CN104374925B - A kind of Creatine kinase MB detection reagent - Google Patents
A kind of Creatine kinase MB detection reagent Download PDFInfo
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- CN104374925B CN104374925B CN201410698140.0A CN201410698140A CN104374925B CN 104374925 B CN104374925 B CN 104374925B CN 201410698140 A CN201410698140 A CN 201410698140A CN 104374925 B CN104374925 B CN 104374925B
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Abstract
The invention discloses a kind of Creatine kinase MB detection reagent.This reagent is by reagent R1 and reagent R2, form according to the ratio of volume ratio 4:1, wherein reagent R1 is made up of imidazole buffer, glucose, nanoparticle, N-acetylcystein, sodium ethylene diamine tetracetate, adenosine diphosphate (ADP), cozymase, ribonucleotide, pyruvic carboxylase, glucose-6-phosphate dehydrogenase, hexokinase, goat-anti people CK-M polyclonal antibody and trehalose, and reagent R2 is made up of imidazole buffer, phosphocreatine, Sodium dodecylbenzene sulfonate and sanitas.Reagent of the present invention be a kind of stable, accurately, the Creatine kinase MB detection reagent of strong interference immunity, there is very high clinical value.
Description
Technical field
The present invention relates to one and relate to biological reagent, be specifically related to a kind of detection reagent, particularly relate to a kind of Creatine kinase MB detection reagent.
Background technology
Creatine kinase (CreatineKinase; CK) in (ATP:CreatineN-phosphotransferaseEC2.7.3.2) cytoplasm of being usually present in the tissues such as the heart of animal, muscle and brain and plastosome; that one operates with intracellular energy, Muscle contraction, adenosine triphyosphate (ATP) regenerate the important kinases 1 having direct relation; 2, its phosphoryl that turns reversibly between catalysis creatine and ATP reacts.Creatine kinase has four kinds of isozyme forms: muscularity (MM), brain type (BB), hydridization type (MB) and Mitochondrial form (MiMi).MM type is mainly present in various muscle cell, and BB type is mainly present in brain cell, and MB type is mainly present in myocardial cell, and MiMi type is mainly present in cardiac muscle and skeletal muscle mitochondrial.The dimer that muscularity creatine kinase molecule is made up of two identical subunits.According to the primary structure of at present rabbit after measured, people, chicken, mouse creatine kinase, M type subunit is made up of 387 amino-acid residues, and quality is about 43KDa, has 8 sulfydryls in molecule, but without disulfide linkage.Giant panda muscularity creatine kinase is also dimer enzyme, and each subunit is made up of 376 amino-acid residues, and molecular weight is 42KDa.
The most important meaning that Serum CK-MB measures is diagnosing acute myocardial infarction.4-6 hour after Acute Myocardial Infarction episode, patients serum CK-MB start to raise prior to gross activity, 12-36 hour peaking; How to recover normal in 72 hours.Its maximum reaches more than control group 4.9-22 times.If 3-4 days, CK-MB still continue not fall after infraction, show that myocardial infarction is still proceeding, if the CK-MB declined raises again, point out former blocking part pathology expand or have new infarction lesion; If chest pain patients not yet occurs that in 48 hours CK-MB raises, or is less than 2 of gross activity, the diagnosis of Acute Myocardial Infarction can be got rid of.Normal using the 3(ion-exchange chromatography method of CK-MB more than CK gross activity clinically) or 10(immunodepression) as the diagnosis basis of Acute Myocardial Infarction.
With must be noted that no matter be the absolute activity of CK-MB or account for gross activity percentile boundary value during CK-MB diagnosing acute myocardial infarction, be not all suitable for the children of less than 14 years old, because the CK-MB of infant and children is all higher than adult.Likely cause Serum CK-MB increased activity after various types of heart operation, between concurrent art or when postoperative myocardial blocks, rising is more remarkable.
CK-MB is made up of CK-M and CK-B subunit.Anti-CK-M antibody completely inhibit the dominant reactive part of CK-MM(creatine kinase) and CK-MB in the activity of CK-M subunit.The vigor detecting CK is again the vigor of remaining CK-B, is equivalent to half CK-MB vigor.So result to be multiplied by the vigor that 2 are CK-MB.The reaction of CK-M viability examination is as follows:
Whole pack is containing two main reagents, be respectively R1 and R2, wherein contain CK-M antibody in R1, the activity of CK-M subunit enzyme can be suppressed completely, CK-B catalysis is utilized to produce the effect of ATP, by a succession of reaction, finally at the content of the wavelength detecting NADH of 340nm, obtain the enzyme activity of CK-MB.Often because the chaff interference such as pyruvic acid, heparin existed in sample when conventional reagent detects, affect the accuracy of detected result, in reagent, anti-CK-M antibody is unstable in reagent simultaneously, causes reagent stability poor, and then have impact on reagent application commercially.
Summary of the invention
For solving the problems of the technologies described above, the invention provides that a kind of accuracy is high, the Creatine kinase MB detection reagent of good stability, strong interference immunity.
A kind of Creatine kinase MB detection reagent, by reagent R1 and reagent R2, forms according to the ratio of volume ratio 4:1;
Described, reagent R1 is grouped into by the one-tenth of following content:
Imidazole buffer 50-100mmo1
Glucose 20-45mmo1
Nanoparticle 20-40mmol
N-acetylcystein 20-30mmo1
Sodium ethylene diamine tetracetate 1-8mmo1
Adenosine diphosphate (ADP) 1-10mmo1
Cozymase 2-6mmo1
Ribonucleotide 10-25mmo1
Pyruvic carboxylase 0.5-2KU
Glucose-6-phosphate dehydrogenase 2-5KU
Hexokinase 2-6KU
Goat-anti people CK-M polyclonal antibody 10-50mL
Trehalose 1-10g
Deionized water adds to 1L;
Described, reagent R2 is grouped into by the one-tenth of following content:
Imidazole buffer 50-100mmo1
Phosphocreatine 120-220mmo1
Sodium dodecylbenzene sulfonate 5-20g
Sanitas 1-10g
Deionized water adds to 1L.
Described, nanoparticle is γ-Fe
2o
3nanoparticle, its particle diameter is 10-40nm.
Described, sanitas is sodiumazide or Proclin300.
Described, the pH value of imidazole buffer is 6.0-7.0.
Preferably, reagent R1 is grouped into by the one-tenth of following content:
Imidazole buffer 80mmo1
Glucose 32mmo1
Nanoparticle 30mmol
N-acetylcystein 25mmo1
Sodium ethylene diamine tetracetate 5mmo1
Adenosine diphosphate (ADP) 5mmo1
Cozymase 4mmo1
Ribonucleotide 15mmo1
Pyruvic carboxylase 1.2KU
Glucose-6-phosphate dehydrogenase 3KU
Hexokinase 4KU
Goat-anti people CK-M polyclonal antibody 30mL
Trehalose 5g
Deionized water adds to 1L.
Preferably, reagent R2 is grouped into by the one-tenth of following content:
Imidazole buffer 70mmo1
Phosphocreatine 170mmo1
Sodium dodecylbenzene sulfonate 10g
Sanitas 6g
Deionized water adds to 1L.
The using method of a kind of Creatine kinase MB detection reagent of the present invention is: reagent preparation R1 and reagent R2, first by reagent R1 in automatic clinical chemistry analyzer 37 DEG C of preincubates 3 minutes, again reagent R2 is added in reagent R1,5 minutes are hatched in automatic clinical chemistry analyzer 37 DEG C after mixing, the volume ratio of reagent R1 and reagent R2 is 4:1, the parameter of adjustment Biochemical Analyzer, places distilled water, Landau calibration object and sample at the correspondence position of sample disc, measures the vigor of Creatine kinase MB.
Advantage of the present invention:
1. the present invention adopts the mode of double reagent, first utilizes the pyruvic carboxylase in reagent R1 to be removed by the interfering substance (as pyruvic acid, heparin) in serum sample, γ-Fe during reaction
2o
3nanoparticle can strengthen the activity of pyruvic carboxylase, makes to react carry out more thorough, effectively removes the interfering substance in serum sample, the accuracy of result when detecting Creatine kinase MB with guaranteed reagent,G.R. R2.
2. with the addition of protein protective agent-trehalose in reagent of the present invention; trehalose is except well protecting the immunocompetence of goat-anti people CK-M polyclonal antibody; protective membrane can also be formed on glucose-6-phosphate dehydrogenase, hexokinase surface; protect the avtive spot of main body reaction enzymes; in reagent react process, the permanent height reaction vigor keeping reagent.
3. reagent of the present invention be a kind of stable, accurately, the Creatine kinase MB detection reagent of strong interference immunity, there is very high clinical value.
Accompanying drawing explanation
Fig. 1 is the relevance detection results of the embodiment of the present invention 1;
Fig. 2 is the relevance detection results of the embodiment of the present invention 2;
Fig. 3 is the relevance detection results of the embodiment of the present invention 3;
Fig. 4 is the relevance detection results of the embodiment of the present invention 4;
Fig. 5 is the Detection of Stability result of the embodiment of the present invention 1 to 4.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
Following examples agents useful for same raw material is commercially available analytical pure.
embodiment 1
A kind of Creatine kinase MB detection reagent is made up of according to the ratio of volume ratio 4:1 reagent R1 and reagent R2;
Reagent R1 is grouped into by the one-tenth of following content:
Imidazole buffer (pH=6.0) 50mmo1
Glucose 45mmo1
Nanoparticle 40mmol
N-acetylcystein 30mmo1
Sodium ethylene diamine tetracetate 8mmo1
Adenosine diphosphate (ADP) 10mmo1
Cozymase 2mmo1
Ribonucleotide 10mmo1
Pyruvic carboxylase 0.5KU
Glucose-6-phosphate dehydrogenase 5KU
Hexokinase 2 KU
Goat-anti people CK-M polyclonal antibody 50mL
Trehalose 10g
Deionized water adds to 1L;
Reagent R2 is grouped into by the one-tenth of following content:
Imidazole buffer (pH=6.0) 100mmo1
Phosphocreatine 120mmo1
Sodium dodecylbenzene sulfonate 20g
Sodiumazide 10g
Deionized water adds to 1L.
Nanoparticle is γ-Fe
2o
3nanoparticle, its particle diameter is 40nm.
The using method of the present embodiment Creatine kinase MB detection reagent is: select Toshiba 40 fully-automatic analyzer with double reagent function, reagent preparation R1 and reagent R2, first by reagent R1 in automatic clinical chemistry analyzer 37 DEG C of preincubates 3 minutes, again reagent R2 is added in reagent R1, 5 minutes are hatched in automatic clinical chemistry analyzer 37 DEG C after mixing, the volume ratio of reagent R1 and reagent R2 is 4:1, the parameter of adjustment Biochemical Analyzer, distilled water is placed at the correspondence position of sample disc, Landau calibration object and sample, first by detecting the absorbancy change of distilled water and the detection of Landau calibration object acquisition calibration object, according to the target value (65U/L) of Landau calibration object Creatine kinase MB reagent, obtain the K factor of reagent, afterwards according to the absorbancy of the sample of automatic clinical chemistry analyzer mensuration, just can obtain the Creatine kinase MB concentration of sample.Concrete operations are as shown in table 1.
Table 1 Creatine kinase MB detection reagent using method
Calculate: Creatine kinase MB vigor (U/L)=(A mensuration/min ÷ A calibration/min) × C calibrates.
embodiment 2
A kind of Creatine kinase MB detection reagent is made up of according to the ratio of volume ratio 4:1 reagent R1 and reagent R2;
Reagent R1 is grouped into by the one-tenth of following content:
Imidazole buffer (pH=6.5) 80mmo1
Glucose 32mmo1
Nanoparticle 30mmol
N-acetylcystein 25mmo1
Sodium ethylene diamine tetracetate 5mmo1
Adenosine diphosphate (ADP) 5mmo1
Cozymase 4mmo1
Ribonucleotide 15mmo1
Pyruvic carboxylase 1.2KU
Glucose-6-phosphate dehydrogenase 3KU
Hexokinase 4KU
Goat-anti people CK-M polyclonal antibody 30mL
Trehalose 5g
Deionized water adds to 1L;
Reagent R2 is grouped into by the one-tenth of following content:
Imidazole buffer (pH=7.0) 70mmo1
Phosphocreatine 170mmo1
Sodium dodecylbenzene sulfonate 10g
Proclin3006g
Deionized water adds to 1L.
Nanoparticle is γ-Fe
2o
3nanoparticle, its particle diameter is 30nm.
A kind of using method of Creatine kinase MB detection reagent is with embodiment 1.
embodiment 3
A kind of Creatine kinase MB detection reagent is made up of according to the ratio of volume ratio 4:1 reagent R1 and reagent R2;
Reagent R1 is grouped into by the one-tenth of following content:
Imidazole buffer (pH=7.0) 100mmo1
Glucose 20mmo1
Nanoparticle 20mmol
N-acetylcystein 20mmo1
Sodium ethylene diamine tetracetate 1mmo1
Adenosine diphosphate (ADP) 1mmo1
Cozymase 6mmo1
Ribonucleotide 25mmo1
Pyruvic carboxylase 2KU
Glucose-6-phosphate dehydrogenase 2KU
Hexokinase 6KU
Goat-anti people CK-M polyclonal antibody 10mL
Trehalose 1g
Deionized water adds to 1L;
Reagent R2 is grouped into by the one-tenth of following content:
Imidazole buffer (pH=6.5) 50mmo1
Phosphocreatine 220mmo1
Sodium dodecylbenzene sulfonate 5g
Proclin3001g
Deionized water adds to 1L.
Nanoparticle is γ-Fe
2o
3nanoparticle, its particle diameter is 10nm.
A kind of using method of Creatine kinase MB detection reagent is with embodiment 1.
embodiment 4
A kind of Creatine kinase MB detection reagent is made up of according to the ratio of volume ratio 4:1 reagent R1 and reagent R2;
Reagent R1 is grouped into by the one-tenth of following content:
Imidazole buffer (pH=6.5) 80mmo1
Glucose 32mmo1
N-acetylcystein 25mmo1
Sodium ethylene diamine tetracetate 5mmo1
Adenosine diphosphate (ADP) 5mmo1
Cozymase 4mmo1
Ribonucleotide 15mmo1
Glucose-6-phosphate dehydrogenase 3KU
Hexokinase 4KU
Goat-anti people CK-M polyclonal antibody 30mL
Deionized water adds to 1L;
Reagent R2 is grouped into by the one-tenth of following content:
Imidazole buffer (pH=7.0) 70mmo1
Phosphocreatine 170mmo1
Sodium dodecylbenzene sulfonate 10g
Proclin3006g
Deionized water adds to 1L.
A kind of using method of Creatine kinase MB detection reagent is with embodiment 1.
dependency inspection experiment
Select same high level serum sample, this serum sample application heparin sodium heparin tube process, determine that in sample, the concentration of Creatine kinase MB is about 1109U/L in inspection body, this sample physiological saline dilutes, and dilution gradient is as shown in table 2.
Table 2 pair sample gradient dilution volume ratio
| Concentration of specimens (X) | 1 | 0.8 | 0.6 | 0.4 | 0.2 | 0 |
| Physiological saline | 0 | 0.2 | 0.4 | 0.6 | 0.8 | 1 |
| Concentration | X | 0.8X | 0.6X | 0.4X | 0.2X | 0 |
Utilize the obtained reagent of embodiment 1 to 4 to detect each concentration gradient sample respectively, result as Figure 1-4.
From Fig. 1-4, embodiment 1,2,3 linearly changes with weaker concn, relation conefficient is respectively r=0.992, r=0.9985, r=0.9977, illustrate that the reagent of embodiment 1,2,3 is all relatively more accurate for the pattern detection result of each concentration, and the sample of 1000U/L and above concentration can be detected.Contrastingly the linearly dependent coefficient of embodiment 4 is that r=0.9723 is less than 0.9900, and linear dependence is very poor, and major cause there is interfering substance in serum sample, have impact on the accuracy of detected result.With the addition of pyruvic carboxylase in the reagent that the present invention obtains, the interfering substance (as pyruvic acid, heparin) in serum sample can be removed by this material, and the homologous pair sexual intercourse achieving dilution gradient and detected result is good, ensure that the accuracy of detected result.
detection of Stability
Be placed on by reagent in 4 DEG C of refrigerators, follow the tracks of the stability of reagent, to same stable sample, utilize the obtained reagent of embodiment 1 to 4 to detect respectively, the per timing first quarter detects once, and in 5 season of tracing detection, detected result as shown in Figure 5.
As shown in Figure 5; As time goes on, the detected result of reagent is in decay, but the reagent stability of embodiment 1,2,3 is obviously good than the reagent of embodiment 4; illustrate and add antibody protective agent trehalose and nanoparticle in reagent, the stability of reagent significantly improves.
It should be appreciated by those skilled in the art that when not departing from the spirit and scope of the present invention be defined by the claims, various amendment and conversion can be carried out to these embodiments.
Claims (5)
1. a Creatine kinase MB detection reagent, is characterized in that, this reagent, by reagent R1 and reagent R2, forms according to the ratio of volume ratio 4:1;
Described reagent R1 is grouped into by the one-tenth of following content:
Imidazole buffer 50-100mmo1/L
Glucose 20-45mmo1
Nanoparticle 20-40mmol
N-acetylcystein 20-30mmo1
Sodium ethylene diamine tetracetate 1-8mmo1
Adenosine diphosphate (ADP) 1-10mmo1
Cozymase 2-6mmo1
Ribonucleotide 10-25mmo1
Pyruvic carboxylase 0.5-2KU
Glucose-6-phosphate dehydrogenase 2-5KU
Hexokinase 2-6KU
Goat-anti people CK-M polyclonal antibody 10-50mL
Trehalose 1-10g
Deionized water adds to 1L;
Described nanoparticle is γ-Fe
2o
3nanoparticle, its particle diameter is 10-40nm;
Described reagent R2 is grouped into by the one-tenth of following content:
Imidazole buffer 50-100mmo1/L
Phosphocreatine 120-220mmo1
Sodium dodecylbenzene sulfonate 5-20g
Sanitas 1-10g
Deionized water adds to 1L.
2. Creatine kinase MB detection reagent according to claim 1, is characterized in that, described sanitas is sodiumazide or Proclin300.
3. Creatine kinase MB detection reagent according to claim 1, is characterized in that, the pH value of described imidazole buffer is 6.0-7.0.
4. Creatine kinase MB detection reagent according to claim 1, is characterized in that, described reagent R1 is grouped into by the one-tenth of following content:
Imidazole buffer 80mmo1/L
Glucose 32mmo1
Nanoparticle 30mmol
N-acetylcystein 25mmo1
Sodium ethylene diamine tetracetate 5mmo1
Adenosine diphosphate (ADP) 5mmo1
Cozymase 4mmo1
Ribonucleotide 15mmo1
Pyruvic carboxylase 1.2KU
Glucose-6-phosphate dehydrogenase 3KU
Hexokinase 4KU
Goat-anti people CK-M polyclonal antibody 30mL
Trehalose 5g
Deionized water adds to 1L.
5. Creatine kinase MB detection reagent according to claim 1, is characterized in that, described reagent R2 is grouped into by the one-tenth of following content:
Imidazole buffer 70mmo1/L
Phosphocreatine 170mmo1
Sodium dodecylbenzene sulfonate 10g
Sanitas 6g
Deionized water adds to 1L.
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| CN106370862B (en) * | 2016-08-30 | 2018-07-20 | 山东博科诊断科技有限公司 | A kind of stabilization, sensitive fibronectin detection reagent |
| CN110554179B (en) * | 2019-09-10 | 2022-06-28 | 四川新健康成生物股份有限公司 | Anti-heparin interference CKMB determination kit |
| CN111057746B (en) * | 2020-01-03 | 2023-10-27 | 浙江夸克生物科技有限公司 | Creatine kinase isoenzyme determination kit |
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| US20040038318A1 (en) * | 2002-08-23 | 2004-02-26 | Bell Michael L. | Creatine kinase isoenzyme determination in multiplexed assays |
| CN102680689A (en) * | 2011-03-10 | 2012-09-19 | 王迎峰 | Kit for detecting creatine kinase isoenzyme and preparation and use methods thereof |
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