CN103278651B - Kit for chemiluminescence immunity quantitative detection of MYO (myohaemoglobinnano) nano magnetic particle and preparation method of kit - Google Patents
Kit for chemiluminescence immunity quantitative detection of MYO (myohaemoglobinnano) nano magnetic particle and preparation method of kit Download PDFInfo
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Abstract
The invention discloses a kit for chemiluminescence immunity quantitative detection of an MYO nano magnetic particle. The kit comprises an MYO calibrator, a nano magnetic particle suspension liquid coupled with streptavidin, bioti-labeled MYO antibodies, MYO abzyme conjugate, an MYO quality control product, a chemiluminescence liquid A and a chemiluminescence liquid B, a 20-time concentrated washing liquor and a reaction tube, wherein for the MYO abzyme conjugate, used enzyme adopts horse radish peroxidase with purity RZ larger than or equal to 3.0 and activity larger than or equal to 250 U/mL. Besides, the invention further discloses a preparation method of the kit. Compared with the conventional kit, the kit provided by the invention has the advantages of high sensibility and test automation, wide measurable concentration range, long useful life of a reagent, simple operation and the like.
Description
Technical field
The present invention relates to field of immunoassay medicine, concrete, the invention provides a kind of myoglobins (MYO) nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box and preparation method thereof.
Background technology
Myoglobins (MYO) is the hemoprotein similar to haemoglobin.Be present in muscle cell, major function stores oxygen, very abundant at myocardium intensive amount.Molecular weight 16700, is formed, in compact spherical containing the polypeptied chain of 153 amino acid residues and a prosthetic heme group by one.Hydrophobic side chain in polypeptied chain on amino acid residue is mostly at intramolecule, and hydrophilic side chain multidigit is in molecular surface, and therefore it is better water-soluble.
Because MYO molecular weight is little, so be comparatively early released into blood circulation after muscle cell injury, 1-2h raises, and 12h reaches peak, declines gradually after 24h, is the leading indicator of early diagnosis acute myocardial infarction AMI (AMI), can win the quality time for the diagnosis of AMI and rescue.
When skeletal muscle disease and renal dysfunction, serum MYO concentration also can raise, therefore MYO is not the Specific marker of myocardial damage.But because it is highly sensitive, generally can be used for negative diagnostic.If 8h after episode and MYO still in normal range, then can get rid of AMI.
Remove from kidney because MYO in patient's blood after AMI is very fast, therefore MYO mensuration contributes to observing in the AMI course of disease with or without infarct or infarct are expanded again again.MYO frequently increases, and points out original myocardial infarction still in continuity.
The sensitivity of Reperfu-sion is determined whether and index accurately in MYO or thromboembolism treatment.
The MYO immunologic detection method of clinical field application mainly contains colloidal gold immunity chromatography (Goldimmnnochromatography GICA), enzymoimmunoassay (Enzyme Immunoassay, EIA) and chemiluminescent immunoassay(CLIA) (CLIA) etc.
Colloidal gold immunity chromatography possess easy and simple to handle, quick, can single part of detection, be convenient to preserve, do not need the advantages such as specific installation, but because sensitivity is not high, be difficult to realize the use that the shortcoming such as quantitative limits this method.
Enzymoimmunoassay is promoted owing to possessing simple to operate, the feature such as the reagent term of validity is long, pollution-free, result available instrumentation mensuration good higher than the susceptibility of collaurum, specificity, but because susceptibility is relatively low, mark enzyme-to-substrate used can quantitative measurement narrow range, and the shortcoming such as Instrument measuring narrow range, limit its application in skeptophylaxis quantitative measurement.
Its research of chemiluminescent immunoassay(CLIA) is started in the beginning of the eighties, and fast development is applied to the nineties, is current microimmuno-assays the most responsive.Principal feature is hypersensitivity, can measure that concentration range is wide, sample can detect without the need to dilution, the reagent term of validity is long, simple to operate, degree of changing into is high automatically, data automatic generating process ability is strong in detection, compatible good, the non-environmental-pollution of determining instrument etc., thus obtains and develops rapidly and application.But detection sensitivity, measured value accuracy are still not high.
Summary of the invention
The problem to be solved in the present invention chemiluminescence immunoassay immue quantitative detection reagent box being to provide myoglobins and preparation method thereof, avoiding colloidal gold strip can not accurate quantitative analysis, euzymelinked immunosorbent assay (ELISA) poor sensitivity, the defects such as sensing range is narrow.
For solving the problems of the technologies described above, the technical solution used in the present invention is: myoglobins (MYO) nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box, comprising: myoglobins calibration object; Coupling has the nano magnetic microparticle suspending liquid of Streptavidin; Biotin labeled myoglobins antibody; Myoglobins abzyme bond, enzyme used is horseradish peroxidase, horseradish peroxidase purity RZ >=3.0, activity >=250U/mL; Myoglobins quality-control product, quality-control product comprises the low value quality-control product of concentration 8-12ng/mL and the high level quality-control product of 400-600ng/mL; Chemical luminescence for liquid A liquid and B liquid, A liquid is the Tris-HCl damping fluid of 5mmol/L, pH8.6, and containing final concentration 0.7g/L luminol and final concentration 0.165g/L p-iodophenol in this damping fluid; B liquid is 0.675g/L urea peroxide; 20 times of concentrated washing lotions; Reaction tube.
Further, described nano magnetic particulate is the tri-iron tetroxide that surface wraps up with amino or carboxyl-reactive group, particle diameter 10-50nm.
Further, the material of described reaction tube is transparent polystyrene, tygon, polypropylene or glass.
The preparation method of kit, comprises the following steps:
(1) preparation of myoglobins calibration object:
Myoglobins antigen lowlenthal serum is mixed with the dense liquid storage of calibration object, calibrates with company standard product, dense liquid storage lowlenthal serum is diluted to working concentration, is respectively 0,5,25,100,250,1000ng/mL;
Company standard product preparation method: with lowlenthal serum by myoglobins antigen according to 1:10,1:10
2, 1:10
3, 1:10
4, 1:10
5, 1:10
6, 1:10
7ratio dilute, use Roche Holding Ag's myoglobins immue quantitative detection reagent box (article No.: 12178214, specification 100 person-portion) to measure, according to measurement result, set up dilution ratio and measure the curve of concentration.Calculate according to curvilinear equation the dilution ratio that concentration is 5,25,100,250,1000ng/mL respectively, by these proportions company standard product, use Roche Products repeatedly to measure, determine company standard product concentration.The company standard product of preparation, with the packing of 0.5mL/ pipe, use freeze drier freeze-drying ,-20 DEG C of preservations.
(2) preparation of myoglobins quality-control product:
With lowlenthal serum, above-mentioned dense liquid storage is diluted to 10ng/mL and 500ng/mL, using 10ng/mL as low value Quality Control, 500ng/mL is as high level Quality Control;
(3) preparation of nano magnetic particulate-Streptavidin suspending liquid:
Prepared by A, ferriferrous oxide nano magnetic particle
Adopt the precipitation method to prepare ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1) by FeCl
36H
2o and FeCl
2.4H
2o joins in distilled water with mol ratio 2:1, dissolved with vigorous agitation; 2) add 0.5M ammoniacal liquor in a nitrogen environment in above-mentioned iron salt solutions, adjust pH9-10, temperature of reaction 65 DEG C, reaction time 45min; 3), after reaction terminates, be washed with distilled water to neutrality, abandon supernatant, in 60 DEG C of oven dry, obtain the ferriferrous oxide nano magnetic particle of 10-50nm;
The coupling of B, nanometer magnetic bead surface carboxyl groups
Dispersion copolymerization method is adopted to carry out coupling, concrete preparation method is as follows: the nano magnetic microparticulate ultrasound getting above-mentioned preparation is dispersed in 10%PEG8000 solution, obtain magnetic fluid solution, in magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume, after stirring 30min, moves into stirrer, condenser pipe, in the three-necked bottle of nitrogen inlet, add crosslinking chemical N, N '-methylene-bisacrylamide; Under the protection of nitrogen, be warming up to 60 ± 1 DEG C, constant temperature stirs 30min, add benzoyl peroxide successively afterwards, consumption is 3% of magnetic fluid consumption, and stirring rate is about 500rpm, volume of styrene is with magnetic fluid solution, and acrylic acid volume is 1/4 of magnetic fluid solution, keeps stream of nitrogen gas, all the other conditions remain unchanged, reaction 8-10h, and products therefrom leaves standstill, use distilled water cyclic washing, regulate pH=1 with hydrochloric acid again, soak 24h, leave standstill; Use distilled water cyclic washing again, remove not coated Fe
3o
4magnetic, puts into dry 24h at vacuum drying chamber 50 DEG C the product precipitated, obtains the nano magnetic particulate that surface is associated with carboxyl;
The preparation of C, nano magnetic particulate-Streptavidin suspending liquid, preparation 1L, method is as follows:
Get 100mL0.1M MES damping fluid, add the nano magnetic particulate that 10mg surface is associated with carboxyl, stirring at room temperature 40min, add 3.5mg Streptavidin afterwards, then 8mg/mLEDC solution is added, after 2-8 DEG C of reaction 1h, with 0.01M PBS buffer solution 3 times, be finally dissolved to 1L with 0.01M PBS;
(4) preparation of biotin labeled myoglobins antibody
Get 0.8mg myoglobins antibody, to dialyse at 2 ~ 8 DEG C 1 ~ 3h with borate buffer solution; Antibody after dialysis is added 30ug biotin, adds dimethyl sulfoxide (DMSO) simultaneously, make dimethyl sulfoxide (DMSO) ultimate density 5-10%, slowly vibrate, lucifuge reaction 3h; 250uL1M ammonium chloride solution is added, reacting at normal temperature without light 30-60min in above-mentioned solution; To dialyse at 2 ~ 8 DEG C 24h with 0.01MPBS solution, period changes liquid 2-4 time;
(5) preparation of myoglobins abzyme bond
After adopting improvement sodium periodate oxidation that myoglobins antibody and horseradish peroxidase are carried out coupling, be diluted to working concentration 1:5000 with enzyme dilution, and added 13% enzyme stabilizers, be stored in 2 ~ 8 DEG C;
Improvement sodium periodate oxidizing process step comprises:
A:HRP activates
1) 10mg/mL HRP solution is configured;
2) 12.8mg/mL sodium periodate NaIO is configured
4solution;
3) by above-mentioned 1) and 2) obtain solution 1:1 mixing by volume, 4 DEG C of lucifuge reaction 30min;
4) configuration concentration is the glycol water of 20uL/mL, with above-mentioned solution 3) mix with same volume, reacting at normal temperature without light 20min, namely activation completes, and puts-20 DEG C of preservations (holding time is no more than 3 months).
B, MYO labeling of monoclonal antibody
1) raw material to be marked is loaded in bag filter, with the carbonate buffer solution of 0.05M pH9.6, dialysis 30min;
2) by the HRP of mark raw material and activation in mass ratio 1:2 mix, during 4 DEG C are dialysed 24h(, change liquid 2-3 time with 0.05M carbonate buffer solution afterwards);
3) configuration concentration is the NaBH of 2mg/mL
4aqueous solution, adds by 1mgHRP the NaBH that 80uL prepares
4the ratio of aqueous solution mixes, and in 4 DEG C of lucifuge reaction 2h;
4) by above-mentioned steps 3) the marking fluid 0.01M PBS that completes in 4 DEG C of dialysis 24h, add equal-volume glycerine ,-20 DEG C of preservations.
Enzyme dilution comprises 10mL/L2M NaOH, 15g/L NaCl, 10g/LBSA, 5g/L glucosan T-2000(Dextran T-2000), 1.05g/L Triton X-100 (Triton X-100), 2.5mL/L gentamicin sulphate, (famille rose is powder solid to 1mL/L famille rose, be mixed with concentration 40mg/mL to use later), 2g/L Tween-20(available from Sigma), 1mL/L ProClin300(available from Sigma);
The preparation of (6) 20 times of concentrated washing lotions
20 times of concentrated washing lotions comprise 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/LTween-20 and 2%Proclin300;
(7) preparation of chemical luminescence for liquid A liquid and B liquid
A liquid is 0.7g/L luminol, 0.165g/L p-iodophenol, and damping fluid is the 5mmol/L Tris-HCl of pH8.6, keeps in Dark Place; B liquid is 0.675g/L urea peroxide, prepares with process water; The 5min mixing before use of A liquid and B liquid;
(8) assemble: mentioned reagent is assembled into box, is stored in 2 ~ 8 DEG C;
(9) to adopting the kit prepared of the method to carry out physical examination, the measured value of linear, precision, the specificity of accuracy, dose-response curve, sensitivity, quality-control product and stability are measured.
Principle of the present invention is, adopt the MYO in double antibody sandwich method mensuration serum or blood plasma, biotin-MYO antibody conjugates is added in Avidin-nano magnetic microparticle suspending liquid, by the compatible reaction of Avidin and biotin, form magnetic particle-Avidin-Biotin-MYO antibody complex, add sample and enzyme, pass through antigen-antibody reaction, form magnetic particle-Avidin-Biotin-MYO antibody-MYO antigen-MYO antibody-HRP compound, with magnetic field, compound is adsorbed on bottom test tube, wash free composition, add substrate working fluid, under oxygenant effect, HRP catalysis luminol generates the aminophthalic acid ion being in excited state, when it returns to ground state, discharge the photon of 425nm, the luminous value RLU of each well is measured in the 5th minute.RLU and the sample MYO concentration of sample are proportionate.MYO concentration in sample is according to the Log(X set up by calibration object MYO concentration and corresponding RLU)-Log(Y) mathematical model is carried out quantitatively, thus the MYO content in detection human serum, blood plasma.
The myoglobins nano magnetic particulate chemistry electrochemiluminescent immunoassay quantitative determination reagent kit of invention, has the following advantages:
(1) highly sensitive, the sensitivity for analysis of this kit is not higher than 1ng/mL.
(2) specificity is good, and this product does not occur cross reaction to haemoglobin (10mg/mL).
(3) accuracy is good, and in batch, imprecision is not higher than 5%, and between batch, imprecision is not higher than 10%.
(4) cost is low, and compare with like product on market, this kit is functional, and cost is low, has clinical value.
(5) have good stability, this product can deposit more than 7 days at 37 DEG C, can deposit 1 year at 2 ~ 8 DEG C.
Accompanying drawing explanation
Fig. 1 is the measurement result comparison diagram that kit measurement myoglobins of the present invention and Roche measure myoglobins, wherein ordinate is the myoglobins value that this kit records, horizontal ordinate is Roche kit measurement myoglobins value, two kinds of method related coefficient (r)=0.9771, straight-line equation y=0.8974x+1.288.
Embodiment
Embodiment 1: prepare myoglobins nano magnetic particulate chemistry electrochemiluminescent immunoassay quantitative determination reagent kit
(1) preparation of myoglobins calibration object:
Myoglobins antigen (purchased from Fitzgerald company) lowlenthal serum (Zhengzhou Yikang bioengineering company limited) is mixed with the dense liquid storage of calibration object, calibrates with company standard product, dense liquid storage lowlenthal serum is diluted to working concentration, be respectively 0,5,25,100,250,1000ng/mL;
(2) preparation of myoglobins quality-control product:
With lowlenthal serum, above-mentioned dense liquid storage is diluted to 10ng/mL and 500ng/mL, using 10ng/mL as low value Quality Control, 500ng/mL is as high level Quality Control;
(3) preparation of nano magnetic particulate-Streptavidin suspending liquid:
Prepared by A, ferriferrous oxide nano magnetic particle
Adopt the precipitation method to prepare ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1) by FeCl
36H
2o and FeCl
24H
2o joins in distilled water with mol ratio 2:1, dissolved with vigorous agitation; 2) add 0.5M ammoniacal liquor in a nitrogen environment in above-mentioned iron salt solutions, adjust pH9-10, temperature of reaction 65 DEG C, reaction time 45min; 3), after reaction terminates, be washed with distilled water to neutrality, abandon supernatant, in 60 DEG C of oven dry, obtain the ferriferrous oxide nano magnetic particle of 10-50nm;
The coupling of B, nanometer magnetic bead surface carboxyl groups
Dispersion copolymerization method is adopted to carry out coupling, concrete preparation method is as follows: the nano magnetic microparticulate ultrasound getting above-mentioned preparation is dispersed in 10% polyglycol (PEG8000) solution, obtain magnetic fluid solution, in magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume, after stirring 30min, moves into stirrer, condenser pipe, in the three-necked bottle of nitrogen inlet, add crosslinking chemical N, N '-methylene-bisacrylamide; Under the protection of nitrogen, be warming up to 60 ± 1 DEG C, constant temperature stirs 30min, add benzoyl peroxide successively afterwards, consumption is 3% of magnetic fluid consumption, and stirring rate is about 500rpm, volume of styrene is with magnetic fluid solution, and acrylic acid volume is 1/4 of magnetic fluid solution, keeps stream of nitrogen gas, all the other conditions remain unchanged, reaction 8-10h, and products therefrom leaves standstill, use distilled water cyclic washing, regulate pH=1 with hydrochloric acid again, soak 24h, leave standstill; Use distilled water cyclic washing again, remove not coated Fe
3o
4magnetic, puts into dry 24h at vacuum drying chamber 50 DEG C the product precipitated, obtains the nano magnetic particulate that surface is associated with carboxyl;
The preparation of C, nano magnetic particulate-Streptavidin suspending liquid, preparation 1L, method is as follows:
Get 100mL0.1M2-morpholino b acid (MES) damping fluid, add the nano magnetic particulate that 10mg surface is associated with carboxyl, stirring at room temperature 40min, add 3.5mg Streptavidin afterwards, then 8mg/mL carbodiimide (EDC) solution is added, after 2-8 DEG C of reaction 1h, with 0.01M PBS buffer solution 3 times, be finally dissolved to 1L with 0.01M PBS;
(4) preparation of biotin labeled myoglobins antibody
Get 0.8mg myoglobins antibody, to dialyse at 2 ~ 8 DEG C 1 ~ 3h with borate buffer solution; Antibody after dialysis is added 30ug biotin, adds dimethyl sulfoxide (DMSO) simultaneously, make dimethyl sulfoxide (DMSO) ultimate density 5-10%, slowly vibrate, lucifuge reaction 3h; 250uL1M ammonium chloride solution is added, reacting at normal temperature without light 30-60min in above-mentioned solution; To dialyse at 2 ~ 8 DEG C 24h with 0.01MPBS solution, period changes liquid 2-4 time;
(5) preparation of myoglobins abzyme bond
After adopting improvement sodium periodate oxidation that myoglobins antibody and horseradish peroxidase are carried out coupling, be diluted to working concentration 1:5000 with enzyme dilution, and added 13% enzyme stabilizers, be stored in 2 ~ 8 DEG C;
Enzyme dilution comprises 10mL/L2M NaOH, 15g/L NaCl, 10g/LBSA, 5g/L Dextran T-2000(available from Sigma), 1.05g/L Triton X-100(available from Sigma), 2.5mL/L gentamicin sulphate, (famille rose is powder solid to 1mL/L famille rose, be mixed with concentration 40mg/mL to use later), 2g/L Tween-20(available from Sigma), 1mL/L ProClin300(available from Sigma)
The preparation of (6) 20 times of concentrated washing lotions
20 times of concentrated washing lotions comprise 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/LTween-20 and 2%Proclin300;
(7) preparation of chemical luminescence for liquid A liquid and B liquid
A liquid is 0.7g/L luminol, 0.165g/L p-iodophenol, and damping fluid is the 5mmol/L Tris-HCl of pH8.6, keeps in Dark Place; B liquid is 0.675g/L urea peroxide, prepares with process water; The 5min mixing before use of A liquid and B liquid;
(8) assemble: mentioned reagent is assembled into box, is stored in 2 ~ 8 DEG C;
(9) to adopting the kit prepared of the method to carry out physical examination, the measured value of linear, precision, the specificity of accuracy, dose-response curve, sensitivity, quality-control product and stability are measured.
Embodiment 2: the inspection of kit of the present invention
(1) physical examination: liquid component should be clarified, without precipitation or floccus; Other components should without packages in damaged condition.
(2) accuracy: kit calibration object and national standard series are carried out analysis simultaneously and measured, and use double-log Model fitting, requires two not obvious parallel deviates of dose-response curve (t check, | t|<2.447); With myoglobins company standard product for reference substance, use double-log Model fitting, the measured value of kit calibration object and the mean value of sign value ratio should in 0.90 ~ 1.10 scopes.
(3) dose-response curve is linear: use double-log Model fitting, and dose-response curve correlation coefficient r absolute value in 5-1000ng/mL concentration range is not less than 0.9900.
(4) sensitivity for analysis: kit assay sensitivity is not higher than 1ng/mL.
(5) precision: 10 hole replicate determination high level and low value quality-control product, calculates the mean concentration of measurement result
with standard deviation (SD), imprecision in batch
use 3 batches of products carry out 3 times test, calculate measurement result mean concentration (
) and standard deviation (SD), imprecision between batch
result should meet batch interior imprecision (CV%) should not higher than 5%; Between batch, imprecision (CV%) should not higher than 10%.(6) measured value of quality-control product: the quality-control product of replicate determination 10 hole high level and low value, with Log (X)-Log (Y) Model fitting, quality-control product measured value should in allowed band, and low value quality-control product measured value is at 8-12ng/mL, and high level quality-control product measured value is at 400-600ng/mL.
(7) specificity:
Cross reaction meets following table and requires:
(8.) stability: place 7 days for 37 DEG C, measured value should meet above-mentioned requirements.
Embodiment 3: the using method of kit of the present invention
(1) kit to be checked is balanced 30 minutes under room temperature (18 ~ 25 DEG C).
(2) washing lotion is prepared: washing lotion will be concentrated by 1:20 dilution (1mL washing lotion adds 19mL distilled water) with distilled water.If concentrated washing lotion has crystallization, concentrated washing lotion can be placed in room temperature or 37 DEG C, dilute again after dissolving to be crystallized.
(3) luminescent solution is prepared: use first 5 minutes and get appropriate luminescent solution A and mix with luminescent solution B equal-volume.
(4) reaction tube is numbered, 25uL calibration object or serum specimen is added successively in test tube, 50uL magnetic-particle-Streptavidin suspending liquid, 50uL biotin-myoglobins antibody conjugates, 100uL myoglobins abzyme bond, oscillating reactions 30min at 37 DEG C, test tube rack is placed on magnetic separator and is separated 5min, then supernatant is poured out, add 500uL washing lotion, after abundant mixing, be separated on magnetic separator, pour out washing lotion, repeat 3 times, Chemoluminescent substrate 100uL is added in each pipe, abundant mixing, secretly put 5min, tube-type chemical light-emitting appearance measures the luminous value (RLU) of each pipe, with the Log value of calibration object concentration for horizontal ordinate, with the Log of luminous value for ordinate, drawing standard curve, the concentration of myoglobins can be calculated according to the luminous value of serum specimen.
Embodiment 4: the evaluation of methodology result of this kit
Sensing range: scope is 5-1000ng/mL, measures after the sample being greater than 1000ng/mL for concentration should first dilute again.
Sensitivity: 1ng/mL.
Precision: be less than 5%.
Accuracy: the mean value of the recovery is in 0.90 ~ 1.10 scope.
Specificity: haemoglobin cross reaction coefficient is less than 1%.
Quality-control product measured value: the measured value of low value quality-control product QcL and high level quality-control product QcH is all in allowed band, and low value quality-control product measured value is at 8-12ng/mL, and high level quality-control product measured value is at 400-600ng/mL.
Stability: reagent component each in kit is placed 7d at 37 DEG C, has good stability.
Embodiment 5: the clinical comparison experiment of this kit
The kit of invention has carried out clinical examination, total sample number 120 example of this clinical testing, first with after the test of myoglobins Roche detection kit, the kit of invention (chemiluminescence) is used to measure again, result shows, straight-line equation is y=0.8974x+1.288, coefficient R=0.9771.Kit prepared by visible this method and hospital's measured value have good consistance.With SPSS13.0 statistical analysis software, t inspection (inspection level α=0.05) is carried out to related coefficient, P<0.001, the related intimate degree of the myoglobins value of two kinds of method mensuration is conspicuousnesses, the myoglobins value that visible two kinds of methods measure is closely related, illustrate that the diagnosis capability of kit is comparatively strong, can clinical practice be promoted.
In order to determine the clinical reference value of this kit, adopt this kit to detect to 256 portions of normal human serums, plasma sample, result shows that the reference value (term of reference) of this kit is 0-100ng/mL.
Claims (1)
1. a myoglobins nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box, described kit comprises:
1) myoglobins calibration object, concentration is 0,5,25,100,250,1000ng/mL;
2) coupling has the nano magnetic microparticle suspending liquid of Streptavidin; Described nano magnetic particulate is the tri-iron tetroxide that surface wraps up with amino or carboxyl-reactive group, particle diameter 10-50nm;
3) biotin labeled myoglobins antibody, described antibody is monoclonal antibody;
4) myoglobins abzyme bond, antibody used is monoclonal antibody, and be not same strain with biotin labeled myoglobins antibody, enzyme used is horseradish peroxidase, horseradish peroxidase purity RZ >=3.0, activity >=250U/mL;
5) myoglobins quality-control product; Quality-control product comprises the low value quality-control product of concentration 10ng/mL and the high level quality-control product of 500ng/mL;
6) chemical luminescence for liquid A liquid and B liquid; A liquid is the Tris-HCl damping fluid of 5mmol/L, pH8.6, and containing final concentration 0.7g/L luminol and final concentration 0.165g/L p-iodophenol in this damping fluid; B liquid is 0.675g/L urea peroxide;
7) 20 times of concentrated washing lotions;
8) reaction tube, the material of described reaction tube is transparent polystyrene, transparent polyethylene, transparent polypropylene or clear glass; It is characterized in that, described kit preparation comprises the following steps:
(1) preparation of myoglobins calibration object:
Myoglobins antigen lowlenthal serum is mixed with the dense liquid storage of calibration object, calibrates with company standard product, dense liquid storage lowlenthal serum is diluted to working concentration, is respectively 0,5,25,100,250,1000ng/mL;
(2) preparation of myoglobins quality-control product:
With lowlenthal serum, above-mentioned dense liquid storage is diluted to 10ng/mL and 500ng/mL, using 10ng/mL as low value Quality Control, 500ng/mL is as high level Quality Control;
(3) preparation of nano magnetic particulate-Streptavidin suspending liquid:
Prepared by A, ferriferrous oxide nano magnetic particle
Adopt the precipitation method to prepare ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1) by FeCl
36H
2o and FeCl
24H
2o joins in distilled water with mol ratio 2:1, dissolved with vigorous agitation; 2) add 0.5M ammoniacal liquor in a nitrogen environment in above-mentioned iron salt solutions, adjust pH9-10, temperature of reaction 65 DEG C, reaction time 45min; 3), after reaction terminates, be washed with distilled water to neutrality, abandon supernatant, in 60 DEG C of oven dry, obtain the ferriferrous oxide nano magnetic particle of 10-50nm;
The coupling of B, nanometer magnetic bead surface carboxyl groups
Dispersion copolymerization method is adopted to carry out coupling, concrete preparation method is as follows: the nano magnetic microparticulate ultrasound getting above-mentioned preparation is dispersed in 10%PEG8000 solution, obtain magnetic fluid solution, in magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume, after stirring 30min, moves into stirrer, condenser pipe, in the three-necked bottle of nitrogen inlet, add crosslinking chemical N, N '-methylene-bisacrylamide; Under the protection of nitrogen, be warming up to 60 ± 1 DEG C, constant temperature stirs 30min, add benzoyl peroxide successively afterwards, consumption is 3% of magnetic fluid consumption, and stirring rate is about 500rpm, volume of styrene is with magnetic fluid solution, and acrylic acid volume is 1/4 of magnetic fluid solution, keeps stream of nitrogen gas, all the other conditions remain unchanged, reaction 8-10h, and products therefrom leaves standstill, use distilled water cyclic washing, regulate pH=1 with hydrochloric acid again, soak 24h, leave standstill; Use distilled water cyclic washing again, remove not coated Fe
3o
4magnetic, puts into dry 24h at vacuum drying chamber 50 DEG C the product precipitated, obtains the nano magnetic particulate that surface is associated with carboxyl;
The preparation of C, nano magnetic particulate-Streptavidin suspending liquid, preparation 1L, method is as follows:
Get 100mL0.1M MES damping fluid, add the nano magnetic particulate that 10mg surface is associated with carboxyl, stirring at room temperature 40min, add 3.5mg Streptavidin afterwards, then 8mg/mLEDC solution is added, after 2-8 DEG C of reaction 1h, with 0.01M PBS buffer solution 3 times, be finally settled to 1L with 0.01M PBS;
(4) preparation of biotin labeled myoglobins antibody
Get 0.8mg myoglobins antibody, to dialyse at 2 ~ 8 DEG C 1 ~ 3h with borate buffer solution; Antibody after dialysis is added 30 μ g biotins, adds dimethyl sulfoxide (DMSO) simultaneously, make dimethyl sulfoxide (DMSO) ultimate density 5-10%, slowly vibrate, lucifuge reaction 3h; 250 μ L1M ammonium chloride solutions are added, reacting at normal temperature without light 30-60min in above-mentioned solution; To dialyse at 2 ~ 8 DEG C 24h with 0.01MPBS solution, period changes liquid 2-4 time;
(5) preparation of myoglobins abzyme bond
After adopting improvement sodium periodate oxidation that myoglobins antibody and horseradish peroxidase are carried out coupling, be diluted to working concentration 1:5000 with enzyme dilution, and added 13% enzyme stabilizers, be stored in 2 ~ 8 DEG C; Enzyme dilution comprises 10mL/L 2M NaOH, 15g/L NaCl, 10g/LBSA, 5g/L Dextran T-2000,1.05g/L Triton X-100,2.5mL/L gentamicin sulphate, 1mL/L is carmine, and famille rose is powder solid, is mixed with concentration 40mg/mL and uses later, 2g/L Tween-20,1mL/L ProClin300;
The preparation of (6) 20 times of concentrated washing lotions
20 times of concentrated washing lotions comprise 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/LTween-20 and 2%Proclin300;
(7) preparation of chemical luminescence for liquid A liquid and B liquid
A liquid is 0.7g/L luminol, 0.165g/L p-iodophenol, and damping fluid is the 5mmol/L Tris-HCl of pH8.6, keeps in Dark Place; B liquid is 0.675g/L urea peroxide, prepares with process water; The 5min mixing before use of A liquid and B liquid;
(8) assemble: mentioned reagent is assembled into box, is stored in 2 ~ 8 DEG C;
(9) to adopting the kit prepared of the method to carry out physical examination, the measured value of linear, precision, the specificity of accuracy, dose-response curve, sensitivity, quality-control product and stability are measured.
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