EP1282818A2 - Methode permettant d'etudier des interactions moleculaires fonctionnelles et reactifs utilises a cet effet - Google Patents

Methode permettant d'etudier des interactions moleculaires fonctionnelles et reactifs utilises a cet effet

Info

Publication number
EP1282818A2
EP1282818A2 EP01922998A EP01922998A EP1282818A2 EP 1282818 A2 EP1282818 A2 EP 1282818A2 EP 01922998 A EP01922998 A EP 01922998A EP 01922998 A EP01922998 A EP 01922998A EP 1282818 A2 EP1282818 A2 EP 1282818A2
Authority
EP
European Patent Office
Prior art keywords
binding
members
label
microspheres
labels
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01922998A
Other languages
German (de)
English (en)
Inventor
Thomas G. c/o Glaxo Wellcome Inc. CONSLER
John G. c/o Glaxo Wellcome Inc. GRAY
Marie A. Glaxo Wellcome Inc. IANNONE
Julie B. Glaxo Wellcome Inc. STIMMEL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Glaxo Group Ltd
Original Assignee
Glaxo Group Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Glaxo Group Ltd filed Critical Glaxo Group Ltd
Publication of EP1282818A2 publication Critical patent/EP1282818A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • 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
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles

Definitions

  • This invention relates generally to a rapid and sensitive method of investigating molecular functional interactions in a multiplexed, high-throughput format.
  • the invention has broad applicability in the pharmaceutical industry as a means for identifying compounds useful in diagnosis or therapy.
  • BACKGROUND ART Molecules typically function by interaction with other molecules, including protein/protein interactions, nucleic acid/protein interactions, and nucleic acid/nucleic acid interactions. Furthermore, these functional interactions are frequently modulated by an additional molecule or molecules to form a macromolecular functional unit. Assays that have traditionally been used to evaluate these functional interactions include enzyme activity assays, ligand binding assays, endpoint assays, kinetic binding assays, competitive binding assays, hybridization reactions, BIACORE, homogeneous time-resolved fluorescence, scintillation proximity assays, and others.
  • this invention in one aspect, relates to a method of identifying, in a single assay, the relative binding of two or more first members of a binding pair to one or more second members of a binding pair, comprising (a) contacting the first members with the second member or members under conditions that allow the first members to bind with the second member or members to form binding pairs, wherein the first members are coupled directly or indirectly to a set of microspheres labeled with a set of first labels, wherein each first label is specific for one first member, wherein each second member is coupled directly or indirectly to a second label, and wherein each second label is specific for one second member; (b)detecting the presence of the first specific labels and the presence or absence of the second labels in a plurality of detection products, wherein each detection product comprises one microsphere; (c) comparing the relative amount of each second label associated with each first label in the detection products, wherein a greater or lesser amount of each second label associated with
  • the invention in another aspect, relates to a method of identifying, in a single assay, the relative modulation, by a modulating agent, of binding between two or more binding pairs, comprising (a) contacting two or more first members and one or more second members of the binding pairs and the modulating agent, under conditions that allow formation of binding pairs; wherein the first members are coupled directly or indirectly to a set of microspheres labeled with a set of first labels; wherein each first label is specific for one first member; wherein the second member is coupled directly or indirectly to a second label; and wherein the second label is specific for one second member; (b) detecting the presence of the first specific labels and the second specific labels in a plurality of detection products, wherein each detection product comprises one microsphere; and (c) comparing, to a control, the relative amount of each second label associated with each first label in the detection products, wherein the control lacks the modulating agent, a greater or lesser amount of each second label associated with each first label in the detection product, indicating that the modulating agent
  • the invention relates to a method of identifying, in a single assay, the relative modulation, by a modulating agent, of binding between the members of two or more binding complexes, comprising (a) contacting two or more first members, one or more second members, a third member of the binding complexes, and the modulating agent, under conditions that allow formation of binding complexes, wherein each binding complex comprises one first and one second member of the binding complex; wherein the first members are coupled directly or indirectly to a set of microspheres labeled with a set of first labels; wherein each first label is specific for one first member; wherein the second member is coupled directly or indirectly to a second label; and wherein the second label is specific for one second member; (b) detecting the presence of the first specific labels and the presence or absence of the second specific labels in a plurality of detection products, wherein each detection product comprises one microsphere; (c) comparing, to a control, the relative amount of each second label associated with each first label in the detection products, wherein
  • the invention relates to a method of screening, in a single assay, for an agent or agents with a selected binding profile, comprising (a) contacting the agent or agents to be screened with two or more putative binding members under conditions that allow the agent or agents to bind with the binding members to form binding pairs, wherein the binding members are coupled directly or indirectly to a set of microspheres labeled with a set of first labels, wherein each first label is specific for one binding member, wherein each agent to be screened is coupled directly or indirectly to a second label, and wherein each second label is specific for one agent to be screened; (b) detecting the presence of the first specific label and the presence or absence of second specific labels in a plurality of detection products, wherein each detection product comprises one microsphere; (c) comparing the relative amount of each second label associated with each first label in the detection products, wherein a greater or lesser amount of each second label associated with each first label in the detection product indicates an agent, indicated by the second specific label, having greater or lesser binding to the binding
  • the invention relates to a method of screening, in single assay, for an agent that modulates binding between two or more first members of a binding pair and one or more second members of a binding pair, comprising (a) contacting two or more first members and one or more second members of the binding pairs and the modulating agent, under conditions that allow formation of binding pairs; wherein the first members are coupled directly or indirectly to a set of microspheres labeled with a set of first labels; wherein each first label is specific for one first member; wherein the second member is coupled directly or indirectly to a second label; and wherein the second label is specific for one second member; (b) detecting the presence of the first specific labels and the presence or absence of second specific labels in a plurality of detection products, wherein each detection product comprises one microsphere; (c) comparing, to a control, the relative amount of each second label associated with each first label in the detection products, wherein the control lacks the agent to be screened, a greater or lesser amount of each second label associated with each first label in the
  • the invention in another aspect relates to a method of screening, in a single assay, for an agent that modulates binding between the members of two or more binding complexes, comprising (a) contacting two or more first members, one or more second members, and a third member of the binding complexes with the agent to be screened, under conditions that allow formation of binding complexes, wherein each binding complex comprises one first and one second member; wherein the first members are coupled directly or indirectly to a set of microspheres labeled with a set of first labels; wherein each first label is specific for one first member; wherein the second member is coupled directly or indirectly to a second label; and wherein the second label is specific for one second member; (b) detecting the presence of the first specific labels and the presence or absence of second specific labels in a plurality of detection products, wherein each detection product comprises one microsphere; and (c) comparing, to a control, the relative amount of each second label associated with each first label in the detection products, wherein the control lacks the agent to be screened,
  • Fig. 1 shows a complex binding profile of estrogen receptor (ER) ⁇ ligand binding domain (LBD) and peoxisome proliferator-activated receptor (PPAR) ⁇ LBD for three LXXLL-motif coactivator peptides.
  • ER estrogen receptor
  • LBD ligand binding domain
  • PPAR peoxisome proliferator-activated receptor
  • Figure 2 shows a complex binding profile for ER ⁇ LBD binding to 34 coactivator peptides in the presence or absence of estradiol (2 ⁇ M).
  • Figure 3 shows a complex binding profile for ER ⁇ LBD binding to 37 coactivator peptides in the presence or absence of tamoxifen (500 nM) or raloxifene (500nM).
  • Figure 4A shows a complex binding profile of coactivator peptides for LBDs derived from Receptor B (lOOnM).
  • Figure 4B shows a complex binding profile, in the presence or absence of a compound designated Agent 1 (200nM), of coactivator peptides for LBDs derived from Receptor C (lOOnM).
  • Figure 4C shows a complex binding profile of coactivator peptides for PPAR ⁇ LBD (1 OOnM) in the presence and absence of five different compounds (designated Agent 2, Agent 3, Agent 4, Agent 5, and Agent 6) at 1 ⁇ M.
  • Figure 5 shows a complex binding profile of binding ER ⁇ LBD with coactivator peptides (SRC-1 (3 (735-759), SRC- 1(626-642), and SRC-l(2)(676-700) and a corepressor peptide(SMRT) (2329-2354) in the absence and presence of various concentrations of estradiol (50, 100, 150, 200 nM).
  • Figure 6 shows the binding profile of ER ⁇ LBD to 34 different coactivator peptides in the absence or presence of estradiol, raloxifene or tamoxifen.
  • first member of a binding pair includes mixtures of first members of binding pairs, and the like.
  • Ranges may be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
  • the invention provides a method of identifying, in a single assay, the relative binding of two or more first members of a binding pair to one or more second members of a binding pair, comprising the steps of (a) contacting the first members with the second member or members under conditions that allow the first members to bind with the second member or members to form binding pairs, wherein the first members are coupled directly or indirectly to a set of microspheres labeled with a set of first labels, wherein each first label is specific for one first member, wherein each second member is coupled directly or indirectly to a second label, and wherein each second label is specific for one second member; (b) detecting the presence of the first specific labels and the presence or absence of the second specific labels in a plurality of detection products, wherein each detection product comprises one microsphere; (c) comparing the relative amount of each second label associated with each first label in the detection products, wherein a greater or lesser amount of each second label associated with each first label in the detection product indicates first members, identified by the specific first label, having greater or lesser binding to
  • a single assay means a single assay volume (e.g., a single tube or well).
  • the single assay can be a multiplexed assay in which simultaneous, or near simultaneous, determinations of binding events can be measured from the same assay process. For example, numerous first members, second members, and additional members of numerous binding complexes can be added to the same assay process for subsequent identification and analysis.
  • this multiplexed format allows a washless format, which improves sensitivity, saves reagents, and promotes efficiency.
  • “Relative binding” means the amount of binding that occurs between one binding pair as compared to another binding pair.
  • one first member may have relatively higher binding to one specific second member than to a different second member.
  • One first member may have higher binding to one second member as compared to the binding between a different first member and a different second member.
  • a specific first and a specific second member may have higher relative binding compared to a different first member and different second member.
  • the amount of binding can include the absence of binding.
  • Such conditions can also include conditions in which some bound first members/second members bind to third members, modulating agents, or agents to be screened. Such conditions do not require that all first and second members combine, only that the conditions allow for specific binding interactions to occur. These conditions include, for example, pH, time, temperature, and buffer composition, which allow binding between the members of interest.
  • binding pairs is meant at least two binding members that have bound together.
  • a modulating agent may bind to either member of the binding pair and may prevent or enhance binding between the first and second members of the binding pair.
  • binding complex is meant at least a binding pair and, optionally, a third member and/or a modulating agent. In some cases the modulating agent may allow binding between two members of a binding pair that do not form a binding pair in the absence of the modulating agent.
  • Modulating agents optionally are identified or classified using peptide sequences that have been identified by phage display. Such peptide sequences can be used as receptor conformation-sensing sequences, which are then used to screen for modulating agents.
  • a “detection product” comprises one microsphere.
  • Each microsphere can have a bound first member which can bind to a second member, a third member, an agent to be screened, or a modulating agent.
  • the microspheres can be identified based on the first label.
  • the "first label” can include one or more labels mixed together that together constitute a specific "first label” for a subset of microspheres.
  • microspheres can be labeled with two or more flurochromes mixed together in varying concentrations, such that each specific label has a specific concentration of each fluorochrome. It is the specific concentrations of the various flurochromes together to provide a spectrum of labels that can be used to distinguish the various subsets of labeled microspheres.
  • One microsphere preferably has only one first member coupled to it, but a plurality of the same first members is preferably coupled to a single microsphere. If a second member or a labeled agent to be screened binds to at least one first member coupled to the microsphere, then a detection product can comprise the microsphere, at least one first member, and at least one second member or agent to be screened. Preferably, a plurality of second members or agents to be screened, which are the same or different than each other but which have specific second labels, bind to the numerous molecules of the first member.
  • Detection products can also comprise additional members (i.e., third members, fourth members, fifth members, etc.) or additional modulating agents (including, for example, first modulators that reduce binding between the first and second members or second modulators that are agonists or antagonists (or modulators) of the first modulators).
  • additional members i.e., third members, fourth members, fifth members, etc.
  • additional modulating agents including, for example, first modulators that reduce binding between the first and second members or second modulators that are agonists or antagonists (or modulators) of the first modulators.
  • a set of microspheres is meant a group of microspheres consisting of subsets of microspheres labeled with distinguishable labels.
  • a specific label for each first member species can be detected.
  • a known amount of coupled microspheres, and consequently a known amount of each first label, is added to each assay process.
  • the first label for uncoupled microspheres is detectable and constant for each set.
  • it is the relative amount of second label as compared to first label in a given detection product that is relevant as the amount of second label varies depending upon binding between the first and second members of the detection product.
  • the microspheres can be coated with strepavidin or maleimide or can be carboxylated, or any modification of strepavidin, maleimide, or carboxylation, and the first members to be coupled to these microspheres can be biotintylated, have one or more free arnine groups, or have one or more free sulfhydryl groups, respectively.
  • the first members to be coupled to these microspheres can be biotintylated, have one or more free arnine groups, or have one or more free sulfhydryl groups, respectively.
  • coupling agents can be used. .
  • WO 99/19515 and WO 99/37814 which are incorporated herein by reference in their entirety for types of functional groups that can be used for coupling the first agent to the microspheres.
  • a linker can be used between the microsphere and the coupling agent.
  • Labels can also be coupled to the microspheres, agents to be screened, or second members using a variety of methods known in the art.
  • label refers to a moiety (e.g., a hapten) that provides a means for labeling, as well as radiolabels, and fluorescent labels.
  • the first and second labels can be radiolabels, dyes, fluorescent labels, or a combination thereof.
  • the microspheres contain the first labels.
  • the label could be attached to the surface of the microsphere.
  • the second label can be attached to the second member of a binding pair or complex or agent to be screened either directly or indirectly.
  • a fluorescent compound such as fluorescein
  • fluorescein can be incorporated into the member or agent to be screened.
  • the second member or agent to be screened can be biotintylated and a subsequent detectable label like a fluorescently labeled strepavidin can be used to indirectly label the second member or agent to be screened.
  • a subsequent detectable label like a fluorescently labeled strepavidin can be used to indirectly label the second member or agent to be screened.
  • fluorochromes could be used as labels in the present method. . See, e.g., WO 99/19515 and WO 99/37814, which are incorporated herein in their entirety for types of fluorescent dyes and fluorochromes that can be used as labels.
  • microspheres can be used. See, e.g., WO 99/19515 and WO 99/37814, which are incorporated herein in their entirety for types of microspheres and methods of making and using same.
  • the microspheres can be polystyrene-divinylbenzene microspheres.
  • detecting the presence of a label means detecting any amount of label.
  • the amount of label may be an absence of the label.
  • a binding profile is a systematic summary of the binding data such that the binding profile indicates the relative binding between each first member and each second member of various binding pairs.
  • a specific binding profile indicates the hierarchical binding of each first member with each second member as compared to different first members and the same or different second members.
  • the binding hierarchy can be a function of both K D or N max for each specific binding pair or binding complex.
  • the data constituting a binding profile can be shown in several different ways. For example, the fluorescence profile can be shown, wherein a shift in the fluorescence profile of a subset of microspheres indicates the presence of a second label. See, for example, Figure 1.
  • the binding profile can be represented by plotting the mean fluorescence per microsphere as mean fluorescence index (MFI) or molecules of equivalent soluble fluorochrome (MESF) for each binding pair and/or each binding complex.
  • the first and second labels are excited by one or more light sources and detected using a detection device, wherein the detection device is capable of detecting and distinguishing each detection product and each label in the same detection product.
  • the light source or sources are lasers.
  • the labels are fluorescent dyes or molecules that can be detected and distinguished from all other first and second labels used in the assay. The first and second labels can be detected and distinguished by passing the microspheres across or through the light emitted by the light source or light sources.
  • the first and second labels are detected and distinguished by passing the emitted light across or through the microspheres.
  • the microspheres can be on a two-dimensional surface, or traditional flow cytometric methods can be used, whereby the microspheres are aligned so that they travel in single file while being hydrodynamically restricted to the central part of a thin column of fluid.
  • the rapidly moving line of microspheres are then passed before a focused beam of laser light, which excites the fluorochromes (i.e., first and second labels) associated with the microspheres.
  • the laser excites the fluorochromes and the emitted fluorescent light is collected for each particle passing the beam, whereby different wavelengths are analyzed simultaneously or nearly simultaneously.
  • the fluorescent light associated with each microsphere is reported in real time and/or stored and analyzed for characteristics that identify and quantify both the microsphere/first member and the second member/agent to be screened. Multiple fluorescence measurements can be made using the methods described in WO
  • the first members are selected from a group consisting of cofactors, receptors, receptor ligands, proteins, peptides, protein domains, oligonucleotides, transcription factors, nucleic acids, small molecules, and small compounds, any of which can be synthetic, modified, or naturally occurring.
  • the second members are selected from a group consisting of cofactors, receptors, receptor ligands, proteins, peptides, protein domains, oligonucleotides, transcription factors, nucleic acids, small molecules, and small compounds, any of which can be synthetic, modified, or naturally occurring.
  • the first members can be cofactors and the second member or members can be receptors.
  • the receptors can be coupled to the microspheres and the cofactors can be in solution.
  • small molecules is meant natural or synthetic organic molecules less than about 1000 daltons and, more preferably, less than about 500 daltons. Small molecules, for example, include estradiol, cyclic nucleotides, retinoic acid, steroid hormones, amino acids, neurotransmitters (e.g., norepinephrine, epinephrine, acetylcholine), and numerous other compounds.
  • the receptors can be, for example, orphan receptors or nuclear receptors.
  • "orphan receptors" are molecules identified as having receptor or receptor-like domains or tertiary structures but lacking a known function. Thus, the present methods can be used to characterize the binding profile of an orphan receptor in an effort to characterize the function of the receptor.
  • Nuclear receptors include, for example, all known receptors identified by the Nuclear Receptors Nomenclature Committee, 1999. See Vincent Laudet, Johan Auwerx, Jan-Ake Gustafsson, and Walter Wahli; A Unified Nomenclature System for the Nuclear Receptor Superfamily, Cell (1999) 97: 161-163, which is incorporated herein in its entirety by reference for the identification of known nuclear receptors.
  • Nuclear receptors also include, for example, receptors that have yet to be identified but have at least one function or have at least one characteristic of known nuclear receptors.
  • Cofactors or “coregulators” as used throughout can refer to coactivators, corepressors, or a combination of coactivators and corepressors.
  • nuclear receptor cofactors include, for example, those identified in Daniel Robyr, Alan P.Wolffe and Walter Wahli, Nuclear Hormone Receptor Coregulators in Action: Diversity for Shared Tasks, Mol.Endocrinol. (2000) 14: 329-347, which is incorporated herein in its entirety for examples of nuclear receptor cofactors.
  • the invention can be used to confirm the results of a phage display that identifies amino acid sequences that bind to form a binding pair or binding complex.
  • the method comprises the steps of (a) expressing the amino acids to form the first members of a binding pair or binding complex; (b) coupling the first members to microspheres labeled with first specific labels, wherein each first label is specific for one first member; (c) contacting the first members with one or more second members, and optionally with a third member of a binding complex, under conditions that allow formation of binding pairs or binding complexes, wherein each second member is coupled directly or indirectly to a second label, and wherein the second label is specific for each second member; and (d) detecting the presence of the first specific labels and the second specific labels in a plurality of detection products, wherein each detection product comprises one microsphere.
  • the invention can be used to confirm the results of a phage display that identifies amino acid sequence of two or more modulating agents of a binding pair or a binding complex
  • Also provided by the present invention is a method of identifying, in a single assay, the relative modulation, by a modulating agent, of binding between two or more binding pairs, comprising (a) contacting two or more first members and one or more second members of the binding pairs and the modulating agent, under conditions that allow formation of binding pairs, wherein the first members are coupled directly or indirectly to a set of microspheres labeled with a set of first labels; wherein each first label is specific for one first member; wherein the second member is coupled directly or indirectly to a second label; and wherein the second label is specific for one second member; (b) detecting the presence of the first specific labels and the presence or absence of the second specific labels in a plurality of detection product, wherein each detection product comprises one microsphere; (c) comparing, to a control, the relative amount of each second label associated with each first label in the detection products, wherein the control lacks the modulating agent, a greater or lesser amount of each second label associated with each first label in the detection product, indicating that the modul
  • relative modulation is meant either attenuation or potentiation in the amount of binding that occurs between one binding pair in the presence or absence of a modulator as compared to the amount of binding that occurs in another binding pair in the presence or absence of the modulator.
  • one first member may have relatively higher binding to one specific second member in the presence of the modulator as compared to binding to the same second member in the absence of the modulator. This modulation may differ when the same first member binds to a different second member or when a different first member binds to the same or a different second member.
  • a specific first and a specific second member may have higher relative binding compared to a different first member and different second member in the presence of a modulator.
  • the present method can further comprise creating a modulating profile for each modulating agent, wherein the modulating profile indicates the relative modulation of binding between each first member and each second member of the binding pairs.
  • modulating profile is meant a systematic summary of the binding data such that the modulating profile indicates the relative modulation by the modulating agent of binding between each first member and each second member of various binding pairs.
  • a specific modulating profile indicates the hierarchical modulation of binding of each first member with each second member as compared to different first members and the same or different second members.
  • the modulating profile may show that a particular modulating agent strongly attenuates binding between certain members of a binding pair but weakly potentiates binding between members of a different binding pair.
  • the modulating agent is selected from a group consisting of cofactors, receptors, receptor ligands, proteins, peptides, protein domains, oligonucleotides, transcription factors, nucleic acids, small molecules, and small compounds, any of which can be synthetic, modified, or naturally occurring.
  • the first members are cofactors
  • the second member or members are receptors
  • the modulating agent is a receptor ligand (e.g., estradiol).
  • a receptor ligand e.g., estradiol
  • the present invention also provides a method of identifying, in a single assay, the relative modulation, by a modulating agent, of binding between the members of two or more binding complexes, comprising (a) contacting two or more first members, one or more second members, a third member of the binding complexes, and the modulating agent, under conditions that allow formation of binding complexes, wherein the binding complex comprises one first and one second member, and optionally, a third member and/or a modulating agent; wherein the first members are coupled directly or indirectly to a set of microspheres labeled with a set of first labels; wherein each first label is specific for one first member; wherein the second member is coupled directly or indirectly to a second label; and wherein the second label is specific for one second member; (b) detecting the presence of the first specific label and the presence or absence of a second specific label in a plurality of detection products, wherein each detection product comprises one microsphere; (c) comparing, to a control, the relative amount of each second label associated
  • condition that allow formation of detection products means conditions in which first members can bind to second members, and preferably conditions in which modulating agents can bind to either the first member, second member, third member, or some combination thereof. Such conditions do not require that all first and second members bind or that all third members bind, only that the conditions allow for specific binding interactions to occur. These conditions include, for example, pH, time, temperature, and buffer composition, which allow binding between the members and agents of interest. Where there is a third member of the binding complex and/or a third member of the detection product, the third member of the binding complex can potentiate or attenuate binding between the first member and second member of the binding complex.
  • the third member can itself be a modulator (e.g., an agonist or antagonist), whose effect is modulated by the modulating agent.
  • the third members and modulating agents are selected from a group consisting of cofactors, receptors, receptor ligands, proteins, peptides, protein domains, oligonucleotides, transcription factors, nucleic acids, small molecules, and small compounds, any of which can be synthetic, modified, or naturally occurring.
  • the first members are cofactors
  • the second member or members are receptors
  • the third member is a receptor ligand.
  • the modulators could result in formation of binding complexes that would not form in the absence of the modulating agent.
  • a modulating agent can bind to the binding pair, the binding complex or any member thereof.
  • the invention further provides a method of screening, in a single assay, for an agent or agents with a selected binding profile, comprising (a) contacting the agent or agents to be screened with two or more putative binding members under conditions that allow the agent or agents to bind with the binding members to form detection products, wherein the binding members are coupled directly or indirectly to a set of microspheres labeled with a set of first labels, wherein each first label is specific for one binding member, wherein each agent to be screened is coupled directly or indirectly to a second label, and wherein each second label is specific for one agent to be screened; (b)detecting the presence of the first and second labels in the same detection products; (c) comparing the relative amount of each second label associated with each first label in the detection products, wherein a greater or lesser amount of each second label associated with each first label in the detection product indicates an agent, indicated by the second specific label, having greater or lesser binding to the binding members, indicated by the first specific label; (d) creating a binding profile for each agent to be screened, where
  • the binding members can be selected from a group consisting of cofactors, receptors, receptor ligands, proteins, peptides, protein domains, oligonucleotides, transcription factors, nucleic acids, small molecules, and small compounds.
  • the agent or agents to be screened are selected from a group consisting of cofactors, receptors, receptor ligands, proteins, peptides, protein domains, oligonucleotides, transcription factors, nucleic acids, small molecules, and small compounds.
  • the binding members are cofactors and the agent or agents to be screened are receptors.
  • the method could be used to screen for orphan receptors or nuclear receptors having a selected binding profile.
  • “putative binding members” is meant binding members that may or may not bind to the agent or agents to be screened to form a binding pair.
  • the method also provides a method of screening, in single assay, for an agent that modulates binding between two or more first members of a binding pair and one or more second members of a binding pair, comprising (a) contacting two or more first members and one or more second members of the binding pairs and the modulating agent, under conditions that allow formation of binding pairs; wherein the first members are coupled directly or indirectly to a set of microspheres labeled with a set of first labels; wherein each first label is specific for one first member; wherein the second member is coupled directly or indirectly to a second label; and wherein the second label is specific for one second member; (b) detecting the presence of the first specific labels and the second specific labels in a plurality of detection products, wherein each detection product comprises one microsphere; (c) comparing, to a control, the relative amount of each second label associated with each first label in the detection products, wherein the control lacks the agent to be screened, a greater or lesser amount of each second label associated with each first label in the detection products, indicating an agent that
  • the method can further comprise creating a modulating profile for each agent to be screened, wherein the modulating profile indicates the relative modulation of binding between each first member and each second member of the binding pairs, and comparing the modulating profile for the agent to be screened to a selected profile.
  • the agent to be screened can be selected from a group consisting of cofactors, receptors, receptor ligands, proteins, peptides, protein domains, oligonucleotides, transcription factors, nucleic acids, small molecules, and small compounds.
  • the first members are cofactors
  • the second member or members are receptors (including, for example, orphan or nuclear receptors)
  • the modulating agent is a small molecule or receptor ligand.
  • the invention also provides a method of screening, in a single assay, for an agent that modulates binding between the members of two or more binding complexes, comprising (a) contacting two or more first members, one or more second members, and a third member of the binding complexes with the agent to be screened, under conditions that allow formation of binding complexes, wherein each binding complex comprises one first and one second member; wherein the first members are coupled directly or indirectly to a set of microspheres labeled with a set of first labels; wherein each first label is specific for one first member; wherein the second member is coupled directly or indirectly to a second label; and wherein the second label is specific for one second member; (b) detecting the presence of the first specific labels and the presence or absence of the second specific labels in a plurality of detection products, wherein each detection product comprises one microsphere; (c) comparing, to a control, the relative amount of each second label associated with each first label in the detection products, wherein the control lacks the agent to be screened, a greater or
  • the screening method can further comprise creating a modulating profile for each agent to be screened, wherein the modulating profile indicates the relative modulation of binding between each first member and each second member of the binding pairs in the presence of the third member, and comparing the modulating profile for the agent to be screened to a selected profile.
  • the detection product can further comprise the third member of the binding complex and/or the modulating agent.
  • the third member of the binding complex can potentiate or attenuate binding between the first member and second member of the binding complex.
  • the third member may be a modulator, and the modulating agent used in this screening method may modulate the attenuation or potentiation caused by the third member.
  • the agent to be screened can be selected from a group consisting of cofactors, receptors, receptor ligands, proteins, peptides, protein domains, oligonucleotides, transcription factors, nucleic acids, small molecules, and small compounds.
  • the first members are cofactors
  • the second member or members are receptors
  • the third member is a small molecule or receptor ligand
  • the modulating agent is a small molecule.
  • the invention also provides a set of microspheres coupled with a set of cofactors, wherein the microspheres are labeled with a label specific for each cofactor of the set.
  • the cofactors coupled to the microspheres can comprise nuclear receptor cofactors, and more specifically coactivators, corepressors, or both coactivators and corepressors.
  • the set of microspheres can be coupled to (1) coactivators comprising a five amino acid motif having, from amino to carboxy end, a leucine residue, two additional amino acid residues of any identity, and two leucine residues, (2) corepressors comprising a five amino acid motif having, from amino to carboxy end, a leucine residue, two additional amino acid residues, and two isoleucine residues, or (3) a combination thereof.
  • the coactivators and corepressors coupled to the microspheres can comprise peptides having SEQ ID NO: 1-43 or any subset thereof, including, for example, SEQ ID NO: 1-5, SEQ ID NO:6-10, SEQ ID NO:l 1-15, SEQ ID NO:16-20, SEQ ID NO:21- 25, SEQ ID NO:26-30, SEQ ID NO:31-35, SEQ ID NO:36-40, SEQ ID NO: 41-43.
  • a "set of microspheres" comprises one or more subsets of microspheres wherein each subset comprises a plurality of microspheres labeled with the same label or combination of labels and wherein each label or combination of labels is specific for that subset.
  • kits for performing the methods of the present invention provides a kit for confirming results of a phage display that identifies the amino acid sequences of two or more agents that bind to one or more specific binding members, comprising: (a) two or more subsets of microspheres having specific first labels, wherein each microsphere having a specific label is separated from microspheres having a different specific label; (b) a means for coupling each set of microspheres selectively to one agent; and (c) the specific binding member or members and a label or labels for coupling directly or indirectly to a specific binding member; or (c') the specific binding member or members, wherein each ligand is coupled directly or indirectly to a second label that is specific for one ligand.
  • the invention also provides a kit for confirming the results of a phage display that identifies amino acid sequences of two or more agents that modulate binding between one or more selected binding pairs or complexes.
  • Peptide sequences with biotin at the amino terminus followed by a 17-27 amino acid sequence containing either an LXXLL or an LXXII-motif sequence were synthesized by Synpep Corporation (Dublin, CA). Each peptide was purified by reverse-phase HPLC. Amino acid composition and peptide concentration were determined by conventional methods for each peptide sequence. Each biotinylated peptide shown in Table 1 was coupled to a specific set of microspheres with a unique fluorescent profile. Lumavidin-coated polystyrene microsphere sets (5.5 mm in diameter) with different ratios of red and orange fluorescence were purchased from the Luminex Corporation (Austin, TX).
  • the Lumavidin-coated microspheres (200,000) were added to 400 ⁇ l buffer (phosphate buffered saline containing 0.02% Tween-20, 0.1% bovine serum albumin, 0.02% sodium azide and 1 mM dithiothreitol).
  • the microspheres were centrifuged and resuspended in 400 ⁇ l fresh buffer and were then incubated with 500 ng biotinylated peptide for 30 min at room temperature in the dark.
  • the microsphere suspension was washed twice and resuspended in 0.1 ml buffer.
  • Free D-biotin 50 ⁇ l of 5 mM D-biotin
  • the microspheres were washed twice in 0.25 ml buffer and resuspended in 0.2 ml buffer.
  • Peptide coupling efficiency was determined by quantifying the biotin-binding capacity of the microsphere sets both before and after peptide coupling. Aliquots of microspheres from above (3,000) in 0.1 ml were incubated with an excess amount of biotin-FITC (5 ⁇ l of 1 mM biotin-FITC) (Molecular Probes, Eugene, OR) for 30 min at room temperature in the dark. The microspheres were washed twice with buffer, then blocked with free biotin as described above.
  • the FITC fluorescence-associated with each microsphere set was measured by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA) and quantified using Quantum Fluorescence Kit for MESF units of FITC calibration particles (SIGMA, St Louis, MO).
  • ER ⁇ LBD Human Estrogen Receptor ⁇ Ligand Binding Domain
  • E.coli strain BL21(DE3) E.coli strain BL21(DE3) as an amino-terminal poly-histidine tagged fusion protein. Expression was under the control of an IPTG inducible T7 promoter. DNA encoding this recombinant protein was subcloned into the pRSET-A expression vector (Invitrogen, Carlsbad, CA). The encoded sequence of the polyhistidine tag (MKKGHHHHG) (SEQ ID NO:44) was incorporated into the 5' PCR amplification primer, which was upstream of DNA encoding residues 250-530 of ER ⁇ . The coding sequence of ER ⁇ LBD was derived from GenBank Accession Number AF051427.
  • cell paste (equivalent to 2-3 liters of the fermentation batch) was resuspended in 300-400 mL TBS, pH 8.0 (25 mM Tris, 150 mM NaCl). Cells were lysed by passing 3 times through a homogenizer (Rannie, Copenhagen, Denmark), and cell debris was removed by centrifugation (30 minutes, 20,000g, 4°C). The cleared supernatant was filtered through coarse pre-filters, and TBS pH 8.0 containing 500 mM imidazole was added to obtain a final imidazole concentration of 50mM.
  • This lysate was loaded onto a column (6 x 8 cm) packed with Sepharose [Ni ++ charged] Chelation resin (Pharmacia, Piscataway, NJ) and pre-equilibrated with TBS pH 8.0/ 50mM imidazole. After washing to baseline absorbance with equilibration buffer, the column was developed with a linear gradient of 50 to 365 mM Imidazole in TBS pH 8.0. Column fractions were pooled and dialyzed against TBS pH 8.0 containing 5% 1, 2-propanediol, 5mM DTT and 0.5mM EDTA.
  • the protein sample was concentrated using Centri-prep 10K (Amicon, Waters, Millipore, Bedford, MA) and subjected to size exclusion chromatography using a column (3 x 90cm) packed with Sepharose S-75 resin (Pharmacia) pre-equilibrated with TBS pH 8.0 containing 5 % 1, 2-propanediol, 5mM DTT and 0.5mM EDTA.
  • Example 5 Biotinylation of ER ⁇ LBD
  • ER ⁇ LBD was buffer exchanged using PD-10 gel filtration columns (Pharmacia) into PBS [lOOmM NaPhosphate, pH 7.2, 150mM NaCTJ.
  • ER ⁇ LBD was diluted to approximately 30 ⁇ M in PBS and five-fold molar excess of NHS-LC-Biotin (Pierce, Rockford, IL) was added in a minimal volume of PBS. This solution was incubated with gentle mixing for 60 minutes at ambient room temperature. The biotinylation modification reaction was stopped by the addition of 2000x molar excess of Tris-HCl pH 8.0.
  • the modified ER ⁇ LBD was dialyzed against 2 buffer changes, each of at least 50 volumes; TBS pH 8.0 containing 5mM DTT, 2mM EDTA and 2% sucrose. This modified protein was distributed into aliquots, frozen on dry ice and stored at -80C.
  • the biotinylated ER ⁇ LBD was subjected to mass spectrometric analysis to reveal the extent of modification by the biotinylation reagent. In general, approximately 95% of the protein had at least a single site of biotinylation; and the overall extent of biotinylation followed a normal distribution of multiple sites, ranging from one to seven.
  • the agents and ligands were prepared according to Example 7. All components (3,500 microspheres of each subset, +/- agent(s), +/- receptor: Alexa 488) were added in a total volume of 0.35 ml buffer. The suspension incubated for 1.5 to 2 hours at room temperature in the dark. The fluorescence associated with each microsphere was measured by flow cytometry.
  • Example 9 Flow cytometric analysis Microsphere fluorescence was measured using a FACSCalibur flow cytometer
  • Example 11 ER ⁇ LBD binding to 34 LXXLL-motif coactivator peptides in the presence or absence of estradiol
  • Example 12 ER ⁇ LBD binding to 37 different coactivator peptides: effect of tamoxifen, and raloxifene
  • Example 13 Binding profile of LBDs derived from Recptor B, Receptor C and PPAR ⁇ LBD for cofactor peptides
  • Microsphere subsets (each subset was coupled to either no peptide, or to one of 40 different coactivator peptides described above) were incubated with PPAR ⁇ LBD or Receptor B or C at 100 nM (receptors were expressed, purified, biotinylated, and coupled to fluorochrome as described above) in the presence or absence of agent (0.2 or luM) for approximately 1.5 hours.
  • agent 0.2 or luM
  • Example 14 ER ⁇ LBD binding to coactivator peptides (src-1) and corepressor peptide (SMRT) in the presence or absence of estradiol
  • Microsphere subsets (coupled to either no peptide, SRC-1(3) (735-759), SRC- 1 (626-642), SRC-1(2) (676-700) or SMRT (2329-2354) as described above)were incubated with ER ⁇ LBD at 100 nM (expressed, purified, biotinylated, and coupled to fluorochrome as described above) in the presence of estradiol, at the indicated concentration, for approximately 1.5 hours.
  • the fluorescence associated with each microsphere was determined by flow cytometric analysis. The data are shown in Figure 5.
  • Example 15 Effect of tamoxifen, raloxifene and GI 165638 on estradiol- enhanced ER ⁇ LBD binding to cofactor peptides
  • Microsphere subsets (coupled to cofactors as described above) are incubated with ER ⁇ LBD at 100 nM (expressed, purified, biotinylated, and coupled to fluorochrome as described above) in the presence of estradiol, and raloxifene, or tamoxifen, for approximately 1.5 hours.
  • the fluorescence associated with each microsphere is determined by flow cytometric analysis.
  • Example 16 Effect of estradiol, raloxifene or tamoxifen on ER ⁇ LBD binding to cofactor peptides
  • Thirty-four fluorescently distinct red-orange Lumavidin-coated microsphere populations (Luminex Corp., Austin, Texas) were separately coupled to 34 biotin- containing peptide sequences and combined. A separate microsphere population was also included that had no peptide coupled to its surface.
  • Each 17-27 amino acid sequence represented a specific coactivator protein containing one unique LXXLL motif as described above.
  • Each coactivator peptide designation on the x-axis of the graph in Figure 6 includes GenBank specified amino acid residues.
  • Biotinylated recombinant ER ⁇ LBD was coupled to Alexa 488 Streptavidin and incubated with the multiplexed set of coactivator peptide-coupled microspheres in the presence or absence of ligand.
  • the green fluorescence associated with each microsphere was analyzed after 1.5 hours of incubation on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, California) equipped with Luminex LabMAP hardware and software. All background fluorescence contributed by the microspheres alone has been subtracted.
  • Figure 6 shows ER ⁇ LBD (100 nM) binding to coactivator peptide- coupled microspheres either in the absence of ligand, or in the presence of estradiol (2 ⁇ M), raloxifene (500 nM) or tamoxifen (500 nM).
  • estradiol (2 ⁇ M) estradiol
  • raloxifene 500 nM
  • tamoxifen 500 nM
  • Estrogen enhanced the ER ⁇ LBD binding to coactivator peptides while the antiestrogenic compounds raloxifene and tamoxifen, generally inhibited ER ⁇ LBD binding to coactivator peptides.
  • the data showed the binding preference of ER ⁇ LBD for certain coactivators such as steroid receptor coactivator- 1 (SRC-1), transcriptional intermediary factor 2 (TIF-2) and RIP 140 over cAMP response element
  • Example 17 Classification of compound activities based on binding patterns to ER ⁇ LBD conformation-sensing peptides
  • Microsphere populations are coupled to synthetic biotinylated conformation-sensing peptide sequences using coupling techniques described above.
  • Nuclear receptor modulators appear to induce very specific conformational changes in nuclear receptor physical structure. Conformation-sensing peptide sequences preferentially bind to a given nuclear receptor (or may decrease binding) upon exposure to ligands that result in certain conformational changes in the nuclear receptor. These peptide sequences may be identified by affinity-selection phage display according to the techniques described by Chang et al.
  • ER ⁇ LBD 10 nM
  • compound 1 mM
  • the advantage of the multiplexed screening approach is that one may characterize a potential nuclear receptor modulator for many conformational states in a single assay volume.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Nanotechnology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Steroid Compounds (AREA)

Abstract

L'invention concerne un dosage multiplex d'interactions moléculaires complexes. Plus particulièrement, l'invention concerne une méthode permettant d'identifier, en un seul dosage, la liaison relative d'au moins deux éléments d'une paire de liaison avec au moins un second élément d'une paire de liaison. L'invention concerne également une méthode permettant d'identifier, en un seul dosage, la modulation relative, au moyen d'un agent de modulation, de la liaison entre au moins deux paires de liaison ou entre au moins deux complexes de liaison. L'invention concerne en outre des méthodes permettant d'effectuer un criblage, en un seul dosage, pour déterminer au moins un agent ayant un profil de liaison choisi ou un agent qui module la liaison entre au moins deux premiers éléments d'une paire de liaison et au moins un second élément d'une paire de liaison ou les éléments d'au moins deux complexes de liaison. L'invention concerne enfin un ensemble de microsphères couplé à un ensemble de cofacteurs, ces microsphères étant marquées avec une étiquette spécifique de chaque cofacteur de l'ensemble.
EP01922998A 2000-03-31 2001-03-30 Methode permettant d'etudier des interactions moleculaires fonctionnelles et reactifs utilises a cet effet Withdrawn EP1282818A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US19382600P 2000-03-31 2000-03-31
US193826P 2000-03-31
PCT/US2001/010506 WO2001075443A2 (fr) 2000-03-31 2001-03-30 Methode permettant d'etudier des interactions moleculaires fonctionnelles et reactifs utilises a cet effet

Publications (1)

Publication Number Publication Date
EP1282818A2 true EP1282818A2 (fr) 2003-02-12

Family

ID=22715172

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01922998A Withdrawn EP1282818A2 (fr) 2000-03-31 2001-03-30 Methode permettant d'etudier des interactions moleculaires fonctionnelles et reactifs utilises a cet effet

Country Status (4)

Country Link
EP (1) EP1282818A2 (fr)
JP (1) JP2003529767A (fr)
AU (1) AU2001249738A1 (fr)
WO (1) WO2001075443A2 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1616179A4 (fr) * 2001-12-21 2006-07-12 Centocor Inc Procede de criblage pour antagoniste moleculaire par cytometrie de flux
AU2003228492A1 (en) * 2002-04-12 2003-10-27 Smithkline Beecham Corporation Compositions and methods for evaluating and designing nuclear receptor ligands that modulate co-regulator affinity
EP1353182A3 (fr) * 2002-04-12 2004-02-04 Smithkline Beecham Corporation Procédé de prédiction des résultats des essais à base de cellules utilisant des profils de liaison

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4510244A (en) * 1980-04-17 1985-04-09 The Board Of Trustees Of The Leland Stanford Jr. University Cell labeling with antigen-coupled microspheres
FI93781C (fi) * 1993-03-18 1995-05-26 Wallac Oy Biospesifinen multiparametrinen määritysmenetelmä
DE69615818T2 (de) * 1995-01-16 2002-06-06 Erkki Soini Ein biospezifisches multiparametrisches testverfahren
US5858648A (en) * 1996-11-04 1999-01-12 Sienna Biotech, Inc. Assays using reference microparticles
US5876946A (en) * 1997-06-03 1999-03-02 Pharmacopeia, Inc. High-throughput assay
WO1999036564A1 (fr) * 1998-01-16 1999-07-22 Luminex Corporation Appareil et procedes d'analyse multiplexee de specimens cliniques

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0175443A3 *

Also Published As

Publication number Publication date
WO2001075443A2 (fr) 2001-10-11
WO2001075443A3 (fr) 2002-11-28
AU2001249738A1 (en) 2001-10-15
JP2003529767A (ja) 2003-10-07

Similar Documents

Publication Publication Date Title
Gonzalez-Gonzalez et al. Nanotechniques in proteomics: protein microarrays and novel detection platforms
Daly et al. Fluorescent ligands, antibodies, and proteins for the study of receptors
US6492125B2 (en) Method to assess library X library interactions
NZ540219A (en) Fret probes and methods for detecting interacting molecules
JP4533536B2 (ja) 複数色の標識
CN104991075A (zh) 使用磁性标记进行的肌钙蛋白i 测定
US6551788B1 (en) Particle-based ligand assay with extended dynamic range
JP2005510706A5 (fr)
US20020004205A1 (en) Method of investigating functional molecular interactions and reagents for use therein
Waller et al. Techniques: GPCR assembly, pharmacology and screening by flow cytometry
WO2001075443A2 (fr) Methode permettant d'etudier des interactions moleculaires fonctionnelles et reactifs utilises a cet effet
Waller et al. High throughput screening of G-protein coupled receptors via flow cytometry
Rinken et al. Dynamics of ligand binding to GPCR: Residence time of melanocortins and its modulation
JP4335526B2 (ja) コロイド構造体および非コロイド構造体による結合スピーシーズの検出
US7018846B1 (en) Display of receptors and analysis of binding interactions and drug libraries
JP4811400B2 (ja) 核内受容体に対する結合物質の検出方法
Schrum Visualization of multiprotein complexes by flow cytometry
KR101492167B1 (ko) 페리틴 단백질을 함유하는 표적-특이적 프로브 및 이를 이용한 바이오마커의 탐지
JP2010210444A (ja) 多抗原同時検出用ナノ粒子
Christoph et al. Novel fluorescence based receptor binding assay method for receptors lacking ligand conjugates with preserved affinity: study on estrogen receptor α
Iannone et al. Effect of microsphere binding site density on the apparent affinity of an interaction partner
US20050064446A1 (en) Detection of binding species with colloidal and non-colloidal structures
Gillies et al. Engineered systems for detection and discovery of nuclear hormone‐like compounds
JP2009250652A (ja) 細胞内移行性速度を測定することによる機能性物質のスクリーニング法
Rothbard et al. Binding of biotinylated peptides to MHC class II proteins on cell surfaces

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20020930

AK Designated contracting states

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

17Q First examination report despatched

Effective date: 20030723

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20040113