Serum total cholesterol (TCH) determination kit

Abstract

The invention provides a serum total cholesterol (TCH) determination kit, the kit comprises a reagent 1 and a reagent 2, the reagent 1 comprises the following main components: a 3-morpholine-2-hydroxypropanesulfonic acid (MOPSO) buffer solution (pH 7.20), cholesterol esterase (CHER), sodium cholate, a surfactant, a stabilizer and a preservative; and the reagent 2 is prepared from the following main components: a 3-morpholine-2-hydroxy propanesulfonic acid (MOPSO) buffer solution (pH is 7.0), cholesterol oxidase (CHOD), peroxidase (POD), sodium cholate, 10-(N-methyl carbamoyl)-3, 7-bis (dimethylamino) phenothiazine (MCDP), a surfactant, a stabilizer and a preservative. By adding the enzyme protective agent and the high-sensitivity pigment source MCTP, the prepared kit is better in stability and wider in linear range.

Description

Technical Field

[0001] The invention belongs to the field of medical examination, and in particular relates to a serum total cholesterol determination kit and a preparation method thereof. Background Art

[0002] Total cholesterol (TCH) refers to the sum of cholesterol contained in various lipoproteins in the blood. It is an important indicator for clinical blood lipid analysis. Plasma cholesterol mainly exists in low-density lipoprotein (LDL), followed by high-density lipoprotein (HDL) and very low-density lipoprotein (VLDL), and the least in chylomicrons (CM). In human plasma, cholesterol exists in two forms, namely cholesterol ester (CE) and free cholesterol (FC), the former accounting for about 70% and the latter accounting for 30%. The cholesterol contained in plasma and tissues is often in an exchange state, and the exchange rate varies with different tissues. Since cholesterol constantly enters and leaves the blood, plasma cholesterol not only reflects the situation of cholesterol uptake and synthesis, but also reflects the synthesis rate of various lipoproteins carrying cholesterol, as well as the situation of receptors that affect lipoprotein metabolism.

[0003] The determination of TCH has important clinical significance for the diagnosis of blood lipid diseases. The determination of blood cholesterol can be used as a diagnostic indicator for liver function, bile function, intestinal absorption function and coronary artery disease. Increased blood cholesterol is common in nephrotic syndrome, diabetes, hypothyroidism, etc. Serum total cholesterol exceeding 5.70mmol / L can be considered hypercholesterolemia, which can easily cause atherosclerosis and cause obstructive cardiovascular and cerebrovascular diseases. A large number of medical studies have shown that the occurrence of cardiovascular diseases is closely related to excessive cholesterol levels in serum. Therefore, accurate determination of cholesterol helps to provide a basis for the diagnosis of cardiovascular diseases and is of great significance for the prevention and control of such diseases.

[0004] The methods for determining total cholesterol include chemical reagent colorimetry, enzyme analysis, gas chromatography and high-performance liquid chromatography. Since gas chromatography and high-performance liquid chromatography have high requirements and the equipment is complex and expensive, they are rarely used in clinical laboratories. The most commonly used methods in clinical laboratories are chemical reagent method and enzyme method. Among them, the chemical reagent method mainly adopts the phosphorus-sulfur-iron method. Since the reagents used in this method are corrosive, organic solvent extraction is required, and the operation steps are cumbersome. At present, the enzymatic method is widely used to detect total cholesterol in serum or plasma. The basic principle of enzymatic determination: the cholesterol ester in the sample is hydrolyzed into free cholesterol by cholesterol esterase (CHER), and together with the free cholesterol present in the sample, it is oxidized by cholesterol oxidase (CHOD) to cholesterenone and hydrogen peroxide (H2O2), and the latter is then catalyzed by peroxidase (POD) to cause 4-aminoantipyrine and phenol to undergo an oxidative condensation reaction to generate red quinone imine (called Trinder reaction, or PAP reaction). The cholesterol content in the sample is proportional to the generated quinone imine. The absorbance of quinone imine is detected at a specific wavelength (505nm), and the total cholesterol content in the specimen can be calculated by comparing it with the calibrant.

[0005]

[0006] During the actual research and testing process, it was found that in the third step of the enzymatic reaction, due to the instability of the product red-purple pigment, the total cholesterol test results were unstable and had poor accuracy. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a serum total cholesterol (TCH) determination kit, the detection result of the kit is more stable and has better accuracy.

[0008] In order to achieve the above object, the present invention discloses a serum total cholesterol (TCH) determination kit, which comprises a reagent 1 and a reagent 2, wherein the volume ratio of the reagent 1 to the reagent 2 is 3:1; the main components of the reagent 1 are: 3-morpholine-2-hydroxypropanesulfonic acid (MOPSO) buffer (pH7.20) with a concentration of 50-100 mmol / L, cholesterol esterase (CHER) with a concentration of 1-5 KU / L, magnesium chloride with a concentration of 2-5 mmol / L, a composite protein protective agent (HPD) with a concentration of 1-10 ml / L, a suitable amount of surfactant, a stabilizer with a concentration of 1-10 g / L, and a preservative with a concentration of 0.1-0.5%; the main components of the reagent 2 are: 3-morpholine-2-hydroxypropanesulfonic acid (MOPSO) buffer (pH7.00) with a concentration of 10-50 mmol / L, a cholesterol esterase (CHER) with a concentration of 1-5 KU / L, a magnesium chloride with a concentration of 2-5 mmol / L, a composite protein protective agent (HPD) with a concentration of 1-10 ml / L, a suitable amount of surfactant, a stabilizer with a concentration of 1-10 g / L, and a preservative with a concentration of 0.1-0.5%. mmol / L, cholesterol oxidase (CHOD) concentration is 1-5KU / L, peroxidase (POD) concentration is 2-4KU / L, 4-aminoantipyrine concentration is 1-10 mmol / L, 3,5-dichloro-2-hydroxybenzenesulfonate sodium concentration is 1-10 mmol / L, 10-(N-methylcarbamoyl)-3,7-bis(dimethylamino)phenothiazine (MCDP) concentration is 1-10mmol / L, magnesium chloride concentration is 2-5mmol / L, surfactant is appropriate, stabilizer concentration is 1-10g / L, preservative concentration is 0.1-0.5%.

[0009] Furthermore, the surfactants in the reagent 1 and the reagent 2 are sodium cholate with a concentration of 2-5 mmol / L and octylphenol polyoxyethylene ether (Triton X-114) with a concentration of 3-6 g / L.

[0010] Furthermore, the stabilizers in the reagents 1 and 2 are selected to have a bovine serum albumin (BSA) concentration of 1-3 g / L and a trehalose concentration of 1-3 g / L.

[0011] Furthermore, the preservative in the reagent 1 and the reagent 2 is proclin-300 with a concentration of 0.3 ml / L.

[0012] The present invention has the following beneficial effects: 1. Based on the enzyme method, the present invention optimizes the reaction system, adds enzyme protectant trehalose, composite protein protectant HPD and stabilizer, which not only protects the activity of the enzyme, but also stabilizes the final product, and significantly enhances the accuracy and stability of the reagent detection. The detection result of the reagent has good accuracy and stability.

[0013] 2. The present invention adds a highly sensitive pigment source 10-(N-methylcarbamoyl)-3,7-bis(dimethylamino)phenothiazine (MCDP) to the kit, and makes the linear range of the total cholesterol detection kit wider, especially improves the accuracy of linear high value detection. 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 method for preparing a serum total cholesterol (TCH) detection kit, the main components of which are as follows:

[0016] (1) Opening stability The reagent bottles were opened and placed in the reagent compartment of the biochemical analyzer, and the stability of the opened bottles was tested on days 0, 2, 4, 6, 8, 10, 12, 16, 20, 24, 28, and 30.

[0017]

[0018] (2) Thermal stability at 37°C After the reagents were packaged, they were placed in a 37°C constant temperature incubator to test the accelerated stability for 7 days.

[0019]

[0020] According to the test results, within 30 days after the reagent was opened, the deviation of the two concentrations of 4.10 mmol / L and 6.20 mmol / L quality control products measured after the kit was calibrated was within 10%. The deviation of the measured value within 7 days after the kit was heated at 37°C was less than 10%, which met the test requirements.

[0021] Linear range and deviation Samples with a theoretical concentration range of 0-10mmol / L were selected to calculate the correlation coefficient r and the deviation between the measured mean and the theoretical concentration. The results are as follows:

[0022] According to the test results, the correlation coefficient R 2 =0.9992, the maximum relative deviation among the 15 groups of data is 2.53%, and the minimum is 0.07%, which is 10% better than the industry standard. The linearity of this kit is good.

[0023] 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 serum total cholesterol (TCH) determination kit, the kit comprising reagent 1 and reagent 2, wherein the volume ratio of reagent 1 to reagent 2 is 3:1; the main components of reagent 1 are: 3-morpholine-2-hydroxypropanesulfonic acid (MOPSO) buffer (pH7.20) with a concentration of 50-100 mmol / L, cholesterol esterase (CHER) with a concentration of 1-5 KU / L, magnesium chloride with a concentration of 2-5 mmol / L, composite protein protective agent (HPD) with a concentration of 1-10 ml / L, a suitable amount of surfactant, a stabilizer with a concentration of 1-10 g / L, and a preservative with a concentration of 0.1-0.5%; the main components of reagent 2 are: 3-morpholine-2-hydroxypropanesulfonic acid (MOPSO) buffer (pH7.00) 10-50 mmol / L, cholesterol oxidase (CHOD) with a concentration of 1-5 KU / L, peroxidase (POD) with a concentration of 2-4 KU / L, 4-aminoantipyrine with a concentration of 1-10 mmol / L, 3,5-dichloro-2-hydroxybenzenesulfonate sodium concentration is 1-10 mmol / L, 10-(N-methylcarbamoyl)-3,7-bis(dimethylamino)phenothiazine (MCDP) concentration is 1-10mmol / L, magnesium chloride concentration is 2-5mmol / L, surfactant is appropriate, stabilizer concentration is 1-10g / L, preservative concentration is 0.1-0.5%.

2. A serum total cholesterol (TCH) determination kit according to claim 1, characterized in that: The surfactants in the reagents 1 and 2 are sodium cholate with a concentration of 2-5 mmol / L and octylphenol polyoxyethylene ether (Triton X-114) with a concentration of 3-6 g / L.

3. A serum total cholesterol (TCH) determination kit as claimed in claim 1, characterized in that: The stabilizers in the reagents 1 and 2 are bovine serum albumin (BSA) with a concentration of 1-3 g / L and trehalose with a concentration of 1-3 g / L.

4. A serum total cholesterol (TCH) determination kit as claimed in claim 1, characterized in that: The preservative in the reagent 1 and the reagent 2 is proclin-300 with a concentration of 0.3 ml / L.

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