CN117025720A - Creatine kinase series standard substance simulating human body composition, method and application - Google Patents

Creatine kinase series standard substance simulating human body composition, method and application Download PDF

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CN117025720A
CN117025720A CN202310870829.6A CN202310870829A CN117025720A CN 117025720 A CN117025720 A CN 117025720A CN 202310870829 A CN202310870829 A CN 202310870829A CN 117025720 A CN117025720 A CN 117025720A
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standard substance
creatine kinase
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胡卫江
胡逸姗
梁加贝
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Yangtze Delta Region Institute of Tsinghua University Zhejiang
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Abstract

The application provides a creatine kinase series standard substance simulating human body composition, a method and application thereof, wherein in the series standard substance, a standard substance I is a creatine kinase isoenzyme standard substance containing normal CK-MB concentration of a human body, a standard substance II is a composite standard substance containing normal total CK concentration of the human body, CK-MM and CK-MB are contained, and the contained CK-MB concentration is equal to the standard substance I; the standard substance III is a creatine kinase isoenzyme standard substance containing abnormal concentration of CK-MB of a human body, the standard substance IV is a composite standard substance containing abnormal concentration of total CK of the human body, the standard substance III contains CK-MM and CK-MB, and the concentration of the contained CK-MB is equal to that of the standard substance III. The content of CK-MB and total CK in the serial standard substances simulates the existence form of serum in human body, the standard substances I and III can be used as clinical calibration and quality control of CK-MB independently, the standard substances II and IV can be used for clinical calibration and quality control of CK-MB and CK-MB, and the standard substances II and IV are respectively compared with the standard substances I and III for verification of methodology and evaluation of accuracy of a detection system.

Description

Creatine kinase series standard substance simulating human body composition, method and application
Technical Field
The application belongs to the technical field of biomedical inspection, and particularly relates to a creatine kinase series standard substance simulating human body composition, a method and application.
Background
In the age of "accurate medical treatment", the accuracy of test results is increasingly being appreciated by clinical testing institutions. The best way to achieve the accuracy of the measurement results is to establish and ensure the traceability of the test results. The standard substance is used as the top end of a traceable chain of the diagnostic reagent industry, and is necessary to ensure that the correct result of clinical examination is obtained. The development of standard substances and the establishment of a standard detection system of important diagnostic reagents based on the standard substances are of great significance.
Creatine Kinase (CK) has four isozymes, respectively muscle type (CK-MM), brain type (CK-BB), hybrid type (CK-MB) and mitochondrial type (CK-Mt). In normal human body, creatine kinase in serum is mostly CK-MM, which accounts for about 95% -99% of total CK, CK-MB accounts for less than 5% of total CK, and CK-BB content is extremely low and almost no creatine kinase exists in serum. According to the existing characteristics of M-type, B-type and MB-type creatine kinase in human bodies, the reference range of the clinical adult male Creatine Kinase (CK) is 46-171 IU/L (37 ℃), the reference range of the female creatine kinase (CK-MB) is 34-145 IU/L (37 ℃), and the upper limit of the reference range of the adult creatine kinase isoenzyme (CK-MB) is 25 IU/L (37 ℃).
CK-MB is a marker of myocardial cell injury, is a "gold standard" for the detection of Acute Myocardial Infarction (AMI), and plays an important role in clinical diagnosis. Clinically, a common method for detecting the enzymatic activity of CK-MB is an immunosuppression method. Based on the premise that CK-BB in a serum sample is negligible, in a CK-MB immunosuppression method reagent, an anti-human CK-M monoclonal antibody inhibits an M subunit, and the activity of CK-MB is obtained by measuring the activity of an uninhibited CK-B monomer in the sample and multiplying 2.
Clinically, the phenomenon of inaccurate CK-MB detection results often occurs, and the CK-MB standard substance has important significance. However, for reasons such as difficulty in obtaining, tracing and accurately determining the value of the high-purity CK-MB, the first in-vitro diagnosis enterprises in global ranking have no calibration materials and standard substances of the CK-MB.
At present, the standard substance database of the International Commission on traceability of medicine (JCTDM), the National Institute of Standards and Technology (NIST) does not contain CK-MB standard substance, and only CK-MM standard substance, such as CK-MM standard substance (ERM-AD 455k/IFCC, activity is 314 IU/L+/-6 IU/L, uncertainty is +/-1.9%), CK-MM standard substance (GBW (E) 091047, activity is 374.8 IU/L+/-9.5 IU/L, uncertainty is +/-2.5%) of the European Union Commission (JRC) and the like.
At present, the international database does not contain CK-MB standard substances, so that the calibration and the traceability of CK-MB can not be carried out, and the problem of inaccurate detection of CK-MB in clinic can not be solved.
Disclosure of Invention
The application aims to provide a creatine kinase series standard substance simulating human body composition, a method and application thereof, and aims to solve the technical problems that the CK-MB standard substance with no accurate fixed value in clinic at present cannot be calibrated and traced to source, and the CK-MB detection is inaccurate in clinic.
To achieve the above object, according to a first aspect of the present application, there is provided a creatine kinase series standard substances simulating human body composition, each comprising a treated stable serum matrix and high-purity CK-MB or high-purity CK-MB and high-purity CK-MM uniformly dispersed in the serum matrix;
the activity of the high purity CK-MB before addition to the serum matrix is 10 5 IU/L or more, the activity of the high purity CK-MM before being added to the serum matrix is 10 5 IU/L is higher than that of the total serum matrix to ensure that the volume of the added enzyme accounts for less than 0.1 per mill;
among the series of standard substances, the standard substance I is a creatine kinase isoenzyme standard substance containing normal CK-MB concentration of a human body, the standard substance II is a creatine kinase composite standard substance containing normal total CK concentration of the human body, CK-MM and CK-MB are contained, and the contained CK-MB concentration is equal to the CK-MB contained in the standard substance I; the standard substance III is a creatine kinase isoenzyme standard substance containing the abnormal concentration of CK-MB of a human body, the standard substance IV is a creatine kinase composite standard substance containing the abnormal total concentration of CK of the human body, the standard substance III contains CK-MM and CK-MB, and the concentration of the contained CK-MB is equal to the concentration of the CK-MB contained in the standard substance III.
Further, total CK and CK-MB viability of the series of standard substances was detected according to the IFCC Creatine Kinase (CK) assay reference method standard protocol.
Further, the activity of CK-MB in the standard substance I is 15-30 IU/L; the total CK activity of the standard substance II is 150-180 IU/L.
Further, the activity of CK-MB in the standard substance III is 50-80 IU/L; the total CK activity of the standard substance IV is 400-450 IU/L.
Furthermore, the serum matrix is from normal human body serum, is negative in infectious disease detection, is subjected to freeze thawing, inactivation, centrifugation and sterile filtration, and is subjected to biochemical index detection, so that common enzymes in the serum matrix are inactivated, and protein substances, ionic substances and micromolecular metabolite substances have no obvious difference from the original serum.
Further, the high purity CK-MB is prepared by the following method:
placing the high-activity CK-MM protein in a first denaturant solution for first denaturation treatment to obtain an inactive denatured CK-MM protein solution; the activity of the highly active CK-MM protein is 10 5 IU/L or more;
placing CK-BB protein with the activity equivalent to that of the high-activity CK-MM protein in a second denaturant solution for second denaturation treatment to obtain an inactive denatured CK-BB protein solution;
mixing the activities of the denatured CK-MM protein solution, the denatured CK-BB protein solution and the like, and placing the mixture in a renaturation agent solution for renaturation treatment to obtain a mixed protein solution;
and purifying the mixed protein solution to obtain CK-MB.
Further, the first denaturant solution is a solution containing 3-6M guanidine hydrochloride, 50-80 mM first buffer solution and 5-10 mM first reducing agent, and the pH value of the solution is 7.0-8.5.
Further, the second denaturant solution is a solution containing 2-4M guanidine hydrochloride, 50-80 mM second buffer solution and 5-10 mM second reducing agent, and the pH value of the solution is 7.0-8.5.
Further, the renaturation agent solution contains 50-80 mM of a third buffer solution, 5-10 mM of a third reducing agent, 0.5-2 mM of a chelating agent, 1-2M of a protective agent, 10-20 wt% of a stabilizer and 1-2 wt% of a surfactant.
Further, the protective agent is at least one of the following: BSA, proline, PEG-1000.
Further, the stabilizer is at least one of the following: trehalose, glycerol, mannitol.
Further, the surfactant is at least one of the following: tween20, tritonX100.
The second aspect of the present application provides a method for preparing the creatine kinase series standard substance simulating human body composition, comprising the following steps:
adding high-purity CK-MB into the serum matrix, and magnetically stirring uniformly at 4 ℃;
dividing the serum added with the high-purity CK-MB into two equal parts, wherein the content of the CK-MB in the two equal parts is consistent, adding the high-purity CK-MM in one part, and not adding the other part as a control; the activity of the high purity CK-MB before addition to the serum matrix is 10 5 IU/L or more, the activity of the high purity CK-MM before being added to the serum matrix is 10 5 IU/L is higher than that of the total serum matrix to ensure that the volume of the added enzyme is less than 0.1 per mill.
In a third aspect of the present application, the application of the creatine kinase series standard substance simulating human body composition in biomedical inspection is provided.
Compared with the prior art, the application has the following technical effects:
the content of CK-MB and total CK in the creatine kinase series standard substances simulating human body composition simulates the existence form of serum in human body, the standard substances I and III can be independently used for clinical calibration and quality control of the CK-MB, the standard substances II and IV can be used for clinical calibration and quality control of the CK-MB and the CK-MB, and the standard substances II and IV respectively form comparison with the standard substances I and III, so that the creatine kinase series standard substances simulating human body composition are used for verification of the total CK and CK-MB methodology and evaluation of accuracy of a detection system.
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In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments or the description of the prior art will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a package diagram of a series of standard substances provided in an embodiment of the present application;
FIG. 2 is a graph showing the molecular sieve purification peaks of CK-MB according to the present application;
FIG. 3 is a SDS-PAGE electrophoresis of the purified CK-MB products according to the present application;
FIG. 4 is a high performance liquid chromatogram of the CK-MB purified product provided in the examples of the present application.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
The embodiment of the application provides a creatine kinase series standard substance simulating human body composition, which comprises a treated stable serum matrix and high-purity CK-MB or high-purity CK-MB and high-purity CK-MM uniformly dispersed in the serum matrix, wherein the total CK is the sum of the CK-MB and the CK-MM.
The creatine kinase series standard substances simulating human body composition comprise a standard substance I, a standard substance II, a standard substance III and a standard substance IV. Wherein the standard substance I is a creatine kinase isoenzyme standard substance containing normal CK-MB concentration of a human body, the standard substance II is a creatine kinase composite standard substance containing normal total CK concentration of the human body, CK-MM and CK-MB are contained, and the contained CK-MB concentration is equal to the CK-MB contained in the standard substance I; the standard substance III is a creatine kinase isoenzyme standard substance containing the abnormal concentration of CK-MB of a human body, the standard substance IV is a creatine kinase composite standard substance containing the abnormal total concentration of CK of the human body, the standard substance III contains CK-MM and CK-MB, and the concentration of the contained CK-MB is equal to the concentration of the CK-MB contained in the standard substance III. Wherein the activity of high purity CK-MM is 10 before adding to serum matrix 5 The activity of high-purity CK-MB before being added into serum matrix is 10 at least 5 IU/L or more.
The development of the creatine kinase series standard substance is based on the actual content condition of creatine kinase isoenzyme in human body: the reference range of the creatine kinase (total CK) of the adult male is 46-171 IU/L (37 ℃), the reference range of the female is 34-145 IU/L (37 ℃), and the upper limit of the creatine kinase isoenzyme (CK-MB) of the adult male is 25 IU/L (37 ℃). According to the actual content of creatine kinase isoenzyme in human body, the total CK activity of the composite standard substance is set to be 150-180 IU/L, and the activity of CK-MB in the composite standard substance is 15-30 IU/L; the total CK activity of the composite standard substance is set to be 400-450 IU/L, and the activity of the CK-MB in the composite standard substance is 50-80 IU/L. Thus, the low value is set at the critical value of the medical reference range, the high value is set at 2.5 times the critical value of the medical reference range, and both normal and abnormal conditions of the patient can be used and are within the detection linear range.
Accordingly, the creatine kinase series standard substance provided by the embodiment of the application comprises:
the standard substance I only contains CK-MB, the activity of the CK-MB is 29.5 IU/L, and the CV is 1.77%; standard substance II containing CK-MB and CK-MM, wherein the activity of the CK-MB is 29.5 IU/L, the total activity of the CK is 160.8 IU/L, the CV is 2.48%, and the total CK is the sum of the CK-MB and the CK-MM;
standard III, which contains only CK-MB, has a CK-MB activity of 74.4. 74.4 IU/L and a CV of 2.39%;
standard IV contains CK-MB and CK-MM, wherein the CK-MB activity is 74.4. 74.4 IU/L, the total CK activity is 435.4. 435.4 IU/L, and the CV is 2.53%.
The uncertainty of the creatine kinase series standard substances provided by the embodiment of the application all meets the standard substance requirements.
The content of CK-MB and total CK in the creatine kinase series standard substances simulating human body composition simulates the existence form of serum in human body, the standard substances I and III can be independently used for clinical calibration and quality control of CK-MB, the standard substances II and IV can be used for clinical calibration and quality control of CK-MB and CK-MB, and the standard substances II and IV are respectively in contrast with the standard substances I and III and are used for verification of the methodologies of total CK and CK-MB and evaluation of the accuracy of a detection system.
In the embodiment of the application, the serum matrix is from normal human body serum, the infectious disease detection is negative, and after freeze thawing, inactivation, centrifugation and sterile filtration, common heteroenzyme in the serum is inactivated through biochemical index detection, and the protein, ionic substances and micromolecular metabolite substances have no obvious difference with the original serum. The serum matrix can be prepared specifically according to the following method:
collecting normal human body serum of a hospital, screening and collecting serum meeting the requirements after detecting indexes such as AIDS, syphilis, hepatitis B, hepatitis C, transaminase and the like, and carrying out the following treatment:
(1) Mixing the collected serum;
(2) Repeatedly freezing and thawing the serum at-80deg.C and 4deg.C for 3 times, centrifuging, and removing macromolecules such as lipid;
(3) The serum is placed in a 60 ℃ oven for 3 hours, common enzymes in a serum matrix are detected, complete inactivation of the common enzymes is ensured, and the enzymes in the original matrix are ensured not to influence the added CK-MM and CK-MB.
(4) The inactivated serum matrix was filtered with a 0.45 um pore size filter as a candidate serum matrix.
(5) The candidate serum matrices were aligned with the original serum: the serum matrix and normal raw serum are subjected to biochemical index detection at the same time, and the result shows that: the common enzymes in the candidate serum matrix are all inactivated, and have no obvious difference from proteins, ionic substances and small molecule metabolites in the original serum (the results are shown in Table 1 in detail), and the serum matrix is suitable to be used as matrix materials of CK and CK-MB standard substances.
TABLE 1 comparison of the serum matrix treated with the raw serum fractions
In the examples of the present application, high purity CK-MB is prepared by the following method:
(10) Placing the high-activity CK-MM protein in a first denaturant solution for first denaturation treatment to obtain an inactive denatured CK-MM protein solution; the activity of the highly active CK-MM protein is 10 5 IU/L or more;
(20) Placing CK-BB protein with the activity equivalent to that of the high-activity CK-MM protein in a second denaturant solution for second denaturation treatment to obtain an inactive denatured CK-BB protein solution;
(30) Mixing the activities of the denatured CK-MM protein solution, the denatured CK-BB protein solution and the like, and placing the mixture in a renaturation agent solution for renaturation treatment to obtain a mixed protein solution;
(40) And purifying the mixed protein solution to obtain CK-MB.
The high activity and high purity CK-MM protein in step (10) and the high activity and high purity CK-BB protein in step (20) can be prepared by the prior art, and are not described herein.
In the step (10), the first denaturant solution is a solution containing 3-6M guanidine hydrochloride, 50-80 mM of first buffer solution and 5-10 mM of first reducing agent, and the pH value of the solution is 7.0-8.5. The first buffer may be selected from at least one of the following: tris-HCl buffer, PIPES buffer, MOPS buffer. The first reducing agent may be selected from at least one of the following: dithiothreitol (DTT), cysteine. By controlling the pH value of the first denaturant solution and the content ranges of the respective components, the CK-MM protein can be denatured with high efficiency. In the embodiment of the present application, the first denaturation treatment condition is selected from the group consisting of standing or stirring at 4 to 25℃for 10 to 24 h, under which denatured CK-M subunits can be obtained with high efficiency.
In the step (20), the second denaturing agent solution is a solution containing 2-4M guanidine hydrochloride, 50-80 mM second buffer solution and 5-10 mM second reducing agent, and the pH of the solution is 7.0-8.5. The second buffer may be selected from at least one of the following: tris-HCl buffer, PIPES buffer, MOPS buffer. The second reducing agent may be selected from at least one of the following: dithiothreitol (DTT), cysteine. By controlling the pH value of the second denaturant solution and the content ranges of the respective components, the CK-BB protein can be denatured with high efficiency. In the embodiment of the present application, the second denaturation treatment condition is optionally that 10-24. 24 h is allowed to stand or stirred at 4-25℃under which denatured CK-B subunits can be obtained with high efficiency.
In the above step (30), the denatured CK-M subunit and CK-B subunit are subjected to a renaturation treatment with a renaturation agent solution to obtain the CK-MB protein. In the implementation of the application, the renaturation agent solution contains 50-80 mM of a third buffer solution, 5-10 mM of a third reducing agent, 0.5-2 mM of a chelating agent, 1-2M of a protective agent, 10-20 wt% of a stabilizer and 1-2 wt% of a surfactant. The third buffer may be selected from at least one of the following: tris-HCl buffer, PIPES buffer, MOPS buffer. The third reducing agent may be selected from at least one of: dithiothreitol, cysteine. The chelating agent may be EDTA. The renaturation treatment condition can be selected from standing or stirring at 4-25deg.C for 10-24 h. The CK-MB protein can be obtained with high efficiency by controlling the content range of each component in the renaturation agent solution and further controlling the renaturation treatment condition.
Wherein the protective agent can be selected from at least one of the following: BSA, proline, PEG-1000. The stabilizer can be at least one of the following: trehalose, glycerol, mannitol. The surfactant may be selected from at least one of the following: tween20, tritonX100.
In the embodiment of the application, the renaturation rate of the obtained CK-MB protein can be improved by adding the protective agent, the stabilizing agent and the surfactant into the renaturation agent solution at the same time, and under the same experimental conditions, the renaturation rate of the CK-MB protein can be improved from 28.2% to 64.52% when the protective agent, the stabilizing agent and the surfactant are added into the renaturation agent solution.
For example, high purity CK-MB can be prepared by the following method:
(a) Will 10 5 The CK-MM protein with IU/L level activity is placed in a 10 mL first denaturant solution for first denaturation treatment to obtain a denatured CK-MM protein solution; the first denaturant solution had a pH of 7.5 and contained the following: 6M guanidine hydrochloride (Gdn-HCl), 50 mM Tris-HCl buffer, 5 mM DTT, the first denaturation treatment conditions were: 24. 24 h was stirred at 4℃and the magnetic stirrer stirring speed was 200 rpm. After the denaturation treatment, full-automatic is usedThe activity of creatine kinase before and after denaturation was measured by biochemical analyzer, and the denaturation rate of CK-MM was 99.7%.
(b) Will 10 5 Placing the IU/L level active CK-BB protein in a 10 mL second denaturant solution for second denaturation treatment to obtain a denatured CK-BB protein solution; the second denaturant solution had a pH of 7.5 and contained the following: 4M guanidine hydrochloride (Gdn-HCl), 50 mM Tris-HCl buffer, 5 mM DTT, the second denaturation conditions being identical to the first denaturation conditions. After the denaturation treatment, the denaturation rate of CK-BB was 98.7% by the same method as in the step (1).
(c) Mixing the denatured CK-MM protein solution and the denatured CK-BB protein solution of 0.5-mL respectively, filling into a 1 mL syringe without needle, slowly dripping the mixture into the renaturation agent solution of 35 mL under the action of gravity, and renaturation treatment to obtain a mixed solution; the renaturation agent solution contains the following components: 50 mM Tris-HCl buffer, 2mM DTT,1 mM EDTA,1M proline, 10% glycerol, 1% Tween, renaturation conditions were: the mixture was stirred at 4℃for 24 and h at 200 rpm. After the renaturation treatment, the renaturation rate of CK-MB is 64.52% by the same method.
(d) The mixed protein solution was concentrated to 1 mL and applied to a Superdex200 Increate 10/300 GL molecular sieve column, and the mobile phase (50 mM Tris-HCl buffer, 300 mM NaCl,5% glycerol, 1 mM DTT) was purified at a flow rate of 0.5 mL/min.
The molecular sieve peak diagram (AKTA peak diagram) of the purified CK-MB is shown in FIG. 2, the peak position of the peak tip is 15.568 mL, and the highest ultraviolet absorption value is 144.842 mAU. Collecting peak position sample, and measuring its CK activity to 8.8X10 5 IU/L. As a result of SDS-PAGE, the purity of the CK-MB sample was very high and no bands were observed as shown in FIG. 3.
A peak position sample was collected and analyzed by high performance liquid chromatography using Agilent-1100, and Superdex5/150 analytical column was selected at a flow rate of 0.5 mL/min and 20. Mu.L, and after passing through a mobile phase (50 mM Tris buffer, pH 8.0, 300 mM NaCl,5% glycerol, 1 mM DTT), the peak pattern was shown in FIG. 4. It was found that it had no impurity peak, indicating that the purity of the obtained CK-MB sample was very high.
The activity value of CK-MB prepared by the embodiment of the application reaches 8.8x10 5 IU/L, and the normal value in human body is 25 IU/L, the CK-MB prepared by the embodiment of the application has very high activity.
The creatine kinase series standard substance of the embodiment of the application is prepared by adopting the prepared high-purity CK-MB and high-purity CK-MM, and specifically comprises the following steps:
1. preparation of creatine kinase series standard substance I and standard substance II
(1) Adding high-purity CK-MB into 1L candidate serum matrix, magnetically stirring at 4deg.C, and detecting with biochemical instrument 7020 and Rayleigh source CK-MB detection kit to make its final concentration reach about 30U/L. The above-mentioned CK-MB-added serum was divided into two equal parts, and the two parts were tested at the same time to confirm that the content of CK-MB was identical.
(2) To one of them, 10 was added 5 High purity CK-MM with IU/L grade viability, only when CK-MM reaches 10 5 The IU/L level vitality is higher than that, the added CK-MM volume is smaller than 0.1 per mill of the total serum matrix volume, and the original system is not influenced. Adding CK-MM to ensure that the total activity of the CK-MM and the CK-MB reaches 150-180 IU/L, thus obtaining a standard substance II. The other serving was used as a control, i.e. standard I.
(3) And subpackaging the standard substance I and the standard substance II. Under magnetic stirring in ice bath, the sample was dispensed into frozen vials with an electronic pipette, 0.6 ml/vial, to ensure uniform dispensing of the target candidate per vial. The packaging diagram after split charging is shown in figure 1.
(4) Standard substance constant value
The viability of the developed standard was tested according to the IFCC Creatine Kinase (CK) assay reference method standard protocol (IFCC Primary ReferenceProcedures for the Measurement of Catalytic Activity Concentrations of Enzymesat ℃, clin Chem Lab Med ℃, 2002;40 (6): 635-642.).
Sample processing: before use, the sample is placed at 4 ℃ for 30 minutes, after re-dissolution, the mixture is gently inverted and mixed for 10 times, and the sample is measured as soon as possible, and the sample is prevented from evaporating in the measuring process.
Sample detection: the samples of each batch were measured 3 times daily for 6 days continuously, and the total of 18 measurement results were statistically calculated and analyzed to obtain the data shown in tables 3 and 4.
Since there is no internationally recognized method for determining the CK-MB value, only the total CK value is determined. The activity of the developed standard substance CK-MB is detected according to the standard operation procedure of the IFCC Creatine Kinase (CK) determination reference method, and the value of the CK-MB in the compound standard substance can be determined by measuring the value of the CK-MB in the control since the added raw materials are pure enough and the total CK value in the control is derived from the CK-MB. And determining the total CK value of the composite standard substance by determining the fixed value and the proportion of the CK-MB/CK.
TABLE 3 detection results of Standard substance I
TABLE 4 detection results of Standard substance II
(5) Uniformity analysis
And (5) carrying out uniformity test on the standard substance by adopting an analysis of variance method. 10 samples are extracted from the standard substance I and the standard substance II, each sample is measured 3 times, 10 groups of data are obtained by measuring the samples with high precision, and statistical calculation is shown in tables 5 and 6:
TABLE 5 uniformity test results for Standard substance I
TABLE 6 uniformity test results for Standard substance II
TABLE 7 statistical analysis of the homogeneity of Standard substance I
Q1/n1 Q2/n2 Evaluation value F Q n 2/Q2 n1 Theoretical F value
Statistical calculation value 0.48 0.30 1.61 2.393
TABLE 8 statistical analysis of Standard substance II homogeneity
Q1/n1 Q2/n2 Evaluation value F Q n 2/Q2 n1 Theoretical F value
Statistical calculation value 0.97 1.39 0.70 2.393
According to statistical method analysis of uniformity analysis, by comparing the inter-group variance with the intra-group variance, and comparing the given significance level α, the ratio of the two is less than the threshold value of the statistical test (see tables 7, 8), and the samples are considered to be uniform from group to group without significant differences. According to the technical requirements of the national first-class standard substance technical specification (JJG 1006-1994) when uniformity test is carried out on single undetermined characteristic values, the experiment obtains the following conclusion: the human serum matrix creatine kinase standard substance developed by the embodiment of the application is uniform and meets the uniformity requirement of the national first-class standard substance technical specification (JJG 1006-1994).
From the calculation results, the creatine kinase/creatine kinase isoenzyme composite standard and the contrast have better uniformity, and the content between bottles has no obvious difference.
2. Preparation of creatine kinase series standard substance III and standard substance IV
(1) Adding high-purity CK-MB into 1L candidate serum matrix, magnetically stirring at 4deg.C, and detecting with biochemical instrument 7020 and Rayleigh source CK-MB detection kit to make its final concentration reach about 80U. The above-mentioned CK-MB-added serum was divided into two equal parts, and the two parts were tested at the same time to confirm that the content of CK-MB was identical.
(2) To one of them, 10 was added 5 High purity CK-MM with IU/L grade viability, only when CK-MM reaches 10 5 The IU/L level vitality is higher than that, the added CK-MM volume is smaller than 0.1 per mill of the total serum matrix volume, and the original system is not influenced. Adding CK-MM to enable the total activity of the CK-MM and the CK-MB to reach 400-450 IU/L, and obtaining a standard substance IV. The other serving as a control, i.e. standard III.
(3) And subpackaging the standard substance III and the standard substance IV. Under magnetic stirring in ice bath, the sample was dispensed into frozen vials with an electronic pipette, 0.6 ml/vial, to ensure uniform dispensing of the target candidate per vial.
(4) Standard substance constant value
The data obtained by the same method as above are shown in tables 11 and 12.
TABLE 11 detection results of Standard substance III
Table 12 results of detection of Standard substance IV
(5) Uniformity analysis
The detection and analysis methods are as above, and the results are shown in tables 13, 14, 15 and 16.
TABLE 13 uniformity test results for Standard substance III
TABLE 14 uniformity test results for Standard substance IV
TABLE 15 statistical analysis of uniformity of Standard substance III
Q1/n1 Q2/n2 Evaluation value F Q n 2/Q2 n1 Theoretical F value
Statistical calculation value 0.49 0.70 2.12 2.393
TABLE 16 statistical analysis of uniformity of Standard substance IV
Q1/n1 Q2/n2 Evaluation value F Q n 2/Q2 n1 Theoretical F value
Statistical calculation value 5.76 31.77 0.18 2.393
According to statistical analysis of uniformity tests, by comparing the inter-group variance with the intra-group variance, the ratio of the two is less than the threshold of the statistical test (see tables 10, 11). According to the technical requirements of the national first-class standard substance technical specification (JJG 1006-1994) when uniformity test is carried out on single undetermined characteristic values, the experiment obtains the following conclusion: the human serum matrix creatine kinase standard substance developed by the embodiment of the application is uniform and meets the uniformity requirement of the national first-class standard substance technical specification (JJG 1006-1994).
From the calculation results, the creatine kinase/creatine kinase isoenzyme composite standard and the contrast have better uniformity, and the content between bottles has no obvious difference.
The foregoing examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of protection of the present application is to be determined by the appended claims.

Claims (10)

1. A creatine kinase series standard substance simulating human composition, wherein the series standard substances each comprise a treated stable serum matrix and high purity CK-MB or high purity CK-MB and high purity CK-MM uniformly dispersed in the serum matrix;
among the series of standard substances, the standard substance I is a creatine kinase isoenzyme standard substance containing normal CK-MB concentration of a human body, the standard substance II is a creatine kinase composite standard substance containing normal total CK concentration of the human body, CK-MM and CK-MB are contained, and the contained CK-MB concentration is equal to the CK-MB contained in the standard substance I; the standard substance III is a creatine kinase isoenzyme standard substance containing the abnormal concentration of CK-MB of a human body, the standard substance IV is a creatine kinase composite standard substance containing the abnormal total concentration of CK of the human body, the standard substance III contains CK-MM and CK-MB, and the concentration of the contained CK-MB is equal to the concentration of the CK-MB contained in the standard substance III;
wherein the high purity CK-MB isThe activity before addition to the serum matrix was 10 5 IU/L or more, the activity of the high purity CK-MM before being added to the serum matrix is 10 5 IU/L is higher than that of the total serum matrix to ensure that the volume of the added enzyme is less than 0.1 per mill.
2. A creatine kinase series standard for simulating human composition according to claim 1, wherein the activity of the total CK and CK-MB of the series standard is detected according to IFCC creatine kinase assay reference method standard protocol;
the activity of CK-MB in the standard substance I is 15-30 IU/L; the total CK activity of the standard substance II is 150-180 IU/L.
3. The creatine kinase series standard substance simulating human body composition according to claim 1, wherein the activity of CK-MB in the standard substance iii is 50-80 IU/L; the total CK activity of the standard substance IV is 400-450 IU/L.
4. A creatine kinase series standard substance for simulating human body composition according to any one of claims 1-3, wherein the serum matrix is derived from normal human body serum, is negative in infectious disease detection, is subjected to freeze thawing, inactivation, centrifugation and sterile filtration, and is subjected to biochemical index detection, and common heteroenzyme in the serum matrix is inactivated, so that proteins, ionic substances and micromolecular metabolite substances are not obviously different from the original serum.
5. A creatine kinase series standard substance for simulating human composition according to any one of claims 1 to 3, wherein the high purity CK-MB is prepared by the following method:
placing the high-activity CK-MM protein in a first denaturant solution for first denaturation treatment to obtain an inactive denatured CK-MM protein solution; the activity of the highly active CK-MM protein is 10 5 IU/L or more;
placing CK-BB protein with the activity equivalent to that of the high-activity CK-MM protein in a second denaturant solution for second denaturation treatment to obtain an inactive denatured CK-BB protein solution;
mixing the activities of the denatured CK-MM protein solution, the denatured CK-BB protein solution and the like, and placing the mixture in a renaturation agent solution for renaturation treatment to obtain a mixed protein solution;
and purifying the mixed protein solution to obtain the CK-MB with high purity and high activity.
6. The creatine kinase series standard substance simulating human body composition according to claim 5, wherein the first denaturant solution is a solution containing 3-6M guanidine hydrochloride, 50-80 mM first buffer solution and 5-10 mM first reducing agent, and the pH value of the solution is 7.0-8.5; and/or
The second denaturant solution is a solution containing 2-4M guanidine hydrochloride, 50-80 mM second buffer solution and 5-10 mM second reducing agent, and the pH value of the solution is 7.0-8.5.
7. The creatine kinase series standard substance simulating human body composition according to claim 5, wherein the renaturation agent solution comprises 50-80 mM of a third buffer solution, 5-10 mM of a third reducing agent, 0.5-2 mM of a chelating agent, 1-2M of a protecting agent, 10-20 wt% of a stabilizing agent and 1-2 wt% of a surfactant.
8. The creatine kinase series standard substance simulating human composition of claim 7, wherein the protective agent is at least one of the following: BSA, proline, PEG-1000; and/or the number of the groups of groups,
the stabilizer is at least one of the following: trehalose, glycerol, mannitol; and/or the number of the groups of groups,
the surfactant is at least one of the following: tween20, tritonX100.
9. A method for preparing a creatine kinase series standard substance for simulating human body composition according to any one of claims 1 to 8, comprising the steps of:
adding high-purity CK-MB into the serum matrix, and magnetically stirring uniformly at 4 ℃;
dividing the serum added with the high-purity CK-MB into two equal parts, wherein the content of the CK-MB in the two equal parts is consistent, adding the high-purity CK-MM in one part, and not adding the other part as a control; the activity of the high purity CK-MB before addition to the serum matrix is 10 5 IU/L or more, the activity of the high purity CK-MM before being added to the serum matrix is 10 5 IU/L is higher than that of the total serum matrix to ensure that the volume of the added enzyme is less than 0.1 per mill.
10. Use of a creatine kinase series standard substance according to any one of claims 1-8 for simulating human composition in biomedical assays.
CN202310870829.6A 2023-07-17 2023-07-17 Creatine kinase series standard substance simulating human body composition, method and application Pending CN117025720A (en)

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