CN107236786A - A kind of preparation method of creatine kinase detection kit - Google Patents
A kind of preparation method of creatine kinase detection kit Download PDFInfo
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- CN107236786A CN107236786A CN201710588830.4A CN201710588830A CN107236786A CN 107236786 A CN107236786 A CN 107236786A CN 201710588830 A CN201710588830 A CN 201710588830A CN 107236786 A CN107236786 A CN 107236786A
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- creatine kinase
- detection kit
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- kinase detection
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- 102000004420 Creatine Kinase Human genes 0.000 title claims abstract description 45
- 108010042126 Creatine kinase Proteins 0.000 title claims abstract description 45
- 238000001514 detection method Methods 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 89
- 239000007788 liquid Substances 0.000 claims abstract description 33
- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 claims abstract description 18
- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000002904 solvent Substances 0.000 claims abstract description 14
- DRBBFCLWYRJSJZ-UHFFFAOYSA-N N-phosphocreatine Chemical compound OC(=O)CN(C)C(=N)NP(O)(O)=O DRBBFCLWYRJSJZ-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000007836 KH2PO4 Substances 0.000 claims abstract description 11
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 11
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 8
- 150000001875 compounds Chemical class 0.000 claims abstract description 6
- 229950007002 phosphocreatine Drugs 0.000 claims abstract description 6
- 238000007789 sealing Methods 0.000 claims abstract description 6
- 108010060059 Sarcosine Oxidase Proteins 0.000 claims abstract description 4
- 102000008118 Sarcosine oxidase Human genes 0.000 claims abstract description 4
- 239000003223 protective agent Substances 0.000 claims abstract description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 16
- 230000002255 enzymatic effect Effects 0.000 claims description 14
- 230000001681 protective effect Effects 0.000 claims description 14
- -1 polyoxyethylene Polymers 0.000 claims description 11
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 9
- 108090000790 Enzymes Proteins 0.000 claims description 9
- 239000005715 Fructose Substances 0.000 claims description 9
- 229930091371 Fructose Natural products 0.000 claims description 9
- POULHZVOKOAJMA-UHFFFAOYSA-M dodecanoate Chemical compound CCCCCCCCCCCC([O-])=O POULHZVOKOAJMA-UHFFFAOYSA-M 0.000 claims description 9
- 150000002148 esters Chemical class 0.000 claims description 9
- 229940070765 laurate Drugs 0.000 claims description 9
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical group [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 claims description 9
- 229940069446 magnesium acetate Drugs 0.000 claims description 9
- 235000011285 magnesium acetate Nutrition 0.000 claims description 9
- 239000011654 magnesium acetate Substances 0.000 claims description 9
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims description 8
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical group CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 claims description 7
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 5
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 5
- DNIAPMSPPWPWGF-GSVOUGTGSA-N (R)-(-)-Propylene glycol Chemical group C[C@@H](O)CO DNIAPMSPPWPWGF-GSVOUGTGSA-N 0.000 claims description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 4
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 claims description 4
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 4
- BJHIKXHVCXFQLS-UYFOZJQFSA-N fructose group Chemical group OCC(=O)[C@@H](O)[C@H](O)[C@H](O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 claims description 4
- 229960003511 macrogol Drugs 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 239000004094 surface-active agent Substances 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000005864 Sulphur Substances 0.000 claims description 3
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 3
- 235000018417 cysteine Nutrition 0.000 claims description 3
- 229960003742 phenol Drugs 0.000 claims description 3
- PBFKVYVGYHNCGT-UHFFFAOYSA-N 1-sulfanylpropane-1,2,3-triol Chemical compound OCC(O)C(O)S PBFKVYVGYHNCGT-UHFFFAOYSA-N 0.000 claims description 2
- HFZWRUODUSTPEG-UHFFFAOYSA-N 2,4-dichlorophenol Chemical class OC1=CC=C(Cl)C=C1Cl HFZWRUODUSTPEG-UHFFFAOYSA-N 0.000 claims description 2
- CSQFODQOQLFYIN-UHFFFAOYSA-N 3-chloro-2-hydroxybenzenesulfonic acid Chemical class OC1=C(Cl)C=CC=C1S(O)(=O)=O CSQFODQOQLFYIN-UHFFFAOYSA-N 0.000 claims description 2
- GIAVHGFPMPSIFI-UHFFFAOYSA-N 3-hydroxy-2,4,6-triiodobenzoic acid Chemical class OC(=O)C1=C(I)C=C(I)C(O)=C1I GIAVHGFPMPSIFI-UHFFFAOYSA-N 0.000 claims description 2
- YCPLPINFTMFQTD-UHFFFAOYSA-N 3-hydroxy-2-iodobenzoic acid Chemical class OC(=O)C1=CC=CC(O)=C1I YCPLPINFTMFQTD-UHFFFAOYSA-N 0.000 claims description 2
- WXNZTHHGJRFXKQ-UHFFFAOYSA-N 4-chlorophenol Chemical class OC1=CC=C(Cl)C=C1 WXNZTHHGJRFXKQ-UHFFFAOYSA-N 0.000 claims description 2
- YTBSYETUWUMLBZ-UHFFFAOYSA-N D-Erythrose Natural products OCC(O)C(O)C=O YTBSYETUWUMLBZ-UHFFFAOYSA-N 0.000 claims description 2
- YTBSYETUWUMLBZ-IUYQGCFVSA-N D-erythrose Chemical compound OC[C@@H](O)[C@@H](O)C=O YTBSYETUWUMLBZ-IUYQGCFVSA-N 0.000 claims description 2
- 206010056474 Erythrosis Diseases 0.000 claims description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 2
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 claims description 2
- PXQLVRUNWNTZOS-UHFFFAOYSA-N sulfanyl Chemical class [SH] PXQLVRUNWNTZOS-UHFFFAOYSA-N 0.000 claims description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims 2
- 230000004913 activation Effects 0.000 claims 2
- 239000003755 preservative agent Substances 0.000 claims 1
- 230000002335 preservative effect Effects 0.000 claims 1
- 239000011814 protection agent Substances 0.000 claims 1
- 102000003992 Peroxidases Human genes 0.000 abstract description 11
- 108040007629 peroxidase activity proteins Proteins 0.000 abstract description 11
- 230000008901 benefit Effects 0.000 abstract description 2
- 238000000034 method Methods 0.000 description 24
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 description 23
- 229960003624 creatine Drugs 0.000 description 11
- 239000006046 creatine Substances 0.000 description 11
- 239000000243 solution Substances 0.000 description 9
- 238000002835 absorbance Methods 0.000 description 6
- 238000003908 quality control method Methods 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- BQODPTQLXVVEJG-UHFFFAOYSA-N [O].C=C Chemical compound [O].C=C BQODPTQLXVVEJG-UHFFFAOYSA-N 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 239000012190 activator Substances 0.000 description 4
- 238000010168 coupling process Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- LRIUKPUCKCECPT-UHFFFAOYSA-N [hydroxy(phenyl)-$l^{3}-iodanyl] 4-methylbenzenesulfonate Chemical compound C1=CC(C)=CC=C1S(=O)(=O)OI(O)C1=CC=CC=C1 LRIUKPUCKCECPT-UHFFFAOYSA-N 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 238000004737 colorimetric analysis Methods 0.000 description 3
- 208000010125 myocardial infarction Diseases 0.000 description 3
- 206010061216 Infarction Diseases 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 230000007574 infarction Effects 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 description 2
- 206010018910 Haemolysis Diseases 0.000 description 1
- 208000009525 Myocarditis Diseases 0.000 description 1
- 201000002481 Myositis Diseases 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 108010077895 Sarcosine Proteins 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- VEQOALNAAJBPNY-UHFFFAOYSA-N antipyrine Chemical compound CN1C(C)=CC(=O)N1C1=CC=CC=C1 VEQOALNAAJBPNY-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
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- 239000004202 carbamide Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
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- 230000008588 hemolysis Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000000021 kinase assay Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000004118 muscle contraction Effects 0.000 description 1
- 201000000585 muscular atrophy Diseases 0.000 description 1
- 229960005222 phenazone Drugs 0.000 description 1
- LFGREXWGYUGZLY-UHFFFAOYSA-N phosphoryl Chemical group [P]=O LFGREXWGYUGZLY-UHFFFAOYSA-N 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 229940043230 sarcosine Drugs 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
- C12Q2326/90—Developer
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Abstract
The invention discloses the preparation method of creatine kinase detection kit, the creatine kinase detection kit includes to be made up of the liquid double reagent of reagent 1 and reagent 2, is comprised the following steps：Compound concentration is the Na of 100mmol/L 200mmol/L concentration respectively2HPO4And KH2PO4Solution; 1mmol/L 10mmol/L 4 amino-antipyrines and chromogen are first added in the solvent of reagent 1; it is sufficiently mixed until being completely dissolved; enzyme-added protective agent, adds 20KU/L 40KU/L kreatinase, 8KU/L 16KU/L sarcosine oxidase, 10KU/L 20KU/L peroxidase afterwards again；260mmol/L 410mmol/L phosphocreatine, 1.5mmol/L 3.2mmol/L adenosine diphosphate (ADP) is first added in the solvent of reagent 2, agent living is added after being completely dissolved, most after the Liquid BPF aN that 0.6g/L 1.0g/L are added in reagent 1 and reagent 23, 28 DEG C of sealings preserve respectively.It is an advantage of the invention that prepared kit is reproducible, the degree of accuracy is high, stability is good.
Description
Technical field
The invention belongs to field of biological, a kind of preparation method of creatine kinase detection kit is specifically referred to.
Background technology
Creatine kinase (Creatine Kinase, CK) is typically found in the thin of the tissues such as the heart, muscle and brain of animal
In endochylema and mitochondria, it is one and regenerates the important kinases for having direct relation with intracellular energy operating, contraction of muscle, ATP, it
Reversibly turn phosphoryl reaction between catalysis creatine and ATP.Creatine kinase is of great significance in clinical diagnosis,
When various lesions include muscular atrophy and miocardial infarction generation, creatine kinase level is improved rapidly in the serum of people, it is now recognized that
The activity that creatine kinase is determined in the diagnosis of miocardial infarction is more more reliable than having an electro-cardiogram.During myocardial infarction, creatine kinase exists
Onset is raised in 6 hours, is reached within 24 hours in peak, 3-4 days and is recovered normal.Creatine kinase because its have important physiological function and
Clinical value has caused people widely to pay attention to and in-depth study, is clinically mainly used in myocardial infarction, the viral heart
The auxiliary diagnosis of myositis.
The method of detection creatine kinase (CK) gross activity has colorimetric method, enzyme coupling method, fluorescence method and biloluminescence method at present,
Wherein colorimetric method and enzyme coupling method is more often used.System, such as B of CN 101063111 are indicated using coenzyme more than enzyme coupling method
(2010.08.25)、CN 1904062 B(2010.11.24)、CN 1916623 B(2013.05.29)、CN 103173519 B
(2014.07.02)、CN 104374905 B(2016.03.30)、CN 104357544 A(2015.02.18).But these sides
Case, which exists, can not effectively eliminate the interference of endogenous creatine, influence accuracy of detection.
The content of the invention
The invention aims to overcome the shortcoming and defect that prior art is present, and provide a kind of creatine kinase
The preparation method of detection kit, a kind of creatine that the reproducible, degree of accuracy is high, stability is good can be stably prepared by the method
Kinase assay kit.
To achieve the above object, the technical scheme is that the creatine kinase detection kit includes by the He of reagent 1
The liquid double reagent composition of reagent 2, comprises the following steps：
Compound concentration is the Na of 100mmol/L-200mmol/L concentration respectively2HPO4And KH2PO4Solution is identical dense by its
Degree correspondence is (if Na2HPO4The concentration of solution is 100mmol/L, then KH2PO4Solution also uses 100mmol/L；If Na2HPO4It is molten
The concentration of liquid is 200mmol/L, then KH2PO4Solution is also with 200mmol/L) PH=7.6 cushioning liquid is modulated into, it is used as reagent
1 solvent；By the Na of correspondence same concentrations2HPO4And KH2PO4Solution is modulated into PH=6.7 cushioning liquid, is used as reagent 2
Solvent；1mmol/L-10mmol/L 4-AA and chromogen is first added in reagent 1, is sufficiently mixed until completely molten
Solution, then enzyme-added protective agent, add afterwards 20KU/L-40KU/L kreatinase, 8KU/L-16KU/L sarcosine oxidase,
10KU/L-20KU/L peroxidase (POD)；260mmol/L-410mmol/L phosphocreatine is first added in reagent 2
(CrP), 1.5mmol/L-3.2mmol/L adenosine diphosphate (ADP), adds agent living, most after in reagent 1 and reagent 2 after being completely dissolved
Add 0.6g/L-1.0g/L Liquid BPF aN3, 2-8 DEG C of sealing preserve respectively.
It is described chromogen 1mmol/L-10mmol/L further to set, the chromogen be the chloro- 2- hydroxy benzene sulfonic acids of 3,5- bis-,
N- ethyls-(2- hydroxyl -3- sulfopropyls) -3,5- dimethoxy-4 's-fluoroaniline, N- (2- hydroxyl -3- sulfopropyls) -3,5- dimethoxy benzenes
Any one in the iodo- 3- hydroxybenzoic acids of amine, 2,4,6- tri-, 4- chlorophenols, 2,4 Dichlorophenols or phenol.
It is that chromogen is 2,4,6- tri- iodo- 3- hydroxybenzoic acids further to set, and its concentration is 6mmol/L.
It is that described enzymatic protective reagent is liquid enzymatic protective reagent further to set, and its addition is 1mL-3mL/100mL；Or
Described enzymatic protective reagent is solid enzymatic protective reagent, and its content is 1g-3g/100mL.
Further set is that liquid enzymatic protective reagent is aliphatic alcohol polyethenoxy (7) ether, alkylphenol-polyethenoxy (10) ether, list
Position is 1mL-3mL/100mL；Described solid enzymatic protective reagent is fructose, Macrogol 6000, PEG 8000, laurate gather
Oxygen ethene (9) ester, unit is 1g-3g/100mL.
It is that the enzymatic protective reagent is fructose and the composition of surfactant further to set, and wherein surfactant is optional
Gather from aliphatic alcohol polyethenoxy (7) ether, laurate polyoxyethylene (9) ester, Macrogol 6000, PEG 8000, alkyl phenol
One or more in oxygen ethene (10) ether.
It is enzymatic protective reagent fructose and laurate polyoxyethylene (9) ester 1 further to set:1 mass ratio is combined.
It is that the activator is magnesium acetate and the composition of mercapto reagent further to set, and wherein mercapto reagent is selected from N- second
It is a kind of in acyl cysteine, mercapto glycerol, dithiothreitol (DTT), two sulphur erythrose alcohol, TGA, mercaptoethanol, cysteine
Or several combinations.
The concentration for being the activator for magnesium acetate and N-acetylcystein composition, wherein magnesium acetate is further set
For 10mmol/L-25mmol/L, N-acetylcystein composition concentration 16mmol/L-30mmol/L.
The operation detection principle of kit prepared by the inventive method is：Creatine kinase (CK) catalytic phosphatase creatine
(CrP) it is transformed into creatine (Cr), while adenosine diphosphate (ADP) (ADP) phosphoric acid chemical conversion atriphos (ATP)；In the catalysis of kreatinase
Lower creatine hydrolysis produces methyl amimoacetic acid and urea；Methyl amimoacetic acid discharges H in the presence of sarcosine oxidase2O2；H2O2With 4- amino
In peroxidase (POD) presence Trinder reactions (coupling terminal colorimetric analysis), reaction equation occur for antipyrine (4-AAP)
It is as follows：
Creatine kinase detection kit prepared by the inventive method, using liquid double reagent, can effectively eliminate endogenous
Property creatine interference, without Sample pretreatment, directly can carry out high-volume pattern detection using automatic clinical chemistry analyzer, operation is simple
Single, the reproducible, degree of accuracy is high, stability is good, is adapted to clinical practice and promotes.
Kit prepared by the inventive method, the double reagent configured using above-mentioned concrete component, so that actually detected
During creatine kinase, it can be mixed using two-step method detection, serum sample with reagent 1 (R1), 37 DEG C of insulation 5min make contained in sample
Side reaction caused by endogenous creatine is finished, and the catalytic reaction that reagent 2 (R2) starts CK is then added, in dominant wavelength
Absorbance is read under 546nm/ auxiliary wavelength 660nm, most goes out CK total activities after 37 DEG C of Response calculations, so as to solve endogenous
The interference that creatine is determined to creatine kinase (CK), the composition distribution of this kit and tie element ratio can be first by serum samples
Original creatine reaction is complete, so as to improve the accuracy, repeatability and stability of testing result.
The present invention is described further with reference to specification drawings and specific embodiments.
Brief description of the drawings
Fig. 1 is in the Detection of Stability knot of 2-8 DEG C and 37 DEG C water-bath one week using reagent made from the embodiment of the present invention 1
Really；
Fig. 2 is in the Detection of Stability knot of 2-8 DEG C and 37 DEG C water-bath one week using reagent made from the embodiment of the present invention 2
Really；
Fig. 3 is in the Detection of Stability knot of 2-8 DEG C and 37 DEG C water-bath one week using reagent made from the embodiment of the present invention 3
Really.
Embodiment
The present invention is specifically described below by embodiment, is served only for that the present invention is further described, no
It is understood that for limiting the scope of the present invention, the technician in the field can be according to the content of foregoing invention to the present invention
Make some nonessential modifications and adaptations.
Embodiment 1
Compound concentration is the Na of 100mmol/L isoconcentrations respectively2HPO4And KH2PO4Solution, is modulated into PH=7.6's
Cushioning liquid, is used as the solvent of reagent 1；PH=6.7 cushioning liquid is modulated into, the solvent of reagent 2 is used as；In reagent 1
The HTIB of the middle 4-AA (4-AAP) and 6mmol/L for first adding 1mmol/L, is sufficiently mixed up to being completely dissolved, then
5g/L fructose and 0.5% laurate polyoxyethylene (9) ester is added, 20KU/L kreatinase, 8KU/L flesh ammonia are added afterwards
The peroxidase (POD) of acid oxidase, 10KU/L；First added in reagent 2 260mmol/ phosphocreatine (CrP),
1.5mmol/L adenosine diphosphate (ADP) (ADP), adds 17mmol/L magnesium acetate and 23mmol/L NAC, finally after being completely dissolved
0.6g/L Liquid BPF aN is added in reagent 1 and reagent 23, 2-8 DEG C of sealing preserve.
Embodiment 2
Compound concentration is the Na of 150mmol/L isoconcentrations respectively2HPO4And KH2PO4Solution, is modulated into PH=7.6's
Cushioning liquid, is used as the solvent of reagent 1；PH=6.7 cushioning liquid is modulated into, the solvent of reagent 2 is used as；In reagent 1
The HTIB of the middle 4-AA (4-AAP) and 6mmol/L for first adding 5mmol/L, is sufficiently mixed up to being completely dissolved, then
10g/L fructose and 1% laurate polyoxyethylene (9) ester is added, 30KU/L kreatinase, 12KU/L flesh ammonia are added afterwards
The peroxidase (POD) of acid oxidase, 15KU/L；First added in reagent 2 330mmol/ phosphocreatine (CrP),
2.3mmol/L adenosine diphosphate (ADP) (ADP), adds 17mmol/L magnesium acetate and 23mmol/L NAC, finally after being completely dissolved
0.8g/L Liquid BPF aN is added in reagent 1 and reagent 23, 2-8 DEG C of sealing preserve.
Embodiment 3
Compound concentration is the Na of 200mmol/L isoconcentrations respectively2HPO4And KH2PO4Solution, is modulated into PH=7.6's
Cushioning liquid, is used as the solvent of reagent 1；PH=6.7 cushioning liquid is modulated into, the solvent of reagent 2 is used as；In reagent 1
The HTIB of the middle 4-AA (4-AAP) and 6mmol/L for first adding 10mmol/L, is sufficiently mixed up to being completely dissolved,
15g/L fructose and 1.5% laurate polyoxyethylene (9) ester are added, 40KU/L kreatinase, 16KU/L is afterwards added
The peroxidase (POD) of sarcosine oxidase, 20KU/L；First added in reagent 2 410mmol/ phosphocreatine (CrP),
3.2mmol/L adenosine diphosphate (ADP) (ADP), adds 17mmol/L magnesium acetate and 23mmol/L NAC, finally after being completely dissolved
1g/L Liquid BPF aN is added in reagent 1 and reagent 23, 2-8 DEG C of sealing preserve.
Embodiment 4
The liquid double reagent prepared using 1-3 of embodiment of the present invention method is subjected to performance evaluation
1. accuracy validation：
From Landau is low, high level quality-control product (1530, lot number：1093UN；1532, lot number：849UE), it is complete using BS-420
Automatic biochemistry analyzer detected, concrete operation method is as described in table one, after being calibrated using One point standard method, CK measured values and
Blank absorbance values can be directly read from instrument, in order to reduce accidental error, be repeated three times and taken average, calculate measured value
With the relative deviation of target value.
The reagent that table 2 is prepared using embodiment 1-3 determines low value Quality Control (1530) degree of accuracy and compared
The reagent that table 3 is prepared using embodiment 1-3 determines high level Quality Control (1532) degree of accuracy and compared
From result above, the average that the liquid double reagent prepared using the method for the present invention determines quality-control product is located at target
In the range of value, relative deviation is obvious<10%, illustrate higher using the liquid double reagent degree of accuracy of method preparation of the invention.
2. Precision Experiment：
From routine clinical sample, the liquid double reagent replication prepared with 1-3 of embodiment of the present invention method 20 times,
The coefficient of variation is calculated, 4 are the results are shown in Table.
The reagent repeatability that table 4 is prepared using embodiment 1-3 is determined
| Determine number of times | Embodiment 1 | Embodiment 2 | Embodiment 3 |
| 1 | 46.3 | 45.6 | 46.2 |
| 2 | 45.3 | 46.1 | 45.4 |
| 3 | 46.3 | 45.5 | 45.3 |
| 4 | 46.3 | 46.8 | 46.4 |
| 5 | 46.3 | 46.3 | 46.1 |
| 6 | 45.6 | 45.6 | 46.2 |
| 7 | 46.3 | 46.3 | 46.6 |
| 8 | 45.4 | 46.4 | 45.4 |
| 9 | 46.3 | 46.8 | 46.7 |
| 10 | 45.2 | 45.2 | 46.6 |
| 11 | 46.2 | 46.7 | 46.1 |
| 12 | 46.2 | 46.2 | 46.1 |
| 13 | 45.0 | 45.5 | 45.6 |
| 14 | 46.3 | 46.5 | 45.8 |
| 15 | 46.3 | 46.1 | 46.4 |
| 16 | 46.1 | 46.1 | 45.7 |
| 17 | 46.2 | 46.2 | 46.1 |
| 18 | 46.2 | 45.6 | 46.5 |
| 19 | 46.2 | 46.4 | 46.5 |
| 20 | 46.2 | 45.9 | 46.2 |
| Average | 46.01 | 46.09 | 46.10 |
| Standard deviation (SD) | 0.4253 | 0.4527 | 0.4189 |
| The coefficient of variation (CV/%) | 0.92% | 0.98% | 0.91% |
From result above, the coefficient of variation (CV) that the liquid double reagent prepared using the method for the present invention is determined is respectively
For 0.92%, 0.98%, 0.91%, substantially<6%, illustrate that the liquid double reagent for using the method for the present invention to prepare has good
Good precision.
3. stability test：
The liquid double reagent prepared using 1-3 of embodiment of the present invention method is subjected to stability checking test.By more than
Three kinds of reagents are every kind of to be divided into two parts, and a as 2-8 DEG C of preservation, another is preserved as in 37 DEG C of water-baths, every other day
Determine once, continuous monitoring one week, observation serum sample measured value (serum definite value 110.60U/L, it is allowed to error range (±
10%)：99.54U/L-121.66U/L), reagent stability is compared by the measure of the degree of accuracy.
The reagent stability data that table 5 is prepared using the method for embodiment 1
The reagent stability data that table 6 is prepared using the method for embodiment 2
The reagent stability data that table 7 is prepared using the method for embodiment 3
It is good using reagent stability made from embodiment 1-3 method it can be seen from Fig. 1, Fig. 2 and Fig. 3.Using reality
Reagent made from the method for a 1-3 is applied in 37 DEG C of water-baths one week, although determining CK concentration has a certain degree of decline, but declines
Amplitude less, in allowed band, illustrate that the suitable enzymatic protective reagent of addition and CK activator can protect the activity of corresponding enzyme,
And then the stability of reagent is improved, the storage life with kit made from this method can be extended.
To sum up performance evaluation is understood, the liquid double reagent prepared using 1-3 of embodiment of the present invention method has the degree of accuracy
The good advantage of high, reproducible, stability, can meet clinical demand, in clinical diagnosis myocardial infarction, vital myocarditis disease
There is good application value in terms of feelings.
Application Example
Creatine kinase (CK) detection kit of the present invention is applied to all kinds of automatic clinical chemistry analyzers, now in full-automatic biochemical
Application on analyzer BS-420, its specifically used method is as follows：
The pattern detection operation sequence of table 1
CK contents (U/L)=Δ A in sampleT/ΔAS× calibration solution concentration
In formula：ΔAT：Using blank tube absorbance as the sample cell absorbance of control；
ΔAS：Using blank tube absorbance as the calibration pipe absorbance of control；
Quality-control product used in the present invention is the high and low value quality-control product of Landau；Institute's test sample product are not haemolysis serum.
Beneficial effect：Kit prepared by the present invention indicates creatine kinase (CK) inspection prepared by system using Trinder
Test agent box is liquid double reagent, using two-step method, effectively eliminates the interference of endogenous creatine, while adding CK activator and corresponding
Enzymatic protective reagent, can improve the degree of accuracy of CK measure and the stability of reagent.
Claims (9)
1. a kind of preparation method of creatine kinase detection kit, it is characterised in that the creatine kinase detection kit includes
The liquid double reagent of reagent 1 and reagent 2, comprises the following steps：
Compound concentration is the Na of 100mmol/L-200mmol/L concentration respectively2HPO4And KH2PO4Solution, by its same concentrations correspondence
Na2HPO4And KH2PO4Solution is modulated into PH=7.6 cushioning liquid, is used as the solvent of reagent 1；Same concentrations are corresponding
Na2HPO4And KH2PO4Solution is modulated into PH=6.7 cushioning liquid, is used as the solvent of reagent 2；First add in the solvent of reagent 1
Enter 1mmol/L-10mmol/L 4-AA and chromogen, be sufficiently mixed until be completely dissolved, then enzyme-added protective agent, it
20KU/L-40KU/L kreatinase, 8KU/L-16KU/L sarcosine oxidase, 10KU/L-20KU/L peroxidating is added afterwards
Thing enzyme；260mmol/L-410mmol/L phosphocreatine, 1.5mmol/L-3.2mmol/L are first added in the solvent of reagent 2
Adenosine diphosphate (ADP), adds agent living, most after the preservative that 0.6g/L-1.0g/L is added in reagent 1 and reagent 2 after being completely dissolved
NaN3, 2-8 DEG C of sealing preserve respectively.
2. a kind of preparation method of creatine kinase detection kit according to claim 1, it is characterised in that：Described color
Former 1mmol/L-10mmol/L, the chromogen is the chloro- 2- hydroxy benzene sulfonic acids of 3,5- bis-, N- ethyls-(2- hydroxyl -3- sulfopropyls) -3,5-
Dimethoxy-4 '-fluoroaniline, N- (2- hydroxyl -3- sulfopropyls) -3,5- dimethoxyanilines, the iodo- 3- hydroxybenzoic acids of 2,4,6- tri-,
Any one in 4- chlorophenols, 2,4 Dichlorophenols or phenol.
3. a kind of preparation method of creatine kinase detection kit according to claim 2, it is characterised in that：Chromogen is 2,
4,6- tri- iodo- 3- hydroxybenzoic acids, its concentration is 6mmol/L.
4. a kind of preparation method of creatine kinase detection kit according to claim 1, it is characterised in that：Described enzyme
Protective agent is liquid enzymatic protective reagent, and its addition is 1mL-3mL/100mL；Or described enzymatic protective reagent is solid enzyme protection
Agent, its content is 1g-3g/100mL.
5. a kind of preparation method of creatine kinase detection kit according to claim 4, it is characterised in that：Liquid enzymes are protected
Shield agent is aliphatic alcohol polyethenoxy (7) ether, alkylphenol-polyethenoxy (10) ether, and unit is 1mL-3mL/100mL；Described solid
Enzymatic protective reagent is fructose, Macrogol 6000, PEG 8000, laurate polyoxyethylene (9) ester, and unit is 1g-3g/
100mL。
6. a kind of preparation method of creatine kinase detection kit according to claim 1, it is characterised in that：The enzyme is protected
The composition that agent is fructose and surfactant is protected, wherein surfactant may be selected from aliphatic alcohol polyethenoxy (7) ether, laurate
One or more in polyoxyethylene (9) ester, Macrogol 6000, PEG 8000, alkylphenol-polyethenoxy (10) ether.
7. a kind of preparation method of creatine kinase detection kit according to claim 6, it is characterised in that：Enzymatic protective reagent
For fructose and laurate polyoxyethylene (9) ester 1:1 mass ratio is combined.
8. a kind of preparation method of creatine kinase detection kit according to claim 1, it is characterised in that：The activation
Agent is magnesium acetate and the composition of mercapto reagent, and wherein mercapto reagent is selected from N-acetylcystein, mercapto glycerol, two sulphur threoses
One or more combination in alcohol, two sulphur erythrose alcohol, TGA, mercaptoethanol, cysteine.
9. a kind of preparation method of creatine kinase detection kit according to claim 8, it is characterised in that：The activation
Agent is magnesium acetate and N-acetylcystein composition, and the wherein concentration of magnesium acetate is 10mmol/L-25mmol/L, N- acetyl half
Cystine composition concentration 16mmol/L-30mmol/L.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107641642A (en) * | 2017-10-25 | 2018-01-30 | 武汉生之源生物科技股份有限公司 | A kind of creatine kinase isozyme double reagent and preparation method thereof |
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| CN107641642A (en) * | 2017-10-25 | 2018-01-30 | 武汉生之源生物科技股份有限公司 | A kind of creatine kinase isozyme double reagent and preparation method thereof |
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