CA1276877C - Multizone analytical element having detectable signal concentration zone - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE A multizone test device for the determination of analyte from a liquid test medium upon contact with the liquid test medium and a labeled reagent comprising a chemical group having a detectable chemical property. The test device preferably comprises multilayers including a reagent layer incorporated with an immobilized reagent and a detection layer incorporated with an immobilized form of an interactive detection reagent for the labeled reagent. The immobilized reagent in the reagent layer and the labeled reagent comprise specific binding patners which will bind to each other dependent upon the amount of analyte present. Labeled reagent which does not become bound to the immobilized reagent in the reagent layer migrates into the detection layer and interacts with the immobilized interactive detection reagent therein which results in the localized generation of a detectable reaction product which preferably is also immobilized in the detection zone. As a result, reverse migration of the labeled reagent, and preferably the detectable reaction product from the detection layer is prevented and the detectable chemical property provided by the label of the labeled reagent is localized in the detection layer for the precise measurement thereof and correlation to the amount of analyte in the test medium.

Description

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- MIJLTI ZONE ANALYTICAL ELEMENT HAVING

-BACKGROUND OF TH:E~ INVENTION
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Field of the Invention The present invention relates to multizone analytical elements which are useful for the determination of an analyte in a liquid test medium~ In particular, ~he present in~ention relates to multilayer immunoassay test devices involving.the use of labeled reagents comprising a chemical group that is detectable based on chemical ~eactivity with another substance to provide a detectable signal, such as fluorescence or color.

Descriptlon of the Prior Art Multizone analytical elements or test devices have been previously proposed and have been applied to binding assays, e.g., lmmunoassays, which depend upon the a~ility of an antibody or antigen to bind to a specific ar.alyte for the determination of the analyte in the liquid test medium. Such assays include those immunoassays where a labeled reagent, such as a labeled form of the analyte or an antibody thereto, par~icipates in an antigen-antibody reaction to form a free species and a : -, .- ~ .

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~%7~77 bound species thereof whereby the amount of the labeled reagent in one of such species can be correlated to the amount of analyte in the liquid test medium. In principle, such assays are refer-red to as heterogeneous immunoassays because the ree and bound species must be separated in order to complete the ass~y.
Multizone, particularly multilayer, analytical elements are now known in the art which inheren~ly perform the required separation step so that no additional manipulations are needed after applica-tion of the liquid test medium. In general, such devices include a plurality of layers having the necessary reagents for carrying out an immunoassay and for accomplishing the necessary separation step incorporated therein. A number of such devices ~- further include a detection layer from which the signal produced by a labeled reagent in either the bound or fre species is detected and measured~
, Detectable signals pro~ided by such devices are usua~ly optical in nature such as color changes, fluorescence, or the like~ Alternatively, detec-tion can be accomplished by electrochemical measure ments using, for example, potentiometric or am-2S pometric techniques.
For example, such multilayer immunoassay ~` ana~ytical elements are described by European Patent Publication No. 97,952 and German Publi-cation NoO DE-OS 3329728 where an immobilized form 3Q o a binding partner, such as an immobilized antibody to an antigen, and an antigen labeled with a detectable substance are incorporated therein.
Upon the application of a liquid test medium to such device, antigen from the test medium competes , :,.
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~t76~37~7 ~ 3 wîth labeled antigen incorporated into the device for binding to the immobilized antibody~ Separa-tion of the bound species from the free species occurs upon migration of the free species of the labeled antigen away from the immobilized zone.
Similarly, European Patent Publication Nos.
51,1~3 and 66,648 disclose such devices where the determination of antigen or antibody in a liquid test medium is dependent upon the competitive binding of the antigen (or antibody) with a labeled orm of the antigen (or antibody) for an immo-bilized form of a binding partner thereof, such as immobilized antibody (or antigen);
Other multilayer immunoassay test devices have also been proposed, such as described in UOS.
Patent No. 4,258,001, which include one or more ~; layers comprising particulate, three dimensional lattices formed by a plurali~y of organopolymeric particles. The particles form interconnected void spaces which a~e claimed to provide for the trans~
port of high molecular weight analytes there~
through. Although not required, it is suggested that interactive compositions, such as antigens or antibodies, can be immobilized onto the particles by providing active l~nking or binding sites on the particles to which such interactive compositions can ~e covalently bonded.
Another of such devices is described in U.S.
Patent No. 4,446,232 which is based on the prin-ciple of competition between bound and free speciesof analyte for a fixed number of recognition sites on an enzyme-labeled antibody. The determination of analyte in a test sample depends upon the bindins of the analyte to enzyme-labeled antibodies :

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~2t7Ei~77 in one zone of the device and which then pass into another zone o~ the device where the enzyme acti-vity of the enzyme-linked antibodies bound to analyte is detected. One of the zones further includes bound and immobilized analyte which competes with analyte from the test sample for binding to the enzyme-labeled antibodies and which bind and immobilize any of the snzyme-labeled antibodies which do not become bound to analyte from the test sample.
A particular disadvantage, however, of such devices is that reverse fluid migration results in reaction products, which have migrated into the lower or detection layer, to migrate back up into the upper layers, resulting in chemical inter-~` ferences and diminished test response. To overcome - this disadvantage, analytical test d~vices have been proposed which at.tempt to localize or other wise prevent such reverse fluid migration o tha reac~ion products.
For example, European Patent Publication Nos.
51,183 and 66,648 suggest layers for collection of the detectable reaction product compxising hydro-philic high molecular weight substances. EP 66,648 further suggests the incorporation o mordanting agents in the detection layer which have a strong interaction with the detectable reaction product in order to collect the detectable reaction product therein. Such mordanting agents include cationic polymers, anionic polymers and quaternary salts.
Similarly, U.S. Patent Nos. 4,144,306 and 4,042,335 disclose multilayer analytical elements which include a registration layer incorporated with ~ mordant for a detectable species in order to ~`' " ` ~.

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collect the detectable species therein and thereby prevent diffusion or migration of the detectable - species out of the registration layer.
A variation of such devices is disclosed by U.S~ Patent No. 4~459,358 which describes a multi-layer element comprising a spreading layer, a reaction layer incorporated with a diffusible label~d antibody, and a registration layer incor-porated with materials adapted to non~specifically bind, immobilize or "mordant" antibodies, such as latex particles. Upon application of a liquid test medium to the device, analyte from the test medium associates with the labeled antibody in the reac-tion layer and immunoprecipitates therein. Any o~
the labeled antibody which does not become bound to the analyte diffuses into the registration layer where it is immobilized by the mordant incorporated therein.
However, the use of mordanting agents can interfere with the pxerequisite reactions which are necessary for the formation or release of the detectable reaction product as a result of non- -specific binding of the mordanting agent. Such interference can make both detection and measure-` 25 ment unreliable, as well as decrease the sensiti-vity of the test device.
In attempts to overcome the disadvantages of mordanting agents in a registration layer, other analytical elements have been proposed employing 3a mordantiny agents in a layer other than a registration layer in order to prevent the migra-tion of a ~ormed detectable reaction product into a layer other than a registration or detection layer which would otherwise render the detectable reac-, .
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~2~ 77 tion product undetectable. Such a device is disclosed by U.S. Patent No. 4,166,093 which includes a species migration-inhibiting layer interposed between a radiation-blocking layer and a reagent layer of a multilayer analytical element.
The detectable specie~ migration-inhibiting layer is permeable to analyte and fixes or otherwise pxevents a significant portion of any detectable species, such as a dye formed in the reagent layer, rom further migrating up into the radiation-blocking layer. Such detectable species migration-inhibiting layer comprises a mordant for the particular detectable species formed in the -reagent layerO ~owever, such an inhibiting layer still presents the disadvantage of a mordanting agent which may interfere with reactions initiated by the presence of analyte and prevent or substan~
~;~ tially inhibit the formation or release of the detectable species.
Still anothex attempt to overcome the problem of reverse fluid migration in multilayer analytical elements is disclosed by International Publication No. WO 84~02193 which provides for a chromogenic ~ support immunoassay which comprises collection of ; 25 an immune complex comprising analyte bound to an enzyme-labeled anti-analyte antibody on a porous or microporous support material. The support func-tions to concentrate the chromatic signal generated by the labe~ component upon reaction with signal ; 30 generating reagents in the support material.
Concentration of the chromatic signal results from covalent attac~ment of the reaction product to the support, and the problem o reverse fluid migration being overcome by providing a single layer. The ~ .
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~ ~7~77 immunoassay, however, requires a number of incuba-tion and washing steps in order to localize and concentrate the signal on the support. Although the immunoassay overcomes reverse fluid migration by providing a single layer-support within which the necessary reactions for pxoduction of the chromatic signal occur~ it still presants the disadvantages of extensive incubation and washing steps which are not necessary with a multilayer analytical element.
Accordingly, it is an ob~ect of the p~esent invention to overcome the aforementioned disadvan-tages by providing a specific binding assay in a multizone r or multilayer, test device which con-centrates the detectable reaction product of the interaction between a chemical label group and an interactive detection reagent therefor without ~; interfering with the specific binding reactions involved in the a say.
Another object of the present invention is to provide, in a multizone, or multilayer, test device, a specific binding assay having an end point in the assay where further migration of the reaction product does not occur.
Further, it is an object of the present invention to provide a sensitive specific binding assay for the highly accurate determination of analyte from a liquid test medium and which has substantially little or no background signal.
Still another object o~ the present invention is to amplify the signal generated by the detect-able reaction product of the interaction betwe~n the chemical group and the interactive detection ~:

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~.~7G877 reagent therefor in order to provide sensitive detection limits.

SUM~RY OF THE INVENTI ON

The present invention provides a mul~izone test device for the determination of analyte fxom a liquid test medium based on interactions among the analyte, a labeled reagent, an immobilized reagent, and an immobilized interactive detection reagent for the labeled reagent.
The present invention derives its principal advanta~es from the use of a labeled reagent which provides a detectable signal in the form of a detectable reaction product as a result of the ~;~ interaction of the labeled reagent with an inter-active detection reagent and which can be rendered immobilized in the detection zone. No separately ~igratable detectable product is generated as with prior art devices and immobilization and concen-tration of the detectable reaction product results from highly specific chemical interactions. The -~ test device co~prises, in fluid flow contact, (1) a reagent zone incorporated with the immobilized reagent which will be an immobilized form of the analyte or a binding analog thereof, or an immo bilized form of a binding partner of the analyte, depending on the immunoassay scheme used, and ~2) a detection zone incorporated with an immobilized form of an interactive detection reagent ~or the labeled reagent. The labeled reagent is a form of 3Q a binding partner of the analyte, or a form of the analyte or a binding analog thereof, which is labeled with a chemical group having a detectable '' .

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chemical property which generates a detectable product upon interacting with the immobilized interactive detection reagent in the detection zone.
The immobilized reagent in the reagent zone and the labeled reagent are selected to comprise specific binding partners which will bind to one another dependent upon the amount of analyte present. When the labeled reagent is a labeled 1~ form of the analyte or an analog thereof, the immobilized reagent will be an immobilized form of a binding partner for the analyte, and the analyte and labeled reagent will compete for binding to the immobilized reagent. When the labeled reagent is a labeled form of a binding partner for the analyte, the immobilized reagent will be an immobilized form of the analyte or an analog thereof, and the labeled reagent that does not become bound to analytP will become immobilized by hinding to the immobilized reagent. Whether labeled analyte or labeled binding partners are in~ol~ed, a portion of the labeled reagent will remain or become unbound to the immobilized reagent depend~nt upon the amount of analyte present.
The resulting labeled reagent which remains or becomes free to migrate within and out of the reagent zone then passes into the detection zone where it interacts with the immobilized interactive ; detection reagent in the detection zone to thereby generate a detectable reaction product. Pre-; ferably, the resulting reaction product is immo-bilized and thereby prevented rom migrating from the detection zone up into the reagent zone. The reaction product can be inherently immobilized by comprising a residue of the immobilized interactive detection reagent or by being a released but insoluble product, or can be immobilized by an .

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~ ~7~7 - lC -immobilized agent having a binding affinity for the reaction product where the reaction pr*duct is generated in a soluble form. The reaction product provides a detectable signal in the detection zone S which is measured and correlated to the amount of analyte in the test medium.

BRIEF DESCRIPTION OF THE DR;~WINGS

FIG. 1 is a sectional view of a multilayer test device having a reagent layer and a detection layer constructed according to the present inven-tion.
FIG. 2 is a sectional view of a multilayer ~-test device having two reagent layers and a de- -tection layer constructed according to the present invention.
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FIG. 3 is a sectional view of a multilayer test device having two reagent layers, a detection ~-l layer, and a support constructed according to the-~' present invention.
.'`', ~ 20 DESCRIPTION OF T~E PREFERRED EMBODIMENTS
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The multizone test device of the present invention provides a specific binding assay in a ;' ' , . ~ , ' . . : . .
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zoned or layered test strip or device. The assay depends upon the partitioning of a labeled reagent, which is either applied to the device or incor-porated within the device, between being retained in the reagent zone by being bound or immobilized to the immobilized reagent and being free to migrate into the detection zone. The labeled reagent which migrates into the detection zone is free to interact with the immobilized int ractive detection reagent which results in the generation of the detectable reaction product therein. The present invention provides an advantageous means for concentrating the detectable reaction product which is generated in the detection zone and thereby amplifies the signal produced thereby.
In order to simplify the disclosure here-inafter, the test device of the present invention will now be desc~ibed principally as comprising a layered structure. It will be understood that other types of zones can accomplish the same result. Also, the labeled reagent will be selected to be a labeled form of a binding partner of the analyte and the immobilized reagent will be se-lected to be an immobilized form of the analyte `~ 25 ~with immobilized analyte being replaceable by an immobilized form of an analog of the analyte as is ; understood in the art) ; In particular, the test device of the present ; ~ invention comprises at least one reagent layer and a detection layer, and, as will be described in greater detail hereinafter, can further include a second reagent layer. The reagent layer is in-corporated with the immobilized reagent which comprises an immobilized form of the analyte which ' ,: ~ . .
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, , ~27~7 is no~ capable of being solubilized or otherwise removed from the reagent layer upon contact with the test medium. The detection layer is incor-porated with an immobilized form of an interactive detection rQagent for the labeled reagent/ which interactive detection reagent is similarly not capable of being solubilized or otherwise removed from the detection layer. Where a second reagent layer is employed, the first reagent layer is incorporated with the lab~led reagent which is solubilized by the test medium when applied there-to, and the second reagent layer is incorporated ` with the immobilized form of the analyte.
It is to be appreciated that according to the teachings of the present invention, the layers which comprise the test device are in fluid contact ; with one another whereby the layers of the test device which axe associated with each other permit the diffusion of a fluid into and ~etween these layers. Such fluid contact permits ~assage of at least some compone~ts of a fluid sample, e.g~, antigens, haptens, and/or antibodies, between the layers of the device and is preferably uniform along the contact interface between the fluid contacting layers. Accordingly, upon application j of the liquid test medium and labeled reagent to the reagent layer, the liquid test medium and labeled reagent are permitted to diffuse and permeate into and through the reagent layer and ; 30 into the detection layer. Where a first and sec~nd .
reagent layer are provided, the liquid test medium is si~ilarly penmitted to diffuse and permeate into and through the first reagent layer whereby the labeled reagent incorporated therein is solubilized .
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and the liquid test medium and the labeled reagent urther diffuse and permeate into and within the second reagent layer and into and within the detection layer.
Once the liquid test medium and the labeled reagent have been applied to and permeate the reagent layer as heretofore described, if the analyte being detected is present in the li~uid test medium, then substantially all of the analyte present is brought into direct fluid contact with and specifically bound to the labeled reagent. As a result of the fluidity between the reagent layer and the detection layer, the resulting analyte-llabeled reagent~ complex thereby formed is free to migrate within and out of the reagent layer and ; into the detection layer. As will be described in ~` greater detail hereinafter, the labeled reagent `-` preferably provides only one available binding site ~ for bi~ding of the analyta to the labeled reagent.
-~ ~ 20 ~s a result, once such available ~inding site has : ~;
been occupied by analyte, the analyte-(labeled `~ reagent) complex is free to migrate within and out of the reagent layer without being immobilized by ` the immobilized analyte incorporated tkerein.
Similarly, where a first and second reagent layer are provided, upon application o~ the liquid test medium to the first reagent layer, the labeled reagent is solubiIized and substantially all of the analyte present is brought into direct fluid 3a contact with and specifically bound to the labeled reagent. The resulting analyte-llakeled reagent) complex thereby formed is permitted to migrate ;~ within and out of the first reagent layer, through ~' the second reagent layer, and into the detection !::
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layer. Any of the labeled reayent which does not become bound to analyte from the test medium is bound to and immobilized by the immobilized analyte in the reagent layer, or, where a first and second reagent layer are provided, immobilized in the second reagent layer.
Once the analyte-(labeled reagent) complex migrates into the detection layer, the labeled reagent of the complex interacts with the immo-bilized interactive detection reagent therein to ~ thereby generate a reaction product as a result of - the interaction therebetween. As will be described in greater detail hereinafter, the reaction product can inherently provide a detectable signal, or can ;; 15 require further interaction with another substance to generate a detectable signal, depending upon the nature of the labeled reagent. It will be appre-~`~ ciated that according to a preferred embodiment of ~he present invention~ the resulting reaction product is immobilized in the detection layer in ~` order to prevent the reverse migration thereof out of the detection layer. Accordingly, localization, .. ~
and preferably immobilization, of the reaction product in the detection layer permits the accurate and sensitive detection and measurement of the detectable reaction product which can be precisely correlated to the amount of analyte in the test . .
medium.

; Labeled Reagent and Detection Systems The labeled reagent of the present invention comprises a binding partner for the analyte under determination, or the analyte or a binding analog :

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thereo, labeled with a chemical group having a detectable chemical or interactive property. It i5 to be appreciated that the chemical group does not generate a detectable product or otherwise provide a detectable signal prior to interacting with an appropriate interactive detection reagent. Accord-ingly, the nature of the chemical group of the labeled reagent and the interacti~e detection reagent necessarily depends upon their interactive properties which generate a reaction product which will provide a detectable signal correlatable to the amount of analyte in a liquid test medium.
A¢cording to the teachings of the present invention, the detection layer is incorporated with an immobilized form of the interactive detection ~- reagent for the labeled reagent which is not capable of being solubilized or otherwise removed fro~ the detection layer upon contact with the liquid test medium or other liquid reagents ~- 20 Immobilization of the interactive detection reagent in the detection layer pxevents migration of the interactive detection reagent into the reagent layer which would result in the interaction thereof with the unreacted labeled reagent immobilized ~ 25 therein. Such interaction would otherwise generate ; an interfering and non-speciic signal. Once theappropriate binding reactions have been initiated as heretofore described, the analyte-(la~eled reagent) comple~ which migrates into the detection 3Q layer is brought into direct fluid contact with by the interactive detection reagent immobilized therein. Accordingly, complete interaction between the chemical group of the labeled reagent and the .,.
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~ ', .', ~27~7 interactive detection reagent is permitted only within the detection zone~ In this respect, the interaction between the chemical yroup and the interactive detection reagent results in the generation of a reaction product which either inherently provides a detectable signal, or re-quires further interaction with another substance or other substances to provide a detectable signal, depending upon the nature of the labeled reagen-t and the interactive detection reagent. It is to be appreciated that the reaction product can also be inherently immobilized as a result of the immobili-~ zation of the labeled reagent and the interactive ; detection reagent, or can be generated in a soluble form which can be immobilized by an immobilizedbinding agent in the detection layer having a binding affinity for the xeaction product. Such ; immobilized binding agent can also be an immo-bilized substance necessa~y for the ge~eration of a detectab~e signal upon interacting with the reac- -tion product w~ere the reaction product does not inherently provide a detectable signal as hereto-fore described.
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~` In general, chemical groups having detectable chemical properties are those groups which are detected on the basis of their own reactivity or interaction with anoth0r substance to provide a detectabIe signal as heretofore described. Such ~` chemical groups have been well developed in the 3Q field of immunoassays and most any such groups employed in immunoassays ca,n be applied to the ~ labeled reagent of the present invention.

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For example, representative chemical groups include enzymatically active groups, such as enzymes (see Clin. Chem. ~1976) 22:1232, U.S.
Reissue Patent No. 31,006, and U.K. Patent No.

2,019,308), enzyme substrates (see British Spec.
No. 1,548,741), enzyme prosthetic groups, coenzymes (see U.S. Pat. ~os. 4,120,797 and 4,238,565) and enzyme cofactors, enzyme inhibitors and activators, chemiluminescent species, chemical catalysts, metal catalysts, members of enzyme channeling, fluorophor-quencher or energy transfer pairs (see U.S. Patent Nos. 3,996,345; 4,174,384; 4,199,559; and 4,233,402), and specifically bindable ligands such as biotin, chelators or a hapten. For example, a cofac~or labeled species can be detected by adding the enz~me for which the label is a cofactor and a ~ substrate or substrates for the enzyme. Also, a - hapten or other specifically bindable ligand (e~g., biotin) labeled species can be detected by adding an antibody to the hapten or a protein (e.g., avidin~ which binds the ligand tagged or labeled with a detectable molecule. Such detectable molecule can be some molecule with a measurable physical property (e.g., fluorescence or absor-ban~e3.
It is to be appreciated that such represen-ative chemical groups possess interactive pro-perties with each other which also permit the use thereof as the interactive detection reagent of the present invention. For example, where the label of the labeled reagent i5 an enzyme substrate, such as umbelliferone galactose, the immobilized detection reagent is an enzyme capable of hydrolyzing the :;
~ substrate, such as ~-galactosidase. Similarly, .
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~ 2~77 where the label is an enæyme cofactor, such as nicotinamide adenine dinucleotide, the immobilized detection reagent is an enzyme, such as lactate dehydrogenase. Other representative groups include enzyme prosthetic groups, such as flavin adenine dinucleotide, and an apoenzyme, such as apoglucose oxidase; and enzyme inhibitors, such as methotrex-ate, and an enzyme which is inhibited by the enzyme inhibitor, s~ch as dihydrofolate reductase. Also, a hapten or other specifically bindable ligand (e.g., biotin) labeled species can be detected with an antibody to the hapten or a protein ~e.g., avidin) which binds the ligand tagged or labeled with a detectable molecule. Such detectable molecule can be some molecule with a measurable physical property (eOg., fluorescence or absor-`~ bance).
Further, the detectable chemical group in the ; lab~led reagen~ can be a polymer residue to which are at~ached internally-quenched multiple fluores-cers. The immobilized i~teractive detection ~ reagent in the detection layer can be an enzyme ;~ which interacts with the fluorescer-polymer residue to cleave and release unquenched fluorescer mole-cules.
As was heretofore described) the reaction product generated by the interaction be~ween the labeled reagent and the interactive detection reagent can (l) inherentl~ provide a detectable signal or (2~ require the interaction with an additional substance to provide a detectable signal. For example, the interaction of an FAD
labeled antibody and an iI~mobilized apoenzyme detection reagent will produce an active enzyme :~
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which will react with glucose to produce hydrogen - peroxide, The latter product can be measured in a coupled enzyme reaction with peroxidase and a chromogen such as tetramethylbenzidine.
Also, the reaction product which is generated can be in an insoluble form or a soluble form, depending upon the nature of the labeled reagent and the interactive detection reagent. In either casel the detectable signal can be inherent or may require the interaction with an additional sub- -stance as heretofore described. Where the reaction product is generated in a soluble form, however, it is preferable to incorporate an immobilized binding agent having a binding affinity for the reaction produc~ in the detection layer i.n order to prevent reverse migration of the reaction product out of the detection layer and into the reagent layer.
~` For example, where the enzyme product formed is an anionic chromophore, the product can be i~mobilized py incorporation of an anion exchange resin into the matrix. Alternatively, the substrate can ~`, contain a derivatized sugar which is attached to ., ~ .
the chromophore and i5 exposed by reaction with the enzyme label. Incorporation of the corresponding immobilized lectin into the matrix, or into an ~ adjacent binding layer, will result in binding and - immobilization of the product therein.

In some instances, however, it may be pre-`~ ferred that the interaction between the labeled reagent and the interactive detection reagent re~ults in the formation of an insoluble reaction product. For example, a number of enzyme ~- substrates are available which generate insoluble products, ~( ~
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such as naphthol AS-Bl phosphate for the enzyme alkaline phosphatase, or naphthol AS-Bl-~-D-galactopyranoside for the enzyme ~-galactosidase~
The detectable signal i~ preferably measured by passing the test device through a zone which is provided with suitable apparatus for detecting the . ultimate optical signal such as by reflection, ; transmission or fluorescence photometry. Such apparatus, for example, directs a source of energy, such as light, on and/or into the test device element~ The light is then reflected from the element back to a detectin~ means where a reflective : support is employed, or passes through the element to a detector in the case of transmission detection where a radiation transmissive or transparent support is employed. Conventional techniques of ~: fluorescence spectrophotometry or luminescence -~ measurements can al80 be employed if desired. In .~ 20 technique~ where an electroactive species is used as a label, detection can be accomplished with ampometric or potentiometric detection devices.

-:~; Multilayer Analytical Elements ~5 ~ Referring now to the drawings, Fig. l ill-.~ \ strates one embodiment of the multilayer test ~; \ device of the present invention which comprises at least one reayent layer and a detection layer which ~; 30 are in fluid contact with one ano~her. The reagent layer is incorporated with the immobilized form of the analyte (represented a~ An"), and the detection layer is incorporated with an immobilized form of an interactive detection reagent for the ~' .
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chemical group label of the labeled reagent (repre-sented as " ~Reagent") as heretofore described.
- Upon application of both the liquid test medium containing analyte and the labeled reagent to the reagent layer, the test medium and labeled reagent difuse into the reagent layer and are thereby brought into fluid contact with the immo-bili2ed analyte in the reagent layer4 In this embodiment, the labeled reagent and the test medium can be applied.independently or together as a mixture, the latter being preferred since such provides equal competition between the labeled reagent and the analyte rom the test medium for ' binding to the immobilized analyte. Accordingly, any of the analyte present in the liquid test medium becomes bound to ~he hinding partner for the ~, analyte of the labeled reagent and the resulting complex thereby formed is free to migrate within and out of the reagent layer and into the detection layer. Any of the excess labeled reagent which does not become bound to analyte from ths test medium becomes bound to the immobilized analyte in ~,, the reagent layer through the binding partner of `~ the analyte of the labeled reagent and prevented :~ 25 from migrating into the detection layer.
Alternatiavely, as is known in the art, rather '~ than adding the labeled reagent as a separate component, whether by addition with the liquid test -~ medium or by being incorporated in a separate reagent zone as described in more detail below, the labeled reagent can b,e prebound to the immobilized reagent in the reagent zone. Since the binding will be reversible, the presence of analyte will ' ' '''.'' .
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reverse some of such binding to release a detect-able amount of the labeled reag~nt.
It is to be appreciated that according to the teachings of the present invention, the binding partner for the analyte preferably has only one specific binding site for the analyte. Preferably, such binding partner is a monovalent fragment of an antibody prepared against the analyte and which is purified or derived from a monoclonal antibody.
Such monovalent antibody fragments can be readily prepared by digestion of normal whole IgG
antibody with a proteolytic enzymej such as papain, to produce antibody fragments commonly referred to in the art as Fab fragments. Alternatively, such monovalent antibody fragmPnts can also be prepared - by digestion of normal whole IgG antibody with a proteolytic enzyme such as pepsin, followed by ~; chemical reduction to produce antibody fragments - commonly referred to in the art as Fabl fragments.
~ 20 However, other binding partners ~an also be - used, preferably o~ course having only one speci-fic, availabl~ binding or recognition site for the analyte under determination. Such other binding partners include whole antibody hybrids, receptor molecules, and the like. For example, a whole antibody hybrid can be used which can be obtained from a number of procedures. Such hybrids can be prepared in vivo from a monoclonal cell line produced by hybridization between a secreting myeloma cell and a splenic cell which secretes the antibody of interest. The resulting cell line can spontaneously produce hybrid molecules consisting of one binding subunit with the specificity of in=erest and a ~econd subunit with the specificity .~
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which is defined by the myeloma cell lone. Such antibody can be isolated from homogeneous imers of the original myeloma antibody or splenic cell by conventional protein purification techniques known in the art. ~ybrids can also be chemically foxmed by co-mixing anti-analyte antibody with a ~econd anti~ody under appropirate denaturing conditions, such as by the addition of urea (8 Molar) and reducing agents such as dithiothreitol, followed by . 10 removal of the denaturing agent ot permit recon-; stitution of the antibody hybrids. Accordingly, a portion of the reconstituted sample will contain hybrids with a binding site for the second carrier antibody which can be further purified by conven-tional protein purification techniques known in the art.
; Accordinglyj once the analyte from the test - medium has become bound to ~he monovalent binding ; ~ partner thereof of the labeled reagent, e.g., the zo monovaIent antibody fragment of the antibody to the analyte, nonspecific immobilization of the result-ing complex by the immobilized analyte in the reagent layer is prevented as a result of the unavailability o~ a binding site on the labeled reagent fox the immobilized analyte. Upon migra tion of the analyte-(labeled reagent) complex into the detection layer, the labelad reagent interacts ~` with the immobilized interactive detection reagent.
Interaction of the analyte-(labeled reagent) 3a complex with the immobilized reagent concentrates or localizes the reaction product in the detection layer for the detection and measurement of the signal produced thereby either visually or with the , use of an appropriate instrument.

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. .' ' ' : . ' '~,, ' ' , ' As will be described in greater detail herein-after, except for reflecting layers and radiation-blocking agents, the various zones or layers and supports of the present invention are radiation-transmissive in most instances~ Such zones or layers and supports permit effective passage of visible light, fluorescent or luminescent emission, radioactive radiation, and the like. The choice of a particular radiation-transmissive material will depend upon the particular radiation selected for use with an element in which the material is to be incorporated. Accordingly, generation of the detectable signal in the detection layer of the device shown in Fig. 1 permits detection of the signal with an appropriate instrument directed either at the detection layer or at the reagent ~; layer. It is to be appreciated that since the labeled reagent in the reagent layer does not exhibit a detectable signal because of the absence of any interaction wi~h the interactive detection reagent in the detection layer, there is no inter-fering signal from the reagent layer when detecting tha detectable signal generated in the detection layer from and through the reagent layer.
Detection of the signal generated by the reaction product from either the reagent layer or the detection layer can be accomplished with the use of an appropriate instrument, such as a spectro-~- photometer, reflectometer, fluorometer or lumino-3Q meter~ For example, where detection is bas~d upon absorbance or fluorescenca, an energy source from such instrument is directed either at and through the reagent layer or at and through the detection layer. On the other hand, where detection is based .

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Referring now to Fig. 2 of the drawings, a test device is illustrated that is similar to the test device of Fig. 1. In this embodiment, the test device further includes a second reagent layer positioned between the first reagent layer and th~
detection layer. The additional reagent layer permits incorporation of a test medium soluble form of the labeled reagent therein which obviates the need for pre-mixing the li~uid test medium and the labeled reagent prior to the application to the test device or the independent application thereof, such as with the test device illustrated in Fig. 1 D
In particular, the first reagen layer is incor-~-~ porated with the test medium soluble labe].ed reagent, (represented as "Labeled Reagent"), which ~`~ is solubilized ~pon fluid contact with the liquid tes~ medium which diffusas therein. The s~c~nd reagënt layer is incorporated with the immobilized form of the analyte (represented as 'I ~An"), and the detection layer is incorporated with the immo~
bilized form of the interactive detection reagent (represented as " ~Reagent") as heretofore de-scribed.
Upon application of the liquid test medium to the first reagent layer, the liquid test medium diffuses into the first reagent layer bringing any 3a analyte from the test medium into direct fluid contact with the labeled reagent therein while, at the same time, solubilizing the labeled reagent.
Accordingly, any analyte from the test medium , "
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-becomes bound to the binding partner thereof of the labeled reagent and the analyte-(labeled reagent) complex thereby formed migrates within and out of the first reagent layer and into the second reagent layer. It is to be appreciated that any of the unbound labeled reagent in ~he first reagent layer, i.e., excess labeled reagent, will also migrate within.and out of the first reagent layer and into : the second reagent layer. Since the binding site of the monovalent binding partner for the analyte ; of the labeled reagent has been occupied by binding to the analyte from the test medium, once within the second reagent layer, the analyte-(labeled reagent) complex is permltted to migrate within and out of the second reagent layer without becoming immobilized, and into the detection layer. Once within the detection layer, the labeled reagent .. interacts with the immobilized interactive ;..
: . detection reagent incorporated therein to produce ~-~he reaction product as h~retofore described.
. However, since any unbound labeled reagent in the .: second reagent layer has an available binding site ~: for the immobilized analyte in the second reagent :~ layer, the labeled reagent becomes bound thereto and immobilized thereby and prevented from further migrating into the detection layer. The resulting ~: signal generated by the reaction product in the detection layer is then detected, measured and correlated ~o the amount of analyte from the test medium as heretofore described.
Although the various layers of the multilayer device of the present invention can be self-supporting, it is preferred that such layers be coated or otherwise positioned onto a support ' .
.

7~

member. The support member can be opaque, reflective, or transparent to light or other energy. A support member of choice for the various layers will be compatible with the intended mode of signal detectionO For example, where the chemistry of the test device generates a gaseous product for detection thereof with a gas sensing elçctrode, the support memb~r is a fluid permeant layer in liquid contact with such electrode. Preferred support lQ members include transparent support ma~erials capable of transmitting electromagnetic radiation - of a wavelength within the region between about 200 nm and about 900 nm. The support need not, of course, transmit over the entire 200-900 nm region, although for fluorometric detection of analytical results through the support it is desirable for the support to transmit over a wider band or, alterna-tively, to transmit at the excitation and emission spectra of the fluorescent materials used for 20- detection. It may also b desirable to have a ~` support that transmits over a narrow wavelength band width and which is reduced transmittance to adjacent wavelengths~ This could be accomplished, for example, by impregnating or coating the support with one or more colorants having suitable absorption characteristics.
Typically, after generation of the detectable signal, the signal is measured with a suitable instrument for reflection, transmission, fluores- -cence or luminescent spectrophotometry. Selection of an appropriate support member will therefore depend upon the method of detection, i.e., trans-missive or reflective.
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A radiation-transmissive or transparent suppoxt member permits a beam of energy, such as light, to pass therethrough. The beam is then reflected, such as from a radiation-blocking layer, back to a sensing component of the instrument.
Ref~ective pigment~, such as titanium dioxide, barium sulfate or ~inc oxide can be used for this purpose. Blush polymers can also be used, either independently, or incorporated with a reflective ; 10 pi~ment to enhance reflectivity or other proper-ties. Such radiation blocking layers and agents -~ are known in the art and include those described inU.S. Pat. Nos. 4,042,335 and 4,255,384. Where an opaque or reflective support member is utilized, a beam of energy is directed through the various layers of the device and reflected by the reflec-tive layer back to a sensing component of the -~ device . :, .
For example, there is illustrated in Fig. 3 a multilayer device having first and second reagent layers and a detection layer mounted or otherwise ~` ~ position d onto a support member. The first reagent layer is incorporated with the labeled rPagent comprising a test medium soluble monovalent antibody fragment of a monoclonal antibody, as heretofore described, having a binding affinity for the analyte under determination and which has been previously labeled with an enzyme, kepresented as ; "Fab-Enz~me"), i.e., having a detectable chemical : 3a property. The second reagent layer is incorporated with an immobilized form of the analyte (repre-sented as " ~An~) which will bind and thereby immobilize any o~ the labeled reagent which is not bound to analyte from the test medium as heretoore , ~

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~27~77 described. The detection layer is incorporated with an immobilized form of a substrate for the enzyme (represented as " ~Substratel') wherein the analyte-(labeled reagent) complex interacts with the immobilized substrate to thereby generate a reaction product. As was heretofore described, the reaction product can be generated as a detectable species, or, it may be gen~rated in a form which requires further interact~on wi.th an additional substance, such as an indicator, to provide a detectable si~nal. In the latter case, it is then preferable to incorporate an immobilized form of such indicator in order to localize the signal produced thereby in the detection layer.
It is to be appreciated that the support member utilized with the multilayer device of the present invention can either be reflective, i.e., radiation-opaque, or transparent, i.e., radiation-- transmissive. For example, to measure the desired enæyme-su~strate reaction where a reflective support member is utilized, a beam of energy is ~` directed through the first and second reagent layers and the detection layer, respectively, where the beam is then reflected back to the sensing means of the instrument by the reflective support member. The nature of the beam which passes through the various layers and reflected by the support member is affected by the amount of the detectable reaction product within the detection 3a layer wherein a detectable change in the beam is correlated to the amount of analyte in the test medium.
Con~ersely, a radiation-transmissive or an~parent support member which permits an energy :, .
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~Z'-g~77 source to pass th0rethrough, requires that the beam be reflected, such as from a radiation-blocking layer or agent, back to a sensing component of the instrumentO Accordingly, where such transparent support member is utilized with the multilayer device of the present invention, it is first of all necessary to incorporate such radiation-blocking layer or.agent into ~he device, preferably, a blockin~ layer between the second reagent layer and ~ 10 the detection layer or a blocking agent incor-: porated into the second reagent layer. In such a ~ device, to measure the desired enzyme-substrate :~ reaction, a source of energy is directed through the transparent support member and into the detec-tion layer, respectively, whereby the energy source is reflPcted back through the detection iayer and support Member by the radiation-blocking layer or agent and back to the sensing means of the instru-ment. The use of such device having a transparent support member and a radiation-blocking layer or ayents is particularly desirable when the liquid ; test medium includes a colored substance, such as réd blood cells where the liquid test medium is whole blood, in which case the radiation-blocking substance or layer prevents intererence of the coloration of re~ blood cells which would be .filtered out by and remain in a layer above the . detection layer.
~ It is to be appreciated that the various :~ 30 layers of the multilayer test device of the pxesent invention are not limited to the layers and con-igurations as heretofore described. Additional layers for use with the multilayer test device have been described and are known in the art which .

, enhance and/or modulate the performance of such tes~ devices. For example, a spreading zone or layer could be included which would be posi~ion~d immediately above and adjacent to the first reagent layer. The spreading zone meters and evenly distributes an applied liquid test sample to the underlying first reagen~ layer. Such spreading zones or layers are known in the art and include those described in U.S. Pat. Nos. 3,992,158 and 4~427,632.
The device can also include an intermediate zone or layer between the various layers which serves as an adhesive or subbing layer to faci-litate adhesion between the layers and to further facilitate adhesion of the layers to a solid suppork member. Intermediate zones or layers can ~` also be employed which, for example, contain reagents for removing interferants which may prevent detection of some of the analyte or, can be :......... . 20 a radiation-blocking zone or layer which masks zones or layers of the device to prevent in~er-ference in detection of the product. 5uch radiation-blocking layers can also be employed which mask the presence of various interfering substances found-in test samples, such as red blood ~:- cells in whole blood.
-; . It is al50 sometimes preferred to provide a timing zone or layer which controls the rate of diffusion of the various reagents incorporated into the multilayer test device through the various layers thereof. Such timing zones or layers are incorporated into khe test device in order to :: . provide controlled incubation times and sequential reactions or to facilitate manufacture of the ~, , ' ~ ' ' .

-device by preventing premature interaction of the reagents in the device.
The device of the present invention can also be a multizone device having reagent zones, detec-tion zones, and the liXe assembled in a configura-tion particularly adapted for chromatographic analysis. Such a device would include an absorbant region which would be immersed into the liquid test medium wherein the test medium would diffuse in an upward direction into the various zones.
~ he zones of such multizone device can be in the form of reagent pads which are mounted on~o a plastic support membex adapted to be immersed or dipped into a liquid test medium. The æone-forming reagent pads are positioned onto the support member in an end to end relationship wherein the ends ; thereof are in fluid flow contact with one another.
~i In particular, such reagent pads include a lower-most, liquid test medium-absorbtive pad or zone, 20 first and second reagent pads or æones, respec-tively, positioned thereabove, and a detection pad or zone positioned above the second reagent zone.
~;~ It is to be appreciated that the reagent and detection zones are incorporated with the various reagents of the multilayer davice previously described and perform the same functions thereof.
In this e~bodiment, however, instead of a liquid test medium sample being applied to the device, the lowermost absorbtive pad of the multizone device is 3a immersed into the liquid test medium. In this manner, the absorbtive pad serves as a wick for the absorbtion of the test medium and the upward diffusion thereof into ~he first reagent zone, the ; second reagent zone, and the detection zone, : ,:
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~Z7g~7 respectively. Devices in configurations such as described in U.S. Pat. Nos. 4,301,139 and 4,361,537 which use a developing fluid ca~ also be adapted to the present invention. As was previously des-cribed, analyte from the test medium which diffusesinto the first reagent zone binds to the labeled reagent incorporated therein and the complex formed thereby continues to migrate through the seco~d reagent zone and into the detection zone where the analyte-tlabeled reagent) complex interacts with the interactive detec~ion reagent immobilized therein to thereby generate the reaction product for the further detection and measurement thereof.
Similarly, any of the labeled reagent in the first reagent zone which is not bound by analyte from the test medium migrates into the second reagent zone where it is immobilized by th~ immobilized form of the analyte incorporated therein.
According to the teachings of the present invention, the v~rious layers described herein preferably comprise a porous matrix which is permeable to at least some components of a fluid sample, e;g., antigens, haptens and/or antibodies, such permeability generally arising from porosity, ability to swell or any other characteristic. The matrix material can include various porous fibrous materials such as cellulose, papers, fleeces, felts, woven fabrics and the like, whether formed from natural or synthetic materials. Such 3Q materialsj for example, are described in U.S.
.
~ Patent Nos. 3,802,842; 3,809,605; 3,897,214 and - 3,987,214. Other porous, but nonfibrous materials ~ include micropoxous polymers such as those referred ; ~o in U.S. Patent No. 3,552,929.

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~27~ 7 Preferably, the matrix-forming materials of the various layers of the multilayer test device of the present invention are permeable materials such as gelatin, agarose and the like. Such materials permit the passage of fluids by diffusion, rather than by capi~lary flow as with fibrous porous materials such as papers or woven materials.
Although the porous, fibrous materials described above can be used, gelatin, agarose and the like are particularly preferred because of their uniform permeability to liquids, as well as their ability to permit the passage of light or other electromagnetic radiation therethrough. Knowing the liquid test medium under analysis, the choice of an appropriate material will be apparent to one skilled in the art.
Various methods known in the art are available for the immobilization o~ analyte in the test device of the present invention, or, a derivative ~; 20 or suitable analog of the anaIyte can be prepared in order to facilitate the immobilization thereof into the test device. Al~hough immobilization through covalent attachment of the analyte or analog thereof is preferred, other means which utilize non-covalent association such as ion exchange or adsorption can also be used. Immo-bilization of analyte can be achieved, for example, by direct incorporation into the carrier matrix of the device, such as cellulose in paper, or into 3a gelatin or agarose in films. Alternatively, the analyte analog can be linked to a polymeric carrier which is then subsequently incorporated into the matrix of the device, the polymer being of suf-ficient size to prevent significant diffusion .
.

, ' -' ~ 35 -between the binding and detection layers. In gelatin, for example, polymers greater than 10,000 ~ in molecular weigh~ will exhibit negligible dif-; fusion through the gelatin matrix~ Similarly~ in agarose, polymers greater than two million in mclecular weight will be restricted from diffusing through the matrix. The analyte can also be linked i directly or through a polymer backbone to very small particles such as polystyrene microbeads ; 10 which can then be subsequently incorporated into the matrices of the device. Such particles are readily available in a range of sizes and include polystyrene, microcrystalline cellulose, cross-linked dextrans and cross-linked agaroses, ion exchange resins, and the like. A wide range of chemistries are available to couple the agents onto the carrier. For example, water soluble carbodi-imides can be used to actiYate ree carboxyl groups . , .
`~ ~or subsequent reaction with nucleophiles including ~-~ 20 various amine compounds; amide residues or beads ~- can be converted by reaction with hydrazine to ., hydrazides which can be further reacted with bifunctional reagents such as glutaraldehyde, 1,5-difluoronitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone, 2,4-dichloro-6-carboxy-methyl-amino-5-triiazine, dimethyladipimidate or dimethylsuberimidate, and the like, foLlowed by reaction with amines or other nucleophiles linked ~; to the analyte or analog of interest; hydrazides can be converted to azide groups by reaction with nitrous acid through a diazotization reaction;
hydrazide~s can be reacted with succinic anhydride to incorporate carboxylate groups with a spacer ~; arm; aliphatic amines or particles can also be ., . ~., ,, ~.
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reacted with bifunctional reagents analogous to the hydrazide chemistry, including the use of hetero-bifunctional crosslinkers which allow attachment to the amines of functional groups with differing specificities such as a maleimid~ group which shows enhanced specificity for sulfhydryl derivatives; -~
hydroxyl group~ can be activated by cyanogen bromide, tosyl chloride, carbonyl diimidazole, or p-nitrophenylchloroformate; pa:rticles such as polystyrene can be nitrated, the nitro groups reduced to aromatic amines, and the aromatic amine~
can be diazolyz~d prior to reaction with a nucleo-philic-analyte/analog of interest. Nitrocellulose, diazobenzoxymethyl (DMB) paperl derivatized nylon mesh, or paper activated with cyanogen bromide, p-nitrophenylchloroformate t or carboxyldiimidazole can also be utilized to link nucleophile reagents or reagents linked to rsactive polymers.
As an a~ternative to directly binding the appropriate binding reagent to a material immo-bilized in the reagent layer, one can also take advantage of specific binding partners to obtain the necessary immobilization ~n ~tu during per formance of the assay. The material to be immo-bilized, i.e., the analyte or analog or bindingpartner, can comprise or be modified to comprise a binding site for a distinct hinding substance which in turn can be immobilized in the reagent layer.
The immobilizable material thus can be situated in any convenient location in the device and upon ~; performance of the assay will result in the appro-priate immobilization. Binding interactions such as described previously ~or immobilizing the ~abe_ed reagent in the ~etection layer can be used.

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Similarly, the methods described above or the immobilization of analyte can also be generally applied for immobilization of the various de ection reagents or derivatives thereof.
The test devic~ of the present invention utilizes multiple reagent layers which are as-sembled to permit fluid contact between adjacent layers as heretofore described. The ~arious layers can be prepared using film formers to prepare consecutive over-laying coatings or prepared by superimposing layers of fibrous reagent matrix such as a filter paper, glass fiber or woven polyester.
Alternatively, adjacent zones can be configured into a chromatography format with each zone at-~- 15 tached on ~he support member with the edges of each reaction zone being in direct fluid contaGt as ` heretofore described.
Mul~iple layers of paper, for example~ can be held in juxtaposition with an enclosing plastic 2~ frame, or alternative~ with a liquid permeant mesh screen, or by incorporation of a water-soluble adhesive between the layers. The casting of m~ltilayer films can be accomplished by a nu~ber of - techniques in the art for casting films, including the use of a doctor blade, extrusion coater, Meyer rod, puddle coater or gravure coater. Alter-~atively, multiple consecutive layers can be cas~
with a cascade coater. Film layers formed by the above procedures can be overlayed with a fabric or 3n mesh material containing reagents which is incu-bated for a predetermined period of time.

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:, ~%7~377 - 3~ -Analyte The present assay can be applied to the detection of any analyte for which there is a binding counterpart available. The analyte usually is a peptide, polypeptide, pro~ein, carbohydrate, glycoprotein, steroid, nucleic acid or other organic molecule or which a binding counterpart exists or which is producihle in hiological systems or can be synthesized. The analyte, in functional terms, is usually selected from the group com-prising antigens and antibodies thereto; haptens and antibodies thereto; complementary polynucleo-tide sequences; and hormones, vitamins, metabolites and pharmacological agents, and their binding counterparts. Usually, the analyte is an immuno-logically-active polypeptide or protein, usually having a molecular weight of between bout 1,000 and about 10,000,000, such as an antibody or antigqnic polypeptide or protein, or a hapten ~` 20 having a molecular weight o at least about 100, and usually less than about 1,500.
Representative polypeptide analytes are angiotensin I and II, C-peptide, oxytocin, vaso-` pressinl neurophysin, gastrin, s~cretin, brady-kinin, and glucagon.
Representative protein analytes include the classes of protamines, mucoproteins, glycoproteins, globulins, albumins, scl~roproteins, phospho~
proteins, histones, lipoproteins, chromoproteins, ; 3Q and nucleoproteins. Exa~ples of speciic proteins are prealbumin, l-lipoproteins, human serum albumin, al-acid glycoprotein, ~l-antitrypsin, al-glycoproteinr transcortin, thyroxine binding ., .

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;~`' , " ' :., '' ~ ~ ' 7~7 globulin, haptoglobin, hemoglobin, myoglobulin, ceruloplasmin, ~2-macroglobulin, ~-lipoprotein, erythropoietin, transferrin, hemopexin, fibrinogen, the immunologublins such as IgG, IgM, IgA, IgD, and IgE, and their fragments, e.g., Fc and Fab, comple-ment factors, prolactin, blood clotting factors such as fibrinogen, thrombin and so forth, insulin, melanotropin, somatotropin, thyrotropin, follicle stimulating hormone, leutinizing hormone, gonado-~ropin, thyroid stimulating hormone, placental lactogen, instxinsic factor, transcobalamin, serum ~
enzymes such as alkaline phosphatase, lactic - -; dehydrogenase, amylase, lipase, phosphatases, cholinesterase, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, and uropepsin, endorphins, enkephalins, protamine, tissue anti-gens, bacterial antigens, and ~iral antigens such as hepatitis associated antigens (e.g., HB~Ag, H~cAg and HBeAg).
; 20 Representative hapten analytes include the general ciasses of drugs, metaboIites, hormones, ~ vitamins; toxins and the like organic compounds.
- Haptenic hormones include thyroxine and triiodo-thyronine. Vitamins include vitamins A, ~, e.g., B12, C, D, E and K, folic acid and thiamine. Drugs include antibiotics such as aminoglycosides, e.g., gentamicin, tobramycin, amikacin, sisomicin, - kanamycin, and netilmicin, penicillin, tetra-cycline, terramycin, chloromycetin, and actino-mycetin: nucleosides and nucleotides such a~
adenosine diphosphate (ADP) adenosine triphosphate (ATP), flavin mononucleotide tFMN), nicotinamide adenine dinucleotide (NAD) and its phosphate derivative (NADP), thymidine, guanosine and ' .
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. ', '"', . . " ', ~27~377 - 4~ -adenosine, prostaglandins; steroids such as the estrogens, e.g., estriol and estradiol, sterogens, androgens, diyoxin, digitoxin, and adrenocortical steriods; and others such as phenobarbital, pheny-toinl primidone, ethosuximide, carbamazepine,valproate, theophylline, caffeine, propranolol, procainamide, quinidine, amitryptiline, cortisol, desipramine, disopyramide, doxepin, doxorubicin, nortryptiline, methotrexate, imipramine, lidocaine, procainamide, N-acetylprocainamide, amphetamines~
catecholamines, and antihistamines. Toxins include acetyl T-2 toxin, alfatoxins, cholera toxin, citrinin, cytochalasins, staphylococcal enterotoxin B, HT-2 toxin, and the like.

Liquid Test Medium - The liquid test medium containing the analyte ~:~ under determination can he a naturally occurring or artiically formed liquid suspected to contain analyte, and is usually a biological fluid or a dilution thereof. Biological fluids from which analyte can be determined include serum, whole blood, plasma, urine, saliva, and amniotic and cerebrospinal fluids.
The present invention will now be illustrated, bu~ is not intended to be limited, by the following examples:

:

~q6~77 Preparation of Enzyme Labeled Ankibody Ascites fluid containing an anti-~igoxin antibody (~6 mg/mL~ is dilutecl five-fold in 0.1 M
citrate buffer, pH 3.5 and incubated with a 1:50 ~w/w) pepsin:antibody solution for 48 hours at 37C. After concentration to ~5 ml by ultra-filtration ovPr an ~ icon PM30 membrane (Amicon Corp., Danvers, MA, USA), the sample is gel fil-tered on a 5eph~cryl S-300 (Pharmacia, Inc., Piscataway, NJ, USA~, column (2.4 x 90 cm) and equilibrated with 10 mM sodium phosphate and 0.15 M
sodium chloride (pH 7~2) to isolate the F~ab')2 fra~ment of the antibody. The antibody is reduced : 15 wi~h 10 mM dithiothreitol and the protein peak is ~ pooled after desalting on a P-6DG polyacxylamide : gel resin ~Bio Rad Co. Richmond, CA 94804) just before reaction with activated ~-galactosidase. ~
. The ~-galactosida~e (type IX, Sigma Chemical Co., 5t. Louis, MO, USA~ is dialyzed against 50 mM
sodium phosphate, p~ 7.4. A solution at lO mg/mL
is reacted with a heterobifunctional cross-linking agent, succinimidyl 4-(N-maleimidomethyl)cyclo-: hexane-l-carboxylate, for two hours at room tem-: 25 peraturej and this material i5 passed over a 1 x 60 .. cm P-6DG column. The activated ~-galactosidase is mixed with the antibody in a 1:3 ratio and reacted for twenty hours at 4C. This material is con-~; centrated approximately ten-fold by ultrafiltration : 30 over an Amicon PM30 membrane. The concentrate is passed over a 1.5 x 110 cm ACA 22 resin (LKB
Instruments, Inc., Gaithersburg, MD, USA) to , ~ ~ Trade Mark .
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separate Fab-~-galactosidase from free antibody fragment (Fab) and from higher substituted oligo- -mers of Fab and ~-galactosidase. The principle enzyme/antibody fractions are pooled and passed over an affinity column containing immobilized ouabainO To prepare the immobilized ouabain column, ouabain is linked to bovine serum albumin similar to the procedure described by Smith ~t al.
[Biochem. 9:331-337~1970)3. Ouabain-BSA is linked to Sephadex~ G-25 (Pharmacia, Inc., Piscataway, NJ, ~SA) after periodate oxidation accordi~g to pre-visouly described procedures ~Wilson, N. and Nakane, P. J. of Immunol. Methods 12, 171-181, (1976)]. The Fab-R-galactosidase containing sample is pa~sed over a 1 x 10 cm column of affinity resin. Free ~-galactosidase is eluted from the : column and is fsllowed by a~ eluting solution containing 20 mM ouabain to release the antibody-enzyme conjugate from the column. ~ive column ~0 volumes are washed through and pooled. The pool is.
concentratPd to 2 mL and dialyzed for twenty hours :~ ayainst ten changes of phosphate saline buffer (50 m~ sodium phosphate, 0.1 ~ sodium chloride, pH
7.~).
' 2 5 _ XAMPLE 2 Preparation of the Immobilized Analyte Layer *Whatman 31-ET (Wha~man, Inc., Clifton, NJ, USA) paper is activated for subsequent derivatiza-tion with para-nitrophenylchloroformate (NPCF).
Paper sheets are immersed for fifteen minutes in distilled water and the water is then decanted and * Trade Mark ~`

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~ ;27gi,~377 - ~3 -the paper rinsed with six successive volumes of acetone to remove free water. The paper is then immersed in a 10~ sol~tion of NPCF in acetone, incubated for six hours, and then unreact~d NPCF
removed by successive rinses with acetone. The rinse solution is tested for the presence of the formate by adding 100 ~L of 1 N NaOH to 300 ~L of the rinse solution. The rinsing is continued with three volumes of acetone until there is no detect . able yellow color, followed ~y washing with 1 L of distilled water and subsequently washed with 5 x ; 100 mL volumes o acetone, and the solvent removed by air drying.

15 Preparation of Immobilized Substrate Layer :' -~ Wha~man 31-ET paper (3.7 g) is incubated with 2 g of 1,1' carbonyldiimidazole in 100 mL of ~ ace~one for one hour at room temperature with - occasional stirring. The paper is washed with 3 x 20 200 mL volumes of acetone and dried at 50C for approximately ten minutes [or until there i5 no detectable acetone odor) and stored with silica gel desiccant at 4C until further use. The paper is subsequently reacted with ~-galactosidase substxate 25 containing an active amine, ~-galactosyl-umbelli-ferone-aminohexyl (see U.S. Pat. No. 4,259,233).
50 mg of the substrate reagent is dissolved in 20 ~; mh of DMSO and added to 1 g of activated paper.
After fifteen minutes, 20 mL isopropanol is added 30 and the reaction contiuned for six hours at room ~ temperature with occasional stirrng. The solvent .~ .
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is decan~ed and the paper is washed with 3 x 100 mL
volumes o isopropanol. A 200 mL aliquot of isopropanol conkaining 2 mL of ethanol-amine is add~d and reacted for thirty minutes at room temperature followed by three 200 mL washes with isopropanol. The paper is left overnight at room temperature in 200 mL of isopropanol~ The next day the solvent is decanted and the residual solvent is removed by drying at 50C for fifteen minutes.

Enzyme-Antibody Conjugate Layer Whatman 54 paper is dipped through a solution containing 10 mg/mL of anti-digoxin Fab-~-galactosidase in a 0.~ M sodium phosphate buffer, p~ 7.4 and dried at 40C for twenty minutes.

~; Assembly of the Multilayer Device ~';
A composite strip device is assembled from the three reagent elements described above. The substrate layer is laminated onto a double-faced adhesive tape (3~ Company, St. Paul, MN, USA) and cut into a 1 cm wide x 12.7 cm long ribbon. This material is then laminated onto and along the length of an edge of one surface of an 8.3 cm wide x 12.7 long clear polystyrene support (Trycite~, Dow Chemical Co~, Midland, MI, USA). A 1-2 mm strip of double-faced adhesive tape is mounted ~` along the back edge of the su~strate layer and a 1 .i :, ' ' .

.

:

~ ;~7~i~37~

cm wide ribbon of reagent paper containing the immobilized analyte analog is mounted thereon by the strip of double-faced adhesi~e tape. The above method is repeated to mount the ribbon of reagent paper containing the enzyme-antibody conjugate.
The resulting multilayer device is slit into 5 mm wide x 8.3 cm lony reagent strips having the various layers mounted to the ends thereof.

';
Operation of the Device A noxmal human serum sample is spiked to 5 nM
with digoxin. A range of concentrations from 0O2 to 5.0 nM digoxin are prepared by dilution of the stock reagen~ with normal human serum. An 80 ~L
aliquot of sample is applied to the test device to initiate the test. The test device is mounted in a reflectance photometer which illuminates the bottom of the reagent pad with a white light from a fiber optic bundle mounted at 45 to the normal of the pad. Reflected light is detected by a fi~er optic bundle mounted normal to the pad which carries the light to a 405 nm interference filter (3 cavity, ~- Ditric Optics, Inc., Hudson, MA, USA) and as-~, sociated detection electronics. The change in-reflectance is followed with time and related to - the concentration of digoxin applied.

.: .

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:. ., , . :,

Claims (29)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A multizone test device for the specific binding assay determination of an analyte in a liquid test medium involving binding between (i) the analyte and a labeled or immobilized form of the analyte or of a binding analog thereof, and (ii) an immobilized or labeled form, respectively, of a binding partner of the analyte, the labeled one of the analyte, analog thereof, or binding partner being a labeled reagent comprising a detectable chemical group which interacts with a detectant composition to produce a detectable signal, the test device comprising, in fluid flow contact, (1) a reagent zone comprising a solid, porous matrix incorporated with the immobilized one of the analyte, analog thereof, or binding partner, and (2) a detection zone comprising a solid, porous matrix for receiving and measuring labeled reagent which migrates into such zone and incorporated with an immobilized form of at least one component of the detectant composition.

2. The test device of Claim 1 wherein the detectant component immobilized in the detection zone participates in a chemical reaction with the labeled reagent to produce a product which provides the detectable signal.

3. The test device of Claim 2 wherein the detectable chemical group in the labeled reagent is an enzymatically active group.

4. The test device of Claim 3 wherein the detectable chemical group in the labeled reagent is (i) an enzyme or (ii) a substrate or cofactor for such enzyme, and wherein the immobilized detectant component is the other thereof.

5. The test device of Claim 4 wherein the detectable chemical group in the labeled reagent is an enzyme and wherein the immobilized detectant component is a substrate therefor.

6. The test device of Claim 5 wherein the substrate is chromogenic.

7. The test device of Claim 5 wherein the substrate is fluorogenic.

8. The test device of Claim 5 wherein the substrate is chemiluminescent.

9. The test device of Claim 4 wherein the detectable chemical group in the labeled reagent is a polymer residue to which are attached internally-quenched multiple fluorescers and wherein the immobilized detectant component is an enzyme which acts on the fluorescer-polymer residue to cleave and release unquenched fluorescer molecules.

10. The test device of Claim 2 wherein the immobilized detectant component undergoes a chemical reaction with the labeled reagent to form an immobilized product which provides the de-tectable signal.

11. The test device of Claim 2 wherein the detectable product is insoluble.

12. The test device of Claim 2 wherein the detectable product is soluble and wherein the detection zone additionally comprises an immo-bilizing agent for such soluble product.

13. The test device of Claim 1 wherein the detectable chemical group in the labeled reagent comprises a fluorescer and wherein the detectant component immobilized in the detection zone is a quencher for the fluorescence of the labeled reagent.

14. The test device of Claim 13 wherein the detectable chemical group in the labeled reagent is a polymer residue bearing multiple fluorescers.

15. The test device of Claim 1 wherein the detectant component is immobilized in the detection zone by being covalently coupled to the matrix comprised therein.

16. The test device of Claim 1 wherein the detectant component is immobilized in the detection zone by being attached to a high molecular weight polymeric substance dispersed in said matrix.

17. The test device of Claim 1 wherein the binding partner of the analyte is an antibody or a fragment thereof.

18. The test device of Claim 1 wherein the reagent and detection zones are in the form of layers in fluid flow contact.

19. The test device of Claim 18 which addi-tionally comprises a reagent layer comprising a solid, porous matrix incorporated with a test medium soluble form of the labeled reagent.

20. The test device of Claim 18 which addi-tionally comprises a support element situated on the opposite side of the detection layer from the reagent layer.

21. The test device of Claim 1 which com-prises a solid, porous chromatographic element and wherein the reagent and detection zones are dis-crete sections of such element.
.

22. A multilayer immunoassay test device for the determination of an analyte in an aqueous liquid medium, which test device provides a de tectable optical signal upon contact with aqueous medium containing analyte, the test device com-prising, in fluid flow contact and in the following ordered sequence, (1) a first reagent layer comprising a solid, porous matrix incorporated with a water soluble form of a labeled reagent com-prising an antibody, or a fragment thereof, for the analyte and an enzyme that acts on an enzyme substrate to transform it into a product which pro-vides the detectable optical signal, (2) a second reagent layer comprising a solid, porous matrix incorporated with an immobilized form of the analyte or a binding analog thereof, (3) a detection layer comprising a solid, porous matrix for receiving and measuring labeled, agent which migrates into such layer and incorporated with an immo-bilized form of said enzyme substrate, and (4) a support element comprising a solid, nonporous substrate.

23. The test device of Claim 22 wherein the substrate is chromogenic.

24. The test device of Claim 22 wherein the substrate is fluorogenic.

25. The test device of Claim 22 wherein the substrate is chemiluminescent.

26. The test device of Claim 22 wherein the antibody fragment is derived from a monoclonal antibody.

27. The test device of Claim 22 wherein the detectant component is immobilized in the detection zone by being covalently coupled to the matrix comprised therein.

28. The test device of Claim 22 wherein the detectant component is immobilized in the detection zone by being attached to a high molecular weight polymeric substance dispersed in said matrix.

29. The test device of Claim 22 wherein said first reagent layer, second reagent layer, and detection layer are transparent to the detectable optical signal, and said support element is opaque to such signal.