EP0648334A1 - Immunoassay using dye complexed enzyme conjugates - Google Patents

Abstract

Target-specific dye labeled complexes are formed by reacting a conjugate of an enzyme and a specific binding substance, such as an antibody or antigen, with an enzyme substrate in a liquid medium under conditions where the substrate is converted to dye molecules which then complex to the conjugate. Such dye labeled complexes are useful in a variety of biological assays, particularly lateral flow assays where the dye labeled complex is immobilized within a label zone (14) which forms part of a flow path for a liquid sample or liquid reagent in a test device (10). Liquid flow through the label zone (14) transports the dye labeled complex to a subsequent capture zone (16) where it is bound in proportion to the presence or absence of a target analyte within the liquid sample.

Description

IMMUNOASSAY USING DYE COMPLEXED ENZYME CONJUGATES

BACKGROUND OP THB INVENTION 1. Pield of the Invention

The present invention relates generally to compositions and methods for their preparation and use in biological assay systems. More particularly, the present invention relates to target-specific labeling complexes comprising a specific binding substance, an enzyme, and dye molecules complexed thereto. The use of chromogenic and fluorescent dyes as

"labels" in biological assay procedures is known. Typical assay protocols call for direct or indirect binding of a dye label to an analyte or analyte analog in a biological sample, where the presence or absence of the dye at a particular stage of the assay can be determined visually and related to the amount of analyte initially present in the sample. A wide variety of specific assay protocols exist.

Of particular interest to the present invention, certain assays utilize naturally colored or dyed particles as a label, where the particles are bound to an antibody or other specific binding substance. Suggested particles include dyed latex beads, dyed liposomes, erythrocytes, metal sols, and the like. The colored particle in such complexes can serve as a visible marker, where separation, capture, or aggregation of the particles is mediated through binding of the antibody or other specific binding substance. The amount of label thus segregated in a particular assay step is related to the amount of analyte initially present in the sample.

A variety of methods for preparing such antibody- particle compositions have been proposed. Such methods generally rely on producing a colored particle, typically by dyeing a latex bead, a liposome, or the like, and subsequently attaching the colored particle to the antibody, typically by passive adsorption or by covalent binding.

While generally useful, methods for preparing such antibody-particle compositions can be relatively complex, usually requiring multi-stage operations including preparati of the particle, coloring of the particle, attachment of the particle to the antibody, and blocking of the particle for u in immunoassays. Moreover, a loss of antibody binding capac can often result from the particle attachment. Sometimes th particles are not compatible with the antibodies selected fo particular application.

It would be desirable to provide improved labeling compositions and methods for their production that would all for any antibody to be used for an application. The compositions should be relatively easy to prepare, with a reduced cost, and have uniform characteristics. In particul the compositions should retain antibody activity to a significant extent. It will be appreciated, however, that t methods and compositions of the present invention need not b superior to the prior art in each or any of these aspects, b rather that these are general advantages that the present invention can provide relative to certain prior art methods products.

2. Description of the Bacfcgτauτ.d ^T*¹ U.S. Patent No. 4,863,875, describes compositions comprising at least ten dye molecules or monomers covalently attached to an antibody through an isocyanate group on the d U.S. Patent No. 4,452,886, describes the covalent attachment photon absorbing or emitting polymers to proteins, such as antibodies and antigens. U.S. Patent No. 4,373,932, describ labeling of a ligand with an aqueous dispersion of a hydrophobic dye or pigment, or a polymer nuclei coated with such a dye or pigment. U.S. Patent No. 4,703,017, describes solid phase assay device which relies on specific binding o ligand-label conjugate on a solid support, where the label disclosed as a particulate, such as a sac, erythrocyte, erythrocyte ghost, liposome, or polymer microcapsule. U.S. Patent No. 4,943,522, describes a solid phase lateral flow assay using erythrocytes as a label. U.S. Patent No. 4,861,711, describes a solid phase lateral flow assay using enzyme antibody conjugate and substrate, each separately held in absorbant pads. See also, U.K. Patent No. 2,204,398; EP Patent No. 306 722; and EP Patent No. 276 152, which relate t lateral flow assays. Enzyme assays and immunohistochemical staining techniques which produce a colored dye by reaction o a substrate with an enzyme bound to a target moiety are known

SUMMARY OF THE INVENTION The present invention provides improved target- specific labeling complexes for use in biological assays, suc as lateral flow assays as described hereinafter. The target- specific complexes comprise an enzyme component bound to a specific binding substance component, such as an antibody or antigen, and dye molecules complexed to the enzyme and/or specific binding substance in an amount sufficient to impart visually discernible color to the complex. The complexes may result from either covalent or non-covalent attachment of the dye to the enzyme component, or both. The complexed dye molecules do not significantly block active site(s) on the specific binding substance which thus remain available for binding to an analyte or other target molecule in the biological assays. Usually, the enzyme will be inactivated that native enzyme activity is removed prior to use as a labeling complex according to the present invention.

Advantageously, the complexed dye molecules are lyophilized remain intact, even when introduced to an aqueous reaction medium, as described hereinafter.

The number of complexed dye molecules or moieties vary wide, from as few as 10 per complex to as many as 10⁷ p complex, with the primary requirement being that there be sufficient dye to render the particle visible in the biologi assay in which it is employed. Usually, each complex will comprise from 10² to 10⁴ dye molecules or moieties, with the individual molecule weight of each moiety being between 100 1000 daltons (although the weight may be as high as 10⁶ daltons) . The aggregate size of the complex will usually li between 10³ and 10¹¹ daltons, more usually being between 10⁶ a 10¹⁰ daltons.

According to a method of the present invention, the target-specific labeling complexes are formed in a aqueous medium by reacting the enzyme and specific binding substance components with an enzyme substrate, where the enzyme convert the substrate to a dye. The dye thus formed in the aqueous medium complexes to the conjugate, and the enzyme reaction is stopped at a time when the color of the labeling complex is sufficiently intense to be readily observable when used in assays as described hereinafter. Conveniently, the reaction may be stopped either by substrate exhaustion and/or by altering the reaction conditions. The method may further comprise inactivating the enzyme and/or lyophilizing the complex after the desired color intensity has been imparted. In another aspect of the present invention, test articles comprise the target-specific labeling complex impregnated, usually by lyophilization, in a support matrix, usually a nonbibulous or bibulous solid matrix capable of conducting liquid flow, preferably in a single direction in matrix. In this way, a liquid sample can be applied to the solid matrix and will flow through the matrix so that impregnated labeling complex can be transported by the liqui flow to a "downstream" location. Advantageously, the labeli complexes of the present invention have been found to remain intact when transported by liquid flow as just described.

Typically, an assay can be run using the test arti based on capture of the labeling complex mediated by the specific binding substance. Usually, a region of the test article will incorporate an immobilized capture reagent capa of directly or indirectly binding the labeling complex. The assay result will then depend on appearance of the label wit this capture zone.

Alternatively, the labeling complex may be initial bound to the capture reagent within the "capture zone" by an antibody or other binding substance specific for an analyte. By then applying a sample to the support matrix, flow of the sample through the capture zone will displace the labeling complex as a result of competitive binding with analyte prese in the sample. Thus, the color initially present in the capture zone will disappear in proportion to the amount of analyte present in the sample. In yet another aspect of the present invention, an assay device for detecting the presence of analyte in a liqui sample comprises a sample receiving zone, optionally a label zone, a capture zone, and an absorbent zone. The sample is applied to the receiving zone and flows through the device where it combines with the labeling complex within the label zone or displaces the labeling complex from the capture zone. In the first case, the labeling complex either binds to or competes with analyte in the sample, and the label is bound within the capture zone depending on the amount of analyte originally present in the sample. In the second case, no labeling zone is necessary and the analyte in the sample displaces the labeling complex into the absorbent zone. In both cases, the absorbent zone acts to receive liquid sample it flows through the previous zones within the device. DESCRIPTION OF THE DRAWINGS

Fig. l illustrates an assay device incorporating t principles of the present invention, specific examples of wh are described in detail in the Experimental section hereinafter. DESCRIPTION OF SPECIFIC EMBODIMENTS

The present invention provides compositions, metho for preparing such compositions, test articles, and test devices for use in performing biological assays, particularl immunoassays. The compositions comprise target-specific labeling complexes which are visually discernible, typically being colored and capable of providing a readily apparent co signal when aggregated or accumulated in a capture zone, as described in more detail hereinafter. The compositions furt comprise a specific binding substance which mediates such capture within the capture zone, and the compositions can be utilized in a wide variety of particular assay formats and protocols. While the compositions are particularly valuable for use in lateral flow assay protocols, they will find use a wide variety of other assay formats as well.

The compositions of the present invention are composed of a conjugate including an enzyme component and a specific binding substance component, where the enzyme component is selected to provide for conversion of appropria substrate(s) into a dye substance which will complex to the conjugate as it is produced in an aqueous medium. Suitable dyes should have a high avidity for the complex, with covalently binding dyes being preferred. Dyes which do not bind covalently should interact with the complex through hydrophobic, ionic, and other non-covalent mechanisms which result in strong binding, particularly to the protein components of the complex. The selection of a particular enzyme and substrate system to produce a particular dye wil depend on the particular application, desired color, and th like.

Exemplary enzyme-substrate systems are set forth Table A.

TAgt-g A

ENZYME SUBSTRATEfS. COLORED PRODUC

OXIDASES:

Glucose Oxidase phenazine ethosulfate formazan tetrazolium salt

PEROXIDASES: Horse radish aminoethylcarbazole; red peroxidase hydrogen peroxide

Horseradish benzidine; brown peroxidase hydrogen peroxide

Horseradish chloronaphthol; blue/gray peroxidase hydrogen peroxide

Horseradish chloronaphtol; purple indamin peroxidase hydrogen peroxide; dye MBTH Horseradish chlornaphthol; red quinoneimi peroxidase hydrogen peroxide; dye 4-aminoantipyrine

PHOSPHATASES: Alkaline indolyl phosphate indigo phosphatase Alkaline azonaphthol phosphate azonaphthol phosphatase

Alkaline indolyl phosphate; formazan-indigo phosphatase tetrazolium complex

Alkaline naphthol phosphate; azo compound phosphatase diazo compound

DIAPHORASE tetrazolium; formazan NADH

GALACTOSIDASES indolyl galactoside indigo azonaphthol galactoside azonaphthol indolyl galactoside; formazan tetrazolium naphthol galactoside; azo diazo compound compound

OXIDOREDUCTASE tetrazolium formazan

The specific binding substance will be a compound having spatial and/or polar features which permit it to bind specifically to another compound. Specific binding substanc useful in the present invention will be selected or prepared specifically bind to particular compositions, such as analyt anti-analyte substances, and other target substances where binding may be advantageously performed as part of a biologi assay procedure. Natural specific binding pairs include antigens and antibodies, lectins and carbohydrates, hormones and hormone receptors, enzymes and enzyme substrates, biotin and avidin, vitamins and vitamin binding proteins, complementary polynucleotide sequences, drugs and receptors, enzymes and inhibitors, apoproteins and cofactors, growth factors and receptors, and the like. Biotin and avidin derivatives may also be used, including biotin analogs/avidi biotin/streptavidin, and biotin analogs/streptavidin. When natural specific binding substance exists, one may be prepar For antigenic and haptenic target substances, antibodies may prepared by well known techniques. For polynucleotides, complementary DNA or RNA fragments may also be prepared by we known synthesis techniques.

The preferred specific binding substances are antibodies, antigen, and haptens, and the remaining discussi will be directed primarily at those substances. It will be appreciated, however, that the preparatory methods can easil be adapted to provide compositions employing other specific binding substances as described above. Additionally, the te articles and test devices of the present invention can easil by modified to employ target-specific complexes having speci binding substances other than antibodies and antigens, and t assay methods of the present invention can also be so modifi

The enzyme will be an oxidase, peroxidase, phosphatase, diaphorase, galactosidase, lytic enzyme, or oxidoreductase, with exemplary enzymes set forth in Table A, above. The enzyme will usually be covalently attached to th specific binding substance, but indirect linkage such as through a biotin-avidin binding or other cognate members of specific binding pairs may also find use. When the specific binding substance is a polypeptide or protein, such as an antibody, the enzyme may be covalently bound through a varie of moieties, including disulfide, hydroxyphenyl, amino, carboxyl, indole, or other functional groups, employing conventional conjugation chemistry as described in the scientific and patent literature. Binding should be effecte in such a way that active site(s) on the specific binding substance are not blocked and remain available for binding t the desired target substance. In the case of antibodies, binding will preferably be effected so that the complementar determining regions remain available for binding to the desi substance. Specific techniques for derivatizing antibodies binding to enzymes are described in Tijssen, "Practice and Theory of Enzyme Immunoassays" in Laboratory Techniques in Biochemistry and Molecular Biology, vol. 15, Burdon and van Knippenberg, eds. 1985, Elsevier, Amsterdam, the disclosure which is incorporated herein by reference.

Once the specific binding substance-enzyme conjuga are formed, they will be reacted with the appropriate enzyme substrate(s) (see Table A) in aqueous medium under conditions sufficient to convert the substrate to a colored dye product. The reaction conditions are further selected so that the dye molecules produced by the enzyme conversion will form a compl with the conjugate, with sufficient dye being incorporated in the complex to impart a visually discernible color.

Reaction conditions necessary to effect complexatio depend on the nature of the enzyme and substrate that are chosen for a particular application. Each enzyme has reactio conditions that are peculiar to it for optimal turnover and generation of dye product. Ideal candidate enzymes, chromogenic substrates, and reaction conditions (such as pH, ionic strength, temperature cofactors, and the like) are thos that are employed for enzyme-antibody mediated histochemical staining, described in chapter 17 of "Practice and Theory of Enzyme Immunoassays," supra.

The enzyme conversion and complexation reaction wil be carried out until sufficient dye has been produced and complexed to the conjugate so that the resulting complex will have a sufficiently intense color to be visually detectable when aggregated or bound within a capture zone, as described hereinafter. The reaction and complexation can be terminated by a variety of approaches. Conveniently, the amount of substrate reacted can be limited so that the substrate will b exhausted when the complex color intensity is sufficient. Alternatively, the enzyme conversion of substrate to dye product can be quenched by altering the reaction conditions o adding an enzyme inhibitor at a time selected to terminate dy production. As a second alternative, it would be possible to simply remove or separate particles from the substrate in the reaction medium in order to stop the reaction and/or remove soluble dye, for example, by using gel permeation chromotography.

Usually, but not necessarily, the enzyme activity will be inhibited after complexation is completed, either by exposure to an enzyme inhibitor, exposure to denaturing conditions (where the conditions selected must not denature o inactivate the specific binding substance) exhaustion of an enzyme cofactor, or the like. It will be appreciated that th ability of the target-specific labeling complex of the presen invention derives from the color provided by the complexed dy molecules, not from the ability of the enzyme to turnover substrate.

Unreacted substrate will preferably be removed fro the labeling complexes after the reaction has been stopped, typically by chromatographic separation techniques, such as permeation. If the enzyme is not inactivated, it is essenti that substantially all substrate be separated in order to prevent additional dye production in the subsequent assay procedure. Usually, however, the enzyme will be inactivated order to further avoid the possibility of unintended dye production. In a preferred technique, the target-specific labeling complexes of the present invention will be applied a solid phase matrix, more preferably being lyophilized with the matrix. The solid phase matrix may be bibulous or nonbibulous (as described in copending application Serial No. 07/639,967, the disclosure of which has previously been incorporated herein by reference) and will generally be able conduct liquid flow therethrough so that the liquid may interact with the impregnated labeling complex.

Such matrices may be composed of a variety of materials of the type generally employed for preparing porou membranes, absorptive pads, and the like, in the immunoassay art. Particularly preferred are fabrics composed of polyest acrylonitrile copolymer, rayon, glass fiber, cellulose, and blends thereof. In certain embodiments it will be desirable treat such fabrics with a blocking agent to render the fabri nonbibulous, as generally described in copending application Serial No. 07/639,967, the disclosure of which has previousl been incorporated herein by reference.

As an alternative to impregnation in a solid phase matrix, the labeling complex particles of the present invent may be dried and lyophilized into a powder form. Such lyophilized labeling complexes are then available for use in assay protocols as generally known in the art. Referring now to Fig. l, an exemplary test device 1 constructed in accordance with the principles of the present invention will be described. Test device 10 will employ nonbibulous sample and reagent flow regions generally as described in copending application Serial No. 07/639,967, the disclosure of which has previously been incorporated herein b reference. It will be appreciated, however, that other test devices employing bibulous sample and reagent flow regions, o combinations of nonbibulous and bibulous regions, may be constructed for use with the target-specific labeling complex of the present invention.

The test device 10 includes a sample receiving zone 12, a label zone 14, a capture zone 16, and an absorbent zone 18. The sample receiving zone 12, label zone 14, and capture zone 16 will be composed of materials capable of receiving liquid samples and other liquid reagents and transporting suc samples and reagents in a lateral direction, i.e. from the receiving zone 12 toward the absorbent zone 18. The absorben zone 18 will be composed of a material capable of receiving a absorbing the same liquid samples and reagents. In this way, liquid sample or other liquid reagent initially applied to th sample zone 12 will be able to flow laterally from the sample receiving zone 12 into and through the label zone 14, into an through the capture zone 16, and finally into the absorbent zone 18 which acts as a wick or sink so that the entire sampl or reagent volume may be flow through the zones 12, 14, and 1 in order to properly complete the assay.

In the preferred case of a nonbibulous assay device 10, both the sample receiving zone 12 and the label zone 14 will be composed of nonbibulous material which permit liquid flow in which all of the dissolved or dispersed component of the liquid are carried at substantially equal rates and with relatively unimpaired flow (in the absence of specific bindin to a capture reagent as discussed hereinafter) . Suitable nonbibulous materials include intrinsically nonbibulous materials, such as porous polyethylene, or intrinsically bibulous materials such as paper, nitrocellulose, polyesters, acrylonitriles, copolymers, rayon, and blends thereof, which have been converted to exhibit nonbibulous flow characterist by the application of blocking agents, such as detergents an proteins which inhibit the molecular forces which contribute the bibulous nature of the material. The sample receiving zone 12 serves to receive a small volume of the sample, typically in the range from 20 μ to 200 μl, and to initiate lateral flow to the label zone 14 The label zone 14 will typically include the target-specific labeling complex of the present invention impregnated or otherwise immobilized therein. Usually, the labeling comple will be lyophilized as described above in connection with preparation of the test article. Flow of the sample or othe reagent will then mobilize (solubilize) the labeling complex and transport the complexes toward and into the capture zone 16.

It will be appreciated that there will be some interaction between the labeling complexes and an analyte within the sample which will mediate binding of the labeling complex within the capture zone 16. Such interaction can ta a variety of forms, typically being "sandwich" labeling (whe the labeling complex includes anti-analyte which binds to th analyte and the capture zone includes immobilized anti-analy which is then able to capture the labeling complex through t analyte) or "competition" labeling (where the labeling compl comprises analyte or analyte analog and binding of the label anti-analyte within the capture zone 16 is inversely proportional to the amount of analyte in the original sample A variety of modifications to the basic sandwich and competitive assay formats are known in the art and can be adapted for use with test devices identical or similar to t device 10 illustrated herein.

The capture zone 16 may also be prepared from bibulous or nonbibulous materials, with nonbibulous materia being preferred. A particularly suitable material for the capture zone is nitrocellulose which has been blocked to prevent bibulous interaction with the sample and other reagents. In use, binding of the target-specific labeling complexes of the present invention within the capture zone 1 will provide a visual signal or indication which represents assay results. Binding of a sufficient amount of the labeli complex within a defined location in the capture zone 16 wil permit a user to visually read the result in the capture zone 16.

The following examples are offered by way of illustration, not by way of limitation. EXPERIMENTAL

The following examples demonstrate preparation of pre-dyed complexes for detection of human chorionic gonadotropin (hCG) in body fluids from an antibody-enzyme conjugate, appropriate enzyme substrate system, and the optional use of an enzyme inhibitor and/or a stabilizer. EXAMPLE 1 1.1 Preparation of Reagents

1.1.1 Antibody Purification and Derivatization

Monoclonal anti-hCG ascites fluid was fractionated 0- °C by delipidation with sodium dextran sulfate and calciu chloride followed by ammonium sulfate treatment at 50% salt saturation and desalted on a G25 column into 10 mM Tris buff (pH 8.0). The antibodies were further purified on a Q- Sepharose® FF resin using a salt gradient of 0 to 0.3 M sodi chloride in the same buffer. Fractionation was monitored at

280 nm, and the protein peak was collected and buffer-exchan on a G25 column into 0.1 M sodium phosphate (pH 7.0). The resultant anti-hCG antibody was concentrated by ultrafiltrat to 5 mg/ml. ^" Maleimide groups were introduced generally as described in Enzyme Immunoassays , Ishikawa et al., eds., Tg Shoin, Tokyo - New York, 1981, into the anti-hCG antibody b adding maleimidobenzoyl-N-hydroxysuccinimide to a final concentration of 100 μg/ml, incubation of the reaction mixt for 45 minutes at 25^βC, and buffer exchange on a G25 column into 0.1 M sodium phosphate (pH 6.0). 1.1.2 Preparation of Antibodv-Enzvme Conjugates

To prepare antibody-enzyme conjugates, the followi enzymes were used: glucose oxidase (GO) , horseradish peroxidase (HRP) , diaphorase I (DIA) , and calf intestine alkaline phosphatase (ALP) . To introduce SH groups, the enzymes were prepared at 5 mg/ml (GO, DIA or ALP) or 10 mg/m (HRP) in 0.1 M sodium phosphate (pH 8.0) containing 0.5 mM 2 mercaptoethanol, incubated at 25°C for 45 min with 2- iminothiolane at a final concentration of 1.28 mg/ml, before being buffer-exchanged on a G25 column into 0.1 M sodium phosphate (pH 7.3).

The ale ide-containing monoclonal anti-hCG antibo (section 1.1 above) and the SH-derivatized enzymes were allo to react for 2 hr. at 25°C followed by binding of the enzyme antibody conjugate on a Sephacryl® S300 HR resin. A 0.45 μm filtered conjugate elution buffer composed of 0.05 M sodium phosphate buffer (pH 7.0) containing 0.1% (w/v) NaN₃, 0.03% (w/v) , MgCl₂-6H₂0, 0.003% (w/v) ZnS0₄-7H₂0, and 0.005% (w/v) Tween^® 20 was used throughout the fractionation. The fractionation was monitored at 280 mm, and the antibody-enzy conjugate fraction pooled. Subsequently, the antibody-enzym conjugates (except for anti-hCG/ALP conjugate) were buffer- exchanged on a G25 column into the following buffers supplemented with 50 μg gentamycin/ml: (i) HRP conjugate: 0.05 M Tris/HCl buffer (pH 7.5); (ii) GO conjugate: 0.1 M

HEPES buffer (pH 8.0); and (iii) DIA conjugate: 0.1 M sodiu phosphate (pH 8.0).

1.2 Pre^paration of Lateral Flow Assay Device for a "Wet" Tw Step Assay

Lateral flow nonbibulous assay test strip were constructed according to the methodology contained in co- pending application Serial No. 07/639,967. The test device (Fig. 1) includes three active zones and fourth absorbent zo which acts as a wick or sink to receive sample flow from the active zones. The active zones comprise a sample receiving zone 12, an intermediate zone 14, and a capture zone 16, as described below. The "wet" assay employs dye-label-complex prepared in a test tube, not illustrated, to which sample wa added. The resulting mixture was then applied to the test strip. Test strips were constructed as follows:

1.2.1 Preparation of Sample Receiving and Intermedia Zones The sample receiving zone and intermediate zones we prepared from Sontara® 0-100 DuPont Orion® spunlace fabric. The fabric was rendered nonbibulous by saturing with methylat bovine serum albumin (BSA) . The conversion to nonbibulous material was achieved by treatment at 38 μl/cm² with a 10 mg/ solution of the BSA at room temperature at five minutes. The pad of Sontara® was then frozen at -70°C for at least an hour The Sontara® fabric was then lyophilized overnight on a Virti Freezemobile. The sample receiving pad and the intermediate zone pad were then cut into 10x4mm rectangles with the spunla fibers being parallel to the longer side of each pad. 1.2.2. Preparation of Capture Zone Membrane

To prepare a nonbibulous capture zone, goat anti-hC serum was fractionated at 0-4°C by delipidation with sodium dextran sulfate and calcium chloride followed by ammonium sulfate treatment at 50% salt saturation and desalted on a G2 column into phosphate-buffered saline (pH 7.2; PBS). The an hCG antibodies were then further purified on an hCG-Sepharos affinity resin using 0.1 M glycine buffer (pH 2.3) as the elution medium. Fractionation was monitored at 230 mm, the onospecific anti-hCG antibody peak collected and buffer- exchanged on a G25 column into PBS. Finally, the antibody w concentrated by ultrafiltration to > 2 mg/ml.

Nitrocellulose having a pore size of 8 μm was affi to an X-Y chart recorder, and hCG capture bands were formed 2-cm spaced parallel lines by 2 mg/ml goat anti-hCG antibody using a plotter pen operated in the manual mode. These line were reactive with hCG contained in a sample. After air dry for 10 min at room temperature, the nitrocellulose membrane placed into a tray containing blocking buffer (10 mg/ml BSA the above Tris buffer) for 15 min at room temperature. The membrane was removed, blotted, allowed to air dry, and subsequently stored in a desiccator at room temperature unti assembly of the device. 1.2.3 Assembly of the Device

A 20 x 4 mm strip of the capture zone membrane was affixed centrally on an adhesive transparency strip. The transparency strip was a 70 x 4 mm strip of overhead projecti transparency film, made adhesive with double-sided adhesive tape. The intermediate zone was then affixed next to the capture zone pad with a 1 mm overlap. The sample receiving p was then placed next to the intermediate pad with 1 mm overla

The device was then provided with an absorbent pad, which was a 20 x 4 mm rectangle of cellulose paper which was affixed to the distal end of the capture zone membrane with mm overlap.

1.3 Preparation of Uninhibited. Pre-dved Label Complex

Containing Antibodv-GO Conjugate for use in a Two-Step "Wet" Assay.

Uninhibited, pre-dyed label complex containing ant hCG monoclonal antibody-GO conjugate was formed by a FAD- mediated electron transfer utilizing phenazine methosulfate a chemical intermediate and a colorless hydrophylic tetrazol salt as an electron acceptor ("GO substrate") . This reactio results in a dyed enzyme-antibody conjugate suitable for detection of the analyte in the body fluid sample.

A 1.0 ml incubation reaction was carried out at 25 by a sequential addition of the following components to 500 of 0.1 M HEPES buffer (pH 8.0) containing 50 μg gentamycin/m

(a) 100 μl of 8 mg MTT tetrazolium salt per ml of 50% (v/v) di ethylformamide (DMF) in deionized water; (b) 50 μl of 0.5

D-(+)- glucose in the same HEPES buffer; (c) 50 μl of 0.5 mg

PMS/ml of deionized water; and finally to initiate the compl formation (d) 300 μl of anti-hCG antibody-GO conjugate at a final concentration of 1.7 to 50 μg/ml in the same HEPES buffer. At the time intervals specified in Table 1, 15 μl multiple aliquots were withdrawn, mixed with 25 μl samples o hCG standard (prepared in phosphate-buffer saline containing 0.5% (w/v) bovine serum albumin (BSA) at either 0 or 800 mlU to give the final hCG concentration of either 0 or 500 mlU hCG/ml, respectively), and immediately applied to 4 mm wide lateral flow assay strips, prepared as described in section

The results based on the appearance of a visually discernib positive signal in the capture zone after 5 minutes are reported in Table l.

Table l GO-Ab, Time allowed (min.) hCG Concentration¹ ug/ml for Complex Formation in Standard

0 mlU/ml 500 mlU/

1.7 10 - - 30 - +/-

5 10 - -

30 - +

.50 10 - -

30 +

¹+ = Clearly discernible appearance of color in capture zone; +/- = Equivocal appearance of color in capture zone; and = No discernable appearance of color in capture zone.

1.4 Preparation of Uninhibited. Pre-dved Label Complex

Containing Antibodv-Alkaline Phosphatase (ALP) Con^ju^gate for use in a Two-Step "Wet" Assay.

Uninhibited, pre-dyed label complex containing anti hCG monoclonal antibody-ALP conjugate was formed by enzyme- catalyzed hydrolysis of a colorless organic phosphate ("ALP substrate") to a colored phenolic product ("ALP product") . 550 μl incubation reaction was carried out at 25^βC by a sequential addition of the following components: to 250 μl 0.1 M AMPD (2-amino-2-methyl-l,3-propancdiol) buffer (pH 10. containing 0.1% (w/v) NaN₃: (a) 200 μl of 5 mg BGP (Fast Bordeaux G diazonium salt) per ml of the same buffer coupled 1-napthol and (b) 45 μl of conjugate elution buffer, and finally to initiate the complex formation (c) 55 μl of anti- hCG-ALP conjugate in the conjugate elution buffer to the fin concentration in the mixture of 2.75 μg or 5 μg/ml. After 1 min. incubation, 15 μl or 30 μl multiple aliquots were withdrawn, mixed with 25 μl or 10 μl aliquots of the hCG standard, respectively, to obtain a final concentration of h of 0 or 500 mlU/ml, and immediately applied to 4 mm wide lateral flow assay strips, prepared for a "wet" assay as described in section 1.2 above. Results based on the appearance of a visually discernible positive signal in the capture zone after 15 minutes are reported in Table 2. Table 2

ALP-Ab, hCG Concentration ug/ml. in standard¹

0 mlU/ml 500 mlU/ml

1.02

1.88 - +

¹+ = Clearly discernible appearance of color in capture zone, and

- = No discernible appearance of color in capture zone.

Uninhibited, pre-dyed label complex containing antibody-ALP conjugate was also prepared by the indigo formation/tetazolium salt technique. A 600 μl incubation reaction was carried out at 25^βC by a sequential addition of the following components to 250 μl of 0.1 M AMPD buffer (pH 10.5) containing 0.1% (w/v) NaN₃: (a) 240 μl of 2.16 mp IP (3-indoxyl phosphate disodium salt) per ml of 0.1 M AMP (2- amino-2-methyl-i-propanol) buffer (pH 10.15) containing 0.1% (w/v) NaN₃; (b) 10 μl of 5 mg MTT/ml of 50% (v/v) DMF in deionized water, and finally to initiate the complex formation, (c) 100 μl of anti-hCG antibody-ALP conjugate in conjugate elution buffer at a final concentration from 0.25 μg/ml to 4.58 μg/ml. After l min incubation, 15 μl or 30 μl multiple aliquots were withdrawn, mixed with 25 μl or 10 μl aliquots of the sample, respectively, to obtain a final concentration of hCG 0 or 500 mlU/ml, and immediately applied to 4 mm wide lateral flow assay strips, prepared for a "wet" assay as described in section 1.2 above. Results based on the appearance of a visually discernible positive signal in the capture zone after 15 minutes reported in Table 3.

Table 3 ALP-Ab, hCG Concentration uσ/ml. in Standard¹

0 mlU/ml 500 mlU/ml

0.313 0.625

^L+ = Clearly discernible appearance of color in capture zone, and

No discernible appearance of color in capture zone. 1.5 Preparation of Uninhibited. Pre-dyed Label Complex

Containing Antibodv-DIA for use in a Two-Step "Wet" Assay.

Uninhibited, pre-dyed label complex containing anti- hCG monoclonal antibody-Diaphorase (DIA) conjugate was formed by enzyme-catalyzed reduction involving NADH cofactor of a suitable organic hydrogen acceptor substrate to a product exhibiting disparate absorption spectrum in a visible region.

A 1677.5 μl incubation was carried out at 25^βC by a sequential addition of the following components to 445 μl of 0.1 M sodium phosphate buffer (pH 8.0) containing 50 μg gentamycin/ml (a) 67.5 μl of 4.1 mg MTT in 15% (v/v) DMF in deionized water; (b) 67.5 μl of 10 mg BSA (bovine serum albumin per ml of the same buffer); (c) 64.5 μl of 10 mM NADH in the same buffer, and, finally to initiate the complex formation (d) 30 μl of 50 μg of anti-hCG monoclonal antibody-DIA conjugate in the same buffer. After 3 min incubation, 15 μl multiple aliquots were withdrawn, mixed with 25 μl samples of hCG standard to give the final hCG concentration of 0, 150 or

500 mlU hCG/ml, and immediately applied to 4 mm wide lateral flow assay strips, prepared for a "wet" assay as described in section 1.2 above. Results based on the appearance of a visually discernible positive signal in the capture zone after three minutes are presented in Table 4.

Table 4 hCG Concentration in the Standard Result³

0 mlU/ml 150 mlU/ml +

500 mlU/ml +

+ = Clearly discernible appearance of color in capture zone, and " ^{= No} discernible appearance of color in capture zone.

^1,6 Preparation of Uninhibited. Pre-dved Label Complex

Containing Antibodγ-HRP Conjugate for use in a Two-Step "Wet" Assay.

Uninhibited, pre-dyed label complex containing anti- hCG monoclonal antibody-HRP conjugate was formed by peroxide- mediated enzymatic conversion of colorless electron donor species to colored products. A 2016 μl incubation reaction was carried out at 25°C by a sequential addition of the following components to 360 μl of 3 mg of 4-chloro-l-naphthol per ml methanol: (a) 54 μl of 0.05 M Tris/HCl buffer (pH 7.5) containing 50 μg gentamycin/ml; (b) 1548 μl of 0.05 M Tris/HCl buffer (pH 7.5) containing 0.02% (v/v) H₂0₂, 0.1 mM EDTA

(ethylenediaminetetracetic acid) , and 50 μg gentamycin/ml; and finally to initiate the complex formation (c) 54 μl of 0.27 mg anti-hCG antibody/HRP conjugate in 0.05 M Tris/HCl buffer (pH 7.5) containing 50 μg gentamycin/ml. After 15 min incubation, the following reagents (chilled to 4^βC) were added: (a) 126 μl of 0.05M Tris/HCl buffer (pH 7.5) containing 50 μg gentaycine/ml; (b) 126 μl of 0.05M Tris/HCl buffer (pH 7.5) containing 0.02% (v/v) H₂0₂, 0.1 mM EDTA and 50 μg gentamycin/ml; and (c) 252 μl 100 mg mBSA/ml in 0.05M Tris/HCl buffer pH 8.0. Multiple 15 μl aliquots were withdrawn, mixed with 25 μl samples of hCG standard to give final concentration of 0, 150 and 500 mlU hCG/ml; and immediately applied to 4 mm wide lateral flow assay strips, prepared for a "wet" assay as described in section 1.2 above. Results based on appearance of a visually discernible positive signal in the capture zone after three minutes are presented in Table 5.

Table 5 hCG Concentration in the Standard Result¹

0 mlU/ml 150 mlU/ml +

500 mlU/ml +

+ - Clearly discernible appearance of color in capture zone, and - = No discernible appearance of color in capture zone.

Uninhibited, pre-dyed complex containing the antibody HRP conjugate was also prepared by reaction with electron-dens carbazole derivatives. HRP substrate was prepared by mixing o 50 μl of 0.40% (w/v) solution at 3-amino-9-ethylcarbazole, dissolved in dimenthylformamide with 940 μl of 50 mM sodium acetate buffer (pH 5.0), followed by addition of 10 μl of 0.3% (v/w) hydrogen peroxide in the same buffer. A 5 μl aliquot of anti-hCG antibody/HRP conjugate (0.34 mg/ml); prepared as described above in this section was added to 228 μl of HRP substrate solution. Following 4 hr. incubation at 25°C, 10 μl aliquots were withdrawn and immediately applied to 4 mm wide lateral flow assay strips assembled for "wet" assays as described in section 1.2 above. 20 μl hCG samples prepared in pooled male urine were added onto the sample receiving strip pad to give a final standardized concentrations of 0, 100 or 400 mlU hCG/ml. Finally, 10 μl portions of unspiked, apparently negative male urine pool were applied to the sample receiving zone to conclude the test. Results based on the appearance of a visually discernible positive signal in the capture zone after 8 minutes are reported in Table 6.

Table 6 hCG Concentration in the Standard Result¹

0 mlU/ml 100 mlU/ml + 400 mlU/ml +

^L+ = Clearly discernible appearance of color in capture zone, and

No discernible appearance of color in capture zone.

EXAMPLE 2

2.1 Preparation of Inhibited Pre-Dyed Label Complex Containing Antibodv-HRP Conjugate for use in a Two-Step "Wet" Assa^y

Inhibited pre-dyed label complexes for detection of hCG in body fluids were constructed to demonstrate that the complexes need not possess enzymatic activity for detection of hCG in the samples once pre-dyed anti-hCG complex was formed. Inhibited pre-dyed complexes are those where the activity of the enzyme in the initial antibody-enzyme conjugate has been inhibited following incubation of the antibody-enzyme conjugate with a suitable enzyme substrate to form the pre-dyed label complexes. Inhibited, pre-dyed label complex containing anti-hCG antibody/HRP conjugate were prepared as follows. A 2016 μl incubation reaction was carried out at 25^βC by a sequential addition of 360 μl of 3 mg of 4-chloro-l-naphthol per ml methanol of the following: (a) 54 μl of 0.05 M Tris/HCl buffer (pH 7.5) containing 50 μg gentamycin/ml; (b) 1548 μl of 0.05 M Tris/HCl buffer (pH 7.5) containing 0.02% (v/v) H₂0₂, 0.1 mM EDTA (ethylenediaminetetracetic acid) and 50 μg gentamycin/ml; and finally to initiate the complex formation (c) 54 μl of 0.27 mg anti-hCG antibody/HRP conjugate in 0.05 M Tris HC1 buffer (pH 7.5) containing 50 μg gentamycin/ml. After 15 min incubation, the following reagents (chilled to 4°C) were added: (a) 125 μl of 2 M NaN₃ in 0.05 M Tris/HCl buffer (pH 7.5) containing 50 μg gentamycin/ml; (b) 126 μl of 10% (w/v) PVP in 0.05 M Tris/HCl buffer (pH 7.5) containing 0.02% (v/v) H₂0₂, 0.1 mM EDTA and 50 μg gentamycin/ml; and 252 μl 100 mg is BSA/ml in 0.05 M Tris/HCl buffer (pH 8.0). Subsequently, 15 μl multiple aliquots were withdrawn, mixed with 25 μl samples of hCG standard to give the final concentration of 0, 150 and 500 lU hCG/ml; and immediately applied to 4 mm wide lateral flow assay strips, prepared for a "wet" assay as described above. Results based on the appearance of a visually discernible positive signal in the capture zone after 3 and 60 minutes are reported in Table 7. Table 7 hCG Concentration Result Result in standard at 3 min.¹ at 60 min.¹ 0 mlU/ml

100 mlU/ml + +

500 mlU/ml + +

¹+ = Clearly discernible appearance of color in capture zone, and

- = No discernible appearance of color in capture zone.

EXAMPLE 3

3.1 Preparation of Lateral Flow Assay Device for a "One-Step" Assay for hCG Utilizing Uninhibited Pre-Dved Label Complex

The test devices for a "one-step" assay were prepared as described above for a "wet" two-step assay by replacing the intermediate zone of the latter with a pre-dyed label pad prepared as follows. Pre-dyed label complex containing antibody-HRP conjugate was prepared in a 2016 μl incubation reaction at 25^βC by a sequential addition to 360 μl of 3 mg of 4-chloro-l-naphthol per ml methanol of the following: (a) 54 μl of 0.05 M Tris/HCl buffer (pH 7.5) containing 50 μg gentamycin/ml; (b) 1548 μl of 0.05 M Tris/HCl buffer (pH 7.5) containing 0.02% (v/v) H₂0₂, 0.1 mM EDTA and 50 μg gentamycin/ml; and finally to initiate the complex formation;

(c) 54 μl of 0.27 mg anti-hCG antibody/HRP conjugate in 0.05 M

Tris/HCl buffer (pH 7.5) containing 50 μg gentamycin/ml. After

15 minutes incubation, the following reagents (chilled to 4°C) were added: (a) 125 μl of 0.05M Tris/HCl buffer (pH 7.5) containing 50 μg gentamycin/ml; (b) 126 μl of 0.05M Tris/HCl buffer (pH 7.5) containing 0.02% (v/v) H₂0₂, 0.1 mM EDTA and 50 μg gentamycin/ml; and (c) 252 μl of 100 mg mBSA/ml in

0.05 M Tris/HCl buffer (pH 8.0).

The mixture was poured onto Sontara spunlace fabric at 38 μl/cm². The matrix was kept at room temperature for 20 minutes and abruptly frozen at -70^βc, along with the lyophilization flask for at least an hour. The resulting composition was lyophilized overnight on Virtis Freezemobile, and the intermediate pre-dyed labeling pads were cut into 10x4 mm rectangles with the spunlace fibers parallel to the longer side of the pad. The pads were then assembled as intermediate zones into a "one-step" device in a manner analogous to that described above for the "wet" two-step assay strips.

Assays were performed by the addition of 40 μl of 0, 150 or 500 mlU hCG/ml to the strip. Results based on the appearance of a visually discernible positive signal in the capture zone after 3 and 60 minutes are reported in Table 8.

Table β hCG Concentration Result Result in Standard at 3 min.¹ at 60 min.¹

0 mlU/ml 100 mlU/ml + +

500 mlU/ml + + + = Clearly discernible appearance of color in capture zone, and

No discernible appearance of color in capture zone. Although the foregoing invention has been described i some detail by way of illustration and example, for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A target-specific labeling complex comprising: an enzyme bound to a specific binding substance; and dye molecules complexed to the conjugate in an amount sufficient to impart a visually discernable color to the complex while leaving an active site of the specific binding substance available for binding to a target molecule, wherein the dye molecules were formed by reaction in aqueous phase of a substrate with the enzyme bound to the specific binding substance.

2. A labelling complex as in claim 1, wherein the specific binding substance is an antibody.

3. A labelling complex as in claim 2, wherein the enzyme is covalently attached to the antibody.

4. A labelling complex as in claim 1, wherein the enzyme is selected from the group consisting of oxidases, peroxidases, phosphatases , diaphorases, galactosidases, and oxidoreductases.

5. A labelling complex as in claim 1, wherein the enzyme is inactivated.

6. A labelling complex as in claim 1, wherein the complex includes from 10 to 10⁷ dye molecules.

7. A labelling complex as in claim 1, wherein the complex is lyophilized.

8. A test article comprising: a support matrix; and a target-specific labeling complex impregnated within the support matrix, wherein the complex comprises:

(i) a conjugate of an enzyme and a specific binding substance; and (ii) dye molecules complexed to the conjugate in an amount sufficient to impart a visually discernable color to th complex while leaving an active site of the specific binding substance available for binding to a target molecule.

9. A test article as in claim 8, wherein the target specific labeling complex is lyophilized within the support matrix.

10. A test article as in claim 8, wherein the suppor matrix is a solid matrix which conducts nonbibulous flow of a liquid sample.

11. A test article as in claim 8, wherein the suppor matrix is a solid matrix which conducts bibulous flow of a liquid sample.

12. A test article as in claim 8, wherein the suppor matrix is a fabric composed of a polyester, an acrylonitrile copolymer, rayon, glass fiber, cellulose, and blends thereof.

13. A test article as in claim 12, wherein the fabri has been treated with a blocking agent to render the fabric nonbibulous.

14. A test article as in claim 8, wherein the specific binding substance is an antibody.

15. A test article as in claim 14, wherein the enzy is covalently attached to the antibody.

16. A test article as in claim 8, wherein the enzym is selected from the group consisting of oxidases, peroxidases phosphatases, diaphorases, galactosidases, lytic enzymes, and oxidoreductases.

17. A test article as in claim 8, wherein the enzym is inactivated.

18. A test article as in claim 8, wherein the complex includes from 10 to 10⁷ dye molecules.

19. A test article as in claim 8, wherein the complex is lyophilized.

20. A method for preparing target-specific labeling complexes, said method comprising. providing a conjugate of an enzyme and a specific binding substance, wherein the enzyme is capable of converting a substrate to a colored dye product and the specific binding substance is capable of binding to a target molecule; and reacting the conjugate with the substrate in an aqueous medium under conditions which result in the complexation of the colored dye product with the conjugate.

21. A method as in claim 20, wherein the reaction conditions are selected to promote turnover of the substrate by the enzyme.

22. A method as in claim 20 wherein reaction between the conjugate and the substrate is stopped after the complex has obtained a desired color intensity.

23. A method as in claim 20, wherein the reaction is stopped by substrate exhaustion.

24. A method as in claim 20, wherein the reaction is stopped by altering the reaction conditions.

25. A method as in claim 20, wherein the reaction is stopped by inactivating the enzyme.

26. A method as in claim 20, wherein the specific binding substance is an antibody.

27. A method as in claim 26, wherein the enzyme is covalently attached to the antibody.

28. A method as in claim 20, wherein the enzyme is selected from the group consisting of oxidases, perosidases, phosphatases, diaphorases, galactosidases, lytic enzymes, and oxidoreductases.

29. In a device for detecting the presence of an analyte in a liquid sample, which device includes:

(a) a sample receiving zone comprising a solid support matrix which conducts lateral flow of liquid sample, in contact with

(b) a labeling zone comprising a solid support matri which conducts lateral flow of liquid sample and at least one assay label which specifically binds to or competes with the analyte, said labeling zone being in contact with (c) a capture zone comprising a solid support matrix which conducts lateral flow and in at least a portion thereof at least one capture reagent capable of binding said assay label or analyte, said capture zone being contiguous with (d) an absorbent zone, the improvement which comprises an assay label comprising: a conjugate of an enzyme and a specific binding substance; and dye molecules complexed to the conjugate in an amount sufficient to impart a visually discernable color to the complex while leaving an active site of the specific binding substance available for binding to a target molecule.

30. A device as in claim 29, wherein the specific binding substance is an antibody.

31. A device as in claim 29, wherein the enzyme is covalently attached to the antibody.

32. A device as in claim 29, wherein the enzyme is selected from the group consisting of oxidases, peroxidases, phosphatases, diaphorases, galactosidases, lytic enzymes, and oxidoreductases.

33. A device as in claim 29, wherein the enzyme is inactivated.

34. A device as in claim 29, wherein the complex includes from 10 to 10⁷ dye molecules.

35. A device as in claim 29, wherein the complex is lyophilized.