WO2003008927A2 - Procedes et substrats servant a surveiller la liaison a une phase solide a l'aide d'analyses d'emission de signaux de proximite - Google Patents

Procedes et substrats servant a surveiller la liaison a une phase solide a l'aide d'analyses d'emission de signaux de proximite Download PDF

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WO2003008927A2
WO2003008927A2 PCT/IL2002/000573 IL0200573W WO03008927A2 WO 2003008927 A2 WO2003008927 A2 WO 2003008927A2 IL 0200573 W IL0200573 W IL 0200573W WO 03008927 A2 WO03008927 A2 WO 03008927A2
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Prior art keywords
pair
signal generation
binding
proximity signal
binding pair
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PCT/IL2002/000573
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English (en)
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WO2003008927A3 (fr
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Avinoam Dukler
Nir Dotan
Avraham Shtavi
Ari Gargir
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Glycominds Ltd.
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Priority to AU2002319882A priority Critical patent/AU2002319882A1/en
Publication of WO2003008927A2 publication Critical patent/WO2003008927A2/fr
Publication of WO2003008927A3 publication Critical patent/WO2003008927A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/12—Libraries containing saccharides or polysaccharides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/10—Methods of screening libraries by measuring physical properties, e.g. mass
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11—Compounds covalently bound to a solid support

Definitions

  • the present invention relates to methods and substrates for monitoring binding to a solid phase, and, more particularly, to methods and devices for monitoring affinity binding of molecules to a solid phase using proximity based signal generation methods, fluorescence resonance energy transfer based methods, in particular.
  • Affinity binding is a wide-spread phenomenon which evolved through years of evolution and is involved in every aspect of life both at the cellular and the physiological level.
  • enzymes affinity bind to their substrates and inhibitors
  • ligands affinity bind to their receptors
  • antibodies and immunoreceptors affinity bind to their antigens
  • single stranded nucleic acids affinity bind to their complementary sequences etc.
  • Affinity binding was recruited into several fields, including medicine, diagnosis, purification procedures and research, to name a few.
  • Affinity binding typically involves the binding of binding members of a binding pair.
  • a first binding member of the binding pair is said to bind a second binding member of the binding pair.
  • binding pairs thus include enzymes and their substrates or inhibitors, receptors and their ligands, antibodies and immunoreceptors and their antigens, and single stranded nucleic acids and their complements.
  • Numerous binding assays were developed employing binding pairs, in which one of the binding members of the binding pair is attached to a solid support and the other binding member of the binding pair is in solution and free to bind to the binding member which is bound to the solid support, wherein the level of binding between the binding members, or in other words the level of binding of the binding member which is in solution to the solid support is monitored.
  • Such monitoring is typically assisted by the use of a label, such as radioactive or luminescencing label which is readily monitored using suitable detectors.
  • a label such as radioactive or luminescencing label which is readily monitored using suitable detectors.
  • regiospecific i.e., characterized by addressable locations
  • a spatially sensitive detector so as to monitor binding in a regiospecific manner.
  • Proximity based signal generation assays offer the advantage of using homogeneous assay formats (i.e., "mix, incubate, and read” or “mix and measure”). Indeed, the high throughput screening (HTS) field is moving rapidly toward the use of proximity based signal generation assays.
  • Proximity based signal generation assays are based on the use of proximity signal generation pairs, for which, when in sufficient proximity, a first member of the proximity signal generation pair causes a second member of the proximity signal generation pair to generate a detectable signal.
  • a further development was to employ a proximity signal generation pair with a binding pair so as to monitor the level of binding between the binding members of the binding pair via the signal generated by the members of the proximity signal generation pair, brought into sufficient proximity due to binding between the binding members of the binding pair.
  • techniques were developed to join together members of binding pairs and members of proximity signal generation pairs in such a way so as not to interfere with binding on the one hand and signal generation on the other hand.
  • linkers of varying lengths and chemical nature are employed to link together members of binding pairs and members of proximity signal generation pairs.
  • proximity based signal generation methods include those methods in which a signal is generated when a first label on a first member of a specific binding pair is brought into close proximity with a second label on a second member of the specific binding pair. Examples of proximity methods include the following. Fluorescence Resonance Energy Transfer (FRET):
  • fluorescence refers to both fast fluorescence and phosphorescence
  • non-radiative processes allow exit from the excited state without direct emission of light by the donor.
  • Non-radiative processes include dissipation of energy in the form of heat, use of the absorbed energy to promote chemical reactions, and transfer of energy to a neighboring molecule.
  • the absorbing molecule enters an excited state and similarly eliminates the energy via radiative or non-radiative processes. Transfer of energy from one molecule to a second molecule occurs if there is sufficient energy state overlap and if the distance between the one energy absorbing donor and one energy absorbing acceptor is in the order of R Q (see below).
  • the area of energy state overlap between the donor emission and the acceptor absorbance is important for energy transfer between the two components.
  • the process of FRET involves illuminating a sample at a wavelength that excites the energy absorbing donor but not the energy absorbing acceptor or that excites the energy absorbing donor to a much greater extent than it excites the energy absorbing acceptor. Excitation of the energy absorbing donor at wavelength I will result in energy being emitted at wavelength II, but not at wavelength III, the emission wavelength of the energy absorbing acceptor. Both the energy absorbing donor and the acceptor should absorb energy at one wavelength and emit energy at a different wavelength. FRET is an all or nothing quantum mechanical event. Therefore, the energy from an individual energy absorbing donor is either transferred to an individual energy absorbing acceptor or it is not. Energy transfer only occurs when the absorption and emission spectra of the energy absorbing donor and energy absorbing acceptor overlap.
  • FRET Fluorescence Activated fluorescence
  • FRET also manifests the lifetime in which the energy absorbing donor remains in an excited state.
  • the number of molecules in an excited state are proportional to the rate at which molecules exit the excited state, plus the sum of radiative and non-radiative states.
  • Fluorescence resulting from FRET is an equilibrium process, the duration of which depends on the time the energy absorbing FRET component remains in an excited state. This, in turn, is a result of competition between the rate at which the energy absorbing FRET component enters the excited state by the incident energy and the sum of the rates at which the energy absorbing FRET component leaves the excited state (by processes such as fluorescence and non-radiative energy transfer).
  • a common form of non-radiative energy transfer is transfer between singlet states.
  • An energy absorbing donor absorbs a photon of energy that alters the charge distribution of the donor from a resting state to an excited state.
  • the excited state charge distribution can be represented as a dipole where one side of the molecule becomes positively charged and the other side of the molecule becomes negatively charged.
  • the energy absorbing donor molecule can return to its resting state by energy transfer from the energy absorbing donor to the energy absorbing acceptor, e.g., by inducing an opposite charged dipole in the energy absorbing acceptor molecule, which consequently enters an excited state.
  • a requisite for energy transfer, for example, by dipole-induced dipole or energy transfer is that the energy absorbing acceptor is in the same vicinity as the energy absorbing donor.
  • the energy absorbing acceptor can return to its resting state by emitting the absorbed energy at a different wavelength or non-radiative energy transfer.
  • a sample can be illuminated at a wavelength that excites the energy absorbing donor but not the energy absorbing acceptor, or that excites the energy absorbing donor to a much greater extend that it does the energy absorbing acceptor.
  • the sample is usually monitored at two separate wavelengths: that of energy absorbing donor emission wavelength and that of energy absorbing acceptor emission wavelength.
  • Et l/[l + (R/Ro)6]
  • R is the separation distance between energy absorbing donor and energy absorbing acceptor
  • Rn. is the distance for half transfer.
  • Rn. is a value that depends upon the overlap integral of the energy absorbing donor emission spectrum and the energy absorbing acceptor excitation spectrum, that index of refraction, the quantum yield of the donor, and the orientation of the donor emission and the acceptor absorbance moments (Forster, Natuforsch. 4A:321-327, 1949; Forster, Disc, Farady Soc. 27:7-17, 1959).
  • Fluorescence can be detected by time resolved techniques, such as gating pulse method, pulse method, and phase modulation.
  • the gating pulse method the sample is excited with a brief pulse of energy, and fluorescence is detected at a selected time period after the initial pulse. For example, if two fluorophores absorb energy and emit the absorbed energy in the form of fluorescence and one fluorophore has an fluorescence lifetime of 10 ns, while the second fluorophore has a fluorescence lifetime of 50 ns, it is possible to selectively detect the fluorescence from separate fluorophores. Detection at time 20 ns after the initial pulse of energy enables the detection of principally the fluorophore with the longer lifetime, i.e., the fluorophore with a fluorescence lifetime of 50 ns.
  • the sample is excited with a brief pulse of energy and the time dependent decay of the fluorescence intensity is measured. For example, if the energy absorbing donor fluorescence decay is being measured, the rate of decay changes depending on energy transfer. If the energy absorbing donor transfers its energy to an energy absorbing acceptor via non-radiative energy transfer, the rate of the energy absorbing donor decay is faster than if the energy absorbing donor decays via radiative energy transfer.
  • Phase modulation is a method whereby the sample is excited with sinusoidal modulated energy and both the phase shift and amplitude of fluorescent light relative to the incident energy, is used to calculate the lifetime. For example, if the sample is excited with a light modulated sinusoidally at a specific frequency, due to the time lag between the absorption and emission, the emission is delayed in phase relative to the incident energy. This phase delay and amplitude modulation are used to calculate fluorescence lifetime.
  • Energy Absorbing FRET Components An energy absorbing FRET component is a donor or an acceptor of non-radiative energy. Both donor and acceptor molecules absorb energy. The function of the donor molecule is to absorb energy at a first wavelength and transmit the absorbed energy via non-radiative energy transfer to the acceptor molecule.
  • the function of the acceptor molecule is to absorb the transmitted energy from the donor molecule. Absorption allows for detection of energy transfer, e.g., by measurement of acceptor emission at a second wavelength.
  • a requirement of the energy absorbing FRET components is that there is sufficient energy state overlap between the two molecules in order for non-radiative energy transfer to occur.
  • Preferred FRET components have the ability to absorb energy at a first wavelength and emit energy as fluorescence at a second wavelength.
  • the FRET components should have energy state wavelengths in a range that avoid background interference from contaminants that may be present in the sample.
  • the FRET components used in biological samples should have a fluorescence that is not quenched by water, since most biological measurements are made in an aqueous solution.
  • the energy absorbing donor absorbs energy at a first wavelength and enters an excited state.
  • the energy absorbing donor leaves the excited state, it can transmit the energy via non-radiative energy transfer. If there is sufficient energy state overlap with the energy absorbing acceptor, the energy can be transferred from the energy absorbing donor to the energy absorbing acceptor. Subsequently, the energy absorbing acceptor enters an excited state by absorbing the energy from the energy absorbing donor.
  • Preferred energy absorbing acceptors emit energy in the form of fluorescent light which is rotated or depolarized with respect to the incident energy resulting in FRET upon leaving the excited state.
  • FRET can be characterized by a decrease in fluorescence intensity at wavelength II (i.e., decreased emission from the energy absorbing donor), an appearance of sensitized fluorescence intensity at III (i.e., an increased emission from the energy absorbing acceptor) and a depolarization of the fluorescence relative to the incident energy.
  • a FRET component is coupled to each member of a specific binding pair.
  • the interaction between the specific binding pairs is responsible for bringing the two energy absorbing FRET donor and acceptor components together.
  • Energy transfer will occur between the energy absorbing FRET donor and the energy absorbing FRET acceptor and FRET will be evidenced, e.g., in the form of decreased fluorescence intensity at wavelength II (i.e., decreased emission from the energy absorbing donor), an appearance of sensitized fluorescence intensity at wavelength III (i.e., increased emission from the energy absorbing acceptor) and depolarization of the fluorescence relative to the incident energy.
  • HTRF Homogeneous Time Resolved Fluorescence
  • Homogenous time resolved fluorescence refers to a method of measuring constituents of a system without prior separation of those constituents.
  • the basic concept of HTRF is based on FRET in that the interaction of two biomolecules can be measured by monitoring the fluorescence energy transfer from the excited donor fluorophore to the acceptor fluorophore when the two biomolecules are in proximity.
  • the fluorescent signal from HTRF is measured after a time delay, thereby eliminating interfering signals.
  • Luminescent Oxygen Channeling Assay involves a photochemical reaction produced by the interaction of two proximity based signal generating label moiety components of the system.
  • the first component a photosensitizer, generates singlet oxygen upon irradiation (e.g., a phthalocyanine containing photosensitizer).
  • the second component is a photochemically activatable chemiluminescent compound (e.g., olefin), which reacts with the singlet oxygen to initiate a delayed luminescence emission.
  • a pair of conjugates should be synthesized, each of the conjugates includes a member of a binding pair linked to a member of a proximity signal generation pair.
  • the present invention is aimed at exploiting the advantages of proximity based signal generation assays while obviating the need of synthesizing a pair of conjugates, while still satisfying the proximity based signal generation assay using a single conjugate of a member of a binding pair linked to a member of a proximity signal generation pair.
  • a method of assaying binding between a first member of a binding pair and a second member of the binding pair comprising attaching the first member of the binding pair to a solid support; attaching to the solid support, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; linking the second member of the binding pair to a second member of the proximity signal generation pair; and assaying binding between the first member of the binding pair and the second member of the binding pair by monitoring signal generation.
  • a method of assaying binding between a first member of a binding pair attached to a solid support and a second member of the binding pair in solution comprising assaying binding between the first member of the binding pair and the second member of the binding pair by monitoring a generation of signal between a first member of a proximity signal generation pair being attached to the solid support, not via the first member or the second member of the binding pair, and a second member of the proximity signal generation pair linked to the second member of the binding pair.
  • a method of assaying binding between a first member of a binding pair and a second member of the binding pair comprising attaching the first member of the binding pair to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; linking the second member of the binding pair to a second member of the proximity signal generation pair; and assaying binding between the first member of the binding pair and the second member of the binding pair by monitoring signal generation.
  • a method of assaying binding between a first member of a binding pair and a second member of the binding pair comprising attaching the first member of the binding pair to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; providing the second member of the binding pair linked to a second member of the proximity signal generation pair; and assaying binding between the first member of the binding pair and the second member of the binding pair by monitoring signal generation.
  • a method of assaying binding between a first member of a binding pair and a second member of the binding pair comprising providing the first member of the binding pair attached to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; providing the second member of the binding pair linked to a second member of the proximity signal generation pair; and assaying binding between the first member of the binding pair and the second member of the binding pair by monitoring signal generation.
  • a method of assaying inhibition of binding between a first member of a binding pair and a second member of the binding pair in a presence of an inhibitor comprising attaching the first member of the binding pair to a solid support; attaching to the solid support, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; linking the second member of the binding pair to a second member of the proximity signal generation pair; and assaying inhibition of binding between the first member of the binding pair and the second member of the binding pair in the presence of the inhibitor by monitoring signal generation.
  • a method of assaying inhibition of binding between a first member of a binding pair attached to a solid support and a second member of the binding pair in solution in a presence of an inhibitor comprising assaying binding between the first member of the binding pair and the second member of the binding pair in the presence of the inhibitor by monitoring a generation of signal between a first member of a proximity signal generation pair being
  • a method of assaying inhibition of binding between a first member of a binding pair and a second member of the binding pair in a presence of an inhibitor comprising attaching the first member of the binding pair to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; linking the second member of the binding pair to a second member of the proximity signal generation pair; and assaying inhibition of binding between the first member of the binding pair and the second member of the binding pair in the presence of the inhibitor by monitoring signal generation.
  • a method of assaying inhibition of binding between a first member of a binding pair and a second member of the binding pair in a presence of an inhibitor comprising attaching the first member of the binding pair to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; providing the second member of the binding pair linked to a second member of the proximity signal generation pair; and assaying inhibition of binding between the first member of the binding pair and the second member of the binding pair in the presence of the inhibitor by monitoring signal generation.
  • a method of assaying inhibition of binding between a first member of a binding pair and a second member of the binding pair in a presence of an inhibitor comprising providing the first member of the binding pair attached to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; providing the second member of the binding pair linked to a second member of the proximity signal generation pair; and assaying inhibition of binding between the first member of the binding pair and the second member of the binding pair in the presence of the inhibitor by monitoring signal generation.
  • a method of assaying activation of binding between a first member of a binding pair and a second member of the binding pair in a presence of an activator comprising attaching the first member of the binding pair to a solid support; attaching to the solid support, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; linking the second member of the binding pair to a second member of the proximity signal generation pair; and assaying activation of binding between the first member of the binding pair and the second member of the binding pair in the presence of the activator by monitoring signal generation.
  • a method of assaying activation of binding between a first member of a binding pair attached to a solid support and a second member of the binding pair in solution in a presence of an activator comprising assaying binding between the first member of the binding pair and the second member of the binding pair in the presence of the activator by monitoring a generation of signal between a first member of a proximity signal generation pair being attached to the solid support, not via the first member or the second member of the binding pair, and a second member of the proximity signal generation pair linked to the second member of the binding pair.
  • a method of assaying activation of binding between a first member of a binding pair and a second member of the binding pair in a presence of an activator comprising attaching the first member of the binding pair to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; linking the second member of the binding pair to a second member of the proximity signal generation pair; and assaying activation of binding between the first member of the binding pair and the second member of the binding pair in the presence of the activator by monitoring signal generation.
  • a method of assaying activation of binding between a first member of a binding pair and a second member of the binding pair in a presence of an activator comprising attaching the first member of the binding pair to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; providing the second member of the binding pair linked to a second member of the proximity signal generation pair; and assaying activation of binding between the first member of the binding pair and the second member of the binding pair in the presence of the activator by monitoring signal generation.
  • a method of assaying activation of binding between a first member of a binding pair and a second member of the binding pair in a presence of an activator comprising providing the first member of the binding pair attached to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; providing the second member of the binding pair linked to a second member of the proximity signal generation pair; and assaying activation of binding between the first member of the binding pair and the second member of the binding pair in the presence of the activator by monitoring signal generation.
  • a method of screening for an effector effecting binding between a first member of a binding pair and a second member of the binding pair comprising attaching the first member of the binding pair to a solid support; attaching to the solid support, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; linking the second member of the binding pair to a second member of the proximity signal generation pair; and assaying binding between the first member of the binding pair and the second member of the binding pair in the presence, absence or varying amounts of the effector by monitoring signal generation.
  • a method of screening for an effector effecting binding between a first member of a binding pair attached to a solid support and a second member of the binding pair in solution comprising assaying binding between the first member of the binding pair and the second member of the binding pair in the presence, absence or varying amounts of the effector by monitoring a generation of signal between a first member of a proximity signal generation pair being attached to the solid support, not via the first member or the second member of the binding pair, and a second member of the proximity signal generation pair linked to the second member of the binding pair.
  • a method of screening for an effector effecting binding between a first member of a binding pair and a second member of the binding pair comprising attaching the first member of the binding pair to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; linking the second member of the binding pair to a second member of the proximity signal generation pair; and assaying binding between the first member of the binding pair and the second member of the binding pair in the presence, absence or varying amounts of the effector by monitoring signal generation.
  • a method of screening for an effector effecting binding between a first member of a binding pair and a second member of the binding pair comprising attaching the first member of the binding pair to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; providing the second member of the binding pair linked to a second member of the proximity signal generation pair; and assaying binding between the first member of the binding pair and the second member of the binding pair in the presence, absence or varying amounts of the effector by monitoring signal generation.
  • a method of screening for an effector effecting binding between a first member of a binding pair and a second member of the binding pair comprising providing the first member of the binding pair attached to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; providing the second member of the binding pair linked to a second member of the proximity signal generation pair; and assaying binding between the first member of the binding pair and the second member of the binding pair in the presence, absence or varying amounts of the effector by monitoring signal generation.
  • a method of assaying binding between a first member of a binding pair attached to a solid support and a second member of the binding pair in solution comprising assaying binding between the first member of the binding pair and the second member of the binding pair by monitoring a generation of signal between a first member of a proximity signal generation pair being attached to the solid support, not via the first member or the second member of the binding pair, and a second member of the proximity signal generation pair linked to the second member of the binding pair.
  • a universal substrate for assaying binding between a first member of a binding pair and a second member of the binding pair, the universal substrate comprising a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair.
  • each of the first member of the binding pair and the first member of the binding pair is selected from the group consisting of an enzyme, a substrate, an inhibitor, a receptor, a ligand, an antibody, an immunoreceptor, an antigen, a nucleic acid, a cell, a bacterium, a virus and a bacteriophge.
  • each of the first member of the binding pair and the first member of the binding pair is selected from the group consisting of a protein, a peptide, a polypeptide, a glycoprotein, a nucleic acid, an oligonucleotide, a monosaccharide, a polysaccharide, a complex carbohydrate, a metabolite and a catabolite.
  • the proximity signal generation pair is selected from the group of fluorescence resonance energy transfer pair, an energy absorbing fluorescence resonance energy transfer pair and a luminescent oxygen channeling pair.
  • the fluorescence resonance energy transfer pair is selected from the group of pairs consisting of indocarbocyanine «indocarbocyanine, e.g., fluorescein ⁇ -rhodamine, NBD N-(7-nitrobenz-2-oxa-l, 3-diazol-3-yI) « rhodamine, fluorescein -eosin, fluorescein ⁇ erythrosin, dansykrhodamine, acridine orange ⁇ rhodamine, pyrene ⁇ -fluorescein, 7-amino-actinomycin-D ⁇ - fluorescein, 7-aminoactinomycin-D ⁇ -R-phycoerythrin, fluorescein
  • indocarbocyanine e.g., fluorescein ⁇ -rhodamine, NBD N-(7-nitrobenz-2-oxa-l, 3-diazol-3-yI) « rhodamine
  • the luminescent oxygen channeling pair is selected from the group of pairs consisting of a photosensitizer paired with an olefin, an ether, an enaminesdioxene, an arylimidazole, luminol, luciferin or aquaphorin.
  • the first member of the binding pair is attached to the solid support via a covalent attachment or via affinity binding using members of an additional binding pair.
  • the second member of the binding pair is linked to the second member of the proximity signal generation pair via a covalent link or via affinity binding using members of an additional binding pair.
  • the signal generation results from a direct interaction between the first member of the proximity signal generation pair and the second member of the proximity signal generation pair.
  • the signal generation results from an indirect interaction between the first member of the proximity signal generation pair and the second member of the proximity signal generation pair, through a third proximity signal generation molecule forming a proximity signal generation pair with each of the first member of the proximity signal generation pair and the second member of the proximity signal generation pair.
  • the solid support is selected from the group consisting of a microscope slide, a microtiter plate, a chip, a lab-on-chip, a column, a membrane, beads, magnetic beads and fibers.
  • the present invention successfully addresses the shortcomings of the presently known configurations by exploiting the advantages of proximity based signal generation assays while obviating the need of synthesizing a pair of conjugates, while still satisfying the proximity based signal generation assay using a single conjugate of a member of a binding pair linked to a member of a proximity signal generation pair, whereas the other member of the proximity signal generation pair and the other member of the binding pair are each independently linked to the solid support so as to allow signal generation upon binding of the binding members of the binding pair.
  • the present invention relates to a combinatorial complex carbohydrate library and a method of its synthesis, which takes advantage of the invention described herein.
  • a combinatorial complex carbohydrate library comprising a plurality of complex carbohydrate structures being attached to a solid support and a first member of a proximity signal generation pair being independently, not through the complex carbohydrate structures, attached to the solid support.
  • a method of producing an addressable combinatorial complex carbohydrate library comprising providing a solid support having a plurality of locations; enzymatically synthesizing a plurality of complex carbohydrate structures, each of the plurality of complex carbohydrate structures being attached to at least one addressed location of the plurality of locations, thereby producing the addressable combinatorial complex carbohydrate library; and attaching to the solid support at each of the addressed locations, not through the complex carbohydrate structures, a first member of a proximity signal generation pair.
  • each of the plurality of complex carbohydrate structures is addressable.
  • each of the addressable complex carbohydrate structures to the solid support is effected by a linker.
  • the linker is cleavable.
  • the linker is cleavable under conditions that are harmless to carbohydrates. According to still further features in the described preferred embodiments the linker is selected so as to allow attaching thereto a p-Nitrophenyl, amine or squaric acid derivative of a sugar.
  • the linker is selected from the group consisting of an amino acid, a peptide, a non-glycosylated protein, a lipid, a ceramide, dolicol phosphate, a cyclodextrin, an oligosaccharide, a monosaccharide, an alkyl chain and a nucleic acid.
  • the linker is of a length of at least 20 Angstrom.
  • the solid support is selected from the group consisting of addressable microparticles, addressable beads and a flat platform.
  • the flat platform is selected from the group consisting of a microtiterplate, a membrane and a chip.
  • the solid support is a chip and further wherein adjacent locations of the plurality of locations are spaced no more than 2.25 mm from one another.
  • the microtiterplate is an addressable microfabricated array of closed reaction chambers supplemented with micro-fluid systems.
  • the closed reaction chambers are arranged at a density of 4-25 per square cm.
  • each of the closed reaction chambers is of 50-1000 nanoliter in volume.
  • the solid support is of a substance selected from the group consisting of polysterene cross-linked with divinylbenzene, polyethylene glycol-polystyrene block copolymer, polyamides, polyacrylamide, polymethacrylamide, silica, glass, quartz, plastic and cellulose.
  • At least one of the plurality of complex carbohydrate structures includes at least two contiguous saccharide units of a single species.
  • At least one of the plurality of complex carbohydrate structures includes at least one branch. According to still further features in the described preferred embodiments at least one of the at least one branch is formed of identical core and branching saccharide units.
  • At least one of the plurality of complex carbohydrate structures includes at least 4 saccharide units.
  • At least one of the plurality of complex carbohydrate structures includes at least 5 saccharide units.
  • At least one of the plurality of complex carbohydrate structures includes at least 6 saccharide units.
  • At least one of the plurality of complex carbohydrate structures includes at least 7 saccharide units. According to still further features in the described preferred embodiments the plurality of complex carbohydrate structures are a representation including non-natural complex carbohydrates.
  • the plurality of complex carbohydrate structures are a representation including natural complex carbohydrates.
  • the natural complex carbohydrates are associated with a condition selected from the group consisting of tumorogenesis, metastasis, pregnancy, vascular disease, heart disease, neurodegenerative disease, autoimmune disease, infertility, allergies, embriogenesis, apoptosis, neurodegenerative disorders and organ transplantation.
  • the natural complex carbohydrates are derived from a human source.
  • the human source is selected from the group consisting of a tissue, cells and body fluids.
  • the plurality of complex carbohydrate structures are a representation of domains of at least one natural complex carbohydrate.
  • the at least one natural complex carbohydrate is derived from a human source.
  • FIG. 1 is a schematic depiction of the concept underlying the present invention.
  • FIG. 2 is a schematic depiction of an optimal hexagonal pack and optimal ratio of first members of a binding pair (open circles) and a first member of a proximity signal generation pair (closed circles), when the latter is a donor.
  • FIG. 3 is a schematic depiction of energy transfer among three molecules.
  • FIG. 4 is a formula of a complex carbohydrate of 14 monosaccharide units of five different types with three branching points.
  • FIG. 5 is a Table representing the stepwise enzymatic synthesis of the complex carbohydrate structure of Figure 4.
  • the enzymatic reaction ER
  • S represents the solid support onto which the complex carbohydrate is immobilized and the synthesis reaction occurs.
  • FIG. 6 is a stepwise depiction of linker synthesis and independent attachment of N-acetylglucose or biotin thereto.
  • FIG. 7 is a plot demonstrating a measured effect of different concentrations of lactose on the inhibition of binding of EcorA to solid support attached galactose ⁇ 1,4 N-acetylglucose measured using FRET according to the present invention.
  • the present invention is of methods and substrates which can be used for monitoring binding to a solid phase. Specifically, the present invention can be used to monitor affinity binding of molecules to a solid phase using proximity based signal generation methods.
  • FIG. 1 illustrates the basic concept underlying the present invention.
  • a solid support 10 is shown to which are independently attached a first member 12a of a binding pair 12, through, for example, a first linker 14, and a first member 16a of a proximity signal generation pair 16, through, for example, a second linker 18, which can be identical to or can differ from linker 14.
  • the attachment of first member 16a of proximity signal generation pair 16 to solid support 10 is via an additional binding pair having its members 20a and 20b one (20a) linked to through linker 18 to solid support 10 and the other (20b) linked to first member 16a of proximity signal generation pair 16, so as to attach first member 16a of proximity signal generation pair 16 to solid support 10.
  • a second member 12b of binding pair 12 is provided linked to a second member 16b of proximity signal generation pair 16 to form a conjugate 20.
  • conjugate 20 When conjugate 20 is contacted with solid support 10 in solution and provided that first member 12a of binding pair 12 is attached to solid support 10, then via specific binding, the surface concentration of conjugate 20 increases until binding equilibrium between members 12a and 12b of binding pair 12 is reached. Since, first member 12a of binding pair 12 is limited to the surface of solid support 10, the surface concentration of conjugate 20 exceeds its volume concentration in solution. As a result, the level of interaction between first member 16a of proximity signal generation pair 16 and second member 16a of proximity signal generation pair 16 increases. Monitoring the signal generated through the interaction of members 16a and 16b of proximity signal generation pair 16 at the surface thus allows monitoring the level of binding between members 12a and 12b of binding pair 12.
  • first member 12a of binding pair 12 and first member 16a of proximity signal generation pair 16 are each independently attached to solid support 10 and not to one another as is characteristic of the prior art.
  • This offers the possibility to manufacture a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair.
  • Such a substrate can be used to thereafter bind thereto one or more molecules of known or unknown structures in regiospecific manner or at random which serve as potential or tested first members of binding pairs without having to first form conjugates therebetween and a member of a proximity signal generation pair.
  • a member of a binding pair includes also a tested or potential member of a binding pair.
  • R is defined as the distance between adjacent members of the proximity signal generation pair.
  • R Q is the distance in which half signal generation occurs, the more efficient signal generation is.
  • Rn. for proximity signal generation typically ranges between 1 nm and 10 nm and is typically about 5 nm.
  • the distance between adjacent members of the proximity signal generation pair should preferably not exceed 1-10 nm, preferably about 5 nm, implying that the distance between the first member of the binding pair and the first member of the proximity signal generation pair should preferably not exceed 1-10 nm, preferably about 5 nm, which is equivalent to a surface concentration of 4- 10 2 molecules of the first member of the binding pair and the first member of the proximity signal generation pair per cm 2 . It will be appreciated in this respect that binding molecules to solid supports can be performed at far higher surface concentrations. The ratio between the members of the proximity signal generation pair should also be considered. Typically a single donor can communicate with several acceptors (see Figure 2).
  • the first member of the proximity signal generation pair i.e., the member attached to the solid support
  • a donor e.g., energy transfer donor
  • the ratio between the first member of the binding pair and the first member of the proximity signal generation pair both are attached to the solid support
  • the first member of the proximity signal generation pair be an acceptor (e.g., energy transfer acceptor) than the ratio between the first member of the binding pair and the first member of the proximity signal generation pair should be smaller than 1, say between 2:1 and 10:1, preferably between 6:1 and 8:1.
  • the former configuration is preferred.
  • This provides another advantage to the present invention according to which the ratio between the first member of the binding pair and the first member of the proximity signal generation pair can be optimized so as to achieve optimization in signal generation.
  • An additional advantage offered by the present invention is the ability to use proximity signal generation pairs in cases where a plurality of tested first members of a binding pair are linked to the solid support.
  • first and second with respect to members of binding and/or proximity signal generation pairs are not intended to provide any functional order and merely serve for distinguishing between the members of a pair.
  • a first member of a proximity signal generation pair can be either the energy donor or the energy acceptor in fluorescence resonance energy transfer between the pair members.
  • a second member of the proximity signal generation pair can be either the energy acceptor or the energy donor, respectively, in the fluorescence resonance energy transfer between the pair members.
  • signal generation refers to either increase of a measurable signal or decrease of a measurable signal.
  • a method of assaying binding between a first member of a binding pair and a second member of the binding pair comprising attaching the first member of the binding pair to a solid support; attaching to the solid support, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; linking the second member of the binding pair to a second member of the proximity signal generation pair; and assaying binding between the first member of the binding pair and the second member of the binding pair by monitoring signal generation.
  • a method of assaying binding between a first member of a binding pair attached to a solid support and a second member of the binding pair in solution comprising assaying binding between the first member of the binding pair and the second member of the binding pair by monitoring a generation of signal between a first member of a proximity signal generation pair being attached to the solid support, not via the first member or the second member of the binding pair, and a second member of the proximity signal generation pair linked to the second member of the binding pair.
  • a method of assaying binding between a first member of a binding pair and a second member of the binding pair comprising attaching the first member of the binding pair to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; linking the second member of the binding pair to a second member of the proximity signal generation pair; and assaying binding between the first member of the binding pair and the second member of the binding pair by monitoring signal generation.
  • a method of assaying binding between a first member of a binding pair and a second member of the binding pair comprising attaching the first member of the binding pair to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; providing the second member of the binding pair linked to a second member of the proximity signal generation pair; and assaying binding between the first member of the binding pair and the second member of the binding pair by monitoring signal generation.
  • a method of assaying binding between a first member of a binding pair and a second member of the binding pair comprising providing the first member of the binding pair attached to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; providing the second member of the binding pair linked to a second member of the proximity signal generation pair; and assaying binding between the first member of the binding pair and the second member of the binding pair by monitoring signal generation.
  • a method of assaying inhibition of binding between a first member of a binding pair and a second member of the binding pair in a presence of an inhibitor comprising attaching the first member of the binding pair to a solid support; attaching to the solid support, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; linking the second member of the binding pair to a second member of the proximity signal generation pair; and assaying inhibition of binding between the first member of the binding pair and the second member of the binding pair in the presence of the inhibitor by monitoring signal generation.
  • a method of assaying inhibition of binding between a first member of a binding pair attached to a solid support and a second member of the binding pair in solution in a presence of an inhibitor comprising assaying binding between the first member of the binding pair and the second member of the binding pair in the presence of the inhibitor by monitoring a generation of signal between a first member of a proximity signal generation pair being attached to the solid support, not via the first member or the second member of the binding pair, and a second member of the proximity signal generation pair linked to the second member of the binding pair.
  • a method of assaying inhibition of binding between a first member of a binding pair and a second member of the binding pair in a presence of an inhibitor comprising attaching the first member of the binding pair to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; linking the second member of the binding pair to a second member of the proximity signal generation pair; and assaying inhibition of binding between the first member of the binding pair and the second member of the binding pair in the presence of the inhibitor by monitoring signal generation.
  • a method of assaying inhibition of binding between a first member of a binding pair and a second member of the binding pair in a presence of an inhibitor comprising attaching the first member of the binding pair to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; providing the second member of the binding pair linked to a second member of the proximity signal generation pair; and assaying inhibition of binding between the first member of the binding pair and the second member of the binding pair in the presence of the inhibitor by monitoring signal generation.
  • a method of assaying inhibition of binding between a first member of a binding pair and a second member of the binding pair in a presence of an inhibitor comprising providing the first member of the binding pair attached to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; providing the second member of the binding pair linked to a second member of the proximity signal generation pair; and assaying inhibition of binding between the first member of the binding pair and the second member of the binding pair in the presence of the inhibitor by monitoring signal generation.
  • a method of assaying activation of binding between a first member of a binding pair and a second member of the binding pair in a presence of an activator comprising attaching the first member of the binding pair to a solid support; attaching to the solid support, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; linking the second member of the binding pair to a second member of the proximity signal generation pair; and assaying activation of binding between the first member of the binding pair and the second member of the binding pair in the presence of the activator by monitoring signal generation.
  • a method of assaying activation of binding between a first member of a binding pair attached to a solid support and a second member of the binding pair in solution in a presence of an activator comprising assaying binding between the first member of the binding pair and the second member of the binding pair in the presence of the activator by monitoring a generation of signal between a first member of a proximity signal generation pair being attached to the solid support, not via the first member or the second member of the binding pair, and a second member of the proximity signal generation pair linked to the second member of the binding pair.
  • a method of assaying activation of binding between a first member of a binding pair and a second member of the binding pair in a presence of an activator comprising attaching the first member of the binding pair to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; linking the second member of the binding pair to a second member of the proximity signal generation pair; and assaying activation of binding between the first member of the binding pair and the second member of the binding pair in the presence of the activator by monitoring signal generation.
  • a method of assaying activation of binding between a first member of a binding pair and a second member of the binding pair in a presence of an activator comprising attaching the first member of the binding pair to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; providing the second member of the binding pair linked to a second member of the proximity signal generation pair; and assaying activation of binding between the first member of the binding pair and the second member of the binding pair in the presence of the activator by monitoring signal generation.
  • a method of assaying activation of binding between a first member of a binding pair and a second member of the binding pair in a presence of an activator comprising providing the first member of the binding pair attached to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; providing the second member of the binding pair linked to a second member of the proximity signal generation pair; and assaying activation of binding between the first member of the binding pair and the second member of the binding pair in the presence of the activator by monitoring signal generation.
  • a method of screening for an effector effecting binding between a first member of a binding pair and a second member of the binding pair comprising attaching the first member of the binding pair to a solid support; attaching to the solid support, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; linking the second member of the binding pair to a second member of the proximity signal generation pair; and assaying binding between the first member of the binding pair and the second member of the binding pair in the presence, absence or varying amounts of the effector by monitoring signal generation.
  • a method of screening for an effector effecting binding between a first member of a binding pair attached to a solid support and a second member of the binding pair in solution comprising assaying binding between the first member of the binding pair and the second member of the binding pair in the presence, absence or varying amounts of the effector by monitoring a generation of signal between a first member of a proximity signal generation pair being attached to the solid support, not via the first member or the second member of the binding pair, and a second member of the proximity signal generation pair linked to the second member of the binding pair.
  • a method of screening for an effector effecting binding between a first member of a binding pair and a second member of the binding pair comprising attaching the first member of the binding pair to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; linking the second member of the binding pair to a second member of the proximity signal generation pair; and assaying binding between the first member of the binding pair and the second member of the binding pair in the presence, absence or varying amounts of the effector by monitoring signal generation.
  • a method of screening for an effector effecting binding between a first member of a binding pair and a second member of the binding pair comprising attaching the first member of the binding pair to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; providing the second member of the binding pair linked to a second member of the proximity signal generation pair; and assaying binding between the first member of the binding pair and the second member of the binding pair in the presence, absence or varying amounts of the effector by monitoring signal generation.
  • a method of screening for an effector effecting binding between a first member of a binding pair and a second member of the binding pair comprising providing the first member of the binding pair attached to a universal substrate which comprises a solid support to which is attached, not via the first member or the second member of the binding pair, a first member of a proximity signal generation pair; providing the second member of the binding pair linked to a second member of the proximity signal generation pair; and assaying binding between the first member of the binding pair and the second member of the binding pair in the presence, absence or varying amounts of the effector by monitoring signal generation.
  • a method of assaying binding between a first member of a binding pair attached to a solid support and a second member of the binding pair in solution comprising assaying binding between the first member of the binding pair and the second member of the binding pair by monitoring a generation of signal between a first member of a proximity signal generation pair being attached to the solid support, not via the first member or the second member of the binding pair, and a second member of the proximity signal generation pair linked to the second member of the binding pair.
  • binding pairs can be used while implementing the present invention, each of which can be the first member, i.e., the member that is attached to the solid support, or he second member, i.e., the member that is linked to the second member of the proximity signal generation pair (i.e., to form conjugate 20, Figure 1), including, but not limited to, enzymes, substrates, inhibitors, receptors, ligands, antibodies, immunoreceptors, antigens, nucleic acids, cells, bacteria, viruses and bacteriophges.
  • Cells, bacteria, viruses and bacteriophges are complex structures which often either naturally or by artificial genetic modification display members of binding pairs.
  • the members of the binding pairs can be of a variety of types of substances, including, but not limited to, proteins, peptides, polypeptides, glycoproteins, nucleic acids, oligonucleotides, monosaccharides, polysaccharides, complex carbohydrates, metabolites, catabolites and small molecules (e.g., below 1000 Daltons) of chemical libraries.
  • the members of the binding pair can be native, synthetic or analogs of native molecular structures. They can be purified or used in context of their source.
  • the proximity signal generation pair can be a fluorescence resonance energy transfer pair or a luminescent oxygen channeling pair.
  • the particulars of these signal generation techniques are further addressed in the Background section above. The following provides a non-limiting list of examples for various pairs.
  • suitable energy absorbing FRET members include fluorophores
  • Dyes useful as energy absorbing FRET donor/acceptor pairs include indocarbocyanine ⁇ -indocarbocyanine, e.g., fluorescei rhodamine, NBD N-(7-nitrobenz-2-oxa-l, 3-diazol-3-yl) ⁇ - rhodamine, fluoresceu eosin, fluorescein ⁇ -erythrosin, dansykrhodamine, acridine orange «rhodamine, pyrene- fluorescein, 7-amino-actinomycin-D ⁇ - fluorescein, 7-aminoactinomycin-D ⁇ cR-phycoerythrin, fluorescein ⁇ -
  • Donor fluorophores useful in homogeneous time resolved fluorescence include rare earth chelates or cryptates, e.g., terbium, europium, dysprosium, samarium or neodynium, preferably europium cryptate [(Eu)kj ⁇ Suitable examples of acceptor fluorophores in HTRF include allophycocyanin (e.g., XL665), allophycocyanin B, phycocyanin C, phycocyanin R, and phthalocyanins.
  • the europium cryptate [(Eu)k] and XL665 FRET pair has several unique properties, for example [(Eu)k] is stable within a pH range of 3 to 8 and has a long lifetime. This allows conventional equipment (such as a microplate fluorometer) to be used for measurement. Cryptate causes an enhancement of the Eu3+ fluorophore, which subsequently transfers the energy to XL665 when the two components are in close proximity. Cryptate also functions to protect EU3+ from fluorescence quenching.
  • the acceptor molecule in HTRF, namely XL665 is a stabilized allophycocyanin molecule that accepts energy transferred from [(Eu)kj.
  • the luminescent oxygen channeling pair includes, for example, photosensitizers that produce singlet oxygen upon irradiation (e.g., light irradiation).
  • Example of photoactivatable photosensitizers includes dyes and aromatic compounds.
  • Examples of chemi-activated photosensitizers include enzymes and metal salts.
  • the photosensitizers compound should absorb energy (e.g., light) in the wavelength range of 200-1100 nm, and the lifetime of the excited state produced must be sufficiently long to permit energy transfer to oxygen. Examples of photosensitizers are described in Turro, "Molecular Photochemistry," page 132, W.A. Benjamin, Inc., New York, NY, 1965.
  • Photochemically activatable chemiluminescent compounds that react with singlet oxygen include olefins (e.g., enol) ethers, enaminesdioxenes, arylimidazoles, luminol, luciferin, and aquaphorin.
  • proximity quenching in which energy emitted from one member of a proximity signal generation pair quenches the fluorescence of the other member of the proximity signal generation pair, can also serve the present invention.
  • the first member of the binding pair can be attached to the solid support via a covalent attachment or via affinity binding using members of an additional binding pair.
  • the second member of the binding pair can be linked to the second member of the proximity signal generation pair via a covalent link or via affinity binding using members of an additional binding pair.
  • the first member of the proximity signal generation pair can be attached to the solid support via a covalent attachment or via affinity binding using members of an additional binding pair.
  • signal generation results from a direct interaction between the first member of the proximity signal generation pair and the second member of the proximity signal generation pair, however, as specifically shown in Figure 3, according to another embodiment of the invention signal generation results from an indirect interaction between the first member 16a of the proximity signal generation pair and the second member 16b of the proximity signal generation pair, through a third proximity signal generation molecule 22 forming a proximity signal generation pair with each of the first member of the proximity signal generation pair and the second member of the proximity signal generation pair.
  • molecule 22 can be either independently attached or co-attached along with either the first member of the binding pair or the first member of the proximity signal generation pair to the solid support, co-linked to the second member of the binding pair along with the second member of the proximity signal generation pair, or be free in solution in a concentration sufficiently high so as to efficiently function in energy transfer at the surface of the solid support.
  • the solid support employed in context of the present invention can be of any type, including, but not limited to, a microscope slide, a microtiter plate, a chip, a lab-on-chip, a membrane, a column, beads, magnetic beads and fibers.
  • nucleic acid solid phase hybridization assays such as Southern, Northern and dot blot assays as well as assays on chip or in lab-on-chip nucleic acid hybridization assays; and antibody-antigen solid phase assays including Western blot, ELISA and on chip or in lab-on-chip antibody- antigen assays.
  • the present invention relates to a combinatorial complex carbohydrate library and a method of its synthesis and screening, which takes advantage of the invention described herein.
  • the invention will now be described with specific emphasis to combinatorial complex carbohydrate libraries and their synthesis.
  • the enzymatic synthesis of complex carbohydrate combinatorial libraries according to the present invention is effected by glycosyltransferases, glycosidases and transglycosidases. These enzymes can be obtained from different sources using different strategies as describe herein below.
  • Enzymes derived from natural sources To date, more than two hundred different kinds of glycosyltransferases, transglycosidases and glycosidases active on a large number of substrates and donors have been extensively characterized. These enzymes are found in mammalians cells, plant cells, invertebrate cells and microorganisms.
  • Recombinant enzymes The coding sequences of many glycosyltransferases and transglycosides have been cloned, and the acceptor substrate specificity of each of the recombinant enzymes encoded thereby have been characterized. Table 1 below lists some of the cloned glycosyltransferases and their respective acceptor substrate specificity. Enzymes for which the coding sequences have been cloned can be produced in sufficient quantities using standard recombinant DNA techniques. Since most of these enzymes require post translational modifications for functionality, expression is preferably effected in insect cell cultures.
  • soluble glycosyltransferases also include secretion from mammalians tissue-cultures (see for example U.S. 5,032,519, which is incorporated by reference as if fully set forth herein).
  • glycosyltransferases which are useful for complex carbohydrate library synthesis, can be identified and isolated from cell types which posses the complex carbohydrate structures typically synthesized by these desired glycosyltransferases.
  • Affinity chromatography techniques with an immobilized acceptor as a ligand are well known in the art and enable a simple one-step separation of a desired glycosyltransferase (see in this respect U.S. Pat. No. 5,288,637, which is incorporated herein by reference).
  • glycosyltransferase Once a glycosyltransferase is identified and isolated, it can be partially sequenced and the gene encoding therefor cloned. Technologies for cloning glycosyltransferases genes are well-established, and many examples and strategies for cloning glycosyltransferase genes are reviewed in the prior art (see, for example, WO 95/02683).
  • sequence alignment techniques can be used for the identification of new glycosyltransferases. For example, 110 distinct cDNAs and genes from animal, yeast, plants and bacteria, whose protein products contain the characteristic "signature sequence" of the UDP glycosyltransferase gene super family were identified. Using these signature sequences or motifs, one skilled in the art can screen relevant databases for novel glycosyltransferases.
  • Enzymes with modified affinities or altered substrate donor or acceptor specificities could also be employed in the synthesis of certain complex carbohydrates.
  • Enzymes with modified affinities or altered substrate donor or acceptor specificities could also be employed in the synthesis of certain complex carbohydrates.
  • the synthesis of complex carbohydrate structures composed of identical repeating monosaccharide units connected in the same regio-specific orientation such as, D-man-o(l,2)-D-Man-o(l,2)-D-man-o(l,2)-R, requires the use of an o-l,2 mannosyltransferase with an acceptor specificity to o-l,2 mannose.
  • Such a modified enzyme would be employed for the addition of a modified GDP-Man to immobilized acceptor D-mano(l,2)-R.
  • the modification of the mannoside moiety of the GDP-Man will then prevent addition of the next mannose moiety since the acceptor to this manosidetransferase is D-man-o(l,2)-R and not D-(modified man)-o(l,2)-D-man-o(l,2)-R.
  • any excess of the modified donor and the enzyme is washed out and the modifying group is removed, thereby enabling the subsequent repeat of the same enzymatic step. This controlled process is continued until the desired number of mannose molecules are assembled into the newly formed carbohydrate.
  • the modifying group can be a chemical residue attached to the donor at any position, but position 1. This modifying group can then be selectively removed by either an enzymatic or chemical reaction, such that the modifying group is released without imposing damage to the complex carbohydrate molecule.
  • enzymes having modified donor and/or acceptor specificities can be prepared using the directed evolution approach.
  • a directed evolution of an enzyme specificity is achieved by random sequential generation of region directed or site directed mutagenesis of the gene or genes encoding the enzyme, followed by selection or screening for clones exhibiting desired specificity and activity.
  • Moore and co-workers performed seven rounds of DNA shuffling to change the substrate specificity of paranitrobenzyl-esterase to a novel antibiotic substrate.
  • Zhang and co-workers performed directed evolution of a fucosidase from galactosidase by DNA shuffling.
  • Enzymatic combinatorial complex carbohydrate library design includes, according to the present invention, determination of the complex carbohydrate constituents included within a specific library in accordance with an envisaged application thereof.
  • the complex carbohydrate members included within a specific library depends upon the desired application for that specific library.
  • the complex carbohydrate members of the library are preferably derivatives or modificants of complex carbohydrates present in human cells. Screening such a library against other molecules derived from other sources, such as specific human cells or pathogens thereof, enables the identification of novel complex carbohydrates that function as receptors for these molecules, functioning in vivo as pathogen receptors, or involved in cell to cell recognition processes.
  • the complex carbohydrate members of the library are preferably synthesized similar or identical to natural complex carbohydrates present in human cells. Screening such a library with drug candidates derived from various natural and synthetic sources, enables the identification of drag candidates which bind to one or more of the complex carbohydrate structures of the library.
  • Another specific library according to the present invention contains complex carbohydrates dedicated for the identification of novel drug candidates.
  • a library of maximized complex carbohydrate diversity which represents, among others, complex carbohydrate structures not found in nature, is generated.
  • Such a library is thereafter screened for potential binding of pathogens or pathogen derived molecules.
  • such a library is thereafter screened for potential binding of other disease inflicting molecules.
  • a library in accordance with the present invention representing all of the possible domains of the complex carbohydrate is prepared. Screening this library for binding or bioactivity enables one to identify the active site domains of the known complex carbohydrate.
  • a complex carbohydrate library of specific combinations of glyco-markers is prepared and screened.
  • one specific library can represent the glyco-markers of several cancer conditions. This library is thereafter screened against antibodies derived from human serum to identify the presence of antibodies against one or more of these glyco-markers.
  • a complex carbohydrate library of specific combinations of carbohydrate members which are structural variations of the glyco-markers normally associated with such conditions is prepared and screened.
  • Other enzymatic combinatorial complex carbohydrate libraries, dedicated at other applications are envisaged and are within the broad scope of the present invention as claimed.
  • Enzymatic modules (EMs) construction includes evaluation of the required enzymatic reactions (ERs), glycosyl donors and acceptors and enzymes that are required for the synthesis of each complex carbohydrate of a library. EMs construction further includes optimization and process development of the required ERs with considerations given to reaction time, temperature and reagent concentrations. EMs construction further includes determination of the specific order in which the ERs should be utilized for every EM.
  • ERs enzymatic reactions
  • glycosyl donors and acceptors enzymes that are required for the synthesis of each complex carbohydrate of a library.
  • EMs construction further includes optimization and process development of the required ERs with considerations given to reaction time, temperature and reagent concentrations. EMs construction further includes determination of the specific order in which the ERs should be utilized for every EM.
  • a specific sequence of enzymatic reactions is determined for each complex carbohydrate constituent of a given library.
  • the ERs sequence follows that of the monosaccharide sequence of such linear non-branched structures in a stepwise fashion.
  • unique synthesis processes should be designed employing unique EMs.
  • the following example provides the rational for selecting particular ERs to provide an EM tailored for the synthesis of a distinct complex carbohydrate.
  • the final complex carbohydrate structure is described by Figure 4 and the design process is described by Figure 5 and Table 3.
  • Such an EM is designed, according to the present invention, for each complex carbohydrate present in a given library. As further detailed hereinunder, consideration is given to efficiency when practically effecting each of the EMs while constructing a library according to the present invention.
  • the first step in the synthesis of the complex carbohydrate shown in Figure 1 is effected, as shown in Figure 4, by attachment of a first building block, GalNAc, onto a solid support (S) via an appropriate linker which is further described herein below.
  • a first building block GalNAc
  • S solid support
  • the second step (D5, see Table 3 and Figure 5 for details) in the synthesis of the complex carbohydrate shown in Figure 1 involves transferring Gal from UDP-Gal to GalNAc-S by a ⁇ (l,3)-galactosyltransferases (E.G. 2.4.1.122).
  • the third step (HI, see Table 3 and Figure 5 for details) in the synthesis of the complex carbohydrate shown in Figure 1 involves the utilization of ⁇ (l,3)N-acetylglucosaminyltransferase (E.C. 2.4.1.146) to transfer an acetylglucoseamine group from UDP-GlcNAc to Gal- ⁇ (l,3)-GalNAc-S. Then, a galactose unit is added to the acceptor (see Figure 4), rather then a fucose unit because the specificity of the enzymes ⁇ (l,3)-fucosyltransferase (E.C.
  • the synthesis process complicates since the galactose units branch into two antennas (branches). Since the structure of these antennas is identical at the branching point, yet different towards their non-reducing ends, an identical stepwise synthesis process that simultaneously forms the identical parts of the two antennas would not enable the subsequent synthesis of a unique reducing end for each of the antennas. Therefor, the synthesis of the two unique portions of each of the antennas proceeds in an independent stepwise fashion. In any case, in the example given, the ⁇ (l,3) branch has to be synthesized first because this antenna requires fucosylation. If the synthesis process would initiate with the other branch, directed fucosylation to the desired branch could not have been effected.
  • the fifth step (H3, see Table 3 and Figure 5 for details) in the synthesis of the complex carbohydrate shown in Figure 1 is effected using ⁇ (l,3)N-acetylglucosaminyltransferase (E.C. 2.4.1.149) to transfer acetylglucosamine from UDP-Glc ⁇ Ac to Gal- ⁇ (l,4)-Glc ⁇ Ac-R.
  • a galactose unit is added to GlcNAc-R using a ⁇ (l,4)galactosyltransferase (E.C. 2.4.1.38).
  • branching is effected 5 by using ⁇ (l,6)N-acetylglucosaminyltransferase on the Gal[Glc ⁇ Ac- ⁇ (l,3)] ⁇ (l,4)-GlcNAc-R acceptor substrate.
  • the ninth step (B2, see Table 3 and Figure 5 for details) in the synthesis of the complex carbohydrates shown in Figure 1 involves two of the four GlcNAc monomers and is effected by o(l,3)fucosyltransferase (E.C. 2.4.1.152) l o which transfers fucose to Gal- ⁇ ( 1 ,4)-GlcNAc-R.
  • the tenth step (D7, see Table 3 and Figure 5 for details) in the synthesis of the complex carbohydrate shown in Figure 1 effects further elongation of the second antenna using an ⁇ (l,4) galactosyltransferase (E.C. 2.4.1.38 ) on the GlcNAc-R acceptor substrate.
  • a sialic acid monomer is appended to the Gal- ⁇ (l,4)GlcNAc-R of the antenna in an o(l,6) orientation using an o(2,3)Sia ⁇ y ⁇ transferases (E.C. 2.4.99.6)
  • this modifying group prevents the unwanted polymerization of multiple monomers of sialic acid, since this enzyme cannot append sialic acid to the acceptor NeuAC(modif ⁇ ed)-o (2,8)-NeuAC-R. In the last step of this ER the modifying group is removed.
  • the modified enzyme o(2,8) polysialyltransferasease is employed along with the modified donor CMP-NeuAC and the acceptor NeuAC(modified)-o(2,8)-NeuAC-R, to thereby generate the complex carbohydrate shown in Figure 1.
  • ERs selection for providing a desired EM is generated using a computer algorithm taking into account the complex carbohydrate structure and the available ERs.
  • Such an algorithm can readily be programmed by one ordinarily skilled in the art, based on the donor-acceptor specificities of the various glycosyltransferases glycosidases and transglycosylases available.
  • Automated synthesis and screening The EMs used in the construction of a combinatorial complex carbohydrate library according to the present invention are executed to produce a combinatorial complex carbohydrate library attached to a solid support using automated technology. Screening to identify bio-active complex carbohydrates cross reactive to a probe of interest is also executed, according to preferred embodiments of the invention, via automated technology.
  • the libraries described by the present invention are preferably generated by a parallel synthesis method, wherein consideration is preferably given to ensure a minimal number of steps executed.
  • a minimal number of steps implies that each of the reagent containers is detailed a minimal number of times.
  • consideration is given to a sequence of synthesis steps that will ensure completion of the synthesis of all of the complex carbohydrates of the library with a minimal number of times each reagent container is detailed.
  • a dedicated algorithm can be readily developed by one ordinarily skilled in the art to design automated library synthesis protocol which will comply with the above requirements.
  • a similar algorithm has already been developed for the parallel addressable synthesis of oligonucleotides and peptides on microchips.
  • Such technologies include, for example, (i) opened reactor systems (e.g., conventional microtiterplates); (ii) closed reactor systems or semi-closed reactor systems (e.g., lab-on-chip); (iii) reaction block systems; (iv) synthesis on polymeric pins; (v) synthesis on polymeric sheets; and (vi) synthesis on a microchip.
  • Solid phase support The libraries according to the present invention are preferably synthesized on a solid phase support.
  • the first building block is provided with a suitable functional group for binding such a support.
  • Suitable binding groups include hydroxyls, carbonyl, carboxyl, amines, halides, thiols, esters, boronates, siloxy, aza, oxo, oxiren, or any unsaturated group.
  • Solid matrix supports are most suitable for generating carbohydrate libraries according to the present invention, such as, but not limited to, polysterene cross-linked with divinylbenzene, polyethylene glycol-polystyrene block copolymer (PEG-PS), polyamides, polyacrylamide, polymethacrylamide and cellulose.
  • PEG-PS polyethylene glycol-polystyrene block copolymer
  • Microfabricated silicon-based arrays produced by standard semi-conductor processing techniques U.S. Pat. Nos. 5,643,738; 5,681,484; and 5,585,069) may also serve as a solid phase support. Linking the first saccharide building block to the solid phase support:
  • the first saccharide building block is preferably covalently attached to the solid phase matrix via a single atom (e.g., the solid phase functional group) or a linker.
  • a linker includes: having bi-functional groups enabling attachment to both the solid support and to the initial building block, and as such to define a structure.
  • the linker is designed cleavable, so as to allow removal of the synthesized oligosaccharide from the matrix post synthesis. This allows for analysis thereof using, for example, mass spectroscopy or any other suitable method. Since enzyme accessibility to the immobilized saccharides is of great importance, the linker length and flexibility are crucial for high yield.
  • linkers suitable for synthesis according to the present invention can include, for example, amino acids, peptides, non-glycosylated proteins, lipids, lipid A, ceramides, dolicol phosphates, cyclodextrins, oligosaccharides, monosaccharides, alkyl chains, nucleic acids, or other spacer molecules.
  • These linkers can be cleavable or non-cleavable and be composed of simple, complex, cyclic or branched entities.
  • the linker is between 3.5 nm and 8 nm in length. It is preferably selected sufficiently hydrophilic so as to stay in solution and to avoid none specific interaction with proteins.
  • the linker is synthesized by elongation cycles using bi-functional building blocks molecules that can form bonds between each other.
  • the starting point can be any functional group which is attached or attachable to the solid support that can react with a bi-functional building block.
  • the linker can be ended by any one of the bi-functional building blocks, there are , as shown in Figures 5a and 5b, two possible ways to connect the first monosaccharide to the linker.
  • the linker is preferably selected cleavable under mild conditions that do not damage carbohydrates.
  • cleavable linkers which comply with all of the above preferred criteria are constructed as described under Example 11 and shown in Figures 5a and 5b.
  • Library arrangement The preferred arrangement of the library constituents according to the present invention is in an array synthesized on a solid phase support in various geometric forms and layouts, such as: two dimensional arrays, multi layer arrays, three dimensional arrays (e.g., stacked microtiters), and arrays which are displayed on spherical disks or cone shapes.
  • the library constituents can be attached to polymer beads in reaction chambers (opened or closed) and arrayed on a two dimensional or a three dimensional support. Any arrangement that enables easy automatic addressable operation of the EMs collection may be used in accordance with the present invention.
  • Microfluid systems can and are preferably employed (U.S. Pat. Nos. 5,643,738; 5,681,484; and 5,585,069).
  • a combinatorial complex carbohydrate library comprising a plurality of complex carbohydrate structures being attached to a solid support and a first member of a proximity signal generation pair being independently, not through the complex carbohydrate structures, attached to the solid support.
  • a method of producing an addressable combinatorial complex carbohydrate library comprising providing a solid support having a plurality of locations; enzymatically synthesizing a plurality of complex carbohydrate structures, each of the plurality of complex carbohydrate structures being attached to at least one addressed location of the plurality of locations, thereby producing the addressable combinatorial complex carbohydrate library; and attaching to the solid support at each of the addressed locations, not through the complex carbohydrate structures, a first member of a proximity signal generation pair.
  • addressable and “addressed” refer to both location and identity.
  • location and identity (composition) of a complex carbohydrate structure of a library according to the present invention are both known in advance and that carbohydrate structure is therefore addressable.
  • a complex carbohydrate structure refers to a plurality of complex carbohydrate molecules all having the same structure and localized at a specific and addressable location on the solid support.
  • the addressable complex carbohydrate structures of a library according to the present invention are preferably attached to the solid support via a linker (spacer).
  • the linker according to preferred embodiments of the invention includes at least two contiguous covalent bonds and it is of a length of at least 20 Angstroms.
  • Suitable linkers include, but are not limited to, an amino acid, a peptide, a non-glycosylated protein, a lipid, a ceramide, dolicol phosphate, a cyclodextrin, an oligosaccharide, a monosaccharide, an alkyl chain, and a nucleic acid (e.g., an oligonucleotide).
  • the solid support onto which complex carbohydrate structures of a library according to the present invention are attached can include addressable microparticles or beads, or a flat platform.
  • the addressable microparticles or beads are arranged, for example, within wells of a microtiter plate.
  • a microtiterplate, a membrane or a chip e.g., silicone chip
  • the solid support is a chip and different complex carbohydrate structures of the plurality of addressable complex carbohydrate structures are formed in patches spaced not more than 2.25 mm from one another (center to center) over the surface of the chip, thereby providing a density of at least 20 different addressable complex carbohydrate structures per square centimeter.
  • the substance of which the solid support is made can be, for example, polysterene cross-linked with divinylbenzene, polyethylene glycol-polystyrene block copolymer, polyamides, polyacrylamide, polymethacrylamide, cellulose, glass, quartz, plastic or silica.
  • At least one, preferably at least three, more preferably at least ten, more preferably, at least 100, more preferably at least 1000 of the plurality of addressable complex carbohydrate structures of a library of the present invention includes at least two, three, four or at least five or more contiguous saccharide units of a single species. As further detailed hereinabove and resolved such a structure is not trivial due to uncontrolled polymerization.
  • At least one, preferably at least three, more preferably at least ten, more preferably, at least 100, more preferably at least 1000 of the plurality of addressable complex carbohydrate structures of a library of the present invention includes at least one, two, three, four or at least five or more branches.
  • At least one, two, three or at least four of the branches are formed of identical core and branching saccharide units.
  • the antennas attached to each of the branches differ in saccharide units composition.
  • At least one, preferably at least three, more preferably at least ten, more preferably, at leas 100, more preferably at least 1000, of the plurality of addressable complex carbohydrate structures includes at least 4 preferably at least 5, more preferably at least 7, more preferably at least 9, more preferably at least 10, more preferably at least 12, more preferably at least 15, 20, 25 or at least 30, more preferably at least 50 or more saccharide units.
  • the plurality of addressable complex carbohydrate structures of a library can be a representation including non-natural or natural complex carbohydrates, e.g., which are derived from a human source, such as tissue, cells or body fluids of a human-being.
  • the plurality of addressable complex carbohydrate structures can be a representation of domains (fragments) of at least one natural complex carbohydrate.
  • Such a library as further detailed herein, can be employed to identify an active site of the natural complex carbohydrate. Further detail relating to the structure, construction and uses of the complex carbohydrate libraries herein described can be found in PCT/IL00/00099 and U.S. Patent Application No. 09/783,083, both are incorporated by reference as if fully set forth herein.
  • the coating of the surface of microtiterplates with N-acetylglucose and galactose ⁇ 1,4 N-acetylglucose was performed employing the following steps (a) synthesis of a linker for attachment of the saccharides to the surface; (b) attachment of N-acetylglucose and biotin to the surface; (c) enzymatic elongation reaction of N-acetylglucose to galactose ⁇ 1,4 N-acetylglucose; and (d) attachment of fluorescent energy transfer donor molecule (Europium) to the surface (via the biotin), whereas the measurement of EcorA binding was performed by the addition of a solution containing EcorA labeled with a suitable fluorescent energy transfer acceptor molecule (allophicocyanin, hereinafter, APC) and the energy transferred from the surface bound donor molecule to labeled lectin bound to the surface via the saccharides was monitored with or without an inhibitor (lac
  • Amino linker synthesis and elongation cycle 100 ⁇ l of a 1,8-diamino 3,6 (Merck, Cat. No. 818116) solution (3 ml in 50 ml 0.1 M carbonate buffer, pH 9.6) or 1,8 diaminooctane (Aldrich, Cat. No. D2, 240-1) solution (100 mg per ml of 0.1 M carbonate buffer, pH 9.6) was added to each well of a cyanuric chloride activated Covalink NH microtiter plate. The wells were sealed and the plate was incubated at 25 °C for 12 hours.
  • Cyanuric chloride activation A solution containing 48 mg of cyanuric chloride (Aldrich, Cat. No. C95501) dissolved in 3 ml of acetone was added, while stirring, to 45 ml of 0.1 M phosphate buffer. An aliquot (200 ⁇ l) of this solution was quickly added (within 2 minutes) to each well of the Covalink NH microtiter plate. The plate was incubated at room temperature for 5 minutes following which the solution was discarded and the plate washed three times with double distilled water and dried at 50 °C for 30 minutes.
  • N-acetylglucose and biotin molecules were linked to the activated plate described above.
  • the following procedure was utilized to effect linking: 100 ⁇ l of solution containing 20 mg of ?-nitrophenyl- ⁇ -acetyl- ⁇ -D-Glc ⁇ Ac (Calbiochem, Cat. No. 487052), 1 mg of ⁇ -nitrophenyl-biotin and 200 mg of sodium dithionite (Fluka, Cat. No. 71700) which were dissolved in 6 ml of double distilled water and titrated to pH 7.5 using 3 ml of 0.1 M sodium carbonate (pH 9.6) were added to each well. The wells were sealed and incubated at room temperature overnight.
  • a solution of EcorA-biotin conjugate (Sigma, Cat. No. L-0893) bound with APC-streptavidin conjugate (Wallac, Cat. No. AD0058) was prepared by the addition of 4 ⁇ g EcorA-biotin and 9.5 ⁇ g APC-streptavidin to 2 ml TBST (1 :1 molar ratio of EcorA-biotin to APC-streptavidin). Following 1 hour incubation, 100 ⁇ l of EcorA-biotin/APC-streptavidin (APC labeled EcorA) solution was added to each well.
  • a solution of EcorA-biotin conjugate (Sigma, Cat. No. L-0893) bound with APC-streptavidin conjugate (Wallac, Cat. No. AD0058) was prepared by the addition of 4 ⁇ g EcorA-biotin and 9.5 ⁇ g APC-streptavidin to 2 ml TBST (1 :1 molar ratio of EcorA-biotin to APC-streptavidin). Following 1 hour incubation, 50 ⁇ l of a lactose solution at different concentrations was added to the wells and thereafter 50 ⁇ l of EcorA-biotin/APC-streptavidin (APC labeled EcorA) solution was added to each well.
  • Table 5 summarizes the fluorescence intensity of the grafted Europium (exitation at 340 nm, emission at 612 nm, average of 4 wells), the fluorescence intensity that resulted from energy transfer from Europium to APC (exitation at 340 nm, emission at 662 nm, average of 4 wells), and the ratio between these intensities.
  • the ratio for galactose ⁇ 1,4 N-acetylglucose was higher then for N-acetylglucose.
  • the results demonstrate that the difference in the measured signal is correlated to the increase in the amount of EcorA bound to the surface of the plate. It substantiates that the affinity of EcorA to Galactose ⁇ 1,4 N-acetylglucose is higher then the affinity of EcorA to N-acetylglucose.
  • Lactose is a known ligand of EcorA.
  • the effect of different concentrations of free lactose on the binding of EcorA to solid support bound galactose ⁇ 1,4 N-acetylglucose measured using fluorescence resonance energy transfer from Europium to APC is described in Figure 7. It is shown that increasing the lactose concentration resulted in a decrease of the fluorescence resonance energy transfer signal, as the amount of bound EcorA-EPC to the solid support decreases.

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Abstract

Cette invention concerne un procédé d'analyse d'une liaison entre un premier élément d'une paire de liaisons fixée à un support solide et un second élément de la paire de liaisons en solution. Ce procédé consiste à analyser la liaison entre le premier élément de la paire de liaisons et le second élément de la paire de liaisons par la surveillance d'une émission de signaux entre un premier élément d'une paire d'émission de signaux de proximité fixée au support solide, et non pas par l'intermédiaire du premier ou du second élément de la paire de liaisons, et un second élément de la paire d'émission de signaux de proximité liée au deuxième élément de la paire de liaisons.
PCT/IL2002/000573 2001-07-17 2002-07-16 Procedes et substrats servant a surveiller la liaison a une phase solide a l'aide d'analyses d'emission de signaux de proximite WO2003008927A2 (fr)

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EP2145895A1 (fr) 2008-07-08 2010-01-20 Commissariat à l'Energie Atomique Processus de fabrication de glycochips
CN103743711A (zh) * 2014-01-01 2014-04-23 桂林理工大学 利用环糊精与荧光共振能量转移技术检测食品中赤霉素的方法
JP2020530112A (ja) * 2017-07-31 2020-10-15 ベステル エレクトロニク サナイー ベ ティカレト エー．エス． 識別タグおよび対象物識別方法

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080261239A1 (en) * 2006-11-13 2008-10-23 Perkinelmer Las, Inc. Detecting molecular interactions
US9146233B2 (en) * 2006-11-13 2015-09-29 Perkinelmer Health Sciences, Inc. Detecting molecular interactions by fluorescence resonance energy transfer on a solid-phase support
EP2145895A1 (fr) 2008-07-08 2010-01-20 Commissariat à l'Energie Atomique Processus de fabrication de glycochips
CN103743711A (zh) * 2014-01-01 2014-04-23 桂林理工大学 利用环糊精与荧光共振能量转移技术检测食品中赤霉素的方法
JP2020530112A (ja) * 2017-07-31 2020-10-15 ベステル エレクトロニク サナイー ベ ティカレト エー．エス． 識別タグおよび対象物識別方法
JP7090692B2 (ja) 2017-07-31 2022-06-24 ベステル エレクトロニク サナイー ベ ティカレト エー．エス． 識別タグおよび対象物識別方法

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