CN103278651A - 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 the immunoassay medical domain, concrete, the invention provides a kind of myoglobins (MYO) nanometer magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof.
Background technology
Myoglobins (MYO) is the hemoprotein similar to haemoglobin.Be present in the muscle cell, major function is to store oxygen, and is very abundant at myocardium intensive amount.Molecular weight 16700 is made of polypeptied chain and a protoheme prothetic group that contains 153 amino acid residues, is tight sphere.Hydrophobic side chain in the polypeptied chain on the amino acid residue is mostly at intramolecule, and the hydrophilic side chain multidigit is in molecular surface, so it is water-soluble better.
Because the MYO molecular weight is little, so early be released into blood circulation after the muscle cell damage, 1-2h raises, and 12h reaches the peak, descend gradually behind the 24h, and be the leading indicator of early diagnosis acute myocardial infarction AMI (AMI), can win the quality time for diagnosis and the rescue of AMI.
Serum MYO concentration also can raise when skeletal muscle disease and renal dysfunction, so MYO is not the specificity marker thing of myocardial damage.But because it is highly sensitive, generally can be used for negative diagnosis.If pectoralgia outbreak back 8h and MYO still in normal range, then can get rid of AMI.
Remove from kidney because MYO is very fast in patient's blood behind AMI, have or not again infarct or infarct to expand again so MYO measures to help to observe in the AMI course of disease.MYO frequently increases, and points out original myocardial infarction still in continuity.
MYO judges the sensitivity have or not perfusion again and index accurately in the thromboembolism treatment.
The MYO immunologic detection method that clinical field is used mainly contain colloidal gold immunity chromatography (Gold immnnochromatography GICA), enzymoimmunoassay (Enzyme Immunoassay, EIA) and chemiluminescent immunoassay(CLIA) (CLIA) etc.
That colloidal gold immunity chromatography possesses is easy and simple to handle, quick, can single part detection, be convenient to preserve, do not need advantage such as specific installation, but because sensitivity is not high, the use of this method that drawbacks limit such as has been difficult to realize quantitatively.
Characteristics such as susceptibility, the specificity that enzymoimmunoassay is simple to operate owing to possessing, the reagent term of validity is long, pollution-free, be higher than collaurum is good, the available Instrument measuring of result are promoted, but because susceptibility is relatively low, but used mark enzyme-to-substrate quantitative measurement narrow range, reach shortcomings such as Instrument measuring narrow range, limited its application in the 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 the most responsive skeptophylaxis determination method.Principal feature is hypersensitivity, can measure that concentration range is wide, sample need not that dilution can detect, the reagent term of validity is long, simple to operate, detection automatically degree of changing into height, data generate automatically that processing power is strong, compatible good, the non-environmental-pollution of determining instrument etc., thereby obtained developing rapidly and used.But detection sensitivity, measured value accuracy are still not high.
Summary of the invention
The problem to be solved in the present invention provides quantitative detection kit of chemiluminescence immunoassay of myoglobins and preparation method thereof, has avoided the colloidal gold strip can not be accurately quantitative, euzymelinked immunosorbent assay (ELISA) poor sensitivity, defective 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) nanometer magnetic microparticle chemiluminescence immune quantitative detection reagent box comprises: the myoglobins calibration object; Coupling has the nanometer magnetic particle suspending liquid of Streptavidin; Biotin labeled myoglobins antibody; Myoglobins abzyme bond, used enzyme are horseradish peroxidase, horseradish peroxidase purity RZ 〉=3.0, activity 〉=250U/mL; Myoglobins quality-control product, quality-control product comprise the low value quality-control product of concentration 8-12ng/mL and the high value quality-control product of 400-600ng/mL; Chemical luminescence for liquid A liquid and B liquid, A liquid are 5mmol/L, the Tris-HCl damping fluid of pH8.6, and contain final concentration 0.7g/L luminol and final concentration 0.165g/L p-iodophenol in this damping fluid; B liquid is the 0.675g/L urea peroxide; 20 times of concentrated washing lotions; Reaction tube.
Further, described nanometer magnetic particle is the tri-iron tetroxide that the surface parcel has 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 may further comprise the steps:
(1) preparation of myoglobins calibration object:
Myoglobins antigen is mixed with the dense liquid storage of calibration object with lowlenthal serum, calibrates with the company standard product, dense liquid storage is diluted to working concentration with lowlenthal serum, be respectively 0,5,25,100,250,1000ng/mL;
Company standard product preparation method: with lowlenthal serum with 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 the quantitative detection kit of Roche Holding Ag's myoglobins (article No.: 12178214, specification 100 person-portions) to measure, according to measurement result, set up dilution ratio and the curve of measuring concentration.Calculate the dilution ratio that concentration is 5,25,100,250,1000ng/mL respectively according to curvilinear equation, in these ratio preparation company standard product, use the Luo Shi product repeatedly to measure, determine company standard product concentration.The company standard product of preparation are used the freeze drier freeze-drying ,-20 ℃ of preservations with the packing of 0.5mL/ pipe.
(2) preparation of myoglobins quality-control product:
With lowlenthal serum above-mentioned dense liquid storage is diluted to 10ng/mL and 500ng/mL, as the low value Quality Control, 500ng/mL is as the Quality Control of high value with 10ng/mL;
(3) preparation of nanometer magnetic particle-Streptavidin suspending liquid:
A, the preparation of ferriferrous oxide nano magnetic particle
Adopt the precipitation method to prepare the ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1) with FeCl
36H
2O and FeCl
2.4H
2O joins in the distilled water with mol ratio 2:1, the vigorous stirring dissolving; 2) under nitrogen environment, add 0.5M ammoniacal liquor in above-mentioned iron salt solutions, transfer pH9-10,65 ℃ of temperature of reaction, reaction time 45min; 3) after reaction finishes, be washed with distilled water to neutrality, abandon supernatant, in 60 ℃ of oven dry, namely get the ferriferrous oxide nano magnetic particle of 10-50nm;
The coupling of B, nanometer magnetic bead surface carboxyl
Adopt dispersion copolymerization method to carry out coupling, concrete preparation method is as follows: get in the ultrasonic 10%PEG8000 of the being dispersed in solution of nanometer magnetic particle of above-mentioned preparation, get magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume in the magnetic fluid solution, stir 30min after, move into and have stirrer, condenser pipe, in the three-necked bottle of nitrogen inlet, add crosslinking chemical N, N '-methylene-bisacrylamide; Under protection of nitrogen gas, be warming up to 60 ± 1 ℃, constant temperature stirs 30min, add benzoyl peroxide afterwards successively, consumption is 3% of magnetic fluid consumption, and stirring rate is about 500rpm, volume of styrene is with magnetic fluid solution, and the 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 the distilled water cyclic washing, regulate pH=1 with hydrochloric acid again, soak 24h, leave standstill; Use the distilled water cyclic washing again, remove the Fe that does not coat
3O
4Magnetic is put into 50 ℃ of following dry 24h of vacuum drying chamber to the product that precipitates, and obtains the nanometer magnetic particle that the surface is associated with carboxyl;
The preparation of C, nanometer magnetic particle-Streptavidin suspending liquid, preparation 1L, method is as follows:
Get 100mL0.1M MES damping fluid, adding 10mg surface is associated with the nanometer magnetic particle of carboxyl, stirring at room 40min, add the 3.5mg Streptavidin afterwards, add 8mg/mLEDC solution then, behind the 2-8 ℃ of reaction 1h, with 0.01M PBS damping fluid washing 3 times, get final product to 1L with 0.01M PBS is fixed molten at last;
(4) preparation of biotin labeled myoglobins antibody
Get 0.8mg myoglobins antibody, with borate buffer solution dialysis 1～3h under 2～8 ℃; Antibody after the dialysis is added the 30ug biotin, add dimethyl sulfoxide (DMSO) simultaneously, make dimethyl sulfoxide (DMSO) ultimate density 5-10%, slowly vibration, lucifuge reaction 3h; In above-mentioned solution, add 250uL1M ammonium chloride solution, reacting at normal temperature without light 30-60min; With 0.01MPBS solution at 2～8 ℃ of following dialysis 24h, during change liquid 2-4 time;
(5) preparation of myoglobins abzyme bond
After adopting the improvement sodium periodate oxidation that myoglobins antibody and horseradish peroxidase are carried out coupling, with the enzyme dilution it is diluted to working concentration 1:5000, and adds 13% enzyme stabilizers, be stored in 2～8 ℃;
Improvement sodium periodate oxidizing process step comprises:
The A:HRP activation
1) configuration 10mg/mL HRP solution;
2) configuration 12.8mg/mL sodium periodate NaIO
4Solution;
3) with above-mentioned 1) and 2) obtain solution 1:1 mixing by volume, 4 ℃ of lucifuges reaction 30min;
4) configuration concentration is the glycol water of 20uL/mL, with above-mentioned solution 3) mix with equal volume, reacting at normal temperature without light 20min, activation is namely finished, and puts-20 ℃ and preserves (holding time is no more than 3 months).
B, MYO labeling of monoclonal antibody
1) raw material to be marked is packed in the bag filter, with the carbonate buffer solution of 0.05M pH9.6, dialysis 30min;
2) the mark raw material is mixed by mass ratio 1:2 with the HRP of activation, during 4 ℃ of dialysis 24h(, change liquid 2-3 time with the 0.05M carbonate buffer solution afterwards);
3) configuration concentration is the NaBH of 2mg/mL
4Aqueous solution adds the NaBH that 80uL prepares by 1mgHRP
4The ratio of aqueous solution is mixed, and in 4 ℃ of lucifuge reaction 2h;
4) with above-mentioned steps 3) marking fluid finished in 4 ℃ of dialysis 24h, adds equal-volume glycerine ,-20 ℃ of preservations with 0.01M PBS.
Comprise 10mL/L2M NaOH in the enzyme dilution, 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 the 1mL/L famille rose, being mixed with concentration 40mg/mL uses later on), 2g/L Tween-20(is available from Sigma company), 1mL/L ProClin300(is available from Sigma company);
The preparation of (6) 20 times of concentrated washing lotions
20 times of concentrated washing lotions comprise the 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 the 0.7g/L luminol, and 0.165g/L p-iodophenol, damping fluid are the 5mmol/L Tris-HCl of pH8.6, keep in Dark Place; B liquid is the 0.675g/L urea peroxide, prepares with process water; A liquid and B liquid 5min before use mix;
(8) assembling: mentioned reagent is assembled into box, is stored in 2～8 ℃;
(9) kit that adopts the preparation of this method is carried out physical examination, measured value and the stability of the linearity of accuracy, dose-response curve, precision, specificity, sensitivity, quality-control product are measured.
Principle of the present invention is, adopt the MYO in double antibody sandwich method mensuration serum or the blood plasma, in Avidin-nanometer magnetic particle suspending liquid, add biotin-MYO antibody conjugates, compatible reaction by Avidin and biotin, form magnetic particle-Avidin-biotin-MYO antibody complex, add sample and enzyme, by antigen-antibody reaction, form magnetic particle-Avidin-biotin-MYO antibody-MYO antigen-MYO antibody-HRP compound, with magnetic field compound is adsorbed on the test tube bottom, wash free composition, add the substrate working fluid, under the oxygenant effect, HRP catalysis luminol generates the aminophthalic acid ion that is in excited state, when it returns to ground state, discharge the photon of 425nm, measured the luminous value RLU of each well in the 5th minute.The RLU of sample and sample MYO concentration are proportionate.MYO concentration in the sample is according to the Log(X that is set up by calibration object MYO concentration and corresponding RLU)-Log(Y) mathematical model is carried out quantitatively, thus detect the MYO content in human serum, the blood plasma.
The myoglobins nanometer magnetic microparticle chemiluminescence immunological quantitative determining kit of this patent 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 cross reaction do not occur to haemoglobin (10mg/mL).
(3) accuracy is good, and imprecision is not higher than 5% in batch, and imprecision is not higher than 10% between batch.
(4) cost is low, compares with like product on the market, and this kit is functional, and cost is low, has clinical value.
(5) have good stability, this product can be deposited more than 7 days at 37 ℃, can deposit 1 year at 2～8 ℃.
Description of drawings
Fig. 1 is the measurement result comparison diagram that kit measurement myoglobins of the present invention and Luo Shi measure myoglobins, the myoglobins value that records for this kit of ordinate wherein, horizontal ordinate is Luo Shi kit measurement myoglobins value, two kinds of method related coefficients (r)=0.9771, straight-line equation y=0.8974x+1.288.
Embodiment
Embodiment 1: preparation myoglobins nanometer magnetic microparticle chemiluminescence immunological quantitative determining kit
(1) preparation of myoglobins calibration object:
Myoglobins antigen (available from Fitzgerald company) is mixed with the dense liquid storage of calibration object with lowlenthal serum (Zhengzhou Yi Kang bioengineering company limited), calibrates with the company standard product, dense liquid storage is diluted to working concentration with lowlenthal serum, 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, as the low value Quality Control, 500ng/mL is as the Quality Control of high value with 10ng/mL;
(3) preparation of nanometer magnetic particle-Streptavidin suspending liquid:
A, the preparation of ferriferrous oxide nano magnetic particle
Adopt the precipitation method to prepare the ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1) with FeCl
36H
2O and FeCl
24H
2O joins in the distilled water with mol ratio 2:1, the vigorous stirring dissolving; 2) under nitrogen environment, add 0.5M ammoniacal liquor in above-mentioned iron salt solutions, transfer pH9-10,65 ℃ of temperature of reaction, reaction time 45min; 3) after reaction finishes, be washed with distilled water to neutrality, abandon supernatant, in 60 ℃ of oven dry, namely get the ferriferrous oxide nano magnetic particle of 10-50nm;
The coupling of B, nanometer magnetic bead surface carboxyl
Adopt dispersion copolymerization method to carry out coupling, concrete preparation method is as follows: ultrasonic being dispersed in 10% polyglycol (PEG8000) solution of nanometer magnetic particle of getting above-mentioned preparation, get magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume in the magnetic fluid solution, stir 30min after, move into and have stirrer, condenser pipe, in the three-necked bottle of nitrogen inlet, add crosslinking chemical N, N '-methylene-bisacrylamide; Under protection of nitrogen gas, be warming up to 60 ± 1 ℃, constant temperature stirs 30min, add benzoyl peroxide afterwards successively, consumption is 3% of magnetic fluid consumption, and stirring rate is about 500rpm, volume of styrene is with magnetic fluid solution, and the 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 the distilled water cyclic washing, regulate pH=1 with hydrochloric acid again, soak 24h, leave standstill; Use the distilled water cyclic washing again, remove the Fe that does not coat
3O
4Magnetic is put into 50 ℃ of following dry 24h of vacuum drying chamber to the product that precipitates, and obtains the nanometer magnetic particle that the surface is associated with carboxyl;
The preparation of C, nanometer magnetic particle-Streptavidin suspending liquid, preparation 1L, method is as follows:
Get 100mL0.1M2-morpholino b acid (MES) damping fluid, adding 10mg surface is associated with the nanometer magnetic particle of carboxyl, stirring at room 40min, add the 3.5mg Streptavidin afterwards, add 8mg/mL carbodiimide (EDC) solution then, behind the 2-8 ℃ of reaction 1h, with 0.01M PBS damping fluid washing 3 times, get final product to 1L with 0.01M PBS is fixed molten at last;
(4) preparation of biotin labeled myoglobins antibody
Get 0.8mg myoglobins antibody, with borate buffer solution dialysis 1～3h under 2～8 ℃; Antibody after the dialysis is added the 30ug biotin, add dimethyl sulfoxide (DMSO) simultaneously, make dimethyl sulfoxide (DMSO) ultimate density 5-10%, slowly vibration, lucifuge reaction 3h; In above-mentioned solution, add 250uL1M ammonium chloride solution, reacting at normal temperature without light 30-60min; With 0.01MPBS solution at 2～8 ℃ of following dialysis 24h, during change liquid 2-4 time;
(5) preparation of myoglobins abzyme bond
After adopting the improvement sodium periodate oxidation that myoglobins antibody and horseradish peroxidase are carried out coupling, with the enzyme dilution it is diluted to working concentration 1:5000, and adds 13% enzyme stabilizers, be stored in 2～8 ℃;
Comprise 10mL/L2M NaOH in the enzyme dilution, 15g/L NaCl, 10g/LBSA, 5g/L Dextran T-2000(is available from Sigma company), 1.05g/L Triton X-100(is available from Sigma company), the 2.5mL/L gentamicin sulphate, (famille rose is powder solid to the 1mL/L famille rose, being mixed with concentration 40mg/mL uses later on), 2g/L Tween-20(is available from Sigma company), 1mL/L ProClin300(is available from Sigma company)
The preparation of (6) 20 times of concentrated washing lotions
20 times of concentrated washing lotions comprise the 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/L Tween-20 and 2%Proclin300;
(7) preparation of chemical luminescence for liquid A liquid and B liquid
A liquid is the 0.7g/L luminol, and 0.165g/L p-iodophenol, damping fluid are the 5mmol/L Tris-HCl of pH8.6, keep in Dark Place; B liquid is the 0.675g/L urea peroxide, prepares with process water; A liquid and B liquid 5min before use mix;
(8) assembling: mentioned reagent is assembled into box, is stored in 2～8 ℃;
(9) kit that adopts the preparation of this method is carried out physical examination, measured value and the stability of the linearity of accuracy, dose-response curve, precision, specificity, sensitivity, quality-control product are measured.
Embodiment 2: the inspection of kit of the present invention
(1) physical examination: liquid component should be clarified, and does not have precipitation or floccus; Other components should not have packages in damaged condition.
(2) accuracy: kit calibration object and national standard product series are carried out assay determination simultaneously, with the match of double-log mathematical model, require two not obvious parallel deviates of dose-response curve (t check, | t|＜2.447); Be reference substance with myoglobins company standard product, with the match of double-log mathematical model, the mean value of the measured value of kit calibration object and sign value ratio should be in 0.90～1.10 scope.
(3) linearity of dose-response curve: with the match of double-log mathematical model, dose-response curve correlation coefficient r absolute value in the 5-1000ng/mL concentration range is not less than 0.9900.
(4) sensitivity for analysis: the kit sensitivity for analysis is not higher than 1ng/mL.
(5) precision: the high value of 10 hole replicate determinations and low value quality-control product, the mean concentration of calculating measurement result
With standard deviation (SD), imprecision in batch
Use 3 batches of products to carry out 3 tests, the mean concentration of calculating measurement result (
) and standard deviation (SD), imprecision between batch
The result should meet batch interior imprecision (CV%) should not be higher than 5%; Imprecision (CV%) should not be higher than 10% between batch.(6) measured value of quality-control product: the quality-control product of the high value in replicate determination 10 holes and low value, with Log (X)-Log (Y) mathematical model match, the quality-control product measured value should be in allowed band, and low value quality-control product measured value is at 8-12ng/mL, and high value quality-control product measured value is at 400-600ng/mL.
(7) specificity:
Cross reaction meets following table and requires:
(8.) stability: placed 7 days for 37 ℃, measured value should meet above-mentioned every requirement.
Embodiment 3: the using method of kit of the present invention
(1) kit to be checked was descended balance 30 minutes in room temperature (18～25 ℃).
(2) preparation washing lotion: will concentrate washing lotion by 1:20 dilution (the 1mL washing lotion adds 19mL distilled water) with distilled water.If concentrate washing lotion crystallization is arranged, can place room temperature or 37 ℃ with concentrating washing lotion, dilute again after the dissolving to be crystallized.
(3) preparation luminescent solution: use and got an amount of luminescent solution A in preceding 5 minutes and mix with luminescent solution B equal-volume.
(4) reaction tube is numbered, in test tube, add 25uL calibration object or serum specimen successively, 50uL magnetic-particle-Streptavidin suspending liquid, 50uL biotin-myoglobins antibody conjugates, 100uL myoglobins abzyme bond, 37 ℃ of following oscillating reactions 30min, test tube rack placed separate 5min on the magnetic separator, pour out supernatant then, add the 500uL washing lotion, fully behind the mixing, on magnetic separator, separate, pour out washing lotion, repeat 3 times, in each pipe, add chemical luminous substrate liquid 100uL, fully mixing is secretly put 5min, measures the luminous value (RLU) of each pipe at the tubular type Chemiluminescence Apparatus, Log value with calibration object concentration is horizontal ordinate, Log with luminous value is ordinate, and the drawing standard curve can calculate the concentration of myoglobins 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 should diluting earlier greater than the sample of 1000ng/mL for concentration again.
Sensitivity: 1ng/mL.
Precision: 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%.
The quality-control product measured value: all in allowed band, low value quality-control product measured value is at 8-12ng/mL for the measured value of low value quality-control product QcL and high value quality-control product QcH, and high value quality-control product measured value is at 400-600ng/mL.
Stability: each reagent component in the kit is placed 7d down in 37 ℃, have good stability.
Embodiment 5: the clinical comparison experiment of this kit
The kit of this patent invention has carried out clinical examination, total sample number 120 examples of this clinical testing, earlier with after the test of myoglobins Luo Shi detection kit, measure with the kit (chemiluminescence) of this patent invention again, the result shows, straight-line equation is y=0.8974x+1.288, coefficient R=0.9771.As seen kit and hospital's measured value of this method preparation have consistance preferably.With the SPSS13.0 statistical analysis software related coefficient is carried out t check (inspection level α=0.05), P＜0.001, the related intimate degree of the myoglobins value of two kinds of method mensuration is conspicuousnesses, as seen the myoglobins value of two kinds of method mensuration is closely related, the diagnosis capability that kit is described is stronger, can promote clinical practice.
In order to determine the clinical reference value of this kit, adopt this kit to detect to 256 parts of normal human serums, plasma samples, the result shows that the reference value (term of reference) of this kit is 0-100ng/mL.
Claims (5)
1. myoglobins nanometer magnetic microparticle chemiluminescence immune quantitative detection reagent box is characterized in that described kit comprises:
1) myoglobins calibration object, concentration are 0,5,25,100,250,1000ng/mL;
2) coupling has the nanometer magnetic particle suspending liquid of Streptavidin;
3) biotin labeled myoglobins antibody, described antibody is monoclonal antibody;
4) myoglobins abzyme bond, used antibody are monoclonal antibody, with biotin labeled myoglobins antibody be not same strain, used enzyme 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 8-12ng/mL and the high value quality-control product of 400-600ng/mL;
6) chemical luminescence for liquid A liquid and B liquid; A liquid is 5mmol/L, the Tris-HCl damping fluid of pH8.6, and contain final concentration 0.7g/L luminol and final concentration 0.165g/L p-iodophenol in this damping fluid; B liquid is the 0.675g/L urea peroxide;
7) 20 times of concentrated washing lotions;
8) reaction tube.
2. myoglobins nanometer magnetic microparticle chemiluminescence immune quantitative detection reagent box according to claim 1 is characterized in that, described myoglobins quality-control product, the preferred 10ng/mL of low value quality-control product, the preferred 500ng/mL of high value quality-control product.
3. myoglobins nanometer magnetic microparticle chemiluminescence immune quantitative detection reagent box according to claim 1 is characterized in that, described nanometer magnetic particle is the tri-iron tetroxide that the surface parcel has amino or carboxyl reactive group, particle diameter 10-50nm.
4. myoglobins nanometer magnetic microparticle chemiluminescence immune quantitative detection reagent box according to claim 1 is characterized in that the material of described reaction tube is transparent polystyrene, tygon, polypropylene or glass.
5. a method for preparing the described kit of the arbitrary claim of described claim 1-3 is characterized in that, may further comprise the steps:
(1) preparation of myoglobins calibration object:
Myoglobins antigen is mixed with the dense liquid storage of calibration object with lowlenthal serum, calibrates with the company standard product, dense liquid storage is diluted to working concentration with lowlenthal serum, 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, as the low value Quality Control, 500ng/mL is as the Quality Control of high value with 10ng/mL;
(3) preparation of nanometer magnetic particle-Streptavidin suspending liquid:
A, the preparation of ferriferrous oxide nano magnetic particle
Adopt the precipitation method to prepare the ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1) with FeCl
36H
2O and FeCl
24H
2O joins in the distilled water with mol ratio 2:1, the vigorous stirring dissolving; 2) under nitrogen environment, add 0.5M ammoniacal liquor in above-mentioned iron salt solutions, transfer pH9-10,65 ℃ of temperature of reaction, reaction time 45min; 3) after reaction finishes, be washed with distilled water to neutrality, abandon supernatant, in 60 ℃ of oven dry, namely get the ferriferrous oxide nano magnetic particle of 10-50nm;
The coupling of B, nanometer magnetic bead surface carboxyl
Adopt dispersion copolymerization method to carry out coupling, concrete preparation method is as follows: get in the ultrasonic 10%PEG8000 of the being dispersed in solution of nanometer magnetic particle of above-mentioned preparation, get magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume in the magnetic fluid solution, stir 30min after, move into and have stirrer, condenser pipe, in the three-necked bottle of nitrogen inlet, add crosslinking chemical N, N '-methylene-bisacrylamide; Under protection of nitrogen gas, be warming up to 60 ± 1 ℃, constant temperature stirs 30min, add benzoyl peroxide afterwards successively, consumption is 3% of magnetic fluid consumption, and stirring rate is about 500rpm, volume of styrene is with magnetic fluid solution, and the 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 the distilled water cyclic washing, regulate pH=1 with hydrochloric acid again, soak 24h, leave standstill; Use the distilled water cyclic washing again, remove the Fe that does not coat
3O
4Magnetic is put into 50 ℃ of following dry 24h of vacuum drying chamber to the product that precipitates, and obtains the nanometer magnetic particle that the surface is associated with carboxyl;
The preparation of C, nanometer magnetic particle-Streptavidin suspending liquid, preparation 1L, method is as follows:
Get 100mL0.1M MES damping fluid, adding 10mg surface is associated with the nanometer magnetic particle of carboxyl, stirring at room 40min, add the 3.5mg Streptavidin afterwards, add 8mg/mLEDC solution then, behind the 2-8 ℃ of reaction 1h, with 0.01M PBS damping fluid washing 3 times, get final product to 1L with 0.01M PBS is fixed molten at last;
(4) preparation of biotin labeled myoglobins antibody
Get 0.8mg myoglobins antibody, with borate buffer solution dialysis 1～3h under 2～8 ℃; Antibody after the dialysis is added the 30ug biotin, add dimethyl sulfoxide (DMSO) simultaneously, make dimethyl sulfoxide (DMSO) ultimate density 5-10%, slowly vibration, lucifuge reaction 3h; In above-mentioned solution, add 250uL1M ammonium chloride solution, reacting at normal temperature without light 30-60min; With 0.01MPBS solution at 2～8 ℃ of following dialysis 24h, during change liquid 2-4 time;
(5) preparation of myoglobins abzyme bond
After adopting the improvement sodium periodate oxidation that myoglobins antibody and horseradish peroxidase are carried out coupling, with the enzyme dilution it is diluted to working concentration 1:5000, and adds 13% enzyme stabilizers, be stored in 2～8 ℃;
The preparation of (6) 20 times of concentrated washing lotions
20 times of concentrated washing lotions comprise the 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 the 0.7g/L luminol, and 0.165g/L p-iodophenol, damping fluid are the 5mmol/L Tris-HCl of pH8.6, keep in Dark Place; B liquid is the 0.675g/L urea peroxide, prepares with process water; A liquid and B liquid 5min before use mix;
(8) assembling: mentioned reagent is assembled into box, is stored in 2～8 ℃;
(9) kit that adopts the preparation of this method is carried out physical examination, measured value and the stability of the linearity of accuracy, dose-response curve, precision, specificity, sensitivity, quality-control product are measured.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102435752A (en) * | 2011-08-31 | 2012-05-02 | 内蒙古科慧生物科技有限责任公司 | Quantitative determination kit for human myoglobin and detection method thereof |
| CN102725633A (en) * | 2009-12-03 | 2012-10-10 | 雅培制药有限公司 | Assay for diagnosis of cardiac myocyte damage |
| CN102998467A (en) * | 2012-11-20 | 2013-03-27 | 博奥赛斯（天津）生物科技有限公司 | Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for beta human chorionic gonadotropin (beta-hCG), and preparation method of kit |
| CN103033627A (en) * | 2011-09-29 | 2013-04-10 | 北京源德生物医学工程有限公司 | Myoglobin enzymatic chemiluminescence immunodetection method and kit |
-
2013
- 2013-06-14 CN CN201310234764.2A patent/CN103278651B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102725633A (en) * | 2009-12-03 | 2012-10-10 | 雅培制药有限公司 | Assay for diagnosis of cardiac myocyte damage |
| CN102435752A (en) * | 2011-08-31 | 2012-05-02 | 内蒙古科慧生物科技有限责任公司 | Quantitative determination kit for human myoglobin and detection method thereof |
| CN103033627A (en) * | 2011-09-29 | 2013-04-10 | 北京源德生物医学工程有限公司 | Myoglobin enzymatic chemiluminescence immunodetection method and kit |
| CN102998467A (en) * | 2012-11-20 | 2013-03-27 | 博奥赛斯（天津）生物科技有限公司 | Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for beta human chorionic gonadotropin (beta-hCG), and preparation method of kit |
Non-Patent Citations (3)
| Title |
|---|
| 王强: "羧基磁性微球检测急性心肌梗塞早期标志蛋白的研究", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》, no. 2, 15 December 2011 (2011-12-15) * |
| 胡建: "Fe3O4/Poly（St-AA）磁性复合高分子微球的制备及生物医学应用", 《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》, no. 12, 15 December 2009 (2009-12-15) * |
| 马艳: "联合检测cTnI、MYO和CK-MB mass对HIE患儿心肌损伤的诊断研究", 《医学研究杂志》, vol. 37, no. 09, 30 September 2008 (2008-09-30), pages 69 - 72 * |
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Address after: 300300 building 14, international medical device Industrial Park, No. 16, Wujing Road, economic and Technological Development Zone, Dongli District, Tianjin Patentee after: Tianjin boasaisi Biotechnology Co.,Ltd. Address before: 300300 No. 10, Siwei Road, development zone, Dongli District, Tianjin Patentee before: BIOSCIENCE (TIANJIN) DIAGNOSTIC TECHNOLOGY Co.,Ltd. |