阅读说明：本技术 一种小而密低密度脂蛋白胆固醇检测试剂盒的制备方法 (Preparation method of small and dense low-density lipoprotein cholesterol detection kit ) 是由 王保全 高洪元 方洪帅 崔学儒 邢瑞 王艳丽 于 2020-07-08 设计创作，主要内容包括：本发明涉及医学检测技术领域,且公开了一种小而密低密度脂蛋白胆固醇检测试剂盒的制备方法,包括以下步骤：S1、获取试剂R1所需要的成分,MES缓冲液一100mmol/L胆固醇酯酶1.6KU/L胆固醇氧化酶0.6KU/L磷脂酶2.7KU/L过氧化氢酶一1.2KU/L Toos 2mmol/L 0.3g/L的琼脂胶；浓度为1g/L的聚乙二醇(PEG)磷钨酸的浓度为4.5g/L；S2、获取试剂R2所需要的成分,MES缓冲液二100mmol/L过氧化物酶二5KU/L 4-氨基安替比林4mmol/L叠氮钠0.05％。本发明用于体外定量测定人血清中小而密低密度脂蛋白胆固醇的含量。本申请的试剂盒中使用的过氧化物酶,提高了检测的灵敏度和特异性。(The invention relates to the technical field of medical detection, and discloses a preparation method of a small and dense low-density lipoprotein cholesterol detection kit, which comprises the following steps: s1, obtaining components required by a reagent R1, MES buffer solution I100 mmol/L cholesterol esterase 1.6KU/L cholesterol oxidase 0.6KU/L phospholipase 2.7KU/L catalase, 1.2KU/L Toos 2mmol/L agar gel 0.3 g/L; the concentration of 1g/L polyethylene glycol (PEG) phosphotungstic acid is 4.5 g/L; s2, obtaining components required by the reagent R2, and adding 0.05% of MES buffer solution of two 100mmol/L peroxidase of two 5 KU/L4-aminoantipyrine of 4mmol/L sodium azide. The invention is used for in vitro quantitative determination of the content of small and dense low-density lipoprotein cholesterol in human serum. The peroxidase used in the kit of the application improves the sensitivity and specificity of detection.)

1. A preparation method of a small and dense low-density lipoprotein cholesterol detection kit is characterized by comprising the following steps: the method comprises the following steps:

s1, obtaining components required by a reagent R1, MES buffer solution I100 mmol/L cholesterol esterase 1.6KU/L cholesterol oxidase 0.6KU/L phospholipase 2.7KU/L catalase, 1.2KU/L Toos 2mmol/L agar gel 0.3 g/L; the concentration of 1g/L polyethylene glycol (PEG) phosphotungstic acid is 4.5 g/L;

s2, obtaining components required by the reagent R2, and adding 0.05% of MES buffer solution of two 100mmol/L peroxidase of two 5 KU/L4-aminoantipyrine of 4mmol/L sodium azide;

s3, preparing a reagent R1, dissolving the raw material of R1 in distilled water, uniformly mixing and fixing the volume;

s4, preparing a reagent R2, dissolving 4-aminoantipyrine in R2 in MES buffer solution II, mixing uniformly, adding peroxidase II and sodium azide into the solution, stirring fully, mixing uniformly, and fixing the volume.

2. The method for preparing a small, dense low-density lipoprotein cholesterol assay kit according to claim 1, wherein: reagent R1 reagent R2 volume ratio is 3: 1.

3. the method for preparing a small, dense low-density lipoprotein cholesterol assay kit according to claim 1, wherein: the distilled water in S3 can be replaced by double distilled water.

4. The method for preparing a small, dense low-density lipoprotein cholesterol assay kit according to claim 1, wherein: the pH of both reagent R1 and reagent R2 was 7.

5. The method for preparing a small, dense low-density lipoprotein cholesterol assay kit according to claim 1, wherein: the enzyme activities of the peroxidase I and the peroxidase II are both 128 KU/g.

Technical Field

The invention relates to the technical field of medical detection, in particular to a preparation method of a small and dense low-density lipoprotein cholesterol detection kit.

Background

Elevated low density lipoprotein cholesterol (LDL-C) levels are an independent atherogenic risk factor [ 1 ], while sdLDL-C, as a major component of LDL, is more likely to cause atherosclerosis than large and light low density lipoprotein (L LDL), due to its high invasiveness into the vessel wall, low affinity to the LDL receptor, longer plasma half-life and low tolerance to oxidative stress.

Recent studies have shown that sdLDL-C is an important marker of cardiovascular disease and can predict the risk of coronary heart disease. Relevant clinical or laboratory testing methods are: an enzymatic method.

The first step is as follows: under the action of cholesterol lipase (CHE), Cholesterol Oxidase (CO), phospholipase and catalase (catalase), the non-sdLDL-C components are cleared firstly, namely: chylomicrons (CM), Very Low Density Lipoproteins (VLDL) and Intermediate Density Lipoproteins (IDL), L LDL and High Density Lipoproteins (HDL).

The second step is that: under the action of sodium azide in the reagent II, catalase is inhibited, sd LDL-C is released under the action of a special surfactant, hydrogen peroxide is generated under the action of cholesterol esterase and cholesterol oxidase, the hydrogen peroxide and chromogen generate reddish purple quinine substances under the action of peroxidase, and the concentration of sdLDL-C is detected.

However, the existing small and dense low-density lipoprotein cholesterol detection kit has the problems of complicated operation, long time consumption, excessive resource waste and unsatisfactory detection sensitivity and specificity in the using process, and is inconvenient for people to use.

Disclosure of Invention

The invention aims to provide a preparation method of a small and dense low-density lipoprotein cholesterol detection kit.

In order to achieve the purpose, the invention provides the following technical scheme: a preparation method of a small and dense low-density lipoprotein cholesterol detection kit comprises the following steps:

s1, obtaining components required by a reagent R1, MES buffer solution I100 mmol/L cholesterol esterase 1.6KU/L cholesterol oxidase 0.6KU/L phospholipase 2.7KU/L catalase, 1.2KU/L Toos 2mmol/L agar gel 0.3 g/L; the concentration of 1g/L polyethylene glycol (PEG) phosphotungstic acid is 4.5 g/L;

s2, obtaining components required by the reagent R2, and adding 0.05% of MES buffer solution of two 100mmol/L peroxidase of two 5 KU/L4-aminoantipyrine of 4mmol/L sodium azide;

s3, preparing a reagent R1, dissolving the raw material of R1 in distilled water, uniformly mixing and fixing the volume;

s4, preparing a reagent R2, dissolving 4-aminoantipyrine in R2 in MES buffer solution II, mixing uniformly, adding peroxidase II and sodium azide into the solution, stirring fully, mixing uniformly, and fixing the volume.

Preferably, the volume ratio of reagent R1 to reagent R2 is 3: 1.

preferably, the distilled water in S3 can be replaced by double distilled water.

Preferably, the pH in both reagent R1 and reagent R2 is 7.

Preferably, the enzymatic activities of the peroxidase I and the peroxidase II are both 128 KU/g.

The invention provides a preparation method of a small and dense low-density lipoprotein cholesterol detection kit. The method has the following beneficial effects:

(1) the small and dense low-density lipoprotein cholesterol detection kit is used for quantitatively determining the content of small and dense low-density lipoprotein cholesterol in human serum in vitro. The peroxidase used in the kit of the application improves the sensitivity and specificity of detection.

(2) Therefore, on the premise of rapid and sensitive determination and high specificity, the whole system has good stability (can be stable for 12 months), is simple and convenient to operate, and can be suitable for being matched with a clinical full-automatic or semi-automatic biochemical analyzer for use.

Detailed Description