A creatine kinase detection kit

Abstract

The present invention relates to the field of medical testing technology, and in particular to a creatine kinase detection kit. The kit of the present invention adds 6-12 g / L of sodium chloride to R1 and uses DTPA instead of EDTA. Experiments have shown that the kit can effectively eliminate the interference of heparin in heparinized plasma samples, eliminating abnormal reaction curves of heparin anticoagulated plasma samples, reducing the deviation between the measured values of heparin anticoagulated samples and serum samples, and achieving more accurate test results.

Description

Technical Field

[0001] The present invention relates to the technical field of medical detection, in particular to a creatine kinase kit. Background Art

[0002] Creatine kinase (CK), also known as creatine phosphokinase, is found most abundantly in skeletal and cardiac muscle, followed by the brain, with lower concentrations in the intestines, kidneys, and lungs. Found primarily in the cytoplasm and mitochondria, CK reversibly catalyzes the conversion of creatine and adenosine triphosphate to creatine phosphate and adenosine diphosphate. Under neutral pH conditions, CK primarily generates ATP to ensure tissue and cellular function. The forward reaction facilitates the oxidation of ATP within the mitochondria, which then enters the cell fluid as creatine phosphate, meeting the needs of cellular physiological activity.

[0003] Clinically, CK is primarily used to diagnose myocardial infarction. In acute myocardial infarction, CK begins to rise 2-4 hours after onset and reaches a peak 12-48 hours after onset. This elevation is greater than that of lactate dehydrogenase (LDH) and occurs earlier, generally consistent with the degree of myocardial damage. CK activity is also elevated in subendocardial myocardial infarction and recurrent myocardial infarction, which are difficult to diagnose on an electrocardiogram. Dynamic monitoring aids in the observation of the condition and prognosis of myocardial infarction. CK also has diagnostic significance for other diseases. CK activity is significantly elevated in severe muscle injury, cerebrovascular accidents, meningitis, and strenuous exercise. Hyperthyroidism, chronic arthritis, and chemotherapy can reduce CK activity.

[0004] Currently, most clinical laboratories test CK activity using the IFCC-recommended method. This method is a three-step reaction: CK catalyzes the reaction of creatine phosphate and ADP to produce ATP and creatine. ATP and glucose, catalyzed by hexokinase (HK), produce glucose-6-phosphate and ADP. Glucose-6-phosphate and oxidized nicotinamide adenine dinucleotide (NADP+) are catalyzed by glucose-6-phosphate dehydrogenase (G6PDH) to produce 6-phosphogluconic acid and reduced nicotinamide adenine dinucleotide (NADPH). The absorbance of NADPH is measured at 340 nm, and its rate of change is positively correlated with CK activity. The reaction equation is as follows:

[0005]

[0006] The CK assay kit recommended by the IFCC has the following drawbacks: when testing heparin-anticoagulated plasma samples, plasma values are generally lower than serum values, and the heparinized plasma sample reaction curve is abnormal. Therefore, providing a CK assay kit with improved heparin resistance is an urgent need. Summary of the Invention

[0007] In view of this, the present invention provides a creatine kinase test kit. The kit can effectively eliminate the interference of heparin in heparinized plasma samples, eliminate abnormal reaction curves of heparinized anticoagulated plasma samples, reduce the deviation between the measured values of heparinized anticoagulated samples and serum samples, and make the test results more accurate and stable.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] A creatine kinase detection kit, consisting of reagent R1 and reagent R2;

[0010] The reagent R1 includes the following components at the following concentrations:

[0011] Buffer 90-120mmol / L, glucose 4-6mmol / L, magnesium acetate 15-17mmol / L, N-acetylcysteine 29-32mmol / L, ADP 2-3mmol / L, AMP 4.5-6.5mmol / L, G6PDH ≥ 3KU / L, NADP + 2-4mmol / L, HK≥5KU / L, sodium sulfite 1.5-2.5mmol / L, DTPA 1.5-2.5mmol / L, preservative 0.5-2g / L, inorganic salt 6-12g / L;

[0012] The reagent R2 includes the following components at the following concentrations:

[0013] Buffer 15-30mmol / L, glucose 45-60mmol / L, creatine phosphate 220-245mmol / L, preservative 1-3g / L.

[0014] Compared with the prior art, the present invention effectively eliminates the interference of heparin in heparinized plasma samples by adding 6-12 g / L of sodium chloride to R1 and using DTPA instead of EDTA, so that the reaction curve of the heparin anticoagulated plasma sample is no longer abnormal, the deviation of the measured values of the heparin anticoagulated sample and the serum sample is reduced, and the accuracy and stability of the detection are significantly improved.

[0015] In the present invention, the inorganic salt is selected from at least one of sodium chloride, potassium chloride, magnesium chloride, calcium chloride and zinc chloride.

[0016] In the present invention, in the R1 reagent, the buffer is at least one of imidazole buffer, MES buffer, and Bis-Tris buffer. In some specific embodiments, the buffer is imidazole buffer with a pH of 6.5.

[0017] In the present invention, the buffer in the R2 reagent is at least one of CAPSO, TRIS buffer, and glycine buffer. In some specific embodiments, the buffer is CAPSO with a pH of 9.0.

[0018] In the present invention, the types of preservatives in R1 and R2 are not particularly limited, including but not limited to at least one of sodium azide and Proclin 300.

[0019] In some specific embodiments of the present invention, in the creatine kinase detection kit, the R1 comprises components with the following concentrations:

[0020] Imidazole buffer at pH 6.5: 115.7 mmol / L, glucose: 5 mmol / L, magnesium acetate: 16.2 mmol / L, N-acetylcysteine: 30.6 mmol / L, ADP: 2.5 mmol / L, AMP: 5.2 mmol / L, G6PDH: 5 KU / L, NADP + 3.3mmol / L, HK10KU / L, sodium sulfite 2mmol / L, DTPA2mmol / L, preservative 1g / L, sodium chloride 6g / L~12g / L.

[0021] The R2 comprises the following components at the following concentrations:

[0022] CAPSO buffer 20 mmol / L, pH = 9.0, glucose 50.5 mmol / L, creatine phosphate 236.8 mmol / L, sodium azide 2 g / L.

[0023] The present invention also provides a method for detecting creatine kinase, which uses the creatine kinase detection kit of the present invention to detect a serum sample to be tested or a heparin anticoagulated plasma sample to be tested.

[0024] In the creatine kinase detection kit provided by the present invention, 6-12 g / L of sodium chloride is added to R1, and DTPA is used instead of EDTA. Experiments have shown that the kit can effectively eliminate the interference of heparin in heparin plasma samples, so that the reaction curve of heparin anticoagulated plasma samples is no longer abnormal, the deviation of the measured values of heparin anticoagulated samples and serum samples is reduced, and the test results are more accurate and stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Shown is the response curve of heparin plasma sample 1;

[0026] Figure 2 The response curve of heparin plasma sample 2 is shown. DETAILED DESCRIPTION

[0027] The present invention provides a creatine kinase detection kit. Those skilled in the art can refer to the contents herein and appropriately modify the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications will be apparent to those skilled in the art and are considered to be encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments, and it is apparent that those skilled in the art can modify or appropriately alter and combine the methods and applications herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0028] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0029] The present invention will be further described below in conjunction with the embodiments:

[0030] Example 1

[0031] In this embodiment, R1 contains sodium chloride (6 g / L), the chelating agent in R1 is DTPA, and the reagent composition is as follows:

[0032] Reagent 1 (R1) ingredients:

[0033]

[0034] Reagent 2 (R2) components:

[0035]

[0036] Example 2

[0037] In this embodiment, R1 contains sodium chloride (12 g / L), the chelating agent in R1 is DTPA, and the reagent composition is as follows:

[0038] Reagent 1 (R1) ingredients:

[0039]

[0040]

[0041] Reagent 2 (R2) components:

[0042]

[0043] Comparative Example 1

[0044] In this embodiment, R1 does not contain sodium chloride, the chelating agent in R1 is EDTA, and the reagent components are as follows:

[0045] Reagent 1 (R1) ingredients:

[0046]

[0047] Reagent 2 (R2) components:

[0048]

[0049] Comparative Example 2

[0050] In this embodiment, R1 does not contain sodium chloride, the chelating agent in R1 is DTPA, and the reagent composition is as follows:

[0051] Reagent 1 (R1) ingredients:

[0052]

[0053]

[0054] Reagent 2 (R2) components:

[0055]

[0056] Comparative Example 3

[0057] In this embodiment, R1 contains sodium chloride (6 g / L), the chelating agent in R1 is EDTA, and the reagent composition is as follows:

[0058] Reagent 1 (R1) ingredients:

[0059]

[0060] Reagent 2 (R2) components:

[0061]

[0062] Test Case

[0063] 1. Specific operation methods

[0064] 1.1 Prepare five reagents according to the formulas of Examples 1 to 2 and Comparative Examples 1 to 3;

[0065] 1.2 Fasting serum and heparinized plasma were collected from 41 healthy subjects for testing;

[0066] 1.3 Testing instrument: Fully automatic biochemical analyzer Canon TBA-120

[0067] 1.4 Detection parameters:

[0068] Table 1

[0069]

[0070] 1.5CK activity (U / L) = (ΔA T / ΔA S )*calibrator activity;

[0071] 1.6 Analyze the deviations of the measured values and the reaction curves of the five reagent plasma and serum tubes.

[0072] Deviation between plasma and serum tube values = (plasma value - serum value) / serum value × 100%

[0073] 2 Results

[0074] 2.1 Response curve of heparin plasma samples

[0075] The results are as follows Figure 1 and 2 .

[0076] The R1 segment only contains the sample and R1, and there should be no reaction, and the reaction curve should be a straight line. It can be seen from the reaction curve that the reaction curve of the R1 segment of Example 1 shows an upward trend, and heparin interference causes a non-specific reaction. Replacing the EDTA in R1 with DTPA slightly improves the reaction curve of the R1 segment of Example 2. Adding sodium chloride to R1 improves the reaction curve of the R1 segment of Example 3 more significantly. Adding 6g / L sodium chloride to R1 and replacing EDTA with DTPA at the same time, the reaction curve of the R1 segment of Example 1 is no longer abnormal. Example 2 is consistent with the phenomenon of Example 1, indicating that adding 6g / L sodium chloride and 12g / L sodium chloride to R1 can improve the abnormal phenomenon of the R1 segment reaction curve, and sodium chloride is better than DTPA. Adding sodium chloride to R1 and replacing EDTA with DTPA can make the R1 segment reaction curve no longer abnormal. In this embodiment, only two heparin plasma sample reaction curves are listed. After research, the test results of other heparin plasma samples are consistent with the test results, and the R1 segment reaction curves are no longer abnormal.

[0077] 2.2 Serum and plasma values are shown in Tables 2 and 3 below:

[0078] Table 2 Serum and plasma values of comparative examples 1, 2 and 3

[0079]

[0080]

[0081]

[0082] Table 3 Serum and plasma values measured in Examples 1 to 2

[0083]

[0084]

[0085] Note: Serum samples and heparin plasma samples with the same serial number are samples from the same person.

[0086] It can be seen from the average deviations of the plasma and serum measurements in Tables 2 and 3 that the average deviation of Comparative Example 2 is smaller than that of Comparative Example 1, indicating that replacing EDTA in R1 with DTPA can improve heparin interference. The average deviation of Comparative Example 3 is smaller than that of Comparative Example 1, indicating that adding sodium chloride in R1 can also improve heparin interference. Compared with Comparative Example 1, the average deviation of the plasma and serum measurements in Example 1 is significantly smaller, indicating that adding sodium chloride in R1 and replacing EDTA with DTPA can effectively improve heparin interference. The results of Example 1 and Example 2 are not much different, indicating that both 6g / L and 12g / L sodium chloride concentrations in R1 can significantly improve heparin interference.

[0087] 2.3 The present invention can effectively improve heparin interference by adding (6-12) g / L sodium chloride to R1 and replacing EDTA in R1 with DTPA, so that the R1 segment reaction curve is no longer abnormal and the deviation between heparin plasma and serum measurements is reduced.

[0088] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A creatine kinase detection kit, characterized in that, It consists of reagent R1 and reagent R2; The reagent R1 consists of the following components: Buffer 90-120 mmol / L, glucose 4-6 mmol / L, magnesium acetate 15-17 mmol / L, N-acetylcysteine 29-32 mmol / L, ADP 2-3 mmol / L, AMP 4.5-6.5 mmol / L, G6PDH ≥3 KU / L, NADP + 2-4 mmol / L, HK ≥5 KU / L, sodium sulfite 1.5-2.5 mmol / L, DTPA 1.5-2.5 mmol / L, preservative 0.5-2g / L, sodium chloride 6-12 g / L; The reagent R2 consists of the following components: Buffer 15-30 mmol / L, glucose 45-60 mmol / L, creatine phosphate 220-245 mmol / L, preservative 1-3g / L.

2. The creatine kinase detection kit according to claim 1, wherein In the R1 reagent, the buffer is at least one of imidazole buffer, MES buffer, and Bis-Tris buffer.

3. The creatine kinase detection kit according to claim 2, wherein In the R1 reagent, the buffer is imidazole buffer with a pH of 6.

5.

4. The creatine kinase detection kit according to claim 1, wherein In the R2 reagent, the buffer is at least one of CAPSO, TRIS buffer, and glycine buffer.

5. The creatine kinase detection kit according to claim 4, characterized in that In the R2 reagent, the buffer solution is CAPSO and the pH value is 9.

0.

6. The creatine kinase detection kit according to claim 1, characterized in that The preservative in R1 and R2 is at least one of sodium azide and Proclin 300.

7. The creatine kinase detection kit according to claim 1, characterized in that The R1 consists of the following components at the following concentrations: Imidazole buffer pH 6.5 115.7 mmol / L, glucose 5 mmol / L, magnesium acetate 16.2 mmol / L, N-acetylcysteine 30.6 mmol / L, ADP 2.5 mmol / L, AMP 5.2 mmol / L, G6PDH 5 KU / L, NADP + 3.3 mmol / L, HK 10 KU / L, sodium sulfite 2 mmol / L, DTPA 2mmol / L, preservative 1g / L, sodium chloride 6~12g / L.

8. The creatine kinase detection kit according to claim 1, characterized in that The R2 consists of the following components at the following concentrations: CAPSO buffer 20 mmol / L, pH 9.0, glucose 50.5 mmol / L, creatine phosphate 236.8 mmol / L, sodium azide 2 g / L.

Images