Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6816—Hybridisation assays characterised by the detection means
  • C12Q1/682—Signal amplification
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6804—Nucleic acid analysis using immunogens
• • 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
  • G01N33/54306—Solid-phase reaction mechanisms
• • 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
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
  • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
  • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Definitions

• the present invention relates generally to methods and compositions for amplifying a detectable signal used to detect the absence or presence of a nucleic acid analyte in a sample.
• diagnostic assays utilize detectable labels to indicate binding events that are indicative of the presence or absence of a target analyte in a sample.
• Typical target analytes include proteins, carbohydrates, or nucleic acids.
• diagnostic assays utilize a target-specific capture molecule that is immobilized on a solid substrate. A sample is placed on the solid substrate and the target analyte, if present, binds to a target-specific capture molecule. The surface-bound target analyte may then be directly modified by binding, directly or indirectly, with a detectable label. Alternatively, a second reagent, modified with a detectable label, may bind to the surface immobilized target.
• the label can be detected directly in the case of radio-labeled or fluorescent labels using devices such as a phosphor-imager or a fluorescence reader, respectively.
• the label may be indirectly detected, for example, by binding the label with an anti-label/enzyme conjugate that is subsequently contacted with an enzyme substrate to produce a signal that can be detected.
• U.S. Pat. No. 5,196,306 discloses a method in which a target-specific, surface-immobilized label is reacted with an amplification polymer to multiply the number of binding sites for a detectable label complex, followed by conjugation with an anti-label antibody conjugate, such as horse radish peroxidase (“HRP”) that is then exposed to a tyramide/label conjugate.
• HRP horse radish peroxidase
• U.S. Pat. No. 5,124,246 discloses amplification of a signal by creating branched layers of DNA hybridization in a target nucleic acid sequence specific manner. The layers culminate in a branched structure that can hybridize to hundreds of labels.
• Other approaches disclosed in U.S. Pat. No. 6,103,474 and U.S. Pat. No. 6,110,682, amplify a signal by targeting homopolymeric regions of a target nucleic acid analyte with multiple-labeled hairpin reporter probes.
• a method has also been developed that amplifies biotin-dependent signaling events (Zhong et al., PNAS (2003) 100:11559-11564).
• biotinylated probes are immobilized on a surface in a target-dependent manner, and are then contacted with an avidin-biotinylated dextran copolymer, resulting in a 50-100 fold increase in assay sensitivity.
• DNA dendrimers have also been used to amplify signals, as disclosed in U.S. Pat. No. 5,175,270, U.S. Pat. No. 5,487,973, and U.S. Pat. No. 6,046,038.
• DNA dendrimers are large cross-linked structures that can be modified to contain up to several hundred label groups. These labels groups include biotin, HRP, streptavidin (“SA”), and fluorescent molecules, as disclosed in U.S. Pat. No. 6,072,043; U.S. Pat. No. 6,110,687; and U.S. Pat. No. 6,762,292.
• DNA dendrimer can contain mixtures of molecules as well, such as SA and HRP. The mixture allows for binding of SA to surface-immobilized biotin, for example. This approach multiplies the number of HRP molecules at the surface of each biotin molecule bound and results in amplification of the signal.
• Another technique employs the targeting of homopolymeric regions of target DNA with multiple-labeled hairpin reporter probes, as disclosed in U.S. Pat. Nos. 6,103,474; 6,110,682.
• Biotinylated probes that were immobilized onto a surface in a target-dependent manner were contacted with an avidin-biotinylated dextran copolymer. This was reported to increase assay sensitivity 50-100 fold increase in assay sensitivity for detection of biotin DNA probes covalently immobilized onto a chip surface.
• the present invention provides improvements in diagnostic assays for detecting and/or quantitating an analyte (such as a nucleic acid or a protein) in a sample.
• the invention provides improved methods and compositions for amplifying a detectable signal used to indicate the presence or absence of the analyte in the sample.
• the present invention provides reagents and methods for improving the sensitivity of a signal generated by means of a plurality of amplification polymers.
• the present invention relates to a method for enhancing the signal of a detection complex using multivalent interactions to stabilize and/or decrease the off-rate of binding of one or more intermediate binding events between the analyte, if present, and the amplification polymer and detection conjugate.
• the amplification polymer binds to one or more non-analyte-specific binding site of the multivalent bridge conjugate, if present on the solid support.
• One specific embodiment of the invention is a method for detecting the presence or absence of an analyte bound to a solid support, comprising:
• the multivalent binding sites on the amplification polymer are present at a density sufficient to enable two or more separate multivalent binding sites of one amplification polymer to bind to two or more non-analyte-specific binding sites of one multivalent bridge conjugate, if present on the solid support.
• the invention relates to a specific order of mixing the multivalent conjugate and amplification polymer, followed by mixing the resulting complex with the sample.
• This aspect may include, for example, a method for detecting the presence or absence of an analyte bound to a solid support, comprising:
• the invention in another aspect, relates to a method in which the multivalent bridge conjugate, amplification polymer and detection conjugate are premixed, and then mixed with the analyte (i.e., applied to a solid substrate to which the analyte is bound), and then detecting the presence, absence or amount that binds to the analyte.
• This aspect may include, for example, a method for detecting the presence or absence of an analyte bound to a solid support, comprising:
• the invention relates to a method for pre-mixing the multivalent bridge conjugate, amplification polymer and detection conjugate (which may later be bound to the analyte).
• This aspect may include, for example, a method for preparing an analyte detection complex having an analyte-specific binding site and improved dissociation properties, comprising combining:
• the invention relates to a method used to detect the presence or absence or amount of an analyte which itself has multiple binding sites.
• This aspect of the invention may include, for example, a method for detecting the presence or absence of a multivalent analyte bound to a solid support, comprising:
• the invention relates to a composition comprising a multivalent bridge conjugate, amplification polymer and detection conjugate (which may be bound to the analyte).
• This aspect of the invention may include, for example, an analyte detection complex having an analyte-specific binding site, wherein the complex comprises:
• the methods and compositions of the invention may further comprise the step of combining the amplification complex or label with one or more solvating compounds in order to increase the number of amplification polymers that form a complex with the detectable labels.
• Alcoholating compound means a compound that reacts with amine groups on the amplification polymer under high salt conditions to acetylate the amine groups not bound with a detectable label complex.
• Amplification polymer means a polymeric compound that specifically binds, either directly or indirectly, to a target nucleic acid analyte and has a plurality of other binding sites that bind to the detection conjugate, which multiplies the number of detectable labels that can be bound to each amplification polymer associated with a given target nucleic acid analyte.
• the amplification polymer may comprise a polymer having a plurality of reactive amine groups to which biotin molecules can be covalently attached. The biotin molecules, when bound to the amplification polymer, can then be used as a binding substrate for a detectable label complex that generates a detectable signal.
• the amplification polymer may be, for example, a dextran copolymer.
• the amplification polymer has a plurality of binding sites, which may include one or more binding sites having different binding specificity to one or more different binding sites of the multivalent binding bridge complex.
• the amplification polymer will also be conformationally flexible, to allow for optimal movement and placement of biotins for a multivalent interactions.
• the amplification polymer will also permit appropriate spacing and density of biotin molecules on the backbone.
• analyte means a molecule, macromolecule, or compound that is the target of an assay. Although “analyte” is often used in the singular in this application, it should be understood that most samples consist of millions or billions of the identical analyte. Examples of analytes include, but are not limited to, proteins or polypeptide molecules, polynucleotide molecules, organic or inorganic compounds, DNA, polymorphisms of DNA, and RNA. An analyte will generally have one or more unique binding sites (or epitopes) to which other molecules may bind. In particular embodiments disclosed herein, the analyte may be bound to a solid support via an analyte-specific capture molecule that is directly or indirectly bound to the solid support, leaving alternative binding sites available for binding to the multivalent binding conjugate.
• alyte-specific means that a compound binds specifically, though not necessarily exclusively, to the analyte in a sample.
• Binds means the formation of an attractive force between two molecules, which includes ionic bonds, covalent bonds, polar covalent bonds, or noncovalent bonds.
• Capture molecule means a label comprising a functional binding group that binds covalently or non-covalently to the analyte, and further comprising a second functional binding group that binds covalently or non-covalently to an amplification polymer or secondary amplification polymer functional binding group.
• Conjugate or “complex” means one or more molecules covalently or non-covalently coupled together.
• Detectable label means a chemical compound that can be either directly or indirectly detected by visual or instrumental means.
• a detectable label may consist of a molecule that itself produces a signal that can be detected, such as a fluorescent, chemiluminescent or radioactive signal.
• the signaling label may comprise a molecule that requires reaction with another molecule to generate a signal that can be detected.
• Detectable labels also include compounds that can be detected visually, for example, colored dyes.
• Detection conjugates and “detectable label complex” means one or more molecules associated together that enable visual or instrumental detection of a detectable label. May be single compound with a detectable label, May be complex of compounds with a detectable label.
• the detectable label complex may comprise molecules that react with other molecules to produce other products that can be detected.
• the detectable label complex may comprise an enzyme that is reacted with a substrate to produce reaction products having a detectable label, or detectable property.
• Label means, in its generic sense, a molecule or binding site of a molecule that is capable of binding either covalently or non-covalently to other molecules, and being used itself as a binding substrate for another molecule or as a signal for detection. Labels often have different chemical functional groups that react with other chemical functional groups on other molecules. A label can also have multiple functions, for example a capture label could also be a signaling label. Labels may be, for example, an enzyme, antibody, or protein. Labels may also be detectable labels that are used to generate a signal that can be detected for purposes of indicating the presence or absence of an analyte of interest in a sample.
• Multivalent bridge conjugate means a compound or complex of compounds having an analyte-specific binding site and one or more non-analyte-specific binding sites.
• the analyte-specific binding site of the multivalent bridge conjugate binds directly or indirectly to an analyte, for example, that is bound to a solid support.
• the non-analyte specific binding sites of the multivalent bridge conjugate bind to the binding sites of the amplification polymer.
• the multivalent binding sites may all have equivalent binding specificity to the same substrate, or they may bind to different substrates.
• a multivalent bridge conjugate may comprise a streptavidin molecule having four equivalent binding sites that bind to biotin, one of which is used to link directly or indirectly to the analyte, and one or more of which is used to link to an amplification polymer.
• the multivalent bridge conjugate may include one or more compounds that bind to the signaling groups of the amplification polymer.
• Signal means a property or characteristic of a detectable label that permits it to be visually or instrumentally detected and/or distinguished. Typical signals include fluorescent signals, dyes, radioactive signals, etc.
• Solid support means a substrate to which an analyte, if present in a sample, binds.
• Specifically binds and “having binding specificity” means that a compound binds specifically, though not necessarily exclusively, to the analyte in a sample.
• High affinity binding interactions between bio-molecules is of critical importance. High affinity binding is particularly important for low amounts or densities of target molecules; higher affinity reagents are capable of binding to lower concentrations of target analyte.
• a method to further enhance the affinity of a bio-molecule is to increase its valency so that more than one interaction is possible per molecule. Multivalent interactions have a strong impact on bio-molecular interactions in multiple ways. First, a higher local concentration of bio-molecule will increase association kinetics. Second, the effective dissociation rate is slowed as well by allowing for rapid rebinding to occur. The net effect is an improved apparent binding constant.
• multiple-labeled biotin polymers will be prepared of sufficient density and size to permit a multivalent interaction with streptavidin; more than one biotin will bind per streptavidin for optimal signal amplification.
• This multivalent binding is a requirement for optimal signal enhancement because the biotinylated polymer will have a very slow off rate, allowing for optimal assay performance.
• Key factors include:
• the embodiments of the invention presented herein demonstrate the principle of multivalent binding to create a highly efficient signal amplification method, termed AMPED.
• the system utilizes two multivalent molecules to amplify signal; the first multivalent molecule binds to a cognate label on immobilized target molecules. This interaction serves as a “bridge” to a second multivalent molecule (amplification polymer) by also binding to labels on the amplification polymer. There are multiple labels on the amplification polymer available to bind to the bridge molecule.
• the binding of the bridge to the amplification polymer may be one or more interactions, and combined with the interaction with the target label, multivalent binding occurs.
• the labels on the amplification polymer may now be detected either directly or indirectly by treatment with anti-label/enzyme conjugates that are subsequently reacted with an enzyme substrate to create a signal.
• the multivalent interactions improve signal amplification in multiple ways: slowed off-rate and improved limits of detection. Slowed off-rate may be accomplished by placing multiple labels on the amplification polymer, rebinding may occur. This slows the effective off-rate, leaving more amplification polymer present to be detected. Limits of detection may be improved by creating a higher affinity interaction with target molecules on the target, lower concentration of target will be bound, improving assay sensitivity.
• the present invention relates to improved methods and compositions for detection of analytes. Improved methods and reagents are disclosed for detection, quantification, and characterization of analytes, such as proteins, carbohydrates, nucleic acids, or other molecules, in a sample. Clinically useful diagnostic methods must be capable of detecting and/or quantifying the presence of an analyte of interest that is present in extremely small quantities in a complex mixture containing similar species. Methods for such diagnostic assays have previously used detectable labels, such as radiolabeling, radiobioassay and immunoassay techniques.
• immunological reagents have been used extensively for detecting and/or quantitating a broad spectrum of molecular species such as proteins, lipids, carbohydrates, steroids, nucleic acids, drugs, carcinogens, antibiotics, inorganic salts etc.
• Polyvalent and monoclonal antibodies are very important diagnostic tools in most areas of clinical medicine today.
• the methods and compositions of the present invention improve upon methods of the prior art by amplifying the signal generated by an analyte-specific detection complex.
• the improved methods disclosed herein increase the number of amplification polymers available for reaction with a detectable label.
• a capture molecule that can recognize specific regions of a target analyte interest is bound to a solid surface and used to capture and immobilize the target analyte on the solid surface.
• the target analyte bound to the solid surface may then be directly or indirectly modified with a detectable label.
• the target analyte may be directly labeled with a detectable label, such as a radio-label or fluorescent label.
• the target analyte may be indirectly labeled, for example, using an anti-label/enzyme conjugate which is then contacted with an enzyme substrate to produce a signal that can be detected.
• an anti-label/enzyme conjugate which is then contacted with an enzyme substrate to produce a signal that can be detected.
• a significant advantage of indirect detection is that intermediate molecules can be conjugated to the target analyte to amplify the number of signals per target analyte bound to the capture molecule.
• the present invention provides improved methods for signal amplification that reduce interference caused by non-specific binding of amplification polymers.
• the present invention relates to methods for detecting an analyte, such as a DNA polymorphism, in a sample.
• An analyte-specific capture label is conjugated to an amplification complex, which comprises a plurality of amplification polymers that are on a polymer or other macromolecule.
• Amplification polymers are also known in the art as “binding sites.” Each polymer or macromolecule has a plurality of amplification groups, such as amine groups or other functional binding groups.
• a detectable label complex is bound to one of the amplification polymers, which can then be detected.
• Amplification polymer complexes may be, such as biotin, will bind to substantially all of the available amine groups on the polymer or macromolecule.
• the detectable label will then either directly or indirectly produce a signal that can be detected.
• the unbound amplification polymers are bound with a capping compound to reduce non-specific binding of the amplification complex.
• the detectable labels are then bound to the to which an analyte is conjugated are separated from the detection labels to which an analyte is not conjugated. If the detectable labels are detected, then the presence of the analyte is inferred.
• the unbound amplification polymers are amine groups on a polymer.
• the capping compound could be an acylating compound that interacts with the amino group and converts it into an acetyl group, which is more stable and less likely to nonspecifically bind to other molecules in the assay.
• the methods of the invention provide a novel approach to amplification of a detectable signal conjugated to an analyte, such as a nucleic acid analyte.
• the invention provides methods for amplification of a detectable signal conjugated to an analyte in high salt concentrations typically used in nucleic acid diagnostic assays.
• the amine groups of the amplification polymer are reacted with an acetylating compound in a salt solution under conditions of ionic strength greater than about 0.5M, to produce an amide group.
• the capping of the amine group by acetylation provides a neutrally charged, water soluble complex.
• Salt solutions compatible with nucleic acid detection are well-known to those in the art.
• the salt solution comprises a salt that is monovalent.
• the salt is selected from the group consisting of NaCl and LiCl.
• the improved methods comprise the step of reacting the amplification polymers with a capping compound that specifically binds the amplification polymers with greater affinity than the detectable label complex. In other embodiments, the improved methods further comprise the step of combining the amplification polymer with a solvating compound.
• the methods of the present invention may be practiced by first capturing an analyte of interest on a solid support.
• a capture molecule that specifically or selectively binds the analyte of interest is first attached to a solid support.
• the present invention can also be practiced with or without a solid support. Without a solid support, for example, a capture label binds to the analyte and an electrophoretic separator can be used to separate bound analyte from unbound analyte.
• a solid support such as a chip, may be more cost-effective and accurate.
• Solid supports include any material that can be used to immobilize an analyte-specific capture label for use in diagnostic tests and in separation procedures.
• Natural or synthesized materials which have or have not been modified chemically, can be used as the solid support, in particular polysaccharides such as cellulose-based materials, for example paper, cellulose derivatives such as cellulose acetate and nitrocellulose, dextran; polymers such as vinyl polychlorides, polyethylenes, polystyrenes, polyacrylates, polyamides, or copolymers based on aromatic vinyl monomers, alkyl esters of alpha-beta unsaturated acids, esters of unsaturated carboxylic acids, vinylidene chloride, dienes or compounds exhibiting nitrile functions (acrylonitrile); polymers of vinyl chloride and propylene; polymers of vinyl chloride and vinyl acetate; copolymers based on styrenes or substituted derivatives of styrene; natural fibers such as cotton and synthetic fibers such as nylon
• the solid support according to the invention can be, in the forms which are customarily suitable, for example, in the form of a chip, microchip, microtitration plate, a sheet, a cone, a tube, a well, beads, particles or the like.
• the choice of a support material can be made, in each particular case, on the basis of simple routine experiments.
• oligonucleotide probes for use in detecting specific nucleic acid sequences in a target nucleic acid.
• oligonucleotides may be immobilized to a solid support by covalent attachment. See, e.g., PCT patent publication Nos. WO 89/10977 and 89/11548. See Chee et al., U.S. Pat. No. 5,837,832. See Strategies for Attaching Oligonucleotides to Solid Supports, Eric J. Devor and Mark A. Behlke, Integrated DNA Technologies (2005).
• the present invention can be used with all of the above methods.
• the present invention provides a method for enhancing a signal in a detection assay by using multivalent interactions between an analyte and a detectable label. Although multivalent interactions have been previously used in other applications, the present invention provides a novel application of multivalent interactions to enhance the signal in a detection assay.
• the invention provides a multivalent bridge conjugate which comprises a complex of one or more molecules, one of which specifically binds to an analyte of interest.
• One or more of the molecules in the complex further serves as a substrate for the binding of a detectable label complex comprising an amplification molecule having a plurality of multivalent binding sites that specifically bind to the non-analyte-specific binding sites of the multivalent conjugate and a plurality of detection conjugate binding groups to which is bound a molecule having a detectable label or that can be used to produce a detectable label.
• the multivalent bridge conjugate may be either directly bound or indirectly bound to the analyte.
• the amplification polymer binds to one or more non-analyte-specific binding site of the multivalent bridge conjugate, if present on the solid support.
• the multivalent binding sites on the amplification polymer are present at a density sufficient to enable two or more separate multivalent binding sites of one amplification polymer to bind to two or more non-analyte-specific binding sites of one multivalent bridge conjugate, if present on the solid support.
• the present invention provides novel complexes for amplifying a signal in a diagnostic assay for a nucleic acid analyte.
• the complexes of the invention may, for example, comprise (i) an amplification polymer bound to an analyte, such as a nucleic acid, (ii) wherein the amplification polymer comprises a plurality of binding sites that bind to a detectable label complex.
• the binding sites may comprise, for example, amine groups to which a biotin molecule (linked to a detectable label or molecules capable of producing a detectable label) can bind.
• the amine groups may also be capped to prevent non-specific binding of other molecules.
• the amine groups may be capped by reacting them with an acetylating compound to produce non-reactive amide groups.
• the analyte-specification detection complex will, in some preferred embodiments, be neutrally charged and water soluble.
• the multivalent bridge conjugate comprises a plurality of binding sites having substantially equivalent binding specificity or binding affinity.
• the multivalent bridge conjugate may be, for example, an antibody, a polyvalent antibody, a multi-subunit protein, a chimeric protein, a glycoprotein, an allosteric aptamer, or a multimeric aptamer.
• the multivalent bridge conjugate may be a unmodified streptavidin molecule.
• the multivalent bridge conjugate may be a protein with repeating subunits. Proteins having repeating subunits include, for example, ferritin and the hepatitis B surface antigen (HBsAg).
• the multivalent bridge conjugate may also comprise an IgG, IgE or IgM antibody.
• the multivalent bridge conjugate may also be an oligosaccharide.
• an analyte of interest is bound to a detection complex that specifically binds to the analyte.
• Appropriate analytes include any substance for which there exists an analyte-specific binding molecule that can be chemically conjugated to other compounds typically used in chemical or biological assays.
• the analyte-specific detection complex may comprise may be a protein or polypeptide molecule, a carbohydrate, a polynucleotide molecule, or an organic or inorganic compound.
• the capture label may be an antibody, a lectin, a DNA repressor protein, a stereospecific receptor-protein, a high affinity enzyme, a sequence specific polynucleotide binding protein, avidin, streptavidin, a hormone or a complementary polynucleotide sequence.
• Target molecules may be any inorganic or organic species that is capable of producing an affinity with a detecting agent.
• analytes that have been disclosed in the prior art are: proteins, lipids, carbohydrates, phospholipids, fats, nucleotides, nucleosides, nucleoside bases, polynucleotides, polypeptides, cancerogenic agents, drugs, antibiotics, pharmaceutical agents, controlled substances, polymers, silicones, organometallic compounds, heavy metals, metal-protein complexes, toxic inorganic salts, and other agents or compounds produced by or having an effect upon a biological organism or material derived from such molecules.
• the present invention could be used with any of the examples from the prior art.
• binding of the multivalent bridge conjugate to the amplification polymer is detected by reacting the amplification polymer with a detection conjugate comprising a detectable label.
• multivalent bridge conjugate to the amplification polymer is detected by reacting an enzyme bound to the amplification polymer with a substrate that produces a product having a detectable label.
• the detection complex may include an analyte-specific molecule, such as an antibody, a lectin, a DNA repressor protein, a stereospecific receptor-protein, a high affinity enzyme, a sequence specific polynucleotide binding protein, avidin, streptavidin, a hormone, a complementary polynucleotide sequence, or some other molecule.
• an analyte-specific molecule such as an antibody, a lectin, a DNA repressor protein, a stereospecific receptor-protein, a high affinity enzyme, a sequence specific polynucleotide binding protein, avidin, streptavidin, a hormone, a complementary polynucleotide sequence, or some other molecule.
• Capture molecules may include, for example, proteins (such as receptor molecules or ligands that bind to a specific cognate molecule), oligonucleotides that specifically hybridize to a complementary polynucleotide sequence, or any other molecule known to bind to a cognate molecule with a high degree of specificity.
• Methods for attaching capture molecules to a solid support are well-known to those skilled in the art, and can be readily selected, as appropriate. See, e.g., Strategies for Attaching Oligonucleotides to Solid Supports, Eric J. Devor and Mark A. Behlke, Integrated DNA Technologies (2005).
• An especially preferred method for detection of target molecules is based upon the foregoing preferred arrangement, but includes a second bridging component.
• the complex i.e., avidin or streptavidin-(biotin ligand)-visualization polymer, is used to complex with a biotin labeled second antibody.
• the second antibody is a general reagent for the first antibody detecting agent which in turn is specific for the target.
• the first antibody is incubated with the target to form an antigen-antibody conjugate.
• the second antibody is incubated with this conjugate.
• the amplification molecule is added which binds to the second antibody and enables detection.
• the detecting agent is a complementary polynucleotide sequence and the target is the corresponding native polynucleotide sequence which will hybridize with the complementary sequence.
• the detecting agent and the visualization polymer are labeled with a biotin or iminobiotin group.
• a complex of avidin or streptavidin-(biotin ligand)-amplification molecule is formed.
• the labeled polynucleotide detecting agent is added to the complex biological mixture containing the native polynucleotide sequence to be detected. Hybridization is allowed to take place, then the complex is added which binds to the hybridized and labeled polynucleotide detecting agent and which provides visualization.
• Multivalent conjugates can be synthesized as described, for example, by Ooya et al (J. Controlled Release (2002) 80: 219-228) in different applications relating to a model system for drug targeting and receptor mediated drug delivery.
• Ooya et al. describe binding of biotin-polyotaxane polymer binding to streptavidin, using a modified polymers containing 11, 35, or 78 biotins per backbone.
• SPR surface plasmon resonance
• the polymer with 78 biotins had an off rate that was 7.5 and 30 times slower than the 35 biotin and 11 biotin polymer, respectively, leading to improved affinity constants with the 78 biotin backbone.
• IgM (on surface)-IgG (secondary in solution) pair was used, the sensitivity was worse (5 ng/mL), but worked at higher HBsAg concentrations (>10 ng/mL).
• the amplification polymer may further comprise a dextran copolymer having a plurality of oligosaccharide chains.
• a dextran copolymer having a plurality of oligosaccharide chains For example, Yoshitani and Takasaki (Analytical Biochemistry (2000) 277:127-134) describe the conjugation of 30 to 180 oligosaccharide chains on a dextran backbone (AsFet-oligo-Dex), which may be used in conjunction with an amplification polymer and a detectable label complex, as described herein.
• multivalent interactions may be facilitated with the use of antibodies having multivalent binding specificity, as described by Zuckier et al (Cancer Res (2000) 60:7008-7013).
• the detection conjugate binding group may be biotin, fluorophores, sugars, nucleotides, and peptides.
• the amplification polymers comprise from about 5 to about 1000 detection conjugate binding groups. In preferred embodiments, the amplification polymers comprise from about 20 to 500 detection conjugate binding groups. In more preferred embodiments, the amplification polymers comprise from about 50 to about 200 detection conjugate binding groups.
• Suitable detection conjugate binding groups include those that do not alter the affinity of the detection conjugate binding groups for the multivalent detection conjugate as a result of the conjugation chemistry used to attach the capture groups to the amplification polymer.
• the detection conjugate binding groups are bound to the amplification polymer via linkage groups having a length ranging from about 14 to about 2000 daltons.
• the detection conjugate binding groups are bound to the amplification polymer via linkage groups comprising, for example, —CH 2 linkages ranging from 1 to 200 units
• the methods of the invention further contemplate the use of an amplification molecule conjugated to the target analyte of interest.
• the amplification molecule comprises, for example, a plurality of amplification polymers.
• Amplification polymers perform the function of providing, for each analyte, multiple bindings sites for a detectable label. Because each analyte is conjugated to multiple binding sites to which a detectable label can be bound, rather than just one binding site for each analyte, the signal associated with each analyte is multiplied or amplified.
• Amplification polymers are typically in the form of macro-molecules, such as polymers, that have multiple binding groups to which other molecules or complexes can bind and be used as a binding substrate for a detectable label or some other signal.
• amplification polymers may be comprised of a biotinylated biomolecule such as an enzyme or protein.
• biotinylated biomolecules are known and available to those skilled in the art.
• biotinylated biomolecules include biotinylated lectins, antibodies, mitogens, DNA, RNA, tRNA, rRNA fragments, nucleosomes, membranes, membrane proteins, glycoproteins, synthetic peptides.
• the polymer or other macromolecule used in the amplification complex can come in many different forms.
• the reactive chemical groups or backbone moieties of polymer subunits have been used to link the detectable label to the polymer or other macromolecule.
• the unit was a protein and was found to contain a dipeptide side chain ending with cysteine
• the mercaptan group of the cysteine was cross-linked to cysteine of another similar protein by reaction with bis (N-butylenylmaleimide).
• the groups and moieties identified may include amine groups, mercaptan groups, carboxyl groups, hydroxyl groups, sugar groups, carbohydrate groups, ester groups, lipid groups, and amide bonds, labile carbon-carbon bonds and carbon-hydrogen bonds.
• Other measurements such as the relation of derivatization and site activity, relation of pH and site activity and type of site reaction produced in the case of an enzyme will help determine a priority for the functional groups based upon the probability of their presence within the vicinity of the active site.
• a typical ranking of priority would be: 1) an epsilon or primary amine group, 2) a sugar group, 3) a carboxyl group, 4) a mercaptan group, 5) a hydroxyl group, and 6) a lipid group.
• Tagged natural or synthetic polypeptide, polyol, polyolefin or carbohydrate have had amplification polymers which are substantially less sensitive to the chemical group/backbone moiety bonding arrangement.
• the fluorescent group, dye, luminescent group, radioactive group or electron dense group which acts as the tag typically have not been subject to variations in activity when adjacent chemical groups or backbone moieties are directly bonded or indirectly linked with coupling agent.
• the prior art has shown that if the tag is to be converted to an active group after the polymer-analyte conjugation is made, then the position of the chemical group or backbone moiety linkage should not interfere with the conversion.
• the amplification complex will be conjugated to multiple detectable labels, either directly interbonded or cross-linked by a coupling agent.
• the structural and functional character of the polymer will be similar to that of the monomer units. The number of units per polymer will depend upon the extent of coupling, the stability of the resulting polymer, the reactivity of the chemical groups or backbone moieties relative to the polymer chain length and the position of the groups or moieties along the unit backbone.
• the number of units incorporated into the polymer may vary from as few as two to thousands per polymer. Higher multiples have been possible when the polymer chain length is not of an order which will render the polymer extremely insoluble in aqueous solution or will be extremely susceptible toward mechanical cleavage.
• the present invention can be used in a similar fashion.
• the polymer may be linear or it may be branched. There may be single or multiple coupling between two adjacent units. Coupling may occur at any point along the unit chain so that adjacent units may lie end to end, or may partially or fully overlap. As a result, the three dimensional structure of the polymer may have all of these features. It may be linear, but more typically, it will be a combination of linear and branching units. Partial overlap will typically occur and multiple coupling will also be present.
• the coupling agent linking units together generally is derived from a bifunctional or multifunctional organic cross-linking reagent.
• the term coupling agent has indicated the group in its coupled form with a chemical group or backbone moiety.
• the term cross-linking reagent has been used to indicate the chemical form of the agent before it is reacted with a chemical group or backbone moiety.
• the choice of the coupling agent/cross-linking reagent has depended upon the choice of the reactive chemical group or backbone moiety to be coupled and the agent chain length which would avoid intraunit interference within the polymer. See “Reagents For Organic Synthesis”, L. Fiezer, M. Fiezer, Vol. 1-8, Wiley & Son; “Cross Linking Reagents” (1980 Ed.), Pierce Biochemical Reagent Catalog, Pierce Chemical Co., Rockford Ill. and references therein, or “Advanced Organic Chemistry” J. March, McGraw Hill (1968).
• the amplification molecules and complexes of the present invention detect and chemically amplify the presence of minute quantities of inorganic or organic target molecules which may be found in biological material. Generally, the detection is based upon interaction between the polymer, its complex and the target molecule to be detected.
• the polymer is carried in a complex carrying arrangement which can bind with specific target molecules and exclude others. Quantitative determination of the target is made by measuring the amount of polymer present in the association formed between the target molecule and carrying arrangement. Signal amplification is provided by the multiple units in the polymer in each association.
• the units of the polymer are an important feature providing visualization of the target carrying arrangement association.
• the units can contain visualization tags or can react with a substrate which can be utilized as a means for quantitative measurement. This measurement may be accomplished by production of a readily identifiable substrate product or production of a spectroscopic signal, as well as other, similar types of nondestructive quantitative analytic methods for measurement.
• the visualization will be based upon the production of color, fluorescence, luminescence, radioactivity, high electron density as well as other forms of spectroscopic measurements.
• the units When the units are enzymes they can generate products which are capable of producing such spectroscopic measurement. For example, they may catalyze reaction of substrates to produce colored, fluorescent, luminescent, electron dense or radioactive products.
• the tagged units may be directly utilized as tools for spectroscopic measurement.
• the natural or synthetic polypeptides, polyols, polyolefins or carbohydrates may be tagged with chemical groups which have coloration, fluorescent, luminescent, electron dense or radioactive properties. These may then be used for spectroscopic measurement.
• Enzymes and tagged polypeptides, polyols, polyolefins or carbohydrates possessing the foregoing properties are well-known as means for spectroscopic quantification.
• the enzymatic substrate or tag When placed in an appropriate spectrometer, the enzymatic substrate or tag will cause a spectrographic change which will indicate the quantity of target present. This process is commonly referred to as visualization and the spectral change is termed the signal produced by the visualization group (the substrate or tag).
• the quantity of target to be detected usually will be minute and if the signal from the complex-target association were produced on an equivalent basis, it also would be extremely weak.
• the carrying arrangement and its visualization polymers chemically amplify the signal so that minute quantities of target will produce a strong, readily determined signal. Amplification is achieved by the polymer because it comprises multiple visualization units. The signal provided by each unit is maintained by the polymer. Consequently, its signal is the sum of the signals of its units.
• the carrying arrangement may contain multiple numbers of polymers. Although it is not necessary, this multiple arrangement is preferred since it provides further amplification.
• the visualization polymer of the invention comprises multiple visualization units monomer directly bonded together or indirectly linked together by a coupling agent bonded to chemical groups or backbone moieties of the units.
• Each unit also possesses a site or sites which provide the visualization signal. That is it may be a site for enzymatic action or a site to which a visualization tag or tags are attached.
• the visualization signal activity of the polymer depends upon production of a signal by each unit. Accordingly, the visualization site or sites should be substantially preserved in its or their original form so that the site activity is not substantially decreased. It follows that chemical modification of the units should be conducted in a manner which does not substantially affect the site or sites.
• the direct bonding or coupling agent linkage should join chemical groups or backbone moieties of the units which are at least one atom and in some embodiments at least 3 to 5 atoms away from the visualization site or sites.
• the choice of chemical groups or backbone moieties for direct bonding or linking with coupling agent should be limited to those which are not present within the site or which are not necessary for site conformation and three dimensional configuration. This choice will be more important for enzyme proteins than for tagged natural or synthetic polypeptides polyols, polyolefins or carbohydrates; however, interference with the production of tag fluorescence, luminescence, coloration, radioactivity or high electron density should also be avoided.
• these site preservation requirements may be met in several ways. If the types of biochemical substructures or chemical residues making up the monomer structure are known, then one which is not part of the visualization site may be chosen as the structure containing the reactive chemical groups or backbone moieties for coupling. Usually, however, a semi-empiric method will be used for choice of the appropriate reactive chemical groups or backbone moieties.
• the unit backbone substituted groups and functional structures such as sugar groups, lipids, oligomer side chains and the like which are not necessary for visualization site action will be identified. Typically, this would be determined by removal modification or modification of such substructures and study of the activity of the resulting product.
• Chemical groups or backbone moieties present primarily within these substructures may then be used for direct bonding or indirect linking with the coupling agent.
• the sugar groups of a glycoprotein which are not necessary for enzymatic activity can be oxidized to dialdehyde groups and reacted with a hydrazine coupling agent to form the visualization polymer.
• the chemical sequence of the unit such as the amino acid sequence of a protein
• this may also be utilized to guide direct bonding or indirect linking Analysis of the sequence for the active site as well as the three dimensional configuration will show which unit structural subunits are not essential to functioning of the site and/or not present within it.
• the reactive chemical groups or backbone moieties of these subunits may be used for bonding or linking with the coupling agent.
• the unit is a protein and it is found to contain a dipeptide side chain ending with cysteine
• the mercaptan group of the cysteine may be cross-linked to cysteine of another similar protein by reaction with bis (N-butylenylmaleimide).
• the reactive chemical groups and backbone moieties of the unit can be determined by appropriate spectrographic and chemical analysis. These include techniques such as NMR, IR, chemical derivatization, electrophoresis, osmometry, amino acid analysis, elemental analysis, mass spectrometry and the like.
• the groups and moieties identified may include amine groups, mercaptan groups, carboxyl groups, hydroxyl groups, sugar groups, carbohydrate groups, ester groups, lipid groups, and amide bonds, labile carbon-carbon bonds and carbon-hydrogen bonds the like [JO needs to clarify this part based on RJ's disclosure] .
• a typical priority will be 1. an epsilon or primary amine group, 2. sugar group, 3. carboxyl group, 4. mercaptan group, 5. hydroxyl group, 6. lipid group. If derivatization of amine groups such as those of lysine residues produces a derivatized product devoid of site activity, then the foregoing priority will change and the amine group will be last.
• the units having visualization sites which are very sensitive to the chemical group/backbone moiety bonding arrangement are enzymes.
• the catalytic site typically will have a conformation closely fitting the substrate and chemical modification which disturbs the three dimensional configuration of the catalytic site may adversely affect the activity of the polymer.
• enzyme site activity can be preserved.
• the enzyme catalytic site may be protected during bonding or linking by reversibly binding it with substrate.
• the units may be any enzyme which will react with an appropriate substrate to produce a colored, fluorescent, luminescent, electron dense or radioactive product. Also, the enzyme may react with a colored, fluorescent or luminescent substrate and quench it. The production or quenching of color, fluorescence or luminescence may result from direct enzyme catalysis or the enzyme may produce an intermediate which enters into a chain of reactions to produce or quench color fluorescence or luminescence.
• the enzyme will act to immobilize it. This may be accomplished by rendering the substrate insoluble, chemically reactive toward the enzyme or otherwise generating an immobilizing physical characteristic. With this type of visualization polymer, the quantity of radioactivity immobilized by the enzymatic reaction or an electron microscopy determination of the quantity of electron dense material present will allow analysis of the minute quantity of target.
• enzymes include peroxidase, alkaline or acidic phosphatase, galactosidase, glucose oxidase, NADPase, luciferase, carboxypeptidase and the like.
• the units may also be natural or synthetic polypeptides, polyols, polyolefins or carbohydrates which are tagged. These may be based upon a polyamide backbone, a polyether backbone, a polyvinyl backbone, or poly (sugar) backbone.
• the amino acid or diamine compound and dicarboxylic acid compound used to make the backbone may be nonfunctional, i.e., composed of a methylene unit chain ending in the appropriate functional groups, or it may be substituted with groups which would provide side chain functionality. Examples would include glycine, alanine, serine, lysine, aspartic acid and the like as amino acids.
• diacids and diamines include arylene or alkylene dicarboxylic acid having at least 6 carbons in the arylene group or 1 to 20 carbons in the alkylene group, and arylene or alkylene diamines having at least 6 carbons in the arylene group and 1 to 20 carbons in the alkylene group.
• examples will include poly(3-aminopropionic acid), polyglycine poly(glycyl-lysine), poly(N-(aminohexyl)alipic amide), poly(N-(aminobutyl)terephthalamide) and the like.
• polyethers epoxides and/or oxacyclic compounds with or without hydroxyl substitution can be used as backbone building blocks. Acidic condensation will couple the oxide compounds.
• the polyols may have a poly(vinyl) backbone with hydroxylic substitution. These may be formed by vinyl/free radical polymerization of alkyl alcohol, butene diol and the like.
• vinyl compounds with or without chemical group substitution may be used as backbone building blocks.
• Vinyl/free radical polymerization of such compounds as acrylamide, acrylic acid, maleic acid, alkyl sulfide, acrylonitrile, methyl acrylate, hydroxyethyl acrylate, alkenyl amine, acrolein, etc. will produce the polyolefin monomers.
• glycosidic linking through hemi-ketal condensation of simple sugar building blocks can be used as the carbohydrate backbone formation process.
• Carbohydrates such as methoxy cellulose, poly(glucose) starch, dextran, polymaltose, amylose, etc. are examples.
• the chemical tags include the known, colored, fluorescent, luminescent, radioactive and electron dense probes which will chemically bond with substituents present in a natural or synthetic polypeptides polyols, polyolefins and carbohydrates. These include probes with carboxylic acid derivative substituents, sulfonic acid substituents, imino ester substituents, maleimide substituents, aldehyde substituents, azide substituents and amine substituents which will react with the appropriate functional group of the unit as outlined in Scheme I and Table 1.
• the probes will be monofunctional rather than difunctional so that they may react only once with a unit chemical group or backbone moiety.
• color tags include azido indigo dye, and congo red with sulfonyl chloride substitution.
• fluorescent tags include fluorescein with an azido or sulfonyl chloride reactive substituent, 3-azido-(2,7)-naphthalene disulfonate and rhodamine.
• radioactive tags include wood reagent (methyl p-hydroxybenzimidate) HCl which can be iodinated, and p-iodobenzenesulfonyl chloride.
• electron dense tags include collodial gold, colloidal silver, ferritin, metal binding proteins and reactive lead salts.
• Isolation and purification of the visualization polymer of the invention may be accomplished by known techniques used for polymer isolations. These include dialyzation, lyophilization, chromatography, electrophoresis, centrifugation, precipitation by electrolyte adjustment or solvent lipophilicity and the like.
• the carrying arrangement of visualization polymer and detecting agent may be direct or indirect.
• the direct carrying arrangement will have the detecting agent covalently bonded to the visualization polymer by a bifunctional or multifunctional cross-linking reagent.
• the bonding will follow Scheme I and method given for linking the visualization units of the polymer. These methods are generally known; for example see K. Peters, et. al., Ann Rev. Biochem., 46, 523-551 (1977); F. Wold, “Methods In Enzymology XXV”, pp 623-651 (1972) or M. Das, et al., Ann Rev. Biophys. Bioeng., 8 165-193 (1979).
• covalent linkage with chemical groups or backbone moieties of the detecting agent should take place in a region of the agent which will not interfere with its ability to detect the target. This may be determined by any of the methods given above, especially the emperic method.
• the indirect carrying arrangement may be of two types.
• the detecting agent may be multivalent and have an affinity for the visualization polymer as well as the target.
• it may be accomplished by employing a multivalent antibody which cross-reacts with the units of the visualization polymer and by utilizing the appropriate amount of antibody and polymer so that at least one of the affinity sites of the antibody remains open.
• the visualization polymer may also be bonded to a ligand which complexes with a multivalent detecting agent. This will accomplish the same kind of carrying arrangement.
• an intermediate ligand binding compound interspersed between the detecting agent and the visualization polymer. It will display a high affinity for specific ligands and will include an antibody, lectin, avidin, streptavidin, a DNA repressor protein, a high affinity enzyme, a sequence specific polynucleotide binding protein or a complementary polynucleotide sequence.
• the agent and polymer will be correspondingly labeled with the appropriate ligand.
• the ligand may be joined to the detecting agent and polymer through a linker similar to a bi or multifunctional cross-linking reagent. Also, the ligand may be substituted for a reactive group of the bi or multifunctional cross-linking reagent.
• the ligand may be covalently bonded directly to the detecting agent and polymer. That is, the ligand may be bonded to a chemical group of the polymer and detecting agent which may include an amine group, mercaptan group, carboxylic acid group, hydroxy group, aldehyde group or a C—H group.
• a chemical group of the polymer and detecting agent which may include an amine group, mercaptan group, carboxylic acid group, hydroxy group, aldehyde group or a C—H group.
• the procedures and reagents for the appropriate reaction will be chosen depending upon the kind of reactive group present on the ligand.
• ligands such as biotin, iminobiotin, polynucleotide sequences, enzyme substrates, sugars, haptenes such as 2,4-dinitrophenol, 2,4-dinitrophenylalkylcarboxylic acid having from 1 to 20 carbons in the alkyl group, and carboxylic acid derivatives thereof
• haptenes include 2,4-dinitrophenylalkylamine having from 1 to 20 carbons in the alkyl, phenylarsenate, inistol and trinetrobenzene.
• An example of this type of carrying arrangement and complex is based upon use of a complementary strand of polynucleotide as a detecting agent for a specific native polynucleotide sequence.
• Avidin or streptavidin is used as the ligand binding compound and a functionalized biotin or imino biotin derivative is used as the ligand. Bonding the biotin or imino biotin to the visualization polymer and polynucleotide detecting agent may be accomplished directly or through use of a linker group.
• the complex includes avidin or streptavidin-(biotin or iminobiotin ligand)-visualization polymer.
• the carrying arrangement in addition includes the biotin or imino biotin labeled polynucleotide detecting agent.
• the method of the invention utilizing this example can be practiced as follows.
• An isolated double strand of native polynucleotide to be detected such as viral DNA, is broken or nicked with a DNAase at random points along each strand.
• Labeled nucleotide monomers are then translated into the nicks using a polymerase enzyme and the other associated strand as a template.
• the complementary strands can be directly labeled with biotin label.
• the labeled complementary pair of polynucleotide strands are then denatured and mixed with a denatured mixture of unknown native polynucleotides, suspected as containing the polynucleotide to be detected. If it is present, hybridization will occur and the labeled double strand may be visualized with the polymer complex.
• a second example of a complex is derived from the methods given in the Background for PAP or ABC complex methods or according to Langer et al., supra.
• avidin or streptavidin is used as the intermediate ligand
• an antibody, lectin, or a sequence specific polynucleotide binding protein is used as the detecting agent and a biotin or imino biotin compound is used as the ligand complexing the visualization polymer and detecting agent with avidin or streptavidin.
• the biotin or imino biotin compound may be directly coupled with amine or hydroxy groups of the polymer and agent through the use of amide bond or ester bond forming coupling reagents respectively. It may also be coupled through a linker group such as that described above.
• the linker group is similar to the bifunctional cross-linking reagent except that one of the two reactive groups will be an amine or acylhydrazide group which is coupled with biotin or iminobiotin.
• the visualization polymer of the present invention may be used to detect minute quantities of target molecules. These molecules may be found in biological material such as tissue and fluid as well as in artificial or synthetic systems. Examples include blood, lymph, urine, feces, organ tissue such as lung, liver, skin, kidney and the like, microorganisms, plant tissue, cultured cells, hybrid cells, cells with recombinant DNA, synthetic mixtures of polypeptides, immobilized enzyme systems, synthesized DNA and other biological material.
• the target molecules may constitute any inorganic or organic species which is capable of producing an affinity with a detecting agent.
• Preferred targets will be found in the foregoing biological material and systems. Examples include proteins, lipids, carbohydrates, phospholipids, fats, nucleotides, nucleosides, nucleoside bases, polynucleotides, polypeptides, cancerogenic agents, drugs, antibiotics, pharmaceutical agents, controlled substances, polymers, silicones, organometallic compounds, heavy metals, metal-protein complexes, toxic inorganic salts, and other agents or compounds produced by or having an effect upon a biological organism or material derived therefrom.
• the visualization polymer for the foregoing detection purposes has advantage since it allows detection of extremely minute quantities of target molecules. It may be employed in medical diagnostic laboratory as an analytical technique for identification of biological products in fluids and tissues which are indicative of a disease state. These would include for example, abnormal amounts of growth hormone, the presence of human gonadotropin indicating cancer, detection of viral invasion, quantification of hormone and regulatory enzyme levels. Also, it may be employed to perform normal fluid and tissue chemistry analyses and may be employed in the biochemical research laboratory as a tool for identification of biochemical substances.
• the visualization polymer may be used in synthetic protein or polynucleotide work to identify synthesized, semisynthetic or native proteins and synthesized, recombinant or native polynucleotides. Applications will be found in the course of preparative or bulk work to produce useful proteins such as insulin, interferon, ACTH, gonadotropin, oxytocin, pituitary hormone, LH, FSH and the like by such techniques as recombinant DNA or hybridomas.
• the carrying arrangement of detecting agent and visualization polymer complex will be the form for use to perform the foregoing analyses. Since the polymer will provide multiple signals from the carrying arrangement association with the target, chemical amplification will result. In the preferred form of the carrying arrangement wherein a complex of polymer and ligand binding compound is employed, the signal amplification by the polymer will be further increased by multivalent liganding of multiple numbers of polymer to each molecule of detecting agent. Accordingly, in the preferred embodiments employing an antibody or complementary polynucleotide sequence detecting agent, biotin or immobiotin labels, on the agent and polymer, and an avidin or streptavidin, detection of femtomole (10-15) quantitites can be achieved.
• the polymer, complex and carrying arrangement of the invention may be formulated as an integral part of a solid or liquid detection system and kit.
• Colorimetric, fluorescent, luminescent and radioactive systems may be prepared in this manner.
• Such systems and kits would include the detecting components, i.e., the polymer, its complex with a ligand, a ligand binding compound, and the detecting agent as well as the appropriate chemicals, reagents and solutions in metered amounts and standardized concentrations also.
• the system and kit will contain the chemicals, substrate and reagents necessary for performing this analysis.
• Fluorimetric, lumimetric and radiometric analyses may be performed in a similar fashion.
• the intensity of fluorescence, luminescence or radioactivity produced by the polymer in the carrying arrangement associated with the target will be measured by the appropriate electronic machine. Necessary reagents and chemicals will also be present. Metered amounts of components will be employed so that the intensity value may be correlated with the quantity of target using a standard Beer's Law mathematical formula.
• a concentration of detecting agent-visualization polymer complex will be used in the test solution which is sufficient to associate with all the target to be detected.
• the concentration will provide an excess amount.
• the target may be grossly separated from other material by sedimentation, by centrifugation, or otherwise separated by such techniques as high pressure liquid chromatography, gel permeation chromatography, electrophoresis, precipitation, thin layer chromatography, paper chromatography or similar techniques. However, this is not necessary for the purposes of this invention.
• the signal producing reaction will be initiated by forming the target-detecting agent conjugate followed by forming the visualization polymer-detecting agent associative arrangement and measuring the visualization signal from this arrangement. Comparison of the signal intensity with a standard graph will yield the quantity of target.
• Other techniques such as conjugate-complex exchange, which are known in the field of immunoanalysis, may also be used.
• Aptamers have some advantages over antibodies, which may not be able to detect low concentrations of analyte if the binding affinity between an antibody and an analyte are too low. Aptamers have been developed to bind specifically to target molecules for purposes of identifying the molecules for disease analysis.
• PCT application number WO 99-07724, by Nextar Pharmaceuticals, Inc. authored by Heilig and Gold, “Nucleic Acid Ligands for Blood-Brain and Cerebrospinal Fluid-Blood Barriers by Tissue SELEX,” published Feb.
• the capture molecule may be directly conjugated with the amplification complex via direct covalent or non-covalent bonding with the polymer, or indirect bonding through an intermediate covalent or non-covalent binding group.
• the capture label may also be conjugated to the polymer or other macromolecule through an intermediate ligand binding complex.
• the capture label acts as a ligand binding compound also and the corresponding ligand is bound to the amplification polymer.
• a first ligand is bound to the agent
• a second ligand is bound to the polymer and they are sandwiched with a ligand binding compound such that the first and second ligands function as bridges that form a complex with the compound.
• Methods for conjugating the amplification complex are well known in the prior art. Examples include use of ligands such as biotin, iminobiotin, polynucleotide sequences, enzyme substrates, sugars, haptenes such as 2,4-dinitrophenol, 2,4-dinitrophenylalkylcarboxylic acid having from 1 to 20 carbons in the alkyl group, and carboxylic acid derivatives thereof. Other examples of haptenes include 2,4-dinitrophenylalkylamine having from 1 to 20 carbons in the alkyl, phenylarsenate, inistol and trinitrobenzene. All of the foregoing examples can be used with the present invention.
• ligands such as biotin, iminobiotin, polynucleotide sequences, enzyme substrates, sugars, haptenes such as 2,4-dinitrophenol, 2,4-dinitrophenylalkylcarboxylic acid having from 1 to 20
• amplification complex may be composed of an avidin or streptavidin-(biotin or iminobiotin ligand)-polymer.
• a biotin or imino biotin compound may be directly coupled with amine or hydroxy groups of the polymer and agent through the use of amide bond or ester bond forming coupling reagents, respectively. It may also be coupled through a linker group such as that described above. The linker group is similar to the bifunctional cross-linking reagent.
• the present invention could be used with biotin or imino biotin compounds.
• detectable label examples include, but are not limited to, biotin or any derivatized form or analog thereof, or any molecule having an affinity for avidin including monomeric avidin, streptavidin, or any protein having biotin-binding properties including recombinant forms of any of the above.
• streptavidin has four binding sites for biotin; thus many examples in the prior art include a biotin-streptavidin-biotin complex.
• Patents and literature are replete with the various biotin compounds including various spacers, linking groups and the like, for use in the present applications. Nonlimiting examples can be found in M. D. Savage, et al.
• a target analyte of interest may be visualized by binding to the target analyte an amplification molecule that amplifies the number of binding sites per target analyte.
• amplification polymer is used to refer to the binding sites of an amplification molecule.
• An amplification molecule may comprise, for example, a polymer having multiple binding sites covalently linked together by polymerization or non-covalently coupled together.
• the amplification molecule binds to the analyte or an intermediate molecule via a binding site on the amplification molecule.
• Each unit of the polymer is coupled to at least one signal label, and the units are linked in a manner which preserves the intrinsic activity of the binding sites or amplification polymers of the units.
• An amplification unit can generate or produce color, fluorescence, luminescence, localization of radioactivity or localization of electron dense material.
• the units may be selected from an enzyme or a tagged natural or synthetic polypeptide, a tagged polyol, tagged polyolefin, or a tagged carbohydrate.
• each amplification molecule that binds to an analyte provides multiple additional binding sites (or “amplification polymers”) to which a detectable label can be bound, thereby providing amplification of the number of signaling events per target molecule bound to the solid substrate.
• the units may be directly linked by polymerization or indirectly linked by a coupling agent.
• the chemical groups or backbone moieties utilized for each unit of polymer will be independently selected from an amine group, an oxidized form of a 1,2-diol group, a carboxy group, a mercaptan group, a hydroxy group or a carbon-hydrogen bond.
• oxidative enzymes such as horseradish peroxidase can be used to polymerize monomer units by oxidative cross-linking.
• a coupling agent may be used, which may be derived from a bifunctional or multifunctional organic cross-linking reagent, bonds with the appropriate chemical group or backbone moiety of the units.
• the term “coupling agent” denotes the linkage group after bonding and the term cross-linking reagent denotes the linkage compound before bonding.
• Bound amplification polymers refers to those functional groups on the polymer that bind to the signal labels.
• Unbound amplification polymers on the amplification polymer such as a dextran polymer, refers to those functional binding groups that could bind to the signal labels, such as biotin, but remain unbound because under experimental conditions, chemical reactions almost never go completely to completion.
• At least some of the plurality of amplification compounds are not bound to a detectable label complex, and the plurality of amplification polymers not bound to a detectable label are reacted with the capping compound.
• the amplification polymer is selected from the group consisting of multi-valent proteins, dimerized proteins, dimerized antibodies, multimerized proteins, multimerized antibodies, and allosteric aptamers.
• the amplification polymers may be comprised of functional binding groups selected from the group consisting of amines, carboxylates, sulfhydryls, arginines, maleimides, or aldehydes.
• the amplification polymer may be selected from the group consisting of the following polymers: dextran, acrylic acid, poly (acrylamide-co-acrylic acid), poly-L-lysine, poly-L-aspartic acid, poly-benzyl-L-glutamate, poly-benzyl-L-aspartate, poly (Arg,Trp), poly (Lys,Phe), polymaleimide and poly-L-glutamic acid.
• the amplification polymer is a dextran polymer, an acrylic acid polymer, or a poly-L-lysine polymer.
• the amplification polymer is biotin-labeled dextran.
• the molecular weight of the amplification polymer ranges from between about 6,000 to about 1,000,000, or alternatively from between about 70,000 to about 500,000.
• the amplification polymer is preferably conformationally flexible.
• the amplification polymer is also preferably soluble in water from 1 fg/ml to 10 mg/mL.
• the amplification polymer is also soluble in 1M monovalent salt from 1 fg/ml to 10 mg/mL.
• the amplification polymer is conjugated to a target analyte, which may, for example, be bound to a solid substrate.
• the methods of the present invention may also employ techniques used for amplifying a signal in a diagnostic assay for a nucleic acid, comprising the steps of:
• the salt may be monovalent.
• the salt may be selected from the group consisting of NaCl and LiCl.
• the capping compounds use in the methods of the invention will not displace the detectable label complex.
• the capping compound may be a stronger base than the functional binding groups to which it binds.
• the capping compound may be an amine-reactive compound, such as a compound that converts the functional binding groups into amides or imides.
• the capping compound may be an acetylating reagent.
• Amine-reactive compounds may include compounds from one or more of the following chemical classes: N-hydroxysuccinimidyl (NHS) esters, imidoesters, aryl halides, acyl halides, isocyanates, isothiocyanates, nitrophenyl esters, carbonyls, carboxylates, and acid anhydrides.
• NHS N-hydroxysuccinimidyl
• Particular amine-reactive compounds may include, for example, any one or more compounds selected from the group consisting of NHS acetate, disuccinimidyl suberate (DSS), succinimidyl-3-(tri-N-butylstannyl)benzoate, methyl N-succinimidyladipate (MSA), mono(latosylamido) mono(succinimidyl)suberate, acetic anhydride, aryl chlorides, acyl chlorides, 2,4-dinitrofluorobenzene (DFNB), sulfonyl halides, aldehydes, 1-ethyl-3-(3-dimethylaminopropyl)-carbodimide (EDC) based activation chemistries, maleic anhydride, succinic anhydride, acetyl chlorides, benzoyl chlorides, propionyl chlorides, butyryl chlorides, and penylethanoyl chlorides.
• NHS
• the capping compound may also be selected from non-acetylating agents, such as diazoacetates, imidoesters, carbodimides, maleimides, ⁇ -haloacetyls, aryl halides, dicarbonyl compounds, sulfhydryls, and hydrazides.
• non-acetylating agents such as diazoacetates, imidoesters, carbodimides, maleimides, ⁇ -haloacetyls, aryl halides, dicarbonyl compounds, sulfhydryls, and hydrazides.
• non-acetylating compounds may be selected from the group consisting of, for example, N-ethylmaleimide, N- ⁇ -maleimidopropionic acid, N- ⁇ -maleimidocaprioic acid, iodoacetic acid, N-[iodoethyl](trifluoroacetamide), 3,4-difluoronitrobenzene (DFNB), sulfonyl halide, (ammonium 4-chloro-7-sulfobenzo-furazan)-chloride (SBF-chloride), glyoxal, phenyglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, 2-mercaptoethanol, dithiothreitol (DTT) followed by sulfhydryl chemistries, (2,4,6-trinitrobenzene sulfonic acid (TNBSA), and 2-mercaptoethanol.
• Reactive amine groups may be capped using various chemical processes.
• an iminoester salt reagent may be reacted with an amine chemical group to produces an amidine coupling agent linkage.
• the reagent may be generated from the acidic alcoholysis of the corresponding nitrile.
• the amidine formation reaction may be conducted in aqueous or polar organic solvent under mild conditions. The methods and procedures are known. See, e.g., Lockhart, et. al., Can. J. Biochem., 53, 861-867 (1975) and Pierce Biochemical Reagent Catalog and references therein, supra.
• Amine groups may also be reacted with an aldehyde reagent to form a bis Schiff base (imine) in a condensation reaction.
• aldehyde reagent examples include glutaraldehyde and other tissue fixing reagents.
• Conditions include use of polar organic solvent and mild temperatures.
• an aldehyde chemical group is reacted with amine groups and amine derivative reagents to form imine and imine derivative compounds.
• These reagents and reactions included primary amine reagents and reagents which react to form a Schiff base (imine).
• other embodiments included substituted hydrazine reagents, which react to form substituted hydrazones, and acyl hydrazide reagents, which also react to form acyl hydrazones.
• the present invention contemplates similar applications.
• capping chemistry options include maleic anhydride, acetic anhydride, succinic anhydride, N-maleimide derivatives, aryl halides, alkyl halides, aldehyde, ketone derivatives, and chemistries that create carboxylates.
• Other substituent groups may be useful for capping functional groups.
• carboxylate reactive chemistries such as diazoacetate, imidoesters, carbodimides
• sulfhydryl reactive chemistries such as maleimides (N-ethylmaleimide, N-beta-maleimidopropionic acid, N-epsilon-maleimidocaprioic acid), alpha-haloacetyls (iodoacetic acid, N-[iodoethyl]trifluoroacetamide), aryl halides (DFNB, sulfonyl halide, SBF-Chloride); arginine reactive chemistries, such as dicarbonyl compounds (glyoxal, phenyglyoxal, 2,3-butanedione, 1,2-cyclohexanedione); maleimide reactive chemistries, such as sulfhydryl
• fluorescent protecting groups such as sulfhydryls (SBF-chloride), amine (TNBSA); aldehyde reactive chemistries, and hydrazides.
• Amine-reactive compounds and compounds that are not amine-reactive may also be used, for example, acetylating reagents, such as NHS-acetate and acetic anhydride.
• acetylating reagents such as NHS-acetate and acetic anhydride.
• Aldehydes, sulfhydryls, and carboxylates may also be used to cap polymers with free reactive groups other than amine groups. Some chemistries can react with multiple groups. For example free-SH and free-NH2 can show similar reactivities depending on the pH of the solution.
• amine groups on the polymer or other macromolecule they could first be protected with a removable protecting group such as a Schiff base, i.e., condensation of the amine groups with an aromatic aldehyde such as p-methoxybenzaldehyde or benzaldehyde which could be removed with dilute hydrogen chloride in acetone.
• a removable protecting group such as a Schiff base, i.e., condensation of the amine groups with an aromatic aldehyde such as p-methoxybenzaldehyde or benzaldehyde which could be removed with dilute hydrogen chloride in acetone.
• amine protecting groups may also be used. These include dinitrofluorobenzene, t-butoxy groups and organosilanes.
• esterification can be conducted using an activated acid reagent.
• Unit residues that have esterified in this fashion have included amino acid residues of serine, threonine, hydroxylysine, tyrosine, thyroxine, hydroxyproline, carbohydrate, starch, lipid and olefinic residues with hydroxyl substitutions, including hexoses, pentoses, dextrans, amyloses, glycerols, fatty acid derivatives, methylhydroxymethacrylate, hydroxymethyl acrylate and similar compounds.
• the present invention could be used with all of the foregoing examples.
• Detectable labels may be selected from the group consisting of biotin, fluorochromes, di-nitro-phenol, and digoxigenin.
• the detectable label may be structurally integrated with a complex that is conjugated to the analyte of interest, or may be a product of the complex.
• the detectable label complex may comprise, for example, biotin molecules to which are conjugated streptavidin and other molecules capable of being used to generate a detectable signal.
• Preferred detection methods and preferred amplification molecule include polymers having multiple units of an enzyme or multiple units of a natural or synthetic polypeptide or polyolefin chemically bonded to a tag selected from a fluorescent group, a dye, a luminescent group or an electron dense group.
• Preferred enzymes include alkaline phosphatase, peroxidase, galactosidase, glucose oxidase, acid phosphatase and luciferase.
• Preferred polypeptides include polyamides of dicarboxylic acids and diamine, polyamides, oligomers and copolymers of alpha amino acids such as glycine, lysine, aspartic acid, cysteine, ornithine and the like.
• Polyolefins include polyacylamide, polyacrylic acid, polymaleic acid, poly(hydroxyethylacrylic ester) and the like. These polypeptides and polyolefins will be tagged with such groups as fluorescein, rhodamine, a diazo dye, colloidal gold, luciferin, radioactive iodine and the like.
• the detectable labels may be directly utilized as tools for spectroscopic measurement.
• the natural or synthetic polypeptides, polyols, polyolefins or carbohydrates may be tagged with chemical groups which have coloration, fluorescent, luminescent, electron dense or radioactive properties. These may then be used for spectroscopic measurement.
• the detectable labels of the units can be sites of biological activity.
• sites for enzymatic action will provide visualization when reacted with an appropriate substrate.
• the visualization sites can be utilized to generate soluble or insoluble bodies of color, fluorescence, luminescence, radioactivity or high electron density which can be measured and correlated with the quantity of target molecules detected.
• the sites may also be created chemically.
• a visualization tag selected from a fluorescent chemical group, a dye, a radioactive group, a photon emitter (a luminescent group) or an electron dense moiety will produce monomer units which can be visualized.
• the detectable label will be present at a ratio greater than one unit of detectable label per target analyte.
• the detectable label may comprise an enzyme, which may be conjugated to a polymer, such that the number of enzyme molecules conjugated to each polymer molecule is, for instance, 1 to 200, 2 to 50, 2 to 25, or some other ratio.
• the secondary amplification polymer may be a gold particle, a radioactive isotope, or a color label, e.g. a low molecular weight fluorophore, and the number of detectable labels conjugated to each polymer molecule is, for instance and not by way of limitation, 1 to 500 or 2 to 200.
• the detectable label may comprise a protein fluorophore.
• the detectable label and may be detected by numerous methods including reflectance, transmittance, light scatter, optical rotation, and fluorescence or combinations hereof in the case of optical labels or by film, scintillation counting, or phosphorimaging in the case of radioactive labels. See e.g., Larsson, 1998, Immunocytochemistry: Theory and Practice, (CRC Press, Boca Raton, Fla.); Methods in Molecular Biology, vol. 80, 1998, John D. Pound (ed.) (Humana Press, Totowa, N. J.). In some embodiments more than one type of detectable label or more than one detectable label may be employed. The present invention contemplates using all of the aforementioned embodiments.
• Isolation and purification of the detectable labels that are conjugated to the analyte may be accomplished by any one of various techniques used for polymer isolation known to those skilled in the art, including dialyzation, lyophilization, chromatography, electrophoresis, centrifugation, precipitation by electrolyte adjustment or solvent lipophilicity and the like.
• Detecting the presence of a detectable label often requires that the detection label be conjugated to some type of label that produces a signal.
• Producing the detectable signal may be performed using any of the methods in the prior art.
• chemical tags include the known, colored, fluorescent, luminescent, radioactive, and electron dense probes which will chemically bond with substituents present in a natural or synthetic polypeptide or carbohydrate. These include probes with carboxylic acid derivative substituents, sulfonic acid substituents, imino ester substituents, maleimide substituents, aldehyde substituents, azide substituents and amine substituents which will react with the appropriate functional group.
• Probes may be monofunctional rather than bifunctional so that they may react only once with a unit chemical group or backbone moiety.
• color tags include azido indigo dye, and congo red with sulfonyl chloride substitution.
• fluorescent tags include fluorescein with an azido or sulfonyl chloride reactive substituent, 3-azido-(2,7)-naphthalene disulfonate and rhodamine.
• radioactive tags include wood reagent (methyl p-hydroxybenzimidate) HCl which can be iodinated, and p-iodobenzenesulfonyl chloride.
• electron dense tags include colloidal gold, colloidal silver, ferritin, metal binding proteins and reactive lead salts. The present invention is contemplates using the foregoing methods
• Immunologic detection methods can utilize direct or indirect visualization techniques for measurement of the formed immune complex. In general, these methods visually indicate the presence of the complex through use of an entity coupled to the complex which produces a detectable, quantifiable signal such as color, fluorescence, radioactivity, enzymatic action and the like. The greater the signal intensity present per complex, the better will be the sensitivity for the presence of a minute quantity of target molecule.
• Enzymes and tagged polypeptides, polyols, polyolefins or carbohydrates are well-known as means for spectroscopic quantification. When placed in an appropriate spectrometer, the enzymatic substrate or tag will cause a spectrographic change which will indicate the quantity of target present.
• a primary antibody or specific ligand-binding protein
• a fluorochrome such as rhodamine or fluorescein which functions as the signal entity.
• Indirect immunofluorescence methods in which a primary antibody is used unmodified and it, in turn, is detected with a fluorescently-labeled secondary antibody, generally will increase the detection sensitivity.
• An additional three to five-fold enhancement in sensitivity has been reported using a “haptene-antibody sandwich” technique. See Cammisuli, et al., J. Immunol., 117,1695 (1976); Wallace, et al., J.
• the enzymatic signal of this method can occur in at least two ways. Enzymatic conversion of a soluble enzyme substrate into an insoluble, colored product permitted the direct localization of the antigen by direct macroscopic visualization, light microscopic examination, or by using other types of apparatus.
• PAP peroxidase-antiperoxidase
• the PAP complex contains two immunoglobulins (antiperoxidase antibodies) and three active peroxidase molecules, the net effect is to provide more enzyme at the antigen site with which to amplify the detection signal.
• the PAP detection system has limitations.
• the secondary “bridge” antibody has to be used at saturating levels to ensure optimal binding of the PAP complex.
• the antiperoxidase and the primary antibody should be of the same, or an immunologically cross-reacting, species so that the secondary antibody will bridge to both.
• Biotin may be covalently conjugated to amino, carboxyl, thiol and hydroxyl groups present in proteins, glycoproteins, polysaccharides, steroids and glycolipids using well established chemical reactions. See Guesdon, et. al., J. Histochem. and Cytochem., 27, 1131 (1979); Sternberger, et. al., J.
• Biotin may also be introduced into other macromolecules, such as DNA, RNA and co-enzymes, by enzymatic methods that utilize biotin-labeled nucleotide precursors. See Langer, et al., Proc. Natl. Acad. Sci. USA, 78, 6633 (1981).
• avidin may be coupled to a host of molecular species by standard chemical reactions. See Sternberger, Immunohistochemistry, 2nd Edition, John Wiley and Sons, N.Y. (1979); Nakane, et. al., J. Histochem. Cytochem., 22, 1084 (1974); Guesdon, et. al., Histochem. and Cytochem., 27, 1131 (1979); Bayer et. al., Methods Biochem. Anal., 26, 1, (1980). This allows for great flexibility in designing detection systems for use in immunology, immunopathology and molecular biology.
• Avidin-biotinylated horseradish peroxidase complex has also been used for antigen detection. Hsu, et. al., Amer. J. Clin. Path., 75, 734 (1981); Hsu, et al., J. Histochem. Cytochem., 29, 577 (1981).
• the primary antibody incubation is followed by an incubation period with a biotin-labeled secondary antibody and then with the ABC complex, formed by preincubating avidin with a titrated amount of biotinylated peroxidase.
• avidin Since avidin has four biotin-binding sites per molecule, at least three peroxidase enzymes can be added to avidin without interfering with its ability to interact with the biotinylated secondary antibody.
• the ABC detection procedure was reported to be 4-8 times more sensitive in detecting antigens in tissues than either the immunoperoxidase or the PAP detection systems.
• the ABC method is four-fold more sensitive for antigen detection using an ELISA system than either the immunoperoxidase or the PAP techniques. Madri, et. al., Lab. Invest., 48, 98 (1983).
• the sensitivity for the ABC method is limited. Typically, only 30 to 100 pg of a target molecule can be detected. This is significantly higher than the upper limit required for detection of a single molecule per cell. Limits for other less sensitive methods are even higher. Accordingly others have developed visualization methods which substantially improve sensitivity over that provided by known visualization techniques.
• the methods of the invention further comprise the step of reacting the amplification polymer and detection complex in the presence of a high molecular weight non-ionic polymer.
• the non-ionic polymers are useful in increasing the detection sensitivity of the assay by reducing background noise from non-specific binding between amplification complexes, detectable labels nucleic acids, etc.
• Non-ionic polymers include, for example, a dextran sulfate, an amino dextran, a polyvinyl pyrollidone (PVP), a polyvinyl sulfate (PVS), a polyethylene glycol (PEG), a carboxymethyl cellulose, a hyaluronic acid or a polyacrylic acid (PAA), or co-polymers such as poly(acrylic acid-co-maleic acid).
• Non-ionic polymers are obtainable in differing degrees of polymerisation, i.e. with different molecular weights.
• polyethylene glycol has a molecular weight of from about 6 kD to about 300 kD, with a molecular weight of about 40 kD being particularly useful.
• Polyvinylpyrrolidone is also useful as non-ionic polymer, having a molecular weight of at least about 40 kD, and up to about 100-750 kD.
• Dextran may be used which has molecular weight of about 200 kD, and up to about 500-1,000 kD.
• concentration of the non-ionic polymer may be, for example, from about 0.5 to about 3% by weight, and can be present as powder, lyophilisate or solution.
• kits for amplifying a detectable signal may include (i) a capture molecule that specifically binds the nucleic acid analyte; (ii) an amplification polymer adapted to be conjugated to the nucleic acid analyte, wherein the amplification polymer comprises a plurality of amine groups; (iii) a conjugation compound capable of conjugating the amplification polymer to the nucleic acid analyte; (iv) an acetylating compound capable of reaction with amine groups on the amplification polymer to create amide groups; and (v) a detectable label complex.
• kits for detecting an analyte in a sample comprising in packaged combination, (a) a multivalent bridge conjugate having an analyte specific binding site and a plurality of non-analyte-specific binding sites, and (b) an amplification polymer having a plurality of multivalent binding sites, wherein the multivalent binding sites are present at a density wherein two or more separate multivalent binding sites of one amplification polymer are bound to two or more non-analyte-specific binding sites of one multivalent bridge conjugate.
• the present invention also provides novel conjugated complexes as intermediates in the methods of the invention.
• the conjugated complexes may, for example, be prepared in advance of testing procedures for inclusion in a kit.
• Such conjugated complexes may comprise (a) a multivalent bridge conjugate having an analyte specific binding site and a plurality of non-analyte-specific binding sites conjugated to (b) an amplification polymer having a plurality of multivalent binding sites, wherein the multivalent binding sites are present at a density wherein two or more separate multivalent binding sites of one amplification polymer are bound to two or more non-analyte-specific binding sites of one multivalent bridge conjugate.
• the invention also provides, for use in conjugating in a complex, novel amplification polymers comprising a plurality of multivalent binding sites having binding specificity to a non-analyte-specific binding sites of the multivalent conjugate and a plurality of detection conjugate binding groups, wherein the amplification polymer binds to one or more non-analyte-specific binding site of the multivalent bridge conjugate, if present on the solid support.
• Chips were purchased from Inverness Medical—Biostar Inc. Surfaces were coated with 5 ug/mL of poly (Lys-Phe) in 1 ⁇ PBS, 2M NaCl pH 6 overnight. Surfaces were washed with water and then coated with 10 uM SFB in 0.1M Borate buffer pH 8.5 for 2 hours at room temperature. Chips were again washed with water, dried with a stream of nitrogen, and stored in a dry box purged with nitrogen and protected from light.
• Biotin Chip dil #1 contained four 120 nL spots of biotinylated probe.
• 5′-Hydrazide-A18 probes with 3′-biotinTEG were diluted with 5′-Hydrazide, un-modified A18 probe to a constant final concentration of 100 nM in 0.1M Sodium Phosphate pH 7.8, 10% glycerol.
• the spots were immobilized to the chip's surface using a non-contact printer, and arranged in a vertical line with the lowest concentration at the top. Each spot corresponded to 110 pM, 330 pM, 1 nM, or 3 nM of biotinylated probe.
• Biotin Chip dil #2 contained five 1000 nL spots of biotinylated probe that were arranged in an “X” pattern on a chip.
• 5′-Hydrazide-A18 probes with 3′-biotinTEG were diluted with 5′-Hydrazide, un-modified A18 probe to a constant final concentration of 100 nM in 0.1M Sodium Phosphate pH 7.8, 10% glycerol.
• the control spot of 100 nM un-labeled A18 was located in the center of the chip. Starting with the highest concentration in the upper left corner and then proceeding from the left to the right, the four remaining spots represented the concentrations of 300 pM, 60 pM, 12 pM, and 2.4 pM of biotinylated probe.
• the MRSA Chip consisted of a chip with two columns of four spots arranged vertically. The left column are fiducial spots of dried latex particles to orient the viewer. The test spots contained probes that specifically recognize sequences in:
• 125 uL of Streptavidin was applied to the biotin chip at a concentration of 1 ⁇ g/mL in 1 ⁇ Hyb buffer (i.e., 5 ⁇ SSC, 0.1% SDS, and 0.1% BlockaidTM) and incubated at room temperature for five minutes. The chip was then washed four times with wash A (i.e., 0.1 ⁇ SSC and 0.1% SDS) and wash B (i.e., 0.1 ⁇ SSC). The sample was then incubated with 125 uL of 1 ⁇ g/mL of the biotin polymer diluted into 1 ⁇ Hyb buffer for five minutes at room temperature. The chip was washed four times with wash B.
• wash A i.e., 0.1 ⁇ SSC and 0.1% SDS
• wash B i.e., 0.1 ⁇ SSC
• pHSA Poly (horse radish peroxidase)-Streptavidin
• anti-biotin horse radish peroxidase conjugate was diluted to 1 ⁇ g/mL in 1 ⁇ Hyb buffer. 125 uL of the diluted anti-biotin/HRP was added to the chip, and incubated at room temperature for 10 minutes. The chip was then washed 6 times with wash B. Finally, TMB was added to each chip. After 10 minutes of incubation, the chip was washed with water, dried, and analyzed.
• Target sequences from the femA gene in Staphylococcus aureus were mixed in water with 20 nM each of two biotinylated detector probe sequences.
• Ten ⁇ L aliquots of the samples were heated to 95° C. for 3 minutes and then diluted into 90 ⁇ L of 1 ⁇ Hyb buffer (i.e., 5 ⁇ SSC, 0.1% SDS, and 0.5% BlockaidTM) that had already been pre-warmed on the surface of the chip.
• the samples were incubated at 53° C. for 30 minutes and then washed with 4 washes each of wash A (0.1 ⁇ SSC, 0.1% SDS) and wash B (0.1 ⁇ SSC).
• Mouse monoclonal anti-biotin/HRP was diluted to 1 ⁇ g/mL in 1 ⁇ Hyb and 125 uL was incubated on the chip for 10 minutes. The chip was washed six times with wash B. Then, 125 uL of a precipitable formulation of the substrate TMB was added to each chip and incubated at room temperature for 10 minutes.
• the following example describes a basic method for forming a capped enhanced detection system (cEDS) by acetylating biotinylated dextran polymers.
• cEDS capped enhanced detection system
• NHS-LC-biotin (Pierce, P/N 21335) was dissolved to a concentration of 10 mM (5.56 mg/ml) in water immediately before use. Varying volumes of the dextran polymer were diluted into 0.1 M borate buffer, pH 8.5. Varying volumes of the NHS-LC-biotin stock were combined with said dextran polymer solutions. The reactions were incubated on a shaker for 3 hours at room temperature.
• NHS-sulfo-acetate (Pierce, P/N 26777) was dissolved in water to form the concentration of 30 mM. An equal volume of diluted NHS-sulfo-acetate was added to an equal volume of NHS-LC-biotin for each reaction. The reactions were incubated with shaking for 3 hours at room temperature. The samples were purified on a PD-10 (Pharmacia) chromatography column.
• the following method describes use of hydrazone chemistry to conjugate hydrazide-biotin to an aldehyde-modified polymer.
• aldehyde dextran polymer 70 kDa, Pierce
• 5 mg of biotin hydrazide (Pierce) was dissolved in 450 uL DMSO.
• 32 mg sodiumcyanoborohydride was dissolved in 0.5 mL PBS.
• 200 uL of the aldehyde dextran was mixed with 30-100 uL of biotin hydrazide (If reduction of bond is sought also add 200 uL sodium borohydride).
• PBS was added to bring the volume of the solution to 800 uL.
• the solution was then reacted overnight at room temperature with agitation.
• the solution was then purified over PD-10 column.
• This example describes a method for determining the extent of biotinylation for various enhanced detection system molecules.
• Levels of biotinylation were determined with a ([2-(4′-hydroxyazobenzene)]benzoic acid) (“HABA”) kit from Pierce.
• HABA formed a HABA-avidin complex, and the biotin in the sample displaced the HABA, causing a change in absorbance when measured at 500 nM. Since the change in absorbance was directly proportional to the amount of biotin, this assay was used to determine the extent of biotinylation per molecule.
• the HABA-avidin mixture was equilibrated to room temperature.
• the spectrophotometer was blanked with 800 ⁇ l of PBS, pH 7.2.
• 100 ⁇ l of ddH 2 O was, first, added to the HABA-avidin microtube and, second, pipetted into a cuvette containing PBS buffer.
• the absorbance at 500 nM (A500) for the HABA-avidin and PBS mixture was recorded as the absorption level for HABA-avididn.
• 100 ⁇ l of biotinylated HRP was added as a positive control to the HABA-avidin cuvette mix and recorded at A500 of HRP+. Steps 1-4 were repeated for each of the biotinylated samples.
• biotinylated sample 100 ⁇ l was added to the HABA-avidin sample; the sample's level of absorption was then recorded. Absorbance is equal to or above 0.3 at steps 5 and 8, if not dilute sample and the A500 dilution was determined. At steps 5 and 8, if the absorption level was below 0.3 absorbance units, then the biotinylated sample was diluted, retested, and A500 was recorded.
• the following chart shows the level of biotinylation that was obtained for various types of EDS.
• a dilution series ranging from high concentration to low concentration was tested on a chip containing model target DNA sequences from the femA gene in methicillin-resistant strains of Staphylococcus aureus (“MRSA”).
• MRSA methicillin-resistant strains of Staphylococcus aureus
• a standard detection assay which used an anti-biotin antibody conjugated to a horse radish peroxidase, was compared to a biotin polymer assay in 1 ⁇ Hyb.
• the standard assay on a thin film biosensor produced a visible signal at 1 pM but did not produce a visible signal at 100 fM.
• the LLOD for the standard detection approach was approximately 300 fM.
• the data for the 500 kD biotin polymer was a solid signal at 30 fM, a faint signal at 3.75 aM, and an even fainter signal at 1.25 fM. Therefore, the 500 kD biotin polymer improved the detection limit to a concentration within the range of 1.25 fM to 3.75 fM, which was an improvement of 80-240 fold in LLOD.
• the chart below outlines the performance for each of the EDS types tested as described:
• biotinylation reagents with appropriate linker length to prevent interference between the biotin on the polymer and strepavidin.
• linkers may provide for better performance based on properties such as flexibility or solubility.
• NHS-LC-biotin Pierce
• NHS-LC-LC-biotin Pierce
• NHS-Chromalink Solulink
• NHS-PEGn-biotin Nektar
• NHS-DNA probes are examples.
• Biotin polymers were created with NHS-LC-biotin ( ⁇ 85 biotins/polymer) and NHS-Chromalink (77 biotins/polymer) with approximately the same number of biotins/polymer.
• the chromalink conjugated polymers were compared to NHS-LC-biotin in Biotin chip dil #1. The data suggested that the polymers have appreciably the same activity for signal enhancement on the biotin chip and that the chemistry options with respect to linker type are numerous.
• the cEDS reagent was compared to the 500 kDa biotin polymer from Molecular Probes on the Biotin chip dil #1. Under the normal test conditions, cEDS were at least 25-fold better than the biotin polymers made by Molecular Probes. The signal was apparent in all four dilutions with the polymer but it was very weak by the 3rd dilution with the Molecular Probes polymer in hybridization buffer. Therefore, the GBS polymer, is at least 25-fold more sensitive than the Molecular Probe. (Both polymers had the same molecular weight and approximately the same number of biotins/polymers. GBS had 85 biotins/polymer and MP polymer had 79 biotins/polymer.
• the cEDS and biotin dextran amplification polymer were compared on an MRSA chip.
• femA target sequences were tested as described.
• the data showed the cEDS can be clearly detected down to 1.25-3.75 fM, whereas the MP biotin dextran has significantly non-specific interactions with the chip surface, making detection of the specific probe untenable.
• the acylation of the remaining amino groups, with NHS-acetate, on the polymer after modification with NHS biotin, and subsequent purification on a size exclusion column improved signal enhancement.
• Signal enhancement which was reproducible, was improved by at least 10-fold.
• Different fractions of 500 kDa biotin dextran polymer were tested on a Biotin chip dil #2, using the following methodology: a. biotinylated polymer untreated, b. biotinylated polymer acetylated with NHS-acetate, c. acetylated, biotinylated polymer passed over a PD10 column to remove excess NHS-acetate.
• the unacetylated gel and the acetylated gel showed spots of similar intensity at 12 pM, 300 pM, and 60 pM concentration of biotinylated probe.
• the results of the gel showed that simply acetylating the polymer had no effect on LLOD.
• signal was also detected at the 2.4 pM spot, which was approximately a ten to twenty fold improvement.
• the acylation further served to mitigate nonspecific interactions of the polymers with the surface.
• the unacetylated fraction of the biotin polymer the results appeared sporadically, which is typical of non-specific binding.
• the sporadic results were most likely caused by the remaining unblocked amino groups on the dextran polymer as they interacted non-specifically with the surface of the chip.
• the results of the unacetylated gel showed spotting at the 300 pM and the 60 pM spots.
• the results of the acetylated gel show an additional faint spot at 12 pM ant 2.4 pM.
• the results of the PD10 purification were similar to the acylated results, except that the intensity of the spotting was increased at the 12 pM concentration. This observation likely accounted for the observation that the amplification polymer cannot be used with the assay enhancer PVP.
• the amplification polymer contains 147 free lysines that can contribute to non-specific interactions.
• polymers were tested as described on Biotin chip dil #1. The results of the experiment were that PVP enhanced the performance of the GBS polymer ⁇ 3-fold, whereas it created a surface passivation with the amplification polymer. Amplification polymers having greater solubility in buffers at basic pH were found to perform better.
• the samples that contained GBS amplification polymer, biotin, dextran, and hybridization buffer gave off a medium-level signal. Exchanging the buffer for buffer 2% PVP resulted in an increase of signal at all 4 spots.
• the sample that contained the commercially obtained amplification (Molecular Probes) polymer, biotin, dextran, and hybridization buffer only produced signal at 1 nM and 3 nM, with a very faint signal at 330 pM.
• non-ionic polymer compound during the cEDS incubation step improved signal enhancement by ⁇ 3-fold compared to cEDS incubated without the compound.
• Other non-ionic polymer compounds may enhance signal also.
• Addition of 2% polyvinyl pyrrolidone (PVP) 40 kD, or 1-2% polyethylene glycol (PEG) 8 kD to the 1 ⁇ Hyb buffer for the cEDS and polyHRP/SA incubations improved signal enhancement by an additional 3-fold above that of cEDS alone.
• PVP polyvinyl pyrrolidone
• PEG polyethylene glycol
• Non-ionic polymer compounds may also work to enhance the performance of the cEDS system.
• the following table summarizes data from the testing of various polymeric buffer additives on Biotin chip dil #1.
• Polyethylene glycol in the range of 8-40 kDa were close in efficacy to 40kDa polyvinyl pyrrolidone.
• Larger molecular weight dextran sulfate and low percentages of carboxy methyl cellulose also had a measurable effect on enhancing cEDS performance.
• alcohol and stearate side groups on polyvinyl backbones did not enhance the performance of the cEDS reagents.

Landscapes

• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Immunology (AREA)
• Organic Chemistry (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Analytical Chemistry (AREA)
• Molecular Biology (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• Biotechnology (AREA)
• Physics & Mathematics (AREA)
• General Health & Medical Sciences (AREA)
• Biophysics (AREA)
• General Engineering & Computer Science (AREA)
• Genetics & Genomics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Pathology (AREA)
• Hematology (AREA)
• Urology & Nephrology (AREA)
• Biomedical Technology (AREA)
• Cell Biology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• General Physics & Mathematics (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Investigating Or Analysing Biological Materials (AREA)

Priority Applications (1)

Applications Claiming Priority (3)

Publications (1)

Family

ID=40679211

Family Applications (4)

Family Applications After (3)

Country Status (4)

Cited By (8)

Families Citing this family (10)

Citations (5)

Family Cites Families (18)

• 2008
  • 2008-11-26 EP EP08855507A patent/EP2225561A4/fr not_active Withdrawn
  • 2008-11-26 CA CA2705985A patent/CA2705985A1/fr not_active Abandoned
  • 2008-11-26 US US12/745,140 patent/US20100304387A1/en not_active Abandoned
  • 2008-11-26 WO PCT/US2008/084990 patent/WO2009070742A2/fr active Application Filing
  • 2008-11-26 US US12/745,130 patent/US8574833B2/en not_active Expired - Fee Related
  • 2008-11-26 WO PCT/US2008/084790 patent/WO2009070640A2/fr active Application Filing
  • 2008-11-26 EP EP08853920A patent/EP2225398A4/fr active Pending
  • 2008-11-26 CA CA2705984A patent/CA2705984A1/fr not_active Abandoned
• 2013
  • 2013-10-03 US US14/045,441 patent/US9200312B2/en not_active Expired - Fee Related
• 2015
  • 2015-11-23 US US14/949,240 patent/US9359637B2/en not_active Expired - Fee Related

Patent Citations (6)

Also Published As

Similar Documents

Legal Events