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及其检测方法 (Small and dense low-density lipoprotein cholesterol detection kit and detection method thereof ) 是由 谢蒙 张闻 陈媛 周海滨 于 2020-06-15 设计创作，主要内容包括：本发明公开了一种小而密低密度脂蛋白胆固醇检测试剂盒及其检测方法。该试剂盒包括试剂1和试剂2,试剂1含GOOD’s缓冲液、保护剂环糊精、胆固醇酯酶、胆固醇氧化酶、磷脂酶、过氧化物酶、稳定剂prionex、防腐剂pc300和色原TOOS；试剂2包括GOOD’s缓冲液、曲拉通X-405和4AAP。试剂盒的检测方法：试剂1和样本按照一定的比例在特定条件下处理一定时间,之后按照一定的比例加入试剂2,同样在特定条件下处理一定时间,根据吸光度值的变化定量测定小而密低密度脂蛋白胆固醇的含量。本发明所述的一种小而密低密度脂蛋白胆固醇检测试剂盒,含有环糊精类保护剂以及特殊的稳定剂,尤其对于低密度脂蛋白胆固醇和小而密低密度脂蛋白胆固醇浓度都比较高的高值样本,检测准确度高,稳定性好,能满足临床检验需要。(The invention discloses a small and dense low-density lipoprotein cholesterol detection kit and a detection method thereof. The kit comprises a reagent 1 and a reagent 2, wherein the reagent 1 contains GOOD's buffer solution, protective agent cyclodextrin, cholesterol esterase, cholesterol oxidase, phospholipase, peroxidase, stabilizer prionex, preservative pc300 and chromogen TOOS; reagent 2 included GOOD's buffer, Triton X-405 and 4 AAP. The detection method of the kit comprises the following steps: treating the reagent 1 and the sample according to a certain proportion for a certain time under a specific condition, then adding the reagent 2 according to a certain proportion, treating for a certain time under the same specific condition, and quantitatively determining the content of the small and dense low-density lipoprotein cholesterol according to the change of the absorbance value. The small and dense low-density lipoprotein cholesterol detection kit contains the cyclodextrin protective agent and the special stabilizer, and particularly has high detection accuracy and good stability for high-value samples with high low-density lipoprotein cholesterol and small and dense low-density lipoprotein cholesterol concentrations, and can meet the requirements of clinical examination.)

1. A small, dense low density lipoprotein cholesterol assay kit comprising a reagent 1 and a reagent 2, wherein said reagent 1 comprises: 100-5000U/L cholesterol esterase, 10-1000U/L cholesterol oxidase, 5-500U/L phospholipase,

0.1 to 10kU/L of peroxidase, 0.1 to 5 percent of stabilizer Prionex by volume percentage,

0.1-10 mM/L chromogen TOOS and 0.1-5% of protective agent cyclodextrin or cyclodextrin derivative by mass percentage; the reagent 2 comprises:

0.1 to 5 volume percent of triton and 0.1 to 10 mM/L4 AAP.

2. The kit of claim 1, wherein the triton comprises triton X-100, X-114, X-200, or X-405.

3. The kit according to claim 1, wherein the reagent 1 further comprises buffer 10-200 mM/L GOOD's, and the reagent 2 further comprises buffer 10-100 mM/L GOOD's, wherein the GOOD's buffer is one of TRIS, MOPS, POPOS, HEPES, or PIPES.

4. The kit of claim 3, wherein the GOOD's buffer is HEPES buffer.

5. The kit according to any one of claims 1 to 4, wherein the reagent 1 further comprises 0.1 to 1ml/L of a preservative ProClin.

6. The kit of claim 5, wherein the preservative ProClin in reagent 1 comprises ProClin150, 200, 300, or 5000.

7. The kit of claim 6, the reagent 1 comprising:

500-1000U/L cholesterol esterase, 50-500U/L cholesterol oxidase, 10-80U/L phospholipase,

1-8 kU/L of peroxidase, 0.2-3% of stabilizer Prionex by volume percentage,

0.5-8 mM/L chromogen TOOS, and 0.5-3% of protective agent cyclodextrin or cyclodextrin derivative by mass percentage; the reagent 2 comprises:

0.5 to 3 volume percent of triton X-405 and 0.1 to 10 mM/L4 AAP.

8. The kit of claim 6 or 7, wherein the protective agent cyclodextrin or cyclodextrin derivative in reagent 1 comprises alpha cyclodextrin, beta cyclodextrin, or gamma cyclodextrin.

9. A method for detecting small and dense low-density lipoprotein cholesterol, which is characterized by comprising the following steps:

S₁: adding the reagent 1 according to any one of claims 1 to 8 to a sample and a standard, respectively, and after reacting at constant temperature for a period of time, measuring the absorbance of the sample and the standard;

S₂: completes operation S₁Thereafter, the reagent 2 according to any one of claims 1 to 8 is further added, and after the isothermal reaction for a certain period of time, the absorbance of the sample and the standard is measured.

10. The detection method according to claim 9, wherein S₁In the step, the volume ratio of the sample to the reagent 1 and the volume ratio of the standard substance to the reagent 1 are both 1: 50; s₂In the step, the volume ratio of the reagent 1 to the reagent 2 is 3: 1.

Technical Field

The invention belongs to the technical field of biochemistry, and particularly relates to a small and dense low-density lipoprotein cholesterol detection kit and a detection method thereof.

Background

Clinically, dyslipidemia fluctuation often leads to the development of coronary heart disease (CAD). Low-density lipoprotein (LDL) is a well-established representative of the atherogenic lipoproteins; all the guidelines for managing hyperlipidemia at home and abroad emphasize that LDL cholesterol (LDL cholesterol, LDL-C) is a key intervention target for reducing the risk of CAD. Recent studies have shown that small, dense LDLs (small dense LDLs) with high density and small particles are more closely related to atherosclerosis among LDL subtypes; sd LDL or sd LDL cholesterol (sd LDL cholesterol, sd LDL-C) levels are more sensitive than LDL or LDL-C as an indicator to assess and predict risk of CAD.

At present, the protective agent in the sd LDL-C detection kit in China is basically a nonionic surfactant, and the principle of the protective agent for protecting small and dense low-density lipoprotein cholesterol by the nonionic surfactant is to reduce the reactivity of enzyme to the small and dense low-density lipoprotein cholesterol but not completely inhibit the enzyme, in the case of the situation, too much enzyme cannot be added, the small and dense low-density lipoprotein cholesterol in a low-value sample can be consumed, so that the result is low, if the enzyme quantity is insufficient, a normal sample has no influence, but when a common low-density lipoprotein cholesterol high-value sample is detected, the small and dense low-density lipoprotein cholesterol cannot be completely consumed, so that the result is high. Similarly, the current stabilizers for measuring low density lipoprotein cholesterol are various and have different stabilizing effects, and false positive can occur due to unstable reagents.

Therefore, the small and dense low-density lipoprotein cholesterol detection kit with a high detection value range and good stability is provided, which meets the urgent need of developing small and dense low-density lipoprotein cholesterol detection in clinic and simultaneously provides support for the prevention and control of arteriosclerotic cardiovascular diseases (ASCVD) and the development of the risk assessment field.

Disclosure of Invention

In order to solve the above problems in the prior art, the present invention aims to provide a small and dense low density lipoprotein cholesterol kit with a high detection range and good stability, and a detection method thereof.

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

a small, dense low density lipoprotein cholesterol assay kit comprising a reagent 1 and a reagent 2, wherein said reagent 1 comprises:

100-5000U/L cholesterol esterase,

10-2000U/L cholesterol oxidase,

5 to 500U/L phospholipase,

0.1 to 10kU/L of peroxidase,

0.1 to 5 percent of stabilizer Prionix,

0.1-10 mM/L chromogen TOOS and 0.1-5% of protective agent cyclodextrin or cyclodextrin derivative by mass percentage; the reagent 2 comprises: 0.1 to 5 volume percent of triton and 0.1 to 10 mM/L4 AAP.

Further, the triton in the reagent 2 comprises triton X-100, X-114, X-200 or X-405.

Further, the reagent 1 further comprises buffer solution 10-200 mM/L GOOD's, and the reagent 2 further comprises buffer solution 10-100 mM/L GOOD's, wherein the GOOD's buffer solution is one of TRIS, MOPS, POPOS, HEPES or PIPES.

Further, the GOOD's buffer is preferably HEPES buffer.

Further, the reagent 1 also comprises 0.1-1 ml/L preservative ProClin.

Further wherein the preservative ProClin in agent 1 comprises ProClin150, 200, 300, or 5000. Further, the kit, preferably reagent 1, comprises:

500-5000U/L cholesterol esterase,

100-1000U/L cholesterol oxidase,

20-200U/L phospholipase, 1-8 kU/L peroxidase,

0.2 to 3 percent of stabilizer Prionix,

0.5 to 8mM/L chromogen TOOS,

And 0.5 to 3 mass percent of protective agent cyclodextrin or cyclodextrin derivative;

the reagent 2 comprises:

0.5 to 3 volume percent of triton X-405 and 0.1 to 10 mM/L4 AAP.

Further, the protective agent cyclodextrin or cyclodextrin derivative in the reagent 1 comprises alpha cyclodextrin, beta cyclodextrin or gamma cyclodextrin.

A method for detecting small and dense low density lipoprotein cholesterol, comprising the steps of:

S₁: respectively adding any one of the reagents 1 into a sample and a standard substance, carrying out heat preservation reaction for a period of time, and then measuring the absorbance of the sample and the standard substance;

S₂: completes operation S₁And then, continuously adding the reagent 2, carrying out heat preservation reaction for a period of time, and then measuring the absorbance of the sample and the standard substance.

Further, wherein S₁In the step, the volume ratio of the sample to the reagent 1 and the volume ratio of the standard substance to the reagent 1 are both 1: 50; s₂In the step, the volume ratio of the reagent 1 to the reagent 2 is 3: 1.

The principle of the low density lipoprotein cholesterol detection kit of the invention is as follows: firstly, the protective agent cyclodextrin or various cyclodextrin derivatives added into the low-density lipoprotein cholesterol detection kit can play a role in protecting sdLDL-C in the first reaction step, and cholesterol esterase, cholesterol oxidase, phospholipase and catalase remove non-small and dense low-density lipoprotein cholesterol which is not protected by the cyclodextrin or the cyclodextrin derivatives as a substrate.

The reagent 2 is added to a sample, the protected sd LDL-C is deprotected, hydrogen peroxide is generated under the action of cholesterol esterase and cholesterol oxidase, and then the hydrogen peroxide is coupled with TOOS to generate a color reaction under the action of peroxidase, and then the color reaction is measured by a colorimetric method.

Calculating by using the absorbance difference, and calculating the concentration of the low-density lipoprotein cholesterol according to the formula 1;

and (3) calibration procedure:

the calibration curve is prepared by using a matched calibrator, and the recalibration is recommended after reagent batch number replacement.

Quality control procedure:

and (3) using a matched quality control product to control the relative deviation within the allowable range of the quality control product.

Method for calculating small and dense low density lipoprotein cholesterol, formula 1:

determination of Δ A: detecting the difference between the absorbance of the sample added with the reagent 1 and the absorbance of the sample added with the reagent 2;

Δ A Standard: the difference between the absorbance of the standard added to reagent 1 and the absorbance of the standard added to reagent 2.

In conclusion, the invention has the beneficial effects that:

the protective agent cyclodextrin or cyclodextrin derivative added into the small and dense low-density lipoprotein cholesterol detection kit can well inhibit the action of small and dense low-density lipoprotein cholesterol with cholesterol esterase, cholesterol oxidase and peroxidase in the reagent 1, and can provide a high-accuracy detection result under the condition of detecting the same high concentration of low-density lipoprotein and small and dense low-density lipoprotein in a sample.

The stabilizer prionex added into the small and dense low-density lipoprotein cholesterol detection kit is a porcine collagen fragment, can improve the stability of cholesterol enzyme, and avoids the condition of false positive caused by unstable reagent or long storage time of the kit.

Drawings

FIG. 1 is a line graph showing the correlation between example 6 and a commercially available kit according to the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.