Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Definitions

• 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.
• target-specific labeling complexes comprising a specific binding substance, an enzyme, and dye molecules complexed thereto.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the enzyme will be inactivated that native enzyme activity is removed prior to use as a labeling complex according to the present invention.
• the complexed dye molecules are lyophilized remain intact, even when introduced to an aqueous reaction medium, as described hereinafter.
• each complex will comprise from 10 2 to 10 4 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 10 6 daltons) .
• the aggregate size of the complex will usually li between 10 3 and 10 11 daltons, more usually being between 10 6 a 10 10 daltons.
• 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.
• 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.
• 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.
• 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.
• the labeli complexes of the present invention have been found to remain intact when transported by liquid flow as just described.
• an assay can be run using the test arti based on capture of the labeling complex mediated by the specific binding substance.
• 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.
• 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.
• 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.
• no labeling zone is necessary and the analyte in the sample displaces the labeling complex into the absorbent zone.
• the absorbent zone acts to receive liquid sample it flows through the previous zones within the device.
• 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.
• 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.
• PEROXIDASES Horse radish aminoethylcarbazole; red peroxidase hydrogen peroxide
• PHOSPHATASES Alkaline indolyl phosphate indigo phosphatase Alkaline azonaphthol phosphate azonaphthol phosphatase
• GALACTOSIDASES indolyl galactoside indigo azonaphthol galactoside azonaphthol indolyl galactoside; formazan tetrazolium naphthol galactoside; azo diazo compound compound
• 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.
• biotin analogs/avidi biotin/streptavidin When natural specific binding substance exists, one may be prepar
• antibodies may prepared by well known techniques.
• 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.
• 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.
• 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.
• the specific binding substance-enzyme conjuga 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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 ⁇ to 200 ⁇ l, 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.
• 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.
• 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.
• 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.
• 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
• glucose oxidase GO
• HRP horseradish peroxidase
• DIA diaphorase I
• ALP calf intestine alkaline phosphatase
• 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 ⁇ m filtered conjugate elution buffer composed of 0.05 M sodium phosphate buffer (pH 7.0) containing 0.1% (w/v) NaN 3 , 0.03% (w/v) , MgCl 2 -6H 2 0, 0.003% (w/v) ZnS0 4 -7H 2 0, and 0.005% (w/v) Tween ® 20 was used throughout the fractionation.
• HEPES buffer pH 8.0
• DIA conjugate 0.1 M sodiu phosphate (pH 8.0).
• 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:
• 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) .
• BSA methylat bovine serum albumin
• the conversion to nonbibulous material was achieved by treatment at 38 ⁇ l/cm 2 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.
• 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 ⁇ m 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.
• 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") .
• GO substrate a colorless hydrophylic tetrazol salt as an electron acceptor
• 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 ⁇ g gentamycin/m
• ALP Antibodv-Alkaline Phosphatase
• 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”) .
• ALP substrate colorless organic phosphate
• ALP product colored phenolic product
• 550 ⁇ l incubation reaction was carried out at 25 ⁇ C by a sequential addition of the following components: to 250 ⁇ l 0.1 M AMPD (2-amino-2-methyl-l,3-propancdiol) buffer (pH 10.
• Uninhibited, pre-dyed label complex containing antibody-ALP conjugate was also prepared by the indigo formation/tetazolium salt technique.
• a 600 ⁇ l incubation reaction was carried out at 25 ⁇ C by a sequential addition of the following components to 250 ⁇ l of 0.1 M AMPD buffer (pH 10.5) containing 0.1% (w/v) NaN 3 :
• 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.
• DIA monoclonal antibody-Diaphorase
• a 1677.5 ⁇ l incubation was carried out at 25 ⁇ C by a sequential addition of the following components to 445 ⁇ l of 0.1 M sodium phosphate buffer (pH 8.0) containing 50 ⁇ g gentamycin/ml (a) 67.5 ⁇ l of 4.1 mg MTT in 15% (v/v) DMF in deionized water; (b) 67.5 ⁇ l of 10 mg BSA (bovine serum albumin per ml of the same buffer); (c) 64.5 ⁇ l of 10 mM NADH in the same buffer, and, finally to initiate the complex formation (d) 30 ⁇ l of 50 ⁇ g of anti-hCG monoclonal antibody-DIA conjugate in the same buffer. After 3 min incubation, 15 ⁇ l multiple aliquots were withdrawn, mixed with 25 ⁇ l samples of hCG standard to give the final hCG concentration of 0, 150 or
• 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 ⁇ l incubation reaction was carried out at 25°C by a sequential addition of the following components to 360 ⁇ l of 3 mg of 4-chloro-l-naphthol per ml methanol: (a) 54 ⁇ l of 0.05 M Tris/HCl buffer (pH 7.5) containing 50 ⁇ g gentamycin/ml; (b) 1548 ⁇ l of 0.05 M Tris/HCl buffer (pH 7.5) containing 0.02% (v/v) H 2 0 2 , 0.1 mM EDTA
• 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 ⁇ l of 0.40% (w/v) solution at 3-amino-9-ethylcarbazole, dissolved in dimenthylformamide with 940 ⁇ l of 50 mM sodium acetate buffer (pH 5.0), followed by addition of 10 ⁇ l of 0.3% (v/w) hydrogen peroxide in the same buffer.
• a 5 ⁇ l aliquot of anti-hCG antibody/HRP conjugate (0.34 mg/ml); prepared as described above in this section was added to 228 ⁇ l of HRP substrate solution. Following 4 hr.
• 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 ⁇ l incubation reaction was carried out at 25 ⁇ C by a sequential addition of 360 ⁇ l of 3 mg of 4-chloro-l-naphthol per ml methanol of the following: (a) 54 ⁇ l of 0.05 M Tris/HCl buffer (pH 7.5) containing 50 ⁇ g gentamycin/ml; (b) 1548 ⁇ l of 0.05 M Tris/HCl buffer (pH 7.5) containing 0.02% (v/v) H 2 0 2 , 0.1 mM EDTA (ethylenediaminetetracetic acid) and 50 ⁇ g gentamycin/ml; and finally to initiate the complex formation (c) 54 ⁇ l of 0.27 mg anti-hCG antibody/HRP conjugate in 0.05 M Tris HC1 buffer (pH 7.5) containing 50 ⁇ g gentamycin/ml.
• 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 ⁇ l incubation reaction at 25 ⁇ C by a sequential addition to 360 ⁇ l of 3 mg of 4-chloro-l-naphthol per ml methanol of the following: (a) 54 ⁇ l of 0.05 M Tris/HCl buffer (pH 7.5) containing 50 ⁇ g gentamycin/ml; (b) 1548 ⁇ l of 0.05 M Tris/HCl buffer (pH 7.5) containing 0.02% (v/v) H 2 0 2 , 0.1 mM EDTA and 50 ⁇ g gentamycin/ml; and finally to initiate the complex formation;
• Tris/HCl buffer pH 7.5 containing 50 ⁇ g gentamycin/ml.
• the mixture was poured onto Sontara spunlace fabric at 38 ⁇ l/cm 2 .
• the matrix was kept at room temperature for 20 minutes and abruptly frozen at -70 ⁇ c, 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.

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