CN103278623A - Kit for chemiluminescence immunity quantitative detection of CK-MB (creatine kinase- isoenzyme) nano magnetic particle and preparation method of kit - Google Patents
Kit for chemiluminescence immunity quantitative detection of CK-MB (creatine kinase- isoenzyme) 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 a CK-MB nano magnetic particle. The kit comprises a CK-MB calibrator; a nano magnetic particle suspension liquid coupled with streptavidin, bioti-labeled CK-MB antibodies, CK-MB abzyme conjugate, a CK-MB 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 CK-MB 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 validity 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 isoenzymes of creatine kinase (CK-MB) nanometer magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof.
Background technology
(Creatine Kinase CK) is present in tissues such as cardiac muscle, skeletal muscle and brain to cretinephosphokinase in a large number, is the important kinases that and intracellular energy running, contraction of muscle, ATP regeneration have direct relation.Natural CK molecule is a chondritic closely, the dimer of being made up of two subunits (M type subunit and Type B subunit).CK has three kinds of main isodynamic enzymes, i.e. CK-MM, CK-MB, CK-BB.Wherein CK-BB mainly is present in brain, prostate, intestines, lung; There are a large amount of CK-MM and a spot of CK-MB in the skeletal muscle; Though and cardiac muscular tissue is based on CK-MM, the CK-MB proportion is far beyond its hetero-organization height.
Because cardiac muscle is the more organ of unique CK-MB of containing, therefore compare the most suitable quantitative observation index as myocardial damage of the mensuration of CK-MB with the enzyme of other several possibility reflecting myocardium damage.CK-MB diagnoses the susceptibility of wall type myocardial infarction and specificity all high, increases behind onset 4h.Reach the peak behind the 16-24h, it is normal that 3-4d recovers.
CK-MB still is kept at high level behind acute myocardial infarction AMI (AMI), mean that then myocardial necrosis also proceeds; Raise again after recovering normally, point out the expansion of former infarct position or new infarct appearance is arranged, because the half life period weak point, CK-MB and hypotype are sent out in the damage more more responsive than troponin again in monitoring, are worth a couple of days because troponin can keep high in blood; Its degree that increases can reflect the scope of infarct more exactly; Whether its peak time of occurrence helps to judge whether success one of thromboembolism treatment in advance.If detect separately, CK-MB is very desirable mark; Carry out joint-detection with cardiac troponin, more help to improve the diagnosis positive rate of AMI.
At present, the CK-MB detection method of clinical field application mainly contains electrophoresis, immunodepression, enzymoimmunoassay and chemiluminescent immunoassay etc.
Electrophoresis and immunodepression all can be subjected to the interference of haemolysis and huge CK, and immunodepression also can be subjected to the interference of CK-BB, have limited the application of these two kinds of detection methods.
Enzymoimmunoassay possesses characteristics such as it is simple to operate, long susceptibility, specificity pollution-free, that be higher than collaurum of the reagent term of validity is good, the available Instrument measuring of result to be 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.Mainly adopt the luminous plaque method at present, the plate differences is big, and measured value repeatability is relatively poor.
Summary of the invention
Defectives such as the problem to be solved in the present invention provides quantitative detection kit of chemiluminescence immunoassay of isoenzymes of creatine kinase and preparation method thereof, has avoided the measured value poor repeatability, and euzymelinked immunosorbent assay (ELISA) sensitivity is low, and sensing range is narrow.
For solving the problems of the technologies described above, the technical solution used in the present invention is: isoenzymes of creatine kinase (CK-MB) nanometer magnetic microparticle chemiluminescence immune quantitative detection reagent box comprises: the isoenzymes of creatine kinase calibration object; Coupling has the nanometer magnetic particle suspending liquid of Streptavidin; Biotin labeled isoenzymes of creatine kinase antibody; Isoenzymes of creatine kinase abzyme bond, used enzyme are horseradish peroxidase, horseradish peroxidase purity RZ 〉=3.0, activity 〉=250U/mL; Isoenzymes of creatine kinase quality-control product, quality-control product comprise the low value quality-control product of concentration 12ng/mL and the high value quality-control product of 300ng/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 isoenzymes of creatine kinase calibration object:
Isoenzymes of creatine kinase antigen is mixed with the dense liquid storage of calibration object with calibration object dilution (phosphate buffer that contains 1%BSA), calibrates with the company standard product, use the calibration object diluted to working concentration dense liquid storage, be respectively 0,3,15,50,200,800ng/mL;
Company standard product preparation method: with the phosphate buffer that contains 1%BSA with CK-MB 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 the CK-MB of Roche Holding Ag (article No.: 11821598, 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 3,15,50,200,800ng/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 isoenzymes of creatine kinase quality-control product:
With the calibration object dilution above-mentioned dense liquid storage is diluted to 12ng/mL and 300ng/mL, 12ng/mL is the low value Quality Control, and 300ng/mL is high-quality Quality Control;
(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 100mL0.1M2-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 isoenzymes of creatine kinase antibody
Get 0.6mg isoenzymes of creatine kinase antibody, with borate buffer solution dialysis 1～3h under 2～8 ℃; Antibody after the dialysis is added the 32ug biotin, add dimethyl sulfoxide (DMSO) simultaneously, making the dimethyl sulfoxide (DMSO) ultimate density is 5-10%, slowly vibration, lucifuge reaction 2.5h; In above-mentioned solution, add 250uL1M ammonium chloride solution, reacting at normal temperature without light 30-60min; With 0.01M PBS solution at 2～8 ℃ of following dialysis 24h, during change liquid 2-4 time;
(5) preparation of isoenzymes of creatine kinase abzyme bond
After adopting the improvement sodium periodate oxidation that isoenzymes of creatine kinase antibody and horseradish peroxidase are carried out coupling, with the enzyme dilution it is diluted to working concentration 1:4000, and adds 10% 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, CK-MB 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/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 ℃, each one bottle of every kind of reagent;
(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 CK-MB in double antibody sandwich method mensuration serum or the blood plasma, in Avidin-nanometer magnetic particle suspending liquid, add biotin-CK-MB antibody conjugates, compatible reaction by Avidin and biotin, form magnetic particle-Avidin-biotin-CK-MB antibody complex, add sample and enzyme, by antigen-antibody reaction, form magnetic particle-Avidin-biotin-CK-MB antibody-CK-MB antigen-CK-MB 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 CK-MB concentration are proportionate.CK-MB concentration in the sample is according to the Log(X that is set up by calibration object CK-MB concentration and corresponding RLU)-Log(Y) mathematical model is carried out quantitatively, thus detect the CK-MB content in human serum, the blood plasma.
The isoenzymes of creatine kinase 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 0.3ng/mL.
(2) specificity is good, and this product is to creatine phosphokinase isoenzyme MM(500ng/mL), creatine phosphokinase isoenzyme BB(500mg/mL) cross reaction do not appear.
(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 isoenzymes of creatine kinase of the present invention and Luo Shi measure isoenzymes of creatine kinase, the isoenzymes of creatine kinase value that records for this kit of ordinate wherein, horizontal ordinate is Luo Shi kit measurement isoenzymes of creatine kinase value, two kinds of method correlation coefficient r=0.9525, straight-line equation y=0.9963x+0.1829.
Embodiment
Embodiment 1: preparation isoenzymes of creatine kinase nanometer magnetic microparticle chemiluminescence immunological quantitative determining kit
(1) preparation of isoenzymes of creatine kinase calibration object:
Isoenzymes of creatine kinase antigen (available from Fitzgerald company) is mixed with the dense liquid storage of calibration object with calibration object dilution (phosphate buffer that contains 1%BSA), calibrate with the company standard product, use the calibration object diluted to working concentration dense liquid storage, be respectively 0,3,15,50,200,800ng/mL;
(2) preparation of isoenzymes of creatine kinase quality-control product:
With the calibration object dilution above-mentioned dense liquid storage is diluted to 12ng/mL and 300ng/mL, 12ng/mL is the low value Quality Control, and 300ng/mL is high-quality Quality Control;
(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 3.5mg Streptavidin (available from Sigma company) 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 isoenzymes of creatine kinase antibody
Get 0.6mg isoenzymes of creatine kinase antibody (available from Fitzgerald company), with borate buffer solution dialysis 1～3h under 2～8 ℃; Antibody after the dialysis is added 32ug biotin (available from Sigma company), add dimethyl sulfoxide (DMSO) simultaneously, making dimethyl sulfoxide (DMSO) final mass concentration is 5-10%, slowly vibration, lucifuge reaction 2.5h; In above-mentioned solution, add 250uL1M ammonium chloride solution, reacting at normal temperature without light 30-60min; With 0.01M PBS solution at 2～8 ℃ of following dialysis 24h, during change liquid 2-4 time;
(5) preparation of isoenzymes of creatine kinase abzyme bond
After adopting the improvement sodium periodate oxidation that isoenzymes of creatine kinase antibody and horseradish peroxidase are carried out coupling, with the enzyme dilution it is diluted to working concentration 1:4000, and adds 10% 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 ℃, each one bottle of every kind of reagent;
(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 isoenzymes of creatine kinase 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 5-500ng/mL concentration range is not less than 0.9900.
(4) sensitivity for analysis: the kit sensitivity for analysis is not higher than 0.3ng/mL.
(5) precision: the high value of 10 hole replicate determinations and low value quality-control product, the mean concentration of calculating measurement result (
) and 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 12ng/mL, and high value quality-control product measured value is at 300ng/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 20uL calibration object or serum specimen successively, 50uL magnetic-particle-Streptavidin suspending liquid, 50uL biotin-isoenzymes of creatine kinase antibody conjugates, 100uL isoenzymes of creatine kinase 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 isoenzymes of creatine kinase according to the luminous value of serum specimen.
Embodiment 4: the evaluation of methodology result of this kit
Sensing range: scope is 5-500ng/mL, measures after should diluting earlier greater than the sample of 500ng/mL for concentration again.
Sensitivity: 0.3ng/mL.
Precision: less than 5%.
Accuracy: the mean value of the recovery is in 0.90～1.10 scope.
Specificity: creatine phosphokinase isoenzyme BB, creatine phosphokinase isoenzyme MM cross reaction coefficient are less than 1%.
The quality-control product measured value: all in allowed band, low value quality-control product measured value is at 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 300ng/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 232 examples of this clinical testing, earlier with after the test of isoenzymes of creatine kinase Luo Shi detection kit, measure with the kit (chemiluminescence) of this patent invention again, the result shows, straight-line equation is y=0.9963x+0.1829, correlation coefficient r=0.9525.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 isoenzymes of creatine kinase value of two kinds of method mensuration is conspicuousnesses, as seen the isoenzymes of creatine kinase 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 400 parts of normal human serums, plasma samples, the result shows that the reference value (term of reference) of this kit is 0-7.0ng/mL.
Claims (4)
1. isoenzymes of creatine kinase nanometer magnetic microparticle chemiluminescence immune quantitative detection reagent box is characterized in that described kit comprises:
1) isoenzymes of creatine kinase calibration object, concentration are 0,3,15,50,200,800ng/mL;
2) coupling has the nanometer magnetic particle suspending liquid of Streptavidin;
3) biotin labeled isoenzymes of creatine kinase antibody, described antibody is monoclonal antibody;
4) isoenzymes of creatine kinase abzyme bond, described antibody is monoclonal antibody, with biotin labeled isoenzymes of creatine kinase antibody be not same strain, used enzyme is horseradish peroxidase, horseradish peroxidase purity RZ 〉=3.0, activity 〉=250U/mL;
5) isoenzymes of creatine kinase quality-control product; Quality-control product comprises the low value quality-control product of concentration 12ng/mL and the high value quality-control product of 300ng/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. isoenzymes of creatine kinase 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.
3. isoenzymes of creatine kinase 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.
4. 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 isoenzymes of creatine kinase calibration object:
With isoenzymes of creatine kinase antigen calibration object dilution, described calibration object dilution is to be mixed with the dense liquid storage of calibration object with the phosphate buffer that contains 1%BSA, calibrate with the company standard product, use the calibration object diluted to working concentration dense liquid storage, be respectively 0,3,15,50,200,800ng/mL;
(2) preparation of isoenzymes of creatine kinase quality-control product:
With human serum the isoenzymes of creatine kinase antigen diluent is mixed with dense liquid storage, and calibrates with enterprise's calibration object, dense liquid storage is diluted to 12ng/mL and 300ng/mL with human serum;
(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 isoenzymes of creatine kinase antibody
Get 0.6mg isoenzymes of creatine kinase antibody, with borate buffer solution dialysis 1～3h under 2～8 ℃; Antibody after the dialysis is added the 32ug biotin, add dimethyl sulfoxide (DMSO) simultaneously, making dimethyl sulfoxide (DMSO) final mass concentration is 5-10%, slowly vibration, lucifuge reaction 2.5h; In above-mentioned solution, add 250uL1M ammonium chloride solution, reacting at normal temperature without light 30-60min; With 0.01M PBS solution at 2～8 ℃ of following dialysis 24h, during change liquid 2-4 time;
(5) preparation of isoenzymes of creatine kinase abzyme bond
After adopting the improvement sodium periodate oxidation that isoenzymes of creatine kinase antibody and horseradish peroxidase are carried out coupling, with the enzyme dilution it is diluted to working concentration 1:4000, and adds 10% 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/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 ℃, each one bottle of every kind of reagent;
(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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| CN105277689A (en) * | 2015-11-17 | 2016-01-27 | 苏州浩欧博生物医药有限公司 | Reagent kit and method for detecting total serum IgE through nano magnetic particle chemiluminescence method |
| CN105435867A (en) * | 2015-10-26 | 2016-03-30 | 深圳华迈兴微医疗科技有限公司 | Magnetic particle chemiluminescent microfluidic chip for detecting creatine kinase isoenzyme in whole blood |
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