US20170320830A1 - Hydrophilic high quantum yield acridinium esters with improved stability and fast light emission - Google Patents

Hydrophilic high quantum yield acridinium esters with improved stability and fast light emission Download PDF

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US20170320830A1
US20170320830A1 US14/901,009 US201414901009A US2017320830A1 US 20170320830 A1 US20170320830 A1 US 20170320830A1 US 201414901009 A US201414901009 A US 201414901009A US 2017320830 A1 US2017320830 A1 US 2017320830A1
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acridinium
acridinium ester
ester according
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Anand Natrajan
David Sharpe
Qingping Jiang
David Wen
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Siemens Healthcare Diagnostics Inc
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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
    • 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
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • C09K11/07—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials having chemically interreactive components, e.g. reactive chemiluminescent compositions
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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

Definitions

  • the present invention relates to hydrophilic, high quantum yield, chemiluminescent acridinium compounds with increased light output, improved stability, fast light emission and low non-specific binding. These compounds because of their enhanced quantum yield and hydrophilic nature, are useful in improving assay sensitivity.
  • the improved stability of these compounds is useful for extending the shelf life of reagents using these compounds as well as for minimizing variation in assay performance with time. Their increased emission kinetics also permits faster light measurements in assays especially in automated analyzers.
  • AEs acridinium esters
  • NSP-DMAE-NHS ester where the N-methyl group has been replaced with an N-sulfopropyl (NSP) group.
  • NSP N-sulfopropyl
  • NSP-DMAE-HEG-Glutarate-NHS (abbreviated as HEG-AE)
  • HEG-AE NSP-DMAE-HEG-Glutarate-NHS
  • a diamino hexa(ethylene) glycol (diamino-HEG) moiety is attached to the phenol to increase the aqueous solubility of the acridinium ester.
  • a glutarate moiety was appended to the end of HEG and was converted to the NHS ester to enable labeling of various molecules.
  • the phenol is the ‘leaving group’ whereas in acridinium sulfonamides, the sulfonamide is the ‘leaving group’ during the chemiluminescent reaction with alkaline peroxide.
  • the overall light output which can also be referred to as the chemiluminescence quantum yield, is a combination of the efficiencies of the chemical reaction leading to the formation of the excited-state acridone and the latter's fluorescence quantum yield.
  • hydrophilic, high quantum yield, chemiluminescent acridinium esters possessing electron-donating functional groups of the form —OG at C2 and/or C7 of the acridinium ring, where G represents a branched hydrophilic substituent provide increased light output, improved stability, fast light emission and/or low non-specific binding in assays.
  • hydrophilic, high quantum yield acridinium esters having the structure of formula (I):
  • R 1 is a methyl or sulfopropyl group
  • G is a branched group independently selected at each occurrence from:
  • R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are independently at each occurrence a methyl group or a group —(CH 2 CH 2 O) n CH 3 , where n is an integer from 1 to 5; and R 12 is an electrophilic or nucleophilic group for conjugating the acridinium compound to an analyte, an analyte analog, or a binding molecule for an analyte.
  • G will be, at one or both occurrences, a group:
  • R 2 and R 3 are independently at each occurrence a methyl group or a group —(CH 2 CH 2 O) n CH 3 , where n is an integer from 1 to 5; and in particular, G may be a group:
  • G may be a group:
  • G is a group:
  • G represents, at one or both occurrences, a group:
  • R 4 , R 5 , R 6 and R 7 are independently at each occurrence a methyl group or a group —(CH 2 CH 2 O) n CH 3 , where n is an integer from 1 to 5.
  • R 4 -R 7 may represent methyl groups, such that G is a group:
  • G is a group:
  • R 12 may be selected, for example, from the group consisting of:
  • R 12 will be —OH, or R 12 will be a group:
  • R 12 will be a group:
  • One acridinium ester according to formula (I) has the following structure:
  • R 12 is an electrophilic or nucleophilic group for conjugating the acridinium compound to an analyte, an analyte analog, or a binding molecule for an analyte.
  • Another acridinium ester according to formula (I) has the following structure:
  • R 12 is an electrophilic or nucleophilic group for conjugating the acridinium compound to an analyte, an analyte analog, or a binding molecule for an analyte.
  • R 12 is an electrophilic or nucleophilic group for conjugating the acridinium compound to an analyte, an analyte analog, or a binding molecule for an analyte.
  • Another acridinium ester according to formula (I) has the following structure:
  • R 12 is an electrophilic or nucleophilic group for conjugating the acridinium compound to an analyte, an analyte analog, or a binding molecule for an analyte.
  • Still another acridinium ester according to formula (I) has the following structure:
  • R 12 is an electrophilic or nucleophilic group for conjugating the acridinium compound to an analyte, an analyte analog, or a binding molecule for an analyte.
  • R 12 represents —OH.
  • an assay for the detection or quantification of an analyte comprising the steps of: (a) providing a conjugate comprising: (i) a binding molecule specific for an analyte; and (ii) a hydrophilic, high quantum yield and fast light emitting acridinium ester according to formula (I); (b) providing a solid support having immobilized thereon a second binding molecule specific for the analyte; (c) mixing the conjugate, the solid phase and a sample suspected of containing the analyte to form a binding complex; (d) separating the binding complex captured on the solid support; (e) triggering chemiluminescence of the binding complex from step (d) by adding chemiluminescence triggering reagents; (f) measuring the amount of light emission with a luminometer; and (g) detecting the presence or calculating the concentration of the analyte by comparing the amount of light emitted from the reaction mixture with a standard
  • an assay for the detection or quantification of an analyte comprising the steps of: (a) providing a conjugate of an analyte with a hydrophilic, high quantum yield and fast light emitting acridinium ester according to formula (I); (b) providing a solid support immobilized with a binding molecule specific for the analyte; (c) mixing the conjugate, solid support and a sample suspected of containing the analyte to form a binding complex; (d) separating the binding complex captured on the solid support; (e) triggering the chemiluminescence of the binding complex from step (d) by adding chemiluminescence triggering reagents; (f) measuring the amount of light with an luminometer; and (g) detecting the presence or calculating the concentration of the analyte by comparing the amount of light emitted from the reaction mixture with a standard dose response curve which relates the amount of light emitted to a known concentration of the analy
  • FIG. 1 illustrates structures of B-AEs with electrophilic N-hydroxysuccinimidyl (NHS) functional groups suitable for preparing conjugates of proteins or other molecules containing nucleophilic functional groups.
  • NHS N-hydroxysuccinimidyl
  • FIG. 2 illustrates B-AE structures with nucleophilic, hexaethylene glycol amine (HEG-NH 2 ) functional groups useful for conjugating the acridinium compound to molecules containing electrophilic functional groups.
  • HEG-NH 2 hexaethylene glycol amine
  • FIG. 3 shows the structures of estradiol conjugates (abbreviated as B-AE-E2) prepared using the B-AEs of FIG. 2 .
  • the introduction of electron-donating functional groups such as OR* at C2 and/or C7 of the acridinium ring increases the quantum yield of the corresponding chemiluminescent acridinium compound.
  • R* group is hydrophilic, such as a sulfopropyl group or methoxy poly(ethylene) glycol
  • the corresponding acridinium compound not only exhibits increased light output but also shows reduced non-specific binding in immunoassays. These two properties in conjunction lead to an increase in the sensitivity of immunoassays.
  • the main objectives of the current invention were to identify structural features of acridinium compounds that result in (a) faster light emission when compared to NSP-DMAE and derivatives as well as HQYAE; (b) improved stability especially when compared to HQYAE; (c) high light output that is comparable to HQYAE and (d) low non-specific binding that is comparable to HQYAE.
  • the hydrophilic acridinium compounds according to the present invention not only show increased light output but also show improved stability and faster light emission.
  • stability we refer to the chemiluminescent activity of the acridinium compounds. An increase in stability is thus manifested as increased retention of chemiluminescent activity as a function of time. Increased stability of acridinium compounds is useful because reagents derived from such compounds are less likely to show a deterioration of assay performance as a function of time and moreover, the shelf life of regents derived from such compounds is likely to be extended thereby leading to less waste.
  • assay reagents derived from acridinium compounds include conjugates of proteins or small molecules.
  • the second property of the acridinium compounds is faster light emission by which is meant that these compounds emit their total light in a significantly shorter period of time compared to acridinium compounds lacking the unique structural features of the acridinium compounds of the current invention.
  • Faster light emission enables faster measurements in assays and has the potential to increase the throughput of automated analyzers.
  • the throughput of automated analyzers is normally defined as the number of tests the analyzer can perform in a given period of time.
  • the third and fourth properties of the acridinium compounds of the current invention are their increased light output and low non-specific binding, both extremely useful for improving assay sensitivity.
  • branched functional groups derived from glycerol, of the type —OG, where G is a branched functional group, at C2 and/or C7 of the acridinium ring significantly increases the stability of the corresponding acridinium compound and leads to faster light emission.
  • the presence of these branched functional groups increases the quantum yield and lowers the non-specific binding of the corresponding acridinium compounds and their conjugates.
  • Non-specific binding in assays using solid phases such as particles or microtiter plates are undesired binding interactions of conjugates to these solid phases. These undesired binding interactions typically increase the background of the assay leading to a net lowering of the signal to background ratio in the assay and thereby decreasing assay sensitivity.
  • the acridinium compounds of the current invention can be represented by the general formula (I):
  • R 1 is a methyl or sulfopropyl (—CH 2 CH 2 CH 2 SO 3 ⁇ ) group; G is defined as
  • acridinium compounds of the present invention can be represented by the following formula:
  • acridinium compounds of the current invention can also be represented by the following formula:
  • FIGS. 1 and 2 illustrate the structures of B-AEs with electrophilic N-hydroxysuccinimidyl (NHS) functional groups whereas FIG. 2 illustrates B-AE structures with nucleophilic, hexaethylene glycol amine (HEG-NH 2 ) functional groups.
  • the former compounds are suitable for preparing conjugates of proteins or other molecules containing nucleophilic functional groups.
  • the latter compounds are also useful for conjugating the acridinium compound to molecules containing electrophilic functional groups.
  • FIG. 3 shows the structures of estradiol conjugates (abbreviated as B-AE-E2) prepared using the B-AEs of FIG. 2 .
  • Estradiol is a steroidal hormone that is commonly measured by immunoassay.
  • TSH thyroid stimulating hormone
  • the acridinium esters of the present invention also show good stability.
  • stability is meant a minimal loss of chemiluminescent activity as measured by the loss of RLUs when the compounds or conjugates are stored in an aqueous solution typically, in the pH range of 7-8, which is within the physiological pH.
  • hydrolysis of the phenolic ester is the main pathway by which chemiluminescent acridinium esters become non-chemiluminescent.
  • Stable conjugates ensure long shelf life for acridinium ester reagents and also ensure that assay performance does not vary greatly over a given period of time.
  • the B-AE conjugates retain a greater proportion of their chemiluminescent activity and are more stable compared to the HQYAE conjugate.
  • the anti-TSH antibody conjugate of HQYAE retains 68% of its chemiluminescent activity after 33 days at 37° C.
  • the B-AE conjugates retain ⁇ 79% of their chemiluminescent activity in the same period of time.
  • estradiol (E2) conjugates where the B-AE conjugates retain ⁇ 75% of their chemiluminescent activity after 33 days at 37° C., whereas the HQYAE conjugate's chemiluminescent activity has dropped to 60% in the same time period.
  • the B-AEs of the present invention also show increased light output that is comparable to or better than HQYAE.
  • Table 5 summarizes the relative quantum yields of the various B-AEs when conjugated to the anti-TSH monoclonal antibody. In this table, the quantum yield of HEG-AE was assigned a value of unity (1) and the quantum yields of all the other conjugates are relative to this conjugate of this compound.
  • the B-AEs of the current invention also show low non-specific binding to solid phases (Table 6).
  • Non-specific binding as described earlier, in assays using solid phases such as particles or microtiter plates are undesired binding interactions of conjugates to these solid phases. These undesired binding interactions typically increase the background of the assay leading to a net lowering of the signal to background ratio in the assay and thereby decreasing assay sensitivity.
  • non-specific binding was measured on two different kinds of particles; paramagnetic particles (PMP) and magnetic latex particles (MLP). These two particles differ in their intrinsic composition.
  • PMPs are made mainly of iron oxide particles with a silane coating containing amines.
  • the amines are used to cross-link proteins to the particle surface using reagents such as glutaraldehyde.
  • MLPs on the other hand are made of polystyrene.
  • the MLPs used in Table 6 contained a thin layer of magnetite to enable magnetic separation and a polyacrylic acid coating for conjugating proteins.
  • the two types of particles were mixed with solutions of the conjugates for a specific period of time and then the particles were magnetically separated, washed once and then the chemiluminescence associated with the particles was measured. (Experimental details can be found in Example 11.)
  • the ratio of this chemiluminescence value in comparison to the total chemiluminescence input is referred to fraction non-specific binding (fNSB).
  • hydrolytically stable, fast light emitting, hydrophilic, high quantum yield acridinium compounds of the invention are useful as labels in assays for the determination or quantitation of analytes.
  • Analytes that are typically measured in such assays are often substances of some clinical relevance and can span a wide range of molecules from large macromolecules such as proteins, nucleic acids, viruses bacteria, etc. to small molecules such as ethanol, vitamins, steroids, hormones, therapeutic drugs, etc.
  • a ‘sandwich’ immunoassay typically involves the detection of a large molecule, also referred to as macromolecular analyte, using two binding molecules such as antibodies.
  • One antibody is immobilized or attached to a solid phase such as a particle, bead, membrane, microtiter plate or any other solid surface.
  • a solid phase such as a particle, bead, membrane, microtiter plate or any other solid surface.
  • Methods for the attachment of binding molecules such as antibodies to solid phases are well known in the art.
  • an antibody can be covalently attached to a particle containing amines on its surface by using a cross-linking molecule such as glutaraldehyde.
  • the attachment may also be non-covalent and may involve simple adsorption of the binding molecule to the surface of the solid phase, such as polystyrene beads and microtiter plate.
  • the second antibody is often covalently attached with a chemiluminescent or fluorescent molecule often referred to as a label.
  • binding molecules such as antibodies and other binding proteins are also well known in the art and are commonly called conjugation reactions and the labeled antibody is often called a conjugate.
  • an amine-reactive moiety on the label reacts with an amine on the antibody to form an amide linkage.
  • Other linkages such as thioether, ester, carbamate, and the like, between the antibody and the label are also well known.
  • the two antibodies bind to different regions of the macromolecular analyte.
  • the macromolecular analyte can be, for example, proteins, nucleic acids, oligosaccharides, antibodies, antibody fragments, cells, viruses, receptors, or synthetic polymers.
  • the binding molecules can be antibodies, antibody fragments, nucleic acids, peptides, binding proteins or synthetic binding polymers.
  • FBP folate binding protein
  • the binding molecules can be antibodies, antibody fragments, nucleic acids, peptides, binding proteins or synthetic binding polymers.
  • FBP folate binding protein
  • Synthetic binding molecules that can bind a variety of analytes have also been disclosed by Mossbach et al. Biotechnology vol. 14, pp. 163-170 (1995).
  • a binding complex is formed between the analyte and the two antibodies.
  • This type of assay is often called a heterogenous assay because of the involvement of a solid phase.
  • the chemiluminescent or fluorescent signal associated with the binding complex can then be measured and the presence or absence of the analyte can be inferred.
  • the binding complex is separated from the rest of the binding reaction components such as excess, labeled antibody prior to signal generation. For example if the binding complex is associated with a magnetic bead, a magnet can be used to separate the binding complex associated with the bead from bulk solution.
  • a ‘dose-response’ curve can be generated using the two antibodies.
  • the dose-response curve correlates a certain amount of measured signal with a specific concentration of analyte.
  • concentration of the analyte in an unknown sample can then be calculated by comparing the signal generated by an unknown sample containing the macromolecular analyte, with the dose-response curve.
  • the two binding components can also be nucleic acids that bind or hybridize to different regions of a nucleic acid analyte.
  • concentration of the nucleic acid analyte can then be deduced in a similar manner.
  • Another class of immunoassays for small molecule analytes such as steroids, vitamins, hormones, therapeutic drugs or small peptides employs an assay format that is commonly referred to as a competitive assay.
  • a conjugate is made of the analyte of interest and a chemiluminescent or fluorescent label by covalently linking the two molecules.
  • the small molecule analyte can be used as such or its structure can be altered prior to conjugation to the label.
  • the analyte with the altered structure is called an analog. It is often necessary to use a structural analog of the analyte to permit the chemistry for linking the label with the analyte.
  • analyte is used to attenuate or enhance its binding to a binding molecule such an antibody.
  • a binding molecule such an antibody.
  • the antibody or a binding protein to the analyte of interest is often immobilized on a solid phase either directly or through a secondary binding interaction such as the biotin-avidin system.
  • the concentration of the analyte in a sample can be deduced in a competitive assay by allowing the analyte-containing sample and the analyte-label conjugate to compete for a limited amount of solid phase-immobilized binding molecule. As the concentration of analyte in a sample increases, the amount of analyte-label conjugate captured by the binding molecule on the solid phase decreases.
  • a dose-response curve can be constructed where the signal from the analyte-label conjugate captured by the binding molecule on the solid phase is inversely correlated with the concentration of analyte.
  • Another format of the competitive assay for small molecules analytes involves the use of a solid phase that is immobilized with the analyte of interest or an analyte analog and an antibody or a binding protein specific for the analyte that is conjugated with a chemiluminescent or fluorescent label.
  • the antibody-label conjugate is captured onto the solid phase through the binding interaction with the analyte or the analyte analog on the solid phase.
  • the analyte of interest present in a sample then “competitively” binds to the antibody-label conjugate and thus inhibits or replaces the interaction of the antibody-label conjugate with the solid phase. In this fashion, the amount of signal generated from the antibody-label conjugate captured on the solid phase is correlated to the amount of the analyte in sample.
  • an assay for the detection or quantification of an analyte comprises, according to one embodiment of the invention, the following steps:
  • step (e) triggering chemiluminescence of the binding complex from step (d) by adding chemiluminescence triggering reagents;
  • an assay for the detection or quantification of an analyte comprising the steps of:
  • step (e) triggering the chemiluminescence of the binding complex from step (d) by adding chemiluminescence triggering reagents;
  • Macromolecular analytes can be proteins, nucleic acids, oligosaccharides, antibodies, antibody fragments, cells, viruses, synthetic polymers, and the like.
  • Small molecule analytes can be steroids, vitamins, hormones, therapeutic drugs, small peptides, and the like.
  • the binding molecules in the assays can be an antibody, an antibody fragment, a binding protein, a nucleic acid, a peptide, a receptor or a synthetic binding molecule.
  • the compound 1,3-Bis(3,6-dioxaheptanyl)glycerol, 2a was synthesized as described by Vacus and Simon in Adv. Mater. 1995, 7, 797-800.
  • Crude 2a (16 g, 0.054 mol) was dissolved in anhydrous pyridine (50 mL) and treated with 4-dimethylaminopyridine (1.32 g, 0.011 mol) followed by p-toluenesulfonyl chloride (12.4 g, 0.065 mol).
  • the crude acridinium ester 2d was suspended in I N HCl (10 mL) and refluxed under a nitrogen atmosphere for 2 hours.
  • HPLC analysis indicated complete conversion to product 2e eluting at 16 minutes.
  • the product was purified by preparative HPLC using an YMC, C 18 30 ⁇ 300 mm column and the same gradient described above at a solvent flow rate of 20 mL/minute and UV detection at 260 nm.
  • 1,3-Bis(3,6,9-dioxadecanyl)glycerol-2-toluenesulfonate, 3b The compound 1,3-Bis(3,6,9-dioxadecanyl)glycerol, 3a, was synthesized as described by Lauter et al. in Macromol. Chem. Phys. 1998, 199, 2129-2140. The alcohol (7 g, 0.0182 mol) was dissolved in anhydrous pyridine (30 mL) and treated with 4-dimethylaminopyridine (0.444 g, 3.6 mmol) and p-toluenesulfonyl chloride (3.85 g, 0.02 mol).
  • the crude acridinium ester 3d was suspended in I N HCl (10 mL) and refluxed under a nitrogen atmosphere for 2 hours.
  • HPLC analysis indicated complete conversion to product 3e eluting at 16.3 minutes.
  • the product was purified by preparative HPLC using an YMC, C 18 30 ⁇ 300 mm column and the same gradient at described above at a solvent flow rate of 20 mL/minute and UV detection at 260 nm.
  • the crude acridinium ester 4d was suspended in I N HCl (10 mL) and refluxed under a nitrogen atmosphere for 2 hours.
  • HPLC analysis indicated complete conversion to product 4e eluting at 17 minutes.
  • the product was purified by preparative HPLC using an YMC, C 18 30 ⁇ 300 mm column and the same gradient at described above at a solvent flow rate of 20 mL/minute and UV detection at 260 nm.
  • the reaction was then cooled to room temperature and partitioned between ethyl acetate (75 mL) and saturated ammonium chloride solution (75 mL). The ethyl acetate layer was separated and the aqueous layer was extracted once more with ethyl acetate (50 mL). The combined ethyl acetate extracts were dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The recovered light brown oil (3.7 g) was used a such in the next reaction.
  • the crude acridinium ester 5e was suspended in IN HCl (10 mL) and refluxed under a nitrogen atmosphere for 2 hours.
  • HPLC analysis indicated complete conversion to product 5f eluting at 17.5 minutes.
  • the product was purified by preparative HPLC using an YMC, C 18 30 ⁇ 300 mm column and the same gradient at described above at a solvent flow rate of 20 mL/minute and UV detection at 260 nm.
  • the labeling reactions were stirred gently at room temperature for 3-4 hours and were then diluted with de-ionized water (1.8 mL). These diluted solutions were then transferred to 2 mL Centricon filters (MW 30,000 cutoff) and centrifuged at 4500 G to reduce the volume to ⁇ 0.2 mL. This process was repeated three more times. The filtered conjugates were finally diluted into a total volume of 200 uL de-ionized water for mass spectral analysis and RLU measurements.
  • Mass spectra were recorded on a Voyager DE MALDI-TOF mass spectrometer and the unlabeled antibody was used as the reference. Approximately 2 uL of the conjugate solution was mixed with 2 uL of sinnapinic acid matrix solution (HP) and the spotted on a MALDI plate. After complete drying, mass spectra were recorded. From the difference in mass values for the unlabeled antibody and the conjugates, the extent of AE incorporation could be measured. Typically, under these labeling conditions, 3-6 AE labels were incorporated in the antibody.
  • acridinium ester-labeled antiTSH (thyroid stimulating hormone) antibody each conjugated to a different acridinium ester were diluted to a concentration of 0.2 nanomolar in Siemens Healthcare Diagnostics TSH3 (thyroid stimulating hormone) Lite Reagent buffer consisting of 0.1 M sodium N-(2-hydroxyethyl) piperazine-N′-2-ethanesulfonate (HEPES), 0.15 M sodium chloride, 7.7 mM sodium azide, 1.0 mM tetrasodium ethylenediaminetetraacetate, (EDTA), 12 mM t-octylphenoxypolyethoxyethanol (Triton X-100), 76 uM bovine serum albumin (BSA), 7 uM mouse immunoglobin (IgG), pH 7.7.
  • TSH3 thyroid stimulating hormone Lite Reagent buffer consisting of 0.1 M sodium N-(2-hydroxyethyl) piperazine-N′-2-ethanesulf
  • Each acridinium ester solution was partitioned into two sets of storage vessels. One set of storage vessels was kept at 4° C. and the other at 37° C. Starting from the day of initial dilution the chemiluminescence from 10 microliters of each acridinium ester-antibody solution was determined under standard conditions on a Berthold Technolgies Autolumat LB953 luminometer with sequeuntial addition of 300 microliters each of Siemens Healthcare Diagnostics Flash Reagent 1 (0.1 M nitric acid and 0.5% hydrogenperoxide) and Siemens Healthcare Diagnostics Flash Reagent 2 (0.25 M sodium hydroxide and 0.05% cetyltrimethylammonium chloride).
  • fractional nonspecific binding of acridinium esters to a solid phase is one parameter by which assay sensitivity is enhanced.
  • the fractional nonspecific bindings of several acridinium esters covalently attached to antiTSH antibody were analyzed for correlation to the molecular structure of the acridinium ester.
  • acridinium ester-labeled antiTSH (thyroid stimulating hormone) antibody each conjugated to a different acridinium ester were diluted to a concentration of 2 nanomolar in Siemens Healthcare Diagnostics TSH3 (thyroid stimulating hormone) Lite Reagent buffer consisting of 0.1 M sodium N-(2-hydroxyethyl) piperazine-N′-2-ethanesulfonate (HEPES), 0.15 M sodium chloride, 7.7 mM sodium azide, 1.0 mM tetrasodium ethylenediaminetetraacetate, (EDTA), 12 mM t-octylphenoxypolyethoxyethanol (Triton X-100), 76 uM bovine serum albumin (BSA), 7 uM mouse immunoglobin (IgG), pH 7.7.
  • TSH3 thyroid stimulating hormone Lite Reagent buffer consisting of 0.1 M sodium N-(2-hydroxyethyl) piperazine-N′-2-ethanesulfon
  • the first solid phase was 200 microliters of Siemens Healthcare Diagnostics ACS PTH (parathyroid hormone) Solid Phase containing 50 micrograms of magnetic latex microparticles (MLP) derivatized with antiPTH antibody.
  • the second solid phase was 200 microliters of Siemens Healthcare Diagnostics ACS TSH3 (thyroid stimulating hormone) Solid Phase containing 60 micrograms of paramagnetic microparticles (PMP) derivatized with antiTSH antibody.
  • the particles were magnetically collected and washed twice with water after an incubation of 10 minutes to allow interaction between the acridinium ester labeled antbodies and the solid phases.
  • the chemiluminescence of acridinium ester associated with the particles was meaured under standard conditions on a Berthold Technolgies Autolumat LB953 luminometer with sequeuntial addition of 300 microliters each of Siemens Healthcare Diagnostics Flash Reagent 1 (0.1 M nitric acid and 0.5% hydrogenperoxide) and Siemens Healthcare Diagnostics Flash Reagent 2 (0.25 M sodium hydroxide and 0.05% cetyltrimethylammonium chloride). Chemiluminescence was measured for 5.0 seconds.
  • Fractional nonspecific binding is calculated as the ratio of particle-bound chemiluminescence to total chemiluminescence input.
  • hydrophobicity of an acridinium ester elevates fNSB and is undesirable when distinguishing small amounts of specific signal
  • hydrophilicity of an acridinium ester lowers fNSB and is desirable when distinguishing small amounts of specific signal.
  • Hastening of acridinium ester chemiluminescence rates is one parameter by which assay throughput rates can be increased.
  • Chemiluminescence kinetics of several acridinium esters covalently attached to anti-TSH antibody were analyzed for the correlation of molecular structure of the acridinium ester to its rate of chemiluminescence light emission.
  • Each acridinium ester labeled antibody was diluted to a concentration of 0.2 nanomolar in a buffer consisting of 0.1 M sodium phosphate, 0.15 M sodium chloride, 6 mM sodium azide and 1 g/L bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the chemiluminescence kinetics for 10 microliters of each acridinium ester-antibody conjugate tested was integrated in 0.1 second intervals for 20 seconds under standard conditions on a Berthold Technolgies Autolumat LB953 luminometer with sequeuntial addition of 300 microliters each of Siemens Healthcare Diagnostics Flash Reagent 1 (0.1 M nitric acid and 0.5% hydrogenperoxide) and Siemens Healthcare Diagnostics Flash Reagent 2 (0.25 M sodium hydroxide and 0.05% cetyltrimethylammonium chloride).
  • Siemens Healthcare Diagnostics Flash Reagent 1 0.1 M nitric acid and 0.5% hydrogenperoxide
  • Siemens Healthcare Diagnostics Flash Reagent 2 (0.25 M sodium hydroxide and 0.05% cetyltrimethylammonium chloride.
  • the chemiluminescence kinetics of the tested acridinium esters were compared for relative rate of light emission.
  • acridinium ester chemiluminescence quantum yield is one parameter by which assay sensitivity can be increased.
  • Chemiluminescence quantum yields of several acridinium esters covalently attached to antiTSH antibody were tested for the correlation of molecular structure of the acridinium esters to the magnitude of their chemiluminescence light output.
  • Each acridinium ester labeled antibody was diluted to a concentration of 0.2 nanomolar in a buffer consisting of 0.1 M sodium phosphate, 0.15 M sodium chloride, 6 mM sodium azide and 1 g/L bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the chemiluminescence kinetics for 10 microliters of each acridinium ester-antibody conjugate tested was measured for 10 seconds under standard conditions on a Berthold Technolgies Autolumat LB953 luminometer with sequeuntial addition of 300 microliters each of Siemens Healthcare Diagnostics Flash Reagent 1 (0.1 M nitric acid and 0.5% hydrogenperoxide) and Siemens Healthcare Diagnostics Flash Reagent 2 (0.25 M sodium hydroxide and 0.05% cetyltrimethylammonium chloride).
  • the chemiluminescence quantum yield was calculated as the ratio of the chemiluminescence to the amount of acridinium ester tested.

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7309615B2 (en) * 2002-09-27 2007-12-18 Siemens Medical Solutions Diagnostic High quantum yield acridinium compounds and their uses in improving assay sensitivity
WO2009067417A1 (en) * 2007-11-20 2009-05-28 Siemens Heathcare Diagnostics Inc. Facile n-alkylation of acridine compounds in ionic liquids
WO2011060228A1 (en) * 2009-11-16 2011-05-19 Siemens Healthcare Diagnostics Inc. Zwitterion-containing acridinium compounds

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US4918192A (en) 1986-10-06 1990-04-17 Ciba Corning Diagnostics Corp. Polysubstituted aryl acridinium esters
US5110932A (en) 1986-10-06 1992-05-05 Ciba Corning Diagnostics Corp. Polysubstituted aryl acridinium esters
ES2063735T3 (es) 1986-10-22 1995-01-16 Abbott Lab Sales de acridinio quimioluminiscentes.
US5656426A (en) 1988-08-01 1997-08-12 Chiron Diagnostics Corporation Functionaized hydrophilic acridinium esters
US6664043B2 (en) 2001-07-03 2003-12-16 Bayer Corporation Acridinium ester labels having hydrophilic modifiers
EP2074101B1 (en) * 2006-10-13 2012-08-08 Siemens Healthcare Diagnostics Inc. Stable acridinium esters with fast light emission

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* Cited by examiner, † Cited by third party
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US7309615B2 (en) * 2002-09-27 2007-12-18 Siemens Medical Solutions Diagnostic High quantum yield acridinium compounds and their uses in improving assay sensitivity
US7785904B2 (en) * 2002-09-27 2010-08-31 Siemens Healthcare Diagnostics Inc. High quantum yield acridinium compounds and their uses in improving assay sensitivity
WO2009067417A1 (en) * 2007-11-20 2009-05-28 Siemens Heathcare Diagnostics Inc. Facile n-alkylation of acridine compounds in ionic liquids
WO2011060228A1 (en) * 2009-11-16 2011-05-19 Siemens Healthcare Diagnostics Inc. Zwitterion-containing acridinium compounds
US8778624B2 (en) * 2009-11-16 2014-07-15 Siemens Healthcare Diagnostics Inc. Zwitterion-containing acridinium compounds

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