WO1990006372A1 - Substrates for peroxidase assaying - Google Patents

Substrates for peroxidase assaying Download PDF

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WO1990006372A1
WO1990006372A1 PCT/US1989/005407 US8905407W WO9006372A1 WO 1990006372 A1 WO1990006372 A1 WO 1990006372A1 US 8905407 W US8905407 W US 8905407W WO 9006372 A1 WO9006372 A1 WO 9006372A1
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c4
c1
alkyl
hydrogen
mono
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PCT/US1989/005407
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French (fr)
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Nicolaas Marthinus Johannes Vermeulen
Charles Robert Petrie
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Microprobe Corporation
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581Chemical 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)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2326/00Chromogens for determinations of oxidoreductase enzymes
    • C12Q2326/50Phenols; Naphthols; Catechols

Abstract

This invention relates to assay methods for in situ detection of analytes bound to a solid support. In particular, this invention relates to a method of assaying peroxidase activity in immunoassays or nucleic acid hybridizations using substrates which form highly colored water-insoluble products after contact with peroxidase enzyme.

Description

SUBSTRATES FOR PEROXIDASE ASSAYING

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to assay methods for in situ detection of analytes bound to a solid support. In particular, this invention relates to a method of assaying peroxidase activity in immunoassays or nucleic acid hybridizations using substrates which form highly colored water-insoluble products after contact with peroxidase enzymes.

Description of the Prior Art

Peroxidases are widely used as signal enzymes in enzyme-lin ed-immunoassays (ELISA) and in nucleic acid hybridization assays. Previously, investigators have emphasized the identification and synthesis of substrates that yield products that are water soluble and suitable for spectrophotometric analysis. (W.D. Goeghegan, in "Enzyme Mediated Immunoassays", T.T. Ngo and H.M. Lenhoff, eds. Plenum Press (1985) pp. 451-465.)

For example, in the peroxidase catalyzed reaction between 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) , selected amines and naphthols as described by Dunford and Adeniran, Arch, of Biochim. Biophys. f 251: 536-542 (1986) , investigators have tried to design compounds that give products with high extinction coefficients that are soluble in water.

One of the most sensitive peroxidase assays using MBTH as a substrate is described by Ngo and Lenhoff, Anal. Bioche . , 105:389-397 (1980). In that reaction, MBTH and 3-(dimethylamino) benzoic acid (DMAB) react in the presence of peroxidase and hydrogen peroxide to yield a indamine dye with a high extinction coefficient. Certain naphthols are known to act as substrates for peroxidase. For example, 4-chloronaphthol functions as substrate that yields a colored precipitate. Elias, Am. J. Clin. Pathol.. 73:797-799 (1980). A recent patent application (EP 167,340) claims 4-methoxynaphthol (MN) as a substrate for peroxidase.

U.S. Patent No. 4,743,541 discloses inhibitors of peroxidase reactions.

Certain indoles are known as substrates for peroxidase. U.S. Pat. No. 4,609,544 and Nelson, D.R. and

Huggins, A.K. The Interaction of 5- Hydroxytryptamine and

Related Hydroxyindoles with Horseradish and Mammalian

Peroxidase Systems, Biochemical Pharmacology. 24:181-192

(1975) . Although others have developed sensitive peroxidase assays that produce water soluble products, there is currently a need for sensitive assays which produce water insoluble products suitable for detection of peroxidase in situ assays and assays where colors are deposited upon a solid support.

SUMMARY OF THE INVENTION

This invention relates to a method of assaying for peroxidase activity which comprises the combining of a hydrazone with either a naphthalene or indole compound through enzymatic catalysis by peroxidase enzymes to give a colored precipitate. More specifically this method comprises the converting of a naphthalene compound of formula I, with H202 to yield a H202 reactive product of formula Ila or lib and converting the reactive product with a hydrazone compound of formula III to yield a colored precipitate, wherein:

R1 is in either the 1 or 2 position and is hydrogen, monosuccinyl, phosphono, sulfo, sulfino, nitro, or an acyl group -C(:0)-R8 wherein R8 is an alkyl (C1-C4) , an aryl (C6-C8) , a carboxyalkyl (C2-C5) , a carboxyaryl

(C7-C11) , or a carboxyalkylaryl (C8-C14) ; R2, R3, R4, R5 can be the same or different and are hydrogen, hydroxy, alkyl (C1-C4) , alkoxy (C1-C4) , alkenyl (C2-C6) , arylalkyl (C7-C14) , halo, nitro, aminocarbonyl, mono or dialkylaminocarbonyl having alkyls of C1-C4, sulfamido or mono or dialkylsulfa ido having alkyls of C1-C4, acyl of R9-C(:0)- wherein R9 is an alkyl (Cl to C4) , mono or dialkylamino having alkyls (C1-C4) , arylalkoxy (C8-C14) or alkoxy (C1-C4) ;

R6 is hydrogen, hydroxy, alkyl (C1-C4) , alkyloxycarboxyl (C2-C5) , alkoxy (C1-C4) , aminocarbonyl, mono and dialkylaminocarbonyl (C1-C4) , sulfamido, mono and dialkylsulfamido having alkyls (C1-C4) , an alkox sulfonyl (C1-C6) , acyl of -C(:0)-R10 wherein R10 is alkyl (C1-C4) , aryl, halo or benzo (fused ring) ; and R7 is an alkyl (C1-C4) , or arylalkyl (C7-C11) .

It is preferred that R1 is hydrogen, nitro, phosphono, sulfo, sulfino, monosuccinyl or an acyl group of —C:0-R8 in either the 1 or 2 position wherein R8 is alkyl (C1-C4) , aryl (C6-C8) , carboxyalkyl (C2-C5) ; carboxyaryl (C7-C11) , or carboxyalkylaryl (C8-C14) . It is more preferred that R1 is hydrogen, succinyl, or an acyl group of -C(:0)-R8 wherein in R8 is methyl. It is most preferred that the above method involve a naphthalene compound of formula I wherein R1 is a hydrogen or succinyl.

Alternatively it is also preferred that the above method be performed using substrates of formula I and III wherein R1 is hydrogen, monosuccinyl, phosphono or an acyl group of -C:θ-R8 wherein R8 is a methyl or carboxyethyl; R2, R3, R4, Rs can be the same or different and are hydrogen, alkyl (C1-C4) , alkoxy (C1-C4) , halogen, hydroxyl, alkyl (C1-C4) , alkoxycarbonyl (C2-C5) , sulfamido, or mono- and dialkylsulfamido having alkyls (C1-C4) ; and R7 is alkyl (C1-C4) . It is particularly preferred wherein R1 is hydrogen or monosuccinyl; R2 is hydroxy, chloro, bromo, methoxy or hydrogen; R3 is hydrogen, hydroxy, chloro, bromo, methoxy, ethoxy or propoxy; R is hydroxy, hydrogen, methoxy, chloro, bromo, or methyl; R5 is hydroxy, bromo, chloro, hydrogen or methoxy; R6 is hydrogen, benzo, sulfamido or alkoxysulfonyl; and R7 is methyl or ethyl. It is especially preferred wherein the naphthalene compound is selected from the group comprising: 4-methoxynaphthol, 4-ethoxynaphthol, 4-propyloxynaphthol, 4-chloronaphthol, l-bromo-2-naphthol, 6-bromo-2-naphthol, 4- methoxynaphthylsuccinate, 4-ethyloxynaphthylsuccinate, 4-propyloxynaphthyl-succinate, 2,6 dihydroxynaphthalene, 1,5 dihydroxynaphthalene, 7-methoxy-2-naphthol, 3- methoxy-2-naphthol or 6-methoxy-2-naphthol. The preferred hydrazone compounds for use in this invention are 3-methyl-2-benzo-thiazolinone hydrazone, 3-ethyl-2- benzo-thiazolinone hydrazone, methyl-2-naphthol- thiazolenone hydrazone, 3-methyl-6-dimethylsulfamido-2- benzothiazolenone or 3-methyl-6-methoxysulfonyl-2- benzothiazolinone hydrazone.

The indoles of this invention are selected from the group represented by formula V wherein R11 is hydrogen, or alkyl (C1-C4) ; R12 is hydrogen, alkyl (Cl- C4) , alkoxycarbonyl (C2-C4) or aminocarbonyl, mono and dialkylaminocarbonyl; R13 is hydrogen, alkyl (C1-C4) , alkoxycarbonyl (C2-C4) , or aminocarbonyl, mono and dialkyl aminocarbonyl; R14 is hydrogen, alkyl (C1-C4) , amino, hydroxy, halo, alkoxy (C1-C4) , alkoxycarbonyl (C2- C4) and R15 is hydrogen, alkyl (C1-C4) , hydroxy, amino, halo, alkoxy (C1-C4) or nitro. The preferred hydrazones for use in this method are as previously described. Preferred indoles include those of formula V wherein R11 is hydrogen; R12 is hydrogen; R13 is hydrogen, methoxycarbonyl; R14 is hydrogen, amino, hydroxy, halo, methoxycarbonyl; and R15 is hydrogen, hydroxy, amino, halo, methoxy or nitro. The preferred assay formats include nucleic acid hybridization sandwich-type assays and enzyme linked immunoassays. The preferred assays include a solid support surface comprising an activated moiety selected from the group consisting of carboxy, amino, thiohydroxy or sulfo. Most preferred are supports comprising carboxy activated latex beads. It is preferred that the concentration of inhibitors to peroxidase catalyzed reactions be minimized. By using known methods, the preferred reactants of this invention can be purified such that assay sensitivity and reproducibility are maximized. Such reagents are substantially free of peroxidase inhibitors. This is empirically determined by the obtention of a reagent with a purity such that additional purification steps yield statistically insignificant improvements in assay sensitivity and reproducibility. For oxidation by-products of naphthols, inhibition appears at concentrations above about 25 μM.

The methods and substrates of this invention are particularly useful for the detection of nucleic acid in biological samples from mammalian tissues. More particularly this invention will provide for the detection of nucleic acid originating from an infectious organism.

Novel substrates that are preferred include: mono(4-ethoxynaphthy1)succinate, mono(4-propoxynaphthyl)- succinate and mono(4-methoxynapthyl)succinate.

The invention disclosed herein can be utilized in the development of a kit for assaying for peroxidase activity in an immunoassay or nucleic acid hybridization assay which involves the combining of a hydrazone with a naphthol compound by the enzymatic catalysis of peroxidases to give a colored precipitate, said kit comprising a solid support, a peroxidase-type enzyme, a solution of hydrazone, naphthol substrate and hydrogen peroxide. The various hydrocarbon moieties are defined as follows: Alkyl refers to aliphatic hydrocarbon radical either branched or unbranched such as methyl, ethyl, n- propyl, isopropyl, or the like.

Arylalkyl refers to an alkyl bound to an aryl moiety where the alkyl functions as a bridge between the aryl and the nucleus.

Alkoxy refers to alkyl radicals which is attached to the remainder of the molecule by oxygen such as methoxy, ethoxy, n-propoxy, isopropoxy or the like. Alkenyl refers to a radical of an aliphatic unsaturated hydrocarbon chain having a double bond and includes both branched and unbranched forms such as vinyl, allyl, isopropenyl or the like.

Aminocarbonyl refers to the radical -C(:θ)-NH Aryl refers to a radical derived from an aromatic hydrocarbon by removal of one hydrogen atom such as phenyl, α-naphthyl or the like.

Benzo, when a moiety on a benzyl ring, refers to a fused carbon bridge that forms a naphthalene ring. Carboxy refers to a radical -C00H.

Carboxyalkyl, carboxyaryl, and the like refer to radicals possessing a carboxyl group (e.g., -CH -COOH) .

Dialkyl substituted moieties may be the same or different unless otherwise stated. Halo refers to halogen radicals which include chloro, bromo, iodo and fluoro.

Phosphono refers to a radical -(0:)P-(OH) .

Sulfonamido refers to monovalent radicals of the formula: -SO 2„NH2 Sulfino refers to monovalent radicals of the formula: : -~SS0022HH

Sulfo refers to monovalent radicals of the formula:: -_SS0033HH Succinyl (succinate) refers to the bivalent radical:

-C(:O)-CH2-CH2-C(:O)- Unless otherwise indicated, in the above description and throughout this document: (a) the parenthetical term (Cn-Cm) is inclusive such that (C1-C4) indicates compounds of one, two, three, and four carbons and their isomeric forms; (b) in groups specifying a carboxy or carbonyl group, such as carboxyalkyl (C2-C4) , the carbonyl atom is included as one of the carbon atoms, thus limiting the total carbons in the alkyl to no more than three; and, (c) where two multiple carbon moieties are present, such as arylalkyl (C7-C14) , the number of carbon atoms in both moieties taken together does not exceed fourteen.

It will be apparent to those skilled in the art that compounds of this invention may contain chiral carbons. All of the racemic mixtures and optically active enantiomers of the compounds are included within the scope of the invention. The invention also includes both the individual isomers and mixtures.

DETAILED DESCRIPTION This invention provides those skilled in the art with a sensitive method of assaying peroxidase activity by the enzymatic oxidation of a hydrazone and a substituted naphthalene compound to form a colored precipitate. By modifying the colored end product to achieve greater insolubility, assay sensitivity is increased dramatically. The present invention is a novel modification of the Ngo/Lenhoff reaction in which MBTH and 3-(dimethylamino) benzoic acid (DMAB) react in the presence of peroxidase and hydrogen peroxide to yield a water soluble indamine dye. In this invention, novel water insoluble secondary substrates such as naphthols or indoles, and aryl amines are substituted for DMAB substrates. The naphthalenes are dissolved in an organic solvent such as dimethyl for amide (DMF) or acetonitrile (CH CN) and added to the enzyme incubation mixture. Enough DMF or CH CN are used to keep the substrates in solution but not to prevent the action of the enzyme.

Reaction conditions in the present invention can be any of those well known in the art for assaying peroxidase activity. Suitable buffers for assaying peroxidase activity include organic or inorganic buffers with a slightly acidic pH. Although preferred buffers are 0.1 M citrate buffer, pH 4.5 to 6.5, and 0.1 M citrate-phosphate, [CAP] buffer pH 6.5, alkaline buffers such as 0.1M carbonate pH 8-9 can also be used. It has been observed the 4-methoxynaphthol, for instance, works best for in situ detection of human papilloma virus in 0.1M citrate pH 5.5 while l-bromo-2-naphthol detects this virus best in 0.1M CAP pH 6.5. Hydrazone concentration can range from 10 to 500 μM. Naphthalene concentration can range from 1 mM to 20 mM. The preferred range is from 5 mM to lOmM. Hydrogen peroxide concentration can range from 0.5mM to 5mM. The preferred concentration is 4mM. Depending on the solubility of the reactants, DMF or CH CN concentration can range from 1% to 20%. The preferred concentration is 4%. Temperature can range from 15°C to 37"C. For the shortest assay time, the preferred temperature is 37°C. The temperature should be carefully maintained at 37"C, however, because the enzyme denatures at 40°c to 50°C.

Any peroxidase enzyme well known in the art can be used in this invention. Suitable enzymes include, but are not limited to, horseradish peroxidase, microperoxidase, lactoperoxidase, catalase and the like. The mechanism of peroxidase activity is not entirely understood. Goeghagan (1985) has suggested that peroxidase catalyzes the oxidation of the hydrazone (for example MBTH) by hydrogen peroxide to yield a reactive cation which reacts with a nucleophilic center on a substituted aromatic a ine, phenol or naphthol. Moreover, certain naphthols can also act as substrates for peroxidase. For example, 4-chloronaphthol functions as substrate that yields a colored precipitate. Elias, Am. J. Clin. Pathol., 73:797-799 (1980). Guilleme et al., Anales de Quimica, 78:83-86 (1982) suggest a radical mechanism for the chemical oxidation of 4-methoxynaphthol (MN) to a dimeric quinone. However, we could show by thin layer chromatography (TLC) that the same dimeric quinone is produced in the enzymatic oxidation of MN.

While not being bound to any specific theory, there is evidence that, even in the presence of a hydrazone, peroxidase catalyzes the oxidation of naphthol compounds to a dimeric quinone (Formula Ila or b) which subsequently reacts with the hydrazone. III, to give a final product (Formula IV) . Compounds of Formula Ila are fairly insoluble in water and can be used to detect peroxidase activity in situ. However, compounds of Formula IV, are much more insoluble and thus decrease background problems in the assay (see Example 2) . The mechanism for reactions involving indoles are also not well understood. It is presumed that the mechanism is similar to that described above for the naphthalenes.

The preferred naphthalene compounds are as described in the summary of the invention. Naphthalene compounds are selected that are soluble in dilute DMF and CH CN solutions and yield insoluble end products. If the naphthalenes are particularly insoluble, they can be derivatized with monosuccinates or other moieties which increase their solubility in the buffered enzyme solution but do not impair their ability to produce insoluble end products. The preferred indoles are as described in the summary of the invention. As with the naphthalene compounds, indoles are selected that are soluble in dilute DMF solutions and which will yield insoluble end products. The preferred hydrazones are as described in the summary of the invention. Hydrazones are selected that react with the naphthalene compounds in the presence of peroxidase and hydrogen peroxide to produce a colored precipitate.

The napthalenes, indoles and hydrazones are either available commercially or prepared using commercially available starting materials using standard processes for organic synthesis. The methoxynaphthols can be synthesized from the dihydroxynaphthalenes using the procedures described by Elsevier's Encyclopedia of Organic Chemistry. Series III, 12B. (Edited by Elsevier Publishing Co. Inc., NY, page 1977 (1950) and G.N. Vyas and N.M. Shah Org. Syn. Coll. Vol. 4:837 (1963).) The hydrazones can be synthesized by the method described by Ebsthorn. Ber Dtsch. Chem. Ges., 43:1524 (1910).

The substrates described in this invention are suitable for any assay procedure that requires an in situ deposit of insoluble colored products which is then visually or mechanically detected. Applications include identification of nucleic acids or proteins bound to membranes after Southern, Northern or Western transfers. The invention is also useful in sandwich immunoassays in which the immunologically active molecule of interest is bound to a solid support.

By the term precipitate, it is meant that the insoluble colored product deposits in a defined region onto the solid support. The color products can be measured using conventional colorimetric devices such as a reflecting spectrophotometer or a densitometer. It is preferred that the colored products be able to withstand several aqueous washes without reduction in color density of no more than about 15%, preferably less than 10%, and most preferably less than 5%. Typical washings comprise 3 one-minute washes without mechanical agitation.

Peroxidase catalyzed reactions are readily inhibited by by-products generated when synthesizing the reactants. Known inhibitors include hydrazine and sulfide ions. Therefore it is optimal to avoid or to eliminate such inhibitors from the reaction components. 4-methoxy naphthol (4-MN) is a preferred reactant which is commercially available. Purifying 4-MN using high pressure liquid chromatography provides a surprisingly reproducible and sensitive assay when compared to the reagent grade 4-MN available commercially. Any of several known purification methods can be used to eliminate these inhibitors, including different conventional chromatographical procedures, and crystallization. It could be shown that some commercial preparations of 4-MN inhibit HRP activity relative to HPLC purified 4-MN. A difference in activity on Pall membranes of 10-250 fold could be detected between different batches. The inhibitor could be isolated from commercially available 4-MN preparations and was identified as 1,4-naphtoquinone. The 1,4-naphtoquinone (NQ) probably formed by oxidation from 1,4- dihydroxynaphthalene, the starting material for the synthesis of 4-MN. Purified 4-MN was spiked with NQ and it was observed that the minimum amount necessary to show inhibition was about 25 μM. Therefore it is useful to maintain the concentration of oxidation contaminants below this concentration level.

The disclosed invention has application in many fields where peroxidase enzymes are involved as a means for detection. Such areas include histological procedures, immunoassays and nucleic acid hybridization assays.

A preferred means for conducting the assays, for which this invention is particularly well suited, involves the use of chemically activated solid supports. The solid supports used in this invention include plastics such as, polystyrene, polypropylene, or polycarbonate or nylon (Nytran™) , and glass or ceramics in any conformation or shape amenable to the desired assay format including a dipstick. Further examples include porous polypropylene frits or porous glass, and nonporous material such as flat polystyrene sheets, microtiter plates and wells, glass tubes and the like. The surfaces can be chemically activated by the addition of moieties such as sulfo groups, amino groups, or carboxy groups. Modification with carboxy groups is preferred. Carboxy groups can be conveniently applied to a solid surface using Carboxy-Modified Tube Coating from Seragen Diagnostics, Inc., Indianapolis, IN.

Target oligonucleotides are covalently bound to the solid supports by activated groups tethered to the oligonucleotides. These methods are known. More specifically, synthetic oligonucleotides of between 20 and 100 bases are obtained using conventional means. During synthesis a linker arm containing a blocked amine group can be coupled using conventional chemistry to the 5•-hydroxy1 group of an oligonucleotide.

The activated oligonucleotides used as starting materials for this invention can be derived through several methods. The reagents for the attachment of primary linker arms terminating in a primary amine are commercially available. A primary amine is the preferred group for attachment to the heterobifunctional reagent, and its attachment via a hexyl arm is preferred. Starting materials suitable for use in this invention are described in PCT U.S. 86/01290; Nucl. Acids Res., 15:3131 (1987); Nucl. Acids Res., 15:2891 (1987); and Nucl. Acids Res., 14:7985 (1986). Alternative means are described in U.S. SN 230,066 filed August 9, 1988 and 148,258, filed January 25, 1988 (see in particular pages 4-8) , both of which are incorporated by reference herein.

Once the oligonucleotide is covalently bound to the solid support surface, it is useful as a probe in nucleic acid hybridization assays of all types. These assays have numerous applications in the medical and biological sciences as well as in numerous industrial settings. A general review of these procedures can be found in Nucleic Acid Hybridization, A Practical Approach, Eds. Hames, B.D. and Higgins, S.J., IRL Press, Washington D.C. (1985) ; Hybridization of Nucleic Acids Immobilized on Solid Supports, Meinkoth, J. and Wahl, G. , Anal. Biochem., 238:267-284 (1984); and U.S. Patent No. 4,358,535.

The detection of peroxidase activity using precipitated color products can be used to develop analytical procedures for the detection of viruses, rickettsia, bacteria, fungi, chromosomal abnormalities, and the like in body tissues and fluids. Nucleic acids from a sample can be hybridized with biotinylated oligonucleotide probes or other oligonucleotide enzyme conjugates according to known techniques.

The subtrates of this invention are useful as a component in a multiple component kit for the purpose of conducting the above-mentioned assays. Such kits will comprise in addition to the peroxidase substrates or this invention, instructions, various containers of buffers for conducting the assays and washing the reactants, reactants which would include a solid support having target molecules (such as antibodies or oligonucleotides) bound to the surface, appropriate positive and negative controls (test molecules) as well as ligands comprising peroxidase for binding to the test molecules or analyte molecule being assayed.

The following examples are offered by way of illustration and are not to be construed as a limitation of the claims.

EXAMPLE 1 Immunoassays on Pall Immunodyne Membranes

Biotinylated anti-goat IgG in 0.5 M sodium phosphate buffer pH 7.0 was applied to a Pall Immunodyne membrane in a Schleicher and Schuell Minifold II slot blot manifold in serial dilutions from 1.0 ng/10 μl to 0.0001 ng/10 μl. The remaining active groups were inactivated with 3% BSA in lx phosphate buffered saline pH 7.0 (PBS) at 65°C for 1 hr. These membranes were then incubated in 3% BSA in lxPBS containing streptavidin- horseradish peroxidase (Bethesda Research Laboratories) in a 1:250 dilution for 30 minutes. The membranes were then washed once with 3% BSA in PBS, and 3x 0.1% Tween, 0.1 M NaCl in PBS and stored until use.

The peroxidase activity on the membranes were determined in either 0.1M citrate buffer, pH 4.5 to 6.5, or a 0.1 M citrate-phosphate (CAP) buffer, pH 5.0 to 7, containing 90 μM 3-methyl-2-benzothiazolinone hydrazone, 6 mM naphthol and 4 mM hydrogen peroxide. The color production is usually complete in 15-30 minutes.

Using the above procedure, the color producing potential of a number of naphtols and phenols were evaluated with the results as reported in Table 1.

Table 1. The sensitivity and color producing ability of HRP substrates.

Figure imgf000017_0001
Compound

2,4-dibromonaphthol

4-amino-l-naphthalenecarbonitril vanallinazine l-bromo-2-naphthyl succinate

6-bromo-2-naphthyl succinate

4-ethoxynaphthol

4-ethoxynaphthol succinate 4-propyloxynaphthol

4-propyloxynaphthol succinate

1,2-dihydroxynaphthalene

1,3-dihydroxynaphthalene

1,5-dihydroxynaphthalene 1,6-dihydroxynaphthalene

2 r3-dihydroxynaphthalene

2,6-dihydroxynaphthalene

2,7-dihydroxynaphthalene

2-methoxy-1-naphthol** 3-methoxy-l-naphthol**

5-methoxy-l-naphthol

6-methoxy-1-naphthol**

3-methoxy-2-naphthol

6-methoxy-2-naphthol 7-methoxy-2-naphthol

Figure imgf000018_0001

* Detection of ng of biotinylated IgG immobilized on the Pall membrane in lOul.

** Mixture of both positional isomers.

EXAMPLE 2

A comparison of the ability of 4-methoxynaphthol in the presence and absence of 3-methyl-2-benzothiazolinone hydrazone to form a color precipitate by the action of peroxidase.

A comparison study of the ability of a) 4- methoxynaphthol(MN) alone; b) 4-methoxy-naphthol/MBTH (MN/MBTH) and c) 4-methoxynaphthyl succinate/MBTH (MNS/MBTH) to detect HRP on Pall membranes as prepared in Example 1 was conducted. The concentrations of MBTH, H202 and naphthyl derivative were lOμM, 4mM and 6 mM respectively in 0.1 M CAP buffer at pH 6.5. The results are summarized in Table 2 and indicate that MN and MBTH work better than MN alone, since less background color is detected in the incubation solution.

While not wishing to be bound by any specific theory, there is evidence to support the hypothesis that, even in the presence of MBTH, the HRP oxidizes the naphthol compounds to a dimer (I) which subsequently reacts with MBTH to give the final color (II) . This hypothesis is supported by the discovery that final color product of the single step reactions described above are identical to the colored product formed in a two step HRP reaction where the naphthol is oxidized first with HRP to give the initial color followed by the addition of MBTH.

Table 2. Comparison of the precipitate and background on Pall membranes as well as the amount of color in the incubation solution.

Color in Solution Substrate(s) Color Background (650 mu)

OD 0.484 0.114

Figure imgf000020_0001
<0.114

***** = severe background

** = acceptable background

* = no significant background

Where the substrates included both MBTH and MN, the enzyme catalyzed a reaction resulting in a strong blue-purple precipitate with very little purple color in solution. This suggests that the proposed end products are less soluble than the napthalene dimer. Furthermore, it could be seen in Table 3 that 4-chloronaphthol (4CN) , l-bromo-2-naphthol(lBr2N) , and 6-bromo-2-naphthol(6Br2N) gave blue-black, yellow and yellow colors respectively as substrates alone in the HRP catalyzed reaction. The same substrates in combination with MBTH gave bright red colors in the HRP catalyzed reaction. In addition, when the three substrates are incubated without the MBTH for any period of time the blue-black and yellow colors could be changed to the bright red colors by the addition of MBTH at a later stage. It is also important to note that the level of sensitivity increases from about 1 to 0.01 μg/lOμl in the case of these three substrates when MBTH is added to form the precipitate on membranes. Table 3. The precipitate colors and sensitivities of naphthols alone and in combination with MBTH on Pall membranes.

Figure imgf000021_0001

EXAMPLE 3

Synthesis of 4-Methoxynaphtyl Succinate (MNS)

In an open beaker 4-methoxy-l-naphthol (0.5g; 2.9mmol), succinic anhydride (1.5g; 14mmol) , and 2- methoxyethyl ether (2ml) were heated on a hot plate for 3 hrs, then cooled. The mixture was adsorbed on silica gel and put on a column of silica gel. The column was eluted using a step gradient from 100% hexane to 40% acetone in hexane in 10% increments. Fractions containing the desired product were pooled and evapoated to dryness under reduced presure to yield 0.4g (55%) of a solid with an mp 119-120°C, which showed one spot on tic(acetone: hexane, 1:1) and gave an elemental analysis which agreed with the assigned structure. MNS was kept in the dark.

EXAMPLE 4 Synthesis of 4-Methoxynaphthyl Acetate

4-Methoxy-1-naphthol (0.5g; 2.9mmol) was added to cold acetic anhydride (5ml) . 4-Dimethylaminopyridine

(0.35g; 2.8mMol) was added with stirring and the reaction mixture was then removed from the ice bath and allowed to come to room temperature, then stirred for 5 hrs. The reaction mixture was then poured into water, extracted with ethyl acetate, and dried over sodium sulfate. Silica gel chromatography using a gradient from 100% toluene to 5% ethyl acetate/95% toluene gave an oily product (0.41g, 59%) which showed one spot on silica gel tic with ethylacetate: toluene (1:5) as mobile phase.

EXAMPLE 5 Purified 4-Methoxynaphthol

4-Methoxynaphthol (90 mg/0.5ml acetonitrile) was separated on a Rainin Dyna ax preparative C-18 column on a Rainin Rabbit-HP HPLC using a flow rate of 20ml/min. Using a gradient of CH3CN/H20 from 40 to 50% in 10 minutes gave satisfactory separation. The major peak was collected, evaporated to dryness and showed one spot on silica TLC in toluene:ethylacetate 5:1. Purified and commercially available 4- methoxynaphthol(Aldrich) was evaluated on Pall membranes for sensitivity. The purified 4-methoxynaphthol showed increased activity of about 10-250 fold over the freshly prepared unpurified compound from different batches.

EXAMPLE 6 a. The Synthesis and Evaluation of 4-Ethoxynaphthol

1,4 dihydroxynaphtalene (3.0g; 1.875 mmol) dissolved in dry ethanol (270 ml) and dry HC1 gas was bubbled through until saturated. (Solution becomes hot) . The reaction solution was stirred for 36 hours when it was added to 500 ml H20 and extracted with ethylacetate. The ethylacetate was removed under vacuum and the product recrystallized from acetic acid water mixture to yield 2.0g, 57% with a p 80-90°C. Elemental analysis agreed with the assigned structure.

4-Ethoxynaphthol [4-EN] was evaluated on Pall membranes with and without MBTH following the procedures of Example 1 substituting 4-EN for 4-MN. It was observed that the precipitate obtained without the MBTH was slightly more sensitive (about 5 fold) than in the presence of MBTH.

b. The Synthesis of 4-Propoxynapthol

4-Propoxynaphthol was synthesized (as described in Example 6a) and similarly recrystallized from acetic acid/H O in a 53.9% yield with a melting point of 92°-

95°C. Elemental analysis agreed with the assigned structure.

EXAMPLE 7 The Detection of Human Papilloma Virus (HPV) in Caski Cervical Tissue Culture Cells Using the MN/MBTH Substrate

Caski cells immobilized on glass slides are fixed in ethanol for 5 minutes and then dried. Hybridization solution pH 8.5 containing 40% deionized formamide, 10% dextrane, 20mM EPPS buffer, 200μg/ml Salmon sperm DNA, 200μg/ml yeast tRNA, 0.05% NaPPi, lx Denharts, 500mM, NaCl and 0.01 to 2μg/ml biotinylated HPV16 probe was added to the slide and placed in a plastic box, covered with saran wrap, denatured for 12 minutes at 90°C and hybridized at 42°C for 60 minutes. The slide was washed for 10 minutes in 50% formamide containing 50mM NaCl and then in lx PBS for 5 minutes. The slide was then incubated for 25 minutes at room temperature in a mixture containing l-50μg/ml streptavidin-horseradish peroxidase in lx PBS, 1% BSA and 5mM EDTA. The slide was then washed for 10 minutes in Tris pH 9.0, followed by 2 washes in PBS for 5 minutes each. The slide was then incubated in substrate solution in the dark containing llμM MBTH, 6mM naphthol and 4mM HO in either 0.1 citrate-phosphate pH6.5 or 0.1M citrate pH 5.5 buffer. The slide is then rinsed in PBS, stained and mounted.

Table 4 lists the amount of probe in μg/ml that gives a strong signal of the granuar HPV integration sites in the nucleus. Table 4. Concentration probe necessary to detect HPV 16 in Caski cells in the presence of different substrates.

Substrate 4-methoxynaphthol 4-methoxynaphthol succinate l-bromo-2-naphthol 6-bromo-2-naphthol 4-chloronaphthol 3-amino-9-ethylcarbazole 5-hydroxynaphtholsulfonamide

Figure imgf000024_0001

Figure imgf000025_0001

Claims

AMENDED CLAIMS
[received by the International Bureau on 21 May 1990 (21.05.90) original claims 1 and 32 cancelled; original claims 10 and 16 amended and renumbered 9 and 15; other claims unchanged but renumbered as claims 2-8, 10-14 and 16-30 (7 pages)]
1. A method for assaying for peroxidase activity which comprises the converting of a naphthalene compound of formula I, with H202 to yield a reactive product of formula Ila and converting the reactive product with a hydrazone compound of formula III to yield a colored precipitate, wherein:
Rl is in either the 1 or 2 position and is hydrogen, monosuccinyl, phosphono, sulfo, sulfino, nitro, or acyl group -C(:0)-R8 wherein R8 is alkyl (C1-C4) , aryl (C6-C8) , carboxyalkyl (C2-C5) , carboxyaryl (C7-C11) , or carboxyalkylaryl (C8-C14) ;
R2, R3, R , R5 can be the same or different and are hydrogen, hydroxy, alkyl (C1-C4) , alkoxy (C1-C4) , alkenyl (C2-C6) , arylalkyl (C7-C14) , halogen, nitro, aminocarbonyl, mono or dialkylaminocarbonyl having alkyls (C1-C4) , sulfamido, mono or dialkylsulfamido having alkyls- (C1-C4) , or acyl of R9-C(:0)- wherein R9 is an alkyl (C1-C4) , mono or dialkylamino having alkyls (Cl- C4) , arylalkoxy (C8-C14) or alkoxy (C1-C4) ;
R6 is hydrogen, hydroxy, alkyl (C1-C4) , alkoxycarbonyl (C2-C5) , carboxyamido, alkoxy (C1-C4) , sulfamido, mono or dialkylsulfamido (C1-C4) , alkoxysulfonyl, mono or dialkylamido having alkyls of Cl- C4, aryl (C6-C8) , acyl of -C(:0)-R10 wherein Rio is alkyl (C1-C4) , or halo; and
R7 is alkyl (C1-C4) or arylalkyl (C7-C11) .
2. A method of claim 1 in which Rl is nitro, phosphono, sulfo, sulfino, monosuccinyl or an acyl group of -C(:0)-R8 in either the 1 or 2 position wherein R8 is alkyl (C1-C4) , aryl (C6-C10) , carboxyalkyl (C2-C5) ; carboxyaryl (C7-C11) , or carboxyalkylaryl (C8-C14) . 3. A method of claim 2 wherein Rl is phosphono, monosuccinyl, sulfino, sulfo or an acyl group of C(:0)- R8 wherein in R8 is methyl.
4. A method of claim 3 wherein Rl is a monosuccinyl.
5. A method of claim 1 wherein:
Rl is hydrogen, monosuccinyl or an acyl group of -C(:0)-R8 wherein R8 is a methyl, carboxyethyl;
R2, R3, R4, R5 can be the same or different and are hydrogen, alkyl (C1-C4) , alkoxy (C1-C4) , halo or acyl groups of -C:0-R9 wherein R9 is an alkyl of C1-C4;
R6 is alkyl (C1-C4) , alkoxycarbonyl (C2-C5) ; or acyl of -C(:O)-R10 wherein RIO is alkyl (C1-C4) ; sulfamido, or dialkylsulfamido having alkyls (C1-C4) ; and R7 is alkyl (C1-C4) .
6. A method of claim 5 wherein: Rl is hydrogen or succinyl;
R2 is chloro, bromo, methoxy, ethoxy, propoxy or hydrogen; R3 is hydrogen, hydroxy, chloro, bromo, methoxy, ethoxy or propoxy;
R4 is hydrogen, hydroxy, chloro, bromo, or methyl;
R5 is bromo, chloro, hydrogen or hydroxy; R6 is hydrogen, sulfamido, dimethylsulfamido, methoxysulfonyl, benzo or halo; and R7 is methyl or ethyl.
7. A method of claim 6 wherein the naphthalene compound is selected from the group comprising: 4- methoxynaphthol, 4-ethoxynaphthol, 4-propyloxynaphthol, 4-chloronaphthol, l-bromo-2-naphthol, 6-bromo-2- naphthol , 4-methoxynaphthylsuccinate 4- ethyloxynaphthylsuccinate, 4-propyloxynaphthyl-succinate,
SUBSTITUTE SHEET 7-methoxy-2-naphthol, 6-methoxy-2-naphthol, 1,5- dihydroxynaphthalene and 2,6-dihydroxynapthalene.
8. A method of claim 7 wherein the hydrazone compound is selected from the group comprising: 3- methyl-2-benzo-thiazolinone hydrazone, 3-ethyl-2-benzo- thiazolinone hydrazone, methyl-2-naphto-thiazolinone hydrazone, 3-methyl-6-dimethylsulfamido-2- benzothiazolinone or 3-methyl-6-methoxysulfonyl-2- benzothiazolinone hydrazone.
9. A method for assaying for peroxidase activity which comprises the converting of an indole compound of formula V with HO to yield a reactive product and converting the reactive product with a hydrazone of formula III to yield a colored precipitate wherein: R6 is hydrogen, hydroxy, alkyl (C1-C4) , alkoxycarbonyl (C2-C5) , alkoxy (C1-C4) , sulfamido, mono or dialkylsulfamido (C1-C4) , alkoxysulfonyl, aminocarbonyl, mono or dialkylaminocarbonyl having alkyls of C1-C4, aryl (C6-C8) , acyl of -C(:O)-R10 wherein RIO is alkyl (C1-C4) , halo, or benzo;
R7 is alkyl (Cl to C4) or arylalkyl (C7-C11) ; Rll is hydrogen, or alkyl (C1-C4) ; R12 is hydrogen, alkyl (C1-C4) , alkoxycarbonyl (C2-C4) , amide or mono or dialkylamide having alkyls Cl to C4;
R13 is hydrogen, alkyl (C1-C4) , alkoxycarbonyl (C2-C4) , aminocarbonyl or mono or dialkylaminocarbonyl having alkyls Cl to C4;
R14 is hydrogen, alkyl (C1-C4) , amino, hydroxy, halogen, alkoxy (C1-C4) , alkoxycarbonyl (C2-C4) ; and
R15 is hydrogen, alkyl (C1-C4) , hydroxy, amino, halo, alkoxy (C1-C4) or nitro.
SUBSTITUTE SHEET 10. A method of claim 9 wherein
R12 is hydrogen, methyl, methoxycarbonyl, aminocarboxyl or dimethylaminocarbonyl;
R13 is hydrogen, methyl, methoxycarbonyl, aminocarboxyl or dimethylaminocarbonyl;
R14 is hydrogen, methyl, amino, hydroxy, halogen, methoxy or methoxycarbonyl; and
R15 is hydrogen, methyl, hydroxy, amino, halo, methoxy or nitro.
11. A method of claim 12 wherein R14 is hydroxy and is located at the 5 position of the indole ring.
12. A method of claim 1 wherein the assay is a nucleic acid hybridization sandwich-type assay.
13. A method of claim 1 wherein the immunoassay is an enzyme linked immunoassay.
14. A method of claim 1 having a solid support surface comprising an activated moiety selected from the group consisting of carboxy, amino, thio or sulfo.
15. A method of claim 1 having a solid support surface comprising amino activated nylon beads.
16. A method of claim 1 for detection of nucleic acid in biological samples from mammalian tissues.
17. A method of claim 1 for detection of nucleic acid originating from infectious organism.
18. A method of claim 17 wherein the virus is human papilloma virus.
SUBSTITUTE SHEET 19. A method of claim 1 wherein the hydrazone, naphthalene, or indole compounds are substantially free of peroxidase inhibiting substances.
20. A compound selected from the group consisting of mono-4-ethoxynapthylsuccinate, mono-4- propoxynaphthylsuccinate and mono-4-methoxynaphthyl- succinate.
21. A kit for assaying for peroxidase activity in an immunoassay or nucleic acid hybridization assay which involves the converting by peroxidase of a hydrazone with either a naphthol or indole compound to give a colored precipitate said . it comprising a solid support, a peroxidase-type enzyme and a solution of hydrazone and naphthol or indole substrate.
22. A kit of claim 21 which involves the converting of a naphthalene compound of formula I, with H O to yield a reactive product and converting the reactive product with a hydrazone compound of formula III to yield a colored precipitate, wherein: Rl is in either the 1 or 2 position and is hydrogen, monosuccinyl, phosphono, sulfo, sulfino, nitro, or an acyl group -C(:0)-R8 wherein R8 is an alkyl (Cl- C4) , aryl (C6-C8) , carboxyalkyl (C2-C5) , carboxyaryl (C7- C10) , or carboxyalkylaryl (C8-C14) ; R2, R3, R4 , R5 can be the same or different and are hydrogen, hydroxy, alkyl (C1-C4) , alkoxy (C1-C4) , alkenyl (C2-C6) , arylalkyl (C7-C14) , halogen, nitro, aminocarbonyl, mono and dialkylaminocarbonyl having alkyls of C1-C4, sulfamido, or mono and dialkylsulfamido having alkyls of C1-C4;
R6 is hydrogen, hydroxy, alkyl (C1-C4) , alkoxycarbonyl (C2-C5) , alkoxy (C1-C4) , sulfamido, mono and dialkylsuflamido (C1-C4) , alkoxysulfonyl, aminocarbonyl, mono or dialkylaminocarbonyl having alkyls of C1-C4, aryl (C6-C8) , acyl of -C(:0)-R10 wherein RIO is alkyl (C1-C4) , halo or benzo; and R7 is alkyl (C1-C4) or arylalkyl (C7-C11) .
23. A kit of claim 22 wherein the assay is a nucleic acid hybridization sandwich-type assay
24. A kit of claim 22 wherein the immunoassay is an enzyme linked immunoassay.
25. A kit of claim 22 having a solid support surface comprising carboxy activated latex beads.
26. A kit of claim 22 for detection of nucleic acid in biological samples obtained from mammals.
27. A kit of claim 21 which involves the converting of an indole compound of formula IV with H O in the presence of a hydrazone of formula III to yield a colored precipitate wherein:
R6 is hydrogen, alkyl (C1-C4) , alkoxycarbonyl
(C2-C5) , alkoxy (C1-C4) , sulfamido, mono or dialkylsulfamido (C1-C4) , an alkoxysulfonyl, a aminocarbonyl, mono or dialkylaminocarbonyl having alkyls of C1-C4, aryl (C6-C8) halo or benzo;
R7 is alkyl (C1-C4) or arylalkyl (C7-C11) ; Rll is hydrogen, or alkyl (C1-C4) ; R12 is hydrogen, alkyl (C1-C4) , alkoxycarbonyl (C2-C4) , aminocarbonyl, or mono or dialkylaminocarbonyl having alkyls of Cl to C4;
R13 is hydrogen, alkyl (C1-C4) , alkoxycarbonyl (C2-C4) , aminocarbonyl, or mono or dialkylaminocarbonyl having alkyls of Cl to C4;
R14 is hydrogen alkyl (C1-C4) , amino, hydroxy, halogen, alkoxy (C1-C4) , or alkoxycarbonyl (C2-C4) ; and R15 is hydrogen, alkyl (C1-C4) , hydroxy, amino, halogen, alkoxy (C1-C4) or nitro.
SUBSTITUTE SHEET 28. A kit of claim 26 wherein the assay is selected from the group of immunoassays or nucleic acid hybridization assays.
29. A kit of claim 26 wherein the assay is a nucleic acid hybridization assay wherein the assay comprises a solid support having carboxy activated latex beads.
30. A kit of claim 21 comprising 4- ethoxynaphthol substantially free of peroxidase inhibiting products.
SUBSTITUTE SHEET STATEMENTUNDERARTICLE19
Claim 1 is cancelled;
Claim 1 (old claim 2) reads "A method for assaying for peroxidase activity which comprises "; "HO to yield a reactive product of formula Ila"; line 6, now reads "formula III";
Claim 9 (old claim 10) , first few lines, reads "A method for assaying for peroxidase activity which comprises the converting of an indole compound of formula V with H202 to yield a reactive product and converting the reactive product with a hydrazone of formula III to yield a colored precipitate wherein:";
Claim 15 (old claim 16) now reads "A method of claim 1 having a solid support comprising amino activated nylon beads;
Claim 32 is cancelled; and
Old claims 3-9, 11-15 and 17-31 are unchanged except for dependency, and are now numbered 2-8, 10-14, and 16-30, respectively.
PCT/US1989/005407 1988-12-01 1989-11-30 Substrates for peroxidase assaying WO1990006372A1 (en)

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