A kind of myoglobin assay kit and its application methodTechnical field
The invention belongs to external diagnosis reagent case field, and in particular to a kind of myoglobin assay kit and its userMethod.
Background technology
Myoglobins (myoglobin, MB) is skeletal muscle and Cardiac-specific oxygen conjugate albumen, it has a polypeptide chainForm, there is 153 amino acid residues and a blood red prothetic group, molecular weight 17.8kD, its have in myocyte transhipment andStore the function of oxygen.It is seldom in blood containing a large amount of MB in normal human's cardiac muscle, skeletal muscle, but when cardiac muscle or skeletal muscle damageWhen hindering, MB can be discharged into circulating from musculature, and through glomerular filtration, is appeared in urine.Therefore, blood and urineIn MB measure can be used for some myopathies and cardiopathic diagnosis, such as acute myocardial infarction AMI (AMI), acute injury of muscle, acute and chronicKidney failure, for a long time severe congestive heart failure, the disease such as shock, muscular dystrophy, amyotrophia and polymyositis.
AMI is a kind of serious disease for jeopardizing human life, when after AMI generations 2~4 it is small when interior, MB in patients serumConcentration increases sharply, and 8~10 peak when small, and can continue until 12 it is small when, and there is higher sensitiveness.Due to boneMuscular diseases, kidney function damage, inflammation, systemic loupus erythematosus, dermatitis can cause the rise of MB, therefore it is as AMI'sDiagnosis index specificity is relatively low, but due to its higher sensitivity, can generally be used as investigation index, assist AMI early diagnosis.Because of the half-life shorts of MB in blood, interior recovery is to normal level when 24 is small after rise, therefore MB can whether there is again as observationInfarct and infarct whether there is the index of extension.
The detection method of existing MB mainly has radioimmunoassays, enzyme-linked immunization, chemoluminescence method, enzyme-linked fluorescence analysisMethod and immunoturbidimetry.Such as：Chinese patent literature CN102565419B discloses a kind of myoglobin assay kit, the reagentBox includes reagent R1 and reagent R2, wherein：R1 is buffer solution, and R2 is to be placed in buffering with MB antibody sensitized polystyrene latex particlesFormed in liquid.For MB in the latex particle and sample of coating MB antibody immune response occurs for the testing principle used in the kitAfterwards, aggregated particle is formed, under certain wavelength, forms caused turbidity by measuring aggregation, you can measure MB in sampleContent.However, technology disclosed in above-mentioned document is coated with emulsion particle using MB antibody, and MB antibody is polyclonal antibody, moreSpecific poor, the polyclonal antibody prepared even with identical antigen of clonal antibody, there is also difference between different batchesIt is different.Therefore, thus obtained kit specificity is undesirable, false positive phenomenon easily occurs, it is impossible to meets the need of clinical diagnosisAsk.In addition, the kit is detected using blood plasma or serum sample, it is necessary to venous blood sampling and blood sampling volume is big, before sample useAlso need to centrifuge, can not be directly using finger micro whole blood as detection sample, detection speed is slow, therefore is not well positioned to meet doctorInstitute's emergency treatment and the demand of outpatient service quick diagnosis.
The content of the invention
Therefore, the technical problem to be solved in the present invention is existing myoglobin assay kit specificity is not high, and adoptIt is sample with blood plasma or serum, the defects of cumbersome and detection range is narrow, so as to provide, a species specificity is high, can directly makeThe myoglobin assay kit of sample is used as by the use of whole blood；Further, make present invention also offers the detection kitUse method.
In order to solve the above technical problem, the present invention provides a kind of myoglobin assay kit, including：
(1) calibration object；
(2) reagent R1；
(3) enzyme conjugates working solution R2；
(4) magnetic bead conjugate working solution M；
(5) cleaning solution；
(6) chemical luminous substrate；
The reagent R1 contains imidazole components, and the cleaning solution contains lauryl sodium sulfate component.
Preferably, the reagent R1 includes：The trishydroxymethylaminomethane of 25~100mmol/L, 150mmol/L NaCl,1~5% sucrose, 1~5% glycerine, 0.1% bovine serum albumin(BSA), 0.4~2mmol/L imidazoles, 0.05~0.2% polysorbas20 and0.05~0.2%Proclin300, pH are 7.0~7.5.
Preferably, the calibration object is to add myoglobins in calibration object dilution to be formulated, and the calibration object is diluteReleasing liquid includes：The 4- hydroxyethyl piperazineethanesulfonic acids of 20~50mmol/L, 150~300mmol/L NaCl, 1% bovine serum albuminIn vain, 0.5~5mmol/L Proclin300, pH are 6.5~7.5.
Preferably, the concentration of the enzyme conjugates working solution R2 is 0.5~2 μ g/mL, and enzyme knot is added by enzyme conjugates solutionCompound diluent preparing forms, wherein, the dilution includes：20~50mmol/L2- (N- morpholines) ethyl sulfonic acid, 150~300mmol/L NaCl, 1% bovine serum albumin(BSA), 5% sucrose, 5% glycerine, 0.1% polysorbas20,0.05~5mmol/LZnCl2, 0.05~5mmol/LMgCl2, 0.05~0.2%Proclin300, pH be 6.0~6.5.
Preferably, the concentration of the magnetic bead conjugate working solution M is 0.1~0.5mg/mL, is added by magnetic bead conjugate solutionEnter magnetic bead diluent preparing to form, wherein, the dilution includes：20~50mmol/L4- hydroxyethyl piperazineethanesulfonic acids, 150~300mmol/L NaCl, 1% bovine serum albumin(BSA), 1% sucrose, 1% gelatin, 0.1% polysorbas20,1% polyvinylpyrrolidone,0.5~5%Proclin300, pH are 7.0~8.0.
Preferably, the main component of the enzyme conjugates is the myoglobins labelled antibody of alkali phosphatase enzyme mark.
Preferably, the main component of the magnetic bead conjugate is the carboxyl magnetic bead of myoglobins coated antibody coupling, describedThe particle diameter of carboxyl magnetic bead is 1.3~4 μm.
Preferably, the myoglobins labelled antibody of the alkali phosphatase enzyme mark is by the way that the myoglobins of activation is markedAntibody and the alkaline phosphatase of activation are 1 in molar ratio：(1~2) mix, in three ethanol that pH is 7.3 under 2~8 DEG C of environment18~24h is reacted in amine buffer solution to obtain.
Preferably, the myoglobins labelled antibody of the activation is anti-by the way that Traut ' s solution and myoglobins are markedLiquid solution is 1 in molar ratio：(15~30) mix, under room temperature pH be 8.5 Triethanolamine buffer in react 12~18min is obtained；The alkaline phosphatase of the activation is by by 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic acidBase succinimide ester sodium salt solution is 1 in molar ratio with alkaline phosphatase enzyme solutions：(10~15) mix, in existing under room temperature12~18min is reacted in N,N-dimethylformamide solution to obtain.
It is highly preferred that the concentration of Traut ' the s solution is 1.3~1.5mg/mL；The myoglobins labelled antibody is moltenThe concentration of liquid is 2~5mg/mL；4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group succinimide ester sodiumThe concentration of salting liquid is 16~18mg/mL；The concentration of the alkaline phosphatase enzyme solutions is 16~20mg/mL.
Preferably, the preparation of the myoglobins labelled antibody of the alkali phosphatase enzyme mark, further includes：In the activationGlycine solution is added in myoglobins labelled antibody, the amount of the material of the glycine is the amount of Traut ' s reagent materials10~20 times, fully mix after 8~12min of reaction at room temperature.
Preferably, the preparation of the myoglobins labelled antibody of the alkali phosphatase enzyme mark, further includes：In the activationAdd glycine solution in alkaline phosphatase, the amount of the material of the glycine be 4- (N- maleimidomehyls) hexamethylene-10~20 times of the amount of 1- carboxylic acid sulfonic group succinimide ester sodium salt materials, fully mix after at room temperature reaction 8~12min。
Preferably, the preparation of the myoglobins labelled antibody of the alkali phosphatase enzyme mark, further includes：In the alkaline phosphorusN-ethylomaleimide solution, the N- ethyls maleoyl- are added in the myoglobins labelled antibody of sour enzyme markThe amount of the material of imines is 5~20 times of the amount of the myoglobins labelled antibody material of the alkali phosphatase enzyme mark, fully mixedIt is even after at room temperature react 25~35min.
Preferably, the preparation of the myoglobins labelled antibody of the alkali phosphatase enzyme mark, further includes：Institute after purificationState and glycerine is added in the myoglobins labelled antibody of alkali phosphatase enzyme mark and is preserved at -25~-15 DEG C.
Preferably, the carboxyl magnetic bead of myoglobins coated antibody coupling is by by myoglobins coated antibody solutionWith carboxyl magnetic bead solution according to mass ratio be 1:(80~120) mix, under room temperature in the 2- (N- morpholines) that pH is 5.016~24h of cross-linking reaction is obtained in ethanesulfonic acid buffer.
It is highly preferred that the concentration of the myoglobins coated antibody solution is 1~5mg/mL；The carboxyl magnetic bead solutionConcentration is 100mg/mL.
Preferably, the preparation of the carboxyl magnetic bead of the myoglobins coated antibody coupling, further includes：To carboxylic before cross-linking reactionBase magnetic bead carries out activation process, and the activation process includes：
(1) carboxyl magnetic bead is placed on magnet stand and stands 2min, Magneto separate abandons supernatant, with the 100mmol/L that pH is 5.0Added after the washing of 2- (N- morpholines) ethanesulfonic acid buffer is secondary in 2- (N- morpholines) ethanesulfonic acid buffer, ultrasound to carboxyl magneticPearl is completely dissolved；
(2) N-N- HOSu NHSs solution and 1- (3- dimethylaminos third are separately added into the system of step (1)Base) -3- ethyl-carbodiimide hydrochloride solution, the quality of the N-N- HOSu NHSs solution is the carboxyl magnetic bead matter0.20~0.25 times of amount, the quality of 1- (3- the dimethylamino-propyls) -3- ethyl-carbodiimide hydrochloride solution is described0.08~0.12 times of carboxyl magnetic bead quality, is uniformly mixed, in 25~35min of stirring reaction under room temperature.
It is highly preferred that the N-N- HOSu NHSs solution concentration is 10mg/mL；1- (the 3- dimethylaminos thirdBase) -3- ethyl-carbodiimide hydrochlorides solution concentration is 10mg/mL.
Preferably, the preparation of the carboxyl magnetic bead of the myoglobins coated antibody coupling, further includes：It is coated with myoglobinsMagnetic bead confining liquid is added in the carboxyl magnetic bead of antibody coupling, in 16~24h of reaction under room temperature.
It is highly preferred that the magnetic bead confining liquid is CE210.
Preferably, the kit further includes myoglobins quality-control product.
Present invention also offers the application method of above-mentioned myoglobin assay kit, including：
(1) three test tubes are taken to be separately added into 25 μ L calibration objects, 25 μ L quality-control products, 25 μ L samples to be tested；
(2) 0~40 μ L reagents R1,50 μ L enzyme conjugates working solution R2 are separately added into every test tube and 50 μ L magnetic beads combineThing working solution M, is sufficiently mixed after 5~10min of reaction at 40~45 DEG C；
(3) Magneto separate, pours out supernatant, 300 μ L cleaning solutions is separately added into every test tube, Magneto separate after mixing, is poured outSupernatant；
(4) 100 μ L chemical luminous substrates are added in every test tube, are mixed, luminous intensity is detected with Chemiluminescence Apparatus.
Technical scheme, has the following advantages that：
(1) myoglobin assay kit provided by the invention, the kit contain reagent R1, and the reagent R1 containsImidazole components, it can quickly eliminate the haemocyte in whole blood, effectively avoid haemocyte from gulping down the possibility into magnetic bead.The examination of the present inventionFinger Peripheral whole blood or anti-freezing venous whole can be used directly as measuring samples in agent box, pre- without being carried out in advance to whole blood sampleProcessing, so that it may be directly detected, substantially increase detection speed, simplify operating procedure, expand the applicable model of kitEnclose；Can automatically, operating in a key, testing result can be gone out within 15 minutes or so, be well positioned to meet hospital emergency and outpatient service is fastThe demand of speed diagnosis, easy to promote and apply on a large scale.
(2) myoglobin assay kit provided by the invention, contains dodecyl sulphur in the cleaning solution of the kitSour sodium component, improves cleaning performance, effectively prevent the non-specific binding of chemical luminous substrate.
(3) myoglobin assay kit provided by the invention, the kit is by chemiluminescence and immune magnetic particleBe combined, there is provided a kind of close to homogeneous reaction system, and employ one-step method reaction pattern so that detection sensitivity andPrecision greatly improves；Using carboxyl magnetic bead as solid phase carrier, the homogeneity of its size and shape, makes target substance rapid and hasIt is incorporated on magnetic bead to effect, and its spherical structure can also eliminate the non-specific binding thing related with irregular shape particle, carryThe high specificity of reagent kit product.
(4) myoglobin assay kit provided by the invention, including enzyme conjugates, the enzyme conjugates main component areThe myoglobins labelled antibody of alkali phosphatase enzyme mark, glycine solution is added in its preparation process to terminate priming reaction, withAnd N-ethylomaleimide solution is closed unnecessary sulfydryl and is reacted with end mark, effectively prevent nonspecific reactionGeneration, ensure that the stability of the myoglobins labelled antibody of alkali phosphatase enzyme mark；Each reactive material in reacting markUsage amount and temperature, pH conditions etc. be defined, optimize the condition of mark reaction.
(5) myoglobin assay kit provided by the invention, the enzyme conjugates, which is placed in glycerine, to be preserved, and is effectively protectedThe activity of enzyme is demonstrate,proved.
(6) myoglobin assay kit provided by the invention, including enzyme conjugates magnetic bead conjugate, the enzyme conjugatesMagnetic bead conjugate is the carboxyl magnetic bead of myoglobins coated antibody coupling, and n-hydroxysuccinimide is first used in its preparation processWith 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochloride activated carboxyl magnetic beads, myoglobins coated antibody is improvedWith the coupling efficiency of carboxyl magnetic bead；The dissolving of carboxyl magnetic bead is accelerated during carboxyl magnetic bead activation process using ultrasonic power, makes itMore uniformly it is dispersed in system, effectively prevent influences to detect signal because of the condensation of magnetic bead, ensure that kit detectsAs a result accuracy；Magnetic bead confining liquid is added after coupling reaction, the probability of cross reaction is significantly reduced, improves examinationThe specificity of agent box, further ensures the accuracy of kit testing result；Use to each reactive material in coupling reactionAmount and temperature, pH conditions are defined, and optimize the condition of coupling reaction.
(7) myoglobin assay kit provided by the invention, each component in the detection kit is the reactionOptimization formula under system, the detection performance to the detection kit provide sound assurance.
(8) myoglobin assay kit provided by the invention, can measure more at the same time on Full-automatic chemiluminescence apparatusA sample, realizes the rapid measure of high throughput of myoglobins, and accuracy and detection efficiency are all greatly improved.
(9) the present invention provides the application method of myoglobin assay kit, this method is the reaction using one-step methodPattern, 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 artEmbodiment or attached drawing needed to be used in the description of the prior art are briefly described, it should be apparent that, in describing belowAttached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative laborPut, 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 will be clearly and completely described below, it is clear that described embodiment is this hairBright part of the embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not havingAll other embodiments obtained under the premise of creative work are made, belong to the scope of protection of the invention.In addition, belowAs long as it is mutual not form conflict each other for involved technical characteristic in described different embodiments of the present inventionWith reference to.
In following embodiments, myoglobins labelled antibody is purchased from medix companies；Myoglobins coated antibody is purchased from medixCompany；Traut ' s Reagent reagents are purchased from Thermo companies；Alkaline phosphatase is purchased from Roche companies；Carboxyl magnetic bead is purchased from dayThis JSR Corp.；CE210 is purchased from Japanese JSR Corp..
The preparation of 1 enzyme conjugates of embodiment
1. weighing 3mg Traut ' s reagents, prepared with the Triethanolamine buffer (pH=8.5 ± 0.05) of 100mmol/LInto the solution that concentration is 1.376mg/mL；0.5mg myoglobins labelled antibodies are weighed, are buffered with the triethanolamine of 100mmol/LLiquid (pH=8.5 ± 0.05) is configured to the solution that concentration is 2mg/mL, and it is 1.376mg/mL's to add concentration theretoThe molar ratio of Traut ' s solution, myoglobins labelled antibody and Traut ' s reagents is 1:15, mix at once, react 15 at room temperatureMinute.The glycine solution of 1mmol/L is added, the amount of the material of glycine is 4- (N- maleimidomehyls) hexamethylene -1-10 times of the amount of carboxylic acid sulfonic group succinimide ester sodium salt material, mix at once, react 10 minutes at room temperature.Desalination, displacementInto the triethanolamine cross-linking buffer of 100mmol/L (pH=7.3 ± 0.05).
2. weighing 3mg 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group succinimide ester sodium salts, useDimethylformamide is configured to the solution that concentration is 17.5mg/mL；Added into the alkaline phosphatase enzyme solutions of 16mg/mL4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group succinimide ester sodium salt solutions of 17.5mg/mL, alkaline phosphorusThe molar ratio of sour enzyme and 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group succinimide ester sodium salts is 1:10,Mix, react 15 minutes at room temperature at once.The glycine solution of 1mmol/L is added, the amount of the material of glycine is Traut ' s examinations10 times of the amount of agent material, mix at once, react 8 minutes at room temperature.Desalination, the triethanolamine of displacement to 100mmol/L are crosslinkedIn buffer solution (pH=7.3 ± 0.05).
3. the activation myoglobins labelled antibody solution of step 1 is mixed with the activated alkaline phosphoric acid enzyme solutions of step 2, it is livingThe molar ratio of the myoglobins labelled antibody of change and the alkaline phosphatase of activation is 1：1, fully mix, and reacted at 2~8 DEG C18 it is small when.
4. weigh 3mg N-ethylomaleimides, with the Triethanolamine buffer of 100mmol/L (pH=7.3 ±0.05) it is made into 12.5mg/mL solution.
5. the N-ethylomaleimide solution in step 4, alkali phosphatase enzyme mark are added into the system of step 3Myoglobins labelled antibody and N-ethylomaleimide molar ratio be 1：10, fully mix, anti-30 points at room temperatureClock, to close unnecessary sulfydryl.After reaction, purified to obtain final alkali phosphatase enzyme mark with the method for sieve chromatographyMyoglobins labelled antibody conjugate.The enzyme conjugates concentrate measured concentration of measure after purification, adds isometric glycerine, fullyMix, -20 DEG C save backup.
The preparation of 2 enzyme conjugates of embodiment
1. weighing 3mg Traut ' s reagents, prepared with the Triethanolamine buffer (pH=8.5 ± 0.05) of 100mmol/LInto the solution that concentration is 1.3mg/mL；0.5mg myoglobins labelled antibodies are weighed, with the Triethanolamine buffer of 100mmol/L(pH=8.5 ± 0.05) is configured to the solution that concentration is 5mg/mL, and it is molten to add the Traut ' s that concentration is 1.3mg/mL theretoThe molar ratio of liquid, myoglobins labelled antibody and Traut ' s reagents is 1:10, mix, react 12 minutes at room temperature at once.AddThe glycine solution of 1mmol/L, the amount of the material of glycine is 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic groups20 times of the amount of succinimide ester sodium salt material, mix at once, react 12 minutes at room temperature.100mmol/L is arrived in desalination, displacementTriethanolamine cross-linking buffer in (pH=7.3 ± 0.05).
2. weighing 3mg 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group succinimide ester sodium salts, useIt is 16mg/mL solution that dimethylformamide, which is configured to concentration,；Add 16mg/mL's into the alkaline phosphatase enzyme solutions of 20mg/mL4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group succinimide ester sodium salt solutions, alkaline phosphatase and 4- (N-Maleimidomehyl) hexamethylene -1- carboxylic acid sulfonic group succinimide ester sodium salts molar ratio be 1:15, mix at once, roomThe lower reaction of temperature 15 minutes.The glycine solution of 1mmol/L is added, the amount of the material of glycine is the amount of Traut ' s reagent materials20 times, mix at once, at room temperature react 12 minutes.Desalination, in the triethanolamine cross-linking buffer for replacing 100mmol/L(pH=7.3 ± 0.05).
3. the activation myoglobins labelled antibody solution of step 1 is mixed with the activated alkaline phosphoric acid enzyme solutions of step 2, it is livingThe molar ratio of the myoglobins labelled antibody of change and the alkaline phosphatase of activation is 1：2, fully mix, and reacted at 2~8 DEG C24 it is small when.
4. weigh 3mg N-ethylomaleimides, with the Triethanolamine buffer of 100mmol/L (pH=7.3 ±0.05) it is made into 12.5mg/mL solution.
5. the N-ethylomaleimide solution in step 4, alkali phosphatase enzyme mark are added into the system of step 3Myoglobins labelled antibody and N-ethylomaleimide molar ratio be 1：20, fully mix, anti-35 points at room temperatureClock, to close unnecessary sulfydryl.After reaction, the flesh for purifying to obtain final alkali phosphatase enzyme mark with ultrafiltration concentration method is redProtein labeling antibody conjugates.The enzyme conjugates concentrate measured concentration of measure after purification, adds isometric glycerine, fully mixedEven, -20 DEG C save backup.
The preparation of 3 enzyme conjugates of embodiment
1. weighing 3mg Traut ' s reagents, prepared with the Triethanolamine buffer (pH=8.5 ± 0.05) of 100mmol/LInto the solution that concentration is 1.5mg/mL；0.5mg myoglobins labelled antibodies are weighed, with the Triethanolamine buffer of 100mmol/L(pH=8.5 ± 0.05) is configured to the solution that concentration is 2.5mg/mL, and adds the Traut ' s that concentration is 1.5mg/mL theretoThe molar ratio of solution, myoglobins labelled antibody and Traut ' s reagents is 1:15, mix, react 18 minutes at room temperature at once.AddEnter the glycine solution of 1mmol/L, the amount of the material of glycine is 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic acid15 times of the amount of base succinimide ester sodium salt material, mix at once, react 10 minutes at room temperature.Desalination, displacement are arrivedIn the triethanolamine cross-linking buffer of 100mmol/L (pH=7.3 ± 0.05).
2. weighing 3mg 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group succinimide ester sodium salts, useDimethylformamide is configured to the solution that concentration is 18mg/mL；18mg/mL is added into the alkaline phosphatase enzyme solutions of 18mg/mL4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group succinimide ester sodium salt solutions, alkaline phosphatase and 4-The molar ratio of (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group succinimide ester sodium salts is 1:12, mix at once,React 12 minutes at room temperature.The glycine solution of 1mmol/L is added, the amount of the material of glycine is Traut ' s reagent materials15 times of amount, mix at once, react 10 minutes at room temperature.Desalination, in the triethanolamine cross-linking buffer for replacing 100mmol/L(pH=7.3 ± 0.05).
3. the activation myoglobins labelled antibody solution of step 1 is mixed with the activated alkaline phosphoric acid enzyme solutions of step 2, it is livingThe molar ratio of the myoglobins labelled antibody of change and the alkaline phosphatase of activation is 1：1.5, fully mix, and it is anti-at 2~8 DEG CAnswer 20 it is small when.
4. weigh 3mg N-ethylomaleimides, with the Triethanolamine buffer of 100mmol/L (pH=7.3 ±0.05) it is made into 12.5mg/mL solution.
5. the N-ethylomaleimide solution in step 4, alkali phosphatase enzyme mark are added into the system of step 3Myoglobins labelled antibody and N-ethylomaleimide molar ratio be 1：5, fully mix, anti-25 points at room temperatureClock, to close unnecessary sulfydryl.After reaction, the flesh for purifying to obtain final alkali phosphatase enzyme mark with ultrafiltration concentration method is redProtein labeling antibody conjugates.The enzyme conjugates concentrate measured concentration of measure after purification, adds isometric glycerine, fully mixedEven, -20 DEG C save backup.
The preparation of 4 magnetic bead conjugate of embodiment
1. 3mg N-N- HOSu NHSs are weighed, with 2- (N- morpholines) ethanesulfonic acid buffer (pH of 100mmol/L=5.0 ± 0.05) it is configured to the solution that concentration is 10mg/mL；It is sub- to weigh 3mg 1- (3- dimethylamino-propyls) -3- ethyls carbon twoAmine hydrochlorate, being configured to concentration with 2- (N- morpholines) ethanesulfonic acid buffers (pH=5.0 ± 0.05) of 100mmol/L isThe solution of 10mg/mL；
2. the carboxyl magnetic bead that particle diameter is 3 μm is placed on magnet stand and stands 2min, Magneto separate abandons supernatant, uses 100mmol/L2- (N- morpholines) ethanesulfonic acid buffer is added after 2- (N- morpholines) ethanesulfonic acid buffers (pH=5.0 ± 0.05) washing is secondaryIn, ultrasound to carboxyl magnetic bead is completely dissolved；Add the n-hydroxysuccinimide solution of 10mg/mL and the 1- (3- bis- of 10mg/mLMethylaminopropyl) -3- ethyl carbodiimide hydrochloride solution, both quality are respectively 0.23 times and 0.1 of carboxyl magnetic bead qualityTimes, it is uniformly mixed, in stirring reaction 30 minutes under room temperature.After reaction, with magnetic sheet precipitation magnetic bead remove supernatant, after againIt is repeated once, the carboxyl magnetic bead after activation is dissolved in 2- (N- morpholines) ethanesulfonic acid buffer (pH=5.0 of 100mmol/L± 0.05) it is stand-by in；
3. 0.5mg myoglobins coated antibodies are weighed, with 2- (N- morpholines) ethanesulfonic acid buffer (pH of 100mmol/L=5.0 ± 0.05) it is configured to the solution that concentration is 2mg/mL；
4. it is 1 in mass ratio by the carboxyl magnetic bead solution in the myoglobins coated antibody solution in step 3 and step 2:100 mixing, when room temperature cross-linking reaction 20 is small on blending instrument.After reaction, supernatant is removed with magnetic sheet precipitation magnetic bead, repeating shouldStep 2 time.Magnetic bead confining liquid CE210 is added, when room temperature reaction 20 is small on blending instrument.After reaction, magnetic bead is precipitated with magnetic sheetRemove supernatant, magnetic bead cleaned 3 times with magnetic bead cleaning solution, be resuspended in obtained magnetic bead magnetic bead preserve liquid (pH=6.0 ± 0.05,30mmol/L 2- (N- morpholines) ethyl sulfonic acid, 200mmol/L NaCl, 1% bovine serum albumin(BSA), 1% sucrose, 1% gelatin,0.1% polysorbas20,1% polyvinylpyrrolidone, 3%Proclin300) in, saved backup in 2~8 DEG C.
The preparation of 5 magnetic bead conjugate of embodiment
1. 3mg N-N- HOSu NHSs are weighed, with 2- (N- morpholines) ethanesulfonic acid buffer (pH of 100mmol/L=5.0 ± 0.05) it is configured to the solution that concentration is 10mg/mL；It is sub- to weigh 3mg 1- (3- dimethylamino-propyls) -3- ethyls carbon twoAmine hydrochlorate, being configured to concentration with 2- (N- morpholines) ethanesulfonic acid buffers (pH=5.0 ± 0.05) of 100mmol/L isThe solution of 10mg/mL；
2. the carboxyl magnetic bead that particle diameter is 1.3 μm is placed on magnet stand and stands 2min, Magneto separate abandons supernatant, uses 100mmol/2- (N- morpholines) ethyl sulfonic acid buffering is added after L 2- (N- morpholines) ethanesulfonic acid buffers (pH=5.0 ± 0.05) washing is secondaryIn liquid, ultrasound to carboxyl magnetic bead is completely dissolved；It is separately added into the n-hydroxysuccinimide solution and 10mg/mL of 10mg/mL1- (3- dimethylamino-propyls) -3- ethyl carbodiimide hydrochloride solution, both quality are respectively the 0.20 of carboxyl magnetic bead qualityTimes and 0.08 times, be uniformly mixed, under room temperature stirring reaction 25 minutes.After reaction, gone with magnetic sheet precipitation magnetic beadClear liquid, after repeat once, the carboxyl magnetic bead after activation is dissolved in 2- (N- morpholines) ethanesulfonic acid buffer of 100mmol/LIt is stand-by in (pH=5.0 ± 0.05)；
3. 0.5mg myoglobins coated antibodies are weighed, with 2- (N- morpholines) ethanesulfonic acid buffer (pH of 100mmol/L=5.0 ± 0.05) it is configured to the solution that concentration is 5mg/mL；
4. it is 1 in mass ratio by the carboxyl magnetic bead solution in the myoglobins coated antibody solution in step 3 and step 2:80 mixing, when room temperature cross-linking reaction 16 is small on blending instrument.After reaction, supernatant is removed with magnetic sheet precipitation magnetic bead, repeating shouldStep 2 time.Magnetic bead confining liquid CE210 is added, when room temperature reaction 16 is small on blending instrument.After reaction, magnetic bead is precipitated with magnetic sheetRemove supernatant, magnetic bead cleaned 3 times with magnetic bead cleaning solution, be resuspended in obtained magnetic bead magnetic bead preserve liquid (pH=6.0 ± 0.05,30mmol/L2- (N- morpholines) ethyl sulfonic acid, 200mmol/L NaCl, 1% bovine serum albumin(BSA), 1% sucrose, 1% gelatin,0.1% polysorbas20,1% polyvinylpyrrolidone, 3%Proclin300) in, saved backup in 2~8 DEG C.
The preparation of 6 magnetic bead conjugate of embodiment
1. 3mg N-N- HOSu NHSs are weighed, with 2- (N- morpholines) ethanesulfonic acid buffer (pH of 100mmol/L=5.0 ± 0.05) it is configured to the solution that concentration is 10mg/mL；It is sub- to weigh 3mg 1- (3- dimethylamino-propyls) -3- ethyls carbon twoAmine hydrochlorate, being configured to concentration with 2- (N- morpholines) ethanesulfonic acid buffers (pH=5.0 ± 0.05) of 100mmol/L isThe solution of 10mg/mL；
2. the carboxyl magnetic bead that particle diameter is 4 μm is placed on magnet stand and stands 2min, Magneto separate abandons supernatant, uses 100mmol/L2- (N- morpholines) ethanesulfonic acid buffer is added after 2- (N- morpholines) ethanesulfonic acid buffers (pH=5.0 ± 0.05) washing is secondaryIn, ultrasound to carboxyl magnetic bead is completely dissolved；It is separately added into the n-hydroxysuccinimide solution of 10mg/mL and the 1- of 10mg/mL(3- dimethylamino-propyls) -3- ethyl carbodiimide hydrochloride solution, both quality are 0.25 times of carboxyl magnetic bead quality respectivelyWith 0.12 times, be uniformly mixed, under room temperature stirring reaction 35 minutes.After reaction, supernatant is removed with magnetic sheet precipitation magnetic beadLiquid, after repeat once, the carboxyl magnetic bead after activation is dissolved in 2- (N- morpholines) ethanesulfonic acid buffer of 100mmol/LIt is stand-by in (pH=5.0 ± 0.05)；
3. 0.5mg myoglobins coated antibodies are weighed, with 2- (N- morpholines) ethanesulfonic acid buffer (pH of 100mmol/L=5.0 ± 0.05) it is configured to the solution that concentration is 5mg/mL；
4. it is 1 in mass ratio by the carboxyl magnetic bead solution in the myoglobins coated antibody solution in step 3 and step 2:120 mixing, when room temperature cross-linking reaction 24 is small on blending instrument.After reaction, supernatant is removed with magnetic sheet precipitation magnetic bead, repeating shouldStep 2 time.Magnetic bead confining liquid CE210 is added, when room temperature reaction 24 is small on blending instrument.After reaction, magnetic bead is precipitated with magnetic sheetRemove supernatant, magnetic bead cleaned 3 times with magnetic bead cleaning solution, be resuspended in obtained magnetic bead magnetic bead preserve liquid (pH=6.0 ± 0.05,30mmol/L 2- (N- morpholines) ethyl sulfonic acid, 200mmol/L NaCl, 1% bovine serum albumin(BSA), 1% sucrose, 1% gelatin,0.1% polysorbas20,1% polyvinylpyrrolidone, 3%Proclin300) in, saved backup in 2~8 DEG C.
The myoglobin assay kit of the present invention of embodiment 7
Myoglobin assay kit, including：Myoglobins calibration object, reagent R1, enzyme conjugates working solution R2, magnetic bead knotCompound working solution M, cleaning solution and chemical luminous substrate.
Wherein：Myoglobins calibration object is to be diluted to the calibration object with traceability by calibration object dilution1500ng/mL, the component of the calibration object dilution are：The 4- hydroxyethyl piperazineethanesulfonic acids of 30mmol/L, 250mmol/LNaCl, 1% bovine serum albumin(BSA), 3mmol/L Proclin300, pH are 7.0 ± 0.05；
Wherein：The specific components of reagent R1 are：Trishydroxymethylaminomethane, 150mmol/L NaCl, 3% sugarcane of 60mmol/LSugar, 3% glycerine, 0.1% bovine serum albumin(BSA), 1.2mmol/L imidazoles, 0.15% polysorbas20 and 0.15%Proclin300, pHFor 7.3 ± 0.05；
Wherein：The concentration of enzyme conjugates working solution R2 is 1.3 μ g/mL, it is by adding enzyme knot in enzyme conjugates solutionCompound diluent preparing forms.The specific component of enzyme combination diluent is：35mmol/L 2- (N- morpholines) ethyl sulfonic acid,250mmol/L NaCl, 1% bovine serum albumin(BSA), 5% sucrose, 5% glycerine, 0.1% polysorbas20,2.5mmol/LZnCl2、2.5mmol/LMgCl2, 0.15%Proclin300, pH be 6.3 ± 0.05；
Wherein：The concentration of magnetic bead conjugate working solution M is 0.25mg/mL, it is by the way that magnetic bead conjugate solution is addedMagnetic bead conjugate diluent preparing forms.The specific component of magnetic bead conjugate dilution is：35mmol/L 4- hydroxyethyl piperazine secondSulfonic acid, 250mmol/L NaCl, 1% bovine serum albumin(BSA), 1% sucrose, 1% gelatin, 0.1% polysorbas20,1% polyvinyl pyrroleAlkanone, 3%Proclin300, pH are 7.5 ± 0.05.
Wherein：The process for preparation of cleaning solution is：Trishydroxymethylaminomethane 1211.4mg, NaCl are added into container successively9g, polysorbas20 1g, lauryl sodium sulfate 2.5g, water 900mL, mix to each component and dissolve, and are adjusted with sodium hydroxide solution moltenLiquid pH to 8.0 ± 0.5, adds water and is settled to 1000mL, mixes 30 minutes, 0.22 μm of filtering obtains cleaning solution.
Wherein：The process for preparation of chemical luminous substrate is：
Accurately weigh 3- (the spiral adamantane of 2-) -4- methoxyl groups -4- (3- phosphorus oxygens acyl)-phenyl -1,2- dioxanes twoSodium salt (AMPPD) 0.5g, trishydroxymethylaminomethane 18g, NaCl 100g, hexadecyltrimethylammonium chloride 0.02g, water are fixedIt is 9.4 ± 0.05 to hold to 1000mL, adjustment chemical luminous substrate pH value of solution；This chemical luminous substrate is for alkaline phosphataseEpidioxy 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.
The myoglobin assay kit of the present invention of embodiment 8
Myoglobin assay kit, including：Myoglobins calibration object, reagent R1, enzyme conjugates working solution R2, magnetic bead knotCompound working solution M, cleaning solution and chemical luminous substrate.
Wherein：Myoglobins calibration object is that the calibration object with traceability is diluted to 1ng/ by calibration object dilutionML, the component of the calibration object dilution are：The 4- hydroxyethyl piperazineethanesulfonic acids of 20mmol/L, 150mmol/L NaCl, 1% NSeralbumin, 0.5mmol/L Proclin300, pH are 6.5 ± 0.05；
Wherein：The specific components of reagent R1 are：Trishydroxymethylaminomethane, 150mmol/L NaCl, 1% sugarcane of 25mmol/LSugar, 1% glycerine, 0.1% bovine serum albumin(BSA), 0.4mmol/L imidazoles, 0.05% polysorbas20 and 0.05%Proclin300, pHFor 7.3 ± 0.05；
Wherein：The concentration of enzyme conjugates working solution R2 is 0.5 μ g/mL, it is by adding enzyme knot in enzyme conjugates solutionCompound diluent preparing forms.The specific component of enzyme combination diluent is：20mmol/L 2- (N- morpholines) ethyl sulfonic acid,150mmol/L NaCl, 1% bovine serum albumin(BSA), 5% sucrose, 5% glycerine, 0.1% polysorbas20,0.05mmol/LZnCl2、0.05mmol/LMgCl2, 0.05%Proclin300, pH be 6.0 ± 0.05；
Wherein：The concentration of magnetic bead conjugate working solution M is 0.1mg/mL, it is by will add magnetic bead conjugate solutionMagnetic bead conjugate diluent preparing forms.The specific component of magnetic bead conjugate dilution is：20mmol/L 4- hydroxyethyl piperazine secondSulfonic acid, 150mmol/L NaCl, 1% bovine serum albumin(BSA), 1% sucrose, 1% gelatin, 0.1% polysorbas20,1% polyvinyl pyrroleAlkanone, 0.5%Proclin300, pH are 7.0 ± 0.05.
In the present embodiment, the component and process for preparation of cleaning solution and chemical luminous substrate are the same as embodiment 7.
The myoglobin assay kit of the present invention of embodiment 9
Myoglobin assay kit, including：Myoglobins calibration object, reagent R1, enzyme conjugates working solution R2, magnetic bead knotCompound working solution M, cleaning solution and chemical luminous substrate.
Wherein：Myoglobins calibration object is to be diluted to the calibration object with traceability by calibration object dilution3000ng/mL, the component of the calibration object dilution are：The 4- hydroxyethyl piperazineethanesulfonic acids of 100mmol/L, 150mmol/LNaCl, 1% bovine serum albumin(BSA), 5mmol/L Proclin300, pH are 7.5 ± 0.05；
Wherein：The specific components of reagent R1 are：The trishydroxymethylaminomethane of 100mmol/L, 150mmol/L NaCl, 5%Sucrose, 5% glycerine, 0.1% bovine serum albumin(BSA), 2mmol/L imidazoles, 0.2% polysorbas20 and 0.2%Proclin300, pH are7.5±0.05；
Wherein：The concentration of enzyme conjugates working solution R2 is 2 μ g/mL, it is combined by adding enzyme in enzyme conjugates solutionThing diluent preparing forms.The specific component of enzyme combination diluent is：50mmol/L2- (N- morpholines) ethyl sulfonic acid,300mmol/L NaCl, 1% bovine serum albumin(BSA), 5% sucrose, 5% glycerine, 0.1% polysorbas20,5mmol/LZnCl2、5mmol/LMgCl2, 0.2%Proclin300, pH be 6.5 ± 0.05；
Wherein：The concentration of magnetic bead conjugate working solution M is 0.5mg/mL, it is by the way that magnetic bead conjugate solution is added magneticPearls knot compound diluent preparing forms.The specific component of magnetic bead conjugate dilution is：50mmol/L 4- hydroxyethyl piperazine second sulphursAcid, 300mmol/L NaCl, 1% bovine serum albumin(BSA), 1% sucrose, 1% gelatin, 0.1% polysorbas20,1% polyvinylpyrrolidineKetone, 5%Proclin300, pH are 8.0 ± 0.05.
In the present embodiment, the component and process for preparation of cleaning solution and chemical luminous substrate are the same as embodiment 7.
The myoglobin assay kit of the present invention of embodiment 10
Myoglobin assay kit, including：Myoglobins calibration object, quality-control product, reagent R1, enzyme conjugates working solutionR2, magnetic bead conjugate working solution M, cleaning solution and chemical luminous substrate.
Wherein：Myoglobins calibration object or quality-control product are by calibration object or matter with traceability by calibration object dilutionControl product are diluted to 3000ng/mL, and the component of the calibration object dilution is：The 4- hydroxyethyl piperazineethanesulfonic acids of 30mmol/L,250mmol/L NaCl, 1% bovine serum albumin(BSA), 3mmol/L Proclin300, pH are 7.0 ± 0.05；
Wherein：The specific components of reagent R1 are：Trishydroxymethylaminomethane, 150mmol/L NaCl, 3% sugarcane of 60mmol/LSugar, 3% glycerine, 0.1% bovine serum albumin(BSA), 1.2mmol/L imidazoles, 0.15% polysorbas20 and 0.15%Proclin300, pHFor 7.3 ± 0.05；
Wherein：The concentration of enzyme conjugates working solution R2 is 1.3 μ g/mL, it is by adding enzyme knot in enzyme conjugates solutionCompound diluent preparing forms.The specific component of enzyme combination diluent is：35mmol/L 2- (N- morpholines) ethyl sulfonic acid,250mmol/L NaCl, 1% bovine serum albumin(BSA), 5% sucrose, 5% glycerine, 0.1% polysorbas20,2.5mmol/LZnCl2、2.5mmol/LMgCl2, 0.15%Proclin300, pH be 6.35 ± 0.05；
Wherein：The concentration of magnetic bead conjugate working solution M is 0.25mg/mL, it is by the way that magnetic bead conjugate solution is addedMagnetic bead conjugate diluent preparing forms.The specific component of magnetic bead conjugate dilution is：35mmol/L 4- hydroxyethyl piperazine secondSulfonic acid, 250mmol/L NaCl, 1% bovine serum albumin(BSA), 1% sucrose, 1% gelatin, 0.1% polysorbas20,1% polyvinyl pyrroleAlkanone, 3%Proclin300, pH are 7.5 ± 0.05.
In the present embodiment, the component and process for preparation of cleaning solution and chemical luminous substrate are the same as embodiment 7.
In the kit detection finger Peripheral whole blood of the present invention of embodiment 11 the step of myoglobins
1. 25 μ L samples, calibration object or quality-control product are added into test tube；
2. adding 20 μ L reagent R1 into test tube, test tube is gently back and forth shaken；
3. 50 μ L enzyme conjugates working solutions R2 are added into test tube；
4. 50 μ L magnetic bead conjugate working solutions M are added into test tube；
5. test tube is prevented from mixing 30 seconds on vortex blending instrument, it is subsequently placed in 42 DEG C and reacts 8 minutes；
6. test tube being placed on after standing 2 minutes on magnet stand and outwelling supernatant, pat dry, add 300 μ L of cleaning solution, the stepIt is repeated 3 times；
It is detected 7. reaction tube is transferred on luminometer, luminometer can add 100 μ L chemiluminescences bottomsThing and the luminous value for reading each test sample.
8. make dose-response curve using the concentration and luminous value of standard items or quality-control product；
9. the luminous value of sample to be tested is substituted into the content for the myoglobins that curve is obtained in sample to be tested.
The myoglobins detection method provided in the present embodiment is equally applicable to the flesh of anti-freezing venous whole, serum or blood plasmaLactoferrin detects.
Experimental example 1
Using 25 μ L anti-freezings venous wholes as sample, detected with the kit of the present invention, luminescence phenomenon is shown, according to this hairThe detecting step of bright embodiment 11 can draw the content of myoglobins in whole blood, and public with Chinese patent literature CN107255723AThe technology for detection opened, does not detect the concentration of myoglobins in whole blood.
2 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 pointMeasure 3 times, is averaged, and as a result makees regression straight line with expected concentration, regression coefficient r=0.9989 ＞ 0.99 are calculated, sayThe dilution good linearity of bright kit provided by the invention.
3 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 productSurvey, 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(1000ng/mL)=3%, CV2(111ngg/mL)=5%.It follows that examination provided by the inventionAgent box has higher precision.
4 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 intoRow 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 inventionMeasured value is approached with target value, illustrates that kit provided by the invention has higher accuracy.
5 sensitivity of experimental example is verified
Take the quality-control product with traceability to be diluted to detection range lower limit (1ng/mL) to be nearby measured, replication 3It is secondary, 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 is approached with target value,Illustrate that kit provided by the invention has higher sensitivity.
6 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 thatIn the range of concentration is 1~300ng/mL, there is luminescence phenomenon, show the kit detection range of the present invention for 1~300ng/mL。
7 relevance verification of experimental example
200 parts of serum samples are taken, kit more of the invention and the myoglobin assay kit of Bake Mann are examinedThe correlation of result is surveyed, the results are shown in Figure 1.Wherein：Abscissa is the testing result of Bake Mann kit, and ordinate isThe testing result of kit of the present invention, coefficient R2=0.9709, regression line equation is：Y=1.0261x+10.8821.ByFig. 1 understands that pattern detection result of the present invention is compared with industry renowned company product testing result, 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 rightFor 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 orChange.There is no necessity and possibility to exhaust all the enbodiments, and the obvious change thus extended out orAmong changing still in the protection domain of the invention.