United States Patent
`
`[191
`
`Eisinger et al.
`
`[111 Patent Number:
`
`4,943,522
`
`[45] _ Date of Patent:
`
`Jul. 24, 1990
`
`422/se
`4.361.537 n/1932 Deutsch etal.
`4.608.246
`3/1935 Bayeretal. ............. .. 424/1:
`4.623.461 11/was I-lossometal”
`422/101x
`4,593,334 9/1931 Hossometal.
`422/1o1x
`4,129,963
`3/1933 Avremeasetal
`435/7x
`
`422/ssx
`4,740,475
`4/1933 Paul
`
`
`
`FOREIGN PATENT DOCUMENTS
`
`1./I98? European Pat. Off.
`0209373
`8303650 5/1988 World Int. Prop. 0.
`
`.......... .. 436./513
`436/518
`
`Primary Exam:‘m2r—Esther M. Kepplinger
`Assistant Exam1'.v:er—Ca.rol A. Spiegel
`Attorney, Agent, or F1'rm—Ireil 8:. Manella
`
`[57]
`
`ABSTRACT
`
`A method and apparatus for conducting specific bind-
`ing pair assays, such as immunoassays. is described. A
`porous membrane capable of non-bibulous lateral flow
`is used as assay substrate; a member of the binding pair
`is affixed in an indicator zone defined in the substrate.
`The sample is applied at a position distant from the
`indicator zone and permitted to flow laterally through
`the zone; any analyte in the sample is complexed by the
`affixed specific binding member, and detected. A novel
`method of detection employs entrapment of observable
`particle in the complex. Blood is a particularly pre-
`ferred sample as the red blood cells can be used as the
`observable particles for detection of the complex.
`
`[54]
`
`[75]
`
`[73]
`
`[21]
`
`[22]
`
`I63]
`
`[51]
`[52]
`
`£53]
`
`£56]
`
`LATERAL FLOW, NON-BIBULOUS
`MEMBRANE ASSAY PROTOCOLS
`
`Inventors: Robert W. Eisinger; Mohauuned H.
`Khalil, both of San Diego; David H.
`Katz, La Jolla; Robert B. Sergeant,
`Ramona, all of Calif.
`
`Assignee: Quidel, San Diego, Calif.
`
`Appl. No.: 230,642
`
`Filed:
`
`Aug. 10, 1988
`
`Related US. Application Data
`Continuation-i.n-part of Ser. No. 51.273, Jun. 1, 1987,
`abandoned. and at continuation-in-part of Ser. No.
`57,271, Jun. I, I937, abandoned.
`
`Int. CL5 ........................................... .. GOIN 33/53
`
`U.S. CL
`435/7; 435/805;
`435/310; 436/512; 436/514; 436/518; 436/520;
`436/523; 43 6/S3 1; 436/5 35; 43 6/307; 436/808;
`436/310; 422/55; 422/56; 422/57; 422/53;
`422/ 101
`Field of Search .................................. .. 422/55-61,
`422/70, 101, 102; 424/ll; 435/7. 5. 805, 310;
`436/514-520, 512, 523, 531, 535, 30?, 808, 810;
`210/431
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`4,323,536 -1-/1982 Columbus
`
`422./56
`
`46 Claims, 4 Drawing Sheets
`
`
`
`ALERE EX. 1009
`ALERE EX. 1009
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`U.S. Patent
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`Jul. 24, 1990
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`Sheet 1 of4
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`4,943,522
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`U.S. Patent
`
`Jul. 24, 1990
`
`Sheet 2 of4
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`4,943,522
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`U.S. Patent
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`Jul. 24, 1990
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`Sheet 3 of4
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`US. Patent
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`Jul. 24, 1990
`
`Sheet 4 of4
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`4,943,522
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`1
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`4,943,522
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`LATERAL FLOW, NON-BIBULOUS MEMBRANE
`ASSAY PROTOCOLS
`
`This is a continuation-in-part of copending applica-
`tion Ser. Nos. 057,273 and 057,271, both filed June 1,
`1987, and now abandoned.
`
`FIELD OF THE INVENTION
`
`This invention relates to immunological and related
`assay methods and apparatus, especially to those for
`blood testing.
`
`BACKGROUND OF THE INVENTION
`
`The technology of specific binding diagnostic reac-
`tions and reagents generally has developed rapidly in
`the past two decades, and continues to develop at a
`rapid pace. Radiommunoassays (RIA.s) and enzyme-
`linked immunosorbent assays (ELISAs), for example,
`have become widely known and are described in nu-
`merous tests,
`treatises, scientific papers and patents.
`ELISAs have become commonplace and achieved
`great importance in medicine and in scientific research
`since the pioneering work begun by Engvall, E., and
`Perlmann, P., et al, Immrmochem (1971) 3:87 I-874; and
`the work of Schuurs and coworkers; see, e.g., Van Wee-
`men, FEBS Letters (1971) 15232-236, and several U.S.
`Patents naming Schuurs et al as inventors; see, e.g., U.S.
`Pat. Nos. Re. 31,006, 3,654,090, 3,839,153, 3,850,752,
`3,862,302, 3,862,923, 3,879,262, and 4,016,043. Mono-
`clonal antibodies in enzyme immunoassays are well
`known; see, e.g., the work of Herzenb-erg and of Eng-
`vall, and others and the later work of David et al, U.S.
`Pat. No. 4,376,110. DNA probes and biochemical and
`biological probes generally exhibit the ability to bind
`specifically and are of great current interest as specific
`binding pairs.
`Ishikawa, M. D.,
`ENZIUUE IMMUNOASSAY.
`Tadashi, Kawai, and Kiyoslu‘, Miyai, eds, IGAKU-
`SHOIN, New York 1981, describes in considerable
`fundamental detail the principles and practices involved
`in enzyme immunoassays. Reference is also made to
`other texts and treatises in the field, such as IMMUNO-
`CHEMICAL METHODS IN THE BIOLOGICAL
`SCIENCES: ENZ YMES AND PROTEINS, Mayer, R.
`1., and Walker, J. H., Academic Press, New York 1980;
`QUANTITATIVE ENZYME IMl'|r.fUNOASSA}’. Eng-
`vall, E., and Peace, A. J., Blackwell Scientific Publica-
`tions, London (Scandinavian Journal of Immunology,
`1978}; THE ENZYME LINKED IMMUNOSORBENT 50
`ASSAY (ELISA) A guide with abstracts of micropicte
`applicnnons, Voller, A., Bidwell, D. E., and Bartlett, A.,
`Dynatech Laboratories, Inc., 1979; and the references
`cited therein for a comprehensive disclosure of the
`principles and usual practices involved in enzyme im-
`munoassay.
`Various approaches have been described for carrying
`out enzyme immunoassays. The early ELISAs were
`what
`is commonly called a “competitive” assay in
`which the enzyme labeled antigen or antibody com-
`peted with the antigen or antibody to be determined for
`a reaction site on a bead, pad or surface to which one
`member of an immunologically coupling pair was at-
`tached. Later, the “sandwic " assay became popular. In
`the sandwich assay, the antibody or antigen to be deter-
`mined was “sandwiched” by an immunochemical reac-
`tion between a solid surface treated with an immunolog-
`ical species reactive with the species to be determined
`
`45
`
`55
`
`2
`and the same or a different reactive immunological
`species which has been coupled to an enzyme label. The
`principles of these types of ELISAS are discussed by
`Belanger, L., Scand J’ Immunol,
`(1978) 8:Suppl. 7,
`33-41; (Chapter 4 in QUANTITA TIVE ENZYME IM-
`MUNOASSA 1’. supra).
`Many forms of solid supports to which one member
`of an immunochemical couple, e.g., antigen-antibody or
`hapten-antibody couple, have been disclosed. A com-
`mon early form of solid support was a plate, tube or
`bead of polystyrene which was well-known from radio-
`immunoassay (KIA) work to bind certain immunologi-
`cal species. Filter paper, glass, various plastics (chemi-
`cal polymers), and other solid support surfaces have
`been used for many years. Examples of such a system
`which used antibody (or antigen) coated polystyrene
`beads are described by Bohn et al, in U.S. Pat. No.
`4,424,279, Jan. 3, 1984; and U.S. Pat. No. 4,458,020, July
`3, 1984, in which the coated beads are utilized in unique
`configurations.
`Several disclosures are directed to assays which em-
`ploy passage of the sample to be tested through a solid
`membrane or support.
`Tom et al, U.S. Pat. No. 4,366,241, disclose an appa-
`ratus for an immunoassay which includes a multiple
`layered construction in which the sample solution flows
`into an enclosure through an immunoabsorbing disk
`which has antibody or antigen bound to it. The solution
`flows from the disk through a membrane spacer which
`is in contact with the disk and into a bibulous strip of
`cellulose or paper which extends through the enclosure
`to a level above the sample into which the apparatus is
`inserted during use.
`U.S. Pat. No. 4,632,901, to Valkirs et al, discloses a
`device and method for immunoassays in which the
`sample flows through the thickness of a membrane to an
`absorbent mass. Antibody is bound to less than the total
`surface of the membrane and binds antigen in the anti-
`body coated area. Conventional ELISA techniques are
`used to detect the sample bound to the supported anti-
`body.
`Various configurations for self-contained assay sys-
`tems have also been described; for example, Deutsch,
`A., and Platt, H. A., U.S. Pat. No. 4,522,923, describe a
`device which comprises a container, at least two water-
`soluble barriers, subdividing the container into at least
`three superimposed chambers, and different biologi-
`cally active substances in each chamber. Upon intro-
`duction of an aqueous biological sample to be tested
`into the topmost chamber,
`the sample successively
`mixes with the contents of the chambers, the contact
`time in each chamber being a function of the water
`solubility of the barriers. The system is designed to give
`a color reading in the final chamber. This provides a
`method for conducting immunochemical reactions in a
`self-contained sealed unit that requires only the addition
`of an unknown sample and water, and thus provides an
`assay system that is safe and accurate even when used
`by an individual who is not technically trained. Specifi-
`cally at least one chamber contains an antigen, antibody,
`or an enzyme, or their conjugates. Preferably the anti-
`body is directed against human chorionic gonadotropin
`hormone. Specifically the substances in the chambers
`represent color-change immunochernical reactions, e.g.,
`home testing of blood or urine for pregnancy.
`Barnett, B., WOB606488, describes a diagnostic test
`kit which has a central well for receiving a sample to be
`analyzed. Several
`reservoirs holding predetermined
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`quantifies of reagents are located in a block which sur-
`rounds the sample well and are connected to it via
`bores. Initially the reagents are retained in the respec-
`tive reservoirs by membranes but the contents of a res-
`ervoir can be discharged by rupturing the membrane.
`The reservoirs are formed by resilient domes which are
`depressed manually to rupture the membrane and serve
`to transfer the reagent to the sample well. The test kit is
`used for screening, chemical or clinical analysis of
`blood. urine, swimming pool water, drinking water or
`soil. The test kit reduces the chance of human error in
`the sequential addition of reagents to a sample.
`Graham, I-I. A., Olelma, D. J., Hawk. J. B., and Ke-
`bles, D. B., EPW922669, describe a test in which red
`blood cells are rapidly tested for the presence of anti-
`gens 0, C, c, E, e or K by mixing them with an antibody
`reagent (A) and, without incubation, examining them
`for agglutination. (A) comprises reduced S-alkylated
`IgG antibody against the appropriate antigen which at
`least meets FDA standards for potency and specificity.
`Deutsch. M. E., and Mead. L. W.. U.S. Pat. Nos.
`4,094,647, 4,235,601 and 4,361,537, describe a test strip
`for determining a characteristic of a sample comprises a
`length of material capable of transporting a developing
`liquid by capillarity and having zones between its ends
`for receiving the sample and holding reagents. The strip
`is used for performing binding assays, particularly those
`in which a radioisotope is used as a label, such as radio-
`irnmunoassays. Minute sample sizes may be used. The
`strip is capable of application to analytical methods
`having sensitivities below 0.1 mg/ml.
`Friedenberg. R. M., FR 2537724, describes a dry
`indicator apparatus for drugs-of-abuse testing of physio-
`logical liquid test solutions. A non-bibulous capillary
`flow membrane vehicle matrix is impregnated with dry
`chemical colorants. When these are placed in contact
`with the test solution the colored reagent indicat the
`type of drug present even in low concentrations. The
`test is a qualitative and quantitative indicator for the
`presence of abused drugs, such as barbiturates, amphet-
`amines, methadone, morphine, cocaine, codeine, di-
`laulid and tranquilizers of the diazepam type. The physi-
`ological
`fluids tested include urine, whole blood,
`plasma, sweat and tears.
`Lipp, V., and Buck, R. L., EP 0206779, describe an
`apparatus for detecting antinuclear antibody in a biolog-
`ical sample comprising a solid support having adhered
`nuclei isolated from eucaryotic cells. Preferably under-
`lying the nuclei on the solid support is a coating, e.g., of
`nuclear antigens, which is unreactive with antibodies to
`non-nuclear antigens and which. like nuclei serves to
`bind antinuclear antibodies in the sample. The apparatus
`permits the screening of human serum for the presence
`of antinuclear antibodies in a system featuring speed,
`simplicity, sensitivity and capacity for automation.
`Medical disorders characterized by the presence of
`antinuclear antibodies include systemic lupus ery'thema-
`tosus, mixed connective tissue disease. Sjogren’s syn-
`drome and scleroderma.
`Deutsch. 2\., Sheets, E. J., and Rhodes, 1., EP
`0139925, describe a kit which comprises (a) a vessel, (b)
`a capillary-active wick extending from the interior of
`the vessel so as to wick a liquid out of the interior of the
`vessel, a portion of the wick carrying an immobilized
`immunological component selected from (i) antigen and
`(ii) antibody, (c) a first reagent comprising an enzyme
`conjugated to an immunological component selected
`from (i) antibody and (ii) antigen specific to (i) or (ii)
`
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`respectively of (b) and (d) a substrate for the enzyme. In
`place of the enzyme and substrate a fluorescent label
`may be used. Antigens which can be tested for include
`dilantin, testosterone and progesterone. If the sample
`contains the antigen, it will combine with the antibody-
`enzyme while moving along the wick so that when this
`mixture subsequently wicks through the antigen-wick,
`there will be no free antibody-enzyme to bind to the
`antigen on the wick so it will pass out of the wick.
`Friedenberg, WPO Int. Pub. No. WO 84/02397, also
`describes an immunoassay in which the reactions occur
`in the liquid phase as the sample moves through a paper
`support.
`the rate of movement being one parameter
`used in identifying constituents.
`Campbell. U.S. Pat. No. 3,893,308, describes a strip of
`filter paper treated in bands with a chemical reagent,
`iodine, into which a sample of gasoline suspected of
`containing lead is wicked from one end and :1 develop-
`ing reagent, dithizone,
`is wicked into the pretreated
`bands.
`Alberty ct al, U.S. Pat. No. 3,395,914, describe an-
`other chemically treated test strip in which chemical
`reagents are applied in bands or zones on a strip for
`detecting barbituric acid.
`'
`While the prior art teaches the use of wiclring bitm-
`lous materials as carriers for specific binding reagents,
`these apparatus and methods rely principally upon the
`ability of the carrier to imbibe the liquid and often to
`enter into the reaction. The use of bibulous materials is
`of great value in some methods. but presents serious
`limitations as well, in reduced sensitivity and in the
`nature of the reagents and analytes which may be used
`or determined. The present invention utilizes a non-
`bibulous material in which the liquid flow is isotropic
`and flows laterally in the material by capillary action,
`thus presenting a system in which the solid membrane
`provides a vessel for the liquid but does not imbibe or
`otherwise enter into or interfere with the specific bind-
`ing reactions.
`
`DISCLOSURE OF THE INVENTION
`
`The invention provides a method and apparatus for
`determining the presence or absence or the amount of
`analyte using a specific binding assay. The apparatus
`comprises a non-bibulous lateral flow membrane which
`has on its surface a sample application zone to receive a
`liquid sample, and, at a lateral distance from the applica-
`tion zone on the surface, at least one indicator zone. In
`the indicator zone is affixed a member of a binding pair;
`the sample contains an analyte which is its complemen-
`tary binding member or an analyte which can be deriva-
`tized so as to bind the fixed member. In one convenient
`configuration the membrane is bound to two substan-
`tially fluid-irnpcrvious sheets, one on either side, with
`openings on one side or both sides to provide definition
`to the application and indicator zones.
`The lateral flow achieved in the method of the inven-
`tion is the result of the properties of the non-bibulous
`lateral flow membrane. The membrane has a much
`smaller thickness than surface dimension and is hydro-
`philic enough to be wetted and thus permit aqueous
`solutions and materials to exhibit lateral flow freely, and
`preferably isotropically, at substantially the same rates
`for various components of a sample.
`Thus, in one aspect, the invention is directed to an
`apparatus for assaying an analyte in a sample to be
`tested by a specific binding reaction which apparatus
`comprises a non-bibulous lateral flow membrane, said
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`which contain the specific binding member of the pair
`pertinent to an antigen prest in the sample. Thus, for
`example, type A blood will be visible in an indicator
`zone containing anti-A; blood lacking this antigen will
`be removed from this zone, which will appear clear.
`In other aspects, the invention relates to specific con-
`figurations of the application and indicator zones on the
`membrane.
`
`5
`membrane having at least one sample application zone
`and at least one indicator zone, said zones laterally
`separated, and wherein in the indicator zone is affixed a
`member of a binding pair.
`The apparatus may further comprise one or more
`breakable containers of buffers or reagents positioned in
`a holder adjacent the membrane. These can be broken
`by a means which may also be included in the holder, to
`provide needed developing reagents or wash solutions
`to the indicator zone when the container is broken.
`Another aspect ofthe invention comprises such designs.
`The membrane may include more than one indicator
`zone, along with control and reference zones. Multiple
`indicator zones may be designed to detect different
`anal)-‘tea or for quantifying the amount of analyte. Mul-
`tiple indicator zones may be in any spaced relationship
`to the application zone, since the membrane itself does
`not provide a barrier to sample flow.
`In the method of the invention, the sample, which
`contains an analyte which is, or which can be deriva-
`tized to include, a first member of a binding pair, is
`applied to the application zone and allowed to be trans-
`ported laterally through the membrane to an indicator
`zone, where there is, affixed to the membrane,
`the
`other, second, member of the binding pair. The first
`member binds,
`in the indicator zone,
`to the second
`member, and the resulting bound complex is detected.
`Detection may use any of a variety of labels and/or
`markers, e.g., enzymes, radioisotopes, liposomes, fluo-
`rescent tags, polymer dyes, or colored particles, etc.,
`and detection is by means of, for example, direct visual
`observation, by developing a color, by radioactive iso-
`tope counting, by fluorescence measurement, or by any
`of many other techniques by which the presence or
`absence of a chemical or biochemical species may be
`detected directly or indirectly.
`In one preferred embodiment, and an additional as-
`pect of the invention, visible particles in, added to, or
`applied before or after, the sample are used for detec-
`tion by being trapped in the indicator zone by the bind-
`ing pair complex. If the analyte itself provides a visible
`particle, for example, in the case of analyte being an
`antigen present on red blood cells, which cells can be
`seen directly, no separate detection means is needed. If
`the visible particles reside in the sample, e.g., the red
`blood cells in whole blood will remain in the indicator
`zone after washing when the binding complex is
`formed.
`Thus, in another aspect, the invention is directed to a
`method to detect the formation of a complex between a
`first binding reaction pair member from a sample and
`the second member of the pair affixed in an indicator
`zone, which method comprises supplying, along with or
`after the sample, detectable particulates which are en-
`trapped by the complex and thus detected in the indica-
`tor zone. The particles may occur naturally in the sam-
`ple as is the case for the red blood cells in whole blood.
`In other instances, the particles may be added artifi-
`cially.
`the invention is directed to a
`In another aspect,
`method of blood typing using the membrane described.
`A blood sample is applied to the application zone and
`permitted to flow through the membrane to one or
`more indicator zones, each of which contains a blood
`typing reagent, such as an antibody to group A antigen,
`to group B antigen or to R}: factor. The blood sample is
`then washed through the membrane so that the red
`blood cells remain visible only in the indicator zones
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is a top plan view of the holder in a preferred
`embodiment of the invention.
`FIG. 2 is an exploded perspective view of the holder
`of FIG. 1 showing the components thereof and the
`relationship of the membrane and absorbent compo-
`nents contained herein.
`FIG. 3 is a side cross—sectional view of the apparatus
`of FIG. 1, take along lines 3-3 of FIG. 1 in the direc-
`tion of the arrows.
`
`FIG. 4 is an exploded view of another preferred em-
`bodiment of the apparatus of this invention which in-
`cludes reagents as well as the membrane and absorbent
`body.
`FIG. 5 is a top plan view of the apparatus of FIG. 4-.
`FIG. 6 is a side elevational view of the apparatus of
`FIG. 5.
`FIG. '7 is a side cross-sectional view of the apparatus
`of FIG. 5 taken along lines 7-? of FIG. 5 in the direc-
`tion of the arrows.
`FIG. 8 is a transverse cross-sectional View of the
`apparatus of FIG. 5 taken along lines 8-8 of FIG. 8 hi
`the direction of the arrows showing the breaking mech-
`anism of the invention.
`FIG. 9 is a plan view of an alternative, simplified
`form of an apparatus suitable for use in this invention,
`especially for blood typing.
`FIG. 10 is a cross-sectional view of the apparatus of
`FIG. 9.
`
`MODES OF CARRYING OUT THE INVENTION
`
`General Description
`An essential feature of the invention is the employ-
`ment of a membrane capable of non-bibulous lateral
`flow. By "non-bibulous” lateral flow is meant liquid
`flow in which all of the dissolved or dispersed compo-
`nents of the liquid are carried at substantially equal rates
`and with relatively unimpaired flow laterally through
`the membrane, as opposed to preferential retention of
`one or more components as would occur, for example,
`in materials capable of adsorbing or "imbibing" one or
`more components. “Bibulous" materials include paper,
`nitrocellulose, nylon and the like, which have the caps-
`bility to effect a chromatographic separation of the
`contained materials.
`An example of the membrane material in which capil-
`lary, non-bibulous lateral flow occurs is the high density
`or ultra high molecular weight polyethylene sheet ma-
`terial manufactured by Porex Technologies Corp. of
`Fairburn, Georgia, USA. This membrane has an open
`pore structure with a typical density, at 40% void vol-
`ume, of 0.57 gm/cc and an average pore diameter of 1
`to 250 micrometers, the average generally being from 3
`to 100 micrometers. The optimum pore diameter for the
`membrane for use in the invention is about 10 to about
`
`50 pm. The membranes are from a few mils (0.001 in) to
`several mils in thickness, typically in the range of from
`5 or 10 mils and up to 200 mils. The membrane may be
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`7
`backed with a generally water impervious layer, or may
`be totally free standing. While membranes made of
`polyethylene have been found to be highly satisfactory,
`lateral flow, non-bibulous membranes formed of other
`olefin or other thennoplastic materials, e.g., polyvinyl
`chloride, polyvinyl acetate, copolymers of vinyl acetate
`and vinyl chloride, polyamide, polycarbonate, polysty-
`rene, etc., can be used. Membranes formed by the classi-
`cal phase inversion process may also be used.
`Thus, the membranes, in general, will have a pore size
`of about 3-100 pin, preferably about l0—50 um; will be
`constructed of an inert material; and will be less than
`20.’) mils in thickness. They are characterized by non-
`bibulous lateral flow. Isotropic flow is preferred. While
`applicants believe this lateral flow to be caused, at least
`in part, by capillary action, they are not bound by any
`particular theory to explain the characteristic nature of
`this non-chromatographic flow.
`In the various apparatus and method embodiments of
`the invtion, the lateral flow non-bibulous membrane
`will contain an application zone and at least one indica-
`tor zone, wherein the indicator zone has affixed to it a
`member of a binding pair. The membrane may be in any
`desired shape and the application and indicator zone
`may have any desired configuration as is convenient for
`use alone or in a particular apparatus.
`To the indicator zone in the apparatus and method of
`the invention is aifixed one of the members of a binding
`pair, which is responsible for the capture of its comple-
`mentary member. By “mixed” in this context is meant 30
`retained in the indicator zone throughout the assay
`procedure—this can be accomplished by covalent
`bonding or, more commonly, by adsorption, e.g., by
`drying. Depending on the nature of the material com-
`prising the membrane, derivatization to permit covalent
`bonding for example, using glutaraldehyde or a carbo-
`cliimide, can be employed.
`Most binding pairs employed in the invention are
`"specific", e.g., antigen-antibody pairs. and other spe-
`cific coupling pairs such as antibody-hapten, antibody— -I-0
`cell, antibody-cell fragment, RNA and DNA probes,
`receptor-receptor
`ligand, enzyme-substrate, enzyme-
`inhibitor and other pairs in which a specific binding
`reaction occurs. However, in some instances the speci-
`ficity of the assay may be conferred in other ways, such
`as by a labeling reagent. The requirement for the affixed
`member is that it must bind the analyte or its derivative.
`Thus, one of the members of the coupling pair is affixed
`physically, chemically, biologically or otherwise to the
`non-bibulous lateral flow membrane indicator zone to
`bind the other member of the pair.
`The affixed member of the pair may bind directly to
`the analytc, or may bind to a derivative thereof. By
`“derivative" is meant any substance whose concentra-
`tion in the sample is directly proportional to analyze.
`For example, the derivative may be a conjugate of the
`analyte with an additional component which, in turn,
`binds to the affixed member, or with an additional com-
`ponent which serves merely to label the analyte, but not
`interfere with the analyte’s ability to bind to the affixed
`member. In another illustration, the derivative might be
`a reaction product formed in stoichiometric relationship
`to analyte in a reaction, wherein the reaction product
`binds to the affixed member. Thus, "derivative" is a
`substance quantitatively related to analyte concentra-
`tion.
`
`brane. The binding pair member may be attached to
`another material wherein said material
`is physically
`entrapped in the indicator zone or otherwise affixed in
`the indicator zone by any physical, chemical or bio-
`chemical means. For example, specific binding mem-
`bers can be attached covalently or passively to beads or
`the like and the beads then affixed on the membrane.
`The method of the invention is conducted by apply-
`ing a liquid sample to the application zone at the surface
`of the membrane in sullicient quantity to pennit the
`sample to flow through at least one, or through at least
`as many indicator zones as desired. Control and blank
`zones may also be defined to receive the sample flow.
`Flow will occur laterally through the membrane due to
`its intrinsic properties; while applicants believe this is
`due to capillary action, there is no intent to be bound to
`any particular theory or explanation. If, along the direc-
`tion of flow, the mhrane terminates at the indicator
`zone, the liquid may flow out of the membrane; if there
`is additional surface beyond the indicator zone in the
`direction of flow, this surface will act as an “absorbent"
`zone, and further encourage the flow of liquid. A bibu-
`low: or nonbibulous material may also be placed in
`contact with the membrane to act as additional absor-
`bent.
`
`In the method, then, the sample flows from the appli-
`cation zone through the indicator zone and, if applica-
`ble, into the absorbent zone. The presence of analyte in
`the sample will cause a detectable reaction in the indica-
`tor zone.
`
`The experimental design protocol or "chemistry” of
`the assays of the invention can be varied as is generally
`known for assays based on specific binding. Most of the
`protocols adapted to other physical formats ofimmuno—
`assay can be used in the apparatus of the invention,
`where the indicator zone fills the role of the binding
`member bound to solid support. For example:
`(1) Analyte in the sample binds specifically to the
`member affixed to the indicator zone; a label is then
`added to detect the bound analyte; i.e., in a sandwich
`immunoassay;
`(2) The sample is spiked with a labeled form of ana-
`lyte and the labeled form bound to the member affixed
`in the indicator zone is detected—i.e., a competition
`3553?;
`(3) Analyte carries with it the means of its own detec-
`tion—the most notable example being analyte conju-
`gated to an observable particle. Also, the analyte may
`first be reacted with label, for example, with an anti-
`body conjugated to enzyme. The label-bearing analyte
`can then be bound specifically to the affixed member. In
`an alternative, 9. labeled analyte may be bound to a
`specific counterpart which is the complement to the
`fixed member. For example, red blood cell-bome ana-
`lyte may be reacted with murine anti-antigen to fonn a
`complex, which is then bound to rabbit antirnurine lg
`affixed in the indicator zone;
`(4) Detectable particles may be used to detect an
`unlabeled complex of analyte with binding pair member
`in the indicator zone.
`
`Typical Analytes
`The analyte may be insoluble or attached to insoluble
`supports or may be soluble.
`Typical cell-bound or solid supported analytes in-
`clude, e.g.,
`Tissue-Specific Cell Surface Markers: Separation of
`cell populations based on these markers has been per-
`
`9 of 23
`90f 23
`
`45
`
`50
`
`55
`
`65
`
`It is not necessary that the binding pair member be
`bound directly, chemically or biologically to the mem-
`
`

`
`9
`»
`formed using lectins (Reisner and Sharon, Trends in
`Biochem Sci (TIBS) 29, 1930), blood leukocyte surface
`glycoproteins (Gahmberg and Anderssen, NYAS (1973)
`312, in Fibroblast Surface Proteins eds. Vahery, Ruslahti
`and Mosher), estrogen steroid receptors (Thompson,
`Cancer Treatment Reports (1978) 63(2) 130, erythrocyte
`insulin receptors (Bhathena et al, Horn: Merab Res
`(1981) l3:l'l'9). or multiple markers as in the case of
`lymphocytes. Further separation of subpopulations is
`possible based on markers identified with specific cell
`functions as in the case of the T lymphocytes (Reinberg
`and Schlossman, N Eng J Med (1980) 3015:1153).
`Tissue-Shared Cell Surface Markers: Some cell sur-
`
`face markers are present on multiple cell types. An
`example of these are the Major Histocompatibility
`Complex Human Lymphocyte Antigen (I-ILA) system,
`LETS protein, p glycoprotein (Kartner et al, Science
`(1983) 22l:1285) and transferrin receptors (Omary et al,
`Nature (London) (1980) 286:888).
`Viral-Associated Cell Surface Markers: Cell mem-
`brane antigens can also result from viral infection. The
`mumps H—N glycoprotein detectable by RIA, immu-
`nofluorescence and hemagglutination inhibition repre-
`sents a viral marker on infected cells (Sever et al, Infect
`dz Immnn (1982) 35(l):l79). Similarly, markers resulting
`from Herpes Simplex 1 and 2 infection are recognizable
`on the host cell
`surface by immunofluorescence
`(Stewart and Herrmann, “Herpes Simplex Virtls” in
`Manual qf'C1inicaI Immunology, 2nd edition, edited by
`N. R. Rose and H. Friedman, American Society for
`Microbiology, Washington. D.C., I980).
`Tumor-Specific Cell Surface Markers: Neoplastic
`and oncogenic transformation results in the alteration of
`the cell phenotype as expressed in cell surface proteins.
`These can be observed as variations in the presence of
`cell surface antigens normally expressed on the cell
`surface, appearance of "altered self antigens," appear-
`ance of embryonic cell surface antigens and the pres-
`ence of tumor specific molecules. Felsted et al (Cane
`Res (1983) 432754) have described cell membrane
`changes during the differentiation of promyelocytic
`leukemia cells. Neoplastic transformation induced
`changes in cell phenotype are p