A creatine kinase detection kit against blood lipid interference and a preparation method thereof

Abstract

This invention discloses a creatine kinase detection kit that counteracts lipemia interference and its preparation method. The kit includes reagent R1 and reagent R2; reagent R1 includes: purified water, imidazole, MgAC, and EDTA Na. 2 Glucose, PC300, Na 2 SO 3 N-acetylcysteine, NADP, AMP, ADP, AP5A, HK, and surfactant; reagent R2 includes: pure water, Capso, creatine phosphate, PEG6000, and NaN. 3 The kit contains BSA and G6PDH. The surfactant is one or a combination of two of Triton 100 and Tween 80. Through the synergistic effect of PEG6000 and the two surfactants Triton 100 and Tween 80, the creatine kinase detection kit significantly improves its resistance to lipemia interference and enhances its detection accuracy. The kit is simple to prepare, has strong resistance to lipemia interference, simple components, and low preparation cost, making it highly promising for market application.

Description

A Creatine Kinase Detection Kit that Counteracts Lipid-Induced Interference and Its Preparation Method Technical Field

[0001] This invention relates to the field of in vitro diagnostic reagent technology, and in particular to a creatine kinase detection kit that resists lipemia interference and its preparation method. Background Technology

[0002] Creatine kinase (CK) is an important kinase directly related to intracellular energy transport, muscle contraction, and ATP regeneration. It reversibly catalyzes the transphosphorylation reaction between creatine and ATP. CK is a dimerizing enzyme with four isoenzyme forms: skeletal muscle type (CK-MM), brain type (CK-BB), cardiac type (CK-MB), and mitochondrial type (CKm). Clinically, the detection of CK and its isoenzyme activities is mainly used for the diagnosis and monitoring of diseases such as myocardial injury. When myocardial injury occurs, CK is released from damaged cells into the blood, causing a rapid increase in serum CK levels. For example, 4 hours after an acute myocardial infarction, serum CK activity begins to rise, peaks at 24 hours, and returns to normal levels within 3-4 days.

[0003] In medicine, lipemia is also known as chylous blood. When people consume high-fat foods, a large number of chylous microparticles enter the bloodstream, causing the serum to become cloudy, a condition known as chylous blood. Lipoemia can significantly interfere with various serum test results, especially those of serum enzymes and other biochemical tests.

[0004] The detection principle of creatine kinase is as follows:

[0005]

[0006]

[0007]

[0008] In principle, the above three reactions are involved, and each step involves enzymes. It is easily affected by lipemia. Studies have shown that high concentrations of lipemia in serum samples can affect the detection results of CK reagent, resulting in lower measured values ​​and statistically significant differences between groups, which is within the clinically unacceptable range.

[0009] Currently, the main focus of publicly available creatine kinase detection kits is to improve the specificity and sensitivity of the detection products. For example, patent application number CN202110467117.0 discloses a kit for specifically detecting creatine kinase isoenzymes. By including an antibody that blocks the B subunit of creatine kinase isoenzyme CK-MB in the kit, it ensures that the antibody that blocks the M subunit of creatine kinase isoenzyme CK-MM in the sample, thus significantly improving the detection specificity. At the same time, blocking CK-MB can improve the detection linear range. Patent application number CN202011373956.8 discloses a high-sensitivity creatine kinase isoenzyme detection kit and its preparation method. The kit includes reagent R1 and reagent R2. In the preparation of reagent R2, sodium periodate is used to oxidize the Fc-terminal glycan chain of anti-mouse IgG-Fc antibody, so that the antibody is directionally labeled on amino latex microspheres. Then, the anti-mouse IgG-Fc antibody is bound to the mouse anti-human CK-MB monoclonal antibody through specific recognition. Compared with other conjugation methods, this method avoids the conjugation of the F(ab)-terminal active site of CK-MB monoclonal antibody to the microspheres, while increasing the amount of CK-MB monoclonal antibody conjugated, thus significantly improving the detection sensitivity of the reagent.

[0010] Therefore, there is currently a lack of effective creatine kinase detection kits that counteract the interference of lipemia, and existing creatine kinase detection kits need further improvement. Summary of the Invention

[0011] To address the above problems, this invention provides a creatine kinase detection kit that resists interference from lipemia. This kit has a good anti-interference effect, effectively reducing the interference of lipemia on creatine kinase test results and ensuring the accuracy of clinical creatine kinase test results.

[0012] Therefore, this application provides the following specific technical solutions:

[0013] This invention provides a creatine kinase detection kit that resists lipemia interference, comprising reagent R1 and reagent R2;

[0014] The reagent R1 includes: pure water, imidazole, MgAC, EDTA Na2, glucose, PC300, Na2SO3, N-acetylcysteine, NADP, AMP, ADP, AP5A, HK, and surfactant.

[0015] The reagent R2 includes: pure water, Capso, creatine phosphate, PEG6000, NaN3, BSA and G6PDH.

[0016] Preferably, the creatine kinase assay kit for lipemia interference includes reagent R1 and reagent R2;

[0017] The reagent R1 comprises the following components in the following amounts:

[0018]

[0019]

[0020] The above-mentioned pH adjustment to 6.87 at 20°C using HAC refers to adjusting the pH to 6.87 when the solution temperature is 20°C, followed by the addition of NADP, AMP, ADP, AP5A, HK, and surfactants.

[0021] The reagent R2 comprises the following components in the following amounts:

[0022]

[0023] The step of adjusting the pH to 9.53 and the temperature to 20°C using HAC refers to the following steps: first, add an appropriate amount of pure water, then add Capso, creatine phosphate, PEG6000 and NaN3 in sequence. After complete dissolution, adjust the pH of the solution to 9.53 and the temperature to 20°C using HAC.

[0024] The Capso mentioned above is 3-(cyclohexylamino)-2-hydroxypropane-1-sulfonic acid.

[0025] Preferably, in the creatine kinase assay kit for lipemia interference, the concentration of PEG6000 is 5 g / L.

[0026] Preferably, in the creatine kinase assay kit for lipemia interference, the surfactant is one or a combination of two of Triton 100 and Tween 80.

[0027] Preferably, in the creatine kinase assay kit for lipemia interference, the surfactant includes Triton 100 and Tween 80, and the volume concentration ratio of Triton 100 to Tween 80 is 0:2-2:0.

[0028] Preferably, the volume concentration ratio of Triton 100 to Tween 80 is 1:1, and the volume concentration of each is 1%.

[0029] Secondly, this application also provides a method for preparing the above-mentioned creatine kinase detection kit that resists lipemia interference, the method being as follows:

[0030] (1) Preparation of reagent R1:

[0031] Weigh an appropriate amount of pure water into a beaker and place it on a magnetic stirrer. Add the corresponding amounts of imidazole, MgAC, EDTANa2, glucose, PC300, Na2SO3, and N-acetylcysteine ​​in sequence. After the raw materials are completely dissolved, adjust the pH of the solution to 6-7 at 20℃. Then add NADP, AMP, ADP, AP5A, HK, and surfactant in sequence. After the raw materials are completely dissolved, make up the volume with pure water to prepare reagent R1.

[0032] (2) Preparation of reagent R2:

[0033] Weigh an appropriate amount of pure water into a beaker and place it on a magnetic stirrer. Add Capso, creatine phosphate, PEG6000, and NaN3 in sequence. After the raw materials are completely dissolved, adjust the pH of the solution to 9-10 at 20℃. Then add BSA and G6PDH in sequence. After the raw materials are completely dissolved, dilute with pure water to obtain reagent R2.

[0034] Preferably, the preparation method of the creatine kinase detection kit is as follows:

[0035] (1) Preparation of reagent R1:

[0036] Weigh an appropriate amount of pure water into a beaker and place it on a magnetic stirrer. Add the following ingredients sequentially: 3.40-6.81g imidazole, 1.28-4.27g MgAC, 0.34-1.68g EDTA Na2, 1.80-7.21g glucose, 0.05-0.15mL PC300, 0.06-0.13g Na2SO3, and 2-6g N-acetylcysteine. After the ingredients are completely dissolved, adjust the pH of the solution to 6.87 at 20℃ using HAC. Then, add 1-4g NADP, 1-5g AMP, 0.9-3g ADP, 0.005-0.018g AP5A, 2-5KU HK, and 10-30mL surfactant. After the ingredients are completely dissolved, bring the volume to 1L with pure water.

[0037] (2) Preparation of reagent R2:

[0038] Weigh an appropriate amount of pure water into a beaker and place it on a magnetic stirrer. Add 2.59-7.78g Capso, 25-75g creatine phosphate, 2.5-10g PEG6000, and 0.25-0.75g NaN3 in sequence. After the raw materials are completely dissolved, adjust the pH of the solution to 9.53 at 20℃ using HAC. Then, add 0.5-1.5g BSA and 6-20KU G6PDH in sequence. After the raw materials are completely dissolved, bring the volume of pure water to 1L.

[0039] When preparing reagents R1 and R2, please note that the next reagent should only be added after the previous one has completely dissolved.

[0040] The present invention has the following beneficial effects:

[0041] 1. This invention provides an effective creatine kinase detection kit that resists lipemia interference. The kit utilizes the synergistic effect of PEG6000 and two surfactants, Triton 100 and Tween 80, to significantly improve the kit's ability to resist lipemia interference and enhance its detection accuracy.

[0042] Specifically, Triton 100 and Tween 80 act on chylomicrons, high-density lipoprotein, low-density lipoprotein, and very low-density lipoprotein in serum, converting them into micronized cholesterol. Meanwhile, PEG6000 has good amphiphilicity and dispersibility, exposing the creatine kinase reaction sites in serum and increasing the sensitivity of the reaction. The combined effect of the two can ensure the accuracy of the measurement.

[0043] 2. The creatine kinase detection kit provided in this application is simple to prepare, convenient to use, and has strong anti-lipidemia interference ability, with an interference of as low as -3.52% against 5% simulated lipemia. In addition, the creatine kinase detection kit has simple components, low preparation cost, and is easy to industrialize, and has a high market application prospect. Attached Figure Description

[0044] Figure 1 shows the standard curve for experimental group 11 in Example 5:

[0045] Figure 2 shows the standard curve for experimental group 14 in Example 5;

[0046] Figure 3 shows the standard curve for experimental group 15 in Example 5. Detailed Implementation

[0047] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0048] Example 1

[0049] 1. This embodiment provides a creatine kinase detection kit that resists lipemia interference. The kit includes reagent R1 and reagent R2. In each embodiment, reagents R1 and R2 are used in a 4:1 ratio.

[0050] (1) Reagent R1 includes the following components:

[0051]

[0052] Adjust the pH to 6.87 using HAC at 20°C.

[0053]

[0054] (2) Reagent R2 includes the following components:

[0055]

[0056] Adjust pH to 9.53 with HAC at 20℃

[0057] BSA 1g / L

[0058] G6PDH 12KU / L

[0059] The solvent is pure water.

[0060] Four experimental groups were set up according to the above formula. The difference between each experimental group was the concentration of PEG6000. The specific concentrations of PEG6000 in experimental groups 1-4 are shown in Table 1 below:

[0061] Table 1. Concentration of PEG6000 used in each experimental group

[0062] PEG6000 concentration experimental groups: 12.5 g / L, 25 g / L, 310 g / L, 415 g / L surface

[0063] 2. The preparation method of the creatine kinase detection kits for each experimental group in this embodiment specifically includes the following steps:

[0064] (1) Preparation of reagent R1:

[0065] Weigh an appropriate amount of pure water into a beaker and place it on a magnetic stirrer. Add the following ingredients sequentially: 6.3g imidazole, 2.68g MgAC, 0.94g EDTA Na2, 5g glucose, 0.1mL PC300, 0.126g Na2SO3, and 4.08g N-acetylcysteine. After the ingredients are completely dissolved, adjust the pH of the solution to 6.87 at 20°C using HAC. Then, add 2.75g NADP, 3.12g AMP, 1.875g ADP, 0.0118g AP5A, and 2.75KU HK. After the ingredients are completely dissolved, bring the volume to 1L with pure water. (Note that each ingredient must be completely dissolved before adding the next.)

[0066] (2) Preparation of reagent R2:

[0067] Weigh an appropriate amount of pure water into a beaker and place it on a magnetic stirrer. Add 4.8g Capso, 50g creatine phosphate, 2.5-15g PEG6000 (2.5g PEG6000 for experimental group 1, 5g for experimental group 2, 10g for experimental group 3, and 15g for experimental group 4), and 0.5g NaN3 in sequence. After the raw materials are completely dissolved, adjust the pH of the solution to 9.53 at 20℃ using HAC. Then, add 1g BSA and 12KU G6PDH in sequence. After the raw materials are completely dissolved, bring the volume to 1L with pure water. (Note that each raw material must be completely dissolved before adding the next.)

[0068] 3. Anti-interference capability test

[0069] (1) Experimental method:

[0070] ① Collect multiple serum samples, prepare a mixed serum pool, add fat emulsion, and prepare simulated lipemia with volume concentrations of 0.5%, 1%, 2%, and 5%, respectively.

[0071] ②The creatine kinase detection kits prepared in experimental groups 1-4 of this embodiment were used to determine the creatine kinase content of four different concentrations of simulated lipemia and raw serum samples without interference on a biochemical analyzer.

[0072] The biochemical analyzer parameters are as follows:

[0073]

[0074] Testing principle:

[0075]

[0076]

[0077]

[0078] The rate of enzyme reaction is directly proportional to the creatine kinase content in the sample. The activity of creatine kinase in the sample can be calculated by measuring the rate of increase in NADPH absorbance at 340 nm.

[0079] Each sample was measured 5 times, and the results were expressed as interference.

[0080] The method for calculating interference is as follows:

[0081] Interference level = (Mean of sample with interference - Mean of sample without interference) / Mean of sample without interference × 100%

[0082] An interference level of ±10% is considered acceptable.

[0083] The mean value of the sample with added interferon refers to the mean value of the creatine kinase content measured five times using this kit after adding lipemia interferon to the original serum sample.

[0084] (2) Experimental Results and Analysis

[0085] Table 2. Interference test results for different experimental groups

[0086] Experimental Group 1 Experimental Group 2 Experimental Group 3 40.5% Simulated Lipids -1.46% -1.33% -1.35% -1.55% 1% Simulated Lipids -2.12% -1.92% -2.03% -2.57% 2% Simulated Lipids -5.75% -4.05% -4.62% -9.26% 5% Simulated Lipids -9.86% -7.96% -9.23% -20.65% surface

[0087] ① As shown in Table 2, experimental group 2, with an addition of 5g of PEG6000, exhibited the best anti-interference effect among the four experimental groups. Therefore, the optimal addition amount of PEG6000 is 5g / L.

[0088] ② In experimental group 4, an excessive amount of PEG6000 was added, which may have inhibited the activity of the enzyme in the reagent. The absolute deviation of the simulated lipemia interference at the four concentrations was the largest among these experimental groups, and the simulated lipemia interference at 5% was unacceptable.

[0089] Therefore, it can be seen that when the amount of PEG6000 added in the reagent is in the range of 2.5-10 g / L, the creatine kinase detection kit has good anti-interference ability.

[0090] Example 2

[0091] 1. Based on the experimental results of Example 1, this embodiment replaces PEG6000 with PEG2000 and PEG8000 to conduct a comparative experiment on anti-lipidemia interference. The molecular weights of PEG2000, PEG6000, and PEG8000 are different, which may lead to differences in physical properties. In this embodiment, PEG2000 and PEG8000 are used instead of PEG6000 while maintaining the same number of moles of PEG per liter of solution.

[0092] Among them, experimental groups 5-7 correspond to experimental groups 1-3 in Example 1; experimental groups 8-10 correspond to experimental groups 1-3 in Example 1, only replacing PEG6000 according to the method in Table 3.

[0093] The preparation of reagents R1 and R2 is the same as in Example 1. The amount of PEG2000 and PEG8000 added to reagent R2 is as shown in Table 3.

[0094] Table 3. Concentrations of PEG2000 and PEG8000 used in each experimental group

[0095] PEG2000 concentration PEG8000 concentration Experimental group 50.83 g / L Experimental group 83.33 g / L Experimental group 61.67 g / L Experimental group 96.67 g / L Experimental group 73.33 g / L Experimental group 1013.33 g / L surface

[0096] 2. Anti-interference capability test

[0097] The method is the same as in Example 1.

[0098] 3. Experimental Results and Analysis

[0099] Table 4. Interference test results for different experimental groups

[0100] Experimental Group 5 Experimental Group 6 Experimental Group 7 Experimental Group 8 Experimental Group 9 100.5% Simulated Lipids -2.36% 2.36% -2.33% -2.12% -2.24% -2.09% 1% Simulated Lipids -3.98% -3.21% -4.18% -3.75% -4.02% -3.99% 2% Simulated Lipids -8.95% -8.88% -9.98% -6.99% -7.59% -7.75% 5% Simulated Lipids -14.66% -19.02% -18.00% -10.23% -11.06% -12.03% surface

[0101] As shown in Table 4 above, experimental groups 5-10, where PEG2000 or PEG8000 were used instead of PEG6000, were less effective at preventing lipemia interference than PEG6000. This indicates that PEG2000, PEG6000, and PEG8000 differ in their physical properties, and that PEG6000 is more effective in preventing lipemia interference in practice.

[0102] Example 3: Further optimization experiments of the creatine kinase detection kit

[0103] 1. Experimental Methods

[0104] This embodiment provides a creatine kinase detection kit that counteracts lipemia interference. The kit includes reagent R1 and reagent R2; in each experimental group, reagents R1 and R2 are used in a 4:1 ratio.

[0105] The creatine kinase detection kits for experimental groups 11-16 were prepared according to the formulations in Table 5 below. The specific combinations and concentrations of the two surfactants Triton 100 and Tween 80 in each experimental group are shown in Table 6.

[0106] Table 5 Formulas for Experimental Groups 11-16

[0107]

[0108]

[0109] Table 6. Dosage of Triton 100 and Tween 80 in each experimental group

[0110] Triton 100: Tween 80 PEG6000 Experimental Group 11 1:1 (1%) 5g / L Experimental Group 12 0:15g / L Experimental Group 13 1:05g / L Experimental Group 14 1:25g / L Experimental Group 15 2:15g / L Experimental Group 16 1:1 (1.5%) 5g / L surface

[0111] 2. The preparation method of the creatine kinase detection kits for each experimental group in this embodiment specifically includes the following steps:

[0112] (1) Preparation of reagent R1:

[0113] Weigh an appropriate amount of pure water into a beaker and place it on a magnetic stirrer. Add 6.3g imidazole, 2.68g MgAC, 0.94g EDTA Na2, 5g glucose, 0.1mL PC300, 0.126g Na2SO3, and 4.08g N-acetylcysteine ​​in sequence. After the raw materials are completely dissolved, adjust the pH of the solution to 6.87 at 20℃ using HAC. Then, add 2.75g NADP, 3.12g AMP, 1.875g ADP, 0.0118g AP5A, and 2.75KU HK in sequence. Finally, add Triton 100 and Tween 80. The amounts of Triton 100 and Tween 80 added to experimental groups 11-16 are as shown in Table 5. After the raw materials are completely dissolved, bring the volume to 1L with pure water. (Note that each raw material should be completely dissolved before adding the next.)

[0114] (2) Preparation of reagent R2:

[0115] Weigh an appropriate amount of pure water into a beaker and place it on a magnetic stirrer. Add 4.8g Capso, 50g creatine phosphate, 5g PEG6000, and 0.5g NaN3 sequentially. After the ingredients are completely dissolved, adjust the pH of the solution to 9.53 at 20°C using HAC. Then, add 1g BSA and 12KU G6PDH sequentially. After the ingredients are completely dissolved, bring the volume to 1L with pure water. (Note that each ingredient must be completely dissolved before adding the next.)

[0116] 3. Anti-interference capability test

[0117] (1) Test method: The test method is performed in accordance with the method in Example 1.

[0118] 4. Experimental Results and Analysis

[0119] The experimental results are detailed in Table 7 below:

[0120] Table 7. Interference test results of creatine kinase assay kits for experimental groups 11-16.

[0121] Experimental Group 11 Experimental Group 12 Experimental Group 13 Experimental Group 14 Experimental Group 15 160.5% Simulated Lipids -1.04% -1.22% -1.23% -1.12% -1.09% -1.05% 1% Simulated Lipids -1.50% -1.85% -1.88% -1.78% -1.76% -1.61% 2% Simulated Lipids -2.98% -3.66% -3.57% -3.56% -3.49% -3.16% 5% Simulated Lipids -3.52% -6.18% -6.35% -5.88% -5.93% -6.03% surface

[0122] (1) As can be seen from the experimental results in Table 7, adding either Triton 100 or Tween 80 surfactants can further improve the anti-interference effect of the kit. Among them, experimental group 11 is the optimal scheme among the 6 experimental groups. When measuring the simulated lipemia at 4 concentrations, the absolute deviation of the interference is the smallest.

[0123] (2) When the amount of the two surfactants Triton 100 and Tween 80 added is in the range of 1-3% (volume percentage concentration), the anti-interference performance of the creatine kinase kit can be improved; and when the amount of surfactants Triton 100 and Tween 80 used is 10 ml / L and the ratio is 1:1, the anti-interference effect of the kit is the best.

[0124] Example 4: Comparison of nonionic surfactants in creatine kinase assay kits

[0125] 1. Experimental Methods

[0126] Triton 100, Tween 80, Tween 20, Brij 35 and Thesit (i.e., polyethylene glycol monolaurate or polyethylene glycol (400) monolaurate) are all nonionic surfactants. To verify whether other nonionic surfactants besides Triton 100 and Tween 80 have the same effect on lipemia interference, experimental groups 17-25 were set up separately.

[0127] The creatine kinase detection kits for experimental groups 17-25 were prepared according to the experimental method in Example 3. The components and contents of the kits were also the same as in Example 3, except that the nonionic surfactant used was different (Tween 80 was replaced in experimental group 11). The specific amounts of Tween 20, Brij 35 and Thesit added to experimental groups 17-25R1 are shown in Table 8 below.

[0128] Table 8 shows the dosage of Tween 20 / Brij 35 / Thesit in experimental groups 17-25R1.

[0129] Triton 100, Tween 20, Brij 35, Thesit. Experimental group 171% 0.5% / / Experimental group 181% 1% / / Experimental group 191% 1.5% / / Experimental group 201% 0.5% / Experimental group 211% 1% / Experimental group 221% 1.5% / Experimental group 231% 0.5% / Experimental group 241% 1% / Experimental group 251% 1.5% surface

[0130] 2. Anti-interference capability test

[0131] (1) Test method: The test method is performed in accordance with the method in Example 1.

[0132] 3. Experimental Results and Analysis

[0133] The experimental results are detailed in Table 9 below, and the analysis is as follows:

[0134] As shown in Table 9, the experimental results indicate that Tween 20, Brij 35, and Thesit, when used to replace Tween 80, did not effectively clear lipoproteins from the blood and cannot be arbitrarily substituted for Tween 80. In the creatine kinase assay kit of this invention, Triton 100 and Tween 80 exhibited the best synergistic effect and formed an organic combination with the other components of this application. These two cannot be arbitrarily replaced with other existing nonionic surfactants.

[0135] Table 9. Interference test results of the creatine kinase assay kit for experimental groups 17-25.

[0136] 0.5% simulated lipemia, 1% simulated lipemia, 2% simulated lipemia, 5% simulated lipemia. Experimental group 17: -1.62% -1.95% -3.92% -6.59%; Experimental group 18: -1.64% -2.26% -4.12% -7.21%; Experimental group 19: -1.73% -2.19% -4.02% -6.99%; Experimental group 20: -1.89% -2.98% -3.78% -6.42%. Experimental Group 21: -1.72% -3.12% -4.03% -8.12% Experimental Group 22: -1.89% -2.85% -4.00% -7.09% Experimental Group 23: -1.96% -3.01% -4.45% -7.08% Experimental Group 24: -1.63% -4.06% -4.21% -8.13% Experimental Group 25: -1.65% -3.26% -4.02% -8.02% surface

[0137] Example 5: Stability testing of the creatine kinase assay kit that counteracts lipemia interference

[0138] 1. Experimental Methods

[0139] In this embodiment, experimental groups 11-16 were subjected to an opening stability test and a 37°C water bath accelerated destructive test.

[0140] (1) Accelerated degradation experiment in 37℃ water bath: The reagent was placed in a 37℃ water bath for 7 days. It was taken out on the 1st, 3rd, 5th and 7th day after degradation and the quality control sample was measured on a biochemical analyzer. The relative deviation between the measured value of the quality control sample and the measured value of the undegraded sample was acceptable if it was within ±5%. On the 7th day, the repeatability and linearity of the measured value were tested.

[0141] Among them, the quality control material used is Landau composite quality control material, which has two levels. Each test is performed 3 times and the average value is taken.

[0142] (2) Opening stability test: An opening stability test was conducted on experimental groups 11 and 14, which could withstand the accelerated damage test in a 37℃ water bath. The specific operation is as follows:

[0143] The reagents were opened and stored in the reagent compartment of the biochemical analyzer for one month. Calibration was performed only on the first day after opening. Thereafter, quality control samples were measured every two days to analyze the measured values ​​of the quality control samples during the opening period. If the relative deviation between the measured value of the quality control sample and the measured value of the quality control sample on the first day was within ±5%, the opening stability was good. The method for measuring the quality control samples was the same as that in (1).

[0144] (3) Repeatability test method: The serum samples of experimental groups 11, 14 and 15 were tested 10 times. The mean and standard deviation (SD) of the measured values ​​were calculated respectively. The coefficient of variation (CV) was calculated according to formula (1). The obtained CV value should be ≤5%.

[0145]

[0146] (4) Linearity Experiment Method: Serum samples close to the upper limit of the linear range were diluted with distilled water to form 5 dilution concentrations. Experimental groups 11, 14, and 15 were tested 3 times for each dilution concentration, and the mean of the test results for each dilution concentration was calculated. The linear regression equation was obtained with the dilution concentration as the independent variable and the mean of the test results as the dependent variable.

[0147] Substitute the dilution concentration into the linear regression equation to calculate the estimated value, and calculate the relative or absolute deviation between the average value and the estimated value according to formula (2) or formula (3).

[0148]

[0149] Absolute deviation = Average value - Estimated value (3)

[0150] Linearity requirements:

[0151] Linear range: 0-1000 U / L;

[0152] Correlation coefficient |r| ≥ 0.9900;

[0153] When the concentration is below 40 U / L, the absolute deviation of linearity should not exceed ±5 U / L;

[0154] Within the range of 40-1000 U / L, the relative deviation of linearity should not exceed ±10%.

[0155] 2. Experimental Results and Analysis

[0156] (1) Results of the destructive stability test of the reagent kit

[0157] As shown in Table 10, the experimental results of Landau composite quality control 1 indicate that the deviation of the measured values ​​of experimental groups 12 and 13 was greater than 5% on day 5 of degradation at 37℃; the deviation of the measured values ​​of experimental group 16 was greater than 5% on day 3 of degradation at 37℃. For experimental groups 11, 14, and 15, the variation of the measured values ​​of the quality control during degradation was within ±5%. Table 11 also shows that the stability of experimental groups 12, 13, and 16 was slightly worse during accelerated degradation at 37℃. The measured values ​​in Tables 10 and 11 are creatine kinase content.

[0158] Table 10. Measured values ​​of Landau composite quality control sample 1 in experimental groups 11-16 during accelerated degradation at 37℃ (unit: U / L).

[0159] Undamaged Day 1 Deviation Day 3 Deviation Day 5 Deviation Day 7 Deviation Experimental Group 1 1 20 5 20 70.98% 2 10 2.44% 2 11 2.93% 2 13 3.90% Experimental Group 1 2 20 8 19 8 -4.81% 2 0 0 -3.85% 2 2 3 7.21% 2 3 0 10.58% Experimental Group 1 20 2 20 93.47% 2 11 4.46% 135.45% 229 13.37% Experimental Group 14 205 2060.49% 2123.41% 2133.90% 2133.90% Experimental Group 15 197 195 -1.02% 200 1.52% 201 2.03% 203 3.05% Experimental Group 16 199 190 -4.52% 187 -6.03% 207 4.02% 215 8.04% surface

[0160] Table 11. Measured values ​​of Landau composite quality control sample 2 in experimental groups 11-16 during accelerated degradation at 37℃ (unit: U / L).

[0161] Undamaged Day 1 Deviation Day 3 Deviation Day 5 Deviation Day 7 Deviation Experimental Group 1 15 23 521 -0.38% 525 0.38% 523 0.00% 529 1.15% Experimental Group 1 25 20 519 -0.19% 533 2.50% 536 3.08% 548 5.38% Experimental Group 1 35 18 523 0.97% 537 3.67% 5455.21% 5495.98% Experimental Group 145265321.14% 5310.95% 5290.57% 5361.90% Experimental Group 155275300.57% 5331.14% 5300.57% 5392.28% Experimental Group 165195240.96% 505-2.70% 5455.01% 5505.97% surface

[0162] Table 12 Repeatability Test Results (Unit: U / L)

[0163] Repeatability mean SDCV Experimental group 11 194 192 194 192 194 196 196 192 194 187 193.10 2.60 1.35% Experimental group 14 195 194 194 193 196 200 196 194 195 192 194.90 2.18 1.12% Experimental group 15 197 195 196 198 196 195 197 190 198 194 195.60 2.37 1.21% surface

[0164] Table 13-1 Linear Experiment Results of Experiment Group 11

[0165]

[0166]

[0167] Table 13-2 Linear Experiment Results of Experiment Group 14

[0168]

[0169] Table 13-3 Linear Experiment Results of Experiment Group 15

[0170]

[0171] The experimental results above show that there is little difference in the repeatability of measurements among experimental groups 11, 14, and 15. However, in terms of linearity, the absolute deviation of the zero concentration point in experimental group 15 is >±5%, which does not meet the requirements, so it is excluded.

[0172] (2) Results of stability test in Kaifeng

[0173] As can be seen from the experimental results in Tables 14 and 15, during the opening period, the relative deviations of the quality control measurements of experimental groups 11 and 14 were both within ±5%, but the relative deviation of experimental group 11 was smaller and its stability was better than that of experimental group 14.

[0174] Table 14. Measured values ​​of Langdao composite quality control sample 1 during the opening period of experimental groups 11 and 14 (unit: U / L).

[0175] Experimental Group 11 Deviation Experimental Group 14 Deviation Day 1 after opening: 202 / 205 / Day 5 after opening: 2030.50% 202-1.46% Day 10 after opening: 2040.99% 203-0.98% Day 15 after opening: 2051.49% 200-2.44% Day 20 after opening: 201-0.50% 2102.44% Day 30 after opening: 200-0.99% 2091.95% Day 40 after opening: 2051.49% 2144.39% surface

[0176] Table 15. Measured values ​​of Langdao composite quality control sample 2 during the opening period of experimental group 11 and 14 (unit: U / L).

[0177] Experimental Group 11 Deviation Experimental Group 14 Deviation Day 1 after opening: 523 / 526 / Day 5 after opening: 525 0.38% 530 0.76% Day 10 after opening: 525 0.38% 528 0.38% Day 15 after opening: 519 -0.76% 530 0.76% Day 20 after opening: 526 0.57% 533 1.33% Day 30 after opening: 529 1.15% 540 2.66% Day 40 after opening: 529 1.15% 549 4.37% surface

[0178] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A creatine kinase assay kit that counteracts lipemia interference, characterized in that, Reagent R1 and reagent R2; 2. The preparation method of the creatine kinase detection kit for lipemia interference as described in claim 1, characterized in that: (1) Preparation of reagent R1: Weigh an appropriate amount of pure water in a beaker, place it on a magnetic stirrer, and add the corresponding amounts of imidazole, MgAC, EDTA Na2, glucose, PC300, Na2SO3, and N-acetylcysteine ​​in sequence; after the raw materials are completely dissolved, adjust the pH of the solution to 6-7 at 20℃; then add NADP, AMP, ADP, AP5A, HK, and surfactant in sequence; after the raw materials are completely dissolved, make up the volume with pure water to prepare reagent R1; (2) Preparation of reagent R2: Weigh an appropriate amount of pure water in a beaker, place it on a magnetic stirrer, and add Capso, creatine phosphate, PEG6000, and NaN3 in sequence; after the raw materials are completely dissolved, adjust the pH of the solution to 9-10 at 20℃; then add BSA and G6PDH in sequence; after the raw materials are completely dissolved, make up the volume with pure water to prepare reagent R2.

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