Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/531—Production of immunochemical test materials
  • G01N33/532—Production of labelled immunochemicals
  • G01N33/533—Production of labelled immunochemicals with fluorescent label
• • 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
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/805—Optical property

Definitions

• compositions containing dendritic polymer chemiluminescent substrate compositions containing dendritic polymer chemiluminescent substrate
• the invention also concerns the use of enhancer substances in combination with the dendritic polymer chemiluminescent substrate conjugates.
• dendritic polymer chemiluminescent substrate conjugates which amplify the detectable chemiluminescent signal by coupling multiple (i.e., from 3 to 3072) enzyme activable chemiluminescent substrates, are useful in detecting the presence
• macromolecules including synthetic polymers, proteins, nucleic acids and the like.
• Dendritic polymers are uniform
• arborols having a central core, an
• the molecular linearity of the polymers produces a heavily entangled
• dendrimer chemistry constructs macromolecules with tight control of size, shape
• these macromolecules start by reacting an initiator core in high-yield iterative
• dendritic wedges are constructed from the periphery inwards towards a
• Dendritic syntheses form concentric layers, known as generations, with each generation doubling the molecular mass and the number of reactive groups at the branch ends so that the end generation dendrimer is a highly
• dendrimer molecular weights range from 300 to 700,000 daltons and the number of surface groups (e.g., reactive sites
• generations 0-3 are dome-shaped,
• generation 4 is a transition generation with an oblate spheriod shape
• generations 5 and greater are symmetrically spherical with a hollow interior and a
• dendritic polymers Dendritic growth, shape and topology are controlled by the core, the interior
• Dendrimers expand symmetrically in a
• dendritic surfaces can have from 3 to 3072 end groups
• dendrimer structure (which defines steric congestion) and the dendrimer
• dendrimers react with, e.g., halosulfonic esters, activated carboxylic
• Astramol PEI dendrimers (NH 2 -terminated), which are shown in Figures 1 A to ID are available from Aldrich Chemical Co., Dendritech Inc. and
• dendritic polymers can be molecularly tailored to meet specialized end uses.
• drugs drugs, toxins, metal ions, radionuclides, signal generators, signal reflectors,
• compositions containing the conjugates modifiers, diagnostic opacifiers, fluorescent moieties and scavenging agents; processes for preparing the conjugates; compositions containing the conjugates;
• U.S. Patent No. 5,482,698 discloses methods for detecting or treating
• lesions that includes a polymer comprising multiple avidin or biotin binding sites that includes a polymer comprising multiple avidin or biotin binding sites
• Dioxetanes developed for this purpose include
• U.S. Patent No. 4,978,614 discloses, among others, 3-(2'-spiroadamantane)4-mefhoxy- 4-(3"-phosphoryloxy)phenyl-l,2-dioxetane, which is commercially available from
• 5,582,980 disclose similar compounds, wherein the adamantyl stabilizing ring is substituted, at either bridgehead position, with a variety of substituents, including hydroxy, halogen, and the like, which convert the otherwise static or passive
• CSPD ® which is a registered trademark of PE Corporation (NY), is a second-generation dioxetane with a
• dioxetanes are used in conjunction with enhancers to detect analytes in concentrations of 10 "12 M or lower.
• Preferred enhancement agents include water-
• TQ poly(vinylbenzyltributylammonium chloride)
• TBQ poly(vinylbenzyltributylammonium chloride)
• BDMQ poly(vinylbenzyldimethylbenzylammonium chloride)
• the enhancement molecules apparently exclude water from the microenvironment
• substrates are used as reporter molecules by acting as substrates for enzymes which
• the enzyme e.g., alkaline
• phosphatase can be covalently linked or otherwise complexed with either an
• antigen or antibody in conventional antigen/antibody ligand binding assays, or a
• nucleic acid probe in nucleic acid assays.
• the enzyme-bearing antigen or antibody, or nucleic acid probe is then admixed with the analyte suspected of containing the
• target antigen or nucleic acid sequence, under conditions which permit complexing or hybridization between the antigen/antibody or probe/nucleic acid sequence.
• the chemiluminescent substrate is added. If the suspected analyte is present, the chemiluminescent substrate is added. If the suspected analyte is present, the chemiluminescent substrate is added. If the suspected analyte is present, the chemiluminescent substrate is added. If the suspected analyte is present, the chemiluminescent substrate is added. If the suspected analyte is present, the chemiluminescent substrate is added. If the suspected analyte is present, the
• the decomposition event is the light-releasing
• microarrays as to improve the overall sensitivity of the assay in order to automate clinical assays and to provide for high throughput screening on high density arrays (microarrays).
• chemiluminescent substrates in which the light signal is amplified by coupling one
• Signal resolution may also improve due to the frictional drag of the high molecular weight dendritic backbone, i.e., slowed molecular diffusion due to increased
• chemiluminescent substrates according to the invention can be further enhanced by linking multiple substrate reporter molecules to an enhancing region of the dendritic polymer. In this manner, more efficient sequestration of the substrate
• incipient reporter can fold into the proximate hydrophobic enhancing region of the
• the invention provides improvements
• the invention provides means for enhanced detection of
• invention further provides improvements in the detectability of electromagnetic (e.g., optically detectable) energy released by the decomposition of
• chemiluminescent substrates used to detect the presence or determine the
• the invention also provides a means for determining concentration of chemical or biological substances by art-recognized immunoassay, chemical assay or nucleic acid probe assay techniques.
• the invention also provides a means for determining concentration of chemical or biological substances by art-recognized immunoassay, chemical assay or nucleic acid probe assay techniques.
• the invention also provides methods of preparing dendritic polymer
• chemiluminescent substrates and intermediates therefor For example, a first process for preparing a conjugate of a dendritic polymer and a chemiluminescent
• substrate comprises reacting the dendritic polymer with the chemiluminescent
• the dendritic in a suitable solvent at a temperature which facilitates the association of the chemiluminescent substrate and the dendritic polymer.
• the present invention is thus directed to dendritic polymer
• compositions containing the conjugates and methods of using the conjugates are provided.
• FIGS. 1A - ID show the structures of a number of commercially available
• FIG. 1A shows a polyamidoamine (PAMAM) dendrimer
• FIG. IB shows a polyamidoamine (PAMAM) dendrimer
• FIG. 1C shows a polyamidoamine (PAMAM) dendrimer with hydroxyl surface groups
• FIG. ID shows a polypropyleneimine
• FIGS. 2 A - 2L show dioxetane coupling precursors according to the
• FIGS. 3A - 30 show dendritic polymer dioxetane conjugates according to
• FIGS. 4 A - 4F show the synthesis scheme for the dendritic polymer
• FIGS. 3A - 3L conjugates shown in FIGS. 3A - 3L wherein: FIG. 4A corresponds to FIG. 3A and FIG. 3G; FIG. 4B corresponds to FIG. 3B and FIG. 3H; FIG. 4C corresponds to FIG. 3C and FIG. 31; FIG. 4D corresponds to FIG. 3D and FIG. 3J; FIG. 4E
• FIG. 3E corresponds to FIG. 3E and FIG. 3K
• FIG. 4F corresponds to FIG. 3F and
• FIG. 3L is a diagrammatic representation of FIG. 3L.
• FIGS. 5A - 51 and FIGS. 6A - 6R show dendritic polymer isoluminol
• FIG. 7 A - 7C show the synthesis of dendritic polymer isoluminol conjugates
• FIG. 8 A - 8F show synthesis of dendritic polymer acridinium ester
• FIG. 9A - 9C show dendritic polymer dioxetane and isoluminol, dioxetane
• FIG. 10 shows linked dioxetanes and quarternary amino enhancers according to the invention.
• FIG. 11 shows dendritic dioxetane enhancer hybrids according to the
• Enzymatic assay design provides significant signal amplification by enzymatic turnover of substrate to generate a signal with increased detection sensitivity.
• the enzyme substrates are usually colorimetric, fluorimetric or
• chemiluminescent enzyme substrates provide a
• the instrumentation for detecting chemiluminescent output is also simpler than that required for fluorescence detection, since no excitation source is
• chemiluminescent substrates include dioxetanes (activated by
• hydrolytic enzymes such as esterases, alkaline phosphatases and glycosidases
• esters acridinium sulfonylamides and luciferins (activated by oxidative enzymes such as horseradish peroxidase, glucose or galactose oxidase and luciferase).
• oxidative enzymes such as horseradish peroxidase, glucose or galactose oxidase and luciferase.
• substrates are enzyme-activated to produce destabilized peroxygenated
• chemiluminescent signal allows detection of enzyme labels in immunoassay
• dendritic polymer According to an embodiment of the invention, dendritic polymer
• chemiluminescent substrates containing one or more dioxetanes, luminols,
• isoluminols, acridinium esters, acridinium sulfonylamides and/or luciferins coupled to the surface of known dendrimer polymers can be synthesized.
• FIGS. 1A - ID examples of some dendrimer starting materials that may be employed to synthesize the dendritic polymer chemiluminescent substrates of the invention are depicted in FIGS. 1A - ID.
• FIG. 1A shows a polyamidoamine (PAMAM) dendrimer with NH,
• FIG. IB shows a polyamidoamine (PAMAM) dendrimer with carboxylic acid surface groups.
• FIG. 1C shows a polyamidoamine (PAMAM)
• FIG. ID shows a polypropyleneimine
• Chemiluminescent substrate coupling precursors that may be employed to
• FIGS. 2 A - 2L the substituents are defined as follows:
• A is H, alkyl, trihaloalkyl or aryl
• B is NA, NC(O)A, O, S or CH 2 wherein A is independently H, alkyl,
• Y is independently H, a hydroxyl group, a halogen, an unsubstituted lower alkyl group, a hydroxy lower alkyl group, a halo lower alkyl group, a phenyl group,
• a halo phenyl group an alkoxy phenyl group, an alkoxy phenoxy group, a hydroxy
• alkoxy group a cyano group, an amide group, an alkoxy group or a carboxyl group
• R is an alkyl group (e.g., a C, - C, 2 alkyl group), a haloalkyl group (e.g., a mono-, di-, or trihaloalkyl), an aryl group or an aralkyl group;
• X is an enzyme-labile group selected from the group consisting of a
• adenosine triphosphate adenosine diphosphate, adenosine monophosphate, adenosine, ⁇ -D-glucoside, ⁇ -D-glucoside, ⁇ -D-glucuronide, ⁇ -D-mannoside, ⁇ -D- fructofuranoside, ⁇ -glucosiduronate; 5-acetamido-3-5-dideoxy- ⁇ -D-glycero-D-
• galacto-2-nonulopyranoside and alkoxy derivatives e.g., 4,7-di-O-methyl
• alkoxy derivatives e.g., 4,7-di-O-methyl
• Z is a halo, alkoxy or alkyl group
• T is H, an electron donating group, an electron withdrawing group, or an organic linker group which may be attached to an ancillary fluorophore or to any biological moiety.
• FIGS. 3A - 30 the first figure.
• FIGS. 3A - 3L the second figure.
• FIGS. 2 A - 2L. N which represents the number of dioxetane moieties conjugated to the dendrimer, is a positive integer. According to a preferred embodiment of the
• N in FIGS. 3A - 3F is from 6 to 768.
• FIG. 3M shows a polyamidoamine (PAMAM) Starburst Dendrimer having
• FIG. 3N shows a polyamidoamine (PAMAM) Starburst Dendrimer having amino terminal groups conjugated with a plurality of dioxetanes
• FIG. 30 shows a
• PEI polypropyleneimine starburst dendrimer having amino surface groups
• Figures 4A - 4F shows synthesis schemes for the dendritic polymer
• the "activator” can be a mixed anhydride, an NHS ester, or another standard functionality used in peptide chemistry to facilitate peptide bond formation.
• the "leaving group” can be a halogen or a sulfonic ester
• the "Aryl" substituent can be a phenyl group or a benzothiazole
• the "Aryl" substituent can be a phenyl group or a benzothiazole as defined in FIGS. 3B or 3H.
• the "Aryl" substituent can be a phenyl group or a benzothiazole as defined in FIGS. 3B or 3H.
• the "Aryl" substituent can be a phenyl group or a benzothiazole as defined in FIGS. 3B or 3H.
• substituent can be a phenyl group or a benzothiazole as defined in FIGS. 3C or 31.
• the "Aryl" substituent can be a phenyl group or a benzothiazole as
• the "Aryl" substituent can be a phenyl group or a benzothiazole as defined in FIGS. 3E or 3K.
• the "Aryl" substituent can be a phenyl group or a benzothiazole as defined in FIGS. 3E or 3K.
• the "Aryl" substituent can be a phenyl group or a benzothiazole as defined in FIGS. 3E or 3K.
• the "Aryl" substituent can be a phenyl group or a benzothiazole as defined in FIGS. 3E or 3K.
• FIGS. 3F or 3L can be a phenyl group or a benzothiazole as defined in FIGS. 3F or 3L.
• FIGS. 5A - 5C show dendritic polymer isoluminol
• N is from 6 to 768.
• the substituent A can be hydrogen or an alkyl
• FIG. 5D shows a polyamidoamine (PAMAM) starburst dendrimer having carboxylic acid surface groups conjugated with a plurality of isoluminol moieties
• FIG. 5E shows a polyamidoamine
• PAMAM starburst dendrimer having amino surface groups conjugated with a
• FIG. 5F shows a polypropyleneimine (PEI) starburst dendrimer having amino surface groups conjugated with a plurality of isoluminol moieties according to a further embodiment of the invention.
• PEI polypropyleneimine
• FIGS. 5G - 51 show conjugates of dendrimers and luciferins according to the
• N is defined as set forth above.
• the substituent A can be H or an alkyl group.
• the substituent D is defined as set forth above.
• the substituent B can be independently NA, O, S or CH 2 wherein the substituent A can be H or an alkyl group.
• FIGS. 6A - 6C show conjugates of dendrimers and acridinium esters
• N is defined as set forth above and
• X is a counterion such as a halide or a trifiate.
• the substituent A can be
• the substituent B can be NA, NH, O, S or CH,
• substituent A can an alkyl or an aryl group.
• FIGS. 6D - 61 show conjugates of dendrimers and acridinium sulfonylamides according to the invention.
• N is defined as set
• X is a counterion such as a halide or a trifiate.
• the substituent A can be H or an alkyl group.
• the substituent R can be an alkyl or an aryl group.
• the substituent B is H or an alkyl group.
• FIG. 6J shows a polyamidoamine (PAMAM) starburst dendrimer having
• FIG. 6K shows a polyamidoamine (PAMAM) starburst dendrimer having carboxylic acid surface groups conjugated with a plurality of acridinium sulfonamide moieties according to
• FIG. 6L shows a polyamidoamine (PAMAM) starburst dendrimer having amino surface groups conjugated with a plurality of acridinium ester moieties according to another embodiment of the invention.
• FIG. 6M shows a polyamidoamine (PAMAM) starburst dendrimer having amino surface groups conjugated with a plurality of acridinium sulfonamide moieties according to another embodiment of the invention.
• FIG. 6N shows a polypropyleneimine (PEI) starburst dendrimer having amino surface groups
• FIG. 60 shows a polypropyleneimine (PEI) starburst
• dendrimer having amino surface groups conjugated with a plurality of acridinium sulfonamide moieties according to a further embodiment of the invention.
• FIGS. 6P - 6R show conjugates of dendrimers and acridan moieties
• the substituent D can be PO 3 X 2 , a
• substituent F can be NA, S or O wherein A can be H or an alkyl group.
• substituent Z can be a halo, alkoxy or alkyl group.
• N is a positive integer. According to a preferred embodiment of the invention, N is an integer from 1 to 10.
• the substituent B can be NA, O, S or CH 2 wherein A can be H or an
• FIGS. 7 A - 7C show the synthesis of dendritic polymer isoluminol
• activator can be a
• FIG. 7A “A” can be H or an alkyl group.
• “B” can be NH or O, and “leaving group” can be a halogen or sulfonic acid ester such as a mesylate, a tosylate or a trifiate.
• N in FIGS. 7 A - 7C is an integer from 6
• FIGS. 8 A - 8F show synthesis schemes for a number of dendritic polymer
• FIGS. 8A - 8C show the synthesis of dendritic polymer acridinium ester conjugates according to the invention and FIGS. 8D - 8F show the
• X represents a counterion such as a halide or a trifiate
• N which represents the number of chemiluminescent substrates conjugated to the
• N in FIGS. 8 A - 8F is an integer from 6 to
• activator can be a mixed anhydride, an NHS
• B can be NH or O; and "leaving group” can represent a halogen or
• sulfonic acid ester such as a mesylate, a tosylate or a trifiate.
• substituent "R” can be an alkyl or an aryl group.
• FIGS. 9 A - 9C enzyme label chemiluminescent substrates are shown in FIGS. 9 A - 9C.
• FIG. 9 A shows a polyamidoamine (PAMAM) starburst dendrimer conjugated with dioxetane
• FIG. 9B shows a polyamidoamine (PAMAM) starburst dendrimer conjugated with dioxetane and acridinium ester moieties according to a further embodiment of the invention.
• FIG. 9C shows a polyamidoamine (PAMAM) starburst dendrimer conjugated with dioxetane and acridinium sulfonamide moieties according to a further embodiment of the invention.
• FIG. 10 shows a polyamidoamine (PAMAM) dendrimer having amino
• enhancer moieties according to an embodiment of the invention. As shown in
• the quarternary ammonium enhancer moieties are perbutylated ammonium moieties.
• FIG. 11 shows a polyamidoamine (PAMAM) 'dendrimer having carboxylic acid surface groups conjugated to both dioxetane moieties and enhancer moieties
• ammonium, phosphonium or sulfonium salts amino terminated quatemized
• a “dendritic polymer” is a polymer exhibiting regular dendritic branching
• dendritic polymer formed by the sequential or generational addition of branched layers to or from a core.
• dendritic polymer encompasses "dendrimers,” which are
• a core characterized by a core, at least one interior branched layer, and a surface branched
• dendrimer is a species of dendrimer having branches emanating from a focal point which is or can be joined to a core, either directly or through a linking moiety to form a dendrimer. Many dendrimers comprise two or more
• dendrons joined to a common core are dendrons joined to a common core.
• dendrimer is used broadly to encompass a single dendron.
• Dendritic polymers include, but are not limited to, symmetrical and unsymmetrical branching dendrimers, cascade molecules, arborols, and the like,
• dendritic polymers are dense star polymers.
• PAMAM dense star dendrimers disclosed herein are symmetric, in that the branch
• the branching occurs at the hydrogen atoms of a terminal -NH 2 group on a preceding generation branch.
• hyperbranched polymers e.g., hyperbranched polyols
• hyperbranched polyols may be equivalent to a
• dendritic polymer where the branching pattern exhibits a degree of regularity
• Topological polymers with size and shape controlled domains, are
• dendrimers that are associated with each other (as an example covalently bridged or
• bridged dendrimers When more than two dense star dendrimers are associated together, they are referred to as “aggregates" or
• dendritic polymers include bridged dendrimers and dendrimer
• Dendritic polymers encompass both generationally monodisperse and generationally polydisperse solutions of dendrimers.
• the dendrimers in a monodisperse solution are substantially all of the same generation, and hence of uniform size and shape.
• the dendrimers in a polydisperse solution comprise a distribution of different generation dendrimers.
• Dendritic polymers also encompass surface modified dendrimers.
• the surface of a PAMAM dendrimer may be modified by the addition of
• amino acid e.g., lysine or arginine
• a coupling agent such as carbodiimides, phosphonium or ammonium
• a second method involves forming ether or amine linkages by nucleophilic
• a third method involves coupling 1-2 dendritic polymer surface groups to cyanuric chloride, with the remaining 1-2 reactive
• a fourth method conjugates avidin- or streptavidin-linked chemiluminescent substrate
• the chemiluminescent substrate precursors can be modified to form the dendritic polymer chemiluminescent substrates. For example, to complete the
• the dendrimer- enol ether conjugates may be peralkylated with an arylalkyl or alkylhalide at any
• the dendritic polymer chemiluminescent substrate conjugates described above show improved light intensity and/or sensitivity, as well as improved signal resolution. These substrate conjugates are specifically prepared for use in enzymatic assays, where hydrolytic enzymatic removal of enzyme labile (e.g., X)
• oxidation e.g., by horseradish peroxidase
• conjugates induces dioxetanone decomposition and chemiluminescence.
• enzyme may be the target analyte in the sample, or may be a reporter molecule
• chemiluminescent substrate conjugates all employing a visually detectable
• chemiluminescent signal to indicate the presence and/or concentration of a
• the sample can be contacted
• dendritic polymer dioxetane conjugates bearing a group capable of being
• the enzyme cleaves the dioxetane' s enzyme
• dioxetane causing the dioxetane to decompose to form a chemiluminescent chromophore that emits light. It is this light emission that is detected as an
• the concentration of the enzyme in the sample can be determined.
• the sample can be contacted with dendritic polymer luminol,
• the enzyme directly oxidizes the
• luminol isoluminol, or luciferin, or indirectly oxidizes the acridinium ester, or
• the concentration of the enzyme in the sample can also be determined.
• chemiluminescent substrate decomposes in protic solvent such as water. Since
• the light-quenching reactions may substantially reduce the chemiluminescence intensity observed. It is
• enhancement molecules including water-soluble polymeric quaternary ammonium, phosphonium or sulfonium salts, and copolymers and/or
• mammalian serum albumins such as bovine serum albumin (BSA) and human
• HSA serum albumin
• water soluble polymeric quaternary ammonium, phosphonium or sulfonium salts poly(vinylbenzyltrimethyl-ammonium chloride)
• fluorescein that accept energy from energy-emitting fluorophores produced by the decomposition of chemiluminescent compounds and in turn emit detectable energy.
• An additional embodiment of the invention thus involves coupling enhancer molecules to the dendritic polymer, or modifying residual reactive surface groups on
• the dendritic polymer to contain enhancer sites.
• the enhancing agent or can be chemically modified to become an enhancing surface group. Or, if required by synthetic constraints, the chemiluminescent substrate
• precursors can be linked to reactive dendritic polymer sites after the enhancer
• the enhancer molecules including, e.g.,
• mammalian serum albumin bovine serum albumin, human serum albumin, Protein A or mammalian IgG
• mammalian serum albumin bovine serum albumin, human serum albumin, Protein A or mammalian IgG
• the enhancer molecules include synthetic macromolecular substances with a reactive terminal
• reactive linker or reactive terminal group include
• poly(vinylarylquatemaryammonium) salts poly-N-vinyloxazolidinones, poryvinylcarbamates, polyhydroxyacrylates and polyhydroxymethacrylates, quatemized amine-containing oligomers (e.g., Jeffamines), synthetic polypeptides (including polylysine and nylons), polyvinylalkylethers, polyacrylamides and
• polymethacrylamides polyvinyl alcohols, poly 2-, 3-, or 4-vinylpyridinium salts, polyvinylalkylpyrrolidinones, polyvinylalkyloxazolidones, quatemized
• polyethyleneimines poly-N-vinylamines, alkylated or arylated polyvinylpiperidine,
• polyacryloyl polymethacryloyl, or 4-vinylbenzoylaminimides, and dendritic or
• chemiluminescence enhancement include peralkylation of terminal and/or interior
• esters with amino-linked ammonium, phosphonium or sulfonium salts According to a fourth approach, a dendritic polymeric chemiluminescent substrate is attached
• a water-solubilizing group (one or more in number), i.e., a substituent which enhances the solubility of the dioxetane in aqueous solution,
• aralkylamides alkyl- or arylurethanes, alkyl- or arylsulfonamides, alkyl- or arylsulfonic acids and quaternary amino salts; the most preferred solubilizing
• substituents are alkyl- or aryloxides, amides, and sulfonamides can be added to the chemiluminescent substrate or the dendrimer to enhance the substrate's solubility in
• the sample which is generally aqueous in nature.
• the identity may affect, in
• Examples of such assays include immunoassays to detect antibodies or antigens,
• viruses e.g., HTLV III or
• cytomegalovirus cytomegalovirus
• bacteria e.g., E. Coli
• cell functions e.g., receptor
• enzyme capable of oxidizing the luminol, isoluminol, acridinium ester, acridinium sulfonylamide or luciferin is preferably bonded to a substance having a specific
• the detectable substance i.e., a substance that binds specifically to the
• detectable substance e.g., the antigen, the antibody or the nucleic acid probe.
• detectable substance e.g., the antigen, the antibody or the nucleic acid probe.
• a ligand can be attached to the
• the ligand is then detected with a high affinity ligand-binding agent labeled with an enzyme capable
• assays are performed as follows. In one example, a sample isoluminol, acridinium ester, acridinium sulfonamide or luciferin. Conventional methods, e.g., carbodiimide coupling, are used to bond the enzyme to the specific affinity substance; bonding is preferably through an amide linkage. In general, assays are performed as follows. In one example, a sample isoluminol, acridinium ester, acridinium sulfonamide or luciferin. Conventional methods, e.g., carbodiimide coupling, are used to bond the enzyme to the specific affinity substance; bonding is preferably through an amide linkage. In general, assays are performed as follows. In one example, a sample isoluminol, acridinium ester, acridinium sulfonamide or luciferin. Conventional methods, e.g., carbodiimide coupling, are used to bond the
• suspected of containing a detectable substance is contacted with a buffered solution containing an enzyme bonded to a substance having a specific affinity for the
• the resulting solution is incubated to allow the detectable substance
• the resulting solution is incubated to allow the detectable substance to
• chemiluminescent substrate having a group cleavable by the enzyme portion of the
• enzyme-labeled ligand binding agent is added. In both examples, the enzyme-
• Luminescence is detected using, e.g., a cuvette, or a light-sensitive film in a camera luminometer, or CCD, or a photoelectric cell or photomultiplier tube, to measure the presence and
• substances with specific affinity for detectable substances in biological samples are arrayed on solid supports in spacially well-defined patterns.
• Solid supports such as glass, plastic, silicon, polymer in planar or
• substances in biological samples may be nucleic acids or proteins.
• the biological sample containing the detectable substance(s) is contacted with the solid phase array
• the detectable substance(s) are prelabeled with detectable
• the bound unlabeled substance is
• Luminescence is detected using a charge-coupled camera (CCD), film or other light
• a coupling agent e.g., dicyclohexylcarbodiimide (DCC, 1.0 eq per carboxylic acid
• amino-terminated dendrimer e.g., Starburst PAMAM dendrimer or polypropylenimine tetrahexacontaamine dendrimer
• PAMAM dendrimer or polypropylenimine tetrahexacontaamine dendrimer in 15 ml CH 2 C1 2 with 0.1 ml triethylamine is added enol ether with a linker terminated
• halide e.g., I or Br
• sulfonic ester e.g., mesylate, tosylate, brosylate.
• linker 0.5 eq per carboxylate end group
• amino-terminated polymer 0.5 eq per carboxylate end group, e.g., polyethylenimine, poly-N-vinylamine or
• EDCI l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
• urea byproduct is removed by filtration or by an aqueous wash (EDCI urea byproduct), the organic solvent is evaporated, and
• alkylhalide e.g., iodomethane to permethylate or butylbromide to perbutylate
• dendritic dioxetane-polyammonium or dendritic dioxetane-polyvinylpiperidinium substrate are dendritic dioxetane-polyammonium or dendritic dioxetane-polyvinylpiperidinium substrate.
• a 0.1 mol solution of amino-terminated dendrimer e.g., Starburst PAMAM
• the biotinylated dendrimer is purified by
• a 0.1 mol solution of amino-terminated dendrimer e.g., Starburst PAMAM
• succinyl-avidin (Sigma) or succinyl-streptavidin in the presence of a coupling agent
• DCC dicyclohexylcarbodiimide
• EDCI l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride
• the avidin-conjugated dendrimer is purified by
• bovine serum albumin (BSA) (present in a 1 : 1 molar ratio) and stirred at ambient
• biotinylated agents to yield dendritic isoluminol-BSA substrate.
• TPP tetraphenylporphine
• a solution of 100 mg amine-terminated dendrimer e.g., PAMAM-NH 2 or
• a coupling agent e.g., dicyclohexylcarbodiimide (DCC, 1.0 eq per carboxylic acid end group) or l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 1.0 eq per carboxylic acid end group)
• DCC dicyclohexylcarbodiimide
• EDCI l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
• a solution of 100 mg amine-terminated dendrimer e.g., PAMAM-NH, or
• a coupling agent e.g., dicyclohexylcarbodiimide (DCC, 1.0 eq per
• phosphatase (at final concentration of 1.05 x 10 "9 M) is added to the test tube and the chemiluminescent signal kinetics is measured in a Turner TD-20E luminometer for 10 to 20 minutes.
• the half- life is calculated from the plot of log RLU versus time. The chemiluminescence half-life is also determined in the presence of
• Sapphire-II TM enhancer in 0.1M diethanolamine, 1 mM MgCl 2 , 10% polyvinylbenzyltributyl ammonium chloride qt 1 mg/mL.
• chemiluminescent signal is measured in a Turner TD-20E luminometer for 10 to 20
• the peak light intensity is recorded.
• the peak light intensity is also
• a dendritic polymer dioxetane conjugate of the invention may be employed
• Mouse monoclonal anti-TSH- ⁇ antibody can be used to coat 1/8 inch beads for analyte capture.
• Mouse monoclonal anti-TSH antibody can be used to coat 1/8 inch beads for analyte capture.
• Mouse monoclonal anti-TSH antibody can be used to coat 1/8 inch beads for analyte capture.
• alkaline phosphatase conjugated with alkaline phosphatase and used as a detection antibody.
• TSH can be obtained from Calbiochem, Catalog No. 609396, and BSA (type
• V— fatty acid free can be obtained from Sigma, Catalog No. A6003.
• the buffer solution used for the analyte and conjugate can contain 0. IM
• reaction tube 200 ⁇ l of the same chemiluminescent compound used in the
• a dendritic polymer dioxetane conjugate of the invention may be any dendritic polymer dioxetane conjugate of the invention.
• the dendritic polymer dioxetane conjugates of the invention may be employed in a nucleic acid hybridization assay as follows:
• CSF cerebrospinal fluid
• cytomegalovirus is collected and placed on a nitrocellulose membrane. The sample
• a DNA probe specific to the viral DNA and labeled with alkaline phosphatase is then chemically treated with urea or guanidinium isothiocyanate to break the cell walls and to degrade all cellular components except the viral DNA.
• the strands of the viral DNA thus produced are separated and attached to the nitrocellulose filter.
• a DNA probe specific to the viral DNA and labeled with alkaline phosphatase is
• a phosphate-containing dendritic polymer dioxetane conjugate is added
• the dendritic polymer dioxetane conjugates of the invention may also be any dendritic polymer dioxetane conjugates of the invention.
• the dendritic polymer dioxetane conjugates of the invention may also be any dendritic polymer dioxetane conjugates of the invention.

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