EP1508045A1 - Procedes de detection de molecules cibles et d'interactions moleculaires - Google Patents

Procedes de detection de molecules cibles et d'interactions moleculaires

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Publication number
EP1508045A1
EP1508045A1 EP03732677A EP03732677A EP1508045A1 EP 1508045 A1 EP1508045 A1 EP 1508045A1 EP 03732677 A EP03732677 A EP 03732677A EP 03732677 A EP03732677 A EP 03732677A EP 1508045 A1 EP1508045 A1 EP 1508045A1
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EP
European Patent Office
Prior art keywords
label
catalytic
substrate
substrate label
interacting
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP03732677A
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German (de)
English (en)
Inventor
Stuart Wilson
Christopher John Stanley
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ISEAO Technologies Ltd
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ISEAO Technologies Ltd
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Application filed by ISEAO Technologies Ltd filed Critical ISEAO Technologies Ltd
Publication of EP1508045A1 publication Critical patent/EP1508045A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification

Definitions

  • the invention relates to methods and kits for detecting target molecules or the interactions between target molecules.
  • the invention relates to methods based on the use of "two-component conjugate" systems comprising a catalytic label and a substrate label.
  • Detection of a target biological molecule in a sample requires sensitive techniques that are capable of discriminating between specific recognition events and non-specific recognition events that might otherwise lead to false positive results.
  • RNA sequence-based amplification (NASBA) (Compton, 1991) and 3SR (Fahy et al . , 1991).
  • Proteins may be detected by well known techniques such as Western Blotting. This technique is very useful but is limited in sensitivity and specificity, due to the lack of an amplification step, and the fact that there is a single entity binding to the target.
  • Target molecules can, however, be detected in a very sensitive and specific manner through the Dual Phage approach, as disclosed in WO 99/63348.
  • the Dual Phage assay two phage are needed in order to generate a signal and the two phage must be brought together or linked through the target molecule. This is most easily achieved by linking the phage to ligands such as antibodies that are specific for the target molecule. In this method it is possible to detect a wide range of target molecules including nucleic acids, proteins and simple or complex molecules .
  • the nucleic acid bridge is unlikely to re-form if only one antibody is bound non- specifically to a molecule other than the intended target.
  • a disadvantage of this approach is the problem of ensuring that all of the nucleic acid bridge molecules are cleaved in the absence of target antigen.
  • the method is complex and involves a number of steps that could involve DNA restriction enzymes, DNA polymerases and DNA ligation enzymes.
  • WO 01/61037 relates to assays for detection of analytes in solution using so-called proximity probes.
  • Said proximity probes consist of a binding moiety and a nucleic acid.
  • the nucleic acids are brought into close proximity and can thus be ligated and then detected, usually by amplification.
  • This technique has the disadvantage that ligation can be an inefficient reaction and the ligase has to be added to the reaction in a high enough concentration to allow efficient ligation of the proximity probes.
  • Sensitive methods are also needed for use in monitoring molecular interactions. Drug discovery and proteomics are just two of the areas of technology that rely on the monitoring of such interactions. A variety of methods are currently in use, which are well known in the art, such as the Scintillation Proximity Assay (SPA) (Bosworth and Towers, 1989) and various Yeast Hybrid methodologies (Ma and Ptashne, 1988; Fields and Sternglanz, 1994).
  • SPA Scintillation Proximity Assay
  • Yeast Hybrid methodologies Ma and Ptashne, 1988; Fields and Sternglanz, 1994.
  • the Dual Phage method can also be applied to the monitoring of molecular interactions.
  • the molecules whose interaction is to be studied each have a ligand-binding site that can bind one phage type either directly or indirectly.
  • the interaction of the molecules is thus able to be monitored through the linking of the two phage types. If the molecules interact the two phage types are brought together but if they do not interact the two phage types remain separate. This approach can be applied to proteomics and drug discovery.
  • FRET fluorescent Resonance Energy Transfer
  • FRET a distance-dependent excited state interaction in which emission of one fluorophore is coupled to the excitation of another.
  • FRET relies on a fluorescence donor which transfers a quantum, or exciton, of energy to a fluorescence acceptor, thus raising an electron in the acceptor to a higher energy state as the photo-excited electron in the donor returns to the ground state.
  • the resonance interaction between donor and acceptor fluorophores occurs over distances that are greater than interatomic.
  • the fluorescent emission spectrum of the donor must overlap the absorption spectrum of the acceptor, and the donor and acceptor transition dipole orientations must be approximately parallel. The probability that energy transfer will occur depends on the sixth power of the distance between the fluorophores.
  • FRET can be detected by the appearance of fluorescence of the acceptor or by quenching of donor fluorescence. If the donor and acceptor are the same, FRET can be detected by the resulting fluorescent depolarization. Energy transfer can be detected by measuring emission from the acceptor fluorophore when excited at the donor fluorophore ' s wavelength. This wavelength does not produce an emission from the acceptor unless FRET has occurred. Alternatively, FRET can be detected by measuring the quenching effect that the acceptor has on donor emission at its excitation wavelength.
  • US 6,251,581 discloses methods for determining an analyte in a medium suspected of containing the analyte.
  • One method comprises treating a medium suspected of containing an analyte under conditions such that the analyte, if present, causes a photosensitizer and a chemiluminescent compound to come into close proximity.
  • the photosensitizer generates singlet oxygen and activates the chemiluminescent compound when in close proximity.
  • the activated chemiluminescent compound subsequently produces light.
  • the amount of light produced is related to the amount of analyte in the medium.
  • the present invention seeks to provide improved methods of detecting target molecules and of monitoring molecular interactions.
  • target detection method a method of detecting a target molecule in a sample comprising the steps of;
  • interaction method a method of detecting interactions between two or more interacting molecules comprising the steps of:
  • detecting the change in the substrate label Both methods rely on specific binding interactions into order to bring into close proximity a "catalytic label” and a "substrate label".
  • the catalytic label is capable of acting directly on the substrate label to generate a detectable change in the substrate label.
  • detectable change means a change in structure and/or activity of the substrate label, caused by the action of the catalytic label, which change in structure and/or activity can be directly or indirectly detected.
  • the “detectable change” in the substrate label is preferably irreversible under the conditions used to detect the change.
  • the "detectable change” is caused by direct action of the catalytic label on the substrate label, meaning that the change in structure and/or activity is brought about by direct contact or physical interaction between the catalytic and substrate labels.
  • direct action excludes changes in structure and/or activity of the substrate label which are brought about solely though the action of a diffusable intermediate, without any requirement for a direct contact or physical interaction between the catalytic and substrate labels.
  • the catalytic and substrate labels are attached to "separate" binding entities/interacting molecules, meaning that no part of the catalytic label is attached to the same binding entity/interacting molecule as any part of the substrate label.
  • the catalytic and substrate labels are brought into close proximity by specific binding of binding entities to a target molecule (in the target detection method) or specific binding of interacting molecules (interaction method) , thus facilitating direct action of the catalytic label on the substrate label.
  • the close juxtaposition of the catalytic and substrate labels as a result of specific binding events results in a rate enhancement that provides an improved signal-to-noise ratio over the background due to random interactions between un-bound catalytic and substrate labels in free solution.
  • the target detection and interaction methods according to the invention are types of biological binding assays and may be carried out in accordance with standard principles known in the art for these types of assays. For example, it is common to include intermediate washing steps between addition of reagents to remove excess/unbound reagents, as illustrated in the accompanying examples.
  • Action of the catalytic label on the substrate label results in a detectable change in structure and/or activity of the substrate label.
  • Changes in the structure of the substrate label may include, for example, physical cleavage of the label .
  • Changes in activity of the substrate label may include, for example, a change in a detectable property such as fluorescence, luminescence, etc, or a change in enzymatic activity, electrochemical activity or redox properties.
  • the change in activity may be accompanied by, or caused by, a change in structure of the substrate label.
  • the "change" in activity of the substrate label may be a change in activity resulting from a discrete switch of the substrate label from an "inactive" to an “active” state (or vice versa) as a result of a discrete action of the catalytic label on the substrate label (an example being a cleavage of the substrate label) .
  • the "change” in activity may be the appearance of an activity which requires continued action of the catalytic label on the substrate label.
  • An example of such a "change” is increased transcriptional activity resulting from interaction between an RNA polymerase catalytic label and a nucleic acid substrate label bearing a promoter specific for the RNA polymerase.
  • Preferred catalytic labels include, but are not limited to, enzymes.
  • the action of the catalytic label on the substrate label may generate a fluorophore, which can subsequently be detected.
  • the substrate label may be formed of a fluorescent moiety linked to a quencher moiety which quenches the fluorescence of the fluorescent moiety by via a linkage which is cleaved by the action of the catalytic label. Cleavage of the linkage releases the quencher moiety and thus increases fluorescence from the fluorescent moiety. This increase in fluorescence may be detected using a suitable measuring instrument.
  • the catalytic label may be an enzyme and the "linkage" may be a molecular structure which is cleaved by the action of the enzyme.
  • the enzyme may be a protease and the linkage a peptide which is cleaved by the action of the enzyme.
  • the precise nature of the linkage is not important, except to the extent that it must enable the fluorescent and quencher moieties to interact when "linked", such that the quencher moiety quenches fluorescence from the fluorescent moiety.
  • the action of the catalytic label on the substrate label may quench a fluorophore .
  • the action of the catalytic label on the substrate label may create an electrochemically active species or group that can subsequently be detected.
  • the catalytic label may be the enzyme alkaline phosphatase and the substrate label may be a phenol phosphate moiety coupled directly to a binding moiety.
  • the enzyme when in close proximity to the substrate, catalyses the removal of the phosphate group, generating an electrochemically active group which may be detected by a redox reaction at an electrode surface.
  • the substrate label may be an inactive molecule which becomes activated by the action of the catalytic label.
  • examples of such labels include, for example, enzyme precursors, zymogens or pro-enzymes.
  • zymogens enzymes are synthesized as inactive precursors, called zymogens or pro-enzymes, which are subsequently activated by cleavage of one or a few specific peptide bonds.
  • This proteolytic activation is an irreversible process.
  • trypsinogen, chymotrypsinogen, proelastase and procarboxypeptidase are all inactive precursors, of the digestive enzymes trypsin, chymotrypsin, elastase and carboxypeptidase, respectively. All of these pro-enzymes become activated by the action of trypsin, which hydrolyses peptide bonds in the pro-enzymes.
  • the catalytic label may be trypsin, or a protease of equivalent proteolytic activity but differing specificity
  • the substrate label may be one of trypsinogen, chymotrypsinogen, proelastase and procarboxypeptidase.
  • enzyme catalytic labels and enzyme precursor substrate labels include enteropeptidase/trypsinogen (cleaved to generate trypsin) , thrombin/fibrinogen (cleaved to generate fibrin) .
  • the "change" brought about by action of the catalytic label on the substrate label may be conveniently detected by a suitable assay specific for the enzyme activity generated by cleavage of the substrate label. It is important that this assay is capable of distinguishing between enzymatic activity generated as a result of cleavage of the substrate label and the enzymic activity of the catalytic label.
  • Dephosphorylation or phosphorylation of the substrate label may, in turn, lead to a detectable change in structure and/or activity of the substrate label.
  • the substrate label which is phosphorylated/dephosphorylated by the kinase/phosphatase may be a protein, however the invention is not limited to only proteins. Any suitable substrate label which can be phosphorylated or dephosphorylated, wherein such phosphorylation or dephosphorylation is accompanied by a detectable change in structure and/or activity, may be used within the scope of the invention.
  • the substrate label may be a nucleic acid and the catalytic label may be a species, typically an enzyme, capable of catalysing a detectable change in the structure and/or activity of the nucleic acid.
  • suitable catalytic labels include, for example, recombinases, ligases, transposases, DNA polymerases, reverse transcriptases or RNA polymerases .
  • changes in the "structure" of a nucleic acid label are taken to include changes in sequence which are detectable, for example with the use of suitable sequence-specific probes and/or nucleic acid amplification techniques. There are many suitable techniques known to those skilled in the art which may be used to detect and differentiate specific nucleic acid sequences.
  • Changes in the "activity" of a nucleic acid label may include, as discussed previously, a change in transcriptional activity, such as may occur if the catalytic label is an RNA polymerase and the substrate label contains a promoter specific for the said polymerase.
  • Preferred RNA polymerase/promoter systems for use in this embodiment of the invention include (but are not limited to) the bacteriophage RNA polymerases, especially T7, T3 and SP6 polymerases, and their cognate promoters. These polymerases are well known in the art and are routinely used for in vi tro transcription.
  • the substrate label may be a reporter gene expression construct comprising a suitable promoter driving expression of a reporter gene. The increase in transcriptional activity may then be monitored by detecting expression of the reporter gene.
  • the substrate label may be formed of two or more component parts, at least one of which is attached to a separate binding entity/interacting molecule to the catalytic label.
  • the component parts of the substrate label may be all attached to a single binding entity/interacting molecule and the catalytic label may be attached to a separate binding entity/interacting molecule.
  • the catalytic label and component parts of the substrate label may each be attached to separate binding entities/interacting molecules.
  • Such a system has the advantage that three species must be brought into close proximity for the interaction between catalytic and substrate labels to take place.
  • target detection method wherein the catalytic label and components of the substrate label are each attached to separate binding entities
  • at least three separate binding events of binding entities to a common target must take place in order to enable the interaction between catalytic and substrate labels.
  • the “interaction method” wherein at least three separate interacting molecules are respectively labelled with a catalytic label and components of the substrate label, then at least three separate species of interacting molecules must come together, in order to enable the interaction between catalytic and substrate labels.
  • the catalytic label and at least one component part of the substrate label may each be attached to separate binding entities/interacting molecules and a further component part of the substrate label may be present in free solution, preferably in an excess amount.
  • a further component part of the substrate label may be present in free solution, preferably in an excess amount.
  • a substrate label formed of two component parts is a substrate label formed of two separate nucleic acid tags, which are capable of interacting, in the presence of a suitable catalytic label, to generate at least one nucleic acid tag having novel sequence.
  • This type of label is particularly useful in an assay system wherein one nucleic acid tag is attached to a binding entity/interacting molecule and another nucleic acid tag is present in free solution in excess.
  • the tags are capable of interacting via recombination to generate at least one tag of novel sequence.
  • the catalytic label comprises an enzyme that catalyses recombination between the nucleic acid tags.
  • Recombination is defined herein to include any exchange of nucleic acid sequence or deletion or insertion of sequences between the nucleic acid tags in order to generate at least one novel sequence that is capable of being detected. Examples include site- specific recombination events (e.g. requiring a specific recombinase) and transposition events (e.g. requiring a specific transposase) .
  • Site-specific recombination events are non- homologous recombination events, in so far as they generally do not require extensive homology between nucleic acid tags. In most cases site-specific recombination requires the presence of short recombination site sequences (generally a few tens of base-pairs) . Many site-specific recombination systems require the presence of identical recombination site sequences. However, in other systems the recombination sites may share little or no sequence homology, as is the case with the integration sites attP and attB, derived respectively from bacteriophage lambda and the E . coli chromosome.
  • the "substrate label” is comprised of two nucleic acid tags, each containing a site-specific recombination sequence recognised by a particular site-specific recombinase enzyme, and the "catalytic label” is the appropriate site-specific recombinase enzyme.
  • Suitable site-specific recombination systems which may be used include the Cre/loxP system, wherein the nucleic acid tags making up the substrate label contain loxP sites, and the catalytic label is Cre recombinase.
  • Another suitable system is the bacteriophage lambda integration system, wherein the nucleic acid tags contain attP and attB recognition sequences or attL and attR sequences, allowing recombination catalysed by an enzyme label which recognises these sites. Recombination between attB and attP sites or between attL and attR sequences is catalysed by the lambda phage enzyme integrase, and requires a host accessory factor IHF.
  • the lambda phage recombination system is well known in the art and the enzymes required for recombination are available commercially (e.g. as components of the GatewayTM cloning system supplied by Invitrogen) . These particular recombination systems are listed by way of example only and it is not intended to limit the invention to the use of these specific systems.
  • the recombinase enzyme may actually be attached to one of the nucleic acid tags making up the substrate label.
  • the two nucleic acid tags making up the recombinase substrate may be attached to separate binding entities/interacting molecules and the recombinase enzyme may be attached to one of the nucleic acid tags.
  • the two nucleic acid tags plus the recombinase will be brought into close proximity, thus enabling the recombination reaction to take place.
  • recombination may depend upon a transposition event and rely upon the use of a transposase as the catalytic label.
  • a suitable example of such a system depends upon Tn5 transposase that recognizes Mosaic Ends recognition sequences.
  • Tn5 transposase that recognizes Mosaic Ends recognition sequences.
  • transposase as the catalytic label in order to generate a "detectable change" by transposition of a nucleic acid sequence between two nucleic acid tags making up the substrate label may or may not require the presence of specific sequences in both the nucleic acid tags in order to allow transposition to take place.
  • the requirements for successful transposition with any particular transposase enzyme/transposable element will generally be appreciated by those skilled in the art.
  • a further preferred embodiment of a system wherein the substrate label is composed of two nucleic acid tags is based on the use of a ligase as the catalytic label.
  • the ligase is capable of ligating together two separate nucleic acid tags, which together make up the substrate label, in order to form a detectable ligation product having novel sequence.
  • this system it is possible to attach one of the nucleic acids to a binding entity/interacting molecule and add the second tag in free solution in an excess amount.
  • the nucleic acid tags used in this embodiment may be formed of double-stranded RNA, enabling ligation by T4 DNA ligase.
  • the tags may have complementary "sticky" ends or blunt ends.
  • the tags may be formed of single-stranded RNA, which can be joined by T4 RNA ligase.
  • Nucleic acid tags having novel sequence such as may be generated by the action of recombinases, transposases or ligases on "substrate" nucleic acid tags may be detected using any suitable technique known in the art.
  • detection of the novel sequence will involve an amplification reaction, for example PCR, NASBA, 3SR or any other amplification technique known in the art. Amplification is achieved with the use of amplification primers specific for the novel sequence.
  • primer binding sites corresponding to a region of completely novel sequence may be selected, or else a novel combination of primer binding sites, not present in the original tags, may be chosen.
  • novel sequence may also include sequences other than primer binding sites which are required for detection of the novel sequence, for example RNA Polymerase binding sites or promoter sequences required for isothermal amplification technologies, such as NASBA or 3SR.
  • detection of the novel sequence is carried out by amplification with "real- time" detection of the products of the amplification reaction. This can be achieved using any amplification technique which allows for continuous monitoring of the formation of the amplification product .
  • a number of techniques for real-time detection of the products of an amplification reaction are known in the art. Many of these produce a fluorescent read-out that can be continuously monitored, specific examples being molecular beacons and fluorescent resonance energy transfer probes. Real-time quantification of PCR reactions can be accomplished using the TaqMan® system (Applied Biosystems) .
  • the method is carried out in real-time, meaning that specific binding of the binding entities to the target molecule (or specific binding of interacting molecules) , action of the catalytic label on the nucleic acid substrate label and detection of the product of the interaction are carried out simultaneously in a single reaction step.
  • Real-time detection requires that the binding step, action of the catalytic label on the substrate label, and detection of the product of the interaction can all be carried out under a single set of reaction conditions, without the need for intermediate washing steps.
  • real-time detection of the novel sequence will preferably be carried out using an isothermal amplification reaction, for example NASBA or 3SR, in order to avoid changes of temperature which might adversely affect the binding of the binding entities to the target molecule/interaction between interacting molecules.
  • an isothermal amplification reaction for example NASBA or 3SR
  • nucleic acid tags includes any natural nucleic acid and natural or synthetic analogues that can be acted upon by a catalytic label to generate novel sequence, for example by recombination.
  • Suitable nucleic acid tags include tags composed of double or single-stranded DNA, double or single-stranded RNA. Tags which are partially double-stranded and partially single- stranded are also contemplated. It is also contemplated to use single-stranded tags in combination with double-stranded tags, i.e. one component labelled with a single-stranded tag and another component labelled with a double-stranded tag capable of interacting with the single-stranded tag.
  • the nucleic acid tags may be composed of any nucleic acid which is capable of participating in the recombination reaction, suitable examples including linear or circular double-stranded DNA (dsDNA) or double-stranded RNA (dsRNA) or mixtures thereof. Most preferably the nucleic acid tags will comprise dsDNA.
  • dsDNA linear or circular double-stranded DNA
  • dsRNA double-stranded RNA
  • the nucleic acid tags will comprise dsDNA.
  • the term “nucleic acid” encompasses synthetic analogues which form a substrate for the catalytic label in an analogous manner to natural nucleic acids, for example nucleic acid analogues incorporating non- natural or derivatized bases, or nucleic acid analogues having a modified backbone.
  • double-stranded DNA or "dsDNA” is to be interpreted as encompassing dsDNA containing non- natural bases.
  • nucleic acid tags are not material to the invention, except to the extent that certain sequences may be required to enable the "action" of the catalytic label on the nucleic acid tags. For example, specific sequences are required to permit site-specific recombination.
  • Two nucleic acid tags making up a "substrate label" will most usually be of different sequence, so that an interaction event between the nucleic acid tags leads to production of at least one novel sequence that can be detected. However, it is not excluded to use tags of identical sequence, provided that the tags are able to interact to generate novel sequence. Most preferably, the action of the catalytic label on the substrate label will lead to the production of two separate nucleic acid tags, each having novel sequence. This has the advantage that the two tags of novel sequence form independently verifiable products.
  • the catalytic label may be formed of two or more component parts, at least one of which is attached to a separate binding entity/interacting molecule to the substrate label.
  • Examples of such catalytic labels may include, for example enzyme/co-enzymes, multi subunit enzymes, etc.
  • the substrate label and component parts of the catalytic label may each be attached to separate binding entities/interacting molecules, or the component parts of the catalytic label may all be attached to a single binding entity/interacting molecule and the substrate label may be attached to a separate binding entity/interacting molecule.
  • the substrate label and catalytic label may each be formed of two or more component parts, wherein component parts of the substrate and catalytic labels are attached to separate binding entities/interacting molecules.
  • all component parts of the substrate label may be attached to a single binding entity/interacting molecule or the component parts may be attached to a number of separate binding entities/interacting molecules.
  • all component parts of the catalytic label may be attached to a single binding entity/interacting molecule or the component parts may be attached to a number of separate binding entities/interacting molecules.
  • the only limitation is that (component parts of) the substrate and catalytic labels must not be attached to the same binding entity/interacting molecule.
  • catalytic label it is generally preferred to start with the catalytic label in an inactivated state and then activate the catalytic label only after binding of the binding entities to the target molecule (or interaction between interacting molecules) has taken place to position the catalytic and substrate labels in close proximity.
  • Activation of the catalytic label may, for example, be achieved by changing the composition, pH or temperature of the reaction medium. This "external activation" step increases the sensitivity of the method by minimising background resulting from non-specific interactions between the catalytic label and substrate label in free solution.
  • step (b) of the target detection method and the equivalent step (a) of the interaction method may be carried out under conditions which do not permit the catalytic label to act on the substrate label.
  • These "conditions” may be, for example, the lack of a key component required for the action of the catalytic label on the substrate label. If the substrate label is formed of two component parts, one of which is to be added in free solution, then the “conditions” could be the absence of this component. After binding of the binding entities to the target molecule (or interaction between interacting molecules) has taken place to position the catalytic and substrate labels in close proximity, the "conditions" may be changed to permit the catalytic label to act on the substrate label.
  • This change could be, for example, addition of the missing component of the substrate label under conditions which permit subsequent reaction between the catalytic label and components of the substrate label.
  • a "change" in reaction medium may easily be achieved with the use of an intermediate washing step . or by simple addition of a missing reaction component (both of which are in accordance with standard principles of biological binding assays) .
  • the substrate label is formed of two (or more) separate nucleic acid tags which are to be brought together in an interaction requiring hybridisation between the two tags
  • the catalytic label and substrate label are directly attached to the binding entities or interacting molecules.
  • Direct linkage may be achieved via a covalent linkage.
  • amine-derivatized nucleic acid tags may be coupled to protein binding entities/interacting molecules using any one of a number of chemical cross- linking compounds . It is also within the scope of the invention for the catalytic or substrate labels to be attached indirectly to the binding entities/interacting molecules. For example, "indirect" attachment may be achieved through linker molecules. Suitable linker molecules include components of biological binding pairs which bind with high affinity, for example biotin/streptavidin or biotin/avidin.
  • the catalytic and substrate labels will be attached to the binding entities/interacting molecules at the start of the reaction, at least before the binding of the binding entities to the target molecule.
  • the binding entities will be supplied pre-labelled with catalytic and substrate labels, or else the labels will be attached in a separate reagent labelling step.
  • the possibility of attaching catalytic and/or substrate labels to the binding entities during the detection reaction itself, i.e. following binding of the binding entities to the target is not excluded.
  • the "target detection method” may be used to detect essentially any target molecule for which it is desired to develop a specific target detection method.
  • the target molecule may comprise a single molecule, a multimer, aggregate or molecular collection or complex.
  • a multimer will generally comprise a number of repeats of a single molecule linked together through covalent or non-covalent interactions.
  • a complex will generally consist of different molecules interacting through covalent or non-covalent interactions.
  • binding entities are defined as any molecule that can bind specifically to a target molecule. Binding entities include, for example, antibodies, lectins, receptors, transcription factors, cofactors and nucleic acids, and fragments thereof which retain target-specific binding activity (e.g. Fab fragments) . This list is merely illustrative and is not intended to be limiting to the invention.
  • the binding entities may bind different regions of a single target molecule.
  • the catalytic and substrate labels will be brought into close proximity when the binding entities bind to their respective regions of the target molecule.
  • the binding entities may bind to equivalent binding sites on the monomeric components of the multimer or units making up the aggregate.
  • the "target detection method” of the invention may be adapted for the detection of essentially any “target molecule” for which suitable "binding entities" of the required specificity are available.
  • the “sample” to be tested using the method may be essentially any material which permits the specific binding reactions that are essential to the operation of the target detection method.
  • target detection method is of use in all areas of technology where it is desirable to provide specific detection of target molecules, in particular target biological molecules such as proteins, nucleic acids, carbohydrates, etc.
  • target biological molecules such as proteins, nucleic acids, carbohydrates, etc.
  • One important area of application of the target detection method is in the field of clinical diagnostics.
  • sample will be a sample of biological fluid, e.g. whole blood, serum, plasma, urine etc, taken from a human patient. Other important applications may include the field of environmental testing and monitoring.
  • the "interaction method” may be used in essentially any field of technology where it is desired to monitor interactions between molecules, and particularly interactions between biological molecules .
  • the interaction method may be used in proteomics in order to investigate molecular interactions.
  • a first interacting molecule may be labeled with either the catalytic or the substrate label, and a library of molecules which may potentially interact with the first interacting molecule may then each be labeled with the other label type. If an interaction occurs between the first interacting molecule and a component from the library of molecules, this brings the catalytic and substrate labels into close proximity, thus allowing interaction to generate a change in structure and/or activity of the substrate label, which can be detected in order to identify interacting partners .
  • a further application is in the field of drug discovery.
  • the interaction method may be used to study interactions between particular combinations of molecules and to identify potential inhibitors or enhancers of molecular interactions. Potential inhibitors of a given interaction could be identified by screening for the ability to reduce the signal detected following interaction of catalytic and substrate labels brought into close proximity by interaction between the interacting molecules.
  • the "interacting molecules” may be essentially any combination of interacting molecules which it is desired to study. These may be, for example, subunits of a multi-subunit complex, a pair of monomers making up a dimer, a ligand and receptor, an enzyme and substrate or inhibitor, etc.
  • the “interaction method” differs from the target detection method only in that the catalytic and substrate labels are attached to the interacting molecules which it is desired to evaluate, rather than to binding entities capable of binding to a target molecule.
  • the interaction method may therefore incorporate analogous features to those described above in connection with the target detection method, as would be apparent to the skilled reader.
  • the invention also relates to reagent kits suitable for use in carrying out the target detection method or the interaction method of the invention.
  • Reagent kits suitable for use in carrying out the "target detection method” may comprise a first binding entity labelled with a catalytic label and a second binding entity labelled with a substrate label, characterised in that the catalytic label is capable of acting directly on the substrate label to generate a detectable change in the substrate label.
  • Reagent kits suitable for use in carrying out the "interaction method” may comprise a first interacting molecule labelled with a catalytic label and a second interacting molecule labelled with a substrate label, characterised in that the catalytic label is capable of acting directly on the substrate label to generate a detectable change in the substrate label.
  • the reagent kits may incorporate any of the preferred features mentioned in connection with the target detection and interaction methods. Preferred combinations of catalytic and substrate labels are as listed above in the description of the target detection and interaction methods.
  • Reagent kits may further include supplies of suitable reaction buffer (s) and also reagents required for detection of the "detectable change" brought about by action of the catalytic label on the substrate label.
  • suitable reaction buffer s
  • reagents required for detection of the "detectable change” brought about by action of the catalytic label on the substrate label.
  • the kit may include assay reagents for use in measuring this enzymatic activity.
  • the kit may also include reagents required for the amplification reaction, for example: primer sets, amplification enzymes, probes for detection of the amplification product (including probes labelled with fluorescent or other revealing labels) , positive control amplification templates, reaction buffers etc.
  • the invention still further provides a reagent labelling kit comprising a catalytic label and a substrate label, characterised in that the catalytic label is capable of acting directly on the substrate label to generate a detectable change in the substrate label, and means for attaching the catalytic label and substrate label to interacting molecules or to binding entities.
  • the means for attaching the tags to interacting molecules or binding entities may be a chemical reagent capable of cross-linking the catalytic label and/or the substrate label to a binding entity or interacting molecule.
  • the "means for attaching the tags" may be an indirect linkage.
  • Preferred types of indirect linkage are provided by components of a biological binding pair, for example biotin/avidin or biotin/streptavidin.
  • the catalytic label and/or substrate label is conjugated with one half of the biological binding pair, enabling linkage to a binding entity or interacting molecule conjugated to the other half of the biological binding pair.
  • the kit may contain a supply of pre-conjugated catalytic and substrate labels, or may include tags which have not yet been conjugated together with means for conjugating the catalytic or substrate labels with half of the binding pair.
  • the kit will further include either binding entity or interacting molecule pre-conjugated with the other half of the binding pair, or else means for conjugating a binding entity or interacting molecule of choice to the other half of the binding pair.
  • the means for attaching half of the biological binding pair to a binding entity or interacting molecule may (depending on the nature of the binding pair) be a chemical cross-linking reagent. However, it may comprise an expression vector which can be used to express the binding entity or interacting protein as a fusion protein, either as a direct fusion with the other half of the binding pair or as a fusion with a polypeptide tag which enables attachment of the other half of the binding pair.
  • vectors for the expression of biotinylated fusion proteins are known in the art and are commercially available (for example the PinPoint vector system from Promega, Madison, WI, USA) .
  • the reagent labelling kit may contain a supply of such a vector, which enables expression of biotinylated binding entities/interacting molecules proteins, plus streptavidin conjugated catalytic and/or substrate labels .
  • Figure 1 is a schematic illustration of a "target detection” assay according to the invention.
  • Figure 2 is a schematic illustration of a further "target detection” assay according to the invention.
  • Example 1 Demonstration of detectable DNA modification by close proximity approach of a binding entity modified with a DNA substrate and a binding entity modified with an enzyme
  • This experiment illustrates that DNA substrates on one binding entity can be modified in a detectable manner by an DNA-specific enzyme on another binding entity under conditions such that the binding entitles are brought into close proximity by binding to a target molecule (see Figure 1) .
  • a double stranded piece of DNA with a single-stranded overhang is linked to a polyclonal anti-hepatitis C core antibody through a biotin-streptavidin bridge.
  • Another antibody with specificity for the core is labelled with T4 DNA ligase.
  • the target antigen Hepatitis C virus core protein
  • the target antigen Hepatitis C virus core protein
  • the labelled antibodies under conditions which allow antibody binding but which do not allow the enzyme on one antibody to modify the DNA on the other.
  • After binding of the antibody conjugates and removal of excess conjugate by washing a DNA oligomer with a complementary single-strand sequence to that in the antibody/DNA conjugate is added in buffer conditions that allow hybridization. If the two antibodies labelled with the DNA and the ligase respectively are in close proximity then the hybridised DNA strands can be ligated to form a stable double stranded DNA molecule which can then be detected by nucleic acid amplification methods, such as PCR.
  • 322S1 was synthesized with a biotin at the 5 ' end to allow conjugation to streptavidin whereas 322AS1 was synthesized with a phosphate at the 5' end to allow subsequent ligation.
  • oligos were mixed at a 50pmol/ ⁇ l concentration in lO M Tris pH 8.0, ImM EDTA, 150mM NaCI and incubated at 37°C for 1 hour.
  • the double-stranded DNA product was complexed with streptavidin to form a conjugate with a 1:1 streptavidin/DNA molar ratio.
  • the reaction was incubated at room temperature (22°C) for 1 hour to allow the complex formation.
  • This double-stranded DNA-streptavidin complex was further complexed to anti-hepatitis C core antibody by adding biotinylated antibody (biotinylated according to standard biotinylation protocols) at a 1:1 streptavidin/antibody molar ratio. The reaction was incubated at room temperature (22°C) for 1 hour to allow the final conjugate formation. In this example the conjugate was then used in the assay procedure without prior removal of any free streptavidin or antibody.
  • biotinylated antibody biotinylated according to standard biotinylation protocols
  • T4 DNA ligase was obtained from Sigma (Poole, UK) and linked to the anti-hepatitis C core antibody using the heterobifunctional reagent SMCC (Pierce Co) following the supplier's recommended standard chemical conjugation procedures. A molar ration of 2 : 1 ligase to antibody was used in the coupling procedure. In this example the conjugate was used in the assay procedure without prior removal of any free ligase or antibody.
  • 322S2 was synthesized with a phosphate at the 5' end to allow subsequent ligation.
  • oligos were mixed at a 50pmol/ ⁇ l concentration in lOmM Tris pH 8.0, ImM EDTA, 150mM NaCI and incubated at 37°C for 1 hour.
  • Microwells in a 96 well microplate were coated with anti-hepatitis C core antibody at 10 ⁇ g/ml in 50 mM carbonate buffer pH 9.0 (see A in Figure 1).
  • the primers 322PCRAS 5'ACGGGTGCGC ATAGAAATTG CATC3 ' and 322PCRS 5'GCGGGATATC GTCCATTCCG ACAG3 ' were used, which amplify across the junction at which the two DNA fragments have been ligated.
  • the amount of DNA amplified in the PCR and the time/cycle of PCR at which the PCR becomes positive is related to the number of ligated DNA fragments present in the reaction, which in turn is related to the number of antibody conjugates brought into close proximity and is therefore a measure of the number of HCV core antigen molecules captured.
  • the time and number of cycles at which the PCR became positive was related to the amount of antigen captured in the well. As little as 0.1 fg of HCV core antigen target could be detected by this method.
  • This experiment shows that antibody conjugates labelled with DNA substrate and a DNA-dependent enzyme that modifies the DNA in a detectable manner can be used to detect antigen in a sensitive and specific manner .
  • This experiment illustrates that two DNA substrates on one binding entity can be modified in a detectable manner by a DNA-specific enzyme on another binding entity under such conditions such that the binding entities are brought into close proximity by binding to a target molecule (see Figure 2) .
  • a target-specific antibody a polyclonal anti-hepatitis C core antibody
  • Another target specific antibody is labelled with T4 DNA ligase.
  • the target antigen is immunocaptured to a solid phase and then incubated with the labelled antibodies under conditions which allow antibody binding but do not allow the enzyme on one antibody to modify the DNA on the other.
  • the buffer conditions are then changed to allow hybridisation. If the two antibody conjugates (conjugated to the two DNA oligomers and the ligase respectively) are in close proximity the two DNA strands can be ligated to yield a stable double- stranded DNA product which can then be detected by nucleic acid amplification methods such as PCR.
  • 322S1 was synthesized with a biotin at the 5 ' end to allow conjugation to streptavidin whereas 322AS1 was synthesized with a phosphate at the 5' end to allow subsequent ligation.
  • oligos were mixed at a 50pmol/ ⁇ l concentration in lOmM Tris pH 8.0, ImM EDTA, 150mM NaCI and incubated at 37°C for 1 hour.
  • 322S2 was synthesized with a phosphate at the 5' end to allow subsequent ligation.
  • oligos were mixed at a 50pmol/ ⁇ l concentration in lOmM Tris pH 8.0, ImM EDTA, 150mM NaCl and incubated at 37°C for 1 hour.
  • T4 DNA ligase was obtained from Sigma (Poole, UK) and linked to the anti-hepatitis C core antibody using the heterobifunctional reagent SMCC (Pierce Co) following the supplier's recommended standard chemical conjugation procedures. A molar ration of 2:1 ligase to antibody was used in the coupling procedure. In this example the conjugate was used in the assay procedure without prior removal of any free ligase or antibody.
  • Microwells in a 96 well microplate were coated with anti-hepatitis C core antibody at 10 ⁇ g/ml in 50 m carbonate buffer pH 9.0 (see A in Figure 2).
  • Serial dilutions of recombinant core antigen were made in PBS buffer pH 7.2, 0.1% (v/v) Tween 20, ImM EDTA and lOO ⁇ l of each dilution incubated in a coated well for 60 min to allow capture (see B in Figure 2) .
  • This experiment shows that antibodies labelled, respectively, with two DNA substrates and a DNA-dependent enzyme that modifies the DNA in a detectable process can be used to detect antigen in a sensitive and specific manner.

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Abstract

L'invention concerne des procédés et des kits permettant de détecter des molécules cibles ou de contrôler des interactions entre celles-ci. L'invention concerne en particulier des procédés fondés sur l'utilisation de systèmes de «conjugué à deux composants» comprenant un marqueur catalytique et un marqueur substrat.
EP03732677A 2002-05-30 2003-05-30 Procedes de detection de molecules cibles et d'interactions moleculaires Ceased EP1508045A1 (fr)

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GBGB0212544.1A GB0212544D0 (en) 2002-05-30 2002-05-30 Methods for detection of target molecules and molecular interactions
PCT/GB2003/002383 WO2003102590A1 (fr) 2002-05-30 2003-05-30 Procedes de detection de molecules cibles et d'interactions moleculaires

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ATE431854T1 (de) * 2003-06-27 2009-06-15 Nanosphere Inc Auf bio-barcodes beruhender nachweis von zielanalyten
JP2007537450A (ja) * 2004-05-12 2007-12-20 ナノスフェアー　インコーポレイテッド バイオバーコードに基づく標的検体の検出
WO2006104979A2 (fr) * 2005-03-29 2006-10-05 Nanosphere, Inc. Methode de detection d'un analyte cible
GB0906643D0 (en) * 2009-04-17 2009-06-03 Wilson Stuart M Detection of bacteria and fungi
WO2017200070A1 (fr) * 2016-05-19 2017-11-23 凸版印刷株式会社 Procédé et kit de détection de molécule cible
US20230088664A1 (en) * 2020-05-29 2023-03-23 Siemens Healthcare Diagnostics Inc. Method of Detecting Analytes in a Sample

Citations (2)

* Cited by examiner, † Cited by third party
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US4275149A (en) * 1978-11-24 1981-06-23 Syva Company Macromolecular environment control in specific receptor assays
US5196306A (en) * 1989-03-29 1993-03-23 E. I. Du Pont De Nemours And Company Method for the detection or quantitation of an analyte using an analyte dependent enzyme activation system

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US3996345A (en) * 1974-08-12 1976-12-07 Syva Company Fluorescence quenching with immunological pairs in immunoassays
US4820630A (en) * 1984-11-23 1989-04-11 Digene Diagnostics, Incorporated Assay for nucleic acid sequences, particularly genetic lesions, using interactive labels
HU204559B (en) * 1987-08-26 1992-01-28 Akad Wissenschaften Ddr Process for producing molecular proobes connected with enzymes and detecting biomolecules with them
US5925517A (en) * 1993-11-12 1999-07-20 The Public Health Research Institute Of The City Of New York, Inc. Detectably labeled dual conformation oligonucleotide probes, assays and kits
GB9400300D0 (en) * 1994-01-10 1994-03-09 Celsis Ltd Hybridisation assay
SE516272C2 (sv) * 2000-02-18 2001-12-10 Ulf Landegren Metoder och kit för analytdetektion mha proximitets-probning

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4275149A (en) * 1978-11-24 1981-06-23 Syva Company Macromolecular environment control in specific receptor assays
US5196306A (en) * 1989-03-29 1993-03-23 E. I. Du Pont De Nemours And Company Method for the detection or quantitation of an analyte using an analyte dependent enzyme activation system

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* Cited by examiner, † Cited by third party
Title
See also references of WO03102590A1 *

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CA2487954A1 (fr) 2003-12-11
WO2003102590A1 (fr) 2003-12-11
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