WO2012028167A2 - Acridinium ester chemiluminescence upon reductive triggering - Google Patents
Acridinium ester chemiluminescence upon reductive triggering Download PDFInfo
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- WO2012028167A2 WO2012028167A2 PCT/EP2010/057017 EP2010057017W WO2012028167A2 WO 2012028167 A2 WO2012028167 A2 WO 2012028167A2 EP 2010057017 W EP2010057017 W EP 2010057017W WO 2012028167 A2 WO2012028167 A2 WO 2012028167A2
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- acridinium
- substituted
- quinones
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- chemiluminescence
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- 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
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- 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
-
- F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
- F21—LIGHTING
- F21K—NON-ELECTRIC LIGHT SOURCES USING LUMINESCENCE; LIGHT SOURCES USING ELECTROCHEMILUMINESCENCE; LIGHT SOURCES USING CHARGES OF COMBUSTIBLE MATERIAL; LIGHT SOURCES USING SEMICONDUCTOR DEVICES AS LIGHT-GENERATING ELEMENTS; LIGHT SOURCES NOT OTHERWISE PROVIDED FOR
- F21K2/00—Non-electric light sources using luminescence; Light sources using electrochemiluminescence
- F21K2/06—Non-electric light sources using luminescence; Light sources using electrochemiluminescence using chemiluminescence
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
Definitions
- This invention involves a method to detect and quantify reaction partners of a
- this invention also relates to the detection and quantification of reducing compounds by recording the chemiluminescence of these reducing compounds in the presence of acridinium ester and certain oxidants.
- oxidative compounds are detected and quantified by recording the chemiluminescence of these oxidizing compounds in the presence of acridinium ester and certain reducing compounds.
- Acridinium esters are well-known in literature and can be prepared by known methods (US 2009/0318627, US 2008/0014660, US6673560, US 6087502, US5879953, US 3539574, Luminescence;2000; 15:311-320, EP0915851). From literature it is well known that acridinium esters (AE) undergo efficient chemiluminescent reaction upon triggering with basic hydrogen peroxide
- the mechanism involves attack by hydrogen peroxide anion on the C-9 position of the acridinium nucleus, followed by intramolecular nucleophilic attack on the carbonyl group. In this way a four membered highly strained dioxetane ring system is formed, decomposition of which is accompanied by light emission.
- Aizawa et al (US RE39,047 E, Mar28, 2006) teach the luminescence by reacting an acridinium ester label in an immunoassay, a hybridization assay, or an immunoblot assay with superoxide in the presence of a flavin compound. Results shown are obtained from electrochemical and enzymatic superoxide production. They do not imply the use of acridinium esters for the detection of superoxide anion or more generally the
- Cooper et al. (MarineChem 2000;70: 191-200) describe a chemiluminescence method for the analysis of hydrogen peroxide in natural waters based on acridinium ester oxidation.
- Cooper et al describe a positive interference in high ferrous iron containing samples caused by the formation of hydrogen peroxide via reduction of molecular oxygen by Fe(II). They treated water with 3 micromolar Fe(II) as ferrous sulphate and found an increase in chemiluminescence of about a factor of 10, compared to blank water. This positive interference could be removed by addition of ferrozine to complex Fe(II) and prevent its reaction with molecular oxygen.
- Kishikawa et al (Anal Bioanal Chem 2009:337-43) report the chemiluminescence occurring from a reaction of certain quinones with dithiothreitol (DTT) as monitored with luminol. The authors implicate a mechanism where superoxide anion reacts with luminol.
- cuprous ions either directly or by reduction from ferric or cupric ions by a suitable reductant like ascorbate or trolox, but also metals like zinc.
- a suitable reductant like ascorbate or trolox, but also metals like zinc.
- ferrous ions can be detected down to less than 3 nanomolar.
- certain acridinium esters can be detected down to less than 3 picogram upon triggering with ferrous ions. This reaction also can be used to determine the reductant with great sensitivity.
- Ascorbate can be detected down to less than
- acridinium ester labeled entities (antigens, 95 antibodies, proteins, DNA, etc) can be detected and quantified under reductive
- a reducing compound e.g. ascorbate, DTT, or TCEP
- an acidic solution of an acridinium e.g. ascorbate, DTT, or TCEP
- an oxidant e.g. ferric or cupric ions or 9,10-phenanthrenequinone (PQ)
- PQ 9,10-phenanthrenequinone
- superoxide anion or superoxide producing systems, e.g. xanthine oxidase/xanthine
- superoxide anion can be detected and quantified by adding an acidic solution of an acridinium ester, followed by triggering the CL reaction by raising the pH.
- the response of this reaction can be discriminated from the hydrogen peroxide induced CL-reaction of acridinium ester by time resolution.
- Reductive conditions are those conditions which allow a compound (a so-called reductant, reducer, or reducing agent) to transfer one or more electrons to an acceptor (a so-called oxidant, oxidizer, or oxidizing agent).
- reductants are metals (potassium, calcium, barium, sodium and magnesium), nascent hydrogen (i.e. formed by reaction of zinc metal and acid), and metal ions like ferrous (Fe(II)), cuprous (Cu(I)), stannous (Sn(II), and the like, thiols e.g.
- Reductive conditions can also be combinations of oxidants e.g. ferric, cupric, quinone, etc. in combination with reductants which are capable of reducing the oxidants e.g. ferrous (Fe(III)) to ferric (Fe(II)), cuprous (Cu(II)) to cupric (Cu(I)), ascorbate to dehydroascorbate, etc, etc.
- reductants which are capable of reducing the oxidants e.g. ferrous (Fe(III)) to ferric (Fe(II)), cuprous (Cu(II)) to cupric (Cu(I)), ascorbate to dehydroascorbate, etc, etc.
- reductants which are capable of reducing the oxidants e.g. ferrous (Fe(III)) to ferric (Fe(II)), cuprous (Cu(II)) to cupric (Cu(I)), ascorbate to dehydroascorbate, etc
- Example 1 Acridinium ester detection under reducing conditions compared with
- Calibration standards of acridinium ester (0-1000 pg/well) were treated with ferrous sulphate (10-lOOuM), a combination of ferric chloride (10-lOOuM) and ascorbate (10- lOOuM), or hydrogen peroxide (100-lOOOuM).
- Chemiluminescence reaction was triggered by the addition of sodium carbonate buffer (pH 9) and the resulting signal accumulated during the first 10 seconds.
- Calibration curves were constructed using the 4-5 seconds integral values of the kinetic profiles and were expressed as signal-background ratios against amounts of acridinium ester. The results are shown in the figure:
- the acridinium ester can be quantified under reducing
- Calibration standards of acridinium ester (0-1000 pg/well) were treated with 145 phenanthrenequinone (PQ) 3, 10, 30, 100 uM). Chemilummescence reaction was triggered by the addition of sodium carbonate buffer (pH 9) containing TCEP (lOOuM) and the resulting signal accumulated during the first 10 seconds. Calibration curves were constructed using the 4-5 seconds integral values of the kinetic profiles and were expressed as signal-background ratios against amounts of acridinium ester. The results for 30 uM PQ are shown in the figure.
- Acridinium ester can be detected and quantified over a large dynamic range with good linearity and detection limit.
- peroxide water was added, while the wells containing PQ were treated with 50 uL of TCEP (lOOuM).
- Acridinium ester (formula E) (100 ng/niL, 10 uL/well) was added.
- Soot extract dilutions were prepared from a stock containing lOOug/mL in water. These dilutions (0.01-lOug/mL, 50uL/well) were treated with TCEP (lOOuM, 50uL/well) during 5- 20 minutes. After that time acridinium ester (formula E) (lOOng/mL, lOuL/well) was added. 180 Chemilummescence reaction was triggered by the addition of sodium carbonate buffer (pH 9) and the resulting signal accumulated during the first 10 seconds. Calibration curves were constructed using the 0-2 seconds integral values of the kinetic profiles and were expressed as signal-background ratios against amounts of soot extract. The results are shown in the figure:
- Example 5 Reductive power detection of urine sample dilution
- Urine sample was diluted 100-10000 fold. To 50uL of a diluted sample either 50uL of a FeC13 solution (lOOuM) or of a PQ solution (lOOuM) was added, followed by lOuL of acridinium 190 ester (formula E) (lOOng/mL). Chemilummescence reaction was triggered by the addition of sodium carbonate buffer (pH 9) and the resulting signal accumulated during the first 10 seconds. The results are shown in the figure 14.
- Urine sample was diluted 100-10000 fold. To 50uL of a diluted sample either 50uL of a CuS04 solution (lOOuM) was added, and incubated at 60oC during 15 minutes. Subsequently, 205 lOuL of acridinium ester (formula E) (lOOng/mL) was added. Chemiluminescence reaction was triggered by the addition of sodium carbonate buffer (pH 9) and the resulting signal accumulated during the first 10 seconds. The results are shown in the figure 9.
- acridinium ester formula E
Abstract
Acridinium esters show strong chemiluminescence under reducing conditions. Reducing conditions involve reduced metal ions or reduced aromatic quinones. This chemistry can be used in the detection and quantification of acridinium ester, (mixtures) of oxidants or (mixtures) of reducing compounds.
Description
ACRIDINIUM ESTER CHEMILUMINESCENCE UPON REDUCTIVE
TRIGGERING
Description of the Invention
This invention involves a method to detect and quantify reaction partners of a
chemiluminescence reaction involving acridinium ester and reductive triggering conditions. Apart from a new method to detect and quantify acridinium esters, in another aspect this invention also relates to the detection and quantification of reducing compounds by recording the chemiluminescence of these reducing compounds in the presence of acridinium ester and certain oxidants. In yet another aspect of this invention oxidative compounds are detected and quantified by recording the chemiluminescence of these oxidizing compounds in the presence of acridinium ester and certain reducing compounds.
Related art
Acridinium esters are well-known in literature and can be prepared by known methods (US 2009/0318627, US 2008/0014660, US6673560, US 6087502, US5879953, US 3539574, Luminescence;2000; 15:311-320, EP0915851). From literature it is well known that acridinium esters (AE) undergo efficient chemiluminescent reaction upon triggering with basic hydrogen peroxide
Contrary to the luminol reaction no catalyst is required. The mechanism involves attack by hydrogen peroxide anion on the C-9 position of the acridinium nucleus, followed by intramolecular nucleophilic attack on the carbonyl group. In this way a four membered highly strained dioxetane ring system is formed, decomposition of which is accompanied by light emission.
INSERT FIGURE 7
Yang et al teach the electrochemiluminescence assay for the detection of acridinium esters (AnalChimActa 2002;461 : 141-6). These authors suggest the intermediacy of nascent oxygen generated on the surface of the working electrode in the course of the oxidation of water as the oxidizer of the acridinium ester.
Aizawa et al (US RE39,047 E, Mar28, 2006) teach the luminescence by reacting an acridinium ester label in an immunoassay, a hybridization assay, or an immunoblot assay with superoxide in the presence of a flavin compound. Results shown are obtained from electrochemical and enzymatic superoxide production. They do not imply the use of acridinium esters for the detection of superoxide anion or more generally the
chemiluminescence of acridinium esters under reductive conditions.
Cooper et al. (MarineChem 2000;70: 191-200) describe a chemiluminescence method for the analysis of hydrogen peroxide in natural waters based on acridinium ester oxidation. In their paper Cooper et al describe a positive interference in high ferrous iron containing samples caused by the formation of hydrogen peroxide via reduction of molecular oxygen by Fe(II). They treated water with 3 micromolar Fe(II) as ferrous sulphate and found an increase in chemiluminescence of about a factor of 10, compared to blank water. This positive interference could be removed by addition of ferrozine to complex Fe(II) and prevent its reaction with molecular oxygen.
From literature (Yildiz and Demiryurek, J PharmacolToxMethods 1998;39: 179-84) it is known that ferrous ion induces luminol chemiluminescence at pH 7.4 but ferrous ion is inactive with lucigenin (bis-N-methylacridinium nitrate). The luminol CL is quenched by low concentrations of ascorbate from which the authors suggest the intermediacy of hydroxyl radicals as the cause of the luminol CL. This CL is quenched by mannitol, a well known quencher of hydroxyl radicals.
In a paper by Li et al (Microchimica Acta 2005;149:205-12) the CL reaction of ascorbic acid in the presence of ferric (Fe(III)) ions with luminol at high pH is described. The authors suggest that ferrous (Fe(II)) ions are intermediates in this reaction.
Zhang and Chen ( Anal Sciences 2000; 16: 1317-21 ) describe CL studies of the oxidation of ascorbic acid by cupric (Cu(II)) ions using luminol in alkaline solution.
Kishikawa et al (Anal Bioanal Chem 2009:337-43) report the chemiluminescence occurring from a reaction of certain quinones with dithiothreitol (DTT) as monitored with luminol. The authors implicate a mechanism where superoxide anion reacts with luminol.
In a paper by Li et al (Microchim Acta 2008;162: 189-98) a secondary chemiluminescence emission of the luminol- ferricyanide system induced by reducing agents is described. These
authors describe that addition of a reducing agents to a reaction mixture in which the CL reaction of luminol and ferricyanide had just finished, results in a strong secondary CL emission. This secondary CL emission could be used to measure reducing agents like caffeine, sulfite, Fe(II), etc in a flow- injection method.
75 The authors don't mention or imply the use of acridinium esters under these conditions.
Detailed description of the invention
It has now been unexpectedly found that certain acridinium esters (FIGURE 8) can be triggered under reductive conditions very efficiently. Reductive conditions include ferrous or
80 cuprous ions, either directly or by reduction from ferric or cupric ions by a suitable reductant like ascorbate or trolox, but also metals like zinc. In this way ferrous ions can be detected down to less than 3 nanomolar. Reversely, certain acridinium esters can be detected down to less than 3 picogram upon triggering with ferrous ions. This reaction also can be used to determine the reductant with great sensitivity. Ascorbate can be detected down to less than
85 100 nanomolar.
In the course of this work other reductive conditions were found like the mixture of certain quinones and organic compounds like dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or reduced glutathione (GSH) which gave chemiluminescence in the presence of certain acridinium compounds.
90 Moreover, kinetic analysis indicate that, depending on the reductive condition, two pathways, the one originating from hydrogen peroxide and the other originating from another form of reduced oxygen, can be seen separately.
This reductive triggering of acridinium esters can be applied in a number of ways:
• Using one aspect of this invention, acridinium ester labeled entities (antigens, 95 antibodies, proteins, DNA, etc) can be detected and quantified under reductive
conditions, e.g. by triggering with ferrous ions.
• Using another aspect of this invention oxidants (or mixtures of oxidants) can be
detected and quantified by adding a reducing compound (e.g. ascorbate, DTT, or TCEP), and after a suitable incubation time adding an acidic solution of an acridinium
100 ester, followed by triggering the CL reaction by raising the pH.
• In another aspect of this invention reducing compounds (or mixtures of reducing
compounds) can be detected and quantified by adding an oxidant (e.g. ferric or cupric ions or 9,10-phenanthrenequinone (PQ)), and after a suitable incubation time adding
an acidic solution of an acridinium ester, followed by triggering the CL reaction by raising the pH.
• In yet another aspect of this invention superoxide anion (or superoxide producing systems, e.g. xanthine oxidase/xanthine) can be detected and quantified by adding an acidic solution of an acridinium ester, followed by triggering the CL reaction by raising the pH. The response of this reaction can be discriminated from the hydrogen peroxide induced CL-reaction of acridinium ester by time resolution.
Definitions
Reductive conditions are those conditions which allow a compound (a so-called reductant, reducer, or reducing agent) to transfer one or more electrons to an acceptor (a so-called oxidant, oxidizer, or oxidizing agent). Examples of reductants are metals (potassium, calcium, barium, sodium and magnesium), nascent hydrogen (i.e. formed by reaction of zinc metal and acid), and metal ions like ferrous (Fe(II)), cuprous (Cu(I)), stannous (Sn(II), and the like, thiols e.g. glutathion, cysteine, dithiothreitol (DTT), TCEP (tris(2-carboxyethyl)phosphine), and the like, vitamins e.g. ascorbate (vitamin C), and vitamin E, urate, trolox, etc, etc.
Reductive conditions can also be combinations of oxidants e.g. ferric, cupric, quinone, etc. in combination with reductants which are capable of reducing the oxidants e.g. ferrous (Fe(III)) to ferric (Fe(II)), cuprous (Cu(II)) to cupric (Cu(I)), ascorbate to dehydroascorbate, etc, etc. In the course of these reactions also reduced forms of molecular oxygen may be formed, e.g. superoxide anion and hydrogen peroxide.
Examples
Example 1: Acridinium ester detection under reducing conditions compared with
H202-triggering
Calibration standards of acridinium ester (formula E) (0-1000 pg/well) were treated with ferrous sulphate (10-lOOuM), a combination of ferric chloride (10-lOOuM) and ascorbate (10- lOOuM), or hydrogen peroxide (100-lOOOuM). Chemiluminescence reaction was triggered by the addition of sodium carbonate buffer (pH 9) and the resulting signal accumulated during the first 10 seconds. Calibration curves were constructed using the 4-5 seconds integral values of the kinetic profiles and were expressed as signal-background ratios against amounts of
acridinium ester. The results are shown in the figure:
INSERT FIGURE 9
140 As can be seen from the figure the acridinium ester can be quantified under reducing
conditions with better S/B ratios than by using hydrogen peroxide.
Example 2: Acridinium ester detection treated with phenanthrenequinone
Calibration standards of acridinium ester (formula E) (0-1000 pg/well) were treated with 145 phenanthrenequinone (PQ) 3, 10, 30, 100 uM). Chemilummescence reaction was triggered by the addition of sodium carbonate buffer (pH 9) containing TCEP (lOOuM) and the resulting signal accumulated during the first 10 seconds. Calibration curves were constructed using the 4-5 seconds integral values of the kinetic profiles and were expressed as signal-background ratios against amounts of acridinium ester. The results for 30 uM PQ are shown in the figure.
150
INSERT FIGURE 10
As can be seen from the figure Acridinium ester can be detected and quantified over a large dynamic range with good linearity and detection limit.
155
Example 3: Comparison of CL-kinetics using hydrogen peroxide trigger or PQ/TCEP trigger
A series of hydrogen peroxide standards (0-88uM) was prepared, 50uL of which was added to the wells of a microtiter plate. In the same microtiter plate dilutions of 9,10-
160 phenanthrenequinone (PQ, 0-1 OuM) were prepared. To the wells containing hydrogen
peroxide water was added, while the wells containing PQ were treated with 50 uL of TCEP (lOOuM). Acridinium ester (formula E) (100 ng/niL, 10 uL/well) was added.
Chemilummescence reaction was triggered by the addition of sodium carbonate buffer (pH 9) and the resulting signal accumulated during the first 10 seconds. The results are shown in the
165 figure:
INSERT FIGURE 11
As can be seen from the figure triggering by hydrogen peroxide gives rise to slower
170 chemilummescence kinetics, while triggering with PQ/TCEP results in faster kinetics.
From the data a calibration curve for PQ could be constructed. This is depicted in Figure 12. PQ can be detected down to 10 nM
INSERT FIGURE 12
175
Example 4: Soot-extract detection of oxidative power
Soot extract dilutions were prepared from a stock containing lOOug/mL in water. These dilutions (0.01-lOug/mL, 50uL/well) were treated with TCEP (lOOuM, 50uL/well) during 5- 20 minutes. After that time acridinium ester (formula E) (lOOng/mL, lOuL/well) was added. 180 Chemilummescence reaction was triggered by the addition of sodium carbonate buffer (pH 9) and the resulting signal accumulated during the first 10 seconds. Calibration curves were constructed using the 0-2 seconds integral values of the kinetic profiles and were expressed as signal-background ratios against amounts of soot extract. The results are shown in the figure:
185 INSERT FIGURE 13
Example 5: Reductive power detection of urine sample dilution
Urine sample was diluted 100-10000 fold. To 50uL of a diluted sample either 50uL of a FeC13 solution (lOOuM) or of a PQ solution (lOOuM) was added, followed by lOuL of acridinium 190 ester (formula E) (lOOng/mL). Chemilummescence reaction was triggered by the addition of sodium carbonate buffer (pH 9) and the resulting signal accumulated during the first 10 seconds. The results are shown in the figure 14.
INSERT FIGURE 14
195
From the figure it is clear that reductive power of urine samples can be detected and quantified using dilute samples.
200
Example 6: Reductive power detection of urine sample dilutions
Urine sample was diluted 100-10000 fold. To 50uL of a diluted sample either 50uL of a CuS04 solution (lOOuM) was added, and incubated at 60oC during 15 minutes. Subsequently, 205 lOuL of acridinium ester (formula E) (lOOng/mL) was added. Chemiluminescence reaction was triggered by the addition of sodium carbonate buffer (pH 9) and the resulting signal accumulated during the first 10 seconds. The results are shown in the figure 9.
INSERT FIGURE 15
210
From the figure it is clear that reductive power with respect to CuS04 of urine samples can be detected and quantified using dilute urine samples.
Claims
Claims
Claim 1
A method of emitting chemiluminescence from an acridinium compound in the presence of reductive conditions, wherein acridinium compound has the formula A
INSERT FIGURE 1 wherein Rl is alkyl, substituted alkyl, aryl, substituted aryl, R2 and R3 are independently chosen from hydrogen, alkyl, substituted alkyl, halogen, cyanide, hydroxyl, alkoxyl, thiol, alkyl mercapto, and wherein Y is a leaving group with a pKa<12, consisting of aryloxy, substituted aryloxy, substituted alkoxy, mercapto, substituted mercapto, sulfonamidyl, substituted sulfonamidyl, N-hydroxylamide, substituted N-hydroxylamide, comprising said acridinium compound reacting under reductive conditions at pH 6-10, wherein reductive conditions are those conditions which allow a compound (a so-called reductant, reducer, or reducing agent) to transfer one or more electrons to an acceptor (a so-called oxidant, oxidizer, or oxidizing agent) such as metals (potassium, calcium, barium, zinc, sodium and
magnesium), nascent hydrogen (i.e. formed by reaction of zinc metal and acid), and metal ions like ferrous (Fe(II)), cuprous (Cu(I)), stannous (Sn(II), and the like, thiols e.g.
glutathione, cysteine, dithiothreitol (DTT), TCEP (tris(2-carboxyethyl)phosphine), and the like, vitamins e.g. aseorbate (vitamin C), and vitamin E, urate, trolox, radical inhibitors like BHT (butylated hydroxytoluene) or combinations of oxidants e.g. ferric, euprie, quinone, etc. in combination with reductants which are capable of reducing the oxidants e.g. ferrous (Fe(III)) to ferric (Fe(II)), cuprous (Cu(II)) to euprie (Cu(I)), aseorbate to dehydroaseorbate, using said chemiluminescence reaction to detect and quantify analytes or groups of analytes in a medium containing said reductive conditions.
Claim 2
A method according to Claim 1 wherein acridinium compound is an acridinium ester, wherein said
INSERT FIGURE 2 acridinium ester has the formula B wherein Rl, R2, and R3 are substituents according to the aforementioned claim 1 , and wherein R4 is chosen from hydrogen, alkyl, aryl, substituted alkyl, or substituted aryl.
Claim 3
A method according to claim 2 wherein acridinium ester has the formula C
INSERT FIGURE 3
Claim 4
A method according to claim 2 wherein acridinium ester has the formula D
INSERT FIGURE 4
Claim 5
A method according to claim 2 wherein acridinium ester has the formula E
INSERT FIGURE 5
Claim 6
A method according to Claim 1 wherein acridinium compound is an acridinium sulfonamide wherein said acridinium sulfonamide has the formula F INSERT FIGURE 6 wherein Rl, R2, and Rl are substituents according to the aforementioned claim 1 and R3 and R4 are independently chosen from alkyl, substituted alkyl, aryl, substituted aryl.
Claim 7
A method according to Claim 1 wherein reductive conditions comprise mixtures of ascorbate and metal ions
Claim 8
A method according to Claim 7 wherein metal ions are ferric (Fe(III)) ions Claim 9
A method according to Claim 7 wherein metal ions are cupric (Cu(II)) ions Claim 10
A method according to Claim 1 wherein reductive conditions comprise mixtures of dithiothreitol (DTT) and quinones
Claim 11
A method according to Claim 10 wherein quinones are phenanthrenequinones Claim 12
A method according to Claim 10 wherein quinones are naphtalenequinones Claim 13
A method according to Claim 10 wherein quinones are benzoquinones Claim 14
A method according to Claim 1 wherein reductive conditions comprise mixtures of Tris(2- carboxyethylphosphine (DECP) and quinones
Claim 15
A method according to Claim 14 wherein quinones are phenanthrenequinones
Claim 16
A method according to Claim 14 wherein quinones are naphtalenequinones
Claim 17
A method according to Claim 14 wherein quinones are benzoquinones
105
Claim 18
A method according to Claim 1 wherein reductive conditions comprise mixtures of dithiothreitol (DTT) and mixtures with oxidative properties
110 Claim 19
A method according to Claim 18 wherein mixtures with oxidative properties comprise particulate matter
Claim 20
115 A method according to Claim 1 wherein a sample is analyzed for the presence and amount of oxidative components by reacting said sample with a reductant during a certain time, followed by addition of an acridinium compound according to the invention, and subsequently triggering the chemiluminescent reaction by raising the pH, and recording the
chemiluminescence and by relating the amount of light generated in the test sample by
120 comparison to a standard curve for said oxidative components
Claim 21
A method according to Claim 20 wherein the reductant is ascorbate 125 Claim 22
A method according to Claim 20 wherein the reductant is dithiothreitol (DTT) Claim 23
A method according to Claim 20 wherein the reductant is tris(2-carboxyethylphosphine 130 (DECP)
Claim 24
A method according to Claim 1 wherein a sample is analyzed for the presence and amount of reductive components by reacting said sample with an oxidant during a certain time, followed 135 by addition of an acridinium compound according to the invention, and subsequently
triggering the chemiluminescent reaction by raising the pH, and recording the
chemiluminescence and by relating the amount of light generated in the test sample by comparison to a standard curve for said reductive components
140 Claim 25
A method according to Claim 24 wherein oxidant is cupric (Cu(II)) ion Claim 26
A method according to Claim 24 wherein oxidant is ferric (Fe(III) ion
145
Claim 27
A method according to Claim 24 wherein oxidant is 9,10-phenanthrenequinone (PQ) Claim 28
150 A method according to Claim 1 wherein a sample is analyzed for the presence and amount of acridinium compound labeled molecule of interest by reacting said sample under reductive conditions according to Claims 8-17 during a certain time, followed by triggering the chemiluminescent reaction by raising the pH, and recording the chemiluminescence and by relating the amount of light generated in the test sample by comparison to a standard curve for
155 said acridinium compound labeled molecule of interest.
Claim 29
A method according to Claim 28 wherein labeled molecule of interest is antibody, antigen, DNA, or R A.
160
Claim 30
Kits containing the necessary components to perform the method as laid out by claims 1-29.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/EP2010/057017 WO2012028167A2 (en) | 2010-05-20 | 2010-05-20 | Acridinium ester chemiluminescence upon reductive triggering |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3441765A1 (en) | 2017-08-08 | 2019-02-13 | Euroimmun Medizinische Labordiagnostika AG | Method for detecting basophil activation |
| WO2021023836A1 (en) | 2019-08-06 | 2021-02-11 | Euroimmun Medizinische Labordiagnostika Ag | An improved autoantibody detection assay |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP3441765A1 (en) | 2017-08-08 | 2019-02-13 | Euroimmun Medizinische Labordiagnostika AG | Method for detecting basophil activation |
| DE102018006212A1 (en) | 2017-08-08 | 2019-02-14 | Euroimmun Medizinische Labordiagnostika Ag | Method for detecting basophil activation |
| US11119109B2 (en) | 2017-08-08 | 2021-09-14 | Euroimmun Medizinische Labordiagnostika Ag | Method for detecting basophil activation |
| WO2021023836A1 (en) | 2019-08-06 | 2021-02-11 | Euroimmun Medizinische Labordiagnostika Ag | An improved autoantibody detection assay |
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| WO2012028167A3 (en) | 2012-05-18 |
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