US007906293B2

(12) United States Patent (10) Patent No.: US 7,906,293 B2 Mattingly et al. (45) Date of Patent: Mar. 15, 2011

(54) ACRIDINIUM PHENYL ESTERS USEFUL IN WO WO96,07912 A1 3, 1996 THEANALYSIS OF BIOLOGICAL WO OO31543 6, 2000 WO WO 2005.005385 A1 1, 2005 WO WO 2005/O15214 A1 2, 2005 (75) Inventors: Phillip G. Mattingly, Third Lake, IL WO WO 2005/O15215 A1 2, 2005 (US); Maciej Adamczyk, Gurnee, IL WO 20060130736 12/2006 (US); Roy Jeffrey Brashear, OTHER PUBLICATIONS Mundelein, IL (US) Adamczyk, et al., Analytica Chimica Acta, “Homogeneous (73) Assignee: Abbott Laboratories, Abbott Park, IL chemiluminescent assays for free choline in human plasma and whole blood”, 579, 61-67 (2006). (US) Adamczyk, et al., Bioorganic & Medicinal Chemistry Letters, "Rapid high-throughput detection of peroxide with and acridinium *) NotOt1Ce: Subjubject to anyy d1Sclaimer,disclai theh term off thithis 9carboxamide: Ahomogeneous chemiluminescent assay for plasma patent is extended or adjusted under 35 choline”. In Press (2006). U.S.C. 154(b) by 380 days. Brown, et al., Analytical Biochemistry, “Employment of a Phenoxy Substituted Acridinium Ester as a Long-Lived Chemiluminescent (21) Appl. No.: 11/697.835 Indicator of Glucose Oxidase Activity and Its Application in an Alkaline Phosphatase Amplification Cascade Immunoassay, 256, 142-151 (1998). (22) Filed: Apr. 9, 2007 McCapra, et al., Photochemistry and Photobiology, “Chemiluminescence Involving Peroxide Decompositions', 4, 11 11 (65) Prior Publication Data 1121 (1965). US 2008/0248493 A1 Oct. 9, 2008 Razavi, et al., Luminescence, Stable and versatile active acridinium esters I, 15, 239-244 (2000). Razavi, et al., Luminescence, Stable and versatile active acridinium (51) Int. Cl. esters II, 15, 245-249 (2000). GOIN33/53 (2006.01) de Silva, et al., 13' Int. Symposium on Bioluminescence and (52) U.S. Cl...... 435/7.1:436/546 Chemiluminescence, Symposium Abstract. “Use of new (58) Field of Classification Search ...... None chemiluminescent reagent in detectionofoxidasse enzymes and their See application file for complete search history. Substrates by a coupled enzyme reaction’. Aug. 2-6, 2004 Yokohama, Japan, (56) References Cited de Silva, et al., 13' Int. Symposium on Bioluminescence and Chemiluminescence, “Use of New Chemiluminescent Reagent in U.S. PATENT DOCUMENTS Detection of Oxidase Enzymes and Their Substrates by a Coupled Enzyme Reaction'. Aug. 2-6, 2004 Yokohama, Japan, Published on 5,241,070 A 8, 1993 Law et al. website of Lumigen Inc. on Jun. 7, 2006, pp. 1-10. 5,340,714 A 8, 1994 Katsilometes 5,491,072 A 2, 1996 Akhavan-Tafiti et al. Waldrop III, et al., Luminescence, “Chemiluminescent determina 5,523,212 A 6, 1996 Akhavan-Tafiti et al. tion of hydrogen peroxide with 9-acridinecarbonylimidazole and use 5,593,845. A 1/1997 Akhavan-Tafiti et al. in measurement of glucose oxidase and alkaline phosphatase activ 5,670,644 A 9, 1997 Akhavan-Tafiti et al. ity” 15, 169-182 (2000). 5,723,295 A 3, 1998 Akhavan-Tafiti et al. ISA/US, International Search Report (Search Completed Jul. 2, 5,750,698 A 5, 1998 Akhavan-Tafiti et al. 2008), International Application PCT/US2008/59710. 6,673,560 B1 1/2004 Sharpe et al. 2002/017724.0 A1* 11/2002 Kundu ...... 436,518 * cited by examiner 2003/0232405 A1 12/2003 Akhavan-Tafiti et al...... 435/25 2008/0199973 A1* 8/2008 Evangelista et al...... 436,536 Primary Examiner — Jacob Cheu FOREIGN PATENT DOCUMENTS (74) Attorney, Agent, or Firm — Audrey L. Bartnicki; Lisa V. EP O 625,510 A2 11/1994 Mueller; Polsinelli Shughart EP O 750 748 B1 5, 2001 EP O 778946 B1 10, 2002 (57) ABSTRACT EP O 625 510 B1 4/2003 WO WO 93.23756 A1 11, 1993 The present invention relates to methods and kits for detect WO WO94,26927 A1 11, 1994 ing an analyte in a test sample using acridinium-9-carboxy WO WO95/23971 A1 9, 1995 late aryl esters. WO WO95/28495 A1 10, 1995 WO 95.29255 11, 1995 8 Claims, 17 Drawing Sheets U.S. Patent Mar. 15, 2011 Sheet 1 of 17 US 7,906,293 B2 U.S. Patent Mar. 15, 2011 Sheet 2 of 17 US 7,906,293 B2

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U.S. Patent Mar. 15, 2011 Sheet 7 Of 17 US 7,906,293 B2

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FIGURE 8 U.S. Patent Mar. 15, 2011 Sheet 8 of 17 US 7,906,293 B2

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FIGURE 10 U.S. Patent Mar. 15, 2011 Sheet 9 Of 17 US 7,906,293 B2

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FIGURE 12 U.S. Patent Mar. 15, 2011 Sheet 11 of 17 US 7,906,293 B2

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FIGURE 13 U.S. Patent Mar. 15, 2011 Sheet 12 of 17 US 7,906,293 B2

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FIGURE 14 U.S. Patent Mar. 15, 2011 Sheet 13 of 17 US 7,906,293 B2

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FIGURE 16 U.S. Patent Mar. 15, 2011 Sheet 14 of 17 US 7,906,293 B2

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FIGURE 1 7 U.S. Patent Mar. 15, 2011 Sheet 15 Of 17 US 7,906,293 B2

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FIGURE 21 U.S. Patent Mar. 15, 2011 Sheet 17 Of 17 US 7,906,293 B2

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FIGURE 22 US 7,906,293 B2 1. 2 ACRIDINUM PHENYLESTERS USEFUL IN SUMMARY OF THE INVENTION THEANALYSIS OF BIOLOGICAL In one embodiment, the present invention relates to a RELATED APPLICATION INFORMATION method of detecting an analyte of interestinatest sample. The 5 method comprises the steps of None. a) processing a test sample to separate the analyte from protein in the test sample; FIELD OF THE INVENTION b) adding an acridinium-9-carboxylate aryl ester to the processed test sample, the acridinium-9-carboxylate aryl The present invention relates to methods for detecting an 10 ester having a formula of analyte in a test sample. Specifically, the methods of the present invention employ certain acridinium-9-carboxylate aryl esters for detecting an analyte in a test sample. Addition RI xe ally, the present invention also relates to kits for detecting an 15 analyte in a test sample. 21 eN1 N R3-- --R2 BACKGROUND OF THE INVENTION Along with the progress in diagnostic or medical technol ogy, various methods for detecting analytes of interest in test O O samples (such as serum, plasma, whole blood, etc.) have been developed and put into use to enable the early detection of R8 R4 various diseases and for confirming the effects of therapy. For the purpose of qualitative or quantitative detection of an ana 25 lyte in a test sample, certain detectable compounds (also R7 R5 known as detectable labels or signal generating compounds) R6 are used. Typically, these detectable compounds are capable of being used to generate detectable signals in the presence of one or more analytes in a test sample. In certain instances, 30 wherein R' is an alkyl, alkenyl, alkynyl, arylalkyl, aryl, these detectable compounds are attached to Substances that sulfoalkyl or carboxyalkyl and R through Rare each inde have a certain affinity for the analyte to be detected and pendently selected from the group consisting of hydrogen, quantified. For example, an antibody can be conjugated to a alkyl, amino, alkoxy, hydroxyl, carboxyl, halide, nitro, cyano, detectable compound (the labeled antibody is referred to - SO, NHC(O)R, C(O)R, C(O)OR, C(O)NHR herein as a "conjugate'). The conjugate can then be used to 35 and—SCN, wherein Risan alkyl, alkenyl, alkynyl, arylalkyl, detect and quantify the amount of an antigen of interest in a aryl, Sulfoalkyl or carboxyalkyl, and X is an anion; test sample. In other instances, however, the detectable com c) adding a basic solution to the processed test sample to pound is simply added to the test sample alone, not attached generate a light signal; and or conjugated to another Substance (such as an antibody). d) quantifying the amount of analyte in the test sample by Regardless of whether a detectable compound is attached or 40 relating the amount of light generated in the test sample by conjugated to another Substance or used alone, once added to comparison to a standard curve for the analyte. the test sample, the compound is activated and the signal In one aspect, the analyte of interest in the test sample detected. As a result, a determination of the presence of an comprises at least one peroxide. In another aspect, the method analyte and the amount of the analyte contained in a test can further comprise the step of adding at least one analyte sample can be readily determined. 45 specific enzyme which produces a peroxide to the test sample A variety of detectable compounds have been developed prior to step a) or after step a). The at least one analyte and used to generate detectable signals. Such compounds specific enzyme which produces a peroxide can be selected include, but are not limited to, radioactive substances, fluo from the group consisting of dismutases, dehydrogenases, rescent Substances, enzymes or metal colloids. In recent oxidases, reductases, synthases and combinations thereof. years, however, chemiluminescence methods using acri 50 Exemplary analyte-specific enzymes which produce a peroX dinium derivatives have drawn attention in view of their high ide are listed in Table 1. sensitivity. Intense luminescence can be generated by reac The test sample used in the above method can be selected tion of acridinium derivatives with hydrogen peroxide under from the group consisting of whole blood, serum, plasma, strong alkaline conditions (See, EP-A 830629 etc.). A number interstitial fluid, saliva, ocular lens fluid, cerebral spinal fluid, of acridinium derivatives are known in the art and are com 55 Sweat, urine, milk, ascites fluid, mucous, nasal fluid, sputum, mercially available. While largely interchangeable, these synovial fluid, peritoneal fluid, vaginal fluid, menses, amni acridinium derivatives can differ to at least some extent in otic fluid and semen. Preferably, the analyte contained in the terms of their physicochemical properties. Such differences test sample is selected from the group consisting of hydrogen can make a particular acridinium derivative either more or peroxide, galactose, glucose, cholesterol, LDL, HDL cho less preferred for the detection of an analyte of interest in a 60 line, lactic acid, uric acid, phosphatidylcholine, acetylcho sample. line, phosphocholine, CDP-choline, lysophosphatidylcho For this reason, there remains a need in the art for acri line, triglycerides, phospholipase A2, phosholipase D. dinium derivatives that can be employed for detecting an lysophosholipase D and sphingomyelin. analyte in a test sample, as well as methods and kits for using In the above method, the test sample is processed to sepa Such acridinium derivatives for the qualitative and/or quanti 65 rate or remove the analyte from protein that may be contained tative detection of analyte. The present invention provides in the test sample. Any process known in the art to separate or among other things such methods and kits. remove protein from a test sample can be used. Specifically, a US 7,906,293 B2 3 4 process that can be used to remove the protein can be selected In the above method, the at one least analyte-specific from the group consisting of ultrafiltration, extraction, pre enzyme which produces a peroxide is an enzyme listed in cipitation, dialysis, chromatography and digestion. Table 1. The basic solution used in the above method is a solution In the above method, the specific binding partner can be an that contains a base and that has a pH greater than or equal to antibody that binds to the analyte. 10, preferably, greater than or equal to 12. The basic solution used in the above method is a solution In the above method, the standard curve used to quantify that contains a base and that has a pH greater than or equal to the amount of analyte in the test sample can be generated from 10, preferably, greater than or equal to 12. In the above method, the standard curve used to quantify Solutions of the analyte of a known concentration. the amount of analyte in the test sample can be generated from In yet another embodiment, the present invention relates to 10 Solutions of the analyte of a known concentration. a method of detecting an analyte in a test sample. The method In yet another embodiment, the present invention relates to comprises the steps of a method of detecting an analyte in a test sample. The method a) contacting the test sample containing the analyte with a comprises the steps of specific binding partner to form an analyte specific binding a) contacting the test sample containing the analyte with a partner complex; 15 specific binding partner having conjugated thereon at least b) separating the analyte specific binding partner complex one Substrate-specific enzyme which produces a peroxide, to from the test sample to form an analyte specific binding form an analyte:Specific binding partner conjugate complex; partner complex sample: b) separating the analyte-specific binding partner conju c) adding at least one analyte-specific enzyme which pro gate complex from the test sample to forman analyte-specific duces a peroxide to the analyte specific binding partner com binding partner conjugate complex sample; plex sample; c) adding at least one Substrate to the analyte-specific bind d) adding an acridinium-9-carboxylate aryl ester to the ing partner conjugate complex sample: analyte specific binding partner complex sample, the acri d) adding an acridinium-9-carboxylate aryl ester to the dinium-9-carboxylate aryl ester having a formula of: analyte-specific binding partner conjugate complex sample, 25 the acridinium-9-carboxylate aryl ester having a formula of RI xe o RI xe 21 N1 N e R3-- --R2 30 21 n1 N R.-- --R2 N 21N 21

O O 35 O O R8 R4 R8 R4

R7 R5 40 R7 R5 R6 R6 wherein R' is an alkyl, alkenyl, alkynyl, arylalkyl, aryl, sulfoalkyl or carboxyalkyl and R through Rare each inde wherein R' is an alkyl, alkenyl, alkynyl, arylalkyl, aryl, pendently selected from the group consisting of hydrogen, 45 sulfoalkyl or carboxyalkyl and R through Rare each inde alkyl, amino, alkoxy, hydroxyl, carboxyl, halide, nitro, cyano, pendently selected from the group consisting of hydrogen, alkyl, amino, alkoxy, hydroxyl, carboxyl, halide, nitro, cyano, –SO, NHC(O)R, C(O)R, C(O)OR, C(O)NHR - SO, NHC(O)R, C(O)R, C(O)OR, C(O)NHR and—SCN, wherein Risan alkyl, alkenyl, alkynyl, arylalkyl, and—SCN, wherein Risan alkyl, alkenyl, alkynyl, arylalkyl, aryl, Sulfoalkyl or carboxyalkyl, and X is an anion; aryl, Sulfoalkyl or carboxyalkyl, and X is an anion; e) adding a basic solution to the analyte specific binding 50 e) adding a basic Solution to the analyte-specific binding partner complex sample to generate a light signal; and partner conjugate complex sample to generate a light signal; f) quantifying the amount of analyte in the analyte specific and binding partner complex sample by relating the amount of f) quantifying the amount of analyte in the test sample by light generated in the test sample by comparison to a standard relating the amount of light generated in the analyte-specific curve for the analyte. 55 binding partner conjugate complex sample by comparison to The test sample used in the above method can be selected a standard curve for the analyte. from the group consisting of whole blood, serum, plasma, The test sample used in the above method can be selected from the group consisting of whole blood, serum, plasma, interstitial fluid, saliva, ocular lens fluid, cerebral spinal fluid, interstitial fluid, saliva, ocular lens fluid, cerebral spinal fluid, Sweat, urine, milk, ascites fluid, mucous, nasal fluid, sputum, Sweat, urine, milk, ascites fluid, mucous, nasal fluid, sputum, synovial fluid, peritoneal fluid, vaginal fluid, menses, amni 60 synovial fluid, peritoneal fluid, vaginal fluid, menses, amni otic fluid and semen. Preferably, the analyte contained in the otic fluid and semen. test sample is selected from the group consisting of galac In the above method, the at least one substrate-specific tose, glucose, cholesterol, LDL, HDL, choline, lactic acid, enzyme which produces a peroxide is one of the enzymes uric acid, phosphatidylcholine, acetylcholine, phosphocho listed in Table 1. In the above method, the at least one sub line, CDP-choline, lysophosphatidylcholine, triglycerides, 65 strate is one of the substrates listed in Table 1. In the above phospholipase A2, phosholipase D, lysophosholipase D and method, the specific binding partner can be an antibody that sphingomyelin. binds to the analyte. US 7,906,293 B2 5 6 The basic solution used in the above method is a solution pendently selected from the group consisting of hydrogen, that contains a base and that has a pH greater than or equal to alkyl, amino, alkoxy, hydroxyl, carboxyl, halide, nitro, cyano, 10, preferably, greater than or equal to 12. - SO, NHC(O)R, C(O)R, C(O)OR, C(O)NHR In the above method, the standard curve used to quantify and—SCN, wherein Risan alkyl, alkenyl, alkynyl, arylalkyl, the amount of analyte in the test sample can be generated from aryl, Sulfoalkyl or carboxyalkyl, and X is an anion; Solutions of the analyte of a known concentration. b. at least one specific binding partner, In another embodiment, the present invention relates to a c. at least one basic Solution; and kit for use in detecting an analyte in a test sample. The kit can d. instructions for detecting an analyte in a test sample. comprise the following: The at least one specific binding partner contained in the kit a.acridinium-9-carboxylate aryl ester having a formula of 10 can be at least one antibody that binds to the analyte. The kit can further comprise at least one analyte-specific enzyme which produces a peroxide. RI xe The specific binding partner contained in the kit can also 15 have conjugated thereon at least one substrate-specific 21 eN1 N enzyme which produces a peroxide. The at least one Sub R3-- --R2 strate-specific enzyme can be an enzyme listed in Table 1. If the specific binding partner has conjugated thereon at least one substrate-specific enzyme which produces a peroxide, then the kit can further comprise at least one substrate. The O O substrate can be a substrate listed in Table 1. R8 R4 BRIEF DESCRIPTION OF THE FIGURES

25 FIG. 1 is a non-limiting illustration of one aspect of the R7 R5 method of the present invention, wherein the analyte of inter R6 est in the test sample is a peroxide, specifically, hydrogen peroxide. The test sample is pretreated prior to the addition of wherein R' is an alkyl, alkenyl, alkynyl, arylalkyl, aryl, the acridinium-9-carboxylate aryl ester. sulfoalkyl or carboxyalkyl and R through Rare each inde 30 FIG. 2 is a non-limiting illustration of another aspect of the pendently selected from the group consisting of hydrogen, method of the present invention, wherein at least one analyte alkyl, amino, alkoxy, hydroxyl, carboxyl, halide, nitro, cyano, specific enzyme which produces a peroxide is added to the test sample containing the analyte of interest. In this aspect, –SO, NHC(O)R, C(O)R, C(O)OR, C(O)NHR the at least one analyte-specific enzyme which produces a and—SCN, wherein Risan alkyl, alkenyl, alkynyl, arylalkyl, 35 aryl, Sulfoalkyl or carboxyalkyl, and X is an anion; peroxide, is added to generate peroxide, specifically, hydro b. at least one basic solution; and gen peroxide. The test sample is pretreated to separate the c. instructions for detecting an analyte in a test sample. analyte of interest from protein in the test sample prior to the At least one of the instructions contained in the test kit addition of the analyte-specific enzyme which produces a describe processing a test sample to separate the analyte from 40 peroxide and the acridinium-9-carboxylate aryl ester. protein contained in the test sample. FIG.3 is a non-limiting illustration of another aspect of the Optionally, the kit can further comprise at least one ana method of the present invention wherein at least one analyte lyte-specific enzyme which produces a peroxide. specific enzyme which produces a peroxide, is added to the In another embodiment, the present invention relates to a test sample containing the analyte of interest. In this aspect, kit for use in detecting an analyte in a test sample. The kit can 45 the at least one analyte-specific enzyme which produces a comprise the following: peroxide, is added to generate peroxide, specifically, hydro a.acridinium-9-carboxylate aryl ester having a formula of gen peroxide. The test sample is pretreated to separate the hydrogen peroxide from protein in the test sample prior to the addition of the acridinium-9-carboxylate aryl ester. RI xe 50 FIG. 4 is a non-limiting illustration of another aspect of the method of the present invention wherein immunoseparation 21 N1e N techniques are used to separate the analyte from protein con R.-- --R2 tained in the test sample. Specifically, an anti-analyte specific N 2N21 binding partner (Analyte Binding Partner') is used. An ana 55 lyte-specific enzyme which produces a peroxide, specifically, hydrogen peroxide and acridinium-9-carboxylate aryl ester O O are added after the analyte is separated from protein in the test sample. R8 R4 FIG. 5 is a non-limiting illustration of another aspect of the 60 method of the present invention wherein immunoseparation techniques are used to separate the analyte from protein con R7 R5 tained in the test sample. In this aspect, the specific binding partner (Analyte Binding Partner) used to separate the ana R6 lyte from the test sample has conjugated thereon at least one 65 Substrate-specific enzyme which produces a peroxide, spe wherein R' is an alkyl, alkenyl, alkynyl, arylalkyl, aryl, cifically, hydrogen peroxide. A substrate for the substrate sulfoalkyl or carboxyalkyl and R through Rare each inde specific enzyme which produces a peroxide, specifically, US 7,906,293 B2 7 8 hydrogen peroxide and an acridinium-9-carboxylate aryl choline; dashed line, 75 uM choline; dotted line, 50 LM ester are added after the analyte is separated from the test choline: -O-, 30 uM choline: -x-, 20 uM choline, --, 10uM sample. choline: -A-, 5uM choline: - () -, OuM choline. FIG. 6 is a non-limiting illustration of another aspect of the FIG. 16 shows a choline assay standard curve for 1 uM of method of the present invention, similar to the method shown 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate in FIG. 5, but in a competitive format. In this aspect, added at pH 4. Abscissa: micromolar choline concentration (“Cho analyte having conjugated thereon at least one Substrate line (LLM)'). Ordinate: maximum relative light units (“RLU specific enzyme which produces a peroxide, specifically, max (0.4s)'). hydrogen peroxide, competes with the analyte of interest in FIG. 17 shows chemiluminescence profiles for 500 nM of the test sample for binding to the analyte-specific binding 10 partner (Analyte Binding Partner) to forman binding part 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate ner complex. A Substrate for the Substrate-specific enzyme at pH 4. Abscissa: time in seconds (“Time(s)). Ordinate: which produces a peroxide, specifically, hydrogen peroxide relative light units (“RLU). Symbols: solid line, 150 uM and an acridinium-9-carboxylate aryl ester are added after the choline; dashed line, 75 uM choline; dotted line, 50 LM binding partner complex is separated from the test sample. 15 choline: -O-, 30 uM choline: -x-, 20 uM choline, --, 10uM FIG.7 shows chemiluminescence profiles for 4 uM 10-me choline: -A-, 5uM choline: - () -, OuM choline. thyl-9-(phenoxycarbonyl)acridinium fluorosulfonate. FIG. 18 shows a choline assay standard Curve for 500 nM Abscissa: time in seconds (“Time(s)). Ordinate: relative of 10-methyl-9-(phenoxycarbonyl)acridinium fluorosul light units (“RLU). Symbols: solid line, 75 uM choline: fonate at pH 4. Abscissa: micromolar choline concentration dashed line, 50 uM choline; dotted line, 30 uM choline: -X- (“Choline(LM)'). Ordinate: maximum relative light units 20 uM choline: - -, 10uM choline: -A-, 5uM choline: - () -, (“RLUmax (0.4 s)). OuM choline. FIG. 19 shows chemiluminescence profiles for 250 nM of FIG. 8 shows a choline assay standard curve for 4 uM 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate. at pH 4. Abscissa: time in seconds (“Time(s)). Ordinate: Abscissa: micromolar choline concentration (“Choline 25 relative light units (“RLU). Symbols: solid line, 150 uM (uM)). Ordinate: maximum relative light units (“RLU choline; dashed line, 75 uM choline; dotted line, 50 LM max). choline: -O-, 30 uM choline: -x-, 20 uM choline, --, 10uM FIG.9 shows chemiluminescence profiles for 2 uM 10-me choline: -A-, 5uM choline: - () -, OuM choline. thyl-9-(phenoxycarbonyl)acridinium fluorosulfonate. FIG. 20 shows a choline assay standard curve for 250 nM Abscissa: time in seconds (“Time(s)). Ordinate: relative 30 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate light units (“RLU). Symbols: solid line, 150 uM choline: at pH 4. Abscissa: micromolar choline concentration (“Cho dashed line, 75uMcholine; dotted line, 50 Micholine: -O-, line(LM)'). Ordinate: maximum relative light units (“RLU 30 uM choline: -x-, 20 uM choline: --, 10uM choline: -A-, max (0.4s)'). 5uM choline: - () -, OuM choline. FIG. 21 shows chemiluminescence profiles for 4 uM FIG. 10 shows a choline assay standard curve for 2 uM of 35 9-(3-carboxypropyl)(4-methylphenyl)sulfonylamino 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate. carbonyl-10-(3-sulfopropyl)acridinium inner salt. Abscissa: Abscissa: micromolar choline concentration (“Choline'). time in seconds (“Time(s)'). Ordinate: relative light units Ordinate: maximum relative light units (“RLUmax (0.4s)'). (“RLU). Symbols: solid line, 150 uM choline; dashed line, FIG. 11 shows chemiluminescence profiles of 2 uM of 75 uM choline; dotted line, 50 uM choline: -O-, 30 uM 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate 40 choline: -x-, 20 uM choline: --, 10 uM choline: -A-, 5uM after overnight storage. Abscissa: time in seconds choline: - O - OuM choline. (“Time(s)). Ordinate: relative light units (“RLU). Symbols: FIG. 22 shows the structure of 9-(3-carboxypropyl)(4- solid line, 150 uMcholine; dashed line, 75uMcholine; dotted methylphenyl)sulfonyl)amino-carbonyl-10-(3-sulfopro line, 50 uM choline: -O-, 30 uM choline: -x-, 20 uM pyl)acridinium inner salt. choline: - -, 10 uM choline: -A-, 5 uM choline: - () -, OuM 45 choline. DETAILED DESCRIPTION OF THE INVENTION FIG. 12 shows a choline assay standard curve for 2 uM of 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate The present invention provides among other things meth after overnight storage. Abscissa: micromolar choline con ods and kits for using acridinium derivatives for the qualita centration (“Choline'). Ordinate: maximum relative light 50 tive and/or quantitative detection of analyte. Unexpectedly units (“RLUmax (0.4s)'). and Surprisingly, the inventors of the present invention found FIG. 13 shows chemiluminescence profiles for 2 uM of that acridinium-9-carboxylate aryl esters are extremely sen 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate sitive to protein contained in test samples. Specifically, the at pH 4. Abscissa: time in seconds (“Time(s)). Ordinate: inventors found that acridinium-9-carboxylate aryl esters lose relative light units (“RLU). Symbols: solid line, 150 uM 55 their intense chemiluminescent properties in the presence of choline; dashed line, 75 uM choline; dotted line, 50 LM protein in a test sample, thus rendering these esters virtually choline;-O-, 30 uMcholine: -x-, 20 uM choline: --, 10uM unusable for use in identifying the presence of an analyte in a choline: -A-, 5uM choline: - () -, OuM choline. test sample. In view thereof, methods for restoring the intense FIG. 14 shows a choline assay standard curve for 2 uM of chemiluminescent properties of the acridinium-9-carboxy 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate 60 late esters make these acridinium-9-carboxylate aryl esters at pH 4. Abscissa: micromolar choline concentration (“Cho useful for use in identifying the presence of an analyte in a test line (LLM)'). Ordinate: maximum relative light units (“RLU sample. This is described in more detail below. max (0.4s)'). As used in this specification and the appended claims, the FIG. 15 shows chemiluminescence profiles for 1 uM of singular forms “a”, “an', and “the include plural references 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate 65 unless the context clearly dictates otherwise. Thus, for at pH 4. Abscissa: time in seconds (“Time(s)). Ordinate: example, references to “the method’ includes one or more relative light units (“RLU). Symbols: solid line, 150 uM methods, and/or steps of the type described herein and/or US 7,906,293 B2 10 which will become apparent to those persons skilled in the art analytes include, but are not limited to, brain natriuretic pep upon reading this disclosure and so forth. tide (BNP) 1-32; NT-proBNP; proBNP; preproBNP; tropo nin I; troponin T; troponin C.; human neutrophil gelatinase Definitions associated lipocalin (hNGAL); tacrolimus; Sirolimus, cyclosporine; ferritin; creatinine kinase MB (CK-MB); As used herein, the term “alkenyl means a straight or digoxin; phenytoin: phenobarbitol; carbamazepine; Vanco branched chain hydrocarbon containing from 2 to 10 carbons mycin; gentamycin; theophylline; Valproic acid; quinidine; and containing at least one carbon-carbon double bond luteinizing hormone (LH); follicle stimulating hormone formed by the removal of two hydrogens. Representative (FSH); estradiol, progesterone; C-reactive protein; lipoca examples of alkenyl include, but are not limited to, ethenyl, 10 lins; IgE antibodies; cytokines; vitamin B2 micro-globulin; 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, glycated hemoglobin (Gly. Hb); cortisol; digitoxin: N-acetyl 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl. procainamide (NAPA); procainamide; antibodies to rubella, As used herein, the term “alkyl means a straight or such as rubella-IgG and rubella IgM; antibodies to toxoplas branched chain hydrocarbon containing from 1 to 10 carbon mosis, such as toxoplasmosis IgG (Toxo-IgG) and toxoplas atoms. Representative examples of alkyl include, but are not 15 mosis IgM (Toxo-IgM); testosterone; Salicylates; acetami limited to, methyl, ethyl, n-propyl, iso-propyl. n-butyl, sec nophen; hepatitis B virus Surface antigen (HbSAg); butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, antibodies to hepatitis B core antigen, such as anti-hepatitis B n-hexyl, 3-methylhexyl, 2,2-dimethylpenty1, 2,3-dimethyl core antigen IgG and IgM (Anti-HBC); human immune defi pentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl. ciency virus (HIV); human T-cell leukemia virus (HTLV); As used herein, the term “alkylcarbonyl.” means an alkyl hepatitis Be antigen (HbeAg); antibodies to hepatitis Be group attached to the parent molecular moiety through a antigen (Anti-Hbe); influenza virus; thyroid stimulating hor carbonyl group. mone (TSH); thyroxine (T4); total triiodothyronine (Total As used herein, the term “alkoxy' means an alkyl group, as T3); free triiodothyronine (FreeT3); carcinoembryonic anti defined herein, appended to the parent molecular moiety gen (CEA); lipoproteins, cholesterol, and triglycerides; through an oxygen atom. Representative examples of alkoxy 25 galactose, glucose, LDL, HDL choline, lactic acid, uric acid, include, but are not limited to, methoxy, ethoxy, propoxy, phosphatidylcholine, acetylcholine, phosphocholine, CDP 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy. choline, lysophosphatidylcholine, phospholipase A2. As used herein, the term “alkynyl' means a straight or phosholipase D, lysophosholipase D and sphingomyelin and branched chain hydrocarbon group containing from 2 to 10 alpha fetoprotein (AFP). Drugs of abuse and controlled sub carbonatoms and containing at least one carbon-carbon triple 30 stances include, but are not intended to be limited to, amphet bond. Representative examples of alkynyl include, but are not amine; methamphetamine; barbiturates, such as amobarbital, limited, to acetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, secobarbital, pentobarbital, phenobarbital, and barbital; ben 2-pentynyl, and 1-butynyl. Zodiazepines, such as propoxy and Valium; cannabinoids, As used herein, the term "amino” means —NRR, Such as hashishand marijuana; cocaine; fentanyl, LSD; meth wherein R, and R, are independently selected from the group 35 aqualone; opiates, such as heroin, morphine, codeine, hydro consisting of hydrogen, alkyl and alkylcarbonyl. morphone, hydrocodone, methadone, oxycodone, oxymor As used herein, the term “analyte' or “analyte of interest' phone and opium; phencyclidine; and propoxyphene. as used interchangeably herein, generally refers to a Sub As used herein, the phrases “analyte-specific enzyme” or stance to be detected. Analytes may include inorganic Sub “substrate-specific enzyme” refer to enzymes which produce stances, including, but not limited to, hydrogen peroxide and 40 a peroxide, including, dismutases, dehydrogenases, oxidases, Sulfite. Analytes may include antigenic Substances, haptens, reductases, synthases or combinations thereof Exemplary antibodies, and combinations thereof. Analytes include, but analyte-specific enzymes which produce a peroxide are listed are not limited to, toxins, organic compounds, DNA, RNA, below in Table 1. Many analyte-specific enzymes/substrate proteins, peptides, microorganisms, amino acids, nucleic specific enzymes that produce a peroxide are known in the art. acids, hormones, steroids, vitamins, drugs (including those 45 For example, analyte-specific enzymes/substrate-specific administered for therapeutic purposes as well as those admin enzymes which produces a peroxide can be conveniently istered for illicit purposes), drug intermediaries or byprod found in on the on the World Wide Web at the Enzyme ucts, bacteria, virus particles and metabolites of orantibodies Nomenclature Database and the Enzyme Database (devel to any of the above Substances. Specific examples of some oped at Trinity College in Dublin, Ireland). TABLE 1.

UBMBENZYME ACCEPTED COMMON NAME NOMENCLATURE PREFERRED SUBSTRATE

(R)-6-hydroxynicotine oxidase EC 1.5.3.6 (R)-6-hydroxynicotine (S)-2-hydroxy acid oxidase EC 1.1.3.15 S)-2-hydroxy acid (S)-6-hydroxynicotine oxidase EC 1.5.3.5 (S)-6-hydroxynicotine 3-aci-nitropropanoate oxidase EC 1.7.3.5 3-aci-nitropropanoate 3-hydroxyanthranilate oxidase EC 1.10.3.5 3-hydroxyanthranilate 4-hydroxymandelate oxidase EC 1.1.3.19 (S)-2-hydroxy-2-(4- hydroxyphenyl)acetate 6-hydroxynicotinate dehydrogenase EC 1.17.3.3 6-hydroxynicotinate Abscisic-aldehyde oxidase EC 1.23.14 abscisic aldehyde acyl-CoA oxidase EC 1.3.3.6 acyl-CoA Alcohol oxidase EC 1.1.3.13 a primary alcohol aldehyde oxidase EC 1.23.1 an aldehyde amine oxidase US 7,906,293 B2 11 12 TABLE 1-continued

UBMBENZYME ACCEPTED COMMON NAME NOMENCLATURE PREFERRED SUBSTRATE amine oxidase (copper-containing) EC 4.3.6 primary monoamines, diamines and histamine amine oxidase (flavin-containing) 4.3.4 a primary amine aryl-alcohol oxidase 1.3.7 an aromatic primary alcohol (2-naphthyl)methanol 3-methoxybenzyl alcohol aryl-aldehyde oxidase 23.9 an aromatic aldehyde catechol oxidase .1.3.14 catechol cholesterol oxidase .1.3.6 cholesterol choline oxidase 1.3.17 choline columbamine oxidase .21.3.2 columbamine cyclohexylamine oxidase 4.3.12 cyclohexylamine cytochrome c oxidase 9.3. D-amino-acid oxidase a D-amino acid D-arabinono-1,4-lactone oxidase 1.3.37 D-arabinono-1,4-lactone D-arabinono-1,4-lactone oxidase 1.3.37 D-arabinono-1,4-lactone D-aspartate oxidase 4.3. D-aspartate D-glutamate oxidase 4.3.7 D-glutamate D-glutamate(D-aspartate) oxidase 4.3. 5 D-glutamate dihydrobenzophenanthridine 5.312 dihydrosanguinarine oxidase dihydroorotate oxidase 3.3. (S)-dihydroorotate dihydrouracil oxidase 3.3.7 5,6-dihydrouracil dimethylglycine oxidase S.3.10 N,N-dimethylglycine D-mannitol oxidase .1.3.40 mannitol ecclysone oxidase .1.3.16 ecclysone ethanolamine oxidase 4.3.8 ethanolamine galactose oxidase 13.9 D-galactose glucose oxidase .1.3.4 B-D-glucose glutathione oxidase 8.33 glutathione glycerol-3-phosphate oxidase .1.3.21 Sn-glycerol 3-phosphate glycine oxidase 4.3.19 glycine glyoxylate oxidase 2.3.5 glyoxylate hexose oxidase 13.5 D-glucose, D-galactose D-mannose maltose actOSe cellobiose hydroxyphytanate oxidase 1.3.27 L-2-hydroxyphytanate indole-3-acetaldehyde oxidase 2.3.7 (indol-3-yl)acetaldehyde lactic acid oxidase Lactic acid L-amino-acid oxidase 4.3.2 an L-amino acid L-aspartate oxidase 4.3.16 L-aspartate L-galactonolactone oxidase 3.312 L-galactono-1,4-lactone L-glutamate oxidase 4.3.11 L-glutamate L-gulonolactone oxidase 13.8 L-gulono-1,4-lactone L-lysine 6-oxidase 4.3.20 L-lysine L-lysine oxidase 4.3.14 L-lysine long-chain-alcohol oxidase 1.3.20 Along-chain-alcohol L-pipecolate oxidase .5.3.7 L-pipecolate L-Sorbose oxidase .1.3.11 L-Sorbose malate oxidase 1.3.3 (S)-malate methanethiol oxidase .8.3.4 methanethiol monoamino acid oxidase N-methyl-lysine oxidase 53.4 6-N-methyl-L-lysine N-acylhexosamine oxidase 1.329 N-acetyl-D-glucosamine N-glycolylglucosamine N-acetylgalactosamine N-acetylmannosamine. NAD(P)H oxidase 6.3.1 NAD(P)H nitroalkane oxidase 7.3.1 a nitroalkane N-methyl-L-amino-acid oxidase S.3.2 an N-methyl-L-amino acid nucleoside oxidase 13.39 adenosine oxalate oxidase .2.3.4 oxalate polyamine oxidase 5.3.11 1-N-acetylspermine polyphenol oxidase .14.18.1 polyvinyl-alcohol oxidase 13.30 polyvinyl alcohol prenylcysteine oxidase 8.35 an S-prenyl-L-cysteine protein-lysine 6-oxidase 4.3.13 peptidyl-L-lysyl-peptide putrescine oxidase 4.3.10 butane-1,4-diamine pyranose oxidase 1.3.10 D-glucose D-xylose L-Sorbose D-glucono-1,5-lactone US 7,906,293 B2 13 14 TABLE 1-continued

UBMBENZYME ACCEPTED COMMON NAME NOMENCLATURE PREFERRED SUBSTRATE pyridoxal 5'-phosphate synthase EC 1.43.5 pyridoxamine 5'- phosphate pyridoxine 4-oxidase EC 1.1.3.12 pyridoxine pyrroloquinoline-quinone synthase EC 1.3.3.11 6-(2-amino-2- carboxyethyl)-7,8-dioxo 1,2,3,4,5,6,7,8- octahydroquinoline-2,4- dicarboxylate pyruvate oxidase EC 1.2.3.3 pyruvate pyruvate oxidase (CoA-acetylating) EC 1.2.3.6 pyruvate reticuline oxidase EC 1.21.3.3 reticuline retinal oxidase EC 1.2.3.11 retinal rifamycin-B oxidase EC 1.10.3.6 rifamycin-B sarcosine oxidase EC 1.5.3.1 sarcosine Secondary-alcohol oxidase EC 1.1.3.18 a secondary alcohol sulfite oxidase EC 18.3.1 sulfite Superoxide dismutase EC 1.15.1.1 Superoxide Superoxide reductase EC 1.15.1.2 Superoxide tetrahydroberberine oxidase EC 1.3.3.8 (S)-tetrahydroberberine thiamine oxidase EC 1.1.3.23 thiamine tryptophan C.3-oxidase EC 1.3.3.10 L-tryptophan urate oxidase (uricase, uric acid EC 1.7.3.3 uric acid oxidase) vanillyl-alcohol oxidase EC 1.1.3.38 vanillyl alcohol Xanthine oxidase EC 1.17.3.2 Xanthine xylitol oxidase EC 1.13.41 xylitol

As used herein, the term “anion” refers to an anion of an ring has one to three double bonds, and each nine-to ten inorganic or organic acid, such as, but not limited to, hydro membered ring has one to four double bonds. Examples of chloric acid, hydrobromic acid, Sulfuric acid, methane Sul 30 cycloalkenyl groups include cyclohexenyl, octahydronaph fonic acid, formic acid, acetic acid, oxalic acid, Succinic acid, thalenyl, norbornylenyl, and the like. The cycloalkenyl tartaric acid, mandelic acid, fumaric acid, lactic acid, citric groups of the present invention can be optionally Substituted acid, glutamic acid, aspartic acid, phosphate, trifluo with one, two, three, four, or five substituents independently romethansulfonic acid, trifluoroacetic acid and fluorosulfonic is selected from the group consisting of alkoxy, alkyl, carboxyl, acid and any combinations thereof. halo, and hydroxyl. As used herein, the term "aryalkyl means an aryl group, as As used herein, the term “cycloalkyl refers to a saturated defined herein, appended to the parent molecular moiety monocyclic, bicyclic, or tricyclic hydrocarbon ring system through an alkyl group, as defined herein. Representative having three to twelve carbonatoms. Examples of cycloalkyl examples of arylalkyl include, but are not limited to, benzyl, 40 groups include cyclopropyl, cyclopentyl, bicyclo3.1.1 hep 2-phenylethyl 3-phenylpropyl, and 2-maphth-2-ylethyl. tyl, adamantyl, and the like. The cycloalkyl groups of the As used herein, the term “aryl' means a phenyl group, or a present invention can be optionally substituted with one, two, bicyclic or tricyclic fused ring system wherein one or more of three, four, or five substituents independently selected from the fused rings is a phenyl group. Bicyclic fused ring systems the group consisting of alkoxy, alkyl, carboxyl, halo, and are exemplified by a phenyl group fused to a cycloalkenyl 45 hydroxyl. group, as defined herein, a cycloalkyl group, as defined As used herein, the term “halide” means a —Cl, —Br. —I herein, or another phenyl group. Tricyclic fused ring systems or —F. are exemplified by a bicyclic fused ring system fused to a As used herein, the term “hydroxyl means an —OH cycloalkenyl group, as defined herein, a cycloalkyl group, as group. defined herein or another phenyl group. Representative 50 As used herein, the term "nitro” means a —NO group. examples of aryl include, but are not limited to, anthracenyl, As used herein, the term "sulfoalkyl refers to an alkyl aZulenyl, fluorenyl, indanyl, indenyl, naphthyl, phenyl, and group to which a Sulfonate group is bonded, wherein the alkyl tetrahydronaphthyl. The aryl groups of the present invention is bonded to the molecule of interest. can be optionally substituted with one-, two, three, four, or As used herein, the phrase “specific binding partner, as five substituents independently selected from the group con- 55 used herein, is a member of a specific binding pair. That is, sisting of alkoxy, alkyl, carboxyl, halo, and hydroxyl. two different molecules where one of the molecules, through As used herein, the term "carbonyl refers to —C(O)—. chemical or physical means, specifically binds to the second As used herein, the term "carboxy” or “carboxyl refers to molecule. Therefore, in addition to antigen and antibody spe —CO.H. cific binding pairs of common immunoassays, other specific As used herein, the term “carboxyalkyl refers to an alkyl 60 binding pairs can include biotin and avidin, carbohydrates group that is Substituted with one or more carboxy groups. and lectins, complementary nucleotide sequences, effector As used herein, the term "cyano' means a —CN group. and receptor molecules, cofactors and enzymes, enzyme As used herein, the term "cycloalkenyl refers to a non inhibitors, and enzymes and the like. Furthermore, specific aromatic cyclic or bicyclic ring system having three to ten binding pairs can include members that are analogs of the carbonatoms and one to three rings, wherein each five-mem- 65 original specific binding members, for example, an analyte bered ring has one double bond, each six-membered ring has analog. Immunoreactive specific binding members include one or two double bonds, each seven- and eight-membered antigens, antigen fragments, antibodies and antibody frag US 7,906,293 B2 15 16 ments, both monoclonal and polyclonal and complexes sample). Methods for separating or removing proteins from thereof, including those formed by recombinant DNA mol test samples are well known to those skilled in the art and ecules. include, but are not limited to, ultrafiltration, extraction, pre As used herein, the term “test sample' generally refers to a cipitation, dialysis, chromatography and digestion. Tech biological material being tested for and/or Suspected of con niques for separating or removing proteins using ultrafiltra taining an analyte of interest. The test sample may be derived tion, extraction, precipitation, dialysis and chromatography from any biological source, such as, a physiological fluid, are well known to those skilled in the art (See, for example, including, but not limited to, whole blood, serum, plasma, Wells, D. (2003) High Throughput Bioanalytical Sample interstitial fluid, saliva, ocular lens fluid, cerebral spinal fluid, Preparation. Methods and Automation Strategies, Elsevier.). Sweat, urine, milk, ascites fluid, mucous, nasal fluid, sputum, 10 synovial fluid, peritoneal fluid, vaginal fluid, menses, amni The amount of protein removed or separated from the test otic fluid, semen and so forth. Besides physiological fluids, sample can be about 40%, about 45%, about 50%, about 55%, other liquid samples may be used such as water, food prod about 60%, about 65%, about 70%, about 75%, about 80%, ucts, and so forth, for the performance of environmental or about 85%, about 90% and about 95%. food production assays. In addition, a Solid material Sus 15 After the separation of protein from the test sample, then pected of containing the analyte may be used as the test the peroxide contained in the processed test sample can be sample. The test sample may be used directly as obtained converted to an end product having a distinct chemilumines from the biological source or following a pretreatment to cent emission. Such an end product is produced by adding to modify the character of the sample. For example, Such pre the processed test sample at least one acridinium ester. Pref treatment may include preparing plasma from blood, diluting erably, the acridinium ester is an acridinium-9-carboxylate viscous fluids and so forth. Methods of pretreatment may also aryl ester has the formula of formula I shown below: involve filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, the addition of reagents, lysing, etc. Moreover, it may also be RI xe beneficial to modify a solid test sample to form a liquid 25 medium or to release the analyte. 21 eN1 N Assay for Detecting an Analyte of Interest R.-- --R2 In general, the present invention relates to an assay for S 2N2 detecting an analyte of interest in a test sample. Specifically, as will be described in more detail below, in the assay of the 30 present invention, the processing of the test sample contain O O ing the analyte of interest to separate or remove protein con tained in the test sample or the removal or separation of the R8 R4 analyte of interest from the test sample (and thus away from protein contained in the test sample) allows for the use of one 35 or more acridinium esters having a particular formula in the R7 R5 quantification of the amount of analyte contained in the test sample. In fact, until the discovery of the present invention, R6 the one or more acridinium esters having the formula described herein typically were not used in the quantification 40 wherein R' is an alkyl, alkenyl, alkynyl, arylalkyl, aryl, of the amount of analyte in the test sample due to sensitivity sulfoalkyl or carboxyalkyl and R through Rare each inde of these acridinium esters in the presence of protein. pendently selected from the group consisting of hydrogen, The assay or method of the present invention involves alkyl, amino, alkoxy, hydroxyl, carboxyl, halide, nitro, cyano, obtaining a test sample from a Subject. A Subject from which - SO, NHC(O)R, C(O)R, C(O)OR, C(O)NHR a test sample can be obtained is any vertebrate. Preferably, the 45 and—SCN, wherein Risan alkyl, alkenyl, alkynyl, arylalkyl, Vertebrate is a mammal. Examples of mammals include, but aryl, Sulfoalkyl or carboxyalkyl, and X is an anion. Examples are not limited to, dogs, cats, rabbits, mice, rats, goats, sheep, of acridinium-9-carboxylate aryl esters having the above for cows, pigs, horses, non-human primates and humans. The test mula that can be used in the present invention include, but are sample can be obtained from the Subject using routine tech not limited to, 10-methyl-9-(phenoxycarbonyl)acridinium niques known to those skilled in the art. Preferably, the test 50 fluorosulfonate (available from Cayman Chemical, Ann sample naturally contains peroxide or contains a compound, Arbor, Mich.). Methods for preparing acridinium 9-carboxy protein or peptide for which there exists one or more analyte late aryl esters are described in McCapra, F., et al., Photo specific enzymes that can be used to generate peroxide, Such chem. Photobiol., 4, 1111-21 (1965); Razavi, Z. et al., Lumi as, at least one dismutase, dehydrogenase, oxidase, reductase nescence, 15:245-249 (2000); Razavi, Zetal. Luminescence, or synthase or a combination of at least one dismutase, dehy 55 15:239-244 (2000); and U.S. Pat. No. 5,241,070. drogenase, oxidase, reductase or synthase. For example, if the Acridinium-9-carboxylate aryl esters having the above for analyte of interest is hydrogen peroxide, no analyte-specific mula I are readily (commercially) available. Additionally, it is enzyme is necessary. However, if the analyte of interest is Surprising that the acridinium-9-carboxylate aryl esters hav choline, then the analyte-specific enzyme could be choline ing the above formula are more efficient chemiluminescent oxidase. Alternatively, if the analyte of interest is lactate, then 60 indicators for hydrogen peroxide produced in the oxidation of the analyte-specific enzyme could be lactic acid oxidase. the analyte by at least one oxidase, at least one both in the In one embodiment, the test sample containing the analyte intensity of the signal and in the rapidity of the signal. By of interest comprises a peroxide, Such as hydrogen peroxide comparison of the acridinium-9-carboxylate aryl ester, (See, FIG. 1). The peroxide containing test sample (contain 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate ing the analyte of interest) is manipulated or processed in Such 65 (FIG. 13). Chemiluminescence profiles for 2 uM of 10-me a manner so as to separate or remove protein that may be thyl-9-(phenoxycarbonyl)acridinium fluorosulfonate at pH contained in the test sample (thus resulting in a processed test 4) and the acridinium-9-carboxamide, 9-(3-carboxypropyl) US 7,906,293 B2 17 18 (4-methylphenyl)sulfonylamino-carbonyl-10-(3-sulfo Either before or after the separation of protein from the test propyl)acridinium inner salt (FIG. 21). Chemiluminescence sample, at least one analyte-specific enzyme, Such as, at least profiles for 4 uM 9-II (3-carboxypropyl)(4-methylphenyl) one dismutase, dehydrogenase, oxidase, reductase or Syn sulfonyl)amino-carbonyl-10-(3-sulfopropyl)acridinium thase or a combination of at least one dismutase, dehydroge inner salt), the former reaches a peak light intensity that is nase, oxidase, reductase or synthase, is added to the test double that of the latter. Further, that increased efficiency is sample. Examples of at least one analyte-specific enzyme that achieved at half the molar concentration. Still further, the can be used are selected from the group consisting of (R)-6- course of the chemiluminescent emission for the acridinium hydroxynicotine oxidase, (S)-2-hydroxy acid oxidase, (S)-6- 9-carboxylate aryl ester is completed rapidly, in under 1 sec hydroxynicotine oxidase, 3-aci-nitropropanoate oxidase, 10 3-hydroxyanthranilate oxidase, 4-hydroxymandelate oxi ond, while the acridinium-9-carboxamide chemiluminescent dase, 6-hydroxynicotinate dehydrogenase, abscisic-aldehyde emission extends over 2 seconds. oxidase, acyl-CoA oxidase, alcohol oxidase, aldehyde oxi After the addition of the acridinium-9-carboxylate aryl dase, amine oxidase, amine oxidase (copper-containing), ester to the processed test sample, at least one basic Solution amine oxidase (flavin-containing), aryl-alcohol oxidase, aryl is added to the test sample in order to generate a detectable 15 aldehyde oxidase, catechol oxidase, cholesterol oxidase, cho signal, namely, a chemiluminescent signal. The basic Solution line oxidase, columbamine oxidase, cyclohexylamine oxi is a solution that contains at least one base and that has a pH dase, cytochrome c oxidase, D-amino-acid oxi greater than or equal to 10, preferably, greater than or equal to dase, D-arabinono-1,4-lactone oxidase, D-arabinono-1.4-lac 12. Examples of basic solutions include, but are not limited to, tone oxidase, D-aspartate oxidase, D-glutamate oxi Sodium hydroxide, potassium hydroxide, calcium hydroxide, dase, D-glutamate(D-aspartate) oxidase, dihydroben ammonium hydroxide, magnesium hydroxide, Sodium car Zophenanthridine oxidase, dihydroorotate oxidase, dihydrou bonate, Sodium bicarbonate, calcium hydroxide, calcium car racil oxidase, dimethylglycine oxidase, D-mannitol oxidase, bonate and calcium bicarbonate. The amount of basic solu ecdysone oxidase, ethanolamine oxidase, galactose oxidase, tion added to the processed test sample depends on the glucose oxidase, glutathione oxidase, glycerol-3-phosphate concentration of the basic solution used in the method. Based 25 oxidase, glycine oxidase, glyoxylate oxidase, hexose oxi on the concentration of the basic solution used, one skilled in dase, hydroxyphytanate oxidase, indole-3-acetaldehyde oxi the art could easily determine the amount of basic solution to dase, lactic acid oxidase, L-amino-acid oxidase, L-aspartate be used in the method. oxidase, L-galactonolactone oxidase, L-glutamate oxi The chemiluminescent signal that is generated can then be dase, L-gulonolactone oxidase, L-lysine 6-oxidase, L-lysine detected using routine techniques known to those skilled in 30 oxidase, long-chain-alcohol oxidase, L-pipecolate oxi the art. Thus, in the assay of the present invention, the chemi dase, L-Sorbose oxidase, malate oxidase, methanethiol oxi luminescent signal generated after the addition of a basic dase, monoamino acid oxidase, N-methyl-lysine oxidase, solution, indicates the presence of the analyte of interest. The N-acylhexosamine oxidase, NAD(P)H oxidase, nitroalkane amount of the analyte in the test sample can be quantified oxidase, N-methyl-L-amino-acid oxidase, nucleoside oxi based on the intensity of the signal generated. Specifically, the 35 dase, oxalate oxidase, polyamine oxidase, polyphenol oxi amount of analyte contained in a test sample is either propor dase, polyvinyl-alcohol oxidase, prenylcysteine oxidase, pro tional or inversely proportional to the intensity of the signal tein-lysine 6-oxidase, putrescine oxidase, pyranose oxidase, generated. For example, in Some instances, a high signal pyridoxal 5'-phosphate synthase, pyridoxine 4-oxidase, pyr intensity may be generated by the lowest concentration of roloquinoline-quinone synthase, pyruvate oxidase, pyruvate analyte in the test sample (in this instance, the amount of 40 oxidase (CoA-acetylating), reticuline oxidase, retinal oxi analyte in the test sample is inversely proportional to the dase, rifamycin-B oxidase, sarcosine oxidase, secondary-al amount of signal generated). Specifically, the amount of the cohol oxidase, Sulfite oxidase, Superoxide dismutase, Super analyte of interest present can be quantified based on com oxide reductase, tetrahydroberberine oxidase, thiamine paring the amount of light generated to a standard curve for oxidase, tryptophan C.B-Oxidase, urate oxidase (uricase, uric the analyte or by comparison to a reference standard. The 45 acid oxidase), Vanillyl-alcohol oxidase, Xanthine oxidase, standard curve can be generated using serial dilutions or xylitol oxidase and combinations thereof Preferably, the Solutions of analyte of interest of known concentration, by amount of at least one analyte-specific enzyme that can be mass spectroscopy, gravimetrically and by other techniques added to the test sample is from about 0.0001 unit/mL to known in the art. about 10,000 units/mL. In a second embodiment, the test sample containing an 50 As alluded to above, the time at which the at least one analyte of interest does not contain or comprise a peroxide. analyte-specific enzyme is added to the test sample is not However, it is preferred that for the analyte of interest to be critical, provided that it is added before the addition of the at detected that there exist one or more analyte-specific least one acridinium ester having the formula of formula I, enzymes that can be added to the test sample in order to which was previously discussed herein. Thus, for example, generate peroxide in the test sample. Preferably, the analyte of 55 the at least one analyte-specific enzyme is added to the test interest is galactose, glucose, cholesterol, LDL, HDL cho sample after the test sample has been processed to separate or line, lactic acid, uric acid, phosphatidylcholine, acetylcho remove the protein (See, FIG. 2). Alternatively, in another line, phosphocholine, CDP-choline, lysophosphatidylcho aspect, the at least one analyte-specific enzyme is added to the line, triglycerides, phospholipase A2, phosholipase D. test sample before the test sample is processed to separate or lysophosholipase D and sphingomyelin. The test sample is 60 remove the protein from the test sample (See, FIG. 3). manipulated or processed in Such a manner so as to separate Preferably, the at least one analyte-specific enzyme is at or remove from the test sample protein that may be contained least one oxidase. Oxidases can be used to generate hydrogen in the test sample (thus forming a processed test sample). The peroxide in a test sample. The peroxide that is generated by methods for separating or removing proteins in the test the addition of the at least analyte-specific enzyme can then sample are the separation methods described above, such as, 65 be converted to an end product having a distinct chemilumi but not limited to, ultrafiltration, extraction, precipitation, nescent emission. Such an end product is produced by adding dialysis, chromatography and digestion. to the processed test sample at least one acridinium ester. US 7,906,293 B2 19 20 Preferably, the acridinium ester is an acridinium-9-carboxy anhydride, N-hydroxysuccinimide and 1-ethyl-3-(3-dim late aryl ester having a formula of formula I, which was ethylaminopropyl)carbodiimide. discussed previously herein. At least one analyte-specific enzyme. Such as at least one After the addition of the acridinium-9-carboxylate aryl dismutase, dehydrogenase, oxidase, reductase or synthase or ester having the formula of formula I to the processed test a combination of at least one dismutase, dehydrogenase, oxi sample, at least one basic solution is added to the test sample dase, reductase or synthase (See, Table 1), is added to the at in order to generate a detectable signal, namely, a chemilu least one analyte-specific binding partner complex sample. minescent signal. The basic solution is the same basic solu The time at which the at least one analyte-specific enzyme is tion discussed previously herein, namely, a solution that con added to the at least one analyte specific binding partner tains at least one base and that has a pH greater than or equal 10 complex sample is not critical, provided that it is added before to 10, preferably, greater than or equal to 12. As also discussed the addition of the at least one acridinium ester having the previously herein, the chemiluminescent signal generated formula of formula I, which was previously discussed herein can be detected using routine techniques known to those (See, FIG. 4). The amount of at least analyte-specific enzyme skilled in the art. that can be added to the analyte specific binding partner In a third embodiment, the test sample contains an analyte 15 complex sample is from about 0.0001 unit/mL to about of interest that does not contain or comprise a peroxide. 10,000 units/mL. However, it is preferred that for the analyte of interest to be Optionally, after the generation of the peroxide, the same detected that there exist one or more analyte-specific specific binding partner (namely, the first specific binding enzymes that can be added to the test sample in order to partner) used to remove or separate the analyte from the test generate peroxide in the test sample, as discussed previously sample, or a second specific binding partner (which is differ herein. In this embodiment, the analyte of interest is prefer ent from the first specific binding partner), can be used to ably, galactose, glucose, cholesterol, LDL, HDL, choline, remove the analyte specific binding partner complex from the lactic acid, uric acid, phosphatidylcholine, acetylcholine, analyte specific binding partner complex sample (thus leav phosphocholine, CDP-choline, lysophosphatidylcholine, ing just the peroxide in the sample). triglycerides, phospholipase A2, phosholipase D, lyso 25 As discussed previously herein, the peroxide that is gener phosholipase D and sphingomyelin. Additionally, in this ated by the addition of the at least one analyte-specific embodiment, the analyte of interest can be removed or sepa enzyme can then be converted to an end product having a rated from the test sample (thus removing the analyte of distinct chemiluminescent emission. Such an end product is interest from protein contained in the test sample) using produced by adding to the analyte-specific binding partner immunoseparation techniques that are well known to those 30 complex sample at least one acridinium ester. Preferably, the skilled in the art. Specifically, in Such techniques, the test acridinium ester is an acridinium-9-carboxylate aryl ester sample containing the analyte of interest is contacted with a having a formula of formula I, which was discussed previ specific binding partner that binds to the analyte of interest ously herein. thus forming an analyte-specific binding partner complex. In After the addition of the acridinium-9-carboxylate aryl one aspect, the specific binding partner can be used in a 35 ester having the formula of formula I to the analyte specific sandwich type format (See, FIG. 4) or a competitive format, binding partner complex sample (which may or may not still the techniques for which are well known in the art. An contain the analyte specific binding partner complex), at least example of a specific binding partner that can be used is an one basic Solution is added in order to generate a detectable antibody, namely, an antibody that binds to the analyte. For signal, namely, a chemiluminescent signal. The basic Solution example, if the analyte of interest is choline, then the specific 40 is the same basic solution discussed previously herein, binding partner is an antibody that is capable of binding to namely, a solution that contains at least one base and that has choline. The analyte specific binding partner complex is then a pH greater than or equal to 10, preferably, greater than or removed or separated from the test sample using routine equal to 12. As also discussed previously herein, the chemi techniques known in the art, such as, but not limited to, luminescent signal generated can be detected using routine washing, thus resulting in an analyte specific binding partner 45 techniques known to those skilled in the art. complex sample. In a fourth embodiment, the test sample contains an analyte The specific binding partner used to remove or separate the of interest and does not contain or comprise a peroxide. analyte of interest can be immobilized on a solid phase. The Preferably, the analytes of interest that can be detected in this Solid phase can be any material known to those of ordinary embodiment are: toxins, organic compounds, DNA, RNA, skill in the art to which the specific binding partners, such as, 50 proteins, peptides, microorganisms, amino acids, nucleic but not limited to, antibodies or antigens, can be attached. acids, hormones, steroids, vitamins, drugs (including those Examples of solid phases that can be used, include, but are not administered fortherapeutic purposes as well as those admin limited to, a test well in a microtiter plate, nitrocellulose, istered for illicit purposes), drug intermediaries or byprod nylon, a bead or a disc (which can be made out of glass, ucts, bacteria, virus particles and metabolites of orantibodies fiberglass, latex, plastic or a paper material), a gel (for 55 to any of the above substances. More preferred analytes example, a gel through which the polypeptides have been run include, but are not limited to, brain natriuretic peptide (BNP) and which is Subsequently dried) or a strip, disc or sheet 1-32; NT-proBNP; proBNP; preproBNP; troponin I; troponin (which can be made out of nitrocellulose, nylon, plastic or T; troponin C.; human neutrophil gelatinase-associated paper). The specific binding partner can be bound to the Solid lipocalin (hNGAL); tacrolimus; Sirolimus, cyclosporine; fer phase by adsorption, by covalent bonding using a chemical 60 ritin; creatinine kinase MB (CK-MB); digoxin; phenytoin: coupling agent or by other means known in the art, provided phenobarbitol; carbamazepine; Vancomycin; gentamycin; that such binding does not interfere with the ability of the theophylline; Valproic acid; quinidine; luteinizing hormone specific binding partner to bind to the analyte of interest. (LH); follicle stimulating hormone (FSH); estradiol, proges Moreover, if necessary, the solid phase can be derivatized to terone; C-reactive protein; lipocalins; IgE antibodies; cytok allow reactivity with various functional groups on any of the 65 ines; vitamin B2 micro-globulin; glycated hemoglobin (Gly. specific binding partner. Such derivatization requires the use Hb); cortisol; digitoxin: N-acetylprocainamide (NAPA): of certain coupling agents such as, but not limited to, maleic procainamide; antibodies to rubella, Such as rubella-IgG and US 7,906,293 B2 21 22 rubella IgM, antibodies to toxoplasmosis, such as toxoplas dase, rifamycin-B oxidase, sarcosine oxidase, secondary-al mosis IgG (Toxo-IgG) and toxoplasmosis IgM (Toxo-IgM); cohol oxidase, Sulfite oxidase, Superoxide dismutase, Super testosterone; salicylates; acetaminophen; hepatitis B virus oxide reductase, tetrahydroberberine oxidase, thiamine Surface antigen (HbSAg); antibodies to hepatitis B core anti oxidase, tryptophan C.B-Oxidase, urate oxidase (uricase, uric gen, Such as anti-hepatitis B core antigen IgG and IgM (Anti acid oxidase), Vanillyl-alcohol oxidase, Xanthine oxidase, HBC); human immune deficiency virus (HIV); human T-cell xylitol oxidase and combinations thereof leukemia virus (HTLV); hepatitis Be antigen (HbeAg); anti The Substrate-specific enzyme can be conjugated to the bodies to hepatitis Be antigen (Anti-Hbe); influenza virus: specific binding partner using routine techniques known to thyroid stimulating hormone (TSH); thyroxine (T4); total those skilled in the art. An example of a specific binding triiodothyronine (Total T3); free triiodothyronine (Free T3); 10 partner that can be used is an antibody, namely, an antibody carcinoembryonic antigen (CEA), lipoproteins, cholesterol, that binds to the analyte. For example, if the analyte of interest and triglycerides; and alpha fetoprotein (AFP). Drugs of is a troponin, then the specific binding partner is an antibody abuse and controlled Substances include, but are not intended that is capable of binding to a troponin. The analyte: specific to be limited to, amphetamine; methamphetamine; barbitu binding partner conjugate complex is then removed or sepa rates, such as amobarbital, secobarbital, pentobarbital, phe 15 rated from the test sample using routine techniques known in nobarbital, and barbital; benzodiazepines, such as propoxy the art, such as, but not limited to, washing, thus resulting in and Valium; cannabinoids, such as hashish and marijuana; an analyte:Specific binding partner conjugate complex cocaine; fentanyl; LSD, methaqualone; opiates, such as sample. heroin, morphine, codeine, hydromorphone, hydrocodone, In order to generate or produce at least one type of peroxide methadone, oxycodone, oxymorphone and opium; phencyc for purposes of detecting the at least one analyte of interest, at lidine; and propoxyphene. least one substrate is added to the analyte:Specific binding In this embodiment, the analyte of interest can be removed partner conjugate complex. Any Substrate that is capable of or separated from the test sample (thus removing the analyte reacting with the Substrate-specific enzyme in the analyte: of interest from protein contained in the test sample) using specific binding partner conjugate complex sample to pro immunoseparation techniques, such as those described pre 25 duce hydrogen peroxide can be used. Examples of Substrates viously herein. Specifically, in Such techniques, the test that can be used include, but are not limited to those listed in sample containing the analyte of interest is contacted with a Table 1. Specifically, the substrate can be selected from the specific binding partner that is conjugated to at least one group consisting of (R)-6-hydroxynicotine, S)-2-hydroxy Substrate-specific enzyme which produces a peroxide, to acid, (S)-6-hydroxynicotine, 3-aci-nitropropanoate, 3-hy form an analyte: specific binding partner conjugate complex. 30 droxyanthranilate, (S)-2-hydroxy-2-(4-hydroxyphenyl)ac The Substrate-specific enzyme which produces a peroxide etate, 6-hydroxynicotinate, abscisic aldehyde, acyl-CoA, a can be selected from the group comprising of those listed in primary alcohol, an aldehyde, primary monoamines, Table 1. Specifically, the at least one substrate-specific diamines and histamine, a primary amine, an aromatic pri enzyme that can be used is selected from the group consisting mary alcohol, (2-naphthyl)methanol, 3-methoxybenzyl alco of (R)-6-hydroxynicotine oxidase, (S)-2-hydroxy acid oxi 35 hol, an aromatic aldehyde, catechol, cholesterol, choline, dase, (S)-6-hydroxynicotine oxidase, 3-aci-nitropropanoate columbamine, cyclohexylamine, a D-amino acid, D-ara oxidase, 3-hydroxyanthranilate oxidase, 4-hydroxymande binono-1.4-lactone, D-arabinono-1.4-lactone, D-aspar late oxidase, 6-hydroxynicotinate dehydrogenase, abscisic tate, D-glutamate, D-glutamate, dihydrosanguinarine, (S)-di aldehyde oxidase, acyl-CoA oxidase, alcohol oxidase, alde hydroorotate, 5,6-dihydrouracil, N,N-dimethylglycine, man hyde oxidase, amine oxidase, amine oxidase (copper 40 nitol, ecdysone, ethanolamine, D-galactose, B-D-glucose, glu containing), amine oxidase (flavin-containing), aryl-alcohol tathione, sn-glycerol 3-phosphate, glycine, glyoxy oxidase, aryl-aldehyde oxidase, catechol oxidase, cholesterol late, D-glucose, D-galactose, D-mannose, maltose, lactose, oxidase, choline oxidase, columbamine oxidase, cyclohexy cello-biose, L-2-hydroxyphytanate, (indol-3-yl)acetalde lamine oxidase, cytochrome c oxidase, D-amino-acid oxi hyde, Lactic acid, an L-amino acid, L-aspartate, L-galactono dase, D-arabinono-1.4-lactone oxidase, D-arabinono-1.4-lac 45 1.4-lactone, L-glutamate, L-gulono-1.4-lactone, L-ly tone oxidase, D-aspartate oxidase, D-glutamate oxi sine, L-lysine. A long-chain-alcohol, L-pipecolate, L-Sorbose, dase, D-glutamate(D-aspartate) oxidase, dihydroben (S)-malate, methanethiol, 6-N-methyl-L-lysine, N-acetyl-D- Zophenanthridine oxidase, dihydroorotate oxidase, dihydrou glucosamine, N-glycolylglucosamine, N-acetylgalac racil oxidase, dimethylglycine oxidase, D-mannitol oxidase, tosamine, N-acetylmannosamine, NAD(P)H, a nitroalkane, ecdysone oxidase, ethanolamine oxidase, galactose oxidase, 50 an N-methyl-L-amino acid, adenosine, oxalate, 1-N-acetyl glucose oxidase, glutathione oxidase, glycerol-3-phosphate spermine, polyvinyl alcohol, an S-prenyl-L-cysteine, pepti oxidase, glycine oxidase, glyoxylate oxidase, hexose oxi dyl-L-lysyl-peptide, butane-1,4-diamine, D-glucose, D-xy dase, hydroxyphytanate oxidase, indole-3-acetaldehyde oxi lose, L-sor-bose, D-glucono-1,5-lactone, pyridoxamine dase, lactic acid oxidase, L-amino-acid oxidase, L-aspartate 5'-phosphate, pyridoxine, 6-(2-amino-2-carboxyethyl)-7,8- oxidase, L-galactonolactone oxidase, L-glutamate oxi 55 dioxo-1,2,3,4,5,6,7,8-octahydroquinoline-2,4-dicarboxy dase, L-gulonolactone oxidase, L-lysine 6-oxidase, L-lysine late, pyruvate, pyruvate, reticuline, retinal, rifamycin-B, Sar oxidase, long-chain-alcohol oxidase, L-pipecolate oxi cosine, a secondary alcohol, Sulfite, Superoxide, Superoxide, dase, L-Sorbose oxidase, malate oxidase, methanethiol oxi (S)-tetrahydroberberine, thiamine, L-tryptophan, uric acid, dase, monoamino acid oxidase, N'-methyl-lysine oxidase, vanillyl alcohol. xanthine and xylitol. N-acylhexosamine oxidase, NAD(P)H oxidase, nitroalkane 60 The amount of substrate added to the analyte: specific bind oxidase, N-methyl-L-amino-acid oxidase, nucleoside oxi ing partner conjugate complex sample is the amount neces dase, oxalate oxidase, polyamine oxidase, polyphenol oxi sary to generated a Sufficient amount of peroxide that is dase, polyvinyl-alcohol oxidase, prenylcysteine oxidase, pro detected using the acridinium esters described herein. The tein-lysine 6-oxidase, putrescine oxidase, pyranose oxidase, amount of substrate to be added can be readily determined by pyridoxal 5'-phosphate synthase, pyridoxine 4-oxidase, pyr 65 one of ordinary skill in the art. roloquinoline-quinone synthase, pyruvate oxidase, pyruvate After the generation of at least one peroxide, at least one oxidase (CoA-acetylating), reticuline oxidase, retinal oxi acridinium ester having the formula of formula I, which was US 7,906,293 B2 23 24 previously discussed herein, can then be added to the analyte: dialysis, chromatography or digestion. The kit can also con specific binding partner conjugate complex sample. As dis tain instructions for generating a standard curve for the pur cussed previously herein, the peroxide that is generated can poses of quantifying the amount of the analyte in the test then be converted to an end product having a distinct chemi sample or a reference standard for purposes of quantifying the luminescent emission. Such an end product is produced by amount of analyte in the test sample. Optionally, the kit may adding to the analyte:Specific binding partner conjugate com also contain at least one analyte-specific enzyme. Such as at plex sample at least one acridinium ester. Preferably, the least one enzyme listed in Table 1. acridinium ester is an acridinium-9-carboxylate aryl ester In another aspect, the kit can contain at least one acri having a formula of formula I, which was discussed previ dinium-9-carboxylate aryl ester having a formula of ously herein. This embodiment can be performed in a sand 10 wich format (See, FIG. 5) or in a competitive format (See, FIG. 6). RI xe After the addition of the acridinium-9-carboxylate aryl e ester having the formula of formula I to the analyte:specific 15 21 N1 N binding partner conjugate complex sample, at least one basic R.-- --R2 Solution is added in order to generate a detectable signal, namely, a chemiluminescent signal. The basic solution is the same basic Solution discussed previously herein, namely, a Solution that contains at least one base and that has a pH O O greater than or equal to 10, preferably, greater than or equal to R8 R4 12. As also discussed previously herein, the chemilumines cent signal generated can be detected using routine tech niques known to those skilled in the art. R7 R5 In another embodiment, the present invention relates to a 25 kit for detecting at least one analyte in a test sample. In one R6 aspect, the kit can contain at least one acridinium-9-carboxy late aryl ester having a formula of: wherein R' is an alkyl, alkenyl, alkynyl, arylalkyl, aryl, 30 sulfoalkyl or carboxyalkyl and R through Rare each inde RI xe pendently selected from the group consisting of hydrogen, alkyl, amino, alkoxy, hydroxyl, carboxyl, halide, nitro, cyano, 21 leN1 N - SO, NHC(O)R, C(O)R, C(O)OR, C(O)NHR R.-- --R2 and—SCN, wherein Risan alkyl, alkenyl, alkynyl, arylalkyl, S 2n 21 35 aryl, Sulfoalkyl or carboxyalkyl, and X is an anion. Additionally, the kit may also contain at least one specific binding partner. Also, the kit can contain at least one basic O O Solution. Additionally, the kit can also contain one or more instructions for detecting an analyte in a test sample. Addi R8 R4 40 tionally, the kit can also contain instructions for generating a standard curve for the purposes of quantifying the amount of the analyte in the test sample or a reference standard for R7 R5 purposes of quantifying the amount of analyte in the test R6 sample. 45 Optionally, the kit can further contain at least one enzyme listed in Table 1. wherein R' is an alkyl, alkenyl, alkynyl, arylalkyl, aryl, Optionally, the at least one specific binding partner con sulfoalkyl or carboxyalkyl and R through Rare each inde tained in the kit can have conjugated thereon at least one pendently selected from the group consisting of hydrogen, Substrate-specific enzyme. The at least one Substrate specific alkyl, amino, alkoxy, hydroxyl, carboxyl, halide, nitro, cyano, 50 enzyme can be at least one enzyme listed in Table 1. If the at - SO, NHC(O)R, C(O)R, C(O)OR, C(O)NHR least one specific binding partner included in the kit has and—SCN, wherein Risan alkyl, alkenyl, alkynyl, arylalkyl, conjugated thereon at least one substrate-specific enzyme, aryl, Sulfoalkyl or carboxyalkyl, and X is an anion. then the kit can further contain at least one substrate. The at Additionally, the kit can contain at least one basic solution. least one substrate that can be included in the kit includes, but Also, the kit can also contain one or more instructions for 55 is not limited to, substrates listed in Table 1. detecting an analyte in a test sample. Preferably, at least one By way of example and not of limitation, examples of the of the instructions in the kit will describe the steps necessary present invention shall not be given. for processing the test sample to separate or remove the analyte of interest from protein contained in the test sample. Example 1 Specifically, the instructions may contain the specific proto 60 cols for separating or removing the analyte of interest from Preparation of protein in the test sample. For example, the instructions may 10-Methyl-9-(Phenoxycarbonyl)Acridinium contain the specific protocols for performing ultrafiltration, Fluorosulfonate Stock Solution extraction, precipitation, dialysis, chromatography or diges tion. Alternatively, the instructions may simply refer to a 65 10-Methyl-9-(phenoxycarbonyl)acridinium fluorosul publication or well-known textbook that describes the proto fonate (Fluka Cat. No. 68617, 25 mg) was dissolved in cols for performing ultrafiltration, extraction, precipitation, degassed anhydrous N,N-dimethylformamide (DMF) (Ald US 7,906,293 B2 25 26 rich Cat. No. 227056, 3 mL) to give a stock solution (20 mM). and 14) was more intense than that recorded for initially for The stock solution was protected from light and stored at 4-8 the solution on day 1 (Example 3), indicating that pseudobase C. when not in use. formation is problematic in neutral Solutions. FIGS. 13-20 show the subsequent response of lower con Example 2 centrations of 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate in pH 4, 0.1% sodium cholate in the choline Preparation of assay. 10-Methyl-9-(Phenoxycarbonyl)Acridinium Fluorosulfonate Working Solutions Example 6 10 10-Methyl-9-(phenoxycarbonyl)acridinium fluorosul Assay of Plasma Samples for Choline using fonate (20 mM in DMF) was diluted into 0.1% (wt/vol) aque 10-Methyl-9-(Phenoxycarbonyl)Acridinium ous sodium cholate (Sigma Cat. No. C6445-25), then serially Fluorosulfonate without Sample Pretreatment diluted to a give working solutions from 250 nM-40 uM. 15 Plasma sample A containing greater than 150 uM choline, Example 3 a low plasma pool (LP) containing 14 LM choline and high Evaluation of plasma pool (HP) containing 40 uM were analyzed as in 10-Methyl-9-(Phenoxycarbonyl)Acridinium Example 3 using 250 nM 10-Methyl-9-(phenoxycarbonyl) acridinium fluorosulfonate, pH 4. While a normal standard Fluorosulfonate in an Assay for Choline curve (See, FIG. 20) was generated, the concentration of Choline standards (0, 5, 10, 20, 30, 50, 75, and 150 uM in choline in the plasma samples was calculated to be less than phosphate buffer, 0.2 M, pH8) and choline oxidase (10U/mL 2 uM in all the samples. in phosphate buffer, 0.2 M, pH8; 0.1% sodium cholate) were prepared as reported in Adamczyk M. Brashear RJ, Mat 25 Example 7 tingly P G. Tsatsos P.H., Homogeneous chemiluminescent assays for free choline in human plasma and whole blood. Assay of Plasma Samples for Choline using Anal Chim Acta. 2006; 579(1):61-7. The assay was run a on a 10-Methyl-9-(Phenoxycarbonyl)Acridinium microplate chemiluminometer (Mithras LB-940, BER Fluorosulfonate with Sample Pretreatment THOLD TECHNOLOGIES U.S.A. LLC, Oak Ridge, Tenn.) 30 equipped with three reagent injectors using 96-well black The experiment of Example 6 was repeated using the same polystyrene microplates (Costar #3792). Choline standards plasma samples that were pretreated by ultrafiltration using a (4 LL) were manually pipetted in quadruplicates into the Nanosep 10K Omega microtube ultrafiltration device (Pall, wells of the microplate. Choline oxidase solution (40 uL), Cat. No. OD0101C35). The plasma sample (100 uL) was 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate 35 added to the device and centrifuged for 5-15 min. The ultra working solution (40 LL) and 0.1 Naqueous Sodium hydrox filtrate was then analyzed. The reported concentration of cho ide (100 uL) were sequentially added and the chemilumines line in each sample read correctly, i.e., sample A (169 uM), LP cent response was recorded for 2 s well by well. (14.62 uM) and HP (41.37 uM). The chemiluminescent response for 10-methyl-9-(phe noxycarbonyl)acridinium fluorosulfonate concentrations 40 Example 8 above 4 uM saturated the chemiluminometer detector. Typi cal responses for lower concentrations and the corresponding Comparison of Chemiluminescent Response in an choline standard curves are shown in FIGS. 7-10. Assay for Choline using 9-(3-Carboxypropyl)(4- methylphenyl)sulfonyl)amino-carbonyl-10-(3-sul Example 4 45 fopropyl)acridinium Inner Salt Effect of Pseudobase Formation with Choline standards were analyzed following the procedure 10-Methyl-9-(Phenoxycarbonyl)Acridinium in Adamczyk, et al. (Adamczyk M. Brashear RJ, Mattingly P Fluorosulfonate in an Assay for Choline G, Tsatsos PH. Homogeneous chemiluminescent assays for 50 free choline in human plasma and whole blood. Anal Chim The experiment in Example 3 was repeated using the 2 uM Acta. 2006; 579(1):61-7). The chemiluminescence profiles at 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate each choline concentration are shown in FIG. 21. In compari working solution that had been stored at 2-8°C. overnight. son to 10-Methyl-9-(Phenoxycarbonyl)Acridinium Fluoro The results shown in FIGS. 11 and 12, show that the chemi sulfonate at 2 uM concentration (See, FIG. 13), the instant luminescent response dropped by 50% due to pseudobase 55 example had a lower response (56%) at double the concen formation compared to Example 3. tration. One skilled in the art would readily appreciate that the Example 5 present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those Recovery from Pseudobase Formation with 60 inherent therein. The molecular complexes and the methods, 10-Methyl-9-(Phenoxycarbonyl)Acridinium procedures, treatments, molecules, specific compounds Fluorosulfonate in an Assay for Choline described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limita The experiment from Example 4 was repeated after acidi tions on the scope of the invention. It will be readily apparent fication of the 2 uM 10-methyl-9-(phenoxycarbonyl)acri 65 to one skilled in the art that varying Substitutions and modi dinium fluorosulfonate working solution to pH 4 with 0.1 N fications may be made to the invention disclosed herein with aqueous nitric acid. The chemiluminescent signal (FIGS. 13 out departing from the scope and spirit of the invention. US 7,906,293 B2 27 28 All patents and publications mentioned in the specification (f) quantifying the amount of analyte in the analyte specific are indicative of the levels of those skilled in the art to which binding partner complex sample by relating the amount the invention pertains. All patents and publications are herein of light generated in the test sample by comparison to a incorporated by reference to the same extent as if each indi vidual publication was specifically and individually indicated standard curve for said analyte. to be incorporated by reference. 5 2. The method of claim 1, wherein the analyte-specific The invention illustratively described herein suitably may enzyme is selected from the group consisting of dismutase, be practiced in the absence of any element or elements, limi dehydrogenase, oxidase, reductase, synthase and combina tation or limitations which is not specifically disclosed herein. tions thereof. Thus, for example, in each instance herein any of the terms 3. The method of claim 2, wherein the analyte-specific “comprising.” “consisting essentially of and "consisting of 10 enzyme is selected from the group consisting of (R)-6-hy may be replaced with either of the other two terms. The terms droxynictotine oxidase, (S)-2-hydroxy acid oxidase, (S)-6- and expressions which have been employed are used as terms hdyroxynicotine oxidase, 3-aci-nitropropanoate oxidase, of description and not of limitation, and there is no intention 3-hydroxyanthranilate oxidase, 4-hydroxymandelate oxi that in the use of such terms and expressions of excluding any dase, 6-hydroxynictonate dehydrogenase, abscisic-aldehyde equivalents of the features shown and described or portions 15 oxidase, acyl-CoA oxidase, alcohol oxidase, aldehyde oxi thereof, but it is recognized that various modifications are dase, amine oxidase, amine oxidase (copper containing), possible within the scope of the invention claimed. Thus, it amine oxidase (flavin-containing), aryl-alcohol oxidase, aryl should be understood that although the present invention has adehyde oxidase, catechol oxidase, cholesterol oxidase, cho been specifically disclosed by preferred embodiments and line oxidase, columbamine oxidase, cyclohexylamine oxi optional features, modification and variation of the concepts dase, cytochrome c oxidase, D-amino-acid oxi herein disclosed may be resorted to by those skilled in the art, dase, D-arabinono-1.4-lactone oxidase, D-aspartate oxi and that Such modifications and variations are considered to dase, D-glutamate oxidase, D-glutamate(-aspartate) oxidase, be within the scope of this invention as defined by the dihydrobenzophenanthridine oxidase, dihydroorotate oxi appended claims. dase, dihydrouracil oxidase, dimethylglycine oxi What is claimed is: dase, D-mannitol oxidase, ecdysone oxidase, ethanolamine oxidase, galactose oxidase, glucose oxidase, glutathione oxi 1. A method of detecting an analyte in a test sample, the 25 dase, glycerol-3-phosphate oxidase, glycine oxidase, glyoxy method comprising the steps of late oxidase, hexose oxidase, hydroxyphytanate oxidase, (a) contacting the test sample containing the analyte with a indole-3-acetaldehyde oxidase, lactic acid oxidase, L-amino specific binding partner to forman analyte specific bind acid oxidase, L-aspartate oxidase, L-galactonolactone oxi ing partner complex; dase, L-glutamate oxidase, L-gulonolactone oxidase, L-lysine (b) separating the analyte specific binding partner complex 30 6-oxidase, L-lysine oxidase, long-chain-alcohol oxi from the test sample to form an analyte specific binding dase, L-pipecolate oxidase, L-Sorbose oxidase, malate oxi partner complex sample: dase, methanethiol oxidase, monoamino acid oxidase, (c) adding at least one analyte-specific enzyme to the ana N-methyl-lysine oxidase, N-acylhexosamine oxidase, NAD lyte specific binding partner complex sample to produce (P)H oxidase, nitroalkane oxidase, N-methyl-L-amino-acid peroxide; 35 oxidase, nucleoside oxidase, oxalate oxidase, polyamine oxi (d) adding an acridinium-9-carboxylate aryl ester to the dase, polyphenol oxidase, polyvinyl-alcohol oxidase, prenyl cysteine oxidase, protein-lysine 6-oxidase, putrescine oxi analyte specific binding partner complex sample, the dase, pyranose oxidase, pyridoxal 5'-phosphate synthase, acridinium-9-carboxylate aryl ester having a formula of pyridoxine 4-oxidase, pyrroloquinoline-quinone synthase, pyruvate oxidase, pyruvate oxidase (CoA-acetylating), retri RI xe 40 culine oxidase, retinal oxidase, rifamycin-B oxidase, sar cosine oxidase, secondary-alcohol oxidase, Sulfite oxidase, 21 eN1 N Superoxide dismutase, Superoxide reductase, tetrahydrober R.-- --R2 berine oxidase, thiamine oxidase, tryptophan C.f3-oxidase, urate oxidase (uricase, uric acid oxidase), Vanillyl-alcohol 45 oxidase, Xanthine oxidase, Xylitol oxidase and combinations thereof. 4. The method of claim 1, wherein the specific binding O O partner is an antibody that binds to the analyte. R8 R4 5. The method of claim 1, wherein the basic solution has a 50 pH greater than or equal to 12. 6. The method of claim 1, wherein the standard curve is R7 R5 generated from Solutions of the analyte of a known concen tration. 7. The method of claim 1, wherein the test sample is whole wherein R' is an alkyl, alkenyl, alkynyl, arylalkyl, aryl, 55 blood, serum, plasma, interstitial fluid, saliva, ocular lens sulfoalkylor carboxyalkyland R through Rare each fluid, cerebral spinal fluid, Sweat, urine, milk, ascites fluid, independently selected from the group consisting of: mucous, nasal fluid, sputum, Synovial fluid, peritoneal fluid, hydrogen, alkyl, amino, alkoxy, hydroxyl, carboxyl, vaginal fluid, menses, amniotic fluid or semen. halide, nitro, cyano. —SO, NHC(O)R, —C(O)R, 8. The method of claim 1, wherein the analyte is selected —C(O)OR, C(O)NHR and - SCN, wherein R is 60 from the group consisting of galactose, glucose, cholesterol, an alkyl, alkenyl, alkynyl, arylalkyl, aryl, Sulfoalkyl LDL, HDL choline, lactic acid, uric acid, phophatidylcho or carboxyalkyl, and X is an anion; line, acetylcholine, phosphocholine, CDP-choline, lyso (e) adding a basic solution to the analyte specific binding phophatidylcholine, triglycerides, phospholipase A2, phos partner complex sample to generate a light signal, pholipase D, lysophospholipase D and sphingomyelin. wherein the light signal is generated in a basic Solution 65 with a pH greater than or equal to 10; and