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AU706232B2 - Novel aryl N-alkylacridancarboxylate derivatives useful for chemiluminescent detection - Google Patents
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AU706232B2 - Novel aryl N-alkylacridancarboxylate derivatives useful for chemiluminescent detection - Google Patents

Novel aryl N-alkylacridancarboxylate derivatives useful for chemiluminescent detection Download PDF

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AU706232B2
AU706232B2 AU46895/97A AU4689597A AU706232B2 AU 706232 B2 AU706232 B2 AU 706232B2 AU 46895/97 A AU46895/97 A AU 46895/97A AU 4689597 A AU4689597 A AU 4689597A AU 706232 B2 AU706232 B2 AU 706232B2
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reagent
acridan
peroxidase
detection
carboxylate
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Hashem Akhavan-Tafti
Zahra Arghavani
Renuka De Silva
Barry Schoenfelner
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Lumigen Inc
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    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
    • C07D219/02—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems with only hydrogen, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the ring system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
    • C07D219/04—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
    • C07D219/04—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
    • C07D219/06—Oxygen atoms
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/968—High energy substrates, e.g. fluorescent, chemiluminescent, radioactive

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Description

NOVEL ARYL N'ALKYLACRIDANCARBOXYLATE DERIVATIVES USEFUL FOR CHEMILUINESCEN T
DETECTION
CROSS REERENCE TO REATE
APLICATION
This application is a continuation-inpart of applicant s co-pending application Serial No. 08/061,810 filed on May 17, 1993.
BACKGROUND OF THE INVENTION FIELD O THE INVENTON This invention relates to chemiluminescent N-alkylacridancarboxylate derivatives hich allow the 1: 0 production of light (chemiluminescence) from the acridan by reaction with a peroxide and peroxidase This invention also relates to an improved method of .:generating light chemically (chemiluminescence) by the action of a peroxidase enzyme and an oxidant such as hydrogen peroxide with a group of N-alkylacridancarboxylate derivatives. The invention also relates to an improved method of enhancing the amount of chemilumines cence produced from this process by the use of specific substances. The invention also relates to the us of this method to detect the peroxidase enzyme. The invention also relates to the use of this method to detect hydrogen peroxide. Further, the invention relates to the use of the method to detect and quantitate various biological molecules. For example, the method may be used to detect haptens, antigens and antibodies by the technique of mmunoassay, proteins by Western blotting DNA and RNA by Southern and Northern blotting, respectively. The method may also be used to detect DNA in DNA sequencing applications. The method -2may additionally be used to detect enzymes which generate hydrogen peroxide such as glucose oxidase,glucose-6-phosphate dehydrogenase, galactose oxidase and the like as are generally known in the art.
DESCRIPTION OF RELATED
ART
The detection and quantitation of biological molecules has been accomplished historically with excellent sensitivity by the use of radiolabeled reporter molecules. Recently numerous non-radioactive methods have been developed to avoid the hazards and inconvenience posed by these materials. Methods based on enzyme-linked analytes offer the best sensitivity since the ability to catalytically turn over substrate to produce a detectable change achieves an amplification. Substrates which generate color, fluorescence or chemiluminescence have been developed, the latter achieving the best sensitivity.
Further increases in assay sensitivity will expand the range of utility of chemiluminescence-based methods by permitting the detection of analytes present in smaller quantities or reducing the amount of time and/or reagents required to perform the assay. A way to increase the speed and sensitivity of detection in an enzymatic chemiluminescent assay is through the use of substrates which generate light with a higher efficiency .or for a greater length of time.
•Among the enzymes used in enzyme-linked detection methods such as immunoassays, detection of oligonucleotides and nucleic acid hybridization techniques, the most extensively used to date has been horseradish peroxidase. Chemiluminescent reagents known in the art do not permit full advantage to be taken of the beneficial properties of this enzyme in analysis mainly due to sensitivity limitations. A reagent which permits the detection of lower amounts of enzyme is needed to enable the use of peroxidase conjugates in applications requiring ultrasensitive detection.
-3- Specifically, reagents are required which gnrt higher levels of chemiluminescence without an accompanying increase in the background or fon-spec if ic chemi luminescenc The increased chemi luminescenc may be accomplished via either a higher maximum intensity or a longer duration than compounds known in the art.
Oxidation of acidan Oxcidation of acrdanby enzyl peroxide in aqueous solution produced chemilUMinescence with very low efficiency 4 cL x 10-7) and a mixture of products including acridine
(S.
Steenken, Photochem. Photobjol. 11, 279-283 (1970)).
N-lMethylacridan is oxidized electrochemialy t N-methylacridiniu ion Hapiot, J. Moiroux, j.
M.
Saveant, J. Am. Chem. Soc.,. 112 1337-43 (1990)
N.
W- Roper,. S. A. Joriker, J. W. Verhoeven, Redl. Tray.
Chim. Pays-Bas, 104(11), 296-302 (1985)). Chemical .oxidation of N-alkylacridan compounds has been performed with ferricyafljd 8 ion Sinha, T. C. Bruice, j. Am.
Chem. So,106(23), 7291-2 (1984)), cranqioe K. CleP. Plank, J. P. Bergsma, R. Lahti, A.
A.
Quesnell A. G. Parsons, Can. J. Chem.., 62(9), 1780-4 (1984)) and lithium nitrite N. Chupakhin, 1. M. *...Sosonkin, A. I. Matern, G. N. Strogov, Doki. Akad. Nauk SSSR, 250(4), 875-7 (1980)). Oxidation of an N-lyardn drvtv has been perf ormed S. Photochemically wihor without a flavincopuda co-oxidant R. Knappe, J. Pharm. Sci., 67(3), 318-20 (1978); G. A.Digenis, S. Shakshir, M. A. IMiyamoto,
H.
B. Kostenbauer, j. Pharm. Sci., 65(2), 247-51 (1976)).
A ry I a nd a1k yl1 esters o f lO-methyacridan 9 aboyi acid undergo autoxidation to N-ehlardn in dipolar aprotic solvents under strongly basic conditions to produce chemiluminescence lcCapra, Accts. Chem. Res.,1 9 2 01-8 (19 76)
F.
McCapra, M. Roth, D. Hysert, K.A. Zaklika in Chemilumi- nescence and Bioluminescence, Plenum Press, New York,' 1973, pp. 313-321; F. McCapra, Prog. Org. Chem., 8, t I) -4- 231-277 (1971); F. McCapra, Pure Appl. Chem., 24, 611-629 (1970); U.S. Patent No. 5,283,334 to McCara).
Chemiluminescence quantum yields ranged from 10 5 to 0.1 and were found to increase as the pKa of the phenol or alcohol leaving group decreased. Quantum yields in aqueous solution were significantly lower due a competing non-luminescent decomposition of an intermediate.
Addition of the cationic surfactant CTAB increased the apparent light yield 130-fold by preventing a competing dark reaction.
Applicants' co-pending application Serial No.
08/061,810 discloses the first use of an enzyme to oxidize substituted and unsubstituted N-alkylacridancarboxylic acid derivatives to generate chemiluminescence.
In the presence of a peroxidase enzyme and a peroxide N-alkylacridancarboxylate derivatives are efficiently oxidized to produce the N-alkylacridone and blue chemiluminescence.
b. Chemiluminescent oxidation of acridinium 20 esters. The chemiluminescent oxidation of aliphatic and aromatic esters of N-alkylacridinium carboxylic acid by H202 in alkaline solution is a well known reaction. The high chemiluminescence quantum yield approaching 0.1 has led to development of derivatives with pendant reactive groups for attachment to biological molecules. Numerous chemiluminescent immunoassays and oligonucleotide probe assays utilizing acridinium ester labels have been reported.
The use of acridinium esters especially when labeled to a protein or oligonucleotide suffers from two disadvantages. The chief problem is limited hydrolytic stability. Acridinium ester conjugates decompose steadily at or slightly above room temperature. Depending on the substitution of the leaving group storage at -20 oC may be required for extended storage.
A second disadvantage of acridinium esters is the tendency to add nucleophiles such as water at the- 9-position to spontaneously form a pseudo-base intermediate which is non-luminescent and decomposes in a pH-dependent manner in a dark process. In practice the pH of solutions containing acridinium esters must be first lowered to reverse pseudo-base formation and then raised in the presence of H0 2 to produce light.
Amides, thioesters and sulfonamides of N-alkylacridinium carboxylic acid have been shown to emit light when oxidized under these conditions
(T.
Kinkel, H. Lubbers, E. Schmidt, P. Molz, H. J.
Skripczyk, J. Biolumin. Chemilumin., 4, 136-139, (i389), G. Zomer, J. F. C. Stavenuiter, Anal. Chim. Acta, 227, 11-19 (1989)). These modifications of the leaving group only partially improve the storage stability performance.
A more fundamental limitation to the use of acridinium esters as chemiluminescent labels lies in the 20 fact that when used as direct labels, only up to at most about 10 molecules can be attached to a protein or oligonucleotide. Coupled with the quantum efficiency for producing a photon (5 an acridinium ester-labeled analyte can generate at most one photon of light. In contrast, enzyme-labeled analytes detected by a chemiluminescent reaction can potentially generate several orders of magnitude more light per analyte molecule detected by virtue of the catalytic action of the enzyme.
An attempt to increase the number of acridinium ester molecules associated with an analyte in an immunoassay was made by constructing an antibody-liposome conjugate wherein the liposome contained an unspecified number of AE's Law, T.
Miller, U. Piran, C. Klukas, S. Chang, J. Unger,
J..
Biolumin. Chemilumin., 4, 88-98, (1989)). This method only produced a modest increase in signal over a -6comparable assay using directly labeled AE's.
c. Chemiluminescent Detection of Horseradish- Peroxidase. Amino-substituted cyclic acylhydrazides such as luminol and isoluminol react with H 2 0 2 and a peroxidase enzyme catalyst (such as horseradish peroxidase, HRP) under basic conditions with emission of light. This reaction has been used as the basis for analytical methods for the detection of H202 and for the peroxidase enzyme. An analog of luminol (8-amino-5-chloro-7-phenylpyrido3,4-dJpyridazine-1,4( 2H, 3H)dione) has been used in an enhanced chemiluminescent assay with HRP Ii, H. Yoshida, y.
Aramaki, H. Masuya, T. Hada, M. Terada, M. Hatanaka,
Y.
Ichimori, Biochem. Biophys. Res. Comm., 193(2), 540-5 (1993)). Application of this compound in an immunoassay led to a two-fold lowering of the detection limit compared to detection using luminol. Another chemiluminescent compound oxidized by a peroxidase enzyme and a peroxide is a hydroxy-substituted 20 phthalhydrazide (Akhavan-Tafti co-pending U. S. patent application No.965,231, filed October 23,1992).
Applicant's co-pending application Serial No. 08/061,810 filed on May 17, 1993 discloses chemiluminescent SN-alkylacridancarboxylic acid esters and sulfonimides which produce light upon reaction with a peroxide and a peroxidase for use in detecting peroxidase enzymes and in assays.
Numerous enhancers have also been employed in conjunction with the use of luminol to increase the intensity and duration of light emitted. These include benzothiazole derivatives such as D-luciferin, various phenolic compounds such as p-iodophenol and p-phenylphenol and aromatic amines Thorpe,
L.
Kricka, in Bioluminescence and Chemiluminescence, New Perspectives, J. Scholmerich, et al, Eds., pp. 199-208 (1987)). For the purposes of the present discussion phenolic compounds are taken to mean hydroxylic aromatic -7compounds which will also include compounds such as 2-naphthol and 6 -bromo-2 -naphthol which are known to enhance other peroxidase reactions in addition to the aforementioned substituted hydroxyphenyl compounds.
Other compounds which function as enhancers of the chemi luminescent oxidation of amino-substituted cyclic acylhydrazides by a peroxidase include 4 4 -hydroxyphenyl)thiazole Ii, H. Yoshida,
Y.
Aramaki, H. Masuya, T. Hada, M. Terada, M. Hatanaka,
Y.
Ichimori, Biochem. Biophys. Res. Comm., 193(2), 540-5 (1 993)), a group of compounds disclosed in U.S. Patent 5,171,668 to Sugiyama, 2 -hydroxy9..fluorenone, and a group of hydroxy-substituted benzoxazole derivatives as disclosed in U. S. Patent No. 5,206,149 to Oyama. The mechanism of oxidation of cyclic acyihydrazides by the combination of a peroxide and a peroxidase enzyme is very complex and remains the subject of intense debate Lundin, L. Hallander, in Bioluminescence and amilumincence, NwPerspectives, J. Scholmerich, e 20 al, Eds.,' pp. 555-558 (1987)); S. Vlasenco, A Arefyev, A. Kilimov B. Kim, E. Gorovits, A. Osipov, E. Gavrilova, A. Yegorov, J. Biolumin. Chenilumin. 4, 164-176 (1989)) This difficulty has hampered the development of new chemiluminescent reactions catalyzed by peroxidases.
25 d. Assays usin HRP. The enzyme horseradish **.peroxidase has found widespread use in enzyme .immunoassays and DNA hybridization assays with chemiluminescent detection using luminol or isoluminol as substrate Thorpe, L. J. Kricjca, S. B. Mosely, T. P. Whitehead Clin. Chem., 31, 1335 (1985), J. A.
Matthews, A. Batici, C. Hynds, L. J. Kricka, Anal.
Biochem.,. 151,205, (1985), P. Walsh, J. Varlaro,
R.
Reynolds, Nuc. Acids Res. 20,(19) 5061-5065 (1992)).
Commercially available kits for conjugation of HRP with enhanced luminol chemiluminescent detection are available. Chemiluminescent assays using a peroxidase enzyme known in the art are not able to detect the I I, -8lowest levels of certain analytes such as the thyroid hormone TSH, mainly due to the inability to detect the enzyme at extremely low levels. A chemiluminescent reagent which permits the detection of lower amounts of enzyme is needed for such assays.
e. Chemiluminescence Enhancement by Surfactants. Enhancement of chemiluminescent reactions using polymeric and monomeric surfactants is known in the art. Enhancement may occur by affecting the outcome of one or more steps e.g. by increasing the fluorescence quantum yield of the emitter, by increasing the percentage of product molecules produced in the excited state, by increasing the fraction of molecules undergoing the chemiluminescent reaction through inhibition of competing side reactions (McCapra Accts.
S. Chem. Res., 201-8 (1976)) or by promoting the action of an enzyme catalyst. No clear or consistent pattern exists concerning the effect of polymeric and monomeric surfactants on chemiluminescent reactions. It 20 is impossible to predict which surfactant compounds, if any, may enhance the chemiluminescence from a particular process and can only be determined by substantial experimentation.
U.S. Patent No. 5,145,772 to Voyta discloses 25 enhancement of enzymatically generated chemiluminescence from 1,2-dioxetanes in the presence of polymeric compounds. Certain cationic polymer compounds were effective chemiluminescence enhancers; nonionic polymeric compounds were generally ineffective and the lone anionic polymer, Example 45, significantly decreased light emission.
U.S. Patent No. 4,927,769 to Chang discloses enhancement by surfactants of the chemical oxidation of acridinium esters with alkaline hydrogen peroxide.
These acridinium ester compounds are discrete from compounds of the present invention in that they react without the use of enzymes. Several of the tested
WMMW
-9surfactants (see Table 2 therein) provide only marginal enhancement.
A report on the effect of surfactants on the firefly luciferin-luciferase reaction J. Kricka,
M.
DeLuca, Arch. Biochem. Biophys., 217, 674 (1983)) discloses enhancement of the light yield with nonionic surfactants by affecting the enzyme reactivity; a cationic surfactant totally extinguished light emission ;by inhibiting the enzyme.
A paper Goto, H. Fukatsu, Tetrahedron.
Lett.,4299 (1969)) teaches chemiluminescence enhancement of the chemical oxidation of Cypridina luciferin in the presence of nonionic and cationic but not anionic surfactants even though the fluorescence quantum yield of the emitter was increased in all three types of surfactants.
A paper Sasamoto, Y. Ohkura, Chem. Pharm.
Bull, 39(2), 411-6 (1991)) discloses enhancement by a cationic surfactant of chemiluminescence from chemical 20 oxidation of a dialkylaminobenzofuranyl-substituted cyclic diacylhydrazide. An anionic surfactant was ineffective at enhancing the chemiluminescence, while a nonionic surfactant diminished light production.
OBJECTS
25 It is therefore an object of the present invention to provide an improved method and N-alkylacridancarboxylate derivatives with superior properties for use in generating chemiluminescence by the action of a peroxidase enzyme for the detection of biological materials and compounds. It is also an object of the present invention to provide an improved method and kit using N-alkylacridancarboxylate derivatives in solution or on membranes for use in generating chemiluminescence by the action of a peroxidase enzyme for the detection of peroxidase
I
enzymes and enzyme-conjugates. Additionally, it is an object of the present invention to provide an improved method and kit using N-alkylacridancarboxylate derivatives for use in generating chemiluminescence by the action of a peroxidase enzyme for use in nucleic acid assays in solution and on surfaces. Further, it is an object of the present invention to provide an improved method and kit using N-alkylacridancarboxylate derivatives for use in generating chemiluminescence by the action of a peroxidase enzyme for detection of proteins in Western blots and DNA in Southern blots and other DNA hybridization assays. Further, it is an object of the present invention to provide an improved method and kit using N-alkylacridancarboxylate derivatives for use in generating chemiluminescence by the action of a peroxidase enzyme for detection of haptens, proteins and antibodies in enzyme immunoassays.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing a comparison of 20 the light emission profiles from a reagent containing 2',6'-difluorophenyl 10-methyl-acridan-9-carboxylate of the present invention, a reagent containing the acridan 4 '-fluorophenyl 10-methylacridan-9-carboxylate and a commercial reagent containing luminol. Forty gL each of three formulations were reacted in separate experiments with 1 pL of a solution containing 1.4 x 10-16 mol of HRP in water. These formulations consisted of: 0.05 mM acridan compound Sa in 0.01 M tris buffer, pH 8.0, 0.4 mM urea peroxide, 0.1 mM p-phenylphenol, 0.025% TWEEN 20, 1 mM EDTA; an identical formulation containing 4'-fluorophenyl 10-methylacridan-9carboxylate in place of 5a and an optimized reagent containing luminol (AMERSHAM ECL, Amersham,
PLC,
Amersham, Figure 1 shows the improved generation of light emission (in Relative Light Units, RLU) using a reagent of the present invention, under these conditions compared to the prior art compounds
I
-11- 4'-fluorophenyl 10-methylacridan-9-carboxylate and luminol.
Figure 2 is a graph showing a comparison of the light emission profiles from a reagent containing 3',5'-difluorophenyl l0-methylacridan-9-carboxylate of the present invention, a reagent containing the acridan 4'-fluorophenyl l0-methylacridan-9-carboxylate and a commercial reagent containing luminol. Forty
ML
each of three formulations were reacted in separate experiments with 1 gL of a solution containing 1.4 x 10-16 mol of HRP in water. These formulations consisted of: 0.05 mM acridan compound 5b in 0.01 M tris buffer, pH 8.0, 0.4 mM urea peroxide, 0.1 mM p-phenylphenol, 0.025% TWEEN 20, 1 mM EDTA; an identical formulation containing 4 '-fluorophenyl 10-methylacridan-9carboxylate in place of 5b and an optimized reagent containing luminol. Figure 2 shows the improved generation of light emission using 5b, a reagent of the 20 present invention, compared to the prior art compounds.
20 Figure 3 is a graph showing a comparison of the light emission profiles from a reagent containing 2' 4'6'-trichlorophenyl l0-methylacridan-9-carboxylate (5c) of the present invention, a reagent containing the acridan 4'-fluorophenyl 10-methylacridan-9-carboxylate 25 and a commercial reagent containing luminol. Forty gL each of three formulations were reacted in separate experiments with 1 AL of a solution containing 1.4 x mol of HRP in water. These formulations consisted of: 0.05 mM acridan compound 5c in 0.01 M tris buffer, pH 8.0, 0.4 mM urea peroxide, 0.1 mM p-phenylphenol, 0.025% TWEEN 20, 1 mM EDTA; an identical formulation containing 4'-fluorophenyl 10-methylacridan-9carboxylate in place of 5c and an optimized reagent containing luminol. Figure 3 shows the improved generation of light emission using 5c, a reagent of the present invention, compared to the prior art compounds.
Figure 4 is a graph showing a comparison of -12the light emission profiles from a reagent containing 2',4 -trichlorophenyl l0-methylacridan-9-carboxylate (Sd) of the present invention, a reagent containing the acridan 4'-fluorophenyl 10-methylacridan-9-carboxylate and a commercial reagent containing luminol. Forty
ML
each of three formulations were reacted in separate experiments with 1 ML of a solution containing 1.4 x 10-16 mol of HRP in water. These formulations consisted of: 0.05 mM acridan compound 5d in 0.01 M tris buffer, pH 8.0, 0.4 mM urea peroxide, 0.1 mM p-phenylphenol, 0.025% TWEEN 20, 1 mM EDTA; an identical formulation containing 4'-fluorophenyl 10-methylacridan-9carboxylate in place of 5d and an optimized reagent containing luminol. Figure 4 shows the improved generation of light emission using 5d, a reagent of the present invention, compared to the prior art compounds.
Figure 5 is a graph showing a comparison of the light emission profiles from a reagent containing 2 0 2' '-trifluorophenyl 10-methylacridan-9-carboxylate (5e) of the present invention, a reagent containing the acridan 4'-fluorophenyl l0-methylacridan-9-carboxylate and a commercial reagent containing luminol. Forty pL each of three formulations were reacted in separate ~experiments with 1 ML of a solution containing 1.4 x 1016 mol of HRP in water. These formulations consisted of: 0.05 mM acridan compound 5e in 0.01 M tris buffer, pH 8.0, 0.4 mM urea peroxide, 0.1 mM p-phenylphenol, 0.025% TWEEN 20, 1 mM EDTA; an identical formulation containing 4'-fluorophenyl 9 -carboxylate in place of 5e and an optimized reagent containing luminol. Figure 5 shows the improved generation of light emission using 5e, a reagent of the present invention, compared to the prior art compounds.
Figure 6 is a graph showing a comparison of the light emission profiles from a reagent containing pentafluorophenyl l0-methylacridan-9-carboxylate (5f) of -13the present invention, a reagent containing the acridan 4'-fluorophenyl l0-methylacridan-9-carboxylate and a commercial reagent containing luminol. Forty gL each of three formulations were reacted in separate experiments with 1 L of a solution containing 1.4 x 10- 6 mol of HRP in water. These formulations consisted of: 0.05 mM acridan compound 5f in 0.01 M tris- buffer, pH 8.0, 0.4 mM urea peroxide, 0.1 mM p-phenylphenol, 0.025% Tween 1 mM EDTA; an identical formulation containing 4 '-fluorophenyl 10-methylacridan-9-carboxylate in place of 5f and an optimized reagent containing luminol.
Figure 6 shows the improved generation of light emission using 5f, a reagent of the present invention, compared to the prior art compounds.
Figure 7 is a graph showing a comparison of the light emission profiles from a reagent containing -trifluorophenyl 3 -methoxy-10-methylacridan_9_ Scarboxylate (5h) of the present invention, a reagent containing the acridan 4 '-fluorophenyl 20 0-methylacridan-9-carboxylate and a commercial reagent containing luminol. Forty gL each of three formulations were reacted in separate experiments with 1 L of a solution containing 1.4 x 10- 16 mol of HRP in water.
These formulations consisted of: 0.05 mM acridan compound 5h in 0.01 M tris buffer, pH 8.0, 04 mM urea peroxide, 0. mM P-phenylphenol, 0.025% TWEEN 20, 1 mM EDTA; an identical formulation containing 4 -fluorophenyl 10-methylacridan-9-carboxylate in place Sof 5h and an optimized reagent containing luminol.
Figure 7 shows the improved generation of light emission using 5h, a reagent of the present invention, compared to the prior art compounds.
Figure 8 is a graph showing a comparison of the linearity of detection of HRP using reagent compositions of the present invention and a commercially available optimized reagent containing luminol. In separate experiments, 40 L of a solution containing -14acridan 5e, 5h or a commercial reagent (AMERSHAM
ECL)
were mixed at room temperature with1 AL aliquots of HRP containing the indicated amounts of enzyme. Light intensities from the compositions containing acridans and 5h were measured at 15 min while data from the ECL reagent represent the maximum light intensity. The term S-B refers to the chemiluminescence signal in RLU in the presence of HRP corrected for background chemiluminescence in the absence of HRP.
Compositions containing acridans 5e and 5h are capable of 100-fold greater sensitivity of detection than the ECL reagent.
Figures 9A, 9B and 9C show the result of three experiments concerning Western blot analysis of human transferrin on PVDF with chemiluminescent detection using fractionated goat anti-human transferrin serum, rabbit anti-goat IgG-peroxidase conjugate. For each experiment, human transferrin loaded into the five slots 2was: 1000 pg, 200 pg, 50 pg, 20 pg, 20 5 pg. Chemiluminescent detection was performed using: a commercial reagent (ECL) containing luminol (Figure 9A); a reagent composition containing the acridan 4'-hydroxyphenyl lO-methylacridan-9-carboxylate previously disclosed in applicants' co-pending application Serial No. 08/061,810 (Figure 9B); and a reagent composition containing the acridan 5e of the present invention (Figure 9C). The blots were exposed to X-OMAT AR X-ray film for 15 sec after a 14 minute i:.ncubation. The image has been scanned and digitally reproduced. The results show the superior image obtained with acridan 5e of the present invention.
Figures 10A, 10B and 10C show the result of a Western blot analysis of human transferrin on nitrocellulose with chemiluminescent detection using fractionated goat anti-human transferrin serum, rabbit anti-goat IgG-peroxidase conjugate. Human transferrin loaded into each slot was 1000 pg, 200 pg, (3) pg, 20 pg, 5 pg. Chemiluminescent detection was performed using: a commercial reagent
(ECL)-
containing luminol (Figure 10A); a reagent composition containing the acridan 4 '-hydroxyphenyl 10-methylacridan-9-carboxylate previously disclosed in applicants' co-pending application Serial No. 08/061,810 (Figure 10B); and a reagent composition containing the acridan 5e of the present invention (Figure 10C). The blots were exposed to X-OMAT AR X-ray film for one minute after a 15 minute incubation. The image has been scanned and digitally reproduced. The results show the superior image obtained with acridan 5e of the present invention.
Figure 11 is a graph showing the linearity of detection of thyroid stimulating hormone (TSH) in a chemiluminescent enzyme immunoassay using a reagent of the present invention containing acridan 5h and a commercial TSH immunoassay kit. The chemiluminescent assay resulted in a linear calibration curve over four 20 orders of magnitude with a lowest detected quantity of 0.003 mIU/L. Excellent linearity and identical analytical sensitivity resulted when light intensity was measured at either 5, 10 or 15 min in each well. The term S-B has the same meaning as in Figure 8. For 25 comparison, the detection limit of the manufacturer's assay using a colorimetric endpoint is 0.05 mIU/L.
Figure 12 is a graph showing the application of a chemiluminescent reagent of the invention containing acridan 5h in an enzyme immunoassay of Figure 11 for human growth hormone (hGH). A commercially available colorimetric assay kit from Sorin Biomedica (Vercelli, Italy) was used according to the kit instructions with substitution of the detection reagent.
The chemiluminescent assay resulted in a nonlinear calibration curve. The term S-B has the same meaning as in Figure 8.
Figure 13 is a graph showing the linearity of -16detection of hGH obtained in the same chemiluminescent enzyme immunoassay by diluting the secondary antibody-HRp conjugate ten-fold. The chemiluminescent assay resulted in a linear calibration curve over three orders of magnitude with a lowest detected quantity of 0.05 ng/mL. Excellent linearity and identical analytical sensitivity resulted when light intensity was measured at either 2.5, 5, 7.5 or 10 min. The term
S-B
has the same meaning as in Figure 8. For comparison, the calculated detection limit of the manufacturer's assay using a colorimetric endpoint is 0.05 ng/mL; the colorimetric assay generates a nonlinear calibration curve.
Figures 14A and 14B show the result of chemiluminescent detection of a Southern blot analysis of EcoRI-restricted mouse genomic DNA on nylon using a fluorescein-labeled v-mos probe and horseradish peroxidase-anti-fluorescein conjugate. In separate experiments, the reagents used for the chemiluminescent 20 detection were: a composition of the present invention containing acridan Se (Figure 14A) and a commercial reagent (ECL) containing luminol (Figure 14B). The blots were exposed to X-OMAT AR X-ray film for 10 min 5 after a 22 min incubation. The image has been scanned and digitally reproduced. The results show the Ssuperior image obtained with acridan 5e of the present invention.
DESCRIPTION OF THE PREFERRED
EMBODIMENTS
The chemiluminescent detection of peroxidase and peroxide with acridans of the formula: -17b
C
wherein R, is selected from alkyl, heteroalkyl and aralkyl groups, wherein R is any group which allows the production of light and a and b are integers between 0 and 4 and wherein y is a leaving group which allows the production of light (chemiluminescence) from the acridan by reaction with a peroxide and a peroxidase were disclosed in applicants' co-pending application Serial No. 08/061,810 filed on May 17, 1993. It has now been discovered that certain acridan derivatives including di- and polyhaloaryl ester derivatives (Formula I, y OArX.) provide superior properties in producing chemiluminescence.
20 The present invention relates particularly to an improved acridan of the formula: 2 I
(II)
0 OAr wherein R, is selected from alkyl, heteroalkyl and aralkyl groups, wherein R is any group which allows the production of light and a and b are integers between 0 and 4 and wherein Ar-O is a leaving group wherein Ar-o is selected from the group consisting of di- and polyhalosubstituted phenoxy groups which allows the production of light from the acridan by reaction with a peroxide and a peroxidase. Ar-O groups containing at least two halogen substituents provide unexpectedly -18superior performance in producing chemiluminescence and in assays.
The present invention relates to an improvement in a reagent composition which generates light in the presence of a peroxidase which comprises: a) an acridan of the formula: I (II) b 0 OAr wherein R, is selected from the group consisting of alkyl, heteroalkyl and aralkyl groups, wherein R is any group which allows the production of light and a and b are integers between 0 and 4 and wherein Ar-O is a leaving group wherein Ar-O is selected from the group consisting of di- and polyhalosubstituted phenoxy groups which allows the production of light from the acridan by 20 reaction with a peroxide and a peroxidase; b) optionally a phenolic compound which enhances light production from the acridan; c) a peroxide compound which participates in the reaction of the acridan with the peroxidase; d) a chelating agent which prevents the peroxide compound from reacting prior to addition of the S* peroxidase to the composition; and e) a surfactant.
The present invention relates to an improved method for producing chemiluminescence which comprises reacting a peroxide compound and a peroxidase enzyme with an acridan of the formula:
(II)
i f n i i -19wherein R, is selected from the group consisting of alkyl, heteroalkyl and aralkyl groups, wherein R is any.
group which allows the production of light and a and b are integers between 0 and 4 and wherein Ar-O is a leaving group wherein Ar-O is selected from the group consisting of di- and polyhalosubstituted phenoxy groups which allows the production of light from the acridan by reaction with a peroxide and a peroxidase.
The present invention also relates to an improved method for detecting a peroxidase enzyme or an analyte linked to or capable of being linked to a peroxidase enzyme in an assay procedure by a chemiluminescent reaction, the improvement which comprises reacting an acridan with a peroxide and a peroxidase enzyme to produce light for detecting the analyte wherein the acridan is of the following formula: (II 20 0 OAr •wherein R, is selected from the group consisting of alkyl, heteroalkyl and aralkyl groups, wherein R is any group which allows the production of light and a and b are integers between 0 and 4 and wherein Ar-O is a leaving group wherein Ar-O is selected from the group consisting of di- and polyhalosubstituted phenoxy groups which allows the production of light from the acridan by reaction with a peroxide and a peroxidase.
The present invention also relates to an improved method for detecting a peroxidase enzyme or an analyte linked to or capable of being linked to a peroxidase enzyme in an assay procedure by a chemiluminescent reaction, the improvement which comprises: a) providing a reagent composition which generates light in the presence of a peroxidase which comprises: an acridan of the formula: p e r o i d as e w h i c h
I
I
N (II) wherein R, is selected from the grou alkyl, heteroalkyl and aralkyl grnoups, in R is any group which allows the production of liwh n R is any are integers between 0 and 4ght and herein is a leaving group wherein Ar-o is selected from the g consisting of di- and polyhalosubstt l ted pom the group which allows the n po l oo..tituted p h enoxy groups which allows the production of light from the acridan by reaction with a peroxide and a peroxidase; a peroxide compound which participates in the reaction of the acridan with the peroxidase n te e a c t io n o f t h 2 0 ai ith Pph eroxidase; an enhancer substance which may be a phenolic compound which enhances the light production from the acridan; a chelating agent which Prevents the peroxide compound from reacting prior to ddition of the peroxidase to the omposition; and a surfactant; and c o stion and a b) adding a peroxidase to the reagent composition so that light is produced for detecting the :analyte.
The present invention also relates to a kit for detecting an analyte in an assay procedure by a 30 chemiluminescent reaction to produce light which comprises in separate containers: -21a) an acridan of the formula:
S(II)
N
(R)
H
O OAr wherein R, is selected from the group consisting of alkyl, heteroalkyl and aralkyl groups, wherein R is any group which allows the production of light and a and b are integers between 0 and 4 and wherein Ar-O is a leaving group wherein Ar-C is selected from the group consisting of di- and polyhalosubstituted phenoxy groups which allows the production of light from the acridan by reaction with a peroxide and a peroxidase; a peroxide; optionally an enhancer substance which may be a phenolic compound which enhances the light production from the acridan; optionally a chelating agent which prevents the 20 peroxide compound from reacting prior to addition of the peroxidase to the composition; and a surfactant; and b) a peroxidase enzyme, wherein the light is detected in the assay procedure by reacting the reagent Scomposition with the peroxidase.
The present invention also relates to an improved method for detecting hydrogen peroxide in an assay procedure by a chemiluminescent reaction, the improvement which comprises reacting hydrogen peroxide and a peroxidase enzyme with an acridan of the formula: I (II) o^ -22wherein R, is selected from the group consisting of alkyl, heteroalkyl and aralkyl groups, wherein R is any group which allows the production of light and a and b are integers between 0 and 4 and wherein Ar-o is a leaving group wherein Ar-o is selected from the group consisting of di- and olyhalosubstituted phe y group which allows the production of light from the acridan by reaction with a peroxide and a peroxidase by The invention involves improved N-alkylacridancarboxylate derivatives with superior properties in one or more of the following characteristics: longer duration of light emission, higher intensity of light emission, faster rate of rise of light emission to the maximum value, lowered background chemiluminescence improved signal/background ratio, extended storage stability of a chemiluminescent detection reagent composition, enhanced light emission on a membrane or other properties.membrane or ther properties. The particular combinations of the Rgroups R and Ar are chosen so as to provide a compound with one or more properties which are ptimal fo particular applications. a r l for onIn particular, N-alkylacridancarboxylate derivatives with Ar groups bearing two or more halogen bstitents are unexpectedly superior in light generating ro e t re It is thought that t generating properties.
It is thought that the incorporation of two or more halogen substituents in the Ar moiety roduces an acridan with a better leaving group thereby accelerating and promoting the chemiluminescent reaction at the expense of competing nn-chemiluminescont ide reactions. side In general when more rapid build-up of light intensity or higher peak light intensity are desired it is advantageous to select the O-Ar group such that the pK. of the conjugate acid (Ar-OH) of the Ar-O- anion is lower than that of Ph-OH, preferably less than about Examples include phenols or phenolic compounds (Ar-OH) which are substituted with PK.-lowering electron -23withdrawing groups such as halogen atoms and hence are more ionized at a given pH. As a result they tend tofunction as better leaving groups in nucleophilic displacement reactions such as the replacement of the OAr group of an ester by a nucleophile such as water, hydroxide ion or peroxy anion. When extended storage stability of a chemiluminescent reagent composition containing an N-alkylacridancarboxylate derivative is desired, one or more of the groups R may be a group such as an alkyl, alkoxy or aryloxy group.
Examples of some preferred compounds are:
I
N
R b -c 0 0-CHX a wherein X is a halogen atom selected from F, Cl, Br or I and m is 2 to Another class of preferred compounds are: 04 9
V
9r 9** 9Y** Rn.
OR
9* 9* 9*
C
more preferred are: wherein X is a halogen atom selected from F, Cl, Br or -24- I and mn is 2 to Additional acridan compounds which have been prepared and tested for light production include phenyl lO-methylacridan9..carboxylate; 2' 6 'dimethylphenyl lO-methylacridan-9-carboxylate; 4 '-fl1uoropheny 1 lO-methylacridan-9-carboxylate; 4' -iodophenyl lO-methylacridan-9-carboxylate; 4 '-phenylphenyl lO-methylacridan-9-.carboxylate; 3' S-dicarbomethoxphenyl lO-methylacridan-9-.carboxy1ate; 4'1 (N-butylamnocarbonyl) phenyl lO-methylacridan9..carboxylate; 4 iethoxyphenyl 1O-methylacridan.9-.carboxyl ate; 2' -dilnethoxyphenyl 10O-methylacridan- 9..cboxy late; 4 '-acetamidophenyl lO-methylacridan.9carboxylate; (succinimidyloxycarbonyl) ethyl)phenyl lO-methylacridan-9 -carboxy late; 2 '-hydroxyphenyl 1O-inethylacri.
dan-9-carboxylate; 3 -hydroxyphenyl lO-methylacrida9.....
carboxylate; 4 1-hydroxyphenyl lO0inethyacridan.9..car.
~boxylate; 6'-dihydroxyphenyl lO-methylacrdan-9.carboxylate; 3 -dihydroxyphenyl lO-methylacridan..9-- 20 carboxylate; 31,5',6 '-tetrafluoro-4 '-hydroxyphenyl lO-methylacridan-9-.carboxylate; naphthyl, 2O-methylacridan-9-carboxylate and 6 '-hydroxynaphthy1 2O-methylacri- @0 dan-9-carboxylate. These compounds were less effective than those of the present invention.
Reaction of certain N-alkylacridancarboylate 0* derivatives of the present invention with a peroxide and a peroxidase enzyme produces chemiluminescence with superior properties for assay applications. The chemiluminescence is believed to arise from the excited state of N-alklyacridone or the substituted N-alklyacrjdone product as shown in the generalized reaction below.
Rb Peroxide Peroxidase Enhancer G 0 H Surfactant C Chelating agent light The present invention involves a method of generating chemiluminescence from the oxidation of N-alkylacridancarboxylic acid derivatives by the action of a peroxidase enzyme, a peroxide compound and enhancers. The invention also relates to the use of this method to detect the peroxidase enzyme with high sensitivity. Further, the invention relates to the use of the method to detect and quantitate various biological molecules which are bound to this enzyme by chemical bonds or through physical interactions.
Further, the invention relates to the use of the method to detect and quantitate various biological molecules which are capable of being bound to this enzyme, for 20 example, by using a biotin-labeled analyte and streptavidin-peroxidase conjugate. Other high affinity binding pairs well known in the art such as fluorescein and anti-fluorescein, digoxigenin and anti-digoxigenin or complementary nucleic acid sequences may also be readily employed as a means of linking a peroxidase enzyme to an analyte for the purpose of practicing this invention. The intensity of the resulting chemiluminescence provides a direct measure of the quantity of labeled organic or biological molecule. For example, the method may be used to detect haptens, antigens and antibodies by the technique of immunoassay, proteins by Western blotting, DNA and RNA by Southern and Northern blotting, respectively. The method may also be used to detect DNA in DNA sequencing applications. The method may additionally be used to detect hydrogen peroxide generated by enzymes such as cholesterol oxidase, glucose oxidase, -26glucose-6-phosphate dehydrogenase, galactose oxidase galactose-6-phosphate dehydrogenase, and amino acid oxidase. The method may also therefore be used as a means to detect the enzymes mentioned above which generate hydrogen peroxide.
The reaction of the present invention may advantageously be carried out in solution such as an aqueous buffer or on the surface of a solid support such as a bead, tube, microwell plate or a membrane.
Suitable buffers include any of the commonly used buffers capable of maintaining a pH in the range of about 7 to about 10 for example, phosphate, borate carbonate, tris(hydroxymethylamino)methane, glycine, tricine, 2 -amino-2-methyl-l-propanol, diethanolamine and the like. The preferred method of practicing the invention in this regard is determined by the requirements of the particular intended use as in for example, immunoassays, Western blotting, Southern blotting etc. When the detection is to be performed on a membrane, said membrane may optionally be provided in a kit.
The detection of chemiluminescence from the oxidation of an N-alkylacridancarboxylate derivative by hydrogen peroxide and a peroxidase enzyme can be 25 accomplished with good sensitivity. Enhancement of this reaction by incorporation of chemiluminescence-enhancing substances permits the measurement of chemiluminescence using still lower levels of the peroxidase enzyme.
Coupling this enzyme to a biological molecule of :30 interest then permits the detection of this biological molecule with great sensitivity.
Incorporation of certain substituted phenolic compounds either alone or in combination with surfactants into the reaction mixture enhances the chemiluminescence produced in the presence of added peroxidase and peroxide. Enhancement may take the form of either a higher light intensity, or light emission of -27longer duration or both. Phenolic compounds which are known to enhance other peroxidase reactions and whichare found to enhance the amount of chemijluminescence in the present invention include but are not limited to: p-phenylphenol, piodophenol,, p-bromophenol, p-hydroxycinnamic acid, 2 -naphthol arnd 6 -bromo-2-naphthol. It will be obvious to one knowledgeable in the art that other phenolic and aromatic amine compounds fall within the scope of this invention. Such compounds include firefly luciferin, 6-hydroxybenzothiazole, 2 -cyano-6-hydroxybenzothiazole 4 4 -hydroxyphenyl)thiazole, p-chlorophenol, 2 4 -dichlorophenol, 2 -chloro-4-phenylphenol, 1-bromo-2-naphthol l, 6 -dibromo-2..naphthol, 2 -hydroxy-9..fluorenone, 6 -hydroxybenzoxazole derivatives, and 4-hydroxy-3- 4 -hydroxyphenyl) 1-oxo-2.-propenylJ 2 H-1-benzopyran2 one as are described in, for example, G. Thorpe, L. Kricka, in Bioluminescence and Chemi luminescence, New Perspectives, J. Scholmerich, et al, Eds., pp. 199-208 (1987), M. Ii, Yoshida, Y. Aramaki, H. Masuya, T. Hada, M. Terada, M. Hatanaka, Y. Ichimori, Biochem. Biophys. Res. Comm., 193(), 40- (193) and in U.S. Patent Nos.5,768 and 5,206,149 which are incorporated herein by reference.
Additives which suppress the generation of *.*.chemi luminescence from the reaction of hydrogen peroxide and N-alky].acridancarboxylate derivatives in the absence of peroxidase enzymes are employed to further improve 30 the utility of the invention. It has also been found that certain compounds including cyclodextrins and surf actants including
C,
2
-C
20 alkyl sulfates and sulfonates, dextran sulfate,
C
12 C 20 alkyltrimethylammonium salts, and nonionic surfactants including polyoxyalkylene alkyiphenols, Polyoxyalkylene alcohols, polyoxyealkylene ethers, polyoxyalkylene sorbitol esters and the like improve the sensitivity of -28detection of the peroxidase enzyme in assays of the present invention by providing a better signal to background ratio. The improvement occurs through minimizing the background chemiluminescence in the absence of added peroxidase, possibly due to a slowing of the autoxidative decomposition of the acridan derivative.
The preferred amounts of the various components of a composition of the present invention are set forth in Table
I.
TABLE I Acridan 5 0.01 10 mM Phenol enhancer 0.001 10 mM Surfactant 0.005 5 Peroxide 0.01 10 mM Chelating agent 0.01 5 mM The present invention involves a solution in an aqueous buffer containing 1) a phenol enhancer or a salt of a phenol enhancer, 2) a peroxide compound wherein the peroxide compound may be hydrogen peroxide urea peroxide, or a perborate salt, 3) an acridan compound of the invention, 4) a cation complexing agent wherein the agent may be selected from the group consisting of chelating agents such as 25 ethylenediaminetetraacetic acid
(EDTA),
diethylenetriaminepentaacetic acid (DTPA), or ethylenebis(oxyethylenenitrilo)-tetraacetic acid (EGTA) and their salts, and 5) a surfactant such as the anionic surfactant sodium dodecyl sulfate (SDS), or preferably 30 a nonionic surfactant such as polyoxyethylenated alkylphenols, polyoxyethylenated alcohols, polyoxyethylenated ethers, polyoxyethylenated sorbitol esters and the like.
In a preferred method of practicing the present invention, an aqueous buffer solution with a pH in the range of 7-10 containing a phenol compound such as p-phenylphenol at a final concentration from about -29- 0.01 M to 1 x 104 M, a nonionic surfactant at a final concentration from about 5 to 0.005 aperoxide source such as hydrogen peroxide or preferably, a perborate salt or urea peroxide and a cation complexing agent such as EDTA at a final concentration from about 1 x 10 3 M to 1 x 10* M is mixed with a second solution containing an acridan compound of the invention to achieve a final concentration from about 0.001 M to 1 x 10- 5 M to form the detection reagent solution. This solution is contacted with the peroxidase enzyme which may either be in solution or adhered to a solid support. Optimum concentrations of reagents must be determined individually for each composition. The concentration of acridan compound and enhancer in particular should be optimized with care for each case in order to produce the maximum enhancement of light emission. The detection reaction may be performed over a range of temperatures including at least the range 20 40 oC.
Detection may be conveniently and advantageously carried out at ambient temperature.
It has further been discovered that the storage life of the detection reagent composition can be significantly extended by excluding oxygen from the solution. Detection reagents of the present invention stored in this manner retain the ability to generate the same quantity of chemiluminescence by the action of a peroxidase enzyme for longer periods of time. Extended storage stability can result in savings in reagents and cost.
Significant advantages of N-alkylacridancarboxylate derivatives and compositions of the present invention containing them are increased sensitivity of detection of the peroxidase enzyme and increased stability of the N-alkylacridancarboxylate derivative to hydrolytic decomposition. Comparative experiments show a 100-fold lowering of the detection limit of HRP using a reagent composition of this invention compared to a detection reagent containing luminol and an enhancer.- An additional advantage is the wider dynamic range of measurement of peroxidase concentration. An additional advantage of N-alkylacridancarboxylate derivatives is their thermal and photochemical stability and ease of purification. The most widely known chemiluminescent substrates for peroxidase enzymes known in the prior art, aminoaryl cyclic diacylhydrazides such as luminol are difficult to prepare and maintain in a state of high purity and must either be protected from light or purified immediately before use A. W. Stott, L. J.
Kricka, Bioluminescence and Chemiluminescence, New Perspectives, J. Scholmerich, et al, Eds., pp. 237-240 (1987)). Still another advantage of the use of certain N-alkylacridancarboxylate derivatives compared to prior compounds is the extended duration of chemiluminescence.
Extending the duration simplifies the measurement by obviating the need for precise reaction timing and increases the sensitivity of detection when using film-based detection methods.
EXAMPLES
Example i. Synthesis of Acridan Derivatives.
Acridancarboxylate derivatives 5a-j were synthesized 25 according to the method shown in Scheme 1 from the corresponding acridine-9-carboxylic acid. In the .ee. structure shown below the groups in formula (II) are all hydrogen except as otherwise specified by the substituent A, the groups (R)b in formula (II) are all 30 hydrogen except as otherwise specified by the substituent
B.
-31- CQW~und
A
H
H
H
H
H
H
2-OCH 3 3-OCH 3 3-00H 3 2-00H 3
H
H
H
H
H
H
H
H
H
7-OCH 3 Ar 2,6-difluorophenyl 3 1 2 ,4,6-tdch lorophenyl 2,4,5-t rich lo rophe nyl 2 3 ,6-trifluorophenyl pentafluorophenyl 2 3 ,6-trifluorophenyl 2 .3,6-trif luorophenyl 2 ,6-diftuorophenyl 2,3 .6-trifluorophenyl Scheme 1.
A-B SOC1 2 N7 ArOH A~ 0~ (3B AQ 0DC3B -OH c-l C -Ar 1* 0 *o R, CF3SO 3 0 3B C0-Ar 0
A
CH3SOiCF,
CH
2
CI,
Zn, H+ -32- The corresponding acridine-9-carboxylic acid compounds 1, when not commercially available, were prepared by the reaction sequence depicted in Scheme 2. Zomer, j.
Stavenuiter, R. Van Den Berg, E. Jansen, In Luminescence Techniques in Chemical and Biochemical Analysis, W. Baeyens, D. De Reuikeleire, K. Korkidis, eds., Dekker, New York, 505-521, (1991); R. Stoll, 3.
Prakt. Chem., 105, 137, (1922)).
N
A-Q Q 0B
C-OH
0 la H
H
-OCHf 3
H-
ii 2-001-3 -33- Scheme 2.
NH 2 AC20 OAC Zn a
C
2 0 2 C' 1 2 AiC1 3 A 0
N
1. RON A-Q@T
~C-OH
0( a.
a.
*eaaaa a -34- General Procedure for synthes oI Cmod3.
Acridine-9-carboxylic acid (Compound la, Aldrich, 0.2-0.5 g) was suspended in excess thionyl chloride (3-10 mL) and reaction mixture was ref luxed for 3 hi. The solvent was removed under reduced pressure to obtain a yellow solid which was dissolved in methylene chloride and pyridine (2-3 eq.) under argon. A solution of the phenol (1-1.5 4eq.) in methylene chloride was added dropwise. The solution was stirred overnight at room temperature then diluted with more methyleno chloride (100 mL) and washed with water (3 x 50 zLL). The organic layer was dried over Na 2
SO
4 and concentrated to obtain the product.
a a 0.60 g 10 mL 2.6-Difluoro- 0.32 g 0.44 g b a 0.25 g 10 mL 3,5-Dlfluoro- 0.169 0.229 C a 0.50 g 7 mL 2.4.6-Trnchloro- 0.50 g 0.52 g d a 0.50 g 7 mL 2A4.-TrIohloro- 0.500 g.52 g o a 0.50 g 5 rL 2,3,6-TrIllluaro- 0.365 g 0.53 g 1 a 0.50 g 10 ML Pentaluoro. 0.4549 0.44 g 9 g 1-20 g 10 mL 2,3,6-Triftuara- 0.80 g 3 mL h h 1.500g 10 mL 2.3.6-Trtuara- 0.57 a 0.7 mL *i h 1.70 g 2-0 mL 2.6-Diftuoro- 1.0Og 2 mL *.25iJ1.0Og 10 mL 2,3,6-Trifluoro- 1.49 g1 mL .General Procedure for Synthesis of Comround 4.
Compound 3 (1-2 mmol) was dissolved in methylens chloride (5-10 al) under argon and methyl trifluoromethanesulfonate (5-10 eq.) was added. The solution was stirred overnight at room temperature to yield a thick yellow precipitate. This precipitate was filtered, washed with ether and dried to obtain the product as yellow crystals.
Compound 4 a b c d e f g h i Compound 3 CHC1l a 0.40 g 5 mL b 0.20 g 5 mL c 0.54 g 10 mL d 0.25 g 10 mL e 0.30 g 25 mL f 0.50 g 15 mL g 0.020 g 2 mL h 0.24 g 3 mL i 0.38 g 5 mL j 0.45 g 10 mL CHOS0 2
CF
3 0.945 mL 7 eq.
0.472 mL 7 eq.
1.5 mL 10 eq.
0.70 mL 10 eq.
0.95 mL 10 eq.
1.0 mL 7 eq.
0.10 mL 19 eq.
0.10 mL 1.4 eq.
1.0 mL 8.8 eq.
1.0 mL 8.8 eq.
S.
S
SSS*
r
S
S
S.
.u S General Procedure for Synthesis of Compound (Method Compound 4 (0.2-0.3 mmol) was suspended in absolute ethanol (15-30 mL) and solution was refluxed for 10 min to obtain a clear solution. Excess ammonium chloride (10-50 eq) was added by portions to the solution followed by zinc (equimolar ratio to the amount of NH 4 C1) causing immediate decolorization of the solution. The colorless solution was refluxed for min. The cooled solution was filtered and the precipitate washed with ethanol (3x20 mL). The solution was concentrated to obtain a creamy solid which was redissolved in methylene chloride and washed with water (3x50 mL). Crude material obtained after evaporation of methylene chloride was chromatographed on silica gel (ethyl acetate/hexane) to yield the pure product as a white solid.
General Procedure for Synthesis of Compound (Method Compound 4 (0.3-0.6 mmol) was dissolved in mL of glacial acetic acid to obtain a yellow solution and zinc was added (100 eq.) causing immediate decolorization of the solution. After 5 min stirring at room temperature, TLC of the reaction mixture showed a nonpolar material. The acetic acid was decanted and the solid washed with methylene chloride. The combined organic solutions were evaporated to obtain a crude ww -36solid which was redissolved in methylene chloride and washed with 2 or 3 50 mL portions of water. The crude material obtained after evaporation of methylene chloride was chromatographed on silica gel (20-30 ethyl acetate/hexane) to yield the pure product as a white solid.
The compounds 5a to 5j and intermediates were prepared as follows: compound compound Method 5 -4 Zinc Ethanol NH.C1 Acetic Acid A a a 0.10g 0.65g 15 niL 0.535 g- B b b 0.20g 1.30g 10 MI B c c 0.34g 3.§9g 10 ML B d d 0.12g 1.4 g 10 ML B e e 0.20g 2.5Sg 10 ML B f f 0.080g 0.4 g 5 ML A g g 0.005g 0.15g 10 niL 0.15 g A h h 0.035g 4.Og 15 mL 4.0 g A i i 0.20g 2.Og 20 mL 2.0Og A j j 0.17g 2.Og 50 m 2.0Og Example 1. Synthesis of Compound 2' 61 -Difluorophenyl acridine-9-carboxcylate (3a).
Product was further purified by chromatography on silica gel (30 ethyl acetate/hexane) to yield the pure product as a creamy solid. 1H NMR (CDCl 3 6 7.13-7.39 (in, 3H), 7.68-8.35 (in, 8H).
S 30 21 61 -Difluorophenyl trif luoromethanesulf onate 1H NMR (acetone-d 6 6 5.28 3H), 7.44-7.68 (in, 3H), 8.32-9.13 (in, 8H).
6 '-Difluorophenyl lO-methylacridan-9-carboxylate (5a) Method A. 'H NMR (CDCl 3 6 3.49 3H) 5. 29 (s, 1H), 6.82-7.10 (in, 7 H) 7 .29-7.41(m, 4H).
Example 2. Synthesis of Compound OS..3' ,5 '-Difluorophenyl acridine-9-carboxylate
H
NMR (CDCl 3 6 6.84-7.09 (mn, 3H) 7.67-8.37 (mn, 8H).
3 5' -Difluorophenyl lO-methylacridinium-9-carboxylate trif luoromethanesulf onate Compound 3b was reacted -37for three days with methyl trifluoromethanesulfonate in dichloromethane. 1H NMR (acetone-d) 45 5.25 3H),- 7.22-7.59 3H), 8.23-9.09 (in, 8H).
3',51Difluoropheny. lO-methylacridan-9.carboxylate (Sb) Method B. 1H NMR (CDC1 3 6 3.44 3H) 5.16 (s, 1H), 6.49-6.65 (in, 3 6.99-7.36-(mn, 8H).
Example 3. Synthesis of Compound 21,41,6 '-Trichiorophenyl acridine-9 carboxylate (3c).
Yellow solid: 1H NMR (CDCl 3 6 7.55 2H) 7.67-8.61 8H).
2' ,4 6' -Trichiorophenyl lO-methylacridinium9..carboxylate trifluoromethanesulfonate 'H NNR Cacetone-d 5.28 3H), 7.93 2H), 8.31-9.13 (in, 8H).
2 4 6 t -Trichlorophenyl IlO-methylacridan-9..carboxy late (Sc) Method B. IH NMR (CDCl 3 6 3.42 3H) 5.27 (s, 1H), 6.97-7.39 10 H).
Example 4. Synthesis of Compound Sd 9 go2',41,5S -Trichiorophenyl acridine-9-carboxylate (3d).
Sc.. Yellow solid: 'H NMR (CDCl 3 6 7.63-8.34 (mn, 10H) got* 2' ,5S -Trichlorophenyl lO-methylacridinium-9-.carboxylate trifluoromethanesulfonate 1H NNR (acetone-d 6 6 5.26 3H), 8.09 1H), 8.26-9.11 (in, 9H).
5* 30 21,41,5' -Trichiorophenyl 10O-methy lacr idan- 9 carboxy late Method B. 'H NMR (CDC1 3 6 3. 43 Cs, 3M) 5. 23 (s, 0g 1H), 6.97-7.42 (in, 10 H).
Example 5. Synthesis of Compound 2 1,31,6 -Trif luorophenyl acridine-9 -carboxy late (3e).
The yellow solid was further washed with ether to remove excess of phenol (82 yield): 'H NMR (CDCl 3 65 7. 08-7.28 -38- (in, 2H) 7.71-8.42 (mn, 8H).
2' ,3,6'-Trifluorophenyl lOmethylacridiniu m-....carboxylate trifluoromethanesulfonate Due to low solubility, compound 3d was suspended in 25 mL of methylene chloride and treated according to the general procedure. IH NMR (acetone-d 6 6 5.29 3H) 7.50-7.67 (in, 2H) 8.26-9.14 8H) -Trifluorophenyl 10O-methylacr idan-9..carboxy late Method B. 'H NMR (CDCl 3 6 3.44 3H) 5.29 (s, 1H), 6.76-6.84 (mn, 2 H) 6.99-7.39 (mn, 8H).
Examp~le 6. Synthesis of Compound Pentafluorofluorophenyl acridine-9-carboxylate (3f).
'H NMR (CDCl 3 6 7.70-8.35 (mn, 8H) Pentafluorofluorophenyl lO0inethylacridiniun.....carboxylate trifluoromethanesulfonate 1H NMR (acetone-d 6 6 5.29 3H), 8.33-9.15 (mn, 8H1).
Pe t f u r f u r p e y 10 m t yl c i an 9 c rb x l t P-etfloarfluroenyOmehcidncarboxylcaidte. H N MeHo B.R 'HD3 6M 4.0C 3 0 6s 3H) 3M),-5.31 (s, 6 47-.43 (in, 4H) 4. sH), 7.32-7.37
WOOMMUS
-39- 2' ,31,6' -Trifluorophenyl 2 -methoxy-10-methylacridan- 9 carboxylate (5g) Method A. 'H NMR (CDC1 3 5 3.414 3H), 3.821 3H), 5.272 1H), 6.78-7.38 (in, 9H).
Examiple-8. Synthesis of Compound 3 -Methoxyacridine-9..carboxylic acid Condensation of the diarylamine with oxalyl chloride produced a mixture of the 3-methoxy and 1-methoxy isomers which were separated after conversion to the esters by column chromatography on silica with 20% ethyl acetate/hexane. 'H N14R (DMSO-d6) 6 4.048 3H), 7.47-8.24 (in, 7H).
2' -Trifluorophenyl 3-ehxardn--abxlt 'H NMR (CDCl 3 6 4.043 3H),7.08-8.2 5 (in, 9H).
21,3'1 6 '-Trifluorophenyl 3 -inethoxy-1o-methyl..
acridinium-9 -carboxy late trif luoroinethanesulf onate (4h) 'H NI4R (DMSO-46) 6 4.288 3M), 4.837 3H) 7.64-8.89 (mn, 9H).
2 0, 3 1 ,6'-Trifluorophenyl 3 -methoxy-1o..
mehlcia--abxlt (5h) Method A. 'H NMRJ (CDC1 3 6 3.422 3M) 3.847 3M) 5.25 1H) 6.54-7.39 (mn, 9H).
Exaile 9. Synthesis of Compound 2' E-Difluorophenyl 3 -methoxyacridine-9-..carboxylate A mixture of the 3-methoxy and 1-methoxy isomers was obtained which was separated by column chromatography on silica with 20% ethyl acetate/hexane.
'H NMR (CDC1 3 6 4.047 3H) 7. 14-8.28 (mn, 10H) 2' ,6 '-Difluorophenyl 9 -carboxylate trifluoroinethanesulfonate 'H NMTR (DMSO-d 6 6 4.289 3H) 4.838 3H) 7.52-8.89 21, 6 -Difluorophenyl 3 -9-carboxylate Method A. 'H NNMR (CDC1 3 6 3.416 3H), 3.843 3H), 5.241 1H), 6.53-7.39 (n Examp~le 1.Synthesis of Compound 2 7 -Dimethoxyacridine-9-.carboxylic acid The acid was formed by the reaction sequence depicted in Scheme 2 with the exception that the condensation of the diarylamine with oxalyl chloride was performed in dichioromethane solvent. IH NMR (DMSO-d.) 6 3.862 (s, 6H), 7.234-7.242 2H), 7.523-7.583 (dd, 2H), 8.103-8.136 2H).
21,3' ,6'-Trifluorophenyl 2 7 -dimethoxyacridine -9-carboxylate 'H NMR (CDC1 3 6 4. 010 6H) 20 7.06-7.25 (in, 2H), 7.379-7.387 2H), 7.436-7.476 (dd, 2H), 8 .125-8.156 2H).
2 1 3 1 ,6'-Trifluorophenyl 2 ,7-dimethoxy-louie.uylarrdniun-9-carboxylate tiluoromethanesulfonate Compound 3 i was reacted for several days with methyl trfurmtansloaei dichorehan.~ :The amine salt formed but did not crystallize from the reaction mixture. 'H NMR (acetone-d 6 6 4.159 6H), 5.184 3H), 7.40-8.98 (in, 8H).
2' -Trifluorophenyl 2, 7 -dimethoxy-1o-methylacridan -9-carboxylate Method A. 'H NMR (CDCl 3 6 3.375 3H), 3.811 3H), 5.230 1H), 6.78-6.96 (in, 8H).
Comparative Example 4 1 -Fluorophenyl acrid ine-9 -carboxy late. (90 yield): -41- 'H NMR (CDC1,) 6 7.21-7.47 4H), 7.67-8.39 8H).
4'-Fluorophenyl 10-methylacridinium-9-carboxylate trifluoromethanesulfonate). 4'-Fluorophenyl acridine-9-carboxylate was reacted for three days with methyl trifluoromethanesulfonate in dichloromethane. 1H NMR (acetone-d) 6 5.23 3H), 7.37-7.81 4H), 8.23-9.08 8H).
4'-Fluorophenyl 10-methylacridan-9-carboxylate. Method B. 'H NMR (CDC 3 1) 6 3.43 3H) 5.17 1H), 6.84-7.38 12H).
Chemiluminescence Measurements The experiments in the following examples were performed using either a Turner Designs TD-20e (Sunnyvale, California) luminometer fitted with neutral density filter for light attenuation or a Labsystems Luminoskan (Helsinki, Finland) luminometer. Data collection, 20 analysis and display were software controlled.
.:Example 11. Comparison of the Light Intensity-Time Profile for Detection of HRP with Compounds 5a-f, h, a Prior Art Acridan and Luminol. In separate experiments, 40 AL volumes of each of three formulations were reacted with 1 AL of a solution containing 1.4 x 10.16 mol of HRP in water. The formulations consisted of: 0.05 mM acridan compound 5a-f or h in 0.01 M tris buffer, pH 8.0, 0.4 mM urea peroxide, 0.1 mM p-phenylphenol, 0.025% Tween 20, 1 mM EDTA; an identical formulation containing the previously reported acridan 4'-fluorophenyl 10-methylacridan-9-carboxylate in place of the acridan 5a-f or h McCapra, Pure Appl. Chem., 24, 611-629 (1970)) and an optimized reagent containing luminol (Amersham ECL formulation and peroxide in separate bottles). Figures 1-7 show the improved generation of light emission using reagents -42containing acridans 5a-f and h of the present invention compared to the prior art reagents under these conditions.
Example 12. Optimization of Formulations. A matrix optimization experiment was done using acridans 5a, and 5h (0.1 mM 0.05 mM) in solutions containing p-iodophenol (0.1-2.25 mM) or p-phenylphenol (0.01-2.25 mM), urea peroxide (0.1 mM 1 mM), Tween 20 in tris buffer, pH 8.0 (0.01-0.2 Sensitivity and dynamic range were evaluated for detection of HRP in the range 1.4 x 10-" 1 to 1.4 x 10-19 mol of enzyme. An especially effective reagent consists of the acridan (0.05 mM), p-iodophenol (1.1 mM) or p-phenylphenol (0.1 mM), urea peroxide (0.4 mM), 1 mM EDTA, Tween (0.025%) in tris buffer, pH 8.0 (0.01 These conditions gave linear assays for HRP over the entire range of enzyme tested for each acridan compound. Light intensity is increased by the incorporation of CaC 2 in 20 the range 0.5-5 jM; higher levels cause background luminescence to increase.
SExample 13. Comparison of the Sensitivity of Detection of HRP with 5e, 5h or Luminol. The linearity of detection of HRP using reagent compositions of the .present invention and a commercially available optimized reagent containing luminol (Amersham ECL) were compared.
Forty ML of a solution containing acridan 5e or 5h as described in example 2 and forty ML of the commercial reagent (Amersham ECL, prepared according to the manufacturer's directions) were mixed at room temperature with 1 gL aliquots of HRP containing between 1.4 x 10-" and 1.4 x 10- 1 9 mol of enzyme. Figure 8 compares the linear range of HRP amount measured. Data from the reagents containing acridans 5e and 5h were measured at 15 min while data from the ECL reagent was measured at the point of maximum light intensity due to -43the faster rise and decay of light intensity for this re.agent. Howeve,. f, teto B e i y o r this intensity with the time to reach the maximu light SECL reagent varies with the a o HRp which makes measurement of the am ou nt of R S difficult. The term S-B refers to the cheMiluminescence signal in RLU in the presence of HRP corrected for background chemiluminescence in the absence of tRp.
Reagents containing acridans se and 5h are capable of 100 -fold greater sensitivity of detection than the ECL reagent. Measurement with the reagents containinge
ECL
acridans so or 5h could be measured at earlier times acridans. or h could t J^ c ntaing^ with equivalent sensitivitym Stability of Horseradish Peroxidase Detection Reagent Containing e The deteion reagents are conveniently stored in two containers, the first comprising an aqueou s buffer solution containing the Peroxide, phenol enhancer, TWEEN 20 and EDTA, the Second solution comprising the acridan compoud 5 in a water-mis.cible organic solvent such as 1:1 ethanol/p-d.ioxane. When stored in this manner, the dcomponents are stable for several months. The tfinal o e t .ction reagent is prepared by mixing appropriate •:q"ntitie Of the two solutions before use. An advantage of acridans of the present invention is their greater stability in the final detection reagel .ixture. Stabil.ty is assessed by measun th eak 1.25 1 pL.oireantd 2 e n 0 -1 M te lih tnt p H 8torefrd nit oX the1 lution rate thperatu se edamount of RP. The pea light 3 intensities from 41 ML of a detection reagent 30 containing compound 5e 0 .05mrM), p-iodophenol (1i. m M), urea peroxide (0.4 mM), TWEEN 20 (0.025%),ET 1i.~ p-dioxane and 1.25 ethanol in 0.01 M tris buffer, pH 8.0 reacted withi 1.4 x i0 tool of HRP at room temperature stored under various conditions are given below.
MMUM
-44- 0 r e i h A r o p d o no 24 no 24 0.2 24 yes Exclusion of xygen from the detection reagent in superior stability for Exame 15-- Stability of Horseradish Detection Reagent Containing 5h A s iilar set of wasiilar se experiments was performed using a reagent with the ame composition containing instead acridan 5h. Stabl he was assessed by measurin* the a c r i d a n 5 lg ht iility an aliquot of the solutio peak light intensity from amount uo of The eaksolution eacted with a specified amount of HRp. The peak light intensities from 41 1 L of a detection reagent c intensities from 41 mM) P-lodophenol ontaining compound 5h (0.05 Podoheno (1.1 M, urea peroxide (0.4 mM), TWEEN 20 EDTA (1 mM), 1.25 p-dioxane and 1.25 ethanol in 0.01 M tris buffe 25 PH 8.0 reacted W2th 1. 4 10 ol f HRP at r b om temperature tored under arious coditions s t o r s are given below ."'Ond Storae Timehr Ar on-.u ed el.
"0 0re no 24 no 24 25 yes 25 o1.0 A detection reagent incorporating acridan Sh shows perior tability pared to previous compounds and is stable for at least one day ithout special precautions. one day without special This solution showed a lower rise time to the peak light intensity.
Effect of pH of the Buffer Detection reagent solutions according the compositi of Example 14 were prepared with either 0.01 M tris in the PH range 7.0-9.0 or 0.01 M Potasiu1 M h at in the range 6.0-6.5 and reacted with HRP phosphate in the pH signal to reagent background resulted The best ratio of d from reagents with a pH in the range 7.5-8.5.
Example 17. Effect of Buffer Salt. Detection reagent solutions according to the composition of example 14 were prepared with substitution of various buffer solutions and reacted with HRP. Useful levels of light intensity compared to reagent background were obtained with reagents prepared from tris hydrochloride, tris acetate, tris malate, potassium phosphate, diglycine-sodium hydroxide and tricine buffers.
Example 18. Effect of Enhancers. Detection reagent solutions according to the composition of example 14 were prepared with substitution of various phenolic enhancers and reacted with HRP. Useful levels of light intensity compared to reagent background were obtained with reagents incorporating p-iodophenol, p-hydroxycinnamic acid and p-phenylphenol.
20 Example 19. Effect of Peroxide. Detection reagent solutions according to the composition of example 14 were prepared with substitution of various peroxides and reacted with HRP. Useful levels of light intensity compared to reagent background were obtained with 25 reagents incorporating hydrogen peroxide, sodium perborate and urea peroxide.
*.Example 20. Effect of Surfactant. Detection reagent solutions according to the composition of example 14 were prepared with substitution of various surfactants and reacted with HRP. Useful levels of light intensity compared to reagent background were obtained with reagents incorporating TWEEN( 20 (Aldrich, Milwaukee, WI), TRITON( X-405 (Aldrich), BRIJ 35 (Aldrich), sodium dodecyl sulfate, cetyltrimethylammonium bromide, f-cyclodextrin and dextran sulfate.
-46- Example 21. Improved Chemiluminescent Detection of Proteins by Western Blot Rabbit anti-goat IgG-peroxidase conjugate was obtained from Cappel Products (Durham, NC). Human transferrin and fractionated goat anti-human transferrin serum were purchased from Sigma Chemical Co. (St. Louis, MO). The IgG sample was centrifuged at 10,000 g for two minutes and the supernatant was used in the immunological reaction. Polyvinylidene difluoride (PVDF) transfer membrane (IMMOBILON P) was obtained from Millipore Corp.
(Bedford, MA). Kodak (Rochester, NY) X-OMAT AR film was used in the assay procedure.
SDS-PAGE was performed utilizing the buffer system described by Laemmli K. Laemmli, Nature (London), 227, 680 (1970)). The stacking gel was 4.38% acrylamide 0.12% bisacrylamide. The separating gel was 6.81% acrylamide 0.19% bisacrylamide. Following electrophoresis the gel was equilibrated for 7-8 minutes with the transfer buffer which contained 20 mM Tris, 153 20 mM glycine and 20% methanol. The gel, sandwiched between a sheet of transfer membrane and a sheet of chromatography paper 3MM (Whatman), was placed in the transfer unit (Bio-Rad Laboratories, Richmond, CA). The proteins in the gel were electroeluted for 25 min at 4 0
C
25 at a 100 V constant voltage. The membrane was then placed in 50 mM Tris-HCl buffered saline at pH 7.4 (TBS) at 40 C overnight. After this period the membrane was washed with TBS for 15 min.
The membrane was treated with 0.05% in 50mM Tris-HCl buffered saline at pH 7.4 (T-TBS) containing 1% non-fat powdered milk (NFM) for one hour at room temperature. This blocked membrane was incubated for 75 minutes at room temperature with primary antibody (1:1500 dilution of goat anti-human transferrin IgG fraction) using T-TBS containing 1% NFM.
The membrane was then rinsed and washed three times for five min each with T-TBS at room temperature.
-47- The washed membrane was incubated for one hour at room temperature with secondary antibody (1:50,000 dilution of rabbit anti-goat IgG peroxidase conjugate) using T-TBS containing 1% NFM. The membrane was rinsed and washed four times for ten minutes each with
T-TBS
followed by a five min wash with
TBS.
The washed membrane was soaked in one of four detection reagents for 5 min, drained and placed between sheets of transparency film. Reagent A was a commercial reagent containing luminol (Amersham ECL). Reagent
B
contained the acridan 4 '-hydroxyphenyl 10-methylacridan-9-carboxylate previously disclosed in applicants' co-pending application Serial No.
08/061,810. Reagent C contained acridan 5e. After an incubation period of 15 min, the X-ray film was exposed to the membrane for varying periods of time and developed. The composition of detection reagent solution containing the acridan compounds was: 20 Tris buffer, pH 8.8 0.1 M .Acridan 0.05 mM p-iodophenol 1.1 mM TWEEN 20 0.5 (w/w) NaBO 3 .4H 2 0 2.5 mM EDTA 0.5 mM P-Dioxane 1.25 Ethanol 1.25 The transferrin standards utilized were clearly visible down to 5 pg/slot over the background after a 15 s exposure to Kodak X-OMAT AR X-ray film. It was possible to make several exposures of the membrane during a period of 24 hours as the membrane continued to emit light. Figure 9 is a digitally scanned image of the X-ray film record of an experiment using a 14 min incubation and 15 s exposure.
Examle 22. Chemiluminescent Detection of Proteins by -48- Western Blot using Nitrocellulose Membrane. A Western blot analysis of human transferrin was conducted by themethod of example 21 with blotting of protein onto nitrocellulose in place of PVDF. The transferrin standards utilized were clearly visible down to pg/slot over the background after a one min exposure to Kodak X-OMAT AR X-ray film. It was possible to make several exposures of the membrane over a period of 12 hours as the membrane continued to emit light. Figure 10 is a digitally scanned image of the X-ray film record of an experiment using a 15 min incubation and 1 min exposure.
Example 23. TSH Enzyme Immunoassay A TSH assay was performed using the components of a COBAS Core enzyme immunoassay kit for TSH from Roche (Basel, Switzerland) and a detection reagent of the present invention. The detection reagent comprised: Tris buffer, pH 8.0 0.01 M 20 Acridan 5h0.05 mM 0.05 mM P-iodophenol 1.1 mM TWEEN N 20 0.025 (w/w) Urea peroxide 0.4 mM EDTA 1 mM p-Dioxane 1.25 1 .25 Ethanol 1.25 and could be prepared up to one day in advance. Samples prepared from the supplied standards were treated according to the manufacturer's instructions up to the detection stage. At this point, beads coated with the primary antibody/TSH/secondary antibody-HRP immunological complex were treated with 100 gL of detection reagent in wells of a white 96 well plate (Dynatech Microlite Chemiluminescence intensities were measured in a Labsystems Luminoskan luminometer every 2.5 min. The assay resulted in a linear calibration curve over four orders of magnitude with a Mww 4 -49lowest detected quantity of 0.003 mIU/L. Excellent linearity and identical analytical sensitivity resulted when light intensity was measured at 5, 10 or 15 min in each well (Figure 11). The term S-B refers to the chemiluminescence signal in RLU in the presence of HRP corrected for background chemiluminescence in the absence of HRP. The quoted analytical sensitivity of the manufacturer's assay which uses a colorimetric endpoint is 0.05 mIU/L.
Example 24. Human Growth Hormone Enzyme Immunoassay An hGH assay was performed using the components of a sandwich enzyme immunoassay kit for hGH from Sorin Biomedica (Vercelli, Italy) and a detection reagent of the present invention. The detection reagent comprised: Tris buffer, pH 8. 0 0.01 M Acridan 5h 0.05 mM P-iodophenol 1.1 mM Tween 20 0.025 (w/w) 20 Urea peroxide 0.4 mM EDTA 1 mM p-Dioxane 1.25 Ethanol 1.25 and could be prepared up to one day in advance. Samples 25 prepared from the supplied standards were treated according to the manufacturer's instructions up to the detection stage. Upon completing the immunological reaction, the streptavidin-coated wells (supplied by the manufacturer) with bound biotin-primary antibody/TSH/secondary antibody-HRP immunological complex were treated with 100 ML of detection reagent.
Chemiluminescence intensities were measured in a Labsystems Luminoskan luminometer every 2.5 min.
A
nonlinear calibration curve resulted which allowed direct measurement of hGH down to 0.05 ng/mL (Figure 12). The term S-B refers to the chemiluminescence signal in RLU in the presence of HRP corrected for background chemiluminescence in the absence of HRP.
The assay was repeated with a modification inwhich the secondary antibody-HRP conjugate supplied in the kit was diluted 10-fold with a dilution buffer supplied by the manufacturer. The assay resulted in a linear calibration curve over three orders of magnitude with a demonstrated detection limit of 0.05 ng/mL of hGH. Excellent linearity and identical analytical sensitivity resulted when light intensity was measured at 2.5, 5, 7.5 or 10 min (Figure 13). Calibration data supplied by the manufacturer for the colorimetric method results in a nonlinear curve covering two orders of magnitude and requires a 30 min detection time. The calculated analytical sensitivity (signal 2 standard deviations of blank) of the manufacturer's assay is 0.05 ng/mL.
Example 25. Chemiluminescent Detection of Southern Blots. Mouse genomic DNA (Clontech Laboratories. Inc., 20 Palo Alto, CA) was cleaved to completion with restriction endonuclease EcoRl (Boehringer-Mannheim) at a concentration of 50 Ig/mL. The restricted DNA was purified by extraction once with phenol/chloroform, once with chloroform and was precipitated with ethanol. The purified DNA was divided into two portions containing and 15 ug of DNA, respectively and was separated by 0.77% agarose gel electrophoresis. The electrophoresis buffer was 40 mM Tris-acetate and 2 mM EDTA (pH After electrophoresis the gel was rinsed with H 2 0 and then soaked in 0.25 N HC1 for 12 min with gentle agitation.
MAGNAGRAPH NYLON (Micron Separations Inc., Westboro, MA) was soaked sequentially in water and SSC (20X SSC is 3 M NaC1, 0.3 M sodium citrate, pH for 2 and 10 min, respectively. The gel was rinsed with water and then treated with 0.5 M NaOH/1.5 M NaCl twice for 15 and 30 minutes, respectively. The gel was rinsed -51with water and then treated with 1 M Tris-HCl (pH 7.5)/1.5 M NaCl three times for 15 min each. The DNA in the gel was transferred onto the membrane by capillary blotting overnight using 10X SSC. The blots were air-dried for 30 min followed by baking at 800C for 2 hours.
The membranes were prehybridized in hybridization buffer (Amersham RPN.3000) containing NaCl and 5% blocking agent (Amersham #RPN.3000) for 60 minutes at 42 0 C with occasional agitation. The hybridization probe, v-mos DNA (Clontech Lab. Inc.) was labeled with HRP according to the manufacturer's instructions (Amersham #RPN.3000) and the hybridization proceeded overnight at 42 oC using a hybridization buffer containing 0.5N NaC1, 5% blocking agent, and 300 ng/mL HRP-labeled v-mos DNA. The membranes were washed sequentially with room temperature 0.5X SSC/0.4% SDS for and 30 min, then again at 550 C three times for 15 min each, followed by two washes with 2X SSC for 5 min each 20 at room temperature.
The membranes were rinsed with water and placed on 3MM blotting paper for one minute to remove excess solution, then transferred to a clean container followed by the addition of the detection reagent of Example 21. After a one minute incubation, excess solution was drained off and the blots were placed between sheets of transparency film followed by exposure to Kodak X-OMAT XAR 5 film.
The reagent of the present invention can be used to detect a single copy gene in mouse genomic
DNA
as shown in Figure 14A. The target restriction fragment is 14 kb providing 70 pg (7 x 10- moles) of target
DNA
in the 15 mg leading tracks. The single copy gene was clearly visible in both tracks of the blot using the detection reagent of the present invention (Figure 14A) while the luminol reagent did not permit the bands to be detected (Figure 14B).
I
-52-- It is intended that the foregoing descripti 0 be only illustrative of the present invention and that the present invention be limited only by the appended claims.
9* 9*
I.
44 .4 4*4*4*
I
53 The Claims defining the invention are as follows,: I- An acridan of the formula:
(II)
000 q 0@ 0.
0* .0 0 00 0 *000 0 00..
0*00 00 0p 00 0000 00 *0 0 0000 00 0 0 0 9 *0 wherein RI is a lower alkyl group, wherein R is selected from lower alkyl and lower alkoxy groups and a and b are integers between 0 and 4 and wherein 0-Ar is of the formula OC6H5-mXm wherein X is a halogen atom selected from F and Cl, and m is 3 to 5 which allows production of chemiluminescence from the acridan by reaction with a peroxide and a peroxidase.
2. The acridan of claim 1 wherein at least one of R is the lower alkoxy group.
3. The acridan of claim I wherein at least one of R is a methoxy group.
4. A compound which is 6 1 -trichlorophenyl-lO-methylacridan.9-carboxylate.
5. A compound which is 5'-trichlorophenyl IO-methylacridan-9-carboxylate.
6. A compound which is 6'-trifluorophenyl lO-methylacridan-9-caboxylate.
7. A compound which is pentafluorophenyl-l0-methylacridan9carboxylate.
8. A compound which is 6 '-trifluorophenyl 2 -methoxy-10-methylacridan9carboxylate.
9. A compound which is 3T, 6 -trifluorophenyl 3 -methoxy-1o-methylacridan-9-.
carboxylate.

Claims (1)

  1. 7-dimethoxy-1O-methylacida.9.. carboxylate. DATED this 31st day of July, 1998 LUMIGEN, INC. By Their Patent Attorneys DAVIES COLLISON CAVE 0* 0 0 09060 0 0000 0.0 ABSTRACT Acridans which are reactable with a peroxidase and peroxide. The acridans are characterized by having an aromatic leaving group ArO which is a di- or polyhalosubstituted phenoxy group. The compounds are useful in assays where one member of a binding pair is linked to the peroxidase and for detecting the peroxidase. The method can also be used to detect hydrogen peroxide. *ooo
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