WO2011051709A1 - Test de ligature de proximité impliquant la génération d'une activité catalytique - Google Patents

Test de ligature de proximité impliquant la génération d'une activité catalytique Download PDF

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Publication number
WO2011051709A1
WO2011051709A1 PCT/GB2010/051797 GB2010051797W WO2011051709A1 WO 2011051709 A1 WO2011051709 A1 WO 2011051709A1 GB 2010051797 W GB2010051797 W GB 2010051797W WO 2011051709 A1 WO2011051709 A1 WO 2011051709A1
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Prior art keywords
assay according
assay
message
catalytic
catalytic activity
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PCT/GB2010/051797
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English (en)
Inventor
Adrian Smith
Prabhjyot Dehal
Lars Gøte Ingemar ØRNING
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Axis-Shield Diagnostics Limited
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Priority to CN2010800467606A priority Critical patent/CN102695803A/zh
Priority to EP10787529A priority patent/EP2494064A1/fr
Publication of WO2011051709A1 publication Critical patent/WO2011051709A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/901—Antibodies with enzymatic activity; e.g. abzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/9015—Ligases (6)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/10—Oligonucleotides as tagging agents for labelling antibodies

Definitions

  • the present invention relates to an assay which detects a recognition event through generation of catalytic activity.
  • immunoassays are well known in the art as methods for detecting and measuring the concentration of analytes in biological samples and other samples.
  • Immunoassays utilise the specific binding of an antibody or other recognition molecule to its antigen to detect an analyte, wherein the analyte may comprise either the antibody or antigen or both. The presence of one or both of the antigen or antibody may then be measured to quantitatively determine the concentration of analyte. In this way it is possible to detect and measure extremely low concentrations of analytes.
  • Immunoassays may be conducted in various manners and formats. Of all the different formats for the application of immunoassays, probably the most widely used are those that employ solid phase separation using immobilised antibodies and/or antigens. This is known in the art as a heterogeneous immunoassay. Separation of immobilised antibodies and/or antigens is needed to effectively measure only the material recognised as the analyte of interest, wherein the solid phase serves as a physical binder which allows washing steps to be performed to remove non-target material. This washing procedure separates the analyte to be measured from the other constituents of complex mixtures that comprise the samples.
  • a detection system In order to detect and measure the concentration of analyte in an immunoassay, a detection system is required. For example, an enzymatic assay may be used, wherein enzymatic action turns an undetectable bound analyte into a detectable and quantitatively measurable signal.
  • Other means for detecting antibody and/or antigen may be achieved by labelling the antibody and/or antigen with detectable labels. Such labels could include colloidal gold labels, radioisotope labels, magnetic labels and fluorescent labels.
  • homogeneous immunoassays An alternative method, used to overcome some of the problems of complexity associated with heterogenous immunoassays, is so-called homogeneous immunoassays. These homogeneous immunoassays may be performed in aqueous solution using one or more additions of reagents but without the need for separation and wash steps. These homogeneous immunoassays have been seen to benefit from higher precision and sensitivity of measurement and make a valuable contribution to the field of analyte measurement.
  • EMIT Enzyme Multiplied Immunoassay Technique technology
  • CEDIA Cloned Enzyme Donor Immunoassay
  • FRET Forster Resonance Energy Transfer
  • chemiluminescent or bioluminescent reporting which have the potential to be very sensitive.
  • PKA Proximity Ligation Assay
  • the product of this operation is a growing strand of DNA of repetitive sequence emanating from each of the assembled analyte- antibody complexes.
  • the incorporation of fluorescent tags into the growing DNA polymer allows for the quantitative measurement of the amount of ligated primer and hence also the quantity of analyte present in the sample.
  • Homogeneous immunoassays could have the potential to be useful in clinical immunoassays.
  • Homogeneous immunoassays can have advantages over heterogeneous immunoassays. However, they also have limitations.
  • the present invention provides a homogenous assay comprising :
  • the present invention also enables disadvantages associated with systems that rely on transcription of a nucleic acid sequence followed by translation to form working enzyme protein through in-vitro protein synthesis to be addressed, wherein the likely complexity in terms of numbers of reagents, timings and potential reagent incompatibilities would be impractical.
  • a coloured product may be produced by the assay which may be measured on clinical chemistry analysers that at the moment are readily available in clinics.
  • the homogenous nature of the assay provides a relatively quick assay because it does not require removal or separation of any substrates.
  • this assay unlike PLA and other methods that rely on nucleotide amplification and fluoroprobes for signal generation (which can require 20- 40 cycles of up to two minutes, as in real time polymerase chain reaction (PCR), to reach a predetermined background signal), has an extra enzyme step whereby it is considered to enable an additional several fold increase in sensitivity, and thus requires fewer amplification cycles to reach the background signal.
  • PCR polymerase chain reaction
  • the assay comprises only two molecules each comprising a recognition component and a partial message.
  • the materials required to perform the assay may be minimised. It will be apparent that the appropriate number of such molecules may be dependent on the nature of the sample that is being assayed and what it is desired to detect.
  • the recognition component is selected from a protein, an antibody, an antibody fragment, molecular imprinted polymer, a complementarity determining region, oligopeptide, olgionucleotide, small organic chemical, peptide, polypeptide, polynucleotide or aptamer.
  • each molecule may be the same or different.
  • they may be the same antibody to the same protein, e.g. one that is part of a multimeric complex or a cell surface component.
  • they could as an example be an antigen for, and antibody directed to, a target antibody of interest .
  • a continuous message results from the partial messages becoming linked or connected in some way.
  • the connection/linkage of the partial messages may be direct or there may be (a) further, intervening message molecule or molecules.
  • the continuous message whose formation is enabled by the partial messages of the molecules comprising recognition components may further comprise one or more linking partial messages. Therefore, optionally the forming of the continuous message is by virtue of the addition of one or more linking partial messages.
  • the partial messages comprise or may be formed from nucleic acid (e.g. DNA or RNA) . They may be of the same or different form.
  • the partial messages are formed from ssDNA but could also be an analogue such as PNA (peptide nucleic acid) .
  • partial messages formed from polynucleotides can be induced to anneal/hybridise to each other under appropriate conditions to enable formation of a continuous message.
  • the continuous message is amplified by isothermal amplification.
  • This form of amplification through avoiding thermal cycling, reduces the steps required to be taken by the person performing the assay and means that the assay may be much more easily conducted without expensive specialised equipment.
  • the isothermal amplification is rolling circle amplification. Rolling circle amplification can be more sensitive and faster then other forms of amplification. Further, it does not require thermal cycling which is not possible in most current clinical chemistry analysers.
  • the assay further comprises a ligation step prior to amplification of the continuous message.
  • a message comprising a nicked sequence to be amplified may be more easily used in the assay.
  • the continuous message is ssDNA comprising a non- circularised message component
  • a standard DNA ligation reaction may be used to circularise the relevant component.
  • a ligation step may be used to circularise the continuous message and enable amplification. It would be apparent to a person skilled in the art how ligation may be performed.
  • the effect of the catalytic entity directly or indirectly on the substrate results in a visible detection product.
  • This visible detection product can then either be viewed without the aid of a machine by the person performing the assay or it can be analysed by a suitable machine (e.g. a spectrophotometer or a colorimetric assay machine) .
  • a suitable machine e.g. a spectrophotometer or a colorimetric assay machine
  • catalytic formation of fluorescent or chemiluminescent products may also be included, by the use of appropriate substrates.
  • the detection product is luminescent, either by fluorescence, phosphorescence or chemiluminescence .
  • the visible detection product is coloured.
  • Enzymes that have been found to be useful in generation of coloured products include ⁇ -galactosidase and ⁇ -lactamase, where an appropriate chromogenic substrate is added to reveal the extent of catalytic activity, which in turn will relate to the quantity of analyte in solution.
  • the catalytic entity comprises DNA (either ssDNA or dsDNA) , RNA or a polypeptide.
  • the catalytic entity can be DNA (either ssDNA or dsDNA) , RNA or a polypeptide. If the catalytic entity is a polypeptide translation of the continuous message if it is DNA or RNA, will need to take place.
  • the assay further comprises at least two complementary catalytic activity subunits which are not in functioning proximity.
  • the subunits might not be in functioning proximity because they are kept spaced apart by an appropriate spacing component (e.g. polypeptide or polynucelotide ) or because they require a suitable linking component or have limited natural affinity for each other.
  • an appropriate spacing component e.g. polypeptide or polynucelotide
  • the at least two complementary catalytic activity subunits are brought into functioning proximity by the catalytic entity. If the subunits are kept apart by an appropriate spacing component, the catalytic entity acts as a ligature thereby drawing the subunits together. If the subunits require a suitable linking component, the catalytic entity acts as a linking component.
  • catalytic activity generation may occur by the labelling of the inactive subunits with lengths of complementary ssDNA such that the required subunits affix to the catalytic entity. In this manner, each catalytic entity will trigger the possibility of a new enzyme unit through a proximity binding occurrence (see figure 4 for illustration) .
  • the intrinsic mutual affinity of each of the complementary catalytic activity subunits of the catalyst is low, so that the new generation of catalytic activity is driven, in the limit, entirely by analyte presence rather than by a default background recombination of the individual catalytic subunits.
  • this embodiment results in guided assembly of incomplete catalytic units to generate de novo catalytic activity.
  • the at least two complementary catalytic activity subunits are an apoenzyme and cofactor.
  • the catalytic entity is a DNAzyme.
  • a DNAzyme would require no translation or transcription to produce a catalytic entity thereby reducing the substrates required in the assay and the time taken to first result.
  • the DNAzyme comprises a G-quadruplex or is G-quadruplex based. Accordingly, preferably the continuous message comprises an anti-sense message for G-quadruplex DNA.
  • the DNAzyme is G-quadruplex.
  • G-quadruplex Various other DNAzymes may be used, but sequences forming G-quadruplex units are especially suitable as these are of low molecular weight and can therefore easily be accommodated within the limited confines of the circular DNA of the RCA (rolling circle amplification) template.
  • the repetitive formation of the said sequence should result in repeated self-assembly of G-quadruplex units, interspersed with linking sections. It has recently been found that the G-quadruplex units actively bind added hemin molecules, the product of which is an efficient peroxidase activity.
  • a DNAzyme that comprises one or more G-quadruplexes that bind hemin to give peroxidase activity may consequently alternatively be thought of as an apo-DNAzyme, and hence hemin may be thought of as a co-factor.
  • the catalytic activity is peroxidase activity .
  • hemin is introduced to the assay.
  • Conveniently hemin and substrate solution may be exposed to the G-quadruplex .
  • G-quadruplex in the presence of hemin has a peroxidase activity and a substrate such as ABTS (2,2 azinobis [ 3-ethylbenzothiazoline-6 sulfonic acid]) or TMB ( tetramethylbenzidine) will produce a visible colour which can be detected.
  • the detected colour will have a linear relationship to the recognition event.
  • the substrate comprises ABTS and/or TMB.
  • the partial messages comprise any one or more of the nucleotide sequences of SEQ ID Nos 1, 2 and 3 or a homologue or functional fragment thereof.
  • one partial message may comprise the nucelotide sequence of SEQ ID No 1 while another partial message comprises the nucelotide sequence of SEQ ID No 3 or SEQ ID Nos 2 and 3, or a homologue or functional fragment thereof.
  • one partial message may consist of the nucelotide sequence of SEQ ID No 1 while another partial message consists of the nucelotide sequence of SEQ ID No 3 or SEQ ID Nos 2 and 3, or a homologue or functional fragment thereof .
  • the assay system of the present invention enables greater sensitivity and hence shorter assay processing times than current protocols, e.g. proximity ligation assays, whereby, for example, in measuring incorporation of a fluorophore there is not a latter additional enzymatic amplification step .
  • a method for detecting an analyte in a sample comprising an assay as described herein wherein the recognition event occurs, either directly or indirectly, between the at least two molecules and the analyte .
  • the analyte is a protein, peptide, polypeptide, antigen antibody, antibody fragment or metabolite of interest.
  • Figure 1 illustrates an example target (analyte) /recognition molecule/continuous message structure.
  • the target (analyte) molecule for measurement in the sample is represented by Streptavidin
  • the recognition components of the (at least) two molecules are each represented by Biotin
  • one of the partial messages is represented by the anchor and template complex (or could otherwise be thought of as the template or relevant part of the template)
  • the other partial message is represented by the primer (or could otherwise be thought of as the relevant part of the primer) .
  • Figure la the partial message (component) that is represented by the template is not circularised (it is nicked) .
  • Figure lb depicts a slightly different situation wherein the template is joined (circularised) instead of nicked.
  • Figures 2 and 3 show typical results from an assay of the present invention measuring the presence of Streptavidin in a sample.
  • the figures show that different concentrations of Hemin (in these cases ⁇ in figure 2 and 47 ⁇ in figure 3) can result in different assay sensitivities.
  • the X-axes show the relevant streptavidin concentrations studied (in pmol) and the Y-axes show the % change in absorbance.
  • the change in absorbances shown in Figures 2 and 3 relate to a mean relative change across the relevant samples in comparison to the mean absorbance obtained for controls where no Streptavidin was present.
  • Figure 4 illustrates the guided assembly of inactive subunits to give an active de novo enzyme.
  • Example 1 Demonstration of Homogeneous Assay Format
  • Primer and anchor DNA conjugated to Biotin were designed for the purposes of this example and can be obtained from commercial sources from companies such as IDT.
  • the template DNA was designed/selected for the purposes of this example.
  • the template sequence comprises an anti-sense message for G-quadruplex DNA.
  • the template DNA in this example had a 3' phosphate to minimise spontaneous circularisation .
  • Those skilled in the art would realise that the phosphorylation is not always necessary and, further, it may be desirable to have a circularised template, as discussed below.
  • Products having a structure akin to the above primer conjugated Biotin and anchor conjugated Biotin molecules may in themselves each represent a molecule comprising a recognition component (e.g. biotin) and a partial message (e.g. anchor/primer).
  • either of the molecules comprising a recognition component and a partial message may be more extensive (as described herein in relation to anchor/template-complex conjugated biotin), whereby the partial message may be thought of as comprising more than one component, e.g. the anchor and template.
  • the template could be thought of as a partial message itself.
  • template DNA diluted to 2pmol/ l in water was mixed with equal volumes of biotin conjugated anchor and incubated at 70°C for a minute (an alternative suitable temperature to reduce potential secondary structure formation but not denature the recognition element can be used) .
  • the mixture was then cooled to facilitate template and biotin conjugated anchor binding/hybridistion . This resulted in formation of a stable biotin anchor-template DNA complex.
  • Streptavidin representing a target within a sample to be detected, was diluted to various concentrations between 0 to 50pmol.
  • ⁇ aliquots of streptavidin were added to reaction tubes. To these reaction tubes was also added 20 ⁇ 1 of the biotin-anchor/template DNA complex. The reaction mixtures were then incubated at room temperature for 5 minutes to allow the biotin-anchor/template DNA complex to bind the streptavidin.
  • the template DNA could be circularised by the ligation of its 3' and 5' ends, or not (as is represented by figure la/b) .
  • a ligation step (such as that described below) may be appropriate, depending on the nature of the partial messages selected.
  • a ligation step could be avoided. In this example however a ligation step was used.
  • ⁇ ligase mix (5 ⁇ 1 ligase buffer + 0.5 ⁇ 1 (1000 units) T4 DNA ligase (e.g. New England Biolabs M0202) + 4.5 ⁇ 1 water) was added to each reaction tube and the reaction mixtures were incubated at room temperature for 2 to 10 minutes to allow nicks between relevant polynucleotide ends
  • streptividin/biotin binding partnership is used as an exemplar for the purposes of the present example where detection of streptavidin as a target analyte is to be shown, but it will be understood by the skilled person that the recognition event to be detected may occur equally between any other binding partnership, such as antibodies (or fragments etc of antibodies) and proteins/peptides etc, so that a target analyte of interest is detected. Further, the recognition components involved in an assay may be different such that there are different binding interactions occurring. [0080] Amplifying the continuous message to form a catalytic entity
  • polymerase master mix (note heat inactivated polymerase was used as a control) was added to each reaction tube. The reaction mixture was then incubated at 30 °C for 30 to 270 minutes to allow the message to amplify.
  • the polymerase master mix consisted of ⁇ (10 units) phi29 DNA polymerase (e.g. as supplied by New England Biolabs; M0269), ⁇ polymerase buffer, 5 ⁇ 1 dNTPs and 3 ⁇ 1 water.
  • the template DNA sequence comprised an anti-sense message for G-quadruplex DNA.
  • the continuous message of a complex of the form of that shown in figure la/b was amplified by rolling circle amplification to generate a continuous DNA polynucleotide which spontaneously folds to form tandem G-quadruplex structures.
  • the incorporation of added hemin into the G-quadruplexes results in a structure with peroxidase activity.
  • partial messages of varying sequences to those indicated in this example could be used.
  • the template message component could be varied to produce different G- quadruplex DNA.
  • Hemin stock solution was prepared by dilution in DMSO to 0.5mM. A further dilution in hemin solution ( 0.1M Tris-HCl, 20mM MgCl 2 , 0.05% Triton X-100) to a concentration of 330 ⁇ was then prepared. An appropriate volume of 330 ⁇ hemin was then added to the reaction tube to give the final concentration of hemin as desired (in this instance ⁇ or 47 ⁇ ) . Experiments using different concentrations of hemin were performed to alter the assay range. The reaction mixture was then incubated at room temperature for up to 2 hours before transferring to a 96 well microtitre plate and adding ⁇ ; TMB substrate solution.
  • the assay principle is that G-quadruplex complexes formed by amplification, that is enabled when a continuous message comprising an anti-sense G-quadruplex sequence is formed, give rise, in the presence of hemin, to peroxidase activity that metabolises TMB to produce a coloured product that can be detected by measuring absorbance.
  • the method has been set out in a staged manner, it will be apparent to the skilled person that various steps could be performed concurrently in order to reduce the time taken to first result. Further, the assay may be performed in the same vessel used for detecting the recognition event.
  • Example 2 Immunocomplex formation with ssDNA-tagged binding partners
  • a cyclic peptide (according to Structure 1 below) known to be immunoreactive against autoantibodies, the presence of which is diagnostic of early rheumatoid arthritis, and possessing a modified ssDNA sequence on the N-terminus was used as one of the two binding partners in this work. Addition of a 22 pM solution of this peptide to a serum sample containing the antibody, from a known positive sample, caused binding to the antibody of the sample as measured by immunoassay after incubation for 10-12 minutes.
  • a further binding partner a solution containing a large molar excess of a modified anti-human IgG specific for the Fc region (obtained from Serotec) was added to the mixture, and allowed to incubate for 15-20 minutes at room temperature.
  • This antibody was modified by covalent linkage with a ssDNA strand on its Fc portion.
  • Verification of the ternary complex was obtained by immunoassay utilising anti-murine IgG specific antibodies bound to the surface of magnetic beads, which when separated from the mixture and allowed to react with alkaline phosphatase bearing covalent ssDNA strand complementary to that of the ssDNA on the cyclic peptide. With subsequent exposure after washing with 1.5 mM p-nitrophenylphosphate in 100 mM diethanolamine buffer, coloured product was developed and the signal read at 450 nm, showing positive binding against a control containing no positive serum.
  • X NH 2 , CH 3 , HCH 3 or N(CH 3 ) 2 ;
  • X citrulline
  • Structure 1 The top structure is a representation of the modified side chain of the arginine (X) seen in the amino acid sequence.
  • Example 3 Demonstration of proximal placement of ssDNA sequences and ligation.
  • the ligation mixture contained ligase enzyme, a ligation template, and ATP.
  • ligase enzyme Ligation mix: volume 5 L, 50 mM KC1, 10 mM Tris-HCl pH 8,3, 3 mM MgCl 2 , 0.8 mM ATP, 20 nM ligation template, 3 Weiss units T4 DNA ligase.
  • PCR PCR mix(TaqMan PCR) : volume 30 L, 50 mM KC1, 10 mM Tris-HCl pH 8.3, 0.4 mM MgCl 2 , 0.33 mM dNTPs , lx buffer A, 1 ⁇ forward primer, 1 ⁇ reverse primer, 83 nM TaqMan probe, 1.56 Units AmpliTaq Gold polymerase (Perkin- Elmer) .
  • the amplification/detection mix contains PCR primers, TaqMan probe, dNTPs and DNA polymerase.
  • Amplification detection mix Probe Ale (SEQ ID NO:4)
  • Probe A2c (SEQ ID NO: 5)
  • Fwd primer (SEQ ID NO: 7; nucleotides 44-65 of SEQ ID NO:4): ATGTGGTCTATGTCGTCGTTCG
  • Rev primer (SEQ ID NO: 8; reverse complement of nucleotides 22-41 of SEQ ID NO : 5 ) :
  • Example 4 Competitive Proximity-probing for Analytes with Only One Binding Site
  • an analyte only has one binding moiety. This can be achieved by using a competitive assay.
  • a purified amount of the analyte itself is conjugated to a nucleic acid and the one existing binding moiety is conjugated with the other reactive nucleic acid.
  • the non- conjugated analyte of unknown amount in the sample will compete for binding to the binding moiety of the proximity- probe thereby decreasing the probability of the conjugated nucleic acids reacting.
  • the signal from the reaction is in this case inversely proportional to the analyte concentration .
  • Example 5 Alternative material preparation and procedural methods .
  • Circular DNA template preparation A circular DNA template was prepared as follows: First, the linear DNA ( 5 ' -GATCCTAACCCAACCCGCCCTACCCAAAACCCA
  • RCA assay Fixed concentrations of the hairpin 4, 2 x 10 "7 M and the circular DNA 2, 2 x 10 "8 M were employed. Polymerase Klenow exo-, 0.4 units/ ⁇ and dNTPs, 0.2 mM were included. The RCA process was performed in a buffer solution consisting of 50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 1 mM DTT and 50 g/ml BSA.
  • the conditions for the absorbance studies are optionally as follows: The experiment was performed in a solution consisting of the products; hemin, 4 x 10 "7 M; H 2 0 2 , 4.4 x 10 "5 M; ABTS2-, 1.82 x 10 "4 M in a buffer solution consisting of 25 mM HEPES, 20 mM KC1 and 200 mM NaCl, pH 7.4, 25°C. Absorbance changes were followed at 415 nm to characterize the rate of the oxidation of ABTS2-.

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Abstract

La présente invention concerne un dosage homogène comprenant les étapes consistant 1) à mettre en contact au moins deux molécules comprenant chacune un composant de reconnaissance et un message partiel avec un échantillon, moyennant quoi le contact des composants de reconnaissance avec l'échantillon entraîne la formation par les messages partiels d'un message continu ; 2) à amplifier le message continu pour former une entité catalytique générant directement ou indirectement une activité catalytique ; à utiliser un substrat permettant la détection d'un événement de reconnaissance entre lesdites deux molécules et l'échantillon par le biais de l'activité catalytique.
PCT/GB2010/051797 2009-10-26 2010-10-26 Test de ligature de proximité impliquant la génération d'une activité catalytique WO2011051709A1 (fr)

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CN2010800467606A CN102695803A (zh) 2009-10-26 2010-10-26 与催化活性的产生相关的邻位连接测试
EP10787529A EP2494064A1 (fr) 2009-10-26 2010-10-26 Test de ligature de proximité impliquant la génération d'une activité catalytique

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GB0918712A GB0918712D0 (en) 2009-10-26 2009-10-26 An assay

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US10620217B2 (en) 2014-11-25 2020-04-14 Ventana Medical Systems, Inc. Proximity assays using chemical ligation and hapten transfer
WO2021021348A1 (fr) * 2019-07-31 2021-02-04 The Regents Of The University Of California Détection in situ de molécules de signalisation par combinaison de la modification chimique de protéines endogènes et du dosage par ligature de proximité

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CN107991274B (zh) * 2017-10-27 2020-11-24 中国农业大学 一种基于铅的功能核酸的比色传感器及其应用
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