WO2007123507A1 - Conjugués hydrosolubles pour la détection électrochimique - Google Patents

Conjugués hydrosolubles pour la détection électrochimique Download PDF

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Publication number
WO2007123507A1
WO2007123507A1 PCT/US2006/010406 US2006010406W WO2007123507A1 WO 2007123507 A1 WO2007123507 A1 WO 2007123507A1 US 2006010406 W US2006010406 W US 2006010406W WO 2007123507 A1 WO2007123507 A1 WO 2007123507A1
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Prior art keywords
water
conjugate
component
soluble
phosphate
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PCT/US2006/010406
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English (en)
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Michael W. Newton
Charles Ajith Wijayawardhana
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Inverness Medical Switzerland Gmbh
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Priority to CN200680053685XA priority Critical patent/CN101395473B/zh
Priority to DE112006003813T priority patent/DE112006003813T5/de
Priority to JP2009501392A priority patent/JP2009530639A/ja
Priority to PCT/US2006/010406 priority patent/WO2007123507A1/fr
Publication of WO2007123507A1 publication Critical patent/WO2007123507A1/fr
Priority to GB0816299A priority patent/GB2449043A/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6883—Polymer-drug antibody conjugates, e.g. mitomycin-dextran-Ab; DNA-polylysine-antibody complex or conjugate used for therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0058—Antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/10—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using coupling agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/548—Carbohydrates, e.g. dextran
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Definitions

  • the present invention relates to compositions of water-soluble conjugates useful in diagnostic assays, methods for preparing and using the conjugates, immunoassays, lateral flow assays, and test devices.
  • EP 0 594 772 Bl relates to water-soluble, polymer-based conjugates comprising moieties derived from divinyl sulfone.
  • EP 0 594 772 Bl describes the possibility of enhancing the attachment of molecular species, such as antibodies and antigens, to a water- soluble carrier molecule by taking advantage of the so-called “salting out” effect. It turned out, however, that by increasing the salt concentration to about 1 M an irreversible precipitate was formed.
  • U.S. Patent No. 6,627,460 to Lihme et al provides methods of water-soluble cross linked conjugates, and methods of their use.
  • the patent provides methods of further increasing the concentration of salt in the reaction mixture, which causes a reversible (i.e. a re-dissolvable) precipitate to form containing a water-soluble conjugate, which is useful in various immunochemical assays such as in lateral flow devices.
  • the present invention provides compositions of water-soluble conjugates for use in diagnostic and detection assays, and methods of their preparation and use.
  • the conjugates are useful in immunoassays and lateral flow assays.
  • the invention provides methods of preparing the conjugates that result in higher yields in the preparation of the conjugates, and higher sensitivities in the assays.
  • the invention also provides water-soluble conjugates that can be produced more economically.
  • the invention also provides devices for use in conducting detection and quantitation assays for a variety of ligands of interest.
  • the invention also provides water-soluble conjugates that utilize electrochemical detection methods and have very high sensitivity.
  • the methods involve a) reacting a water-soluble intermediate conjugate having a carrier component, a linking component, a spacer component, and a signal component (the signal component being covalently attached to the spacer component and the spacer component being covalently attached, via the linking component, to the carrier component), with at least one primary targeting component (a targeting element for a ligand to be detected or ligand to be detected).
  • the reaction occurs with unreacted reactive moieties derived from the linking component, in an aqueous solution. The conditions are such that a reversible precipitate is formed.
  • the reversible precipitate containing the water-soluble conjugate is re-dissolved in an aqueous medium; and c) optionally, the water-soluble cross-linked conjugate is subjected to a purification step.
  • Further details of the reaction parameters are provided in U.S. Patent No. 6,627,460, which is hereby incorporated by reference in its entirety, including all tables, figures, and claims.
  • the conjugates can be cross-linked to one another to form larger conjugate molecules.
  • the targeting element can be attached to the carrier via the linker, or can be attached to the spacer or to a non-specific protein, as described below.
  • the signal component can be attached to the carrier, or to the spacer, or even to the targeting element. The precise arrangement of components can be varied in any manner to result in a water-soluble conjugate that functions as a reagent and the assay is performed and provides a useful result.
  • the invention provides methods for preparing a water- soluble conjugate involving a) preparing a water-soluble conjugate having at least one carrier, at least one linker, at least one signal component, and at least one targeting element for a ligand to be detected or a ligand to be detected, as a reversible precipitate in a suspension.
  • the suspension is subjected to sonication to form a sonicated formulation, and a supernatant is separated from the formulation containing the water-soluble conjugate.
  • the water-soluble conjugate can be purified from the supernatant.
  • the water- soluble conjugate is purified by gel filtration (or size exclusion) chromatography.
  • the gel filtration chromatography is performed using a medium with an average size exclusion of 30OkD.
  • the water- soluble conjugate can also contain a spacer component.
  • the carrier is covalently attached to the linker and the signal component is covalently attached to the spacer.
  • the water-soluble conjugate can be prepared by contacting a water-soluble intermediate conjugate with the ligand to be detected or with the targeting element for a ligand in the presence of a lyotropic salt at a concentration of at least about 1.25 M. In other embodiments the concentration of the lyotropic salt can be at least about 1.5 M or at least about 1.75 M or at least about 2.0 M or at least about 2.5 M.
  • a “water-soluble conjugate” contains a carrier, a linker, a targeting element for a ligand to be detected or a ligand to be detected, a signal component, and can optionally also contain a spacer component.
  • water-soluble intermediate conjugate is meant a molecule containing a carrier, a linker, and a signal component.
  • a water-soluble intermediate conjugate may also contain a spacer component.
  • water-soluble intermediate precursor is meant a molecule having any two or more of the components of a water-soluble conjugate and that is not a water-soluble conjugate or intermediate conjugate.
  • the water soluble intermediate precursor contains a carrier and a linker.
  • the precursor contains the carrier, linker, and spacer component.
  • the water-soluble intermediate conjugate contains the carrier, linker, signal component, and spacer components.
  • Sonication refers to the known technique used in chemistry and biology of exposure to a high frequency sound energy. It is also sometimes referred to as ultrasonication. The sonication can performed at any appropriate power, e.g., at least about 300 watts, or at least about 500 watts, or at least about 700 watts, or at least about 900 watts, or at least about 1000 watts, or at greater than 1000 watts. Any desirable frequency can also be used such as (for example) from 20 to 24 kHz. As used herein, “about” means plus or minus 10%.
  • reversible precipitate indicates that the precipitate formed is capable of being re- dissolved upon dilution with aqueous solution at 25 °C.
  • the lyotropic salt can contain components such as sulphates, phosphates, citrates and tartrates of lithium, sodium, potassium, calcium and ammonium, and can be present at a concentration of about 2.5 M.
  • the salt is potassium phosphate or sodium phosphate.
  • water soluble when used in connection with the conjugates means that the conjugates obtained should be soluble in an aqueous medium, such as water, at room temperature, i.e. the cross-linked conjugates obtained by the methods disclosed herein should give rise to a solution which is substantially clear and homogenous as judged by visual inspection of the sample.
  • the conjugates obtained have a water solubility of at least 0.1, or at least 0.2 or at least 0.5, or at least 1, or at least 3, or at least 5, or at least 7, or from 5 to 10, or from 4 to 11, or at least 10, or at least 20, or at least 30, or at least 40, or at least 50, or at least 100, and in particular at least 200 mg dry conjugate per ml water at 25 °C.
  • the present invention also provides water-soluble conjugate prepared according to any method of the invention.
  • the present invention provides methods of preparing a water- soluble conjugate involving preparing a water-soluble conjugate as described herein as a precipitate in a suspension.
  • a pellet containing the water-soluble conjugate is separated from the suspension, and the pellet washed with an aqueous solution to form a second suspension.
  • a pellet is separated from the second suspension containing the water-soluble conjugate.
  • the water-soluble conjugates prepared according to these methods can have the same structure as those conjugates described herein.
  • the conjugates can further contain a spacer, and the carrier can be covalently attached to the linker, and the signal component covalently attached to the spacer.
  • the water-soluble conjugate is purified by the process of separating the precipitate from a supernatant, forming a suspension of the precipitate in an aqueous solution, and separating the precipitate from a supernatant.
  • the conjugates can contain a non-specific protein attached to the carrier via the linker (e.g., bovine serum albumin, an immunoglobulin).
  • the targeting element is an antibody that has been treated with a reducing agent.
  • reducing agent is meant a substance that chemically reduces other substances by donating an electron or electrons. Examples of reducing agents include beta-mercaptoethanol, dithiothreitol, and 2-iminothiolane.
  • washing the pellet is meant that the pellet is placed into contact with the aqueous solution and agitated.
  • the agitation can be by any method, for example by vortexing or stirring or shaking the container. Portions of the pellet may break off of the initial pellet during the agitation, and can be re-pelleted by centrifugation or another method. In another embodiment no further purification step is performed on the water-soluble conjugate after the washing with the aqueous solution.
  • the water-soluble conjugate is separated from the supernatant by centrifugation, although centrifugation is not necessary to practicing the method.
  • the conjugate can also be purified from the supernatant by any convenient techniques, such as by gel filtration.
  • supernatant is meant the liquid portion of a sample.
  • the present invention provides water-soluble conjugates containing a carrier, a linker covalently bound to the carrier, a signal component, a targeting element for a ligand to be detected or a ligand to be detected, and a non-specific protein.
  • the non-specific protein is covalently bound to the carrier via the linker.
  • the non-specific protein is bound to the carrier via the linker and to no other component (other than the linker) of the conjugate.
  • at least 2% or at least 3% or at least 5% or at least 10% or at least 15% or at least 20% of the non-specific proteins are bound to the carrier via the linkers and to no other component (other than the linker) of the conjugate.
  • At least 50% or at least 60% or at least 70% or at least 75% or at least 80% or at least 85% or at least 90% or at least 95% of the non-specific proteins are bound to the carrier via the linker and to no other component (other than the linker) of the conjugate.
  • all the same percentages of linkers recited above are bound to a non-specific protein, and the non-specific protein is bound to the linker and to no other component of the water-soluble conjugate.
  • Any of the water-soluble conjugates can also contain a spacer component.
  • the "non-specific protein” is a protein that does not have a binding specificity or target within the context that it is used.
  • the non-specific protein is typically linked to the water-soluble conjugate by a linking chemistry.
  • Bovine serum albumin, immunoglobulins, keyhole limpet hemocyanin, and other proteins are examples of non-specific proteins.
  • the non-specific protein is a protein other than the one used as a spacer (when a spacer is present), but the spacer and non-specific protein can also be the same protein used to accomplish different functions.
  • the non-specific protein can contain amino groups, which are used to covalently bind the non-specific protein to the conjugate, although other suitable linking chemistries may also be used.
  • the spacer and the nonspecific protein are independently and covalently attached to the carrier via the linker; the signal component is covalently attached to the spacer; and the ligand to be detected or targeting element for a ligand to be detected is covalently attached to the carrier.
  • the signal component is attached to either or both of the spacer and/or the non-specific protein. It is also possible to attach the non-specific protein and spacer to the carrier via the linker, and attach the targeting element or ligand, and signal component to either or both of the non-specific protein and spacer.
  • the present invention provides methods for preparing a water-soluble conjugate involving a) contacting a water-soluble intermediate conjugate having a carrier, a linker, a spacer, and a signal component, with i) a targeting element for a ligand to be detected or ii) a ligand to be detected, to form a suspension containing a precipitate comprising the water-soluble conjugate.
  • the method also involves extracting the water-soluble conjugate from the suspension.
  • the targeting element for a ligand to be detected or the ligand to be detected is pre-treated with a reducing agent prior to contact with the water-soluble intermediate conjugate.
  • the extraction can be performed by any suitable method.
  • the water-soluble conjugate is extracted by centrifugation.
  • pre-treat is meant that the composition is contacted or incubated with the reducing agent.
  • the reducing agent is dithiothreitol, which can be used at any suitable concentration.
  • the pre-treatment can be with at least about 15 mg of dithiothreitol/ 100 ul, or at least about 10 mg/100 ul, or at least about 5 mg/100 ul, or at least about 20 mg/100 ul. Equivalent quantities of other reducing agents can also be used.
  • the pre-treatment can be conducted for any suitable period of time, for example, 5 minutes or 10 minutes or 15 minutes, or 20 minutes, or longer than 20 minutes.
  • the present invention provides methods of preparing a water-soluble conjugate involving contacting a water-soluble intermediate precursor having at least one carrier and at least one linker, with at least one targeting element for a ligand to be detected or a ligand to be detected, and a non-specific protein to form a water-soluble intermediate conjugate.
  • a signal component is attached to the water-soluble intermediate conjugate to form the water-soluble conjugate.
  • a suspension is formed containing a precipitate of the water-soluble conjugate, and the water-soluble conjugate is extracted from the suspension.
  • the non-specific protein can be bovine serum albumin, an immunoglobulin, or keyhole limpet hemocyanin.
  • the carrier and linkers can also contain the signal component before addition of the targeting element or ligand, and non- specific protein, or the signal component can be added after attaching the targeting element or ligand and non-specific protein.
  • the targeting element or ligand, and nonspecific protein are contacted, added to, attached to, or incubated with the water-soluble intermediate precursor simultaneously.
  • the water-soluble intermediate conjugate precursor consists of the carrier and the linker before addition of the targeting element or ligand.
  • the targeting element or ligand and non-specific protein can be attached to the intermediate precursor at a salt concentration of at least 1.6 M or at least 1.7 M or at least 1.8 M or at least 1.9 M or at least 2.0 M or at least 2.2 M or at about 2.5 M.
  • the nonspecific protein can be reacted with the precursor at ratios of 1 : 1 or greater (precursor to nonspecific protein) or 1:5 or greater, or 1:7 or greater, or 1:10 or greater, or 1 :12 or greater, or 1 :15 or greater, or 1 :20 or greater.
  • the method produces a water-soluble conjugate as described herein.
  • the conjugate can be created by attaching the nonspecific protein to the carrier via the linker so that all linkers are blocked.
  • the targeting element or ligand can then be attached to the precursor via the non-specific protein.
  • the signal component can be attached either with the targeting element or ligand and non-specific protein, or can be attached in a later step to form the final conjugate.
  • the present invention provides methods of preparing a water- soluble conjugate.
  • the methods involve incubating a water-soluble conjugate precursor containing a carrier, a linker covalently bound to the carrier, and a signal component with a ligand to be detected or a targeting element for a ligand to be detected and a non-specific protein.
  • a water-soluble conjugate having the non-specific protein covalently bound to the carrier via the linker is thus prepared.
  • the non-specific protein is bound to the carrier via the linker, and to no other component of the water-soluble conjugate.
  • the targeting element for a ligand to be detected (or a ligand to be detected) and the non-specific protein are incubated simultaneously with the precursor.
  • the invention provides a device containing a water-soluble conjugate of the invention.
  • the conjugate is located on a test strip, which is a porous carrier material having a sample zone and a detection zone. Liquid sample applied to the sample zone flows to the detection zone.
  • the test strip also has a second targeting element selective for a targeting element bound to the ligand or for a ligand suspected to be present in the liquid sample, applied to the detection zone of the test strip.
  • porous carrier is meant a bibulous material through which fluid can move by capillary force.
  • An example of such a material is nitrocellulose, although persons of ordinary skill in the art will identify other bibulous materials that also function in the invention, for example, polyamide, and pretreated papers.
  • sample zone is the area of the test strip where sample to be tested is applied.
  • a “reagent zone” is an area where reagents are contained on the test strip. The reagents can be present in a dried form, and can be movably present on the strip.
  • a “detection zone” is the area of the test strip where a measurement is taken to determine the presence, absence, or amount of a ligand suspected to be present in the sample.
  • the device can also have a "label zone” where label is movably applied to the test strip.
  • Label zone refers to the interfacial forces that act among liquids in a capillary or in a porous medium, and which cause liquid to move through the capillary or porous medium.
  • movably is meant that the reagents or other composition can be moved along the device from one zone to another by the flow of liquid through the test strip.
  • the water-soluble conjugate is in a dry form on the test strip, upstream of the detection zone and downstream of the sample zone.
  • the test strip can also have a control zone, which can be located downstream from the detection zone.
  • the "control zone' contains a targeting element, and binding at the control zone indicates that the assay is functioning as designed.
  • the device also has a casing, which envelopes the test strip and defines the sample zone.
  • the porous carrier can be backed with a moisture-impervious material, which is placed in contact with the inside of the casing.
  • the device also has a cap, which is selectively received over one end of the casing and covers the sample zone of the device.
  • the casing can be made of plastic or another suitable material.
  • the test strip also contains a filter, which is situated upstream from the detection zone, and can be a part of the porous carrier material. The filter serves to remove any contaminating matter that may be present in the applied sample.
  • the test strip can also be prepared so it has a portion of the binding sites within the test strip blocked with a blocking protein or polyvinyl alcohol.
  • the blocking protein can be bovine serum albumin, milk protein, or another material having an equivalent effect on the assay.
  • the sample can be urine, serum, plasma, blood, semen, sputum, or another body fluid or other fluid of biological origin to be tested.
  • the device has a test strip, a casing, and a portion of the test strip protrudes from the casing. For example, a 1 cm or less portion of the test strip protrudes from the casing for receiving sample.
  • the present invention provides methods for preparing a water-soluble conjugate involving a) contacting a water-soluble intermediate precursor containing a carrier, a linker, a spacer, and a targeting element for a ligand to be detected or a ligand to be detected, with a signal component to form a suspension containing a precipitate comprising the water-soluble conjugate. Water-soluble conjugate is then extracted from the suspension.
  • the present invention provides a water soluble conjugate containing at least one carrier component, at least one linking component, at least one electrochemical signal component covalently attached to the carrier component through the linking component; and at least one targeting element for a ligand to be detected or at least one ligand to be detected.
  • the conjugate can also contain a spacer component, which can be attached to the carrier component through the linking component.
  • the electrochemical signal component being attached "through" the linking component is meant either that it is directly attached to the linking component without an intervening molecule, or that the signal component is attached to a component that is itself attached to the linking component.
  • the signal component can be attached to the spacer component, which is attached to the linking component.
  • the electrochemical signal component is an enzyme that converts a substrate or a reaction mediator into an electrochemically detectable species.
  • the electrochemical signal component can be alkaline phosphatase, horseradish peroxidase, or another enzyme
  • the substrate can be any that is appropriate for the enzyme, for example, 1-naphthyl phosphate, or hydroquinone, or other appropriate substrates for the enzymes.
  • the electrochemically detectable species can therefore be 1-naphthol, or benzoquinone, or another species that results from the action of enzyme on the substrate used.
  • the electrochemical signal component is covalently attached to the spacer, and the ligand to be detected or targeting element for a ligand to be detected is covalently attached to the carrier through the linking component.
  • the present invention provides methods of preparing a water- soluble conjugate.
  • the methods involve contacting a carrier component bearing at least one linking component with at least one electrochemical signal component to form a water- soluble intermediate conjugate, and contacting the water-soluble intermediate conjugate with a ligand or targeting element for a ligand to be detected to form a water-soluble conjugate.
  • the molar ratio of carrier component to electrochemical signal component is 1:10 or greater, or 1:15 or greater, or about 1 :20, or 1 :20 or greater.
  • the step of contacting of the water-soluble precursor with the targeting element to form the water-soluble conjugate results in the water-soluble conjugate being formed as a precipitate.
  • the method can also include the step of subjecting the precipitate to sonication after forming the water-soluble conjugate.
  • the present invention provides methods of detecting the presence or amount of an analyte in a sample.
  • the methods involve contacting a surface coated with a specific binding molecule to the analyte to be detected with the sample, contacting the surface coated with the specific binding molecule to the analyte to be detected with a water-soluble conjugate as described herein, forming a binding complex of the specific binding molecule coated on the surface, the analyte, and the water-soluble conjugate, contacting the binding complex with a substrate for the electrochemical signal enzyme to form a product solution, and determining the presence or absence of analyte in the sample.
  • the specific binding molecule is an antibody or fragment thereof.
  • the binding complex is the complex of the antibody attached to the surface, the analyte, and the conjugate.
  • the surface can be an electrode, a magnetic bead, or another surface.
  • the method can further comprise contacting the product solution with an electrode to determine the presence or amount of analyte in the sample.
  • Figure 1 provides a graphical depiction of water-soluble conjugates and general techniques in their preparation to assist the reader in visualizing the conjugates and in following the procedures described in the specification.
  • Figure 2 provides a comparison of responses of traditional HRP-dextran conjugate detection methods versus the present method.
  • the methods of the present invention allow higher product yields than previously have been obtainable by appropriate sonication of the water-soluble conjugate after cross-linking.
  • the sonication process produces a clear solution, meaning that it contains no non-liquid matter visible with the unaided eye, or produces minimal non-liquid matter. Normally, after following this procedure, further centrifugation is unnecessary.
  • One aspect of the invention involves the washing of a pellet produced by centrifugation of a formed water-soluble conjugate, which is present as a precipitate in the reaction product.
  • the supernatant is separated from the pellet and the pellet washed with an aqueous solution or buffer to form a second suspension.
  • a second pellet is separated, and the wash step can be repeated 1-2 more times, as necessary.
  • the steps of washing the pellet solubilized free (unreacted) targeting element e.g., an antibody
  • unreacted targeting element can then be easily disposed of.
  • the steps of washing eliminated the need to perform further purification of the product.
  • the expensive step of purification of the product on, for example, a gel filtration (S-300) column was eliminated.
  • the present invention provides water-soluble conjugates (and methods of their preparation) which contain a carrier, a linker, optionally a spacer component, a signal component, a targeting element for a ligand to be detected or a ligand to be detected, and a non-specific protein covalently bound to the carrier via the linker.
  • a carrier e.g., a carrier for a ligand to be detected or a ligand to be detected
  • a non-specific protein covalently bound to the carrier via the linker.
  • large quantities of targeting element (or ligand) molecules were utilized to ensure sufficient coupling and cross-linking to produce the water-soluble conjugate.
  • substantially less targeting element or ligand is necessary to ensure sufficient cross-linking than is used by binding to all available sites on the carrier.
  • the user is able to reduce the amount of targeting element (or ligand) utilized in the preparation procedure, and therefore substantially reduce the cost of producing the product.
  • the present invention provides methods of preparing a water- soluble conjugate by contacting a water-soluble intermediate conjugate with a targeting element or ligand to form a suspension containing a precipitate that contains the water- soluble conjugate, extracting the water-soluble conjugate from the suspension, where the targeting element or ligand is pre-treated with a reducing agent prior to contact with the water-soluble intermediate conjugate.
  • Pre-treatment of the targeting element with a reducing agent allows a higher binding rate of the targeting element to the carrier, resulting in an increase in the sensitivity of the assay.
  • Example 5 provides a practical application of this aspect of the invention.
  • Any reducing agent can be used, for example, dithiothreitol, beta- mercaptoethanol, Traut's Reagent (2-iminothiolane), or another reducing agent.
  • the present invention provides methods for determining the presence an analyte in a liquid sample.
  • the method involve contacting a liquid sample to a portion of the test device of the invention, the portion being located upstream from the detection zone; allowing the liquid sample to flow to the detection zone; and determining the presence, absence, or amount of the analyte in the liquid sample by observing the detection zone.
  • the detecting step can be visually observing the signal in the detection zone.
  • the Carrier is the Carrier
  • carrier in the context of the present invention is used to denote the “backbone” of the conjugate, i.e. the carrier component functions as a backbone on which various components may be attached.
  • the water-soluble polymers which function as the carrier component in the method for the preparation of conjugates may be chosen from a wide variety of types of polymers, including: natural and synthetic polysaccharides, as well as derivatives thereof, for example dextrans and dextran derivatives, starches and starch derivatives, cellulose derivatives, amylose and pectin, as well as certain natural gums and derivatives thereof, such as gum arabic and salts of alginic acid; homopoly(amino acid)s having suitable reactive functionalities, such as polylysines, polyhistidines or polyornithines; natural and synthetic polypeptides and proteins, such as bovine serum albumin (BSA) and other mammalian albumins; and synthetic polymers having nucleophilic functional groups, such as polyvinyl alcohols
  • Very suitable polymers for the purposes of the invention are polysaccharides and derivatives thereof, for example: dextrans, carboxymethyl-dextrans, hydroxyethyl- and hydroxypropyl-starches, glycogen, agarose derivatives, and hydroxyethyl- and hydroxypropyl-celluloses.
  • dextrans have proved to be particularly suitable polymers in connection with the invention.
  • a suitable polymeric carrier component for use in the method of the invention is, in its free state, substantially linear and substantially uncharged at a pH in the range of about 4 to about 10, the latter pH interval being the interval of practical relevance for the vast majority of immunochemical procedures, hybridization procedures and other applications of conjugates.
  • polymers which meet this criterion are, for example, numerous polysaccharides and polysaccharide derivatives, e.g. dextrans and hydroxyethyl- and hydroxypropylcelluloses.
  • the conjugates may be based on water-soluble polymeric carrier components having a range of molecular weights.
  • the polymeric carrier component may have a peak molecular weight in the range of about 40,000 to about 40,000,000 (prior to reacting the water-soluble polymeric carrier components with linker reagent such as DVS (divinyl sulfone) or EPCH (epichlorhydrin), or reacting resulting water-soluble intermediate precursor with a spacer or signal component for the eventual formation of cross-linked conjugate and cross-linked conjugate complexes).
  • linker reagent such as DVS (divinyl sulfone) or EPCH (epichlorhydrin
  • Peak molecular weights which are of considerable interest are peak molecular weights in the range of 100,000 to 10,000,000, such as in the range from 500,000 to 8,000,000, or in the range from 500,000 to 4,000,000, e.g. in the range from 500,000 to 2,000,000.
  • Peak molecular weights of particular interest notably in the case of dextrans as polymeric carrier components, are peak molecular weights of about 500,000, about 1,000,000, about 1,500,000, about 2,000,000, 2,500,000, about 3,000,000, about 3,500,000 and about 4,000,000.
  • dextrans in the molecular weight ranges of 20,000 to 2,000,000 are suitable as starting carrier components.
  • 20,000 Da dextrans are suitable for, but not restricted to, conjugates and/or complexes using streptavidin as the primary or secondary target.
  • 500,000 Da dextrans are suitable for, but not restricted to, conjugates and/or complexes using certain dyes, enzymes, and with certain specific binding molecules as the primary or secondary target.
  • 2,000,000 Da dextrans are suitable for, but not restricted to, certain other dyes.
  • the carrier can be any suitable carrier molecule, such as, for example, dextran, starch, glycogen, agarose, cellulose, natural gum, or mixtures thereof.
  • peak molecular weight denotes the molecular weight of greatest abundance, i.e. that molecular weight, among a distribution of molecular weights, which is possessed by the greatest number of molecules in a given sample or batch of the polymer. It is quite normal to characterize numerous types of polymers in this manner, owing to the difficulty (particularly for the highest molecular weights) of obtaining or preparing polymer fractions of very narrow molecular weight distribution.
  • peak molecular weight data (determined, for example, by gel-permeation chromatography) which can provide a basis for the selection of the proper fraction of the polymeric carrier component.
  • peak molecular weight values when used in connection with the carrier component cited in the present specification and claims refer to the peak molecular weight of the free polymer in question, and take no account of, for example, the possible formation of cross-linked polymer units, e.g.
  • cross-linked units will, on average, have higher molecular weights than the individual free polymer molecules from which they are formed.
  • linking component is intended to cover bi-functional molecules capable of establishing covalent links between other— typically larger—molecules.
  • linking components suitable for the method according to the invention are e.g. molecules comprising a bi-functional reactivity such as glutaraldehyde, carbodiimides, N,N'-phenylenedimaleimide, N-succinimidyl 3-(2- pyridylthio)propionate, p-benzoquinone, bis-oxiranes, divinyl sulfone (DVS) and epoxide derivatives, such as epoxides of the general formula I:
  • Ri is hydrogen or Ci- 4 -alkyl
  • n is an integer in the range from 1-4, i.e. 1, 2, 3 or 4
  • X is a leaving group such as tosyl, mesyl, or halogen such as fluorine, chlorine, bromine, or iodine.
  • Ci -4 -alkyl designates a straight or branched saturated hydrocarbon group having from 1 to 4 carbon atoms, such as methyl ethyl, n- propyl, n-butyl, isopropyl, isobutyl, etc.
  • EPCH epichlorohydrin
  • the linking component should be stable in an aqueous environment and, accordingly, the linking component EPCH constitutes together with the linking component DVS a very useful linking components for use in the methods of the invention.
  • the spacer component is, via reaction with the linking component, covalently attached to the water-soluble intermediate precursor, thereby forming a second water-soluble intermediate precursor.
  • the "spacer component” is covalently attached, via the linking group, to the carrier component.
  • the term "spacer component” when used in the present context indicates a protein or a polypeptide which has a plurality of sites available for covalent attachment of signal components, such as dyes (vide infra). Spacer components are useful in any of the water-soluble conjugates used herein and in any of the methods of preparing water-soluble conjugates, although it is also possible for the conjugates to function without a spacer component.
  • One purpose for the incorporation of a spacer component, and particularly for a spacer having a plurality of sites available for covalent attachment of signal components, is that this method provides for a suitable means of increasing the number of signal components which can be attached to the conjugate (i.e. the "load" of the signal component in the water-soluble intermediate conjugate, vide ante), and thereby increasing the sensitivity of such conjugates when employed in various assays, e.g. immunochemical assays and in the lateral flow devices described herein (vide infra).
  • the number of moles of spacer per mole of starting dextran ranges from 1 to 500, particularly from 2 to 100, most frequently from 5 to 75.
  • the second water-soluble intermediate can be characterized by, e.g., the number (moles) of spacer component attached per mole carrier component.
  • the spacer component can be a protein such as, for example, bovine serum albumin (BSA), ovalbumin, globulin, etc. or a polypeptide such as homopolypeptides, e.g. polylysines, polyhistidines, polyornithines, etc.
  • BSA bovine serum albumin
  • the choice of spacer component will depend on the employed signal component (e.g. the actual dye employed in a particular conjugate) as well as the employed linking component.
  • the molecular weight of the spacer component e.g. a protein
  • the molecular weight of the spacer component can be at least 2,500 Da, or at least 5,000 Da, or at least 10,000 Da, or in the range of 10,000- 2,000,000, such as in the range of 20,000-500,000.
  • the one of the features of the introduced spacer components is to multiply the number of available positions for introduction of the signal components, it is furthermore desirable that the number of available functional groups for attachment of signal components is at least 5 per molecule of spacer component, for example, 10-1,000, in particular 10-500.
  • the spacer component can be a polysaccharide or polynucleic acid. Chemical modifications of these polymers may be required prior to the preparation of the water-soluble intermediate conjugate.
  • a reactive functionality such as a nucleophilic functionality
  • Suitable spacer components will then be, for example, those with nucleophilic functional groups such as: --O " (e.g. deprotonated phenolic hydroxy groups, such as deprotonated aromatic hydroxy groups in tyrosine residues of polypeptides or proteins), — S ' (e.g.
  • deprotonated thiol groups on aromatic rings or aliphatic groups such as deprotonated thiol groups in cysteine residues of polypeptides or proteins
  • -OH e.g. aliphatic hydroxy groups present in certain amino acid residues of polypeptides or proteins, such as serine or threonine residues
  • --SH e.g. thiol groups in cysteine residues of polypeptides or proteins
  • primary amino groups e.g. in lysine or ornithine residues of polypeptides or proteins
  • secondary amino groups e.g. in histidine residues of polypeptides or proteins
  • the obtained second water-soluble intermediate precursor may be purified by the methods already discussed in connection with the purification step, i.e. in connection with the purification of the water-soluble intermediate precursor.
  • a suitable method for purifying the obtained second water-soluble intermediate precursor is gel-filtration.
  • the signal component is, via reaction with the spacer component, covalently attached to the second water-soluble intermediate precursor, thereby forming a water-soluble intermediate conjugate.
  • the term "signal component” refers to moieties which are directly physically detectable or which are precursors for or produce such physically detectable moieties.
  • the signal component functions as a label or a marker which can be readily measured by some physical technique known in art, e.g. by means of optical methods, such as spectrophotometry, fluorescence, luminescence, phosphorescence or other methods such as those described in e.g. "Instrumental Methods of Chemical Analysis” G. W. Ewing, 5th Ed., McGraw-Hill Book Company, New York, 1988.
  • the signal component can also be detected by visual observation with the unaided eye.
  • the signal component may—as indicated above—be a precursor for such a physically detectable component.
  • a precursor for such a physically detectable component is an enzyme which upon action on a suitable substrate is capable of generating species, for example colored species, which can be detected by one or more of the physical methods mentioned above.
  • the signal component may be selected from substances such as dyes; fluorescent, luminescent, phosphorescent and other light-emitting substances; metal-chelating substances, including iminodiacetic acid, ethylenediaminetetraacetic acid (EDTA), diethylene triaminepentaacetic acid (DTPA) and desferoxamine B; substances labelled with a radioactive isotope; substances labelled with a heavy atom; and mixtures thereof.
  • substances such as dyes; fluorescent, luminescent, phosphorescent and other light-emitting substances; metal-chelating substances, including iminodiacetic acid, ethylenediaminetetraacetic acid (EDTA), diethylene triaminepentaacetic acid (DTPA) and desferoxamine B; substances labelled with a radioactive isotope; substances labelled with a heavy atom; and mixtures thereof.
  • substances such as dyes; fluorescent, luminescent, phosphorescent and other light-emitting substances; metal-chelating substances, including iminodia
  • fluorescent substances may be selected from, e.g., fluorescein (suitably as fluorescein isothiocyanate, FITC), fluoresceinamine, 1- naphthol, 2-naphthol, eosin, erythrosin, morin, o-phenylenediamine, rhodamine and 8- anilino-1-naphthalenesulfonic acid.
  • Radioactive isotopes of relevance may be selected, for example, among isotopes of hydrogen (i.e.
  • Heavy atoms of relevance may be selected, for example, among Mn, Fe, Co, Ni, Cu, Zn, Ga, In, Ag, Au, Hg, I, Bi, Y, La, Ce, Eu and Gd. Gold (Au) is a particularly useful heavy atom in many cases.
  • signal components can be the non-particulate labels, for example, non-particulate dyes.
  • dye is intended to mean any spectrophotometrically detectable dye molecule or derivative thereof.
  • Dyes useful for incorporation into the conjugates prepared by the methods according to the invention include those derived from visual dyes, phosphorescent dyes, fluorescent dyes, laser dyes, infrared dyes and lanthanide chelates.
  • Dyes which are particular interesting are visual dyes, including soluble visual dyes, such as pigments, vat dyes, sulphur dyes, mordant dyes, leucovat dyes and species such as fluorescein, rhodamine and derivatives thereof (such as sulphorhodamine, rhodamine-hydride and rhodamine hydrazide), as well as oxazine dyes, cyanine dyes and azol dyes.
  • soluble visual dyes such as pigments, vat dyes, sulphur dyes, mordant dyes, leucovat dyes and species
  • fluorescein, rhodamine and derivatives thereof such as sulphorhodamine, rhodamine-hydride and rhodamine hydrazide
  • oxazine dyes such as sulphorhodamine, rhodamine-hydride and rhodamine hydrazide
  • non- particulate label is one where the basis of detection of the label is other than the detection of a solid, whether the solid is the signal component (e.g., latex or other particles) or whether a solid precipitate is produced that is the basis for detection.
  • dyes which are useful as signal components for the purposes of the present invention, are all well-known in the art and it will be clear to the skilled person that other dyes can be used as signal components for the purposes of the present invention.
  • Other examples of dyes to be used as signal components are e.g. such dyes as mentioned in "Dyeing and Chemical Technology of Textile Fibers", Trotman, 34th Ed., C. Griffin & Co., London and “The Chemistry of Synthetic Dyes", Vankataramon (Ed.), Academic Press, New York, 1979, the disclosures of which are incorporated herein by reference.
  • the signal component can be capable of reacting with a protein, such as BSA and/or, for alternative embodiments described below, capable of reacting with an unreacted reactive moiety of a linker component.
  • a protein such as BSA and/or, for alternative embodiments described below
  • the signal component upon reacting or binding to the spacer, should not confer any undesirable properties of the resulting water-soluble intermediate conjugate, i.e. the signal component should not promote any uncontrollable non-specific binding nor inhibit the activity of the targeting components (e.g. antibodies) bound to the conjugate.
  • the signal component should not reduce the water solubility of the conjugate significantly.
  • only a small fraction of the reactive moieties of the linking component of the second water-soluble intermediate reacts with the signal component in the formation of the water-soluble intermediate conjugate.
  • the spacer component, and on the linker component, after reacting the signal component, and relative to the amount of unreacted reactive linking component available in the second water-soluble intermediate precursor from 50 to 100% of the unreacted reactive moieties of the linker component, for example 60-100%, particularly 70-100%, such as ranging from 80-100% and notably 90-100% remain unreacted (N.B. as compared to the second water-soluble intermediate precursor).
  • the conjugate prepared by the method of the invention reflects, scatters, or emits photons in the visible range, in the UV range or in the near infrared range.
  • a visual dye such as rhodamine will cause the conjugate of the invention to reflect or scatter photons in the visible region (e.g. blue), resulting in the transmission of the complementary wavelength of color (e.g. red) to an observer.
  • the use of a fluorescent dye will (when radiated) cause the conjugate of the invention to emit photons at a specific wavelength due to the return of electrons to the ground state.
  • a "visual dye” is a dye that reflects or scatters light in the visible range.
  • the signal component is a donor/acceptor dye pair.
  • Donor/acceptor dye pairs are known in the chemical arts. In resonant energy transfer, the donor molecule absorbs a photon and initiates energy transfer to the acceptor. The acceptor receives the energy transfer and emits a photon. The donor dye and acceptor dye can perform fluorescent resonance energy transfer (FRET) upon excitation.
  • FRET fluorescent resonance energy transfer
  • Suitable donor/acceptor pairs are ⁇ -carboxyfluorescein/ ⁇ -carboxy-X-rhodamine (FAM-ROX), 3- (epsilon-carboxypenty ⁇ -S'-ethyl-S ⁇ '-dimethyloxacarbocyanine/ o-carboxy-X-rhodamine (CYA-ROX), and the 4,4-difluoro-4-bora-3 alpha,4 alpha-diaza-s-indacene-3 -propionic acid (BODIPY) derivatives, 5,7-dimethyl-BODIPY/5-(4- ⁇ henyl-l,3-butadienyl) BODIPY (BODIPY503/512-BODIPY581/591).
  • FAM-ROX ⁇ -carboxyfluorescein/ ⁇ -carboxy-X-rhodamine
  • CYA-ROX o-carboxy-X-rhod
  • FRET fluorescent resonance energy transfer, which is the transfer of the excited state energy from a donor to an acceptor.
  • the spacer is covalently attached to the carrier via the linker
  • the signal component is a dye (e.g., a member of a donor/acceptor pair) covalently attached to the spacer
  • the ligand or targeting element for a ligand is covalently attached to the carrier.
  • the carrier is dextran
  • the linker is divinylsulfone
  • the spacer is bovine serum albumin.
  • the methods are also suitable for the preparation of water-soluble conjugates wherein the signal component is covalently attached to the linking component, which in turn is attached to the carrier component, i.e. no protein or polypeptide spacer component is incorporated in the conjugate (vide infra). Further details are available in U.S. Patent No. 6,627,460, column 12 inter alia.
  • the signal component is an electrochemical signal component, which can be covalently attached to the carrier through the linker.
  • the conjugate consists of a carrier molecule, linking component, and the electrochemical signal component bound to the linking component.
  • the electrochemical signal component can be an enzyme that reacts with a substrate to produce an electrochemically detectable species.
  • the electrochemically detectable species can be converted from the substrate, or can be a bi-product of the reaction, e.g., a reaction mediator.
  • Examples of enzymes that can be used in the present invention include alkaline phosphatase, horseradish peroxidase, glucose 6-phosphate dehydrogenase, acetylcholinesterase, galactosidase, glucose oxidase, catalase, and choline oxidase.
  • a combination of enzymes can also be used, whether combining enzymes that act independently, or a bi-enzymatic or multi-enzymatic system.
  • a bi-enzymatic system is NADH oxidase and alcohol dehydrogenase.
  • Another bi-enzymatic system that can function in the present invention is tyrosinase and glucose dehydrogenase.
  • Bi-enzymatic systems can use oxygen sensors for detection.
  • the electrochemical signal component is an enzyme (or combination of enzymes) it is termed an electrochemical signal enzyme.
  • Substrates or reaction mediators that can be used to generate the electrochemical signal include any substrate or reaction mediator that is appropriate for the enzyme selected, and which will produce an electrochemically detectable product or species.
  • substrates include 4-aminophenyl phosphate, 1-naphthyl phosphate, glucose-6- phosphate, 4-hydroxynaphthyl-l -phosphate, 3-indoxyl phosphate, phenyl phosphate, 5- bromo-4-chloro-3-indolyl phosphate ester, 6-(N-ferrocenoylamino)-2,4-dimethylphenyl phosphate, paracetamol phosphate, 3,3',5,5'-tetramethylbenzidine (TMB), hydroquinone, redox Os +2 -based polymer, NAD + and glucose-6-phosphate, acetylthiocholine iodide, A- aminophenyl-beta-D-galactopyranoside
  • reaction mediators examples include ferricyanide, ferrocene, and ferrocene derivatives. These lists merely provide examples as many other substrates and reaction mediators can also be used.
  • a "reaction mediator” is a substance that is distinct from the substrate and is utilized in the enzymatic reaction. Reaction mediators can be converted into electrochemically detectable species during the enzymatic reaction.
  • An "electrochemically detectable species” is a substance that is detectable by at least one of the electroanalytical techniques of voltammetry, potentiometry, or conductimetry.
  • electrochemically detectable species include 4-aminophenol, 1- naphthol, glucose, dihydroxy naphthalene, indigo blue, phenol, H 2 O 2 , 6-(N- ferrocenoylamino)-2,4-dimethylphenol, 4-acetamidophenol (TMB ox), benzoquinone, ferricyanide, oxidized ferrocene and ferrocene derivatives, Os +3 , NADH, thiocholine, A- aminophenol (PAP), gluconolactone and a reduced mediator, and betaine.
  • TMB ox 4-acetamidophenol
  • Voltammetry is the electrochemical measuring technique used for electrochemical analysis or for the determination of the kinetics and mechanism of electrode reactions.
  • the potential of the working electrode is controlled (typically with a potentiostat) and the current flowing through the electrode is measured.
  • Potentiometry is the field of electroanalytical chemistry in which potential is measured under the conditions of no current flow. The measured potential may then be used to determine the analytical quantity of interest, generally the concentration of some component of the analyte solution.
  • the potential that develops in the electrochemical cell is the result of the free energy change that would occur if the chemical phenomena were to proceed until the equilibrium condition has been satisfied.
  • Conductimetry is the field of scientific measurement of solution conductance. Ligands and Targeting Elements for a Ligand to be Detected
  • targeting element refers to molecules, especially molecules of biological origin, which are capable of binding to or reacting with, a complementary molecule or a complementary structural region of a material of biological origin.
  • the targeting element is a targeting element for the ligand to be detected, the targeting element binds to or reacts with the ligand to be detected.
  • Examples of relevant targeting elements for a ligand to be detected are, for example: monoclonal and polyclonal antibodies, gene probes, natural and synthetic oligo- and polynucleotides, natural and synthetic mono-oligo- and polysaccharides, lectins, avidin, streptavidin, biotin, growth factors, hormones, receptor molecules, protein A and protein G; and mixtures thereof.
  • anti-human chorionic gonadotropin anti hCG
  • LH luteinizing hormone
  • Rabbit anti human CRP streptavidin
  • avidin anti-HIV
  • anti hepatitis C anti Chlamydia
  • anti-herpes anti-thyroid stimulating hormone (anti TSH)
  • anti- Listeria anti-salmonella
  • anti- mononucleosis anti-HBeAb
  • anti-HBsAb anti-HBsAb
  • anti-H anti-H.
  • Ligands are molecules to which targeting elements for the ligand will bind.
  • ligands useful in the present invention are antigens and haptens, but can include any ligand of interest in the detection.
  • hormones as ligands are hormones (e.g. estrogen, estradiol, progesterone, human chorionic gonadotropin (hCG), luteinizing hormone, follicle stimulating hormone, cortisone, T3, T4), amino acid hormones (e.g. thyroxine) and peptide and protein hormones (e.g. vasopressin, bombesin, gastrin or insulin), and drags of abuse.
  • hormones e.g. estrogen, estradiol, progesterone, human chorionic gonadotropin (hCG), luteinizing hormone, follicle stimulating hormone, cortisone, T3, T4
  • amino acid hormones e.g. thyroxine
  • peptide and protein hormones e.g.
  • ligands include cardiac markers such as Troponin I, Troponin T, high sensitivity C-Reactive Protein (hsCRP), CK-MB, myoglobin, NT-proBNP, B-type natriuretic peptide (BNP), atrial natriuretic peptide (ANP), and myeloperoxidase; and cancer markers such as prostate specific antigen (PSA), carcinoembryonic antigen (CEA), and alpha-fetoprotein (AFP).
  • a "drug of abuse” is a drag that is taken for non-medicinal reasons (usually for mind- altering effects). The abuse of such drags can lead to physical and mental damage and (with some substances) dependence and addiction.
  • drugs of abuse examples include cocaine, amphetamines (e.g., black beauties, white bennies, dextroamphetamines (dexies, beans), methamphetamines (crank, meth, crystal, speed)), barbiturates, lysergic acid diethylamide (LSD), depressants, sedatives (e.g., selective serotonin reuptake inhibitors), phencyclidine (PCP), tetrahydrocannabinol (THC), and opiates (e.g., morphine, opium, codeine, and heroin).
  • amphetamines e.g., black beauties, white bennies, dextroamphetamines (dexies, beans), methamphetamines (crank, meth, crystal, speed)
  • barbiturates examples include lysergic acid diethylamide (LSD), depressants, sedatives (e.g., selective seroton
  • the present methods and compositions are useful in a variety of assay formats.
  • some formats utilize an antibody that is specific for the ligand suspected of being present in the sample.
  • reagents can be movably located on the test strip and sample is applied to the sample zone. Sample then migrates through the reagent zone where reagents can bind to ligand suspected of being present in the sample, and arrives at the detection zone where a targeting element is applied to the zone and binds to the ligand suspected to be present or the targeting element bound to the ligand, or even to another component of the conjugate.
  • selective binding is meant that the targeting element distinguishes between the ligand of interest from any other ligand likely to be present in the sample, so that the assay functions as intended.
  • a targeting element that binds selectively can still bind to more than one ligand.
  • specific binding is meant that the targeting element binds to its target ligand and to no other ligand that may be present in the sample.
  • two antibodies can be used which have a lower selectivity for the ligand of interest, and therefore can bind not only to the ligand of interest, but also to a second molecule present in the sample.
  • a scavenger antibody is used that binds to the binding sites on the second molecule, thus blocking these sites and leaving the two antibodies to bind only to the ligand of interest.
  • more than one scavenger antibody can be used and more than two antibodies can be used.
  • the invention can also be applied in a direct sandwich assay format.
  • sample is applied to a sample zone, and flows through a label zone containing a label (e.g., dextran-BSA-anti-hCG Antibody-rhodamine) and if the ligand to be detected is present the label binds to the ligand.
  • a label e.g., dextran-BSA-anti-hCG Antibody-rhodamine
  • Sample then continues to flow to the detection zone (containing, for example, anti-hCG antibody affixed to the test strip).
  • the detection zone containing, for example, anti-hCG antibody affixed to the test strip.
  • labeled ligand is bound to the detection zone and observation of the detection zone provides the result of the assay.
  • sample is applied to a sample zone and migrates through a reagent zone containing a targeting element specific for the ligand to be detected movably present in the reagent zone (e.g. biotin-hCG Ab).
  • a targeting element specific for the ligand to be detected movably present in the reagent zone (e.g. biotin-hCG Ab).
  • the sample then continues to migrate through a label zone, which contains a conjugate of the invention that specifically binds to the ligand (e.g., the beta-portion of hCG) or to a targeting element bound to the ligand.
  • a signal component is attached to the ligand to be detected.
  • the sample then continues to migrate to the detection zone, where there is a bound a targeting element for the sample (e.g., streptavidin-IgG or streptavidin-BSA).
  • a targeting element for the sample e.g., streptavidin-IgG or streptavidin-BSA.
  • Visual observation of the detection zone reveals the presence, absence, or amount of ligand present.
  • the present invention can be applied in the formats described in the following references: 5,602,040, 5,622,871, 5,656,503, 6,187,598, 6,228,660, 6,352,862, 2001/0,008,774, 2001/0,041,368, 5,714,389, 5,989,921, 6,485,982, 5,252,496, 5,559,041, 5,728,587, 6,027,943, 6,506,612, 6,541,277, 2002/0,160,525, 5,073,484, 5,654,162, 6,020,147, 5,120,643, 5,578,577, 6,534,320, 4,703,017, 4,743,560, 5,591,645, 3,011,874, 3,641,235, 4,094,647, 4,168,146, 4,373,932, 4,477,575, 4,722,889, 4,861,711, 4,943,522, 4,978,503, 5,571,726, EP 0,149,168, EP 0,170,375, EP 0,192,320, EP 0,250,137, EP
  • ECI enzyme electrochemical immunoassay
  • the optimum electrode potential for detection is chosen by obtaining the current response produced by the analyte as a function of the applied potential.
  • This current response for an electroactive species in solution usually has three distinctive regions of behavior. One, a region of potential where the compound is not electroactive and current is negligible. Two, a region of rising current response defined by the Nernst equation. Third, a limiting current plateau that is independent of potential. The best potential for detection is along this limiting current plateau where analyte is being electrolyzed at the limit of mass transport to the electrode and small changes in the applied potential do not significantly affect the current measurement.
  • E-S-P enzyme-specific phosphatase
  • PAPP 4-aminophenyl phosphate
  • PAP oxidation potential
  • I-naphthyl phosphate The less costly and more stable ALP substrate I-naphthyl phosphate has also been used. This E-S pair has shown to be especially good with screen-printed electrode based immunosensors. Other enzymes used in ECI are described herein.
  • E-S pair is the most common practice in enzyme immunoassays
  • bi-enzymatic or multi-enzymatic systems can also be used.
  • One detection scheme involves using tyrosinase to oxidize phenol first to catechol and then to O-quinone.
  • O-quinone becomes a mediator in the enzymatic dehydrogenation of glucose and is reconverted to catechol.
  • Quantitation of ALP is done indirectly by measuring the loss of O 2 in the oxidation of phenol.
  • SPEs Screen-printed electrodes
  • fabricated with thick-film technology using a graphite powder-based ink to print electrodes on a polystyrene surface are adapted for immunosensors chiefly by passive adsorption of antibodies to the electrode surface.
  • SPE-based immunosensors can be used in a number of applications for the assays described herein.
  • compositions and methods of the invention can be used in any convenient assay format. However, two formats are well suited for electrochemical detection reagents.
  • magnetic beads are treated with specific binding molecules (e.g., antibodies) to an analyte to be detected and placed into a vessel.
  • a sample to be analyzed is contacted with the beads. If analyte is present in the sample it will be bound by the specific binding molecules on the beads.
  • a composition of the present invention having an electrochemical signal component is added, and the mixture reacted. After appropriate rinsing, a substrate for the electrochemical signal component is placed into contact with the beads. This mixture can be reacted in a capillary tube for very small volumes.
  • specific binding molecules to an analyte are adsorbed to an electrode.
  • sample solution is placed in contact with the electrode. If analyte is present in the sample, it will be bound by the specific binding molecules.
  • a composition of the present invention is placed into contact with the electrode to form a complex of the antibody to the analyte, the analyte, and the conjugate of the invention. After appropriate rinsing, a substrate for the electrochemical signal component is placed into contact with the electrode. After reaction, a reading is taken, for example using a voltammetry, potentiometry, or conductimetry, and the presence or amount of the analyte in the sample is determined.
  • specific-binding molecule is meant a molecule that will selectively bind, through chemical or physical means to a detectable substance present in a sample.
  • selectively bind is meant that the molecule binds preferentially to the target of interest or binds with greater affinity to the target than to other molecules.
  • the specific binding molecule is an antibody or a fragment of an antibody.
  • Antibody refers to an immunoglobulin, whether natural or partially or wholly synthetically produced. The term also includes derivatives thereof which maintain specific binding ability. The term also covers any protein having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced.
  • An antibody may be monoclonal or polyclonal, and can be a member of any immunoglobulin class (or combination of classes), including any of the human classes: IgG, IgM, IgA, IgD, IgG, and IgE.
  • An "antibody fragment” is any derivative of an antibody which is less than full-length. The antibody fragment can retain at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , scFv, Fv, dsFv diabody, and Fd fragments.
  • a "derivative" is any molecule having the same basic structure as the parent compound.
  • the antibody fragment may be produced by any means.
  • the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody or it may be recombinantly produced from a gene encoding the partial antibody sequence.
  • the antibody fragment may be wholly or partially synthetically produced.
  • the antibody fragment may optionally be a single chain antibody fragment.
  • the fragment may comprise multiple chains which are linked together, for instance, by disulfide linkages.
  • the fragment may also optionally be a multimolecular complex.
  • a functional antibody fragment will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids.
  • Single-chain Fvs are recombinant antibody fragments consisting of only the variable light chain (VL) and variable heavy chain (VH) covalently connected to one another by a polypeptide linker.
  • V L or V H may be the NH 2 -terminal domain.
  • the polypeptide linker may be of variable length and composition so long as the two variable domains are bridged without serious steric interference.
  • the linkers are comprised primarily of stretches of glycine and serine residues with some glutamic acid or lysine residues interspersed for solubility.
  • “Diabodies” are dimeric scFvs. The components of diabodies typically have shorter peptide linkers than most scFvs and they show a preference for associating as dimers.
  • An "Fv” fragment consists of one V H and one VL domain held together by noncovalent interactions.
  • the term “dsFv” is used herein to refer to an Fv with an engineered intermolecular disulfide bond to stabilize the VH -VL pair.
  • a F(ab') 2 fragment is an antibody fragment essentially equivalent to that obtained from immunoglobulins (typically IgG) by digestion with an enzyme pepsin at pH 4.0-4.5. The fragment may be recombinantly produced.
  • a Fab' fragment is an antibody fragment essentially equivalent to that obtained by reduction of the disulfide bridge or bridges joining the two heavy chain pieces in the F(ab') 2 fragment. The Fab' fragment may be recombinantly produced.
  • a “Fab” fragment is an antibody fragment essentially equivalent to that obtained by digestion of immunoglobulins (typically IgG) with the enzyme papain.
  • the Fab fragment may be recombinantly produced.
  • the heavy chain segment of the Fab fragment is the Fd piece.
  • Active fragments of antibodies preferably include the Fv region of an antibody. Active fragments of antibodies can be made using methods known in the art, such as proteolytic digestion of samples including antibodies. Antibodies may be polyclonal or monoclonal, unless otherwise specified.
  • a preparation of antibodies can be crude, such a whole blood or serum or plasma, or can be partially purified, such as by crude separation methods such as molecular weight purification or ammonium sulfate precipitation, or can be substantially purified, such as by affinity chromatography for a class of antibody, subclass of antibody, or by binding with a particular antigen or epitope. Methods for such purification are known in the art, such as provided by Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press (1988).
  • This embodiment of the preparation procedures involves four steps: activation of the dextran with di vinyl sulfone, attaching BSA to the activated dextran, incorporating the rhodamine dye to the BSA portion of the dextran-BSA backbone, and cross-linking the antibody to the dextran-BSA-Rhodamine backbone.
  • the activation conditions were 10 mg/ml final concentration of dextran, 0.25 M potassium phosphate buffer, 0.25 mg/ml final concentration of sodium borohydride and 5% DVS. The entire operation was done in a fume hood. The dextran, distilled water and potassium phosphate buffer were combined initially and allowed to mix for 10-15 minutes. The sodium borohydride was added and followed immediately by the DVS. The timer was started from the first drop of DVS added and the DVS was added in a dropwise fashion within 2 minutes. After the entire amount of DVS was added, the solution was continually stirred for up to 30-35 minutes. After the 30-35 minute incubation, the activation was stopped by adjusting the pH to 7 with 25% HCl. The activated dextran was dialysed extensively against distilled water, with the water being changed twice a day for four days. The dialysate was collected, and chlorobutanol added at a final concentration of 0.01%.
  • bovine serum albumin in 0.1 M sodium chloride, 0.4 M potassium phosphate pH 10.4 and 0.1 M sodium chloride.
  • the conjugation conditions were: 1 :25 activated dextran to BSA molar ratio, 0.010M K 2 HPO 4 , pH 10.4, 30 0 C, and 22 hours.
  • the activated dextran, BSA solution and potassium phosphate buffer were added together.
  • the pH of the mixture was adjusted to 10.4 with IM HCl.
  • the mixture was placed in a 30°C oven for 22 hours. After the 22 hours incubation, the pH of the mixture was lowered to 6.5 with 1 M HCl. Then the mixture was purified using a S300 size exclusion column with 0.1 M sodium chloride as the running buffer. The first peak was collected and used for the next step.
  • the conjugation conditions were 100-200 ug dye/mg BSA, 0.1 M sodium bicarbonate, pH 8.0, 30°C, and 1 hour.
  • the dextran-BSA, rhodamine solution, and sodium bicarbonate buffer were added together.
  • the pH was adjusted to 8.0 with 1 M HCl.
  • the mixture was incubated in a 30°C oven for 1 hour. After the incubation, the mixture was dialysed extensively against 10 niM K 2 HPO 4 pH 7.2 (2 changes per day for 4 days). The dialysate was collected and Bronidox added at a final concentration of 0.05%.
  • the components needed for the cross-linking are: antibody solution,
  • the cross-linking conditions were 1 :2.5 to 1 :5 dextran-BSA-rhodamine to antibody molar ratio, 30 °C, 18-22 hours and 2.5 M K 2 HPO 4 salt molarity.
  • the dextran- BSA-rhodamine was centrifuged at 4000 g to remove any particulates.
  • the antibody solution, dextran-BSA-rhodamine and K 2 HPO 4 were combined together.
  • the mixture was incubated in a 3O 0 C oven for 18-22 hours. After the incubation, cysteine was added at 1/10 the total volume.
  • the salt concentration was adjusted from 2.5 M to 1.75 M by adding distilled water.
  • the mixture was centrifuged at 9,333 g to pellet the water soluble conjugate.
  • the pellet was resuspended in distilled water at 1 A the original volume of dextran- BSA-rhodamine used for the cross-linking.
  • the resuspended pellet was centrifuged at 327g for 5 minutes.
  • the supernatant was purified in a S300 gel filtration column using 50 mM Tris/O.IM NaCl/0.02% sodium azide as the running buffer. The first peak was collected and used as a label conjugate.
  • the example illustrates the use of sonication in the methods.
  • the cross- linking conditions were 1:2.5 dextran-BSA-Rhodamine to antibody molar ratio, 3O 0 C, 18-22 hours and 2.5M salt molarity.
  • the dextran-BSA-Rhodamine was centrifuged at 4000 g to remove any particulates.
  • the antibody solution, dextran-BSA-Rhodamine and K 2 HPO 4 were combined together. The mixture was incubated in a 30°C oven for 18-22 hours. After the incubation, cysteine was added at 1/10 the total volume.
  • the salt concentration was adjusted from 2.5 M to 1.75 M by adding distilled water.
  • the mixture was then centrifuged at 9,333 g to pellet the water soluble conjugate.
  • the pellet was resuspended in distilled water at 1 A the original volume of dextran-BSA-Rhodamine used for the cross-linking.
  • the resuspended pellet was sonicated (power set at 700 watts, 5 second /cycle, 10 cycles, 10 second pause between cycles) and then centrifuged at 327 g for 5 minutes.
  • the supernatant was purified in a S300 gel filtration column using 50 mM Tris/0.1 M NaCl/0.02% sodium azide as the running buffer. The first peak was collected and used as a label conjugate.
  • the concentration of phosphate buffer in solution was adjusted to 1.75 M by addition of de-ionized water to the solution. Solution was then spun for 5 minutes at 10,000 rpm and the supernatant, which was clear and almost colorless, was carefully aspirated with a pipette.
  • This example illustrates the preparation of a water-soluble conjugate using pre-treatment of antibody (targeting element) with dithiothreitol.
  • NC FHC 102 stripped with Monoclonal anti alpha hCG, Aeon Bio
  • This example illustrates one alternate method of preparing the water- soluble conjugate.
  • the signal component is linked to the targeting element prior to the combination with the water-soluble intermediate conjugate to form the water- soluble conjugate.
  • the bovine serum albumin was conjugated to activated dextran.
  • the composition was purified to separate the free BSA.
  • the hCG antibody was then conjugated to dextran-BSA at a molar ratio of 5:1 in 0.1 M potassium phosphate, pH 9.6, at 30 °C for 18 hours.
  • the composition was again purified to separate the free antibody.
  • Rhodamine dye was conjugated to the dextran-BSA-antibody at a ratio of 150 ug dye/mg protein in 0.1 M sodium bicarbonate, pH 8.0 at 30 0 C for 3 hours.
  • the reaction was stopped with cysteine and dialysed extensively again 10 mM K 2 HPO 4 pH 7.2.
  • the antibody was cross-linked to the dextran-BSA-antibody-dye at a ratio of 2.5:1 in 2.5 M K 2 HPO 4 , pH 10.6 at 30°C for 18 hours.
  • the conjugate was then purified to separate the free antibody.
  • This example illustrates the use of the invention in an indirect assay format.
  • the water soluble conjugate was prepared according to the procedures described above, except that after the first centrifugation, the pellet was washed three times in distilled water. The final pellet was then resuspended in distilled water. The solution was sonicated using 5- second cycles for 10 cycles with 10-sec pause between cycles. A label pad was made at OD 550 45.
  • the label pad was evaluated according to this configuration: a test strip containing a sample zone, a biotinylated alpha-hCG antibody in the reagent zone, a label pad, and Streptavidin-IgG striped down on the nitrocellulose and absorbent in the detection zone.
  • the test strip was placed inside a plastic housing. The test device was tested with different levels of hCG concentration, negative urine and distilled water.
  • This example illustrates the conjugation of HRP to DVS-activated dextran of MW 500,000.
  • Activated dextran with a molecular weight of 500,000 with an extent of activation of 26% was added to HRP solution, in a molar ratio of 1 :20 (dextran:HRP).
  • the coupling buffer was 10 mM phosphate, pH 10.4. Coupling continued for 22 hours at 30 °C using HRP in 40 mg/ml. After removal of the bottle from the 30 0 C incubator, the pH was titrated to 6.5 with 1 M HCl. The solution was separated on a Sephacryl ® S-200 gel filtration column.
  • the column was equilibrated in 0.1 M NaCl, which was degassed before use.
  • the purification was performed by isocratic elution with 0.1 M NaCl as eluent. HRP elution was detected at 403 nm.
  • This example illustrates the synthesis of an anti-hCG conjugate by conjugation of an anti-hCG antibody to divinyl sulfone-activated dextran-HRP, utilizing precipitation in a high salt buffer.
  • Anti-beta-hCG (R006003, 6.5 mg/ml in PBS) is conjugated to an activated dextran-HRP-conjugate via free divinylsulfone (DVS) groups on the dextran. After coupling and precipitation, any free un-reacted VS groups are blocked by addition of cysteine. The precipitate is pelleted by centrifugation and resuspended by sonication in deionized water. The dextran-HRP/anti-hCG conjugate is separated from unbound antibody by gel filtration on Sephacryl ® S-300. The purified conjugate is measured at 280 nm.
  • DVD free divinylsulfone
  • a molar ratio of 2 moles of antibody per 1 mole of dextran-HRP was used. 0.46 ml of Dextran-HRP with 0.95 ml of anti-beta-hCG (6.5 mg/ml) in phosphate-buffered saline (PBS). 1.9 ml OfK 2 HPO 4 (3 M 5 pH 9.0) was added dropwise to bring the phosphate concentration to 2 M, while swirling in a 15 ml conical tube. The total volume was 3.31 ml. The tube was incubated with mild shaking at 125 rpm at 30 °C for 18 hours. After 18 hours the precipitate is accumulated near the solution surface.
  • PBS phosphate-buffered saline
  • the tube was sonicated in a cuphorn sonicator, maintaining ice in the cuphorn bath.
  • the controller of the sonicator was set for 5 second pulses, 90% maximum output.
  • the polypropylene plastic tube was pressed against the sonic probe during the sonication to achieve maximum energy transfer at 4 °C. Sonication was performed in 3 minute intervals. The tube remained on ice and was pressed against the sonicator four times for the 3 minute intervals. This procedure was found to discourage micro-heating of sample and to maximize re-solubilization of sample.
  • the pellet was again spun down and the supernatant was saved.
  • the pellet was resuspended in 0.35 ml of DI water and the sonication procedure repeated. The cycles of sonication and spinning were repeated until at least 90% of the pellet was dissolved. The solutions were then combined.
  • the dissolved conjugate was concentrated to a volume of approximately 1 ml using a spin concentrator with a molecular weight cutoff of 30,000.
  • the conjugate was then applied to an S-300 column (at least 31 ml of bed volume), pre-equilibrated with 50 mM Tris, 0.1 M NaCl, pH 7.2. Elution was at a flow rate of 1 ml/min with Tris buffered saline, and the first peak was collected into one or two fractions. The conjugate eluted in the first peak, and peak fractions were combined.
  • This example illustrates the preparation of antibody coated carbon electrodes to be used as an affinity sensor for electrochemical detection of hCG.
  • a screen-printed carbon electrode was used having a Melinex ® ST328 polyester film substrate. The electrodes were printed using graphite ink and silver chloride ink. The screen printer was an SMT Optiprint, model 1616, PD-F.
  • the electrode was soaked in coating buffer with 100 ⁇ g/ml anti- ⁇ hCG antibody for 2 hours at room temperature, followed by soaking in a blocking buffer for 1 hour at room temperature. After washing and drying, the electrodes were stored dessicated.
  • the coated electrodes were incubated in sample matrix or PBS buffer containing known concentrations of hCG, such as 0, 2, 5, or 50 mIU/ml hCG for 30 minutes at room temperature. After washing, the electrodes were soaked in anti- ⁇ hCG label conjugate (3 ⁇ g/ml) in label conjugate buffer for 30 minutes at room temperature. After washing, 20 ⁇ l of substrate was applied to the electrodes (20 mM naphthol phosphate, 0.1 M NaCl, 0.1 M diethanolamine pH 9.6, for ALP conjugate), and incubated for 10 minutes at room temperature. Signal was then recorded using differential pulse voltammetry.
  • the capture surface was prepared by mixing 50 ul of Dynabeads® M-280 Streptavidin and 133 ul of 100 ug/ml biotinylated 6002 hCG capture antibody to a vessel, and mixing for 50 minutes.
  • the beads were washed with Rinse Buffer (80 ul of phosphate buffer (pH 7.2) with 0.5% BSA and 0.5% Tween). 80 ul of 1% casein was added and the mixture incubated for 2 hours. The mixture was then washed with 1% casein, and re-suspended in 166 ul of 1% casein, and placed in the refrigerator at 4 °C.
  • Tube A The beads in Tube A were transferred to Tube B, and washed with phosphate-buffered saline (PBS). The beads were then separated with a magnet and the supernatant removed. After washing 15 ul of enzyme substrate mixture (10 mM hydroquinone with freshly mixed H 2 O 2 to give a final concentration of 10 mM), mixing for 20 minutes was performed. The beads were separated, and 5 ul of beads were pipetted onto an electrode to run differential pulse voltammetry. [00135] For comparison, in a separate experiment the capture surface was prepared by mixing 30 ul of Dynabeads® M-280 Streptavidin with 80 ul of 100 ug/ml biot-6002 antibody.
  • PBS phosphate-buffered saline
  • the mixture was mixed for 45 minutes, and washed Ix with 80 ul of phosphate buffer (pH 7.2) with 0.5% BSA and 0.5% Tween. 80 ul of 1% casein was added and incubated for 2 hours. The mixture was washed with 80 ul of casein, and re-suspended in 100 ul of 1% casein.
  • the labeled antibody was alkaline phosphatase-6003 conjugate antibody prepared as described herein.
  • hCG standards were prepared as follows:
  • the hCG standards were prepared along one column of a micro-titer plate, and 5 ul of labeled antibody per well was added. At 2 minutes time, 50 ul of hCG standards was rapidly transferred from column 12 to 1, and mixed and pre-incubated for 2 minutes. 10 ul of beads was added per well while mixing on a plate shaker, and incubated for 8 minutes. The beads were separated with a magnet and the supernatant was removed from each well. The beads were rinsed 2x with 70 ul of phosphate buffer (pH 7.2) with 0.5% BSA and 0.5% Tween. 70 ul of the phosphate buffer was then added to each well, and the beads transferred to adjacent wells in column 2.
  • phosphate buffer pH 7.2
  • the beads were again rinsed with 70 ul of the phosphate buffer. 15 ul of substrate (20 mM 1-naphthyl phosphate in 100 mM diethanolamine, pH 9.6) was added, and the mixture incubated for 25 minutes. The beads were held and 8 ul of substrate was transferred directly onto a screen printed electrode for differential pulse voltammetric measurements.
  • Figure 2 shows that, when electrochemical detection was utilized according to the present invention, a several-fold higher sensitivity was obtained compared to traditional electrochemical methods. When one corrects for the nearly twice as long incubation time (20 min) used in the traditional methods versus 11 minutes for the present method, the difference is even more dramatic.
  • the invention illustratively described herein may be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein.
  • the terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.

Abstract

La présente invention concerne des conjugués hydrosolubles et leurs procédés d'utilisation dans des dosages diagnostiques et de détection. L'invention concerne également des dispositifs pour réaliser de dosages de détection et de quantification. Dans divers modes de réalisation, les conjugués sont utilisés dans des dosages immunologiques et des dosages de flux latéral. L'invention concerne en outre des procédés de préparation des conjugués donnant lieu à des rendements plus élevés et à des sensibilités supérieures pour les dosages. L'invention concerne enfin des conjugués hydrosolubles mettant en oeuvre des composants à signal électrochimique capables de détecter des analytes avec une très grande sensibilité.
PCT/US2006/010406 2006-03-20 2006-03-20 Conjugués hydrosolubles pour la détection électrochimique WO2007123507A1 (fr)

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CN200680053685XA CN101395473B (zh) 2006-03-20 2006-03-20 利用水溶性轭合物的电化学检测方法
DE112006003813T DE112006003813T5 (de) 2006-03-20 2006-03-20 Wasserlösliche Konjugate zur elektrochemischen Detektion
JP2009501392A JP2009530639A (ja) 2006-03-20 2006-03-20 電気化学的検出のための水溶性コンジュゲート
PCT/US2006/010406 WO2007123507A1 (fr) 2006-03-20 2006-03-20 Conjugués hydrosolubles pour la détection électrochimique
GB0816299A GB2449043A (en) 2006-03-20 2008-09-05 Water soluble conjugates for electrochemical detection

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CN101923092A (zh) * 2010-06-28 2010-12-22 宁波大学 丝网印刷电极的癌胚抗原工作电极的制备方法
JP2012530253A (ja) * 2009-06-16 2012-11-29 ベー．エル．アー．ハー．エム．エス ゲゼルシャフト ミット ベシュレンクテル ハフツング ペルオキシレドキシン４の診断的使用
CN101825627B (zh) * 2009-03-02 2013-10-02 江苏迈迪基因生物科技有限公司 心力衰竭的生物标志物的联合并行检测方法及诊断试剂盒
US8609433B2 (en) 2009-12-04 2013-12-17 Rapid Pathogen Screening, Inc. Multiplanar lateral flow assay with sample compressor
US8669052B2 (en) 2008-06-10 2014-03-11 Rapid Pathogen Screening, Inc. Lateral flow nucleic acid detector
CN103712982A (zh) * 2013-12-13 2014-04-09 山东博科生物产业有限公司 一种高灵敏度的tmb显色液及其制备方法
US8815609B2 (en) 2008-05-20 2014-08-26 Rapid Pathogen Screening, Inc. Multiplanar lateral flow assay with diverting zone
US8962260B2 (en) 2008-05-20 2015-02-24 Rapid Pathogen Screening, Inc. Method and device for combined detection of viral and bacterial infections
US9068981B2 (en) 2009-12-04 2015-06-30 Rapid Pathogen Screening, Inc. Lateral flow assays with time delayed components
US9121849B2 (en) 2008-06-10 2015-09-01 Rapid Pathogen Screening, Inc. Lateral flow assays
US10379121B2 (en) 2008-05-20 2019-08-13 Rapid Pathogen Screening, Inc. Method and device for combined detection of viral and bacterial infections
US10808287B2 (en) 2015-10-23 2020-10-20 Rapid Pathogen Screening, Inc. Methods and devices for accurate diagnosis of infections
EP3985392A4 (fr) * 2019-06-12 2023-06-28 GMD Biotech, Inc Conjugué pour immunodétection basé sur un dosage à écoulement latéral, et procédé d'immunodétection l'utilisant

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101581694B (zh) * 2009-06-20 2012-05-09 西北师范大学 氢醌的电化学检测方法
ES2410754T3 (es) * 2010-05-25 2013-07-03 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Procedimiento para la detección electroquímica de reacciones de unión
US9347946B2 (en) * 2010-09-23 2016-05-24 Biocept, Inc. Methods and reagents for signal amplification
KR101177353B1 (ko) * 2010-10-08 2012-11-15 주식회사 메디센서 진단 장치 및 이를 이용한 진단 방법
JP6455149B2 (ja) * 2013-01-11 2019-01-23 コニカミノルタ株式会社 センサーチップの製造方法
WO2018135651A1 (fr) * 2017-01-20 2018-07-26 国立大学法人埼玉大学 Conjugué anticorps-polysaccharide et procédé de dosage immunologique à haute sensibilité l'utilisant
TWI655288B (zh) * 2017-10-26 2019-04-01 國立中興大學 Method of detecting a target in a sample
CN112630283B (zh) * 2020-12-18 2023-01-17 河南城建学院 (e, e)-1,1′-双(2-吡啶乙烯基)二茂铁作为电化学传感器的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5543332A (en) * 1991-07-04 1996-08-06 Immunodex K/S Water-soluble, polymer-based reagents and conjugates comprising moieties derived from divinyl sulfone
US6627460B1 (en) * 1998-07-30 2003-09-30 Amdex A/S Method for preparing water-soluble cross-linked conjugates

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FI792576A (fi) 1979-08-20 1981-02-21 Orion Yhtymae Oy Foerbaettrad metod att utfoera enzymimmunologiska bestaemningar
EP0149168B1 (fr) 1983-12-19 1991-04-24 Daiichi Pure Chemicals Co. Ltd. Dosage immunologique
CA1231136A (fr) 1984-06-13 1988-01-05 Ian A. Shanks Dispositif d'essai chimique agissant par capillarite
GB8500698D0 (en) 1985-01-11 1985-02-13 Unilever Plc Preparation of reagents
AU602694B2 (en) 1986-06-09 1990-10-25 Ortho Diagnostic Systems Inc. Improved colloidal gold membrane assay
AU590071B2 (en) 1987-02-25 1989-10-26 Genesis Labs, Inc. Dry test strips having a red blood cell exclusion layer preventing interference by red blood cells in analyte detection visualization
DE3887771C5 (de) 1987-04-27 2009-06-04 Inverness Medical Switzerland Gmbh Immunoassays und Vorrichtungen dafür.
AU2684488A (en) 1988-06-27 1990-01-04 Carter-Wallace, Inc. Test device and method for colored particle immunoassay
GB8923868D0 (en) * 1989-10-26 1989-12-13 Immunosens Spa Method and apparatus for electrochemical immunoassay
CA2112992C (fr) 1991-07-04 2002-06-11 Allan O. F. Lihme Reactifs hydrosolubles a base de polymere et composes conjugues comprenant des fractions derivees du divinylsulfone
CN1046531C (zh) * 1994-07-25 1999-11-17 伯伦格·曼海姆有限公司 有带电连接基的金属配合物、轭合物及其用途和制备方法
ATE468531T1 (de) * 2001-05-30 2010-06-15 I Sens Inc Biosensor
JP4086277B2 (ja) * 2001-12-27 2008-05-14 栄研化学株式会社 水溶性担体−抗体複合体の製造方法および使用方法
EP1512009A2 (fr) * 2002-05-08 2005-03-09 Yissum Research Development Company Of The Hebrew University Of Jerusalem Procede et dispositif a controle magnetique pour la determination d'un analyte dans un support liquide

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5543332A (en) * 1991-07-04 1996-08-06 Immunodex K/S Water-soluble, polymer-based reagents and conjugates comprising moieties derived from divinyl sulfone
US6627460B1 (en) * 1998-07-30 2003-09-30 Amdex A/S Method for preparing water-soluble cross-linked conjugates

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8815609B2 (en) 2008-05-20 2014-08-26 Rapid Pathogen Screening, Inc. Multiplanar lateral flow assay with diverting zone
US10408835B2 (en) 2008-05-20 2019-09-10 Rapid Pathogen Screening, Inc. Method and device for combined detection of viral and bacterial infections
US10379121B2 (en) 2008-05-20 2019-08-13 Rapid Pathogen Screening, Inc. Method and device for combined detection of viral and bacterial infections
US8962260B2 (en) 2008-05-20 2015-02-24 Rapid Pathogen Screening, Inc. Method and device for combined detection of viral and bacterial infections
US8669052B2 (en) 2008-06-10 2014-03-11 Rapid Pathogen Screening, Inc. Lateral flow nucleic acid detector
US8822151B2 (en) 2008-06-10 2014-09-02 Rapid Pathogen Screening, Inc. Lateral flow nucleic acid detector
US9121849B2 (en) 2008-06-10 2015-09-01 Rapid Pathogen Screening, Inc. Lateral flow assays
CN101825627B (zh) * 2009-03-02 2013-10-02 江苏迈迪基因生物科技有限公司 心力衰竭的生物标志物的联合并行检测方法及诊断试剂盒
JP2012530253A (ja) * 2009-06-16 2012-11-29 ベー．エル．アー．ハー．エム．エス ゲゼルシャフト ミット ベシュレンクテル ハフツング ペルオキシレドキシン４の診断的使用
US8609433B2 (en) 2009-12-04 2013-12-17 Rapid Pathogen Screening, Inc. Multiplanar lateral flow assay with sample compressor
US9068981B2 (en) 2009-12-04 2015-06-30 Rapid Pathogen Screening, Inc. Lateral flow assays with time delayed components
US9939434B2 (en) 2009-12-04 2018-04-10 Rapid Pathogen Screening, Inc. Multiplanar lateral flow assay with sample compressor
CN101923092A (zh) * 2010-06-28 2010-12-22 宁波大学 丝网印刷电极的癌胚抗原工作电极的制备方法
CN103712982A (zh) * 2013-12-13 2014-04-09 山东博科生物产业有限公司 一种高灵敏度的tmb显色液及其制备方法
CN103712982B (zh) * 2013-12-13 2016-02-03 山东博科生物产业有限公司 一种高灵敏度的tmb显色液及其制备方法
US10808287B2 (en) 2015-10-23 2020-10-20 Rapid Pathogen Screening, Inc. Methods and devices for accurate diagnosis of infections
EP3985392A4 (fr) * 2019-06-12 2023-06-28 GMD Biotech, Inc Conjugué pour immunodétection basé sur un dosage à écoulement latéral, et procédé d'immunodétection l'utilisant

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JP2009530639A (ja) 2009-08-27
CN101395473B (zh) 2012-09-19

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