CN113866410A - Kit for determining activity of creatine kinase isoenzyme and determination method thereof - Google Patents

Kit for determining activity of creatine kinase isoenzyme and determination method thereof Download PDF

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CN113866410A
CN113866410A CN202111301783.3A CN202111301783A CN113866410A CN 113866410 A CN113866410 A CN 113866410A CN 202111301783 A CN202111301783 A CN 202111301783A CN 113866410 A CN113866410 A CN 113866410A
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齐文闯
蒋析文
吴润锋
简俊兴
段少卿
卢华丹
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Guangzhou Da'an Gene Co ltd
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Abstract

The embodiment of the application belongs to the field of medical examination and determination, and relates to a kit for determining creatine kinase isozyme activity, which comprises a first reagent and a second reagent, wherein the first reagent comprises a first buffer solution, adenosine diphosphate, disodium ethylene diamine tetraacetate, magnesium acetate, an activator, adenosine monophosphate, an anti-human CK-MM antibody, AP5A, glucose and NAD (P)+And a first preservative, the components of the second reagent including a second buffer, creatine phosphate, magnesium acetate, hexokinase, glucose-6-phosphate dehydrogenase, a tetrazolium salt, diaphorase, and a second preservative. The application also relates to a method for measuring the activity of the creatine kinase isoenzyme. The technical scheme provided by the application can improve the test sensitivity.

Description

Kit for determining activity of creatine kinase isoenzyme and determination method thereof
Technical Field
The application relates to the technical field of medical examination and determination, in particular to a kit for determining creatine kinase isoenzyme activity and a determination method thereof.
Background
Creatine Kinase (CK) has three major isoenzymes, namely CK-MM, CK-MB, CK-BB. Muscle type (MM) is mainly present in various muscle cells, brain type (BB) is mainly present in brain cells, hybrid type (MB) is mainly present in cardiac muscle cells, and creatine kinase MB isozyme (CK-MB) is the most specific enzyme in the cardiac muscle zymogram. Creatine kinase MB isozyme (CK-MB) is mainly distributed in cardiac muscle, and the content change of the CK-MB can reflect the state of cardiac muscle, so that the CK-MB can be used for diagnosing and prognosing Acute Myocardial Infarction (AMI), diagnosing and prognosing myocarditis, diagnosing muscle diseases, diagnosing brain diseases such as cerebral infarction, meningitis, encephalitis and the like. The majority of the normal human serum is the activity of CK-MM (the main active part of creatine kinase), only a small amount of CK-MB is contained, the CK-MB accounts for less than 5 percent of the total activity of CK, the absolute value does not exceed 25U/L, and the measured value of the CK-MB is increased along with the increase of the measured value of the CK. When myocardial cells are damaged or necrosed due to inflammation or infarction and the like, a large amount of CK is released into blood, the CK-MB in the blood serum accounts for more than 5 percent of the total CK activity and is positive, and the highest value reaches 12 to 38 percent. CK-MB can be increased in serum 3 hours after acute myocardial infarction, and the peak value can be reached within 12-24 hours. Therefore, the increased CK-MB activity is a specific index of myocardial damage and has important value in early diagnosis and determination of myocardial necrosis in myocardial infarction.
The current mainstream creatine kinase isoenzyme method is that the reagent contains an antibody which binds to the M subunit of creatine kinase in a serum sample, and thus can inhibit the activity of the M subunit. The B subunit of the enzyme remains free and is able to act on the substrate present in R2. Creatine kinase isozymes can reversibly catalyze the transfer of phosphate groups from phosphocreatine to Adenosine Diphosphate (ADP), producing creatine and Adenosine Triphosphate (ATP). The ATP formed serves to convert glucose into glucose-6-phosphate and ADP. This reaction is catalyzed by Hexokinase (HK), requiring magnesium ions for maximum activity. Under the action of glucose-6-phosphate dehydrogenase (G6PDH), glucose-6-phosphate is oxidized while the coenzyme nicotinamide adenine dinucleotide phosphate (NADP +) is reduced, yielding NADPH and 6-phosphogluconate. The increase in absorbance at 340nm due to NADPH formation is proportional to the activity of creatine kinase isoenzyme (CK-MB) in the sample. The principle reaction diagram is as follows:
Figure BDA0003338691700000021
Figure BDA0003338691700000022
Figure BDA0003338691700000023
because the content of creatine kinase isoenzyme (CK-MB) in the serum of normal human is low, in order to meet the sensitivity requirement of clinical use, the sample volume is generally increased to improve the detection sensitivity, and the interference factor caused by increasing the sample volume is increased to influence the accuracy, in addition, the molar absorption coefficient of NADPH is low, the change rate of the determination is not obvious, and the sensitivity of the determination method is also low.
Drawings
In order to illustrate the solution of the present application more clearly, the drawings that are needed in the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and that other drawings can be obtained by those skilled in the art without inventive effort.
Fig. 1 is a flowchart of an example of a method for measuring creatine kinase isoenzyme activity according to the present application.
Disclosure of Invention
The technical problem to be solved by the embodiments of the present application is that in the related art, the detection sensitivity is improved by increasing the sample size, resulting in a reduction in the detection accuracy.
In order to solve the above technical problems, embodiments of the present application provide a kit for determining creatine kinase isoenzyme activity, which adopts the following technical scheme:
a first reagent and a second reagent;
the components of the first reagent comprise a first buffer solution, adenosine diphosphate, disodium ethylenediamine tetraacetate, magnesium acetate, an activator, adenosine monophosphate, an anti-human CK-MM antibody, AP5A, glucose, NAD (P) + and a first preservative;
the components of the second reagent comprise a second buffer solution, creatine phosphate, magnesium acetate, hexokinase, glucose-6-phosphate dehydrogenase, a tetrazolium salt, diaphorase and a second preservative.
Further, the first buffer solution is one of imidazole buffer solution, 2- (N-morpholine) ethanesulfonic acid buffer solution and 1, 4-piperazine diethylsulfonic acid buffer solution.
Further, the activator is one of thioglycerol, N-acetyl-L-cysteine and L-cysteine.
Further, the first preservative is one of sodium azide, methylisothiazolinone and Proclin series preservatives.
Further, the second buffer solution is one of a tris buffer solution, an imidazole buffer solution and a 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution.
Further, the tetrazolium salt is WST-1 or WST-3.
Further, the second preservative is one of sodium azide, methylisothiazolinone and Proclin series preservatives.
Further, the first reagent is prepared by adopting the following process:
according to the formula proportion corresponding to the first reagent, purified water is respectively added into each component of the first reagent to be mixed and stirred evenly, and the pH value is adjusted to prepare the first reagent.
Further, the second reagent is prepared by adopting the following process:
according to the corresponding formula proportion of the second reagent, purified water is respectively added into the components of the second reagent to be mixed and stirred evenly, and the pH value is adjusted to prepare the second reagent.
In order to solve the above technical problems, embodiments of the present application further provide a method for measuring activity of creatine kinase isozyme, wherein the kit for measuring activity of creatine kinase isozyme described above is applied, and the following technical scheme is adopted:
preparing a sample of creatine kinase isoenzyme;
adding a first reagent in the kit into the sample, uniformly mixing, and incubating at 37 ℃ for 5min to obtain a mixed sample;
adding a second reagent in the kit into the incubated mixed sample, uniformly mixing, and delaying for 1.5min to obtain a sample to be detected;
and continuously monitoring the absorbance change rate of the sample to be detected for 2-3 min under the detection wavelength of a detector, and calculating the absorbance change rate to obtain the activity of the creatine kinase isoenzyme.
Compared with the prior art, the embodiment of the application mainly has the following beneficial effects:
under the action of an anti-human creatine kinase CK-M antibody, the isozyme (CK-MB) of creatine kinase catalyzes the transfer of phosphate from phosphocreatine to Adenosine Diphosphate (ADP) to generate creatine and Adenosine Triphosphate (ATP). The ATP formed serves to convert glucose into glucose-6-phosphate and ADP. This reaction is catalyzed by Hexokinase (HK), requiring magnesium ions for maximum activity. Under the action of glucose-6-phosphate dehydrogenase (G6PDH), glucose-6-phosphate is oxidized, coenzyme nicotinamide adenine dinucleotide phosphate (NADP +) is reduced at the same time, NADPH and 6-phosphogluconate are generated, under the action of tetrazolium salt and diaphorase, the NADPH generates formazan and NADP +, and the activity of isozyme of creatine kinase can be indirectly reflected by measuring the generation rate of the formazan at a specific wavelength. Compared with the existing determination method, the method has the advantage that the test sensitivity is higher by more than 3-4 times.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "including" and "having," and any variations thereof, in the description and claims of this application and the description of the above figures are intended to cover non-exclusive inclusions. The terms "first," "second," and the like in the description and claims of this application or in the above-described drawings are used for distinguishing between different objects and not for describing a particular order.
Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the application. The appearances of the phrase in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. It is explicitly and implicitly understood by one skilled in the art that the embodiments described herein can be combined with other embodiments.
In order to make the technical solutions better understood by those skilled in the art, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings.
The embodiment of the application provides a kit for determining creatine kinase isozyme activity, which comprises a first reagent R1 and a second reagent R2, wherein the components of the first reagent R1 comprise a first buffer solution, adenosine diphosphate, disodium ethylene diamine tetraacetate, magnesium acetate, an activator, adenosine monophosphate, an anti-human CK-MM antibody, AP5A, glucose, NAD (P) +, and a first preservative, and the components of the second reagent R2 comprise a second buffer solution, creatine phosphate, magnesium acetate, hexokinase, glucose-6-phosphate dehydrogenase, tetrazolium salt, diaphorase, and a second preservative.
In some alternative implementations of this embodiment, the components and concentrations of the first reagent R1 and the second reagent R2 in the kit can be found in table 1.
TABLE 1
Figure BDA0003338691700000051
Figure BDA0003338691700000061
Aiming at the kit, the technical principle adopted by the application is as follows: under the action of an anti-human creatine kinase CK-M antibody, the isozyme (CK-MB) of creatine kinase catalyzes the transfer of phosphate from phosphocreatine to Adenosine Diphosphate (ADP), producing creatine and Adenosine Triphosphate (ATP). The ATP formed serves to convert glucose into glucose-6-phosphate and ADP. This reaction is catalyzed by Hexokinase (HK), requiring magnesium ions for maximum activity. Under the action of glucose-6-phosphate dehydrogenase (G6PDH), glucose-6-phosphate is oxidized, coenzyme nicotinamide adenine dinucleotide phosphate (NADP +) is reduced at the same time, NADPH and 6-phosphogluconate are generated, the NADPH generates formazan and NADP + under the action of tetrazolium salt and diaphorase, the generation rate of the formazan is measured at a specific wavelength, and the activity of creatine kinase isozyme can be indirectly reflected. The principle reaction formula is as follows:
Figure BDA0003338691700000062
Figure BDA0003338691700000063
Figure BDA0003338691700000064
Figure BDA0003338691700000065
the formazan has high molar absorptivity, and the absorptivity change rate of the formazan is measured, so that the detection sensitivity can be improved.
In this embodiment, the buffer type of the first buffer in the first reagent R1 may be one of imidazole buffer, 2- (N-morpholine) ethanesulfonic acid (MES) buffer, and 1, 4-piperazine diethylsulfonic acid (PIPES) buffer, and is preferably imidazole buffer.
In this embodiment, the activator in the first reagent R1 may be one of thioglycerol, N-acetyl-L-cysteine and L-cysteine, preferably thioglycerol.
In this embodiment, the preservative in the first reagent R1 may be one of sodium azide, methylisothiazolinone and Proclin series preservatives, and is preferably sodium azide.
In this embodiment, the buffer type of the second buffer of the second reagent R2 may be one of Tris buffer, imidazole buffer and 4-hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES), and is preferably Tris buffer.
In this example, the tetrazolium salt of the second reagent R2 was WST-1 or WST-3.
WST (a water-soluble tetrazolium salt) is produced by introducing a positive or negative charge and a hydroxyl group into the benzene ring of the tetrazolium salt, and WST-1 is a compound similar to MTT that can be reduced by some dehydrogenases in mitochondria in the presence of an electron coupling reagent to produce orange-yellow formazan. The more rapid the cell proliferation, the darker the color; the more cytotoxic, the lighter the color.
In this example, the diaphorase of the second reagent R2 is derived from Bacillus megaterium.
In this embodiment, the preservative of the second reagent R2 may be one of sodium azide, methylisothiazolinone and Proclin series preservatives, and is preferably sodium azide.
In this example, the first reagent R1 and the second reagent R2 were prepared by a conventional method, in which purified water was added to each component of the first reagent R1 and the second reagent R2 to a predetermined concentration, respectively, according to the formulation ratio of table 1 above, and each was mixed and stirred to adjust the pH.
The application also provides a method for measuring the activity of the creatine kinase isoenzyme, and the test conditions are as follows:
the temperature is 37 ℃; the optical path of the cuvette was 1.0 cm. And detecting the main wavelength of 400-480 nm and the auxiliary wavelength of 700 nm.
The method for determining the activity of the creatine kinase isoenzyme (CK-MB) in the sample by using the creatine kinase isoenzyme determination kit comprises the following steps:
step S10, preparing a sample of creatine kinase isoenzyme;
step S20, adding the first reagent in the kit into the sample, mixing uniformly, and incubating for 5min at 37 ℃ to obtain a mixed sample;
step S30, adding a second reagent in the kit into the incubated sample, uniformly mixing, and delaying for 1.5min to obtain a sample to be detected;
and step S40, continuously monitoring the absorbance change rate of the sample to be detected for 2-3 min under the detection wavelength of the detector, and obtaining the activity of the creatine kinase isoenzyme by calculating the absorbance change rate.
In this example, 12. mu.L of a sample of creatine kinase isoenzyme was prepared and placed in a sample tube, and a calibration tube and a blank tube were prepared, wherein the calibration tube used a calibrator as a sample and the blank tube used purified water as a sample. Respectively adding 160 mu L of first reagent R1 into a sample tube, a calibration tube and a blank tube, uniformly mixing, incubating for 5min at 37 ℃, respectively adding 40 mu L of second reagent R2 into the sample tube, the calibration tube and the blank tube, uniformly mixing, delaying for 1.5min, continuously monitoring the absorbance change rate for 2-3 min under the measurement wavelength of a detector, calculating the absorbance change rate delta A/min, and obtaining the activity of the creatine kinase isoenzyme through the calculated absorbance change rate.
The activity of creatine kinase isoenzyme (CK-MB) in a sample measured by the kit is calculated according to the following formula:
Figure BDA0003338691700000081
wherein, Delta ASample (I)The absorbance change rate of the sample tube; delta AStandard of meritTo calibrate the absorbance change rate of the tube; delta ABlank spaceThe absorbance change rate of the blank tube.
The kit of the present invention is applicable to fully automatic biochemical analyzers such as Hitachi 7600/7180, Merrill BS800, Dirrill CS600, Toshiba TBA40FR, Orlinbas AU2700, Beckman 680/5800, Siemens ADVIA2400, Roche P800, and the like.
Example 1
This example formulated a first reagent R1 and a second reagent R2 according to the components and concentrations in table 2 below.
TABLE 2 composition and concentration tables for first reagent R1 and second reagent R2
Figure BDA0003338691700000082
Figure BDA0003338691700000091
The preparation method of the reagent is a conventional method, namely the components of the reagent are respectively added into purified water, then are respectively mixed and stirred evenly, and the pH value is adjusted.
Example 2
This example formulated a first reagent R1 and a second reagent R2 according to the components and concentrations in table 3 below.
TABLE 3 composition and concentration tables of first reagent R1 and second reagent R2
Figure BDA0003338691700000092
Figure BDA0003338691700000101
The preparation method of the reagent is a conventional method, namely the components of the reagent are respectively added into purified water, then are respectively mixed and stirred evenly, and the pH value is adjusted.
A pair of control examples were also provided, and reagents R1 and R2 of the control examples were prepared according to the components and concentrations shown in Table 4 below.
TABLE 4 composition and concentration tables for first reagent R1 and second reagent R2
Figure BDA0003338691700000111
Comparative example kit components the diaphorase and tetrazolium salts of example 1 were prepared as in example 1.
The measurement methods of the above examples 1 and 2 are as follows:
the temperature is 37 ℃; the optical path of the cuvette was 1.0 cm. The main wavelength is 450nm and the sub-wavelength is 700 nm.
A12. mu.L sample of creatine kinase isoenzyme was placed in a sample tube, and a calibration tube and a blank tube were prepared, wherein the calibration tube contained a calibrator as a sample and the blank tube contained purified water as a sample. Respectively adding 160 mu L of first reagent R1 into a sample tube, a calibration tube and a blank tube, uniformly mixing, incubating for 5min at 37 ℃, respectively adding 40 mu L of second reagent R2 into the sample tube, the calibration tube and the blank tube, uniformly mixing, delaying for 1.5min, continuously monitoring the absorbance change rate for 2-3 min under the wavelength measurement, and calculating the absorbance change rate delta A/min.
Measurement parameters of the comparative example: the temperature is 37 ℃; the optical path of the cuvette was 1.0 cm. Detecting the main wavelength of 340nm and the sub wavelength of 700nm, preparing a sample tube, putting a 12 mu L sample, and preparing a calibration tube and a blank tube, wherein the calibration tube takes a calibrator as a sample, the blank tube takes purified water as a sample, 160 mu L of reagent R1 is respectively added into the sample tube, the calibration tube and the blank tube to be mixed uniformly, after incubation at 37 ℃ for 5min, 40 mu L of reagent R2 is added to be mixed uniformly, the delay is 1.5min, under the measurement wavelength, the absorbance change rate is continuously monitored for 2-3 min, and the delta A/min is calculated.
In examples 1-2 and the comparative example, the sample amount of the machine parameters was the same as the amount of the reagents.
The measurements were carried out on Hitachi 7180 fully automatic biochemical analyzer according to the above measurement settings, and the Δ A/min values of 40 clinical specimens were recorded, and the results are shown in Table 5.
TABLE 5 values of Delta A/min for clinical samples of examples 1-2 and comparative example 40
Figure BDA0003338691700000121
Figure BDA0003338691700000131
Figure BDA0003338691700000141
The results show that the delta A/min values measured in the examples 1 and 2 are respectively 3 times and 4 times of the values measured in the comparative example, and the activity measurement sensitivity of the creatine kinase isoenzyme (CK-MB) is obviously improved.
In conclusion, the kit for determining the activity of the creatine kinase isozyme can indirectly reflect the activity of the creatine kinase isozyme by determining the generation rate of the formazan at a specific wavelength, and compared with the existing determination method, the kit is higher in test sensitivity by more than 3-4 times.
It is to be understood that the above-described embodiments are merely illustrative of some, but not restrictive, of the broad invention, and that the appended drawings illustrate preferred embodiments of the invention and do not limit the scope of the invention. This application is capable of embodiments in many different forms and is provided for the purpose of enabling a thorough understanding of the disclosure of the application. Although the present application has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that the present application may be practiced without modification or with equivalents of some of the features described in the foregoing embodiments. All equivalent structures made by using the contents of the specification and the drawings of the present application are directly or indirectly applied to other related technical fields and are within the protection scope of the present application.

Claims (10)

1. A kit for measuring the activity of creatine kinase isoenzyme, which comprises:
a first reagent and a second reagent;
the components of the first reagent comprise a first buffer solution, adenosine diphosphate, ethylene diamine tetraacetic acid disodium, magnesium acetate, an activator, adenosine monophosphate, an anti-human CK-MM antibody, AP5A, glucose, NAD (P)+And a first preservative;
the components of the second reagent comprise a second buffer solution, creatine phosphate, magnesium acetate, hexokinase, glucose-6-phosphate dehydrogenase, a tetrazolium salt, diaphorase and a second preservative.
2. The kit of claim 1, wherein the first buffer is one of an imidazole buffer, a 2- (N-morpholine) ethanesulfonic acid buffer, and a 1, 4-piperazine diethylsulfonic acid buffer.
3. The kit of claim 1, wherein the activator is one of thioglycerol, N-acetyl-L-cysteine, and L-cysteine.
4. The kit according to claim 1, wherein the first preservative is one of sodium azide, methylisothiazolinone and Proclin series preservatives.
5. The kit of claim 1, wherein the second buffer is one of tris buffer, imidazole buffer, and 4-hydroxyethylpiperazine ethanesulfonic acid buffer.
6. The kit of claim 1, wherein the tetrazolium salt is WST-1 or WST-3.
7. The kit according to claim 1, wherein the second preservative is one of sodium azide, methylisothiazolinone and Proclin series preservatives.
8. The kit of claim 1, wherein the first reagent is prepared by the following process:
according to the formula proportion corresponding to the first reagent, purified water is respectively added into each component of the first reagent to be mixed and stirred evenly, and the pH value is adjusted to prepare the first reagent.
9. The kit of claim 1, wherein the second reagent is prepared by the following process:
according to the corresponding formula proportion of the second reagent, purified water is respectively added into the components of the second reagent to be mixed and stirred evenly, and the pH value is adjusted to prepare the second reagent.
10. A method for measuring the activity of creatine kinase isoenzyme, using the kit for measuring the activity of creatine kinase isoenzyme according to any one of claims 1 to 9, characterized in that the method comprises the steps of:
preparing a sample of creatine kinase isoenzyme;
adding a first reagent in the kit into the sample, uniformly mixing, and incubating at 37 ℃ for 5min to obtain a mixed sample;
adding a second reagent in the kit into the incubated mixed sample, uniformly mixing, and delaying for 1.5min to obtain a sample to be detected;
and continuously monitoring the absorbance change rate of the sample to be detected for 2-3 min under the detection wavelength of a detector, and calculating the absorbance change rate to obtain the activity of the creatine kinase isoenzyme.
CN202111301783.3A 2021-11-04 2021-11-04 Kit for determining activity of creatine kinase isoenzyme and determination method thereof Pending CN113866410A (en)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4360413A (en) * 1980-12-29 1982-11-23 Beckman Instruments, Inc. Creatine kinase isoenzyme electrophoretic technique and improved creatine kinase isoenzyme reagent for use therein
US5817467A (en) * 1995-11-16 1998-10-06 Kyowa Medex Co., Ltd. Method for quantitatively determining creatinine kinase and a reagent therefor
US6130054A (en) * 1997-12-19 2000-10-10 Unitika Ltd. Test strip for creatine kinase activity measurement
CN102154443A (en) * 2011-03-25 2011-08-17 浙江东瓯诊断产品有限公司 Creatine jubase MB isozyme activity detection reagent and preparation method thereof
CN104357544A (en) * 2014-12-05 2015-02-18 重庆中元生物技术有限公司 High-stability creatine kinase detection kit
CN109517879A (en) * 2018-11-08 2019-03-26 东软威特曼生物科技(南京)有限公司 A kind of creatine kinase and its isoenzyme determination reagent and its kit

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4360413A (en) * 1980-12-29 1982-11-23 Beckman Instruments, Inc. Creatine kinase isoenzyme electrophoretic technique and improved creatine kinase isoenzyme reagent for use therein
US5817467A (en) * 1995-11-16 1998-10-06 Kyowa Medex Co., Ltd. Method for quantitatively determining creatinine kinase and a reagent therefor
US6130054A (en) * 1997-12-19 2000-10-10 Unitika Ltd. Test strip for creatine kinase activity measurement
CN102154443A (en) * 2011-03-25 2011-08-17 浙江东瓯诊断产品有限公司 Creatine jubase MB isozyme activity detection reagent and preparation method thereof
CN104357544A (en) * 2014-12-05 2015-02-18 重庆中元生物技术有限公司 High-stability creatine kinase detection kit
CN109517879A (en) * 2018-11-08 2019-03-26 东软威特曼生物科技(南京)有限公司 A kind of creatine kinase and its isoenzyme determination reagent and its kit

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