Small and dense low-density lipoprotein cholesterol detection kit

Abstract

The invention provides a small and dense low-density lipoprotein cholesterol detection kit, which comprises a reagent 1 and a reagent 2, wherein the reagent 1 comprises the following main components: a 2-(N-morpholino) ethanesulfonic acid buffer solution (pH 6.3), cholesterol oxidase, cholesterol esterase, catalase, phospholipase, N-ethyl-N-(2-hydroxy-3-benzenesulfonic acid)-3-methylaniline sodium salt, a surfactant (ethanol polyether-4-carboxylic acid or naphthalene sulfonic acid formaldehyde polymer) and a preservative (HOPO); and the reagent 2 comprises the following main components: a 2-(N-morpholino) ethanesulfonic acid buffer solution (pH 6.3), peroxidase, 4-aminoantipyrine, Triton X-100 and a preservative (HOPO). The problem of low reaction degree of the reagent is solved by adjusting the surfactant; the accuracy and the stability of the reagent are further improved by screening preservatives having no inhibition effect on enzyme activity.

Description

Technical Field

[0001] The invention belongs to the technical field of medical detection, and in particular relates to a small and dense low-density lipoprotein cholesterol detection kit. Background Art

[0002] Cholesterol is an important substance that is indispensable to the human body. It participates in the formation of plasma membranes and is the raw material for the synthesis of bile acids, vitamin D and steroid hormones. Cholesterol and other lipids combine with apoproteins in serum to form lipoproteins. According to the differences in their physical properties, they can be divided into chylomicrons (CM), very low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL). Low-density lipoproteins (LDL) are heterogeneous. Among them, LDL with smaller particles and higher density is called small dense Low-Density Lipoprotein (sdLDL), LDL with larger particles and lower density is called large light low-density lipoprotein, and the subcomponent between the two is called intermediate-density low-density lipoprotein. In contrast, small and dense low-density lipoprotein (sdLDL) has a stronger ability to cause atherosclerosis and is more likely to cause cardiovascular diseases.

[0003] The detection methods of small and dense low-density lipoprotein cholesterol include density gradient ultracentrifugation, gradient gel electrophoresis, high-performance liquid chromatography and enzymatic method. Among them, the enzymatic method has a short analysis time and high accuracy, and is widely used in clinical practice. Other methods are cumbersome to operate and cannot meet the needs of clinical batch testing.

[0004] The enzymatic detection of small and dense low-density lipoprotein cholesterol is based on a protective agent that inhibits the reaction of small and dense low-density lipoprotein cholesterol with cholesterol esterase and cholesterol oxidase in the reagent components. Due to different inhibitory effects, the accuracy of the test results is greatly affected. The types and ratios of protective agents will affect the test results and the stability of the reagent. The following problems exist in the traditional commercially available enzymatic small and dense low-density lipoprotein cholesterol detection kits. First, the protective agent ratio is complex and the effect is poor. Some small and dense low-density lipoprotein cholesterol has reacted with cholesterol esterase and cholesterol oxidase in the reagent components, thereby reducing the reactivity of the entire reagent, resulting in low accuracy of the final test results. Second, the commonly used preservative in the kit is Proclin-300. Because it is reducing, it has a certain inhibitory effect on the activity of the enzyme in the reagent component, resulting in low accuracy of the final test result and poor stability of the reagent. In clinical testing, a small and dense low-density lipoprotein cholesterol detection kit with a more reasonable protective agent component, more accurate test results, and higher stability is required. Summary of the invention

[0005] The present invention aims at the deficiencies of the prior art and provides a small and dense low-density lipoprotein cholesterol detection kit, which has more reasonable protective agent components, more accurate detection results, higher stability, and is more suitable for clinical auxiliary diagnosis of arteriosclerosis.

[0006] The technical solution adopted by the present invention is: A small and dense low-density lipoprotein cholesterol detection kit, the kit comprises reagent 1 and reagent 2, wherein the main components of reagent 1 are: 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.3), cholesterol oxidase (COD), cholesterol esterase (CHE), catalase (CAT), phospholipase, N-ethyl-N-(2-hydroxy-3-benzenesulfonic acid)-3-methylaniline sodium salt (TOOS), surfactant, and preservative; the main components of reagent 2 are: 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.3), peroxidase (POD), 4-aminoantipyrine (4-AAP), Triton X-100, and preservative.

[0007] Preferably, in reagent 1, the concentration of 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.3) is 50-500 mmol / L, the concentration of cholesterol oxidase (COD) is 0.4-2.0 KU / L, the concentration of cholesterol esterase (CHE) is 2.5-5.0 KU / L, the concentration of catalase (CAT) is 1.0-2.0 KU / L, the concentration of phospholipase is 1.5-3.0 KU / L, the concentration of N-ethyl-N-(2-hydroxy-3-benzenesulfonic acid)-3-methylaniline sodium salt (TOOS) is 1.0-2.0 g / L, the concentration of surfactant is 0.5-1.0 g / L, and the concentration of preservative is 0.2-0.4‰; in reagent 2, the concentration of 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.3) is 50-500 mmol / L, the concentration of peroxidase (POD) is 4.0-5.0 KU / L, 4-aminoantipyrine (4-AAP) concentration was 2.0-4.0 mmol / L, TritonX-100 concentration was 0.5-1.0 g / L, and preservative concentration was 0.2-0.4‰.

[0008] Preferably, in the reagent 1, the concentration of 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.3) is 100 mmol / L, the concentration of cholesterol oxidase (COD) is 1.0 KU / L, the concentration of cholesterol esterase (CHE) is 5.0 KU / L, the concentration of catalase (CAT) is 1.2 KU / L, the concentration of phospholipase is 2.5 KU / L, the concentration of N-ethyl-N-(2-hydroxy-3-benzenesulfonic acid)-3-methylaniline sodium salt (TOOS) is 1.2 g / L, the concentration of surfactant is 1.0 g / L, and the concentration of preservative is 0.2‰; the main components of reagent 2 are: the concentration of 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.3) is 100 mmol / L, the concentration of peroxidase (POD) is 5.0 KU / L, the concentration of 4-aminoantipyrine (4-AAP) is 4.0 mmol / L, and the concentration of Triton X-100 is 1.0 g / L, the preservative concentration is 0.2‰.

[0009] Preferably, the surfactants in the reagent 1 are ethanol polyether-4-carboxylic acid and naphthalenesulfonic acid formaldehyde polymer, and their concentrations are 0.5 g / L respectively.

[0010] Preferably, the preservative in the reagent 1 and the reagent 2 is 2-hydroxypyridine-N-oxide (HOPO).

[0011] Preferably, the kit can be used to detect the content of small dense low-density lipoprotein cholesterol in human serum, and the ratio of reagent to sample is: reagent 1: reagent 2: sample = 300: 100: 3.

[0012] The present invention has the following beneficial effects: 1. By adding surfactants ethanol polyether-4-carboxylic acid and naphthalenesulfonic acid formaldehyde polymer to reagent 1, the false negative caused by the reaction of small and dense low-density lipoprotein cholesterol with cholesterol esterase and cholesterol oxidase in the reagent components is inhibited, the problem of low reactivity is solved, and the accuracy of the reagent is improved.

[0013] 2. The preservative in reagents 1 and 2 is 2-hydroxypyridine-N-oxide (HOPO). Compared with the conventional preservative Proclin-300, HOPO has no reducing property and no inhibitory effect on enzyme activity, and has a better preservative effect, which can improve the accuracy and stability of the reagents and is beneficial to the storage of the reagents. DETAILED DESCRIPTION

[0014] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

[0015] Example 1 The present invention provides a small and dense low-density lipoprotein cholesterol detection kit, comprising reagent 1 and reagent 2, the main components of which are shown in Table 1: Table 1 Reagent components of Example 1

[0016] According to different surfactants, they were divided into control group and experimental group. The components and concentrations are shown in Table 2: Table 2 Surfactant control group Group Surfactants concentration Comparison 1 Ethanol polyether-4-carboxylic acid 1.0 g / L Comparison 2 Naphthalenesulfonic acid formaldehyde polymer 1.0 g / L Experimental Group Ethanol polyether-4-carboxylic acid naphthalenesulfonic acid formaldehyde polymer 0.5 g / L0.5 g / L Example

[0017] The present invention provides a small and dense low-density lipoprotein cholesterol detection kit, comprising reagent 1 and reagent 2, the main components of which are shown in Table 3: Table 3 Reagent components of Example 2

[0018] A control group and a test group were set up according to the different preservatives. The preservative in the control group was 0.2‰ Proclin-300.

[0019] Detection method: The fully automatic biochemical analyzer Hitachi 7180 was used for detection. The detection wavelength was: main wavelength 546nm, sub-wavelength 700nm. The accuracy and stability of the reagents were tested. The specific operation is shown in Table 4: Table 4 Operation steps

[0020] Test results: 1. Accuracy The low-density lipoprotein cholesterol samples with two levels of concentration, 3.39 and 5.24 mmol / L, were measured and repeated three times. The average value of the test results was recorded as (M), the target value of the reference substance was recorded as (T), and the relative deviation was recorded as (B), B=(MT) / T×100%. The accuracy of Example 1 was tested, and the test results are shown in Table 1.

[0021] Table 5. Accuracy test results By comparing the test results of different groups, it can be seen that when ethanol polyether-4-carboxylic acid and naphthalenesulfonic acid formaldehyde polymer are prepared separately, the relative deviation value of the low-density lipoprotein cholesterol detection reagent is greater than ±10%, which does not meet the technical requirements; when ethanol polyether-4-carboxylic acid and naphthalenesulfonic acid formaldehyde polymer are added at the same time, the low-density lipoprotein cholesterol detection value is close to the target value of the quality control product, and the relative deviation value is within ±10%, which meets the technical requirements.

[0022] 2. Stability The kit described in Example 2 was placed in front of a 37°C constant temperature drying oven, and the two levels of low-density lipoprotein cholesterol samples were measured repeatedly 10 times (the concentrations were 3.39 and 5.24 mmol / L, respectively). The average value of the test results was used as the initial value (0th day), and then placed in a 37°C constant temperature drying oven for an accelerated test. The test was performed once each after 1d, 3d, 5d, 7d, and 9d of acceleration. Each sample was measured repeatedly 10 times and the average value was taken. The data were recorded. The test results are shown in Table 6.

[0023] Table 6. Stability test results

[0024] By comparing the measurement results of the above different groups, it can be seen that after adding the preservative Proclin-300, the measured value of the quality control product decreases, and in the acceleration process, the measured value gradually decreases. After acceleration for ≥3d, the relative deviation is greater than ±10%, which does not meet the technical requirements; while the preservative 2-hydroxypyridine-N-oxide (HOPO) has no effect on the measured value, and in the acceleration process, the relative deviation is less than ±10%, which meets the requirements for the stability of the reagent shelf life.

[0025] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A small and dense low-density lipoprotein cholesterol detection kit, characterized in that: The kit includes reagent 1 and reagent 2, wherein the main components of reagent 1 are: 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.3), cholesterol oxidase (COD), cholesterol esterase (CHE), catalase (CAT), phospholipase, N-ethyl-N-(2-hydroxy-3-benzenesulfonic acid)-3-methylaniline sodium salt (TOOS), surfactant, and preservative; the main components of reagent 2 are: 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.3), peroxidase (POD), 4-aminoantipyrine (4-AAP), Triton X-100, and preservative.

2. A small and dense low-density lipoprotein cholesterol detection kit as claimed in claim 1, characterized in that: In reagent 1, the concentration of 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.3) is 50-500 mmol / L, the concentration of cholesterol oxidase (COD) is 0.4-2.0 KU / L, the concentration of cholesterol esterase (CHE) is 2.5-5.0 KU / L, the concentration of catalase (CAT) is 1.0-2.0 KU / L, the concentration of phospholipase is 1.5-3.0 KU / L, the concentration of N-ethyl-N-(2-hydroxy-3-benzenesulfonic acid)-3-methylaniline sodium salt (TOOS) is 1.0-2.0 g / L, the concentration of surfactant is 0.5-1.0 g / L, and the concentration of preservative is 0.2-0.4‰; the concentration of 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.3) in reagent 2 is 50-500 mmol / L, peroxidase (POD) concentration is 4.0-5.0KU / L, 4-aminoantipyrine (4-AAP) concentration is 2.0-4.0 mmol / L, Triton X-100 concentration is 0.5-1.0 g / L, and preservative concentration is 0.2-0.4‰.

3. A small and dense low-density lipoprotein cholesterol detection kit as claimed in claim 1 or 2, characterized in that: In the reagent 1, the concentration of 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.3) is 100 mmol / L, the concentration of cholesterol oxidase (COD) is 1.0 KU / L, the concentration of cholesterol esterase (CHE) is 5.0 KU / L, the concentration of catalase (CAT) is 1.2 KU / L, the concentration of phospholipase is 2.5 KU / L, the concentration of N-ethyl-N-(2-hydroxy-3-benzenesulfonic acid)-3-methylaniline sodium salt (TOOS) is 1.2 g / L, the concentration of surfactant is 1.0 g / L, and the concentration of preservative is 0.2‰; the main components of reagent 2 are: the concentration of 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.3) is 100 mmol / L, the concentration of peroxidase (POD) is 5.0 KU / L, the concentration of 4-aminoantipyrine (4-AAP) is 4.0 mmol / L, and the concentration of Triton X-100 is 1.0 g / L, the preservative concentration is 0.2‰.

4. A small and dense low-density lipoprotein cholesterol detection kit as claimed in claim 1, 2 or 3, characterized in that: The surfactants in the reagent 1 are ethanol polyether-4-carboxylic acid and naphthalenesulfonic acid formaldehyde polymer, and the concentrations thereof are 0.5 g / L respectively.

5. A small and dense low-density lipoprotein cholesterol detection kit as claimed in claim 1, 2 or 3, characterized in that: The preservative in the reagent 1 and the reagent 2 is 2-hydroxypyridine-N-oxide (HOPO).

6. A small and dense low-density lipoprotein cholesterol detection kit, characterized in that: This kit can be used to detect the content of small, dense low-density lipoprotein cholesterol in human serum. The ratio of reagent to sample is: reagent 1: reagent 2: sample = 300: 100: 3.

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