CN115478096A - Creatine kinase detection kit - Google Patents

Abstract

The invention relates to the technical field of medical detection, in particular to a creatine kinase detection kit. According to the kit, 6-12g/L of sodium chloride is added into R1, and meanwhile, EDTA (ethylene diamine tetraacetic acid) is replaced by DTPA (diethylenetriamine pentaacetic acid), and experiments show that the kit can effectively eliminate the interference of heparin in a heparin plasma sample, so that the reaction curve of the heparin anticoagulated plasma sample is not abnormal, the deviation between the measured values of the heparin anticoagulated sample and a serum sample is reduced, and the detection result is more accurate.

Description

Creatine kinase detection kit

Technical Field

The invention relates to the technical field of medical detection, and particularly relates to a creatine kinase kit.

Background

Creatine Kinase (CK), also known as creatine phosphokinase, is present in the greatest amounts in skeletal muscle and cardiac muscle, followed by brain tissue with lesser amounts in the intestinal tract, kidneys and lungs. CK is mainly present in cytoplasm and mitochondria, can reversibly catalyze creatine and adenosine triphosphate to generate phosphocreatine and adenosine diphosphate, mainly generates ATP under the condition of neutral pH value to ensure the functions of tissue cells, and makes the ATP generated by oxidation in mitochondria enter cell sap in the form of phosphocreatine so as to meet the requirements of cell physiological activities.

CK is mainly used for diagnosing myocardial infarction clinically, CK begins to rise in 2-4h of morbidity when acute myocardial infarction occurs, reaches a peak value in 12-48h, is higher than Lactate Dehydrogenase (LDH), appears early and basically accords with the degree of myocardial damage, CK activity is also raised when endocardial myocardial infarction and recurrent myocardial infarction which are difficult to diagnose through electrocardiogram are caused, and dynamic monitoring is helpful for disease observation and prognosis estimation of myocardial infarction. CK has certain diagnostic significance for other diseases, CK activity is obviously increased when muscles are seriously injured, CK activity is increased when cerebrovascular accidents, meningitis, strenuous exercise and the like are caused, and CK activity can be reduced by hyperthyroidism, chronic arthritis, chemotherapy and the like.

At present, the method for detecting CK activity in clinical laboratories is mostly an IFCC recommendation method, and the method is a three-step reaction, wherein CK catalyzes creatine phosphate and ADP to generate ATP and creatine, ATP and glucose generate glucose-6-phosphate and ADP under the catalysis of Hexokinase (HK), glucose-6-phosphate and oxidized nicotinamide adenine dinucleotide (NADP +) generate 6-phosphogluconate and reduced Nicotinamide Adenine Dinucleotide (NADPH) under the catalysis of glucose-6-phosphate dehydrogenase (G6 PDH), the reaction absorbance of NADPH is detected at 340nm, the change rate of the reaction absorbance is positively correlated with the CK activity, and the reaction equation is as follows:

the CK determination kit of the IFCC recommendation method has the following defects: when testing heparin anticoagulated plasma samples, plasma measurements are generally lower than serum measurements, and the heparin plasma sample response curves are abnormal. Therefore, the problem to be solved is to provide a CK kit with good anti-heparin performance.

Disclosure of Invention

In view of this, the invention provides a creatine kinase kit. The kit can effectively eliminate the interference of heparin in a heparin plasma sample, so that the reaction curve of the heparin anticoagulated plasma sample is not abnormal, the deviation of the measured value of the heparin anticoagulated sample and the measured value of the serum sample is reduced, and the detection result is more accurate and stable.

In order to achieve the above object, the present invention provides the following technical solutions:

a creatine kinase detection kit, which consists of a reagent R1 and a reagent R2;

the reagent R1 comprises the following components in concentration:

90-120mmol/L buffer solution, 4-6mmol/L glucose, 15-17mmol/L magnesium acetate, 29-32mmol/L N-acetylcysteine, 2-3mmol/L ADP, 4.5-6.5mmol/L AMP, G6PDH not less than 3KU/L, NADP ⁺ 2-4mmol/L, HK is more than or equal to 5KU/L, sodium sulfite is 1.5-2.5mmol/L, DTPA is 1.5-2.5mmol/L, preservative is 0.5-2g/L, and inorganic salt is 6-12g/L;

the reagent R2 comprises the following components in concentrations:

15-30mmol/L of buffer solution, 45-60mmol/L of glucose, 220-245mmol/L of creatine phosphate and 1-3g/L of preservative.

Compared with the prior art, the method has the advantages that 6-12g/L of sodium chloride is added into R1, and the DTPA is used for replacing EDTA, so that the interference of heparin in the heparin plasma sample is effectively eliminated, the reaction curve of the heparin anticoagulated plasma sample is not abnormal, the deviation between the measured values of the heparin anticoagulated sample and the serum sample is reduced, and the accuracy and the stability of detection are obviously improved.

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

In the invention, in the R1 reagent, the buffer solution in the R1 reagent is at least one of imidazole buffer solution, MES buffer solution and Bis-Tris buffer solution. In some embodiments, the buffer is an imidazole buffer, and the pH is 6.5.

In the invention, in the R2 reagent, the buffer solution is at least one of CAPSO, TRIS buffer solution and glycine buffer solution. In some embodiments, the buffer is CAPSO at a pH of 9.0.

In the present invention, the kind of the preservative in R1 and R2 is not particularly limited, and includes, but is not limited to, at least one of sodium azide and Proclin 300.

In some embodiments of the present invention, in the creatine kinase detection kit, the R1 includes the following components at the following concentrations:

imidazole buffer 115.7mmol/L, glucose 5mmol/L, magnesium acetate 16.2mmol/L, N-acetylcysteine 30.6mmol/L, ADP2.5mmol/L, AMP5.2mmol/L, G6PDH5KU/L, NADP ⁺ 3.3mmol/L, HK10KU/L, sodium sulfite 2mmol/L, DTPA2mmol/L, preservative 1g/L, sodium chloride 6 g/L-12 g/L.

The R2 comprises the following components in concentration:

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

The invention also provides a method for detecting creatine kinase, which is used for detecting a serum sample to be detected or a heparin anticoagulant plasma sample to be detected by using the creatine kinase detection kit.

In the creatine kinase detection kit provided by the invention, 6-12g/L of sodium chloride is added into R1, and meanwhile, DTPA is used for replacing EDTA (ethylene diamine tetraacetic acid), and experiments show that the kit can effectively eliminate the interference of heparin in a heparin plasma sample, so that the reaction curve of the heparin anticoagulated plasma sample is not abnormal, the deviation between the measured values of the heparin anticoagulated sample and a serum sample is reduced, and the detection result is more accurate and stable.

Drawings

FIG. 1 shows the response curve of heparin plasma sample 1;

figure 2 shows the response curve of heparin plasma sample 2.

Detailed Description

The invention provides a creatine kinase detection kit. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1

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

reagent 1 (R1) composition:

reagent 2 (R2) composition:

example 2

In this example, R1 contains sodium chloride (12 g/L), and the chelating agent in R1 is DTPA, and the reagent comprises the following components:

reagent 1 (R1) composition:

reagent 2 (R2) composition:

comparative example 1

In this embodiment, R1 does not contain sodium chloride, the chelating agent in R1 is EDTA, and the reagent comprises the following components:

reagent 1 (R1) composition:

reagent 2 (R2) composition:

comparative example 2

In this example, R1 does not contain sodium chloride, the chelating agent in R1 is DTPA, and the reagent comprises the following components:

reagent 1 (R1) composition:

reagent 2 (R2) composition:

comparative example 3

In this example, R1 contains sodium chloride (6 g/L), R1 contains chelating agent EDTA, and the reagent comprises the following components:

reagent 1 (R1) composition:

reagent 2 (R2) composition:

test example

1. Specific operating method

1.1 five reagents were prepared according to the formulations of examples 1-2 and comparative examples 1-3;

1.2 extracting fasting serum and heparin anticoagulation plasma of 41 healthy people for detection;

1.3 detection instrument: canon TBA-120 of full-automatic biochemical analyzer

1.4 detection parameters:

TABLE 1

1.5CK Activity (U/L) = (Δ A) _T /ΔA _S ) Calibrator activity;

1.6 analyzing the deviation of measured values of five reagent blood plasma and serum tubes and reaction curves.

Plasma to serum tube measurement deviation = (plasma value-serum value)/serum value × 100%

2 results

2.1 reaction curves for heparin plasma samples

The results are shown in FIGS. 1 and 2.

The R1 section only comprises a sample and R1, no reaction is required, and the reaction curve is a straight line. As can be seen from the reaction curves, the reaction curve of the R1 section of comparative example 1 is ascending, and the nonspecific reaction occurs due to heparin interference. The reaction curve of comparative example 2, in which EDTA in R1 was replaced by DTPA, was slightly improved in the section R1. The reaction curve of the section R1 of the comparative example 3 is improved more obviously by adding sodium chloride into the section R1. When 6g/L of sodium chloride is added to R1 and EDTA is replaced by DTPA, the reaction curve of the R1 section of the example 1 is not abnormal. The phenomenon of example 2 is consistent with that of example 1, and the results show that 6g/L sodium chloride and 12g/L sodium chloride can be added into R1. As can be seen from comparison of reaction curves, the phenomenon of abnormal reaction curve of the R1 section can be improved by adding sodium chloride into the R1 or replacing EDTA with DTPA, and the effect of the sodium chloride is better than that of the DTPA. The addition of sodium chloride to R1 and the replacement of EDTA with DTPA made the response curve no longer abnormal in the R1 region. In the embodiment, only two heparin plasma sample reaction curves are listed, and through research, other heparin plasma samples are consistent with the detection result, and the reaction curve of the R1 section is not abnormal any more.

2.2 serum and plasma values are given in tables 2 and 3 below:

table 2 serum and plasma measurements of comparative examples 1, 2 and 3

TABLE 3 serum and plasma values for examples 1-2

Note: the same serial number serum sample and heparin plasma sample are the same human sample.

As can be seen from the mean deviation of the plasma and serum measurements in tables 2 and 3, the mean deviation is smaller in comparative example 2 compared to comparative example 1, which shows that the heparin interference can be improved by replacing EDTA in R1 with DTPA. Comparative example 3 shows that the average deviation is smaller than that of comparative example 1, and that the heparin interference can be improved by adding sodium chloride to R1. Example 1 mean deviation of plasma and serum measurements was significantly smaller than comparative example 1, indicating that addition of sodium chloride to R1 and replacement of EDTA with DTPA is effective in improving heparin interference. The results of the embodiment 1 and the embodiment 2 are not very different, which shows that the concentration of sodium chloride in R1 is 6g/L and 12g/L, which can obviously improve the heparin interference.

2.3 the invention can effectively improve heparin interference by adding (6-12) g/L sodium chloride in R1 and replacing EDTA in R1 with DTPA, so that the reaction curve of the R1 section is not abnormal any more, and the deviation of the measured value of heparin plasma and serum is reduced.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims (9)

1. The creatine kinase detection kit is characterized by consisting of a reagent R1 and a reagent R2;

the reagent R1 comprises the following components in concentrations:

90-120mmol/L buffer solution, 4-6mmol/L glucose, 15-17mmol/L magnesium acetate, 29-32mmol/L N-acetylcysteine, 2-3mmol/L ADP, 4.5-6.5mmol/L AMP, G6PDH not less than 3KU/L, NADP ⁺ 2-4mmol/LHK is more than or equal to 5KU/L, sodium sulfite is 1.5-2.5mmol/L, DTPA is 1.5-2.5mmol/L, preservative is 0.5-2g/L, and inorganic salt is 6-12g/L;

the reagent R2 comprises the following components in concentrations:

15-30mmol/L of buffer solution, 45-60mmol/L of glucose, 220-245mmol/L of creatine phosphate and 1-3g/L of preservative.

2. The creatine kinase detection kit of claim 1, wherein the inorganic salt is at least one selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, calcium chloride, and zinc chloride.

3. The creatine kinase detection kit of claim 1, wherein in the R1 reagent, the buffer is at least one of imidazole buffer, MES buffer, bis-Tris buffer.

4. The creatine kinase detection kit of claim 3, wherein the buffer solution in the R1 reagent is imidazole buffer solution and the pH is 6.5.

5. The creatine kinase detection kit of claim 1, wherein in the R2 reagent, the buffer solution is at least one of CAPSO, TRIS buffer solution, and glycine buffer solution.

6. The creatine kinase assay kit of claim 5, wherein the buffer solution in the R2 reagent is CAPSO and the pH is 9.0.

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

8. The creatine kinase detection kit according to claim 1, wherein R1 comprises the following components in concentration:

imidazole buffer 115.7mmol/L, grape of pH6.5Sugar 5mmol/L, magnesium acetate 16.2mmol/L, N-acetylcysteine 30.6mmol/L, ADP2.5mmol/L, AMP5.2mmol/L, G6PDH5KU/L, NADP ⁺ 3.3mmol/L, HK10KU/L, sodium sulfite 2mmol/L, DTPA2mmol/L, preservative 1g/L, and sodium chloride 6-12 g/L.

9. The creatine kinase detection kit of claim 1, wherein R2 comprises the following components in the concentrations:

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

Images