CN107918022A - A kind of cTnI detection kits and its application method - Google Patents

A kind of cTnI detection kits and its application method Download PDF

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CN107918022A
CN107918022A CN201711146344.3A CN201711146344A CN107918022A CN 107918022 A CN107918022 A CN 107918022A CN 201711146344 A CN201711146344 A CN 201711146344A CN 107918022 A CN107918022 A CN 107918022A
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ctni
solution
magnetic bead
antibody
enzyme
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CN107918022B (en
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王保君
汤双双
欧卫军
徐艳
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NANTONG EGENS BIOTECHNOLOGY CO Ltd
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NANTONG EGENS BIOTECHNOLOGY CO Ltd
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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00—Magnetic particle immunoreagent carriers
    • G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
    • G01N2446/90—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids characterised by small molecule linker used to couple immunoreagents to magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction

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Abstract

The invention discloses a kind of cTnI detection kits, including calibration object, cleaning solution, substrate solution, pretreatment fluid, enzyme conjugates working solution and magnetic bead conjugate working solution；Contain imidazoles in the pretreatment fluid, the cTnI antibody containing enzyme mark in the enzyme conjugates working solution, the magnetic bead containing cTnI antibody mark in the magnetic bead conjugate working solution.Above-mentioned cTnI detection kits can realize the Accurate Determining to cTnI in whole blood sample, simplify detecting step, improve detection efficiency.Kit lowest detection is limited to 0.02ng/ml, and the range of linearity is 0.02 50ng/ml, and detection sensitivity is high, and the range of linearity is wide, and testing result is accurate.The invention also discloses a kind of application method of above-mentioned cTnI detection kits, it is simple using step, effectively shortens the detection time of cTnI, is advantageously implemented quick, the Sensitive Detection of cTnI.

Description

A kind of cTnI detection kits and its application method
Technical field
The invention belongs to immunochemistry detection technique field, and in particular to a kind of cTnI detection kits and its user Method.
Background technology
Angiocardiopathy is to endanger the great fatal disease of the health of our people and life, is counted according to the Ministry of Public Health, In China city, just there are 235 people to die of cardiovascular and cerebrovascular disease in every 100,000 people, account for dead caused by various diseases First place, and every year with 2% speed increase.Acute myocardial infarction AMI (Acute Myocardial Infarction, AMI) is then Cause cardiovascular patient main causes of death.AMI is a kind of clinically sudden illness, it is often accompanied by pectoralgia, dislikes when falling ill The a series of symptoms such as the heart, fever, arrhythmia cordis, blood serum designated object rise, as can simultaneously interventional treatment is made a definite diagnosis early, it is dying to saving Cardiac muscle, improvement prognosis, reduction acute stage case fatality rate etc. are of great significance.
Troponin (Tn) is cardiac muscle and the contraction regulatory protein of skeletal muscle, and cTn is cardiac troponin, by cTnI, Tri- subunit's compositions of cTnT and cTnC.CTnI, cTnC and cTnT play important regulative in muscle diastole and contraction process, But cTnC is generally not used for myocardial damage detection without Cardiac-specific.CTnI and cTnT are unable to penetration cell under normal condition Film enters blood, so cTnI and cTnT are extremely low in Healthy People blood；Such as myocardial cell damage, cTnI and cTnT are into people's cell interstitial And blood.But the content of cTnT is in the diseases such as kidney failure, pneumonia and septicemia in blood, can also occur significantly to carry Rise, therefore with the specific relatively low of cTnT detections AMI.CTnI is raised when 3~5 is small after AMI morbidities, and 15~24 reach height when small Peak, the duration is long, can be down to after 5~10 days normal.At present, it is to examine with cTnI diagnosing myocardial infarctions and myocardial cell injury " goldstandard " new disconnected AMI.
NT-proBNP methods for measuring have electrophoresis, ion-exchange chromatography method, ELISA method and radioimmunology etc..Often Rule are more using electrophoresis and ELISA method, but electrophoresis can not be applied to automatic clinical chemistry analyzer, and instrument price is held high It is expensive, so can not popularize；ELISA method operating process is complicated, detection time is long, is unfavorable for realizing the quick inspection of clinic of cTnI Survey.
Chinese patent literature CN104330575A discloses a kind of Troponin I detection reagent, utilizes the even phase of colloidal gold Grain characteristic, colloid gold particle surface is marked on by specific cTnI antibody, when in detecting system or detection environment there are during cTnI, The antibody on colloid gold particle surface forms antigen-antibody complex i.e. by corresponding antigen capture, in turn results in part The polymerization or accumulation of colloid gold particle, make the even phase reagent transmittance spectrum of colloidal gold be moved from red to blue color spectrum, this movement Key reaction is the reduction and the rise of absorbance at 660nm of absorbance at 540nm, so as to reach cTnI in quantitative detection human body The purpose of antigen.Above-mentioned cTnI detection reagents refer to detection speed and the sensitivity of cTnI to a certain extent, but detect Reagent is simply possible to use in cTnI contents in detection serum or blood plasma, can not realize accurate, the quick measure to cTnI contents in whole blood. , it is necessary to which the whole blood sample collected is centrifuged in actual clinical practice, make that the detecting step of cTnI is cumbersome, inspection Cost is surveyed to increase；On the other hand, there is detection poor repeatability, be unfavorable for Quality Control in colloidal gold detection method, easily occur misreading erroneous judgement etc. Phenomenon.
The content of the invention
Therefore, the technical problem to be solved in the present invention is to overcome cTnI detections of the prior art can not realize to whole blood The defects of pattern detection.
For this reason, the present invention provides a kind of cTnI detection kits, including：Calibration object, cleaning solution, substrate solution, pre- place Manage liquid, enzyme conjugates working solution and magnetic bead conjugate working solution；Contain imidazoles, the enzyme conjugates work in the pretreatment fluid CTnI antibody containing enzyme mark in liquid, the magnetic bead containing cTnI antibody mark in the magnetic bead conjugate working solution.
Above-mentioned cTnI detection kits, the cTnI antibody of the enzyme mark is the cTnI antibody of alkali phosphatase enzyme mark.
Above-mentioned cTnI detection kits, the cTnI antibody of the alkali phosphatase enzyme mark are prepared by the following method：
A. cTnI antibody is activated
1. adding the cTnI antibody of 2mg/ml into the TCEP solution of 0.5M, to the final concentration of 1.25mM of TCEP, mix, Reaction is stood at room temperature；
2. by the antibody-solutions desalination after reaction, in the Triethanolamine buffer for replacing pH 7.3,100mM；
B. activated alkaline phosphatase
3. adding the Sulfo-SMCC solution of 17.5mg/mL into alkaline phosphatase enzyme solutions, mix, stand at room temperature；Its In, Sulfo-SMCC solution is that Sulfo-SMCC reagents are added in dimethylformamide to be formulated, Sulfo-SMCC examinations The molar ratio that agent is added with alkaline phosphatase is (10~15)：1；
4. continuously adding the glycine solution of 1M, mix, stand reaction at room temperature；Wherein, glycine and Sulfo-SMCC The molar ratio of reagent dosage is (10~20)：1；
5. by the alkaline phosphatase enzyme solutions desalination after reaction, in the Triethanolamine buffer for replacing pH 7.3,100mM；
C. the cTnI antibody-solutions activated in step A and the alkaline phosphatase enzyme solutions activated in step B are uniformly mixed, 2 18~24h of reaction is stood at a temperature of~8 DEG C；Wherein, cTnI antibody and the molar ratio that alkaline phosphatase adds are 1：2；
D. the N-ethylomaleimide solution of 12.5mg/ml is added in the solution after being reacted into step C, is mixed It is even, stand react at room temperature, solution after purification reaction, obtains the cTnI antibody of alkali phosphatase enzyme mark.
Above-mentioned cTnI detection kits, the cleaning solution are the Tris containing SBS, Tween-20 and Proclin-300 Buffer solution, the substrate solution are enzyme-catalyzed chemical luminescence substrate solution, calibration object calibration object diluent preparing The HEPES buffer solution of cTnI.
Above-mentioned cTnI detection kits, further include：Quality-control product, quality-control product calibration object diluent preparing The HEPES buffer solution of cTnI.
Above-mentioned cTnI detection kits, the calibration object dilution include：The BES of 20~50mM, 150~300mM NaCI, 1% horse serum, the mannitol of the Proclin-300 of 0.5~5mM and 1%, the pH of the calibration object dilution is 6.5 ~7.5.
Above-mentioned cTnI detection kits, the concentration of the imidazoles is 0.8~1.5mM, the cTnI antibody of the enzyme mark Concentration be 0.6~1.8 μ g/ml, the concentration of the cTnI antibody of the marked by magnetic bead is 0.2~0.5mg/ml.
Above-mentioned cTnI detection kits, the pretreatment fluid further include：The NaCl of Tris, 150mM of 25~100mM, 1 ~5% sucrose, 1~5% glycerine, 0.1% BSA, 0.05~0.2% Tween-20 and 0.05~0.2% Proclin-300, the pH of the pretreatment fluid is 7.0~7.5；
The enzyme conjugates working solution further includes：The Tris of 20~50mM, the NaCl of 150~300mM, 1% BSA, 5% Sucrose, 5% glycerine, 0.1% Tween-20, the ZnCl of 0.02~5mM2, 0.02~5mM MgCl2With 0.05~ 0.2% Proclin300, the pH of the enzyme conjugates working solution is 7.0~9.0；
The magnetic bead conjugate working solution further includes：The NaCl of the Tris of 20~50mM, 150~300mM, 1% BSA, 1% sucrose, 1% gelatin, 0.1% Tween-20, the Proclin300 of 1% PVP360 and 0.05~0.2%, it is described The pH of magnetic bead conjugate working solution is 7.0~9.0.
Above-mentioned cTnI detection kits, the particle diameter of the magnetic bead is 3 μm.
Above-mentioned cTnI detection kits, the magnetic bead of the cTnI antibody mark are prepared by the following method：
E. the MES buffer solutions of pH5.0,100mM are added into cTnI antibody, the concentration to antibody-solutions is 1~5mg/ml；
F. activated magnetic beads
1) the magnetic bead solution of 100mg/ml is placed on magnet stand and stood, Magneto separate abandons supernatant；Wherein, magnetic bead solution with The mass ratio of cTnI antibody is 100：1；
2) supernatant is abandoned with the magnetic bead obtained in the MES buffer solution steps 1) of pH5.0,100mM, Magneto separate；
3) magnetic bead obtained in step 2) is dissolved in the MES buffer solutions of 100mM, continuously adds 0.23 times of magnetic bead weight The EDC solution of the NHS solution of 10mg/ml and the 10mg/ml of 0.1 times of magnetic bead weight, at room temperature oscillating reactions；Wherein, NHS solution To be dissolved in NHS reagents in the MES buffer solutions of pH5.0,100mM to obtain, EDC solution be by EDC reagents be dissolved in pH5.0, Obtained in the MES buffer solutions of 100mM；
4) the magnetic bead solution Magneto separate after reaction in step 3) is abandoned into supernatant, is then dissolved in the MES buffer solutions of 100mM；
G. the cTnI antibody-solutions obtained in step E are mixed with the magnetic bead solution activated in step F, crosslinking is anti-at room temperature 16~24h is answered, then Magneto separate abandons supernatant, continuously adds magnetic bead confining liquid CE210, when room temperature reaction 16~24 is small after mixing；
H. supernatant will be abandoned after the magnetic bead solution Magneto separate obtained in step G, then cleans magnetic bead, obtain cTnI antibody mark Magnetic bead.
The application method of above-mentioned cTnI detection kits, comprises the following steps：
(1) pretreatment fluid is added into sample to be tested, magnetic bead working solution, enzyme conjugates working solution are added after mixing, is mixed Reacted afterwards at a temperature of 37 DEG C~42 DEG C；Wherein, the volume ratio that the pretreatment fluid is added with the sample to be tested≤ 4, the volume ratio that the pretreatment fluid, the magnetic bead working solution and the enzyme conjugates working solution add is 1：5：5；
(2) reaction solution obtained in step (1) is subjected to Magneto separate, collects magnetic bead；
(3) magnetic bead is cleaned, then adds enzyme-catalyzed chemical luminescence substrate solution, is mixed, detects luminous value.
The present invention has the following advantages that compared with the prior art：
1st, cTnI detection kits provided by the invention, including calibration object, cleaning solution, substrate solution, pretreatment fluid, enzyme knot Compound working solution and magnetic bead conjugate working solution；Contain imidazoles in the pretreatment fluid, contain in the enzyme conjugates working solution The cTnI antibody of enzyme mark, the magnetic bead containing cTnI antibody mark in the magnetic bead conjugate working solution.
Above-mentioned cTnI detection kits for sample to be tested when detecting, the cTnI antibody and cTnI antibody marks of enzyme mark The magnetic bead of note is combined by cTnI antibody with the different epitopes of cTnI antigens in sample to be tested respectively, forms " sandwich " structure. The Direct precipitation in externally-applied magnetic field, it is i.e. separable to be not required to centrifugation.Remove supernatant, clean the compound of precipitation, then add with Enzyme-catalyzed chemical luminescence substrate.Substrate, by catalytic pyrolysis, forms unstable excitation state intermediate, works as excitation state under the action of enzyme Intermediate just sends photon when returning to ground state, form luminescence-producing reaction, you can using the luminous intensity of light-emitting appearance detection reaction, shines Intensity is directly proportional to the cTnI concentration in sample to be tested, using the detection to luminous intensity, can realize in sample to be tested The Accurate Determining of cTnI contents.CTnI detection kits provided by the invention divide chemiluminescence immunoassay and magnetic particle It is combined from technology, high sensitivity, high specificity, the testing result of detection are accurate.
CTnI detection kits provided by the invention, contain imidazoles in pretreatment fluid, it can be quickly eliminated in whole blood Haemocyte, effectively avoids haemocyte from gulping down the possibility into magnetic bead.Finger Peripheral whole blood or anti-can be used directly in the kit of the present invention Solidifying venous whole is as measuring samples, without being pre-processed in advance to whole blood sample, so that it may be directly detected, greatly improve Detection speed, simplifies operating procedure, expands the scope of application of kit；Can automatically, operating in a key, 15 minutes left sides The right side can go out testing result, be well positioned to meet the demand of hospital emergency and outpatient service quick diagnosis, easy to promote and apply on a large scale.
It is provided by the invention including calibration object, calibration object is the cTnI standard samples of series concentration gradient, utilizes the present invention The kit of offer detects the chemiluminescence intensity under different cTnI concentration, and the standard that can draw to obtain kit detection is bent Line.Follow-up kit can be calculated in sample when for detecting the cTnI antigens in sample to be tested according to standard curve CTnI contents, realization accurately quantitatively detect.
2nd, pretreatment fluid provided by the invention, the concentration of enzyme conjugates working solution and magnetic bead conjugate working solution, component and The magnetic bead of each component content, the cTnI antibody that can be marked for enzyme and cTnI antibody mark provides the working environment of stabilization, in sample It can realize specific recognition and the detection to cTnI when cTnI antigen concentrations are 0.02ng/ml in this, detection range is up to 0.02 ~50ng/ml, is advantageously implemented high sensitivity and the wide scope detection to cTnI；Meanwhile improve the stabilization of kit detection Property, the repeatability of kit testing result is high, reduce be likely to occur during AMI diagnosis misread erroneous judgement.In addition, above-mentioned is molten Liquid system ensure that the high catalytic activity of enzyme, make enzymatic reaction substrate being capable of long-acting illuminating under its effect.It is advantageously implemented The high detection performance of cTnI detection kits.
3rd, the present invention provides alkali phosphatase enzyme mark cTnI antibody preparation method, utilize Sulfo-SMCC activation alkali Acid phosphatase, TCEP reduction cTnI antibody, makes to carry the sulfydryl that can be combined with the alkaline phosphatase of activation on cTnI antibody, By the combination of sulfydryl and amino, the coupling of antibody and enzyme is realized.The present invention realizes alkaline phosphatase and cTnI using reduction method The coupling of antibody, has high coupling efficiency, it is possible to increase the selectivity of coupling reaction and the homogeneity of coupled product；Reactant Triethanolamine buffer is used in liquid, triethanolamine has tertiary amine groups, without primary amine groups, will not coupling process produce interference, be The coupling of alkaline phosphatase and NT-ProBNP antibody provides stable coupling environment.The alkalescence obtained using above-mentioned preparation process The cTnI antibody of phosphatase enzyme mark has high enzymatic performance and the joint efficiency with cTnI antigens, so that containing alkaline phosphorus The enzyme conjugates working solution of the cTnI antibody of sour enzyme mark for the detection of cTnI antigens be with high sensitivity, high specificity and The advantages such as detection range is wide.
Glycine solution is added in preparation process to terminate priming reaction, and N-ethylomaleimide solution envelope Close unnecessary sulfydryl to react with end mark, effectively prevent the generation of nonspecific reaction, ensure that cTnI antibody and enzyme The stability of conjugate；The usage amount of each reactive material and temperature, pH conditions etc. are defined in reacting mark, are optimized Mark the condition of reaction.
4th, the present invention provides cTnI antibody mark magnetic bead preparation method, first lived using NHS and EDC in preparation process Change magnetic bead, improve the coupling efficiency of cTnI antibody and magnetic bead；The scattered steady of magnetic bead is ensure that using MES buffer solution magnetic beads Qualitative, effectively prevent influences to detect signal because of the condensation of magnetic bead, ensure that the accuracy of kit testing result；Kit During for whole blood test, complicated component in blood, preparation method provided by the invention adds after by cTnI antibody and magnetic bead coupling Enter magnetic bead confining liquid, effectively close the not site with antibody binding on magnetic bead, when avoiding late detection whole blood sample there may be Nonspecific combination, further ensure the accuracy of testing result.Realized at the same time to magnetic using magnetic bead confining liquid CE210 Effectively closing and ensure cTnI antibody activities for pearl, improves the detection performance of kit；To each reactive material in coupling reaction Usage amount and temperature, pH conditions are defined, and optimize the condition of coupling reaction.
5th, cTnI detection kits provided by the invention, can measure multiple samples at the same time on Full-automatic chemiluminescence apparatus This, realizes the rapid measure of high throughput of cTnI, and accuracy and detection efficiency are all greatly improved.
6th, the present invention provides the application method of cTnI detection kits, this method is the reaction pattern using one-step method, Reaction system is homogeneous, greatly improves the speed of reaction.
Brief description of the drawings
, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution of the prior art Embodiment or attached drawing needed to be used in the description of the prior art are briefly described, it should be apparent that, in describing below Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor Put, other attached drawings can also be obtained according to these attached drawings.
Fig. 1 is that the kit of the present invention detects the correlation of clinical serum with Bake Mann kit.
Embodiment
Technical scheme is clearly and completely described below in conjunction with attached drawing, it is clear that described implementation Example is part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ordinary skill Personnel's all other embodiments obtained without making creative work, belong to the scope of protection of the invention.
Illustrate embodiments of the present invention below by way of specific embodiment, unless otherwise stated, disclosed in this invention Experimental method use the art routine techniques, the percentage that the present invention uses is mass percent.Following implementations CTnI antibody in example is purchased from Hytest companies；Alkaline phosphatase is purchased from Roche companies；Carboxyl magnetic bead is purchased from JSR plants of formulas of Japan Commercial firm；CE210 is purchased from Japanese JSR Corp..
Embodiment 1
The present embodiment provides a kind of preparation method of the cTnI antibody of alkali phosphatase enzyme mark, comprise the following steps：
1st, the preservation concentration of cTnI labelled antibodies (namely cTnI antibody) is 2mg/mL, is added into cTnI labelled antibodies The TECP solution of 0.5M mixes, 25 DEG C stand reaction 60 minutes immediately to the final concentration of 1.25mM of TECP labelled antibodies.
2nd, the cTnI labelled antibodies solution obtained in step 1 carries out desalination, three ethanol of displacement to pH7.3,100mM immediately Amine aqueous solution, the concentration of determined by ultraviolet spectrophotometry antibody-solutions.
3rd, appropriate Sulfo-SMCC reagents are weighed, it is 17.5mg/mL solution to be made into concentration with dimethylformamide DMF.
4th, it is molten that the Sulfo-SMCC that step 2 is prepared is added into alkaline phosphatase enzyme solutions (AP solution, concentration 20mg/ml) The molar ratio 1 of liquid, alkaline phosphatase and Sulfo-SMCC：10, mix at once, 25 DEG C stand reaction 15 minutes.
5th, the glycine solution of 1M, glycine and Sulfo-SMCC are added in the alkaline phosphatase enzyme solutions obtained to step 4 The molar ratio of reagent is 10:1, mix at once, 25 DEG C stand reaction 10 minutes.
6th, the alkaline phosphatase enzyme solutions desalination at once for obtaining step 5, the triethanolamine of displacement to pH7.3,100mM are molten Liquid, ultraviolet spectrophotometry measure the concentration of alkaline phosphatase enzyme solutions；
7th, the antibody-solutions that step 2 has activated are mixed with the alkaline phosphatase enzyme solutions that step 6 has activated, the cTnI of activation The molar ratio of labelled antibody and the alkaline phosphatase of activation is 1：2, fully mix, when 2 DEG C of standing reactions 24 are small.
8th, appropriate N-ethylomaleimide (NEM) reagent is weighed, is matched somebody with somebody with the triethanolamine solution of pH7.3,100mM Into the NEM solution of 12.5mg/mL.
9th, the NEM solution that 1 percent debulking steps 8 are prepared is added in the solution terminated to step 7 cross-linking reaction, fully Mix, 25 DEG C stand reaction 30 minutes to close unnecessary sulfydryl.
The 10th, cross-linking agent solution that step 9 is terminated to reaction end is purified by modes such as dialysis, desalination or ultrafiltration concentrations.
11st, by the cTnI antibody concentrated solution measured concentrations of alkali phosphatase enzyme mark after purification, isometric glycerine is added, fully Mix, -20 DEG C save backup.
Embodiment 2
The present embodiment provides a kind of preparation method of the cTnI antibody of alkali phosphatase enzyme mark, comprise the following steps：
1st, the preservation concentration of cTnI labelled antibodies (namely cTnI antibody) is 2mg/mL, is added into cTnI labelled antibodies The TECP solution of 0.5M mixes, 25 DEG C stand reaction 60 minutes immediately to the final concentration of 1.25mM of TECP labelled antibodies.
2nd, the cTnI labelled antibodies solution obtained in step 1 carries out desalination, three ethanol of displacement to pH7.3,100mM immediately Amine aqueous solution, the concentration of determined by ultraviolet spectrophotometry antibody-solutions.
3rd, appropriate Sulfo-SMCC reagents are weighed, it is 17.5mg/mL solution to be made into concentration with dimethylformamide DMF.
4th, it is molten that the Sulfo-SMCC that step 2 is prepared is added into alkaline phosphatase enzyme solutions (AP solution, concentration 20mg/ml) The molar ratio 1 of liquid, alkaline phosphatase and Sulfo-SMCC：15, mix at once, 25 DEG C stand reaction 15 minutes.
5th, the glycine solution of 1M, glycine and Sulfo-SMCC are added in the alkaline phosphatase enzyme solutions obtained to step 4 The molar ratio of reagent is 10:1, mix at once, 25 DEG C stand reaction 10 minutes.
6th, the alkaline phosphatase enzyme solutions desalination at once for obtaining step 5, the triethanolamine of displacement to pH7.3,100mM are molten Liquid, ultraviolet spectrophotometry measure the concentration of alkaline phosphatase enzyme solutions；
7th, the antibody-solutions that step 2 has activated are mixed with the alkaline phosphatase enzyme solutions that step 6 has activated, the cTnI of activation The molar ratio of labelled antibody and the alkaline phosphatase of activation is 1：2, fully mix, when 8 DEG C of standing reactions 18 are small.
8th, appropriate N-ethylomaleimide (NEM) reagent is weighed, is matched somebody with somebody with the triethanolamine solution of pH7.3,100mM Into the NEM solution of 12.5mg/mL.
9th, the NEM solution that 1 percent debulking steps 8 are prepared is added in the solution terminated to step 7 cross-linking reaction, fully Mix, 25 DEG C stand reaction 30 minutes to close unnecessary sulfydryl.
The 10th, cross-linking agent solution that step 9 is terminated to reaction end is purified by modes such as dialysis, desalination or ultrafiltration concentrations.
11st, by the cTnI antibody concentrated solution measured concentrations of alkali phosphatase enzyme mark after purification, isometric glycerine is added, fully Mix, -20 DEG C save backup.
Embodiment 3
The present embodiment provides a kind of preparation method of the magnetic bead of cTnI antibody mark, comprise the following steps：
CTnI coated antibodies (namely cTnI antibody) the 1, are stored in the MES buffer solutions of pH5.0,100mM, antibody concentration is 1mg/mL；
2nd, the carboxyl magnetic bead solution of 100 times of antibody weight is taken, carboxyl magnetic bead solution concentration is 100mg/ml, carboxyl magnetic bead grain Footpath is 3 μm, and Magneto separate abandons supernatant；
3rd, wash：The magnetic bead that above-mentioned steps obtain is dissolved again with the MES buffer solutions of pH5.0,100mM, and Magneto separate is abandoned Clearly.
4th, repeat step 3 is once.
5th, the magnetic bead that the MES buffer solution steps 4 of appropriate 100mM obtain is added.
6th, appropriate NHS (n-hydroxysuccinimide) reagent is weighed, with the MES buffer solutions of pH5.0,100mM into dense Spend the solution for 10mg/mL；
7th, appropriate EDC (1- (3- dimethylamino-propyls) -3- ethyl carbodiimides) reagent is weighed, with pH5.0,100mM Solution of the MES buffer solutions into concentration for 10mg/mL；
8th, the magnetic bead solution obtained into step 5 adds the NHS solution and 0.1 times of magnetic of the step 6 of 0.23 times of magnetic bead weight The EDC solution of the step 7 of pearl weight, reacts 30 minutes for 25 DEG C on blending instrument.
9th, magnetic bead solution Magneto separate after reaction, abandons supernatant.
10th, the magnetic bead that the MES buffer solution steps 9 of appropriate 100mM obtain is added.
11st, the cTnI coated antibodies of step 1 are added in the magnetic bead that step 10 obtains, cTnI coated antibodies and carboxyl magnetic Pearl solution quality ratio is 1:100, when room temperature cross-linking reaction 16 is small on blending instrument.
12nd, after being crosslinked, Magneto separate, supernatant, which is collected, surveys crosslinking rate.Repeat the above steps 3 times.
13rd, the magnetic bead obtained to step 12 adds magnetic bead confining liquid, when room temperature reaction 24 is small on blending instrument.
14th, above-mentioned magnetic bead solution Magneto separate after reaction, abandons supernatant.
15th, magnetic bead is washed 3 times with magnetic bead cleaning solution.
16th, the magnetic bead after washing is resuspended in magnetic bead and preserves in liquid, and 2 DEG C save backup.
Embodiment 4
The present embodiment provides a kind of preparation method of the magnetic bead of cTnI antibody mark, comprise the following steps：
CTnI coated antibodies (namely cTnI antibody) the 1, are stored in the MES buffer solutions of pH5.0,100mM, antibody concentration is 1mg/mL；
2nd, the carboxyl magnetic bead solution of 100 times of antibody weight is taken, carboxyl magnetic bead solution concentration is 100mg/ml, carboxyl magnetic bead grain Footpath is 3 μm, and Magneto separate abandons supernatant；
3rd, wash：The magnetic bead that above-mentioned steps obtain is dissolved again with the MES buffer solutions of pH5.0,100mM, and Magneto separate is abandoned Clearly.
4th, repeat step 3 is once.
5th, the magnetic bead that the MES buffer solution steps 4 of appropriate 100mM obtain is added.
6th, appropriate NHS reagents are weighed, the solution for being 10mg/mL into concentration with the MES buffer solutions of pH5.0,100mM；
7th, appropriate EDC reagents are weighed, the solution for being 10mg/mL into concentration with the MES buffer solutions of pH5.0,100mM；
8th, the magnetic bead solution obtained into step 5 adds the NHS solution and 0.1 times of magnetic of the step 6 of 0.23 times of magnetic bead weight The EDC solution of the step 7 of pearl weight, reacts 30 minutes for 25 DEG C on blending instrument.
9th, magnetic bead solution Magneto separate after reaction, abandons supernatant.
10th, the magnetic bead that the MES buffer solution steps 9 of appropriate 100mM obtain is added.
11st, the cTnI coated antibodies of step 1 are added in the magnetic bead that step 10 obtains, cTnI coated antibodies and carboxyl magnetic Pearl solution quality ratio is 1:100, when room temperature cross-linking reaction 24 is small on blending instrument.
12nd, after being crosslinked, Magneto separate, supernatant, which is collected, surveys crosslinking rate.Repeat the above steps 3 times.
13rd, the magnetic bead obtained to step 12 adds magnetic bead confining liquid, when room temperature reaction 16 is small on blending instrument.
14th, above-mentioned magnetic bead solution Magneto separate after reaction, abandons supernatant.
15th, magnetic bead is washed 3 times with magnetic bead cleaning solution.
16th, the magnetic bead after washing is resuspended in magnetic bead and preserves in liquid, and 8 DEG C save backup.
Embodiment 5
The present invention provides a kind of cTnI detection kits, including：Pretreatment fluid, enzyme conjugates working solution and magnetic bead conjugate Working solution, cleaning solution, substrate solution, calibration object and quality-control product.The component of each reagent is as follows in cTnI detection kits：
1st, calibration object and quality-control product
By cTnI antigens by definite concentration of tracing to the source, cTnI calibration objects and cTnI Quality Controls are prepared with calibration object diluted Product.Such as the concentration of calibration object is respectively：0.05,0.5,1,5,25,50ng/ml, quality-control product concentration is 5ng/ml.
The specific component of calibration object dilution is：The BES (double (2- the ethoxys) -2-aminoethanesulfonic acids of N, N-) of 20mM, The NaCl of 300mM, 1% horse serum, the mannitol of the Proclin-300 of 0.5mM and 1%, the pH of calibration object dilution are 6.5。
2nd, pretreatment fluid
Imidazoles containing 1.5mM in pretreatment fluid；Additionally include：The NaCl of Tris, 150mM of 25mM, 1% sugarcane Sugar, 5% glycerine, 0.1% BSA, the Proclin-300 of 0.05% Tween-20 and 0.2%, the pH of pretreatment fluid are 7.0。
3rd, enzyme conjugates working solution
The cTnI antibody of alkali phosphatase enzyme mark containing 0.6 μ g/ml in enzyme conjugates working solution, alkali phosphatase enzyme mark CTnI antibody be made using the preparation method shown in embodiment 1.
Further included in enzyme conjugates working solution：The NaCl of Tris, 150mM of 50mM, 1% BSA, 5% sucrose, 5% Glycerine, the ZnCl of 0.1% Tween-20,0.2mM2, 5mM MgCl2With 0.05% Proclin300, enzyme conjugates work The pH for making liquid is 7.0.
4th, magnetic bead conjugate working solution
The cTnI antibody of marked by magnetic bead containing 0.5mg/ml in magnetic bead conjugate working solution, the cTnI antibody of marked by magnetic bead It is made using the preparation method shown in embodiment 3.
Magnetic bead conjugate working solution further includes：The NaCl of the Tris of 20mM, 300mM, 1% BSA, 1% sucrose, 1% Gelatin, 0.1% Tween-20, the Proclin300 of 1% PVP360 and 0.05%, the pH of magnetic bead conjugate working solution be 7.0。
5th, substrate solution
Accurately weigh 3- (the spiral adamantane of 2-) -4- methoxyl groups -4- (3- phosphorus oxygens acyl)-phenyl -1,2- dioxanes two Sodium salt (AMPPD) 0.5g, trishydroxymethylaminomethane 18g, NaCl 100g, hexadecyltrimethylammonium chloride 0.02g, water are fixed It is 9.4 ± 0.05 to hold to 1000mL, adjustment chemical luminous substrate pH value of solution；This chemical luminous substrate is for alkaline phosphatase Epidioxy ethane substrate, it is necessary to be kept in dark place under the conditions of 2~8 DEG C, the used time slowly mixes again.
6th, cleaning solution
Tris 1211.4mg, NaCl 9g, SBS 3g, Tween-20 1g, Proclin-300 are added into container successively 1g, water 900mL, mix to each component and dissolve, and adjust pH value of solution to 8.0 ± 0.5 with sodium hydroxide solution, add water and be settled to 1000mL, mixes 30 minutes, 0.22 μm of filtering obtains cleaning solution.
Embodiment 6
The present invention provides a kind of cTnI detection kits, including：Pretreatment fluid, enzyme conjugates working solution and magnetic bead conjugate Working solution, cleaning solution, substrate solution, calibration object and quality-control product.The component of each reagent is as follows in cTnI detection kits：
1st, calibration object and quality-control product
By cTnI antigens by definite concentration of tracing to the source, cTnI calibration objects and cTnI Quality Controls are prepared with calibration object diluted Product.Such as the concentration of calibration object is respectively：0.05,0.5,1,5,25,50ng/ml, quality-control product concentration is 5ng/ml.
The specific component of calibration object dilution is：The NaCl of the BES of 50mM, 150mM, 1% horse serum, 5mM The mannitol of Proclin-300 and 1%, the pH of calibration object dilution is 7.5.
2nd, pretreatment fluid
Imidazoles containing 0.8mM in pretreatment fluid；Additionally include：The NaCl of Tris, 150mM of 100mM, 5% sugarcane Sugar, 1% glycerine, 0.1% BSA, the Proclin-300 of 0.2% Tween-20 and 0.05%, the pH of pretreatment fluid are 7.5。
3rd, enzyme conjugates working solution
The cTnI antibody of alkali phosphatase enzyme mark containing 1.8 μ g/ml in enzyme conjugates working solution, alkali phosphatase enzyme mark CTnI antibody be made using the preparation method shown in embodiment 2.
Further included in enzyme conjugates working solution：The NaCl of Tris, 300mM of 20mM, 1% BSA, 5% sucrose, 5% Glycerine, the ZnCl of 0.1% Tween-20,5mM2, 0.02mM MgCl2With 0.2% Proclin300, enzyme conjugates work The pH for making liquid is 9.0.
4th, magnetic bead conjugate working solution
The cTnI antibody of marked by magnetic bead containing 0.2mg/ml in magnetic bead conjugate working solution, the cTnI antibody of marked by magnetic bead It is made using the preparation method shown in embodiment 4.
Magnetic bead conjugate working solution further includes：The NaCl of the Tris of 50mM, 150mM, 1% BSA, 1% sucrose, 1% Gelatin, 0.1% Tween-20, the Proclin300 of 1% PVP360 and 0.2%, the pH of magnetic bead conjugate working solution be 9.0。
5th, substrate solution
Accurately weigh 3- (the spiral adamantane of 2-) -4- methoxyl groups -4- (3- phosphorus oxygens acyl)-phenyl -1,2- dioxanes two Sodium salt (AMPPD) 0.5g, trishydroxymethylaminomethane 18g, NaCl 100g, hexadecyltrimethylammonium chloride 0.02g, water are fixed It is 9.4 ± 0.05 to hold to 1000mL, adjustment chemical luminous substrate pH value of solution；This chemical luminous substrate is for alkaline phosphatase Epidioxy ethane substrate, it is necessary to be kept in dark place under the conditions of 2~8 DEG C, the used time slowly mixes again.
6th, cleaning solution
Tris 1211.4mg, NaCl 9g, SBS 3g, Tween-20 1g, Proclin-300 are added into container successively 1g, water 900mL, mix to each component and dissolve, and adjust pH value of solution to 8.0 ± 0.5 with sodium hydroxide solution, add water and be settled to 1000mL, mixes 30 minutes, 0.22 μm of filtering obtains cleaning solution.
Embodiment 7
The present embodiment provides the step using cTnI antigens in the cTnI detection kits measure whole blood provided in embodiment 5 Suddenly, finger Peripheral whole blood is taken as sample to be tested：
1st, cTnI calibration objects, cTnI quality-control products and the sample to be tested of 10 μ l is added respectively into three reaction tubes；
2nd, the pretreatment fluid that 40 μ l are added in each reaction tube mixes；
3rd, mixed after 50 μ l enzyme conjugates working solutions and 50 μ l magnetic bead working solutions are added in each test tube；42 DEG C of reactions 5min；
4th, the solution after being reacted in step 3 is subjected to Magneto separate, collects magnetic bead；300 μ l cleanings are added in each reaction tube Liquid cleans magnetic bead, repeats 3-5 times, removes cleaning solution；
5th, 100 μ l substrate solutions are added in each reaction tube, luminous value is detected after mixing.
6th, mark curve is drawn using the concentration and luminous value of standard items；
The 7th, the luminous value of sample to be tested is substituted into the content for the cTnI that standard curve is obtained in sample to be tested.
The cTnI detection methods provided in the present embodiment are equally applicable to the cTnI inspections of anti-freezing venous whole, serum or blood plasma Survey.
Embodiment 8
The present embodiment provides the step using cTnI antigens in the cTnI detection kits measure whole blood provided in embodiment 6 Suddenly, finger Peripheral whole blood is taken as sample to be tested：
1st, cTnI calibration objects, cTnI quality-control products and the sample to be tested of 10 μ l is added respectively into three reaction tubes；
2nd, the pretreatment fluid that 25 μ l are added in each reaction tube mixes；
3rd, mixed after 50 μ l enzyme conjugates working solutions and 50 μ l magnetic bead working solutions are added in each test tube；37 DEG C of reactions 10min；
4th, the solution after being reacted in step 3 is subjected to Magneto separate, collects magnetic bead；300 μ l cleanings are added in each reaction tube Liquid cleans magnetic bead, repeats 3-5 times, removes cleaning solution；
5th, 100 μ l substrate solutions are added in each reaction tube, luminous value is detected after mixing.
6th, mark curve is drawn using the concentration and luminous value of standard items；
The 7th, the luminous value of sample to be tested is substituted into the content for the cTnI that standard curve is obtained in sample to be tested.
The cTnI detection methods provided in the present embodiment are equally applicable to the cTnI inspections of anti-freezing venous whole, serum or blood plasma Survey.
1 linear verification of experimental example
Standard items or quality-control product with traceability are taken, concentration is as far as possible high, sample is diluted with physiological saline, every point Measure 3 times, is averaged, and as a result makees regression straight line with expected concentration, regression coefficient r ＞ 0.99 are calculated, illustrate the present invention The dilution good linearity of the kit of offer.
2 precision of experimental example is verified
The serum high level quality-control product with traceability, each portion of low value quality-control product are taken, 10 inspections are carried out to every part of quality-control product Survey, totally 10 testing results will calculate average value, standard value.According to coefficient of variation CV=(standard deviation/average value) × 100%, it is calculated：CV1(25ng/mL)=4%, CV2(5ng/mL)=2%.It follows that kit provided by the invention With higher precision.
3 accuracy validation of experimental example
Take the serum high level quality-control product with traceability, each portion of low value quality-control product, with the present invention kit respectively into Row detection, it is each to detect 5 times, average value is calculated, is compareed afterwards with quality-control product target value.The results show that the kit inspection of the present invention Measured value is approached with target value, illustrates that kit provided by the invention has higher accuracy.
4 sensitivity of experimental example is verified
Take the quality-control product with traceability to be diluted to detection range lower limit (0.02ng/mL) to be nearby measured, repeat to survey It is 3 times fixed, average value is calculated, is compareed afterwards with quality-control product target value.The results show that the kit detected value of the present invention connects with target value Closely, illustrate that kit provided by the invention has higher sensitivity.
5 detection range of experimental example is verified
The standard items with traceability are taken, luminescence phenomenon has been detected whether respectively after diluting different multiples, the results show that In the range of concentration is 0.02~50ng/ml, there is luminescence phenomenon, show the kit detection range of the present invention for 0.02~ 50ng/ml。
6 relevance verification of experimental example
200 parts of serum samples are taken, kit more of the invention and the cTnI detection kits detection of Bake Mann are tied The correlation of fruit, the results are shown in Figure 1.Wherein：Abscissa is the testing result of Bake Mann kit, and ordinate is this hair The testing result of bright kit, coefficient R2=0.9774, as shown in Figure 1, pattern detection result of the present invention is known with the industry Name Products testing result is compared, as a result no significant difference.
Obviously, the above embodiments are merely examples for clarifying the description, and the restriction not to embodiment.It is right For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of change or Change.There is no necessity and possibility to exhaust all the enbodiments, and the obvious change thus extended out or Among changing still in the protection domain of the invention.

Claims (11)

  1. A kind of 1. cTnI detection kits, it is characterised in that including：Calibration object, cleaning solution, substrate solution, pretreatment fluid, enzyme knot Compound working solution and magnetic bead conjugate working solution；Contain imidazoles in the pretreatment fluid, contain in the enzyme conjugates working solution The cTnI antibody of enzyme mark, the magnetic bead containing cTnI antibody mark in the magnetic bead conjugate working solution.
  2. 2. cTnI detection kits according to claim 1, it is characterised in that the cTnI antibody of the enzyme mark is alkalescence The cTnI antibody of phosphatase enzyme mark.
  3. 3. cTnI detection kits according to claim 2, it is characterised in that the cTnI of the alkali phosphatase enzyme mark resists Body is prepared by the following method：
    A. cTnI antibody is activated
    1. adding the cTnI antibody of 2mg/ml into the TCEP solution of 0.5M, to the final concentration of 1.25mM of TCEP, mix, room temperature It is lower to stand reaction；
    2. by the antibody-solutions desalination after reaction, in the Triethanolamine buffer for replacing pH 7.3,100mM；
    B. activated alkaline phosphatase
    3. adding the Sulfo-SMCC solution of 17.5mg/mL into alkaline phosphatase enzyme solutions, mix, stand at room temperature；Wherein, Sulfo-SMCC solution is that Sulfo-SMCC reagents are added in dimethylformamide to be formulated, Sulfo-SMCC reagents with The molar ratio that alkaline phosphatase adds is (10~15)：1；
    4. continuously adding the glycine solution of 1M, mix, stand reaction at room temperature；Wherein, glycine and Sulfo-SMCC reagents The molar ratio of addition is (10~20)：1；
    5. by the alkaline phosphatase enzyme solutions desalination after reaction, in the Triethanolamine buffer for replacing pH 7.3,100mM；
    C. the cTnI antibody-solutions activated in step A and the alkaline phosphatase enzyme solutions activated in step B are uniformly mixed, 2~8 DEG C At a temperature of stand reaction 18~24h；Wherein, cTnI antibody and the molar ratio that alkaline phosphatase adds are 1：2；
    D. the N-ethylomaleimide solution of 12.5mg/ml is added in the solution after being reacted into step C, is mixed, room Lower stand of temperature is reacted, and solution after purification reaction, obtains the cTnI antibody of alkali phosphatase enzyme mark.
  4. 4. according to claim 1-3 any one of them cTnI detection kits, it is characterised in that the cleaning solution be containing The Tris buffer solutions of SBS, Tween-20 and Proclin-300, the substrate solution is enzyme-catalyzed chemical luminescence substrate solution, described Calibration object is the HEPES buffer solution with the cTnI of calibration object diluent preparing.
  5. 5. according to claim 1-4 any one of them cTnI detection kits, it is characterised in that further include：Quality-control product, it is described Quality-control product is the HEPES buffer solution with the cTnI of calibration object diluent preparing.
  6. 6. cTnI detection kits according to claim 4 or 5, it is characterised in that the calibration object dilution includes：20 The BES of~50mM, the NaCI of 150~300mM, 1% horse serum, the mannitol of the Proclin-300 of 0.5~5mM and 1%, The pH of the calibration object dilution is 6.5~7.5.
  7. 7. according to claim 1-6 any one of them cTnI detection kits, it is characterised in that the concentration of the imidazoles is 0.8~1.5mM, the concentration of the cTnI antibody of the enzyme mark is 0.6~1.8 μ g/ml, the cTnI antibody of the marked by magnetic bead Concentration is 0.2~0.5mg/ml.
  8. 8. according to claim 1-7 any one of them cTnI detection kits, it is characterised in that the pretreatment fluid also wraps Include：The NaCl of Tris, 150mM of 25~100mM, 1~5% sucrose, 1~5% glycerine, 0.1% BSA, 0.05~ 0.2% Tween-20 and 0.05~0.2% Proclin-300, the pH of the pretreatment fluid is 7.0~7.5；
    The enzyme conjugates working solution further includes：The Tris of 20~50mM, the NaCl of 150~300mM, 1% BSA, 5% sugarcane Sugar, 5% glycerine, 0.1% Tween-20, the ZnCl of 0.02~5mM2, 0.02~5mM MgCl2With 0.05~0.2% Proclin300, the pH of the enzyme conjugates working solution is 7.0~9.0；
    The magnetic bead conjugate working solution further includes：The NaCl of the Tris of 20~50mM, 150~300mM, 1% BSA, 1% Sucrose, 1% gelatin, 0.1% Tween-20, the Proclin300 of 1% PVP360 and 0.05~0.2%, the magnetic bead The pH of conjugate working solution is 7.0~9.0.
  9. 9. according to claim 1-8 any one of them cTnI detection kits, it is characterised in that the particle diameter of the magnetic bead is 3 μ m。
  10. 10. cTnI detection kits according to claim 1, it is characterised in that the magnetic bead of the cTnI antibody mark leads to Cross following methods preparation：
    E. the MES buffer solutions of pH5.0,100mM are added into cTnI antibody, the concentration to antibody-solutions is 1~5mg/ml；
    F. activated magnetic beads
    1) the magnetic bead solution of 100mg/ml is placed on magnet stand and stood, Magneto separate abandons supernatant；Wherein, magnetic bead solution resists with cTnI The mass ratio of body is 100：1；
    2) supernatant is abandoned with the magnetic bead obtained in the MES buffer solution steps 1) of pH5.0,100mM, Magneto separate；
    3) magnetic bead obtained in step 2) is dissolved in the MES buffer solutions of 100mM, continuously adds 0.23 times of magnetic bead weight The EDC solution of the NHS solution of 10mg/ml and the 10mg/ml of 0.1 times of magnetic bead weight, at room temperature oscillating reactions；Wherein, NHS solution To be dissolved in NHS reagents in the MES buffer solutions of pH5.0,100mM to obtain, EDC solution be by EDC reagents be dissolved in pH5.0, Obtained in the MES buffer solutions of 100mM；
    4) the magnetic bead solution Magneto separate after reaction in step 3) is abandoned into supernatant, is then dissolved in the MES buffer solutions of 100mM；
    G. the cTnI antibody-solutions obtained in step E are mixed with the magnetic bead solution activated in step F, at room temperature cross-linking reaction 16 ~24h, then Magneto separate abandon supernatant, continuously add magnetic bead confining liquid CE210, after mixing react at room temperature 16~24 it is small when；
    H. supernatant will be abandoned after the magnetic bead solution Magneto separate obtained in step G, then cleans magnetic bead, obtain the magnetic of cTnI antibody mark Pearl.
  11. 11. the application method of claim 1-10 any one of them cTnI detection kits, it is characterised in that including following step Suddenly：
    (1) pretreatment fluid is added into sample to be tested, magnetic bead working solution, enzyme conjugates working solution are added after mixing, after mixing Reacted at a temperature of 37 DEG C~42 DEG C；Wherein, volume ratio≤4 that the pretreatment fluid is added with the sample to be tested, institute It is 1 to state the volume ratio that pretreatment fluid, the magnetic bead working solution and the enzyme conjugates working solution add：5：5；
    (2) reaction solution obtained in step (1) is subjected to Magneto separate, collects magnetic bead；
    (3) magnetic bead is cleaned, then adds enzyme-catalyzed chemical luminescence substrate solution, is mixed, detects luminous value.
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CN112014575A (en) * 2020-09-03 2020-12-01 武汉生之源生物科技股份有限公司 CYFRA21-1 determination kit and preparation method thereof
CN112014575B (en) * 2020-09-03 2023-08-08 武汉生之源生物科技股份有限公司 CYFRA21-1 determination kit and preparation method thereof
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