A creatine kinase assay kit and method of use thereof

Abstract

The application discloses a creatine kinase determination kit and a use method thereof. The creatine kinase determination kit comprises an R1 component and an R2 component. The R1 component comprises oxidized glutathione and auxiliary material 1. The R2 component comprises reduced coenzyme II, glucose-6-phosphate dehydrogenase, creatine phosphate, glutathione reductase and auxiliary material 2. The oxidized glutathione (GSSG) is reduced into reduced glutathione (GSH) by catalytic degradation of glutathione reductase (GR), and the reduced glutathione contains -SH, thereby activating CK. By the above method, the -SH compound N-acetyl-L-cysteine does not need to be added, thereby fundamentally solving the problem that -SH is gradually oxidized and deactivated, greatly improving the long-term stability of the reagent, and prolonging the effective period of the reagent.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, specifically to a creatine kinase assay kit and its usage method. Background Technology

[0002] Creatine kinase (CK), also known as creatine phosphokinase, is abundant in skeletal muscle, cardiac muscle, and smooth muscle, followed by brain tissue, and less abundant in the gastrointestinal tract, lungs, and kidneys. It is primarily found in the cytoplasm and mitochondria and is a crucial kinase directly related to intracellular energy transport, muscle contraction, and ATP regeneration. Creatine kinase activity assays can be used for the diagnosis of skeletal muscle and cardiac diseases.

[0003] Creatine kinase has four isoenzyme forms: muscle type (MM), brain type (BB), hybrid type (MB), and mitochondrial type (MiMi). MM isoenzymes are mainly found in various muscle cells, BB is mainly found in brain cells, MB is mainly found in cardiomyocytes, and MiMi is mainly found in the mitochondria of cardiomyocytes and skeletal muscle. The muscle type creatine kinase molecule is a dimer composed of two identical subunits.

[0004] Creatine kinase isoenzymes play a crucial role in clinical diagnosis. In various diseases, including muscle atrophy and myocardial infarction, serum creatine kinase levels rise rapidly. Currently, measuring creatine kinase activity is considered more reliable than electrocardiogram (ECG) in diagnosing myocardial infarction. During myocardial infarction, creatine kinase levels rise within 6 hours of onset, peak at 24 hours, and return to normal within 3-4 days. Among these isoenzymes, creatine kinase-MB (CK-MB) has the highest diagnostic specificity. Due to its important physiological functions and clinical applications, creatine kinase has attracted widespread attention and in-depth research.

[0005] CK (creatine oxalate) is deactivated in blood and requires activation by compounds containing -SH (thiocyanate-containing compounds) for CK activity assays. Currently, this is typically achieved by adding -SH-containing compounds as activators to reagents; commonly used activators include N-acetyl-L-cysteine ​​(NAC), mercaptoethanol, and thioglycerol. However, CK activators are unstable; during long-term storage, the -SH in the activator is gradually oxidized, forming disulfide bonds and thus losing its CK-activating effect. Therefore, it is usually necessary to add chelating agents and reducing substances to the reagent to improve the stability of -SH. However, these methods cannot completely inhibit the oxidation of -SH, resulting in a short reagent shelf life. Summary of the Invention

[0006] The technical problem to be solved by the present invention is that the stability of -SH is usually improved by adding chelating agents and reducing substances to the reagent. However, the above methods cannot fundamentally and completely inhibit the oxidation of -SH, and the shelf life of the reagent is short.

[0007] The first objective of this invention is to provide a creatine kinase assay kit, comprising a component R1 and a component R2, wherein the component R1 comprises oxidized glutathione and excipient 1, and the component R2 comprises reduced coenzyme II, glucose-6-phosphate dehydrogenase, phosphocreatine, glutathione reductase and excipient 2.

[0008] Using the above technical solution, oxidized glutathione (GSSG) is reduced to reduced glutathione (GSH) by glutathione reductase (GR) catalysis. Reduced glutathione contains -SH, which activates CK. This eliminates the need to add the -SH compound N-acetyl-L-cysteine, thus fundamentally solving the problem of -SH being gradually oxidized and deactivated, significantly improving the long-term stability of the reagent and extending its shelf life.

[0009] As one possible design, the concentration of oxidized glutathione in component R1 is 0.80 g / L to 1.12 g / L.

[0010] As one possible design, the concentration of oxidized glutathione in component R1 is 1.00 g / L.

[0011] As one possible design, the R1 component is acidic.

[0012] As one possible design, the concentration of reduced coenzyme II in component R2 is 0.71 g / L to 0.90 g / L, the concentration of glucose-6-phosphate dehydrogenase is 25 to 34 kDa / L, the concentration of glutathione reductase is 0.5 to 1.3 kDa / L, and the concentration of creatine phosphate is 46 to 53 g / L.

[0013] As one possible design, the concentrations of reduced coenzyme II, glucose-6-phosphate dehydrogenase, glutathione reductase, and creatine phosphate in the R2 component are 0.80 g / L, 30 kDa / L, 1 kDa / L, and 51 g / L, respectively.

[0014] As one possible design, the pH value of component R1 is 5 to 6.8.

[0015] As one possible design, the excipient 1 includes disodium EDTA, imidazole, magnesium chloride, PLD-8, sodium azide, nicotinamide adenine dinucleotide, adenosine monophosphate, endogenous dinucleotide pentaphosphate, and hexokinase; the concentrations of imidazole, disodium EDTA, magnesium chloride, PLD-8, sodium azide, nicotinamide adenine dinucleotide, adenosine monophosphate, endogenous dinucleotide pentaphosphate, and hexokinase in component R1 are 6.05~8.42 g / L, 1.02~2.31 g / L, 9.23~11.57 g / L, 1.2~2.3 g / L, 0.2~0.8 g / L, 1.15~2.30 g / L, 1.94~2.31 g / L, 0.0038~0.0079 g / L, and 8~13 kDa / L, respectively.

[0016] As one possible design, the excipient 2 includes trihydroxymethylaminomethane, sodium azide, PLD-8, glucose, and adenosine diphosphate; the concentrations of trihydroxymethylaminomethane, sodium azide, PLD-8, glucose, and adenosine diphosphate in component R2 are 8~12 g / L, 0.3~0.6 g / L, 1.5~2.3 g / L, 4.3~7.5 g / L, and 6.2~8.5 g / L, respectively.

[0017] The second objective of this invention is to provide a method for using a creatine kinase assay kit, wherein components R1 and R2 are mixed and then added to the test solution.

[0018] Using the above technical solution, the method is simple and can successfully obtain reduced glutathione (GSH). The presence of -SH in reduced glutathione can activate CK. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments. The illustrative embodiments and descriptions of this invention are only used to explain this invention and are not intended to limit this invention.

[0020] Currently, the detection of creatine kinase is usually achieved by adding compounds containing -SH as activators to the reagent. However, CK activators are unstable. During long-term storage, the -SH in the activator is easily oxidized to form disulfide bonds, thus losing its CK activation function. Therefore, it is usually necessary to add chelating agents and reducing substances to the reagent to improve the stability of -SH. However, this method cannot completely inhibit the oxidation of -SH, and the reagent has a short shelf life.

[0021] To address the above problems, the present invention provides a creatine kinase assay kit, comprising component R1 and component R2. Component R1 comprises oxidized glutathione (GSSG) and excipient 1, and component R2 comprises reduced coenzyme II (NADPH), glucose-6-phosphate dehydrogenase (G6PDH), creatine phosphate, glutathione reductase (GR), and excipient 2.

[0022] The oxidized glutathione (GSSG) is reduced to reduced glutathione (GSH) by glutathione reductase (GR) catalysis. Reduced glutathione contains -SH, which activates CK. This eliminates the need to add the -SH compound N-acetyl-L-cysteine, fundamentally solving the problem of gradual oxidation and inactivation of -SH, significantly improving the long-term stability of the reagent and extending its shelf life.

[0023]

[0024] In this invention, the concentration of oxidized glutathione in component R1 is generally 0.80 g / L to 1.12 g / L, for example: 0.85 g / L, 0.92 g / L, 0.96 g / L, 0.99 g / L, 1.02 g / L, 1.08 g / L and 1.10 g / L, etc.; preferably 1.00 to 1.12 g / L, more preferably 1.00 g / L.

[0025] In this invention, the concentration of reduced coenzyme II in component R2 is 0.71 g / L to 0.90 g / L, for example: 0.74 g / L, 0.79 g / L, 0.83 g / L, 0.88 g / L, etc., preferably 0.80 g / L; the concentration of glucose-6-phosphate dehydrogenase is 25 to 34 kDa / L, for example: 25 kDa / L, 27 kDa / L, 29 kDa / L, 32 kDa / L, and 34 kDa / L, etc., preferably 30 kDa / L; the concentration of glutathione... The concentration of glutathione reductase is 0.5~1.3 kDa / L, for example: 0.5 kDa / L, 0.8 kDa / L, 0.9 kDa / L, 1.0 kDa / L, 1.2 kDa / L and 1.3 kDa / L, etc., preferably 1 kDa / L; the concentration of creatine phosphate is 46~53 g / L, for example: 46 g / L, 47 g / L, 48 g / L, 49 g / L, 50 g / L, 51 g / L, 52 g / L and 53 g / L, etc., preferably 51 g / L.

[0026] In this invention, the R1 component is generally acidic, specifically 5 to 6.8, for example: 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, and 6.8, etc., preferably 6.0; it is adjusted by common acidity regulators, such as succinic acid, oxalic acid, etc., and succinic acid is preferred in this invention.

[0027] In this invention, the excipient 1 includes disodium ethylenediaminetetraacetate (EDTA-2NA), imidazole, magnesium chloride, PLD-8, sodium azide (NaN3), and nicotinamide adenine dinucleotide (NAD). + ), adenosine monophosphate, endogenous dinucleotide pentaphosphate (AP5A), and hexokinase.

[0028] The concentrations of imidazole, disodium EDTA, magnesium chloride, PLD-8, sodium azide, nicotinamide adenine dinucleotide, adenosine monophosphate, endogenous dinucleotide pentaphosphate, and hexokinase in component R1 are 6.05~8.42 g / L, 1.02~2.31 g / L, 9.23~11.57 g / L, 1.2~2.3 g / L, 0.2~0.8 g / L, 1.15~2.30 g / L, 1.94~2.31 g / L, 0.0038~0.0079 g / L, and 8~13 kDa / L, respectively; preferably, 7.08 g / L, 2 g / L, 10.6 g / L, 2.0 g / L, 0.5 g / L, 1.75 g / L, 2.26 g / L, 0.0065 g / L, and 10 kDa / L.

[0029] The excipient 2 includes tris(hydroxymethyl)aminomethane (Tris), sodium azide (NaN3), PLD-8, glucose, and adenosine diphosphate (ADP).

[0030] The concentrations of trihydroxymethylaminomethane, sodium azide, PLD-8, glucose, and adenosine diphosphate in component R2 are 8-12 g / L, 0.3-0.6 g / L, 1.5-2.3 g / L, 4.3-7.5 g / L, and 6.2-8.5 g / L, respectively; preferably, 10 g / L, 0.5 g / L, 2.0 g / L, 6.0 g / L, and 7.5 g / L.

[0031]

[0032] In practical applications, simply mix components R1 and R2, and then add the solution to be tested. Example

[0033] This embodiment discloses a creatine kinase assay kit, which includes component R1 and component R2, wherein the composition of component R1 is shown in Table 1.

[0034] Table 1

[0035] The composition of component R2 is shown in Table 2.

[0036] Table 2

[0037] Before actual use, components R1 and R2 are stored separately; when using, components R1 and R2 are mixed, and then the solution to be tested is added to them. Example

[0038] This embodiment discloses a creatine kinase assay kit, which includes component R1 and component R2, wherein the composition of component R1 is shown in Table 3.

[0039] Table 3

[0040] The composition of component R2 is shown in Table 4.

[0041] Table 4

[0042] Before actual use, components R1 and R2 are stored separately; when using, components R1 and R2 are mixed, and then the solution to be tested is added to them. Example

[0043] This embodiment discloses a creatine kinase assay kit, which includes component R1 and component R2, wherein the composition of component R1 is shown in Table 5.

[0044] Table 5

[0045] The composition of component R2 is shown in Table 6.

[0046] Table 6

[0047] Before actual use, components R1 and R2 are stored separately; when using, components R1 and R2 are mixed, and then the solution to be tested is added to them.

[0048] Comparative Example 1 This comparative example discloses a creatine kinase assay kit, including R 11 Components and R 22 Components, wherein R11 The composition of the components is shown in Table 7.

[0049] Table 7

[0050] The composition of component R22 is shown in Table 8.

[0051] Table 8

[0052] Before actual use, R 11 Components and R 22 The components are stored separately; when using, R 11 Components and R 22 After the components are mixed, the test solution can be added to them.

[0053] Experimental Example Creatine kinase assay kit detection method 1. Instruments It has fully automated or semi-automated biochemical analyzers with wavelengths of 340nm and 405nm.

[0054] 2. Parameter settings and operation Table 9

[0055] The specific procedures for sample determination are shown in Table 10.

[0056] Table 10

[0057] 3. Calculation formula The F-value and calibration curve are automatically generated on the fully automated biochemical analyzer based on the concentration value of the calibrator and the rate of change of absorbance.

[0058] Sample CK activity (U / L) = ΔA / min × F For use in the creatine kinase assay kit obtained in Example 1, the sample CK activity (U / L) = △A / min × F.

[0059] Experimental Example 2 Other performance evaluations of the creatine kinase assay kit 1. Repeatability test: Using clinical high-value and low-value samples as test samples, the test was repeated 10 times using the reagents of Example 1 and Comparative Example 1 of this invention, and the coefficient of variation (CV) was calculated.

[0060] Table 11

[0061] As shown in Table 11, the reaction system using GR to catalyze the degradation of GSSG to reduce it to GSH has good precision compared to the reaction system containing NAC.

[0062] 2. Linear Range Detection: Take a linear sample with a concentration of approximately 1500 U / L, dilute it to at least 5 different concentrations (xi), and measure each concentration 3 times using the reagent of this invention according to the detection method, and calculate the average value (yi). Using xi as the independent variable and yi as the dependent variable, derive the linear regression equation and calculate the linear regression correlation coefficient r; substitute xi into the linear regression equation to calculate the estimated value of yi and the deviation between yi and the estimated value.

[0063] Table 12

[0064] Table 13

[0065] As shown in Tables 12 and 13, the reaction system of GR catalytic degradation of GSSG to GSH has comparable linear performance to the reaction system containing NAC.

[0066] 3. Long-term stability: The reagent kit was stored in a sealed environment protected from light at 2–8°C. The reagent performance indicators were tested at 0, 1, 6, 12, 18 and 24 months, and the reactivity and reagent blank changes were calculated.

[0067] Table 14

[0068] Figure 15

[0069] As shown in Tables 14 and 15, the long-term stability of the reaction system that catalyzes the degradation of GSSG to GSH is significantly better than that of the reaction system containing NAC, and since the raw materials are readily available, it is worth promoting its use.

[0070] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A creatine kinase assay kit, characterized by, It includes component R1 and component R2. Component R1 includes oxidized glutathione and excipient 1. Component R2 includes reduced coenzyme II, glucose-6-phosphate dehydrogenase, creatine phosphate, glutathione reductase and excipient 2. The excipient 1 includes hexokinase, and the excipient 2 includes glucose and adenosine diphosphate.

2. The creatine kinase assay kit according to claim 1, characterized in that, The concentration of oxidized glutathione in component R1 is 0.80 g / L to 1.12 g / L.

3. The creatine kinase assay kit according to claim 2, characterized in that The concentration of oxidized glutathione in component R1 is 1.00 g / L.

4. The creatine kinase assay kit according to claim 1, characterized in that, The R1 component is acidic.

5. The creatine kinase assay kit according to claim 1, characterized in that The concentration of reduced coenzyme II in component R2 is 0.71 g / L to 0.90 g / L, the concentration of glucose-6-phosphate dehydrogenase is 25 to 34 kDa / L, the concentration of glutathione reductase is 0.5 to 1.3 kDa / L, and the concentration of creatine phosphate is 46 to 53 g / L.

6. The creatine kinase assay kit according to claim 5, characterized in that The concentrations of reduced coenzyme II, glucose-6-phosphate dehydrogenase, glutathione reductase, and creatine phosphate in the R2 component are 0.80 g / L, 30 kDa / L, 1 kDa / L, and 51 g / L, respectively.

7. The creatine kinase assay kit according to claim 4, characterized in that The pH value of component R1 is 5 to 6.

8.

8. The creatine kinase assay kit according to claim 1, characterized in that The excipient 1 includes disodium EDTA, imidazole, magnesium chloride, PLD-8, sodium azide, nicotinamide adenine dinucleotide, adenosine monophosphate, endogenous dinucleotide pentaphosphate, and hexokinase; the concentrations of imidazole, disodium EDTA, magnesium chloride, PLD-8, sodium azide, nicotinamide adenine dinucleotide, adenosine monophosphate, endogenous dinucleotide pentaphosphate, and hexokinase in component R1 are 6.05~8.42 g / L, 1.02~2.31 g / L, 9.23~11.57 g / L, 1.2~2.3 g / L, 0.2~0.8 g / L, 1.15~2.30 g / L, 1.94~2.31 g / L, 0.0038~0.0079 g / L, and 8~13 kDa / L, respectively.

9. The creatine kinase assay kit according to claim 1, characterized in that The excipient 2 includes trihydroxymethylaminomethane, sodium azide, PLD-8, glucose, and adenosine diphosphate; the concentrations of trihydroxymethylaminomethane, sodium azide, PLD-8, glucose, and adenosine diphosphate in component R2 are 8~12 g / L, 0.3~0.6 g / L, 1.5~2.3 g / L, 4.3~7.5 g / L, and 6.2~8.5 g / L, respectively.

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