sample enters the support. the complex is too big to
`tlow. so it remains at the front end of the support. The
`particles I:hat are “free” flow up the strip. Any particles that
`have anti-analyte bound to them will get trapped by the line
`of analyte. 'I'he rermtinder of the particles can be trapped by
`the avidin (or neutravidin or strepavidin). The more “free”
`particles there are. the darker the avirlin line(s) become. If
`there are multiple avidin lines. one can also count lines as a
`measure of concentration of analyte in the sample. The
`analyte can. as stated above. be any small molecule or family
`of molecules. such as polychlorinated biphenyls {PCB}.
`drugs of abuse. steroid hormones. etc. Ifthere are analogs of
`analyte that bind to anti-analyte antibody at a lower aflinity
`than the analyte does. they can be placed on the particle
`surface to maximize the interaction with the analyte in the
`sample and the anti-analyte antibody. rather than the anti-
`body binding to the particles.
`If there is no analyte in the sample. all of the particles are
`bound to the complex. andlor get trapped by the line of
`analyte in the capture area on the solid support. If this line
`is hidden (i.e.. under the plastic housing as shown in FIG. 2}.
`
`6 of 8
`6of8
`
`

`
`5.793.273
`
`6
`(shown in FIG. 30). There is a second read—out of
`lesser intensity than the control line.
`iii. One control line plus two read-out lines (equal or
`greater than conn-ol line in intensity ,1: 50 ppm PCB
`(shown in FIG. 19). FIG. 3b shows results for
`between (J and 5 ppm analyte: and FIG. 3d shows
`results for between 5 and 50 ppm analyte.
`B. Single line visual read (this version would have a
`single read—out zone (line) within the read—otrt area)
`i. One control line visible: 0 ppm PCB
`ii. The intensity of the read—out line was compared to an
`intensity chart.
`C. Single line instrumented read (this version would have
`a single read—out zone (line) within the read-out area
`with or without a control line in the read-out area)
`I. The reflectance of the read-out line was determined
`with a reflectance spectrophotometer.
`The concentration of PCB (0. 0.5. 3. 5. 10. and 2.2 ppm)
`was read. The absorbance obtained from the Read-out line 1
`from each strip was examined with Sigma Scan Image. A
`pixel value was detennined for each strip and plotted versus
`the log of the PCB (ppm). The data are shown in the
`following Table I.
`
`TABLE 1
`
`
`
`Pixel Value
`PCB ppm
`16.3
`0.5
`50.4
`3
`48.7
`3
`54.3
`5
`63.8
`I
`10
`81.5
`1.342
`12
`
`
`1.34222 'l'8.7
`
`Log [PCB]
`-.30!
`.-677
`.471‘
`.699
`
`These data are plotted on the graph shown in FIG. 4. A
`linear regression of X=log (PCB) and Y=pixel value yielded
`r=0.99.
`What is claimed is:
`1. A process for determining the presence or amount of an
`analytic in a sample comprising:
`a} contacting said sample with a preparation containing
`i) anti-analyte antibodies. which bind to analyte or to
`both analyte and an analyte analog. and
`ii) an assay tracer. wherein said assay tracer comprises
`a detectable particrdate substance having i.mmobi-
`lized on its surface. analyte or analyte analog and a
`first member of a binding pair. to provide a sample!
`preparation mixture containing analytefanti-analytc
`antibody complexes andfor assay lracerfanti-analyte
`complexes:
`b} contacting said samplclpreparation mixture with a
`sample addition area of a lateral flow device. wherein
`said device comprises a solid support containing. in
`sequence.
`i) said sample addition area.
`ii) a capture area. wherein analyte or analyte analog is
`immobilized.
`to capture said analyter'anti—analyte
`antibody complexes andlor assay u'acere'anti-analyte
`complexes.
`least one zone.
`iii) a read-out area comprising at
`wherein a second member of said binding pair.
`which binds to said first member. is immobilized. to
`bind assay tracer not captured in said capture area. in
`proportion to the amount of analyte in said sample;
`c) allowing said sarnplefpreparation mixture to flow
`through said lateral flow device; and
`
`7 of 8
`7of8
`
`5
`
`one reads no signal in the read—out area and the zone(s)
`therein. As the analyte in the sample increases. less complex
`is formed. and more particles are “free“. causing a positive
`read. The advantage in using a binder such as biotin to etfect
`the trapping of the "free" particles is that it binds to a
`molecule such as avidin with an extremely high aflinity and
`that binding occurs very quickly. Since the analyte to be
`determined is. for this assay.
`intended to be a small
`molecule. it may be chemically attached to a carrier for
`attachment to the particle surface andfor the analyte line in
`the capture area on the solid support. The following
`Example is intended to be demonstrative and is not intended
`to in any way limit the present invention.
`
`EXAMPLES
`
`Example I
`
`REAGENTS
`
`Direct Read PCB Assay
`
`1. A conjugate of PCB analog and rabbit gamma globulin
`(Conjl) was prepared.
`2. The conjugate was coupled to carboxy blue latex particles
`by EDC (1-ethyl-3(3 dimetltylaminopropyl) u1rbodiin1-
`ide. The latex suspension was labeled with biotin by
`reaction with N-hydroxysuccinimidobiotin.
`3. Anti-PBC antibody was ptnified from serum by protein A
`agarose ohrornatography.
`4. A solution of purified antivhOG antibody was prepared for
`the control system.
`5. hCG was coupled to carboxy blue latex particles for the
`control system
`6. A 1% solution of erylhosin B was prepared for an
`end-of-assay dye indicator.
`PROCEDURE
`1. A strip of ninocellulose membrane 0.7x3.2 cm was cut.
`2. One line of Conjl was applied to the nitrocellulose
`membrane (capture area}.
`3. One line of anti-hCG antibody solution was applied to the
`nitrocellulose membrane (control zone within the read-
`o-ut area).
`. Two lines of Conjl mixed with neutravidin were applied
`to the nitrocellulose membrane (two zones of read-out
`area).
`5. One line of 1% etytllosin B solution was applied close to
`the distal end of the strip for end-of-assay indicator.
`. The strip was dried for 60 minutes at 45° C. and a foam
`pad was attached at the sample end.
`7. The strip was inserted into a housing with a hole located
`at
`the sample area. a window at
`the read—out area
`(including the control zone and two other zones). and a
`window at the distal end of the strip for reading the end-of
`assay marker.
`8. 20 uL of ethanol extract were pipetted into a tube.
`9. 50 1.tL of anti-PCB antibody in aqueous buffer were
`pipetted into the tube and mix.
`10. 10 |JL of a suspension of hCG latex particles and
`Conj 1-biotin latex particles were pipetted into the tube.
`mixed and incubated 5 minutes at room temperature.
`11. The entire suspens ion was added to the sample area of
`the strip (in the housing).
`12. After the endwof assay window filled with red color. the
`results were read as follows:
`
`35
`
`45
`
`55
`
`A. Multi-line visual read {shown in FIG. 3).
`i. One control line visible: 0 ppm PCB (shown in FIG.
`3a).
`ii. One control line plus one read-out line (equal or
`greater than control line in intensity): 5 ppm PCB
`
`65
`
`

`
`5.798.273
`
`7
`
`5
`
`d) detecting the presence or amount of assay tracer bound
`in said at least one zone of said read-out area as a
`measure of analyte in said sample.
`2. The process of claim 1. wherein said assay tracer is a
`colored particle.
`3. The process of claim 2. wherein said colored particle is
`a colored liposome or a colored polymeric bead.
`4. The process of claim 3. wherein said colored polymeric
`head is a colored polystyrene bead
`5. The process of claim 1. wherein said solid support
`comprises nitrocellulose.
`6. The process of claim 1. wherein said first member of
`said binding pair.
`on said assay tracer. is biotin.
`7. The process of claim 1. wherein said second member of
`said binding pair. immobilized in said read-out zone. is
`avidin. streptavidin. or neutravidin.
`8. The process of claim 1. wherein:
`a) said lateral flow device ftmher comprises a housing
`which covers the entire lateral flow device. wherein
`said housing has
`i) an opening over said sample addition area.
`ii) a first window for viewing said read-out area. and
`iii) a second window for viewing a distal end of said
`device: and
`
`b} said process further comprises contacting said sample!
`preparation mixture with said sample addition area by
`pouring said saruplcfpreparation mixture through said
`opening.
`9. The process of claim 8. wherein an endcf-assay tracer
`is added to said samplelpreparation mirtture for viewing
`through said second window.
`10. The process of claim 1. wherein:
`a) said lateral flow device further comprises a second zone
`in said read-out area. which functions as a control.
`wherein an ant:i-control-antigen antibody is immobi-
`lized; and
`
`35
`
`b) said process further comprises contacting a control
`tracer with said sample in step a). wherein said control
`tracer comprises a detectable particulate substance hav-
`ing
`on its surface a control antigen which
`binds to said anti-control-antigen antibody.
`11. The process of claim 10. whaein said assay tracer and
`said control tracer are colored particles.
`12. The process of claim 11. wherein said colored par-
`ticles are colored liposomes or colored polymeric beads.
`13. The process of claim 12. wherein said colored poly-
`maic beads are colored polystyrene beads.
`14. The process of claim 10. wherein said solid support
`comprises n.it.rocel.ltJlose.
`15. The process of claim 10. wherein said first member of
`said binding pair. immobilized on said assay tracer. is biotin.
`16. The process of claim 10. wherein said second member
`of said binding pair. immobilized in said read-out zone. is
`avidin. streptavidin. or neutravidin.
`17. The process of claim 10. wherein:
`a) said lateral flow device further comprises a housing
`which covers the entire lateral flow device. wherein
`said housing has
`i) an opening over said sample addition area.
`ii) a first window for viewing said read-out area. and
`iii) a second window for viewing a distal end of said
`device; and
`
`b) said process further comprises contacting said sample)‘
`preparation mixture with said sample addition area by
`pouring said samplelpreparation mixture through said
`opening.
`
`8
`18. The process of claim 1'1. wherein an end-of-assay
`tracer is added to said samplefpreparation mixture for view-
`ing through said second window.
`19. A kit for determining the presence or amount of an
`analyte in a sample. comprising:
`a) a preparation containing
`i) anti-analyte antibodies. which bind to analyte or to
`both analyte and an analyte analog. and
`ii) an assay tracer. wherein said assay tracer comprises
`a detectable particulate substance having irn.mobi-
`lized on its surface. analyte or analyte analog and a
`first member of a binding pair; and
`b) a lateral flow device. comprising a solid support
`containing. in sequence.
`i) said sample addition area.
`ii) a capture area. wherein analyte or an aualyte analog
`is irnrnobiiized. and
`iii) a read-out area comprising at least one zone.
`wherein a second member of said binding pair.
`which binds to said first member. is immobilized.
`20. The kit of claim 14. wherein said assay tracer is a
`colored particle.
`21. The lcit of claim 20. wherein said colored particle is a
`colored liposome or a colored polymeric head.
`22. The Irit of claim 21. wherein said colored polymeric
`head is a colored polystyrene bead.
`23. The kit of claim 19. wherein said solid support
`comprises nitrocellulose.
`24. The kit of claim 19. wherein said second rrtembu of
`said binding pair.
`in said read—out zone. is
`avidin. streptavidin. or neutravidin.
`25. The kit of claim 19. further comprising:
`a housing which covers the entire lateral flow device.
`wherein said housing has
`i) an opening over said sample addition area.
`ii) a first window for viewing said read-out area. and
`iii) a second window for viewing a distal end of said
`device.
`26. The kit of claim 19. further comprising:
`a) a second zone in said read—out area. which funrxions as
`a control. wherein an anti-control-antigen antibody is
`imraobilized; and
`
`b) a control tracer comprising a detectable particulate
`substance having
`on its surface a control
`antigen which binds to said a.nti-control-antigen anti-
`body.
`27. The kit of claim 26. wherein said assay tracer and said
`control tracer are colored particles.
`28. The kit of claim 27. wherein said colored particles are
`colored liposornes or colored polymeric beads.
`29. The kit of claim 28. wherein said colored polymeric
`beads are colored polystyrene beads.
`30. The kit of claim 26. wherein said solid support
`comprises nitrocellulose.
`31. The kit of claim 26. wherein said second member of
`said binding pair. immobilized in said read-out zone. is
`avidin. streptavidin. or neutravidin.
`32. The kit of claim 26. further comprising:
`a housing which covers the entire lateral flow device.
`wherein said housing has
`i) an opening over said sample addition area.
`ii) a firs! window for viewing said read-out area. and
`iii) a second window for viewing a distal end of said
`device.
`
`SS
`
`60
`
`65
`
`8 of 8
`8of8