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WO2019104529A1 - 均相检测方法 - Google Patents

均相检测方法 Download PDF

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Publication number
WO2019104529A1
WO2019104529A1 PCT/CN2017/113570 CN2017113570W WO2019104529A1 WO 2019104529 A1 WO2019104529 A1 WO 2019104529A1 CN 2017113570 W CN2017113570 W CN 2017113570W WO 2019104529 A1 WO2019104529 A1 WO 2019104529A1
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Prior art keywords
enzyme
aptamer
analyte
enzymatic reaction
donor
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PCT/CN2017/113570
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English (en)
French (fr)
Inventor
张建亮
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Shenzhen Langji Life Science And Technology Co Ltd
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Shenzhen Langji Life Science And Technology Co Ltd
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Priority to PCT/CN2017/113570 priority Critical patent/WO2019104529A1/zh
Priority to CN201780001782.2A priority patent/CN110100180B/zh
Priority to US16/768,258 priority patent/US20200371108A1/en
Publication of WO2019104529A1 publication Critical patent/WO2019104529A1/zh
Anticipated expiration legal-status Critical
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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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • 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
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • 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
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
    • G01N2333/938—Hydrolases (3) acting on glycosyl compounds (3.2) acting on beta-galactose-glycoside bonds, e.g. beta-galactosidase

Definitions

  • the invention belongs to the field of biological detection technology, and in particular relates to a homogeneous detection method.
  • LETIA latex-enhanced immunoturbidimetric technology
  • CLIA chemiluminescence immunoassay
  • LETIA binds the monoclonal antibody conjugated to the microspheres to the antigen in a short time by the monoclonal antibody cross-linked on the surface of the polymer latex microspheres, thereby changing the absorbance of the reaction solution;
  • the change in absorbance of the reaction solution has a linear relationship with the concentration of the antigen to be tested, and thus can be used to reflect the concentration of the antigen to be tested.
  • CLIA is an immunoassay method for directly labeling antigens or antibodies with chemiluminescent agents with high sensitivity and a good linear range.
  • the chemical small molecule assay technique has a cloned enzyme donor test (cloned enzyme donor).
  • CEDIA Enzyme Amplification Immunoassay
  • EMIT Enzyme Amplification Immunoassay
  • FPI A Fluorescence Polarization Immunoassay
  • CEDIA is produced using recombinant DNA technology (two fragments of 3-galactosidase: a large fragment called an enzyme acceptor (EA), a small fragment called an enzyme donor (ED), two fragments themselves) They are not enzymatically active, but have enzymatic activity when bound together under suitable conditions.
  • EA enzyme acceptor
  • ED enzyme donor
  • the homogeneous enzyme immunoassay established by the characteristics of the two-phase fragments is called a cloning enzyme donor immunoassay.
  • the ED-labeled hapten is bound to the antibody. Later, due to steric hindrance, it can no longer be combined with EA.
  • CEDIA is mainly used for the determination of drugs and small molecules. It also has a patent to link specific epitopes of protein molecules to ED fragments, thus enabling the detection of biological macromolecules.
  • CEDIA technology is currently tested using competition methods. The concentration of the analyte is directly proportional to the enzyme activity.
  • the basic principle of EMI T is that the hapten and the enzyme bind to the enzyme-labeled hapten, retaining the activity of the hapten and the enzyme.
  • the labeled enzyme is in close contact with the antibody, so that the active center of the enzyme is affected and the activity is inhibited.
  • the sample contains the hapten to be tested, it will compete with the ELISA hapten to bind the antibody, so that Inhibition of enzyme activity is restored.
  • EMIT technology is tested using a competition method, and the concentration of the analyte and the enzyme activity are large. ⁇ 0 2019/104529 ⁇ (:17 ⁇ 2017/113570
  • the reagents of the VIII are fluorescein-labeled small molecule analytes and anti-drug antibodies, and the mode is a homogeneous competition method.
  • the principle of fluorescence polarization immunoassay is as follows. After the fluorescent substance is irradiated by a single plane of polarized blue light (wavelength 485!1111), the absorbed light energy jumps into the excited state; when it returns to the ground state, it releases energy and emits a single plane polarization ⁇ . Light (wavelength 52511111). The intensity of the polarization quenching is inversely proportional to the rate at which the twilight material is excited when excited.
  • the macromolecular substance rotates slowly, and the emitted polarized fluorescence is strong; the small molecular substance rotates fast, and its polarization fluorescence is weak.
  • the small molecule to be tested is attached to the fluorescent molecule, and an antibody against the small molecule to be tested is added in the detection environment. When the detection environment contains the small molecule to be tested, the small molecule to be tested is to be tested with the labeled fluorescent molecule. The small molecule competes for binding to the antibody, and a part of the analyte to which the fluorescent molecule is labeled is released, and the intensity of the polarized light in the detection environment is decreased.
  • the method is a competition method test, and the concentration of the analyte is inversely proportional to the intensity of the polarized light. Using this phenomenon, a fluorescence polarization immunoassay is established for the determination of small molecular substances, particularly drugs.
  • the method is a heterogeneous test.
  • the test involves a washing and separating step, the test speed is slow, the test cost is high, and the heterogeneous reaction leads to poor repeatability of the test, and the above method requires the use of antibodies.
  • the object of the present invention is to overcome the above-mentioned deficiencies of the prior art, and to provide a homogeneous detection method, which aims to solve the technical problem that the prior detection method has poor sensitivity and repeatability and high cost, so that its application is limited.
  • the present invention provides a homogeneous detection method, which includes the following steps:
  • the aptamer specifically recognizing the analyte, and the aptamer is linked to the ⁇ 0 2019/104529 ⁇ (:17 ⁇ 2017/113570
  • the homogeneous detection method provided by the present invention is a novel homogeneous non-competition method, which uses an aptamer that can specifically recognize a test substance to label an enzyme, first according to a standard solution of the test object.
  • the enzymatic reaction signal of the enzymatic reaction, the equation of the relationship between the enzymatic reaction signal and the content of the analyte is derived, and then the enzymatic reaction signal of the enzymatic reaction in the sample solution is detected according to the equation, and the sample solution can be calculated.
  • the invention since the enzymatic reaction signal is proportional to the content of the analyte, the content of the analyte can be quickly calculated based on the enzymatic reaction signal in the sample solution. Therefore, compared with the prior art, the invention has the characteristics of high sensitivity, good repeatability, strong anti-interference ability, fast detection speed and low cost, and can detect various biomolecules, and is widely used.
  • Example 1 is a schematic diagram of (:-reactive protein detection in Example 1 of the present invention.
  • Example 2 is a schematic diagram of (:-reactive protein detection) in Example 2 of the present invention.
  • Embodiments of the present invention provide a homogeneous detection method, where the homogeneous detection method includes the following steps: ⁇ 0 2019/104529 ⁇ (:17 ⁇ 2017/113570
  • [0017] 802 configuring different concentrations of the standard solution of the analyte, adding the aptamer-enzyme complex and the substrate of the enzyme to the standard solution of the analyte to perform an enzymatic reaction, and measuring Enzymatic reaction signal, obtaining an equation of the enzymatic reaction signal and the content of the analyte;
  • [0018] 803 adding the aptamer-enzyme complex and the substrate to a sample solution containing the analyte to perform an enzymatic reaction, measuring an enzymatic reaction signal, and calculating the equation according to the equation The content of the analyte in the sample solution.
  • the above-mentioned homogeneous detection method provided by the embodiment of the present invention is a novel homogeneous non-competition method, which uses an aptamer that can specifically recognize a test object to mark an enzyme, first according to the object to be tested.
  • the enzymatic reaction signal of the enzymatic reaction in the standard solution and an equation for deriving the relationship between the enzymatic reaction signal and the content of the analyte (in a specific embodiment, may be a linear equation or a nonlinear equation), and then according to the equation,
  • an equation for deriving the relationship between the enzymatic reaction signal and the content of the analyte in a specific embodiment, may be a linear equation or a nonlinear equation
  • a specific aptamer is linked to the enzyme or a complementary active enzyme fragment (enzyme donor or enzyme receptor), the aptamer itself does not affect the activity of the enzyme or The complementary activity of the enzyme fragment does not affect the activity of the enzyme or the complementary activity of the enzyme fragment after being linked to the enzyme, but when the sample solution contains the analyte, the analyte and the aptamer directly bind specifically, due to the spatial position. The resistance effect, the activity of the enzyme or the complementary activity of the enzyme fragment is lost. The higher the concentration of the analyte, the lower the enzyme activity, and the concentration of the analyte is inversely proportional to the enzyme activity.
  • the homogeneous detection method has the characteristics of high sensitivity, good repeatability, strong anti-interference ability, fast detection speed and low cost, and can detect various biomolecules, and is widely used.
  • the enzyme is a catalytically active enzyme, that is, the enzyme is a holoenzyme, and may be an enzyme donor and an enzyme receptor having complementary activities (enzyme).
  • the donor and the enzyme receptor constitute a catalytically active holoenzyme;
  • the enzyme may be a natural enzyme or an artificial enzyme, and the artificial enzyme may be genetically engineered to modify the above whole enzyme or enzyme fragment to change its Vitality, specificity, to optimize the sensitivity, linear range, detection specificity, stability and other properties of this test method ⁇ 0 2019/104529 ⁇ (:17 ⁇ 2017/113570
  • the aptamer is ligated to the holoenzyme to obtain the aptamer-enzyme complex in step 301, followed by an enzymatic reaction.
  • the enzyme is an enzyme donor and an enzyme receptor having complementary activities
  • the aptamer can be ligated to the enzyme donor or the enzyme receptor separately, and then subjected to homogeneous detection, and the above three cases can be obtained.
  • the effects of the present invention are all within the scope of the present invention. Specifically, two cases in which an aptamer is ligated to an enzyme donor or an enzyme receptor are as follows:
  • a homogeneous detection method includes the following steps:
  • a homogeneous detection method includes the following steps:
  • an aptamer-enzyme receptor complex is obtained;
  • step 3011 when the aptamer is attached to the enzyme donor, the aptamer-enzyme donor complex and the enzyme receptor in step 3012 or 3013 are combined in the reaction solution. , the aptamer in 301 is formed - ⁇ 0 2019/104529 ⁇ (:17 ⁇ 2017/113570
  • step 3021 when the aptamer is ligated to the enzyme receptor, the aptamer-enzyme receptor complex and the enzyme donor in step 02 2 or 3023 are combined in the reaction solution. An aptamer-enzyme complex in 301 was formed.
  • the aptamer comprises at least one of a nucleic acid aptamer, a polypeptide aptamer, and a peptide nucleic acid aptamer.
  • the aptamer molecular weight is generally less than In the embodiment of the invention, it is preferred that the aptamer has a molecular weight less than The aptamer performs best in this range.
  • the aptamer is a polypeptide aptamer, the amino acid sequence of which is
  • the above aptamer is a modified aptamer, and the modification means at least one of a cyclization modification (e.g., formation of an intramolecular disulfide bond), a methylation modification, and a phosphorylation modification. Modification of the aptamer can further enhance the stability, affinity or specificity of the aptamer. Of course, if it is an antagonistic small molecule or a derivative thereof of the analyte, it also has the same function as the aptamer, and it is also within the scope of the aptamer in the embodiment of the present invention.
  • the analyte includes at least one of a protein, a liposome, a hormone, a nucleic acid, a virus, a bacterium, a fungus, a cell, and a tissue;
  • the homogeneous detection method can detect various biological macromolecules, cell bodies or tissues.
  • the molecular weight of the analyte is generally greater than 101 ⁇ 03. The larger the molecular weight, the higher the detection sensitivity, and further preferably the analyte is larger than the molecular weight. Substance, its detection sensitivity is better.
  • the enzyme includes glucose-6-phosphate dehydrogenase, (3-galactosidase, peroxidase, luciferase, alkaline phosphatase, and fluorescence) At least one of a protein (such as green fluorescent protein and red fluorescent protein, etc.), but is not limited to the above.
  • the substrate for the enzyme is any one of a chromogenic substrate, a luminescent substrate, and a fluorescent substrate. Specifically, a suitable substrate can be selected according to different sensitivity, linear range, etc.
  • the enzymatic reaction signal can be any one of a colorimetric signal, an illuminating signal, and a fluorescent signal.
  • the corresponding substrates are o-nitrobenzene (3-galactopyranoside and glucose-6-phosphate, respectively, and (3-galactosidase) Divided into fragments and fragments, Fragment and aptamer fusion expression, glucose-6-phosphate dehydrogenase as a holoenzyme linked to an aptamer.
  • the enzyme is linked to the aptamer by any of chemical coupling, affinity adsorption (such as biotin-avidin linkage) and gene fusion expression.
  • affinity adsorption such as biotin-avidin linkage
  • a fragment of an enzyme or an enzyme can be obtained by chemical synthesis or gene expression, preferably a gene fusion expression method to link the enzyme to the aptamer, and even more preferably, the enzyme can be linked to the aptamer via a linker peptide, ie, A ligation peptide is ligated between the aptamer and the holoenzyme or enzyme fragment, which allows the aptamer to maintain its independent biological properties with the holoenzyme or enzyme fragment.
  • the fusion protein is a fusion of the £0 fragment of 3-galactosidase with the recognition (:-reactive protein aptamer, with a linker peptide added between the two (sequence: .
  • the enzyme is linked to one or more of the aptamers. That is, the enzyme can be linked to one or more of the aptamers for the same analyte to achieve detection of one analyte, and one or more aptamers labeled for the same analyte are labeled.
  • the method has obvious technical advantages in preventing false negatives; of course, different enzymes or enzyme fragments can be labeled for aptamers of different analytes to realize simultaneous detection of multiple analytes. The above methods are all within the scope of the invention and are protected by the present invention.
  • a chemical substance including, but not limited to, a surfactant, a cyclodextrin, and bovine serum albumin is further added.
  • a chemical substance including, but not limited to, a surfactant, a cyclodextrin, and bovine serum albumin is further added.
  • a chemical substance including, but not limited to, a surfactant, a cyclodextrin, and bovine serum albumin is further added.
  • 83 VIII at least one of casein, amino acid, chelating agent, nucleotide, hydrophilic polymer, reducing agent, oxidizing agent, antiseptic salt, buffering salt, polysaccharide, alcohol and metal ion.
  • the addition of the above substances may change the complementary activity of the aptamer-labeled enzyme fragment or change the affinity between the aptamer-labeled enzyme and the analyte to reduce the interference caused by the analog of the analyte, or Improve the stability, anti-interference ability and other properties of the prepared reagents.
  • the homogeneous detection includes any one of tubular detection, plate detection, microfluidic detection, and chromatographic detection.
  • a kit in the form of a liquid, a dry powder, or a combination of both may be prepared according to the test 1 used in the homogeneous detection method.
  • two fragments of the enzyme can be replaced with two different fluorescent groups capable of undergoing fluorescence resonance energy transfer (donor fluorescent group).
  • donor fluorescent group capable of undergoing fluorescence resonance energy transfer
  • the group and the acceptor fluorophore can also achieve the same technical effects as the aforementioned detection methods.
  • the alternative homogeneous detection method includes the following steps:
  • the aptamer can specifically recognize the analyte; ⁇ 0 2019/104529 ⁇ (:17 ⁇ 2017/113570
  • Fluorescence resonance energy transfer refers to the emission spectrum of one fluorophore (donor! ⁇ ! ⁇ ) and another base fluorescence resonance energy transfer group (receptor) in two different fluorophores.
  • the absorption spectrum has a certain overlap.
  • the distance between the two fluorophores is appropriate (generally less than 100)
  • the phenomenon that the fluorescence energy is transferred from the donor to the acceptor is observed, that is, the former group
  • the excitation wavelength is excited, the fluorescence emitted by the latter group can be observed.
  • one or more aptamers are respectively labeled, and when the analyte is added, the aptamers on the two fluorophores form a sandwich structure with the analyte. Bringing the two fluorophore groups closer together, fluorescence resonance energy transfer occurs, and the fluorescence intensity of the fluorophore emission is detected according to the fluorescence resonance energy transfer signal, and the fluorescence intensity is in an equation relationship with the concentration of the analyte, so that the calculation can be performed quickly The concentration of the analyte to be measured.
  • the donor fluorophore and the acceptor fluorophore are any one of a paired fluorescent protein, a paired organic dye, and a paired semiconductor quantum dot.
  • a paired fluorescent protein such as the pairing of cyan fluorescent protein and yellow fluorescent protein
  • organic dyes such as the pairing of cyan fluorescent protein and yellow fluorescent protein
  • semiconductor quantum dots etc.
  • the homogeneous detection method according to the fluorescence resonance energy transfer signal is the same in aptamer selection as the foregoing method according to the enzymatic reaction signal, and the aptamer may include a nucleic acid aptamer, a polypeptide At least one of the aptamer and the peptide nucleic acid aptamer may also be modified.
  • the selection of the analyte may also be the same, including at least one of a protein, a liposome, a hormone, a nucleic acid, a virus, a bacterium, a fungus, a cell, and a tissue.
  • the manner in which the donor fluorophore and the acceptor fluorophore are linked to the aptamer may also be any one of chemical coupling, affinity adsorption (such as biotin-avidin linkage) and gene fusion expression, of course, if Semiconductor quantum dots are chemically coupled or affinity adsorbed.
  • affinity adsorption such as biotin-avidin linkage
  • gene fusion expression of course, if Semiconductor quantum dots are chemically coupled or affinity adsorbed.
  • the choice of other steps can be the same except that no enzyme substrate is required.
  • the present invention has been subjected to a number of tests in succession, and a part of the test results are now described in further detail as a reference, and will be described in detail below in conjunction with specific embodiments.
  • a C-reactive protein detection method the reagents and steps used are as follows:
  • Reagent 1 200 mM sodium phosphate, adjusted to pH 7.3, 5 mM EDTA, 0.1% pr oclin-300, 1% BSA, 0.05 uM ED-CA fusion protein.
  • Reagent 2 200 mM sodium phosphate, adjusted to pH 7.3, 5 mM EDTA, 0.1% pr oclin-300, 1% BSA, 0.05 uM EA enzyme fragment, 0.2 g/L ONPG.
  • C-reactive protein standard solution standard solution of four concentration gradients of lmg/L, 10 mg/L, 100 mg/L, and 1000 mg/L.
  • the ED-CA fusion protein is an aptamer that recognizes an ED fragment of 3-galactosidase and recognizes a C-reactive protein
  • SEQ ID NO: l EWACNDRGFNCQLQR
  • Fusion expression ED-CA
  • a ligation peptide sequence: GGGGS
  • Detection step In the reaction vessel, first add 2 ul of C-reactive protein standard solution, then add 100 ul of reagent 1, incubate at 37 ° C for 5 minutes, add 100 ul of reagent 2, and carry out enzymatic reaction at 37 ° C
  • the change in absorbance at a wavelength of 415 nm is detected, and the rate of change in absorbance (reactivity) is inversely proportional to the concentration of C-reactive protein, and a standard curve (ie, a relationship equation) is established by the concentration of the C-reactive protein standard solution and the degree of reactivity.
  • Test the reactivity of the sample solution to be tested, and calculate the C-reactive protein concentration in the sample solution according to the standard curve.
  • a C-reactive protein detection method the reagents and steps used are as follows:
  • TRIS trishydroxymethylaminomethane, purchased from Aladdin
  • EDTA purchased from Sinopharm
  • glucose-6-phosphate dehydrogenase purchased from Roche
  • glucose-6-phosphate purchased from Roche
  • oxidized coenzyme II purchased from Roche
  • preservative proclin-300 purchased from sigma
  • MES 2-morpholineethanesulfonic acid, purchased from si gma
  • C-reactive protein purchased from Nanzu Liding
  • EDC 1-(3 dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, available from sigma).
  • G6PD-CA Aptamer labeled glucose-6-phosphate dehydrogenase
  • G6PD-CA Prepare 5g/L MES buffer, adjust pH to 6.5, add 50mg/L glucose-6-phosphate dehydrogenase and 2mg /L C-reactive protein aptamer (SEQ ID NO: l: EWACNDRGFNCQLQR) After stirring, add 100 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) The reaction was carried out at 25 ° C for 1 hour, and 10 g/L of BSA was added to terminate the reaction, and dialysis was carried out overnight using a 20 kd dialysis bag to remove the unreacted C-reactive protein aptamer.
  • EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • Reagent 1 200 mM TRIS buffer, adjusted to pH 8.0, 10% of the above labeled G 6PD-CA, 0.1% proclin-300, 1% BSA was added.
  • Reagent 2 2 g/L of glucose-6-phosphate, 4 g/L of oxidized coenzyme II, and 0.1% of proclin-300 were added to pure water.
  • C-reactive protein standard solution standard solution of four concentration gradients of lmg/L, 10 mg/L, 100 mg/L, and 1000 mg/L.
  • Detection step In the reaction vessel, first add 2 ul of C-reactive protein standard solution, then add 200 ul of reagent 1 , incubate at 37 ° C for 5 minutes, add 50 ul of reagent 2, and carry out the reaction at 37 ° C to detect 340 nm.
  • the change in absorbance at the wavelength, the rate of change in absorbance (reactivity) is inversely proportional to the concentration of C-reactive protein, and the standard curve (ie, the existence equation) is established by the concentration of C-reactive protein standard solution and the degree of reactivity.
  • the reactivity of the sample to be tested is tested, and the concentration of C-reactive protein in the sample solution can be calculated from the standard curve.
  • FIG. 2 The principle of this embodiment is shown in FIG. 2:
  • the aptamer recognizing C-reactive protein is passed through a coupling agent such as 1-(3-dimethyl). ⁇ 0 2019/104529 ⁇ (:17 ⁇ 2017/113570

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Abstract

一种均相检测方法，该均相检测方法包括如下步骤：提供适配体和酶，适配体可特异性识别待测物，将适配体连接到酶上，得适配体-酶复合物；配置不同浓度的待测物标准溶液，将适配体-酶复合物和酶作用的底物加入到待测物标准溶液中进行酶促反应，测得酶促反应信号，获取酶促反应信号与待测物含量的方程；将适配体-酶复合物和底物加入到含有待测物的样品溶液中进行酶促反应，测得酶促反应信号，根据方程计算样品溶液中的待测物含量。该均相检测方法具有灵敏度高、重复性好、抗干扰能力强、检测速度快和成本低的特点，可检测各种生物分子，应用非常广泛。

Description

发明名称:均相捡测方法
技术领域
[0001] 本发明属于生物检测技术领域， 具体涉及一种均相检测方法。
背景技术
[0002] 目前， 生物大分子测定技术有胶乳增强免疫比浊技术 （LETIA） 、 化学发光免 疫分析技术 （CLIA） 。 LETIA通过在高分子乳胶微球表面交联的单克隆抗体， 在抗原存在的条件下， 偶联微球的单克隆抗体与抗原在短时间内迅速结合聚集 在一起， 从而改变反应溶液的吸光度； 反应液吸光度的改变与被测抗原的浓度 在一定范围内具有线性关系， 从而可用来反映被测抗原的浓度。 CLIA是用化学 发光剂直接标记抗原或抗体的免疫分析方法， 该方法具有很高的灵敏度和良好 的线性范围。
[0003] 化学小分子的测定技术有克隆酶供体免疫测试 （cloned enzyme donor
immunoassay, CEDIA） 、 酶放大免疫测试 （EMIT） 、 荧光偏振免疫测试 （FPI A） 。 CEDIA是利用重组 DNA技术制备（3 -半糖苷酶的两个片段： 大片段称为酶受 体 （enzyme acceptor, EA） ， 小片段称为酶供体 （enzyme donor, ED） ， 两个 片段本身均不具酶活性， 但在合适的条件下结合在一起就具有酶活性， 利用这 两相片段的特性建立的均相酶免疫测定称为克隆酶供体免疫测定。 ED标记的半 抗原与抗体结合后由于空间位阻， 不再能与 EA结合。 当样品中含有待测半抗原 时， 会与 ED标记的半抗原竞争结合抗体， 使被抑制的 ED恢复自由状态， 能够与 EA互补， 酶活性恢复。 CEDIA主要用于药物和小分子物质的测定， 也有专利将 蛋白分子的特定表位连接到 ED片段上， 从而实现了对生物大分子的检测， 但目 前 CEDIA技术都是使用竞争法进行测试， 待测物浓度与酶活力大小成正比。 EMI T的基本原理是半抗原与酶结合成酶标半抗原， 保留半抗原和酶的活性。 当酶标 半抗原与抗体结合后， 所标的酶与抗体密切接触， 使酶的活性中心受到影响而 活性被抑制， 当样品中含有待测半抗原时， 会与酶标半抗原竞争结合抗体， 使 被抑制的酶活性恢复。 EMIT技术使用竞争法进行测试， 待测物浓度与酶活力大 \¥0 2019/104529 卩（：17 \2017/113570
2 小成正比。 ？?1八的试剂为荧光素标记的小分子待测物和抗药物的抗体， 模式为 均相竞争法。 荧光偏振免疫测试的原理如下， 荧光物质经单一平面的偏振光蓝 光 （波长 485!1111） 照射后， 可吸收光能跃入激发态； 在恢复至基态时， 释放能量 并发出单一平面的偏振変光 （波长 52511111） 。 偏振変光的强度与変光物质受激发 时分子转动的速度成反比。 大分子物质旋转慢， 发出的偏振荧光强； 小分子物 质旋转快， 其偏振荧光弱。 将待测小分子连接到荧光分子上， 同时在检测环境 中添加针对待测小分子的抗体， 当检测环境中含有待测小分子时， 该待测小分 子会与标记了荧光分子的待测小分子竞争结合抗体， 使部分标记了荧光分子的 待测物释放出来， 检测环境中的偏振光强度下降。 该方法为竞争法测试， 待测 物浓度与偏振光强度呈反比， 利用这一现象建立了荧光偏振免疫测定用于小分 子物质特别是药物的测定。
[0004] 上述方法中， 1^11八技术最高可以只能实现 0.111¾ 的分析灵敏度， 但对于一 些灵敏度要求更高的分析就无能为力了。 （:
Figure imgf000004_0001
方法是一种非均相的测试， 测试 过程中涉及到清洗分离步骤， 测试速度较慢， 测试成本很高， 且非均相反应导 致测试的重复性较差， 而且以上方法由于需要使用抗体， 还会存在类风湿因子 （1^） 、 人抗动物免疫球蛋白 等干扰， 均可能严重影响测试结果的 准确性。
Figure imgf000004_0002
最适合用来测试小分子物质， 检测大分子时非常受限， 且同等情况下， 竞争法灵敏度较非竞争法低。 因此， 现有技术有待改进。
技术问题
[0005] 本发明的目的在于克服现有技术的上述不足， 提供一种均相检测方法， 旨在解 决现有检测方法灵敏度和重复性较差、 成本高， 以致使其应用受限的技术问题
问题的解决方案
技术解决方案
[0006] 为实现上述发明目的， 本发明采用的技术方案如下：
[0007] 本发明提供一种均相检测方法， 该均相检测方法包括如下步骤：
[0008] 提供适配体和酶， 所述适配体可特异性识别待测物， 将所述适配体连接到所述 \¥0 2019/104529 卩（：17 \2017/113570
3 酶上， 得适配体 -酶复合物；
[0009] 配置不同浓度的待测物标准溶液， 将所述适配体-酶复合物和所述酶作用的底 物加入到所述待测物标准溶液中进行酶促反应， 测得酶促反应信号， 获取酶促 反应信号与待测物含量的方程；
[0010] 将所述适配体-酶复合物和所述底物加入到含有所述待测物的样品溶液中进行 酶促反应， 测得酶促反应信号， 根据所述方程计算所述样品溶液中的待测物含 量。
发明的有益效果
有益效果
[0011] 本发明提供的均相检测方法， 是一种全新的均相非竞争法， 该方法利用可特异 性识别待测物的适配体对酶进行标记， 先根据待测物标准溶液中酶促反应的酶 促反应信号， 推导出酶促反应信号与待测物含量之间关系的方程， 然后根据该 方程， 检测样品溶液中酶促反应的酶促反应信号， 就可以计算出样品溶液中的 待测物含量； 由于该酶促反应信号与待测物含量成一定比例， 所以根据样品溶 液中的酶促反应信号可以快速计算出待测物含量。 因此， 本发明与现有技术相 比， 具有灵敏度高、 重复性好、 抗干扰能力强、 检测速度快和成本低的特点， 可检测各种生物分子， 应用非常广泛。
对附图的简要说明
附图说明
[0012] 图 1为本发明实施例 1中的（:-反应蛋白检测的原理图；
[0013] 图 2为本发明实施例 2中的（:-反应蛋白检测的原理图。
发明实施例
本发明的实施方式
[0014] 为了使本发明要解决的技术问题、 技术方案及有益效果更加清楚明白， 以下结 合附图和实施例， 对本发明进行进一步详细说明。 应当理解， 此处所描述的具 体实施例仅仅用以解释本发明， 并不用于限定本发明。
[0015] 本发明实施例提供了一种均相检测方法， 该均相检测方法包括如下步骤： \¥0 2019/104529 卩（：17 \2017/113570
4
[0016] 801： 提供适配体和酶， 所述适配体可特异性识别待测物， 将所述适配体连接 到所述酶上， 得适配体 -酶复合物；
[0017] 802： 配置不同浓度的待测物标准溶液， 将所述适配体-酶复合物和所述酶作用 的底物加入到所述待测物标准溶液中进行酶促反应， 测得酶促反应信号， 获取 酶促反应信号与待测物含量的方程；
[0018] 803： 将所述适配体-酶复合物和所述底物加入到含有所述待测物的样品溶液中 进行酶促反应， 测得酶促反应信号， 根据所述方程计算所述样品溶液中的待测 物含量。
[0019] 本发明实施例提供的上述均相检测方法， 是一种全新的均相非竞争法， 该方法 利用可特异性识别待测物的适配体对酶进行标记， 先根据待测物标准溶液中酶 促反应的酶促反应信号， 推导出酶促反应信号与待测物含量之间关系的方程 （ 在具体实施例中， 可以是线性方程或非线性方程） ， 然后根据该方程， 检测样 品溶液中酶促反应的酶促反应信号， 就可以计算出样品溶液中的待测物含量； 由于该酶促反应信号与待测物含量成一定比例， 所以根据样品溶液中的酶促反 应信号可以快速计算出待测物含量。
[0020] 因适配体分子量较低， 将一段特异性的适配体连接到酶或具有互补活性的酶片 段 （酶供体或酶受体） 上， 适配体本身不影响酶的活性或酶片段的互补活性， 与酶连接后也不会影响酶的活性或酶片段的互补活性， 但当样本溶液中含有待 测物时， 待测物与适配体直接特异性结合， 由于空间位阻效应， 酶的活性或酶 片段的互补活性丧失， 待测物浓度越高酶活力越低， 待测物浓度与酶活力成反 比， 因此检测到酶促反应信号 （与酶活力对应） 后可以快速计算出样品溶液中 的待测物浓度。 该均相检测方法具有灵敏度高、 重复性好、 抗干扰能力强、 检 测速度快和成本低的特点， 可检测各种生物分子， 应用非常广泛。
[0021] 具体地， 本发明实施例的上述均相检测方法中， 该酶是具有催化活性的酶， 即 该酶是全酶， 也可以是具有互补活性的酶供体和酶受体 （酶供体和酶受体组成 一个具有催化活性的全酶） ； 另外， 该酶可以是天然酶或人工酶， 人工酶是可 以通过基因工程的方法对以上全酶或酶片段进行改造， 以改变其活力、 特异性 ， 来对本检测方法的灵敏度、 线性范围、 检测特异性、 稳定性等性能进行优化 \¥0 2019/104529 卩（：17 \2017/113570
5
[0022] 当所述酶为全酶时， 将所述适配体连接到所述全酶上， 得到步骤301中的所述 适配体 -酶复合物， 然后进行酶促反应。 当然， 当所述酶是具有互补活性的酶供 体和酶受体时， 适配体可单独连接在酶供体或酶受体上， 后续再进行均相检测 ， 以上三种情况都可以取得本发明的效果， 都在本发明的保护范围内。 具体地 ， 适配体单独连接在酶供体或酶受体上的两种情况如下：
[0023] 一种均相检测方法， 包括如下步骤：
[0024] 8011： 提供适配体和酶， 所述适配体可特异性识别待测物， 所述酶包括具有互 补活性的酶供体和酶受体， 将所述适配体连接到所述酶供体上， 得适配体 -酶供 体复合物；
[0025] 8012： 配置不同浓度的待测物标准溶液， 将所述适配体-酶供体复合物、 所述 酶受体和所述酶作用的底物加入到所述待测物标准溶液中进行酶促反应， 测得 酶促反应信号， 获取酶促反应信号与待测物含量的方程；
[0026] 8013： 将所述适配体-酶供体复合物、 所述酶受体和所述底物加入到含有所述 待测物的样品溶液中进行酶促反应， 测得酶促反应信号， 根据所述方程计算所 述样品溶液中的待测物含量。
[0027] 一种均相检测方法， 包括如下步骤：
[0028] 8021： 提供适配体和酶， 所述适配体可特异性识别待测物， 所述酶包括具有互 补活性的酶供体和酶受体， 将所述适配体连接到所述酶受体上， 得适配体 -酶受 体复合物；
[0029] 8022： 配置不同浓度的待测物标准溶液， 将所述适配体-酶受体复合物、 所述 酶供体和所述酶作用的底物加入到所述待测物标准溶液中进行酶促反应， 测得 酶促反应信号， 获取酶促反应信号与待测物含量的方程；
[0030] 8023： 将所述适配体-酶受体复合物、 所述酶供体和所述底物加入到含有所述 待测物的样品溶液中进行酶促反应， 测得酶促反应信号， 根据所述方程计算所 述样品溶液中的待测物含量。
[0031] 具体地， 在步骤 3011中， 所述适配体连接到所述酶供体上时， 步骤 3012或3013 中的适配体-酶供体复合物和酶受体在反应液中结合， 就形成了301中的适配体- \¥0 2019/104529 卩（：17 \2017/113570
6 酶复合物。 同理， 在步骤 3021中， 所述适配体连接到所述酶受体上时， 步骤 02 2或3023中的适配体-酶受体复合物和酶供体在反应液中结合， 就形成了 301中的 适配体 -酶复合物。
[0032] 进一步地， 在上述均相检测方法中， 所述适配体包括核酸适配体、 多肽适配体 和肽核酸适配体中的至少一种。 优选地， 适配体分子量一般小于
Figure imgf000008_0001
本发明 实施例中， 优选适配体分子量小于
Figure imgf000008_0002
该范围内适配体性能最佳。 在一具体 实施例中， 适配体为多肽适配体， 其氨基酸序列如
Figure imgf000008_0003
NO:l所示： EWACNDRGFNCQLQR。 更进一步优选地， 上述适配体为修饰的适 配体， 修饰方式包括环化修饰 （如形成分子内二硫键） 、 甲基化修饰和磷酸化 修饰中的至少一种。 将适配体进行修饰， 可以进一步以提高适配体的稳定性、 亲和力或特异性。 当然， 如果是待测物的拮抗小分子或其衍生物， 也具有适配 体的相同功能， 也属于本发明实施例中的适配体范围内。
[0033] 进一步地， 在上述均相检测方法中， 所述待测物包括蛋白质、 脂质体、 激素、 核酸、 病毒、 细菌、 真菌、 细胞和组织中的至少一种； 即本方面实施例的均相 检测方法可检测各种生物大分子、 细胞体或组织， 优选地， 待测物的分子量一 般大于 101^03, 分子量越大， 检测灵敏度越高， 进一步优选待测物为分子量大于 的物质， 其检测灵敏度更佳。
[0034] 进一步地， 在上述均相检测方法中， 所述酶包括葡萄糖 -6 -磷酸脱氢酶、 （3 -半乳 糖苷酶、 过氧化物酶、 荧光素酶、 碱性磷酸酶和荧光蛋白 （如绿色荧光蛋白和 红色荧光蛋白等） 中的至少一种， 但不限于以上几种。 所述酶作用的底物为显 色底物、 发光底物和荧光底物中的任意一种， 具体可以根据不同灵敏度、 线性 范围等要求从中选择合适的底物。 而酶促反应信号可为比色信号、 发光信号和 荧光信号中的任意一种。 在本发明实施例中， 优选（3 -半乳糖苷酶和葡萄糖 -6 -磷 酸脱氢酶， 对应的底物分别为邻硝基苯（3 -半乳吡喃糖苷和葡萄糖 -6 -磷酸， 同 时， 将（3 -半乳糖苷酶分为 片段和 片段，
Figure imgf000008_0004
片段和适配体融合表达， 葡萄糖 -6 -磷酸脱氢酶作为全酶与适配体连接。
[0035] 进一步地， 在上述均相检测方法中， 所述酶连接所述适配体的方式为化学偶联 、 亲和吸附 （如生物素-亲和素连接） 和基因融合表达中的任意一种。 适配体、 \¥0 2019/104529 卩（：17 \2017/113570
7 酶或酶的片段可以通过化学合成或基因表达获得， 优选基因融合表达方式将酶 连接适配体， 更进一步优选地， 所述酶可通过连接肽与所述适配体相连， 即可 以在适配体与全酶或酶片段之间添加一段连接肽进行连接， 该连接肽可以使适 配体与全酶或酶片段保持其独立的生物学属性。 在本发明一实施例中，
Figure imgf000009_0001
融合蛋白即为将（3 -半乳糖苷酶的£0片段与识别（: -反应蛋白的适配体融合表达， 两者中间增加一段连接肽 （序列为：
Figure imgf000009_0002
。
[0036] 进一步地， 在上述均相检测方法中， 所述酶连接有一种或多种所述适配体。 即 所述酶可以连接针对同一待测物的一株或多株所述适配体， 实现对一种待测物 的检测， 采用一株或多株针对同一待测物的适配体标记的方法， 在防止假阴性 时具有明显的技术优势； 当然， 也可以选择针对不同待测物的适配体对不同的 酶或酶片段进行标记， 实现多重待测物的同步检测。 以上方法都在本发明保护 范围内， 都受本发明保护。
[0037] 进一步地， 在上述均相检测方法中， 在进行所述酶促反应的过程中， 还加入了 化学物质， 该化学物质包括但不限于表面活性剂、 环糊精、 牛血清白蛋白 （：83八 ） 、 酪蛋白、 氨基酸、 螯合剂、 核苷酸、 亲水高分子、 还原剂、 氧化剂、 防腐 齐 ^ 缓冲盐、 多糖、 醇和金属离子中的至少一种。 上述物质的加入， 或可改变 适配体标记的酶片段的互补活性， 或改变适配体标记的酶与待测物之间的亲和 性， 以减少待测物类似物导致的干扰， 或改善所制备试剂的稳定性、 抗干扰能 力等性质。
[0038] 进一步地， 在上述均相检测方法中， 所述均相检测包括管式检测、 板式检测、 微流控检测和层析检测中的任意一种。 同时， 根据该均相检测方法中使用的试 齐 1 可以制备成液体、 干粉或者两者组合的形式的试剂盒。
[0039] 另外， 本发明实施例可以将酶的两个片段 （酶供体片段和酶受体片段） 分别替 换成两个不同的、 可发生荧光共振能量转移的荧光基团 （供体荧光基团和受体 荧光基团） ， 也能起到前述检测方法相同的技术效果。
[0040] 具体地， 该替代的均相检测方法， 包括如下步骤：
[0041] £01： 提供适配体、 可发生荧光共振能量转移的供体荧光基团和受体荧光基团
， 所述适配体可特异性识别待测物； \¥0 2019/104529 卩（：17 \2017/113570
8
[0042] £02： 配置不同浓度的待测物标准溶液， 将所述供体荧光基团和所述受体荧光 基团均连接所述适配体后， 加入到所述待测物标准溶液中， 测得所述供体荧光 基团的荧光强度， 获取荧光强度与待测物含量的方程；
[0043] £03： 将连接有所述适配体的供体荧光基团和连接有所述适配体的受体荧光基 团加入到含有所述待测物的样品溶液中， 测得所述供体荧光基团的荧光强度， 根据所述方程计算所述样品溶液中的待测物含量。
[0044] 荧光共振能量转移是指在两个不同的荧光基团中， 如果一个荧光基团 （供体 !!〇!·） 的发射光谱与另一个基荧光共振能量转移团 （受体
Figure imgf000010_0001
的吸收光谱 有一定的重叠， 当这两个荧光基团间的距离合适时 （一般小于 100人） ， 就可观 察到荧光能量由供体向受体转移的现象， 即以前一种基团的激发波长激发时， 可观察到后一个基团发射的荧光。 在供体荧光基团和受体荧光基团上， 分别标 记一种或多种适配体， 当加入待测物时， 两个荧光基团上的适配体与待测物形 成夹心结构， 并使两个荧光基团距离拉近， 发生荧光共振能量转移， 根据该荧 光共振能量转移信号检测荧光基团发射的荧光强度， 该荧光强度与待测物浓度 呈方程关系， 因此， 可以快速计算出待测物浓度。
[0045] 进一步地， 在该均相检测方法中， 所述供体荧光基团和所述受体荧光基团为配 对的荧光蛋白、 配对的有机染料和配对的半导体量子点中的任意一种。 现在市 场上已经有很多商品化的荧光染料， 可作为本均相检测方法中配对的两个荧光 基团， 包括但不限于荧光蛋白 （如青色荧光蛋白与黄色荧光蛋白的配对， ） 、 有机染料 （如，
Figure imgf000010_0002
公司的
Figure imgf000010_0003
系列染料） 、 半导体量子点等。
[0046] 该依据荧光共振能量转移信号的均相检测方法中， 与前述依据酶促反应信号的 方法相比， 在适配体选择上是一样的， 适配体可以包括核酸适配体、 多肽适配 体和肽核酸适配体中的至少一种， 也可进行修饰。 待测物的选择也可以是一样 的， 包括蛋白质、 脂质体、 激素、 核酸、 病毒、 细菌、 真菌、 细胞和组织中的 至少一种。 供体荧光基团和受体荧光基团连接适配体的方式也可以是化学偶联 、 亲和吸附 （如生物素-亲和素连接） 和基因融合表达中的任意一种， 当然如果 是半导体量子点， 则是化学偶联或亲和吸附。 总之， 除了不需要添加酶底物， 其他步骤的选择都可以一样。 [0047] 本发明先后进行过多次试验， 现举一部分试验结果作为参考对发明进行进一步 详细描述， 下面结合具体实施例进行详细说明。
[0048] 实施例 1 :
[0049] 一种 C-反应蛋白检测方法， 使用的试剂和步骤如下：
[0050] 实验材料： 磷酸钠 （购自国药） 、 EDTA （乙二胺四乙酸， 购自国药） 、 ONP G （邻硝基苯（3-D-半乳卩比喃糖 I， 购自 sigma） 、 吐温 -20 （购自国药） 、 防腐剂 p roclin-300 （购自 sigma） 、 C-反应蛋白 （购自南京立顶） 、 ED-CA融合蛋白 （本 公司构建表达， 序列为 SEQ ID NO:2） 、 EA酶片段 （本公司构建表达， 在野生 型（3-半乳糖苷酶基础上敲除了 11-41位氨基酸序列） 。
[0051] 试剂 1的配制： 200mM的磷酸钠， 调整 pH到 7.3， 加入 5mM的 EDTA、 0.1%的 pr oclin-300、 1%的 BSA、 0.05uM的 ED-CA融合蛋白。
[0052] 试剂 2的配制： 200mM的磷酸钠， 调整 pH到 7.3， 加入 5mM的 EDTA、 0.1%的 pr oclin-300、 1%的 BSA、 0.05uM的 EA酶片段、 0.2g/L的 ONPG。
[0053] C-反应蛋白标准液： 酉己制 lmg/L、 10mg/L、 100mg/L、 1000mg/L四个浓度梯度 的标准液。
[0054] 其中， ED-CA融合蛋白为将（3-半乳糖苷酶的 ED片段与识别 C-反应蛋白的适配体
（SEQ ID NO: l： EWACNDRGFNCQLQR） 融合表达 （ED-CA） ， 两者中间增 加一段连接肽 （序列为： GGGGS） ， SEQ ID NO:2的序列如下
[0055] ITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQ
LRSLNG GGGGSEWACNDRGFNCOLOR。
[0056] 检测步骤： 在反应容器中， 先加入 2ul的 C-反应蛋白标准液， 再加入 lOOul的试 剂 1， 37°C孵育 5分钟， 加入 lOOul的试剂 2， 37°C下进行酶促反应， 检测 415nm波 长下吸光度的变化， 吸光度变化速度 （反应度） 与 C-反应蛋白的浓度呈反比， 以 C-反应蛋白标准液的浓度与反应度建立标准曲线 （即存在关系方程） 。 测试 待测样本溶液的反应度， 根据标准曲线可计算出样品溶液中的 C-反应蛋白浓度
[0057] 本实施例的原理见图 1 : 由于在 ED片段上增加的适配体氨基酸很少， 不影响 ED -CA和 EA互补形成有活力的酶， 当检测环境中含有 C-反应蛋白时， C-反应蛋白 与 ED-CA结合， 由于 C-反应蛋白分子量约为 l lOkd， 两者结合的空间位阻， 阻碍 了: ED-CA与 EA片段的结合， 导致两者无法形成有活力的酶， C-反应蛋白浓度越 高无活力的酶越多， 酶促反应的信号值越低， 根据酶促反应的信号值可以计算 出 C-反应蛋白的浓度。
[0058] 实施例 2:
[0059] 一种 C-反应蛋白检测方法， 使用的试剂和步骤如下：
[0060] 实验材料： TRIS （三羟甲基氨基甲烷， 购自阿拉丁） 、 EDTA （购自国药） 、 葡萄糖 -6 -磷酸脱氢酶 （购自罗氏） 、 葡萄糖 -6 -磷酸 （购自罗氏） 、 氧化型辅酶 I I （购自罗氏） 、 防腐剂 proclin-300 （购自 sigma） 、 MES （2 -吗啉乙磺酸， 购自 si gma） C-反应蛋白 （购自南足立顶） 、 EDC （1-（3二甲氨基丙基）-3 -乙基碳二 亚胺盐酸盐， 购自 sigma） 。
[0061] 适配体标记葡萄糖 -6 -磷酸脱氢酶 （G6PD-CA） : 配制 5g/L的 MES缓冲液， 调 整 PH到 6.5， 加入 50mg/L的葡萄糖 -6 -磷酸脱氢酶和 2mg/L的 C-反应蛋白适配体 （ SEQ ID NO: l： EWACNDRGFNCQLQR） 搅拌均匀后加入 100mg的 1-（3 -二甲氨 基丙基）-3 -乙基碳二亚胺盐酸盐 （EDC） ， 25°C下反应 1小时， 加入 10g/L的 BSA 终止反应， 使用 20kd的透析袋过夜透析， 去掉未反应的 C-反应蛋白适配体。
[0062] 试剂 1的配制： 200mM的 TRIS缓冲液， 调整 pH到 8.0， 加入 10%上述标记好的 G 6PD-CA、 0.1 %的 proclin-300、 1 %的 BSA。
[0063] 试剂 2的配制： 纯水中加入 2g/L的葡萄糖 -6 -磷酸、 4g/L的氧化型辅酶 II、 0.1%的 proclin-300。
[0064] C-反应蛋白标准液： 酉己制 lmg/L、 10mg/L、 100mg/L、 1000mg/L四个浓度梯度 的标准液。
[0065] 检测步骤： 反应容器中， 先加入 2ul的 C-反应蛋白标准液， 再加入 200ul的试剂 1 ， 37°C孵育 5分钟， 加入 50ul的试剂 2， 37°C下进行反应， 检测 340nm波长下吸光 度的变化， 吸光度变化速度 （反应度） 与 C-反应蛋白的浓度呈反比， 以 C-反应 蛋白标准液的浓度与反应度建立标准曲线 （即存在关系方程） 。 测试待测样本 溶液的反应度， 根据标准曲线可以计算出样品溶液中的 C-反应蛋白浓度。
[0066] 本实施例的原理见图 2: 将识别 C-反应蛋白的适配体通过偶联剂， 如 1-（3 -二甲 \¥0 2019/104529 卩（：17 \2017/113570
11 氨基丙基）-3 -乙基碳二亚胺盐酸盐 （£0（：） ， 按照一定比例， 偶联到葡萄糖 -6 -磷 酸脱氢酶上， 形成酶-适配体复合物
Figure imgf000013_0001
， 由于适配体很小， 对酶的催 化活性影响很小。 当检测环境中含有（:-反应蛋白时， （: -反应蛋白与
Figure imgf000013_0002
合， 由于（:-反应蛋白分子量约为
Figure imgf000013_0003
覆盖了酶的催化中心， 导致酶活力降低 ， （:-反应蛋白浓度越高， 酶活力越低， 酶促反应的信号值越低， 根据酶促反应 的信号值可以计算出（: -反应蛋白的浓度。
[0067] 以上所述仅为本发明的较佳实施例而已， 并不用以限制本发明， 凡在本发明的 精神和原则之内所作的任何修改、 等同替换和改进等， 均应包含在本发明的保 护范围之内。

Claims

\¥0 2019/104529 卩（：17 \2017/113570 12 权利要求书
[权利要求 1] 一种均相检测方法， 其特征在于， 包括如下步骤：
提供适配体和酶， 所述适配体可特异性识别待测物， 将所述适配体连 接到所述酶上， 得适配体 -酶复合物；
配置不同浓度的待测物标准溶液， 将所述适配体 -酶复合物和所述酶 作用的底物加入到所述待测物标准溶液中进行酶促反应， 测得酶促反 应信号， 获取酶促反应信号与待测物含量的方程； 将所述适配体 -酶复合物和所述底物加入到含有所述待测物的样品溶 液中进行酶促反应， 测得酶促反应信号， 根据所述方程计算所述样品 溶液中的待测物含量。
[权利要求 2] 如权利要求 1所述的均相检测方法， 其特征在于， 所述酶为全酶， 将 所述适配体连接到所述全酶上， 得所述适配体 -酶复合物。
[权利要求 3] 如权利要求 1所述的均相检测方法， 其特征在于：
所述酶包括具有互补活性的酶供体和酶受体， 将所述适配体连接到所 述酶供体上， 得适配体-酶供体复合物；
配置不同浓度的待测物标准溶液， 将所述适配体-酶供体复合物、 所 述酶受体和所述酶作用的底物加入到所述待测物标准溶液中进行酶促 反应， 测得酶促反应信号， 获取酶促反应信号与待测物含量的方程； 将所述适配体-酶供体复合物、 所述酶受体和所述底物加入到含有所 述待测物的样品溶液中进行酶促反应， 测得酶促反应信号， 根据所述 方程计算所述样品溶液中的待测物含量。
[权利要求 4] 如权利要求 1所述的均相检测方法， 其特征在于：
所述酶包括具有互补活性的酶供体和酶受体， 将所述适配体连接到所 述酶受体上， 得适配体-酶受体复合物；
配置不同浓度的待测物标准溶液， 将所述适配体-酶受体复合物、 所 述酶供体和所述酶作用的底物加入到所述待测物标准溶液中进行酶促 反应， 测得酶促反应信号， 获取酶促反应信号与待测物含量的方程； 将所述适配体-酶受体复合物、 所述酶供体和所述底物加入到含有所 \¥0 2019/104529 卩（：17 \2017/113570
13 述待测物的样品溶液中进行酶促反应， 测得酶促反应信号， 根据所述 方程计算所述样品溶液中的待测物含量。
[权利要求 5] 如权利要求 1-4任一项所述的均相检测方法， 其特征在于， 所述适配 体包括核酸适配体、 多肽适配体和肽核酸适配体中的至少一种。
[权利要求 6] 如权利要求 1-4任一项所述的均相检测方法， 其特征在于， 所述适配 体为修饰的适配体， 所述修饰包括环化修饰、 甲基化修饰和磷酸化修 饰中的至少一种。
[权利要求 7] 如权利要求 1-4任一项所述的均相检测方法， 其特征在于， 所述适配 体的分子量＜ 101^¾; 和/或所述待测物的分子量 ñ 101^¾。
[权利要求 8] 如权利要求 1-4任一项所述的均相检测方法， 其特征在于， 所述待测 物包括蛋白质、 脂质体、 激素、 核酸、 病毒、 细菌、 真菌、 细胞和组 织中的至少一种。
[权利要求 9] 如权利要求 1-4任一项所述的均相检测方法， 其特征在于， 所述酶包 括葡萄糖 -6 -磷酸脱氢酶、 （3 -半乳糖苷酶、 过氧化物酶、 荧光素酶、 碱 性磷酸酶和荧光蛋白中的至少一种； 和/或
所述酶包括天然酶或人工酶； 和/或
所述底物为显色底物、 发光底物和荧光底物中的任意一种。
[权利要求 10] 如权利要求 1-4任一项所述的均相检测方法， 其特征在于， 所述酶连 接所述适配体的方式为化学偶联、 亲和吸附和基因融合表达中的任意 一种。
[权利要求 11] 如权利要求 1-4任一项所述的均相检测方法， 其特征在于， 所述酶连 接有一种或多种所述适配体； 和/或
所述酶通过连接肽与所述适配体相连接。
[权利要求 12] 如权利要求 1-4任一项所述的均相检测方法， 其特征在于， 在进行所 述酶促反应的过程中， 还加入化学物质， 所述化学物质包括表面活性 剂、 环糊精、 牛血清白蛋白、 酪蛋白、 氨基酸、 螯合剂、 核苷酸、 亲 水高分子、 还原剂、 氧化剂、 防腐剂、 缓冲盐、 多糖、 醇和金属离子 中的至少一种。 \¥0 2019/104529 卩（：17 \2017/113570
14
[权利要求 13] 如权利要求 1-4任一项所述的均相检测方法， 其特征在于， 所述酶促 反应信号为比色信号、 发光信号和変光信号中的任意一种。
[权利要求 14] 如权利要求 1-4任一项所述的均相检测方法， 其特征在于， 所述均相 检测包括管式检测、 板式检测、 微流控检测和层析检测中的任意一种
[权利要求 15] 一种均相检测方法， 其特征在于， 包括如下步骤：
提供适配体、 可发生荧光共振能量转移的供体荧光基团和受体荧光基 团， 所述适配体可特异性识别待测物；
配置不同浓度的待测物标准溶液， 将所述供体荧光基团和所述受体荧 光基团均连接所述适配体后， 加入到所述待测物标准溶液中， 测得所 述供体荧光基团的荧光强度， 获取荧光强度与待测物含量的方程； 将连接有所述适配体的供体荧光基团和连接有所述适配体的受体荧光 基团加入到含有所述待测物的样品溶液中， 测得所述供体荧光基团的 荧光强度， 根据所述方程计算所述样品溶液中的待测物含量。
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