阅读说明：本技术 一种高密度脂蛋白3胆固醇检测试剂盒、制备方法及用途 (High-density lipoprotein 3 cholesterol detection kit, preparation method and application ) 是由 梁艳 龚婷 吴年芬 舒芹 张雪娇 赵愿安 于 2020-08-06 设计创作，主要内容包括：本发明提供了一种高密度脂蛋白3胆固醇检测试剂盒,属于疾病检测试剂领域,包括试剂1和试剂2,试剂1包括：Good’s缓冲液、4-氨基安替比林、Proclin 300、胆固醇脂酶、胆固醇氧化酶、磷脂酶、过氧化氢酶、抗坏血酸氧化酶和环糊精；试剂2包括：Good’s缓冲液、叠氮钠、TOOS、亚铁氰化钾、α-淀粉酶和过氧化物酶。该试剂盒能提高HDL3-c检测的准确度,并且不影响其它性能,如：精密度、线性、稳定性。本发明还提供了一种高密度脂蛋白3胆固醇检测试剂盒的制备方法及用途。(The invention provides a high-density lipoprotein 3 cholesterol detection kit, which belongs to the field of disease detection reagents and comprises a reagent 1 and a reagent 2, wherein the reagent 1 comprises: good's buffer solution, 4-aminoantipyrine, Proclin300, cholesterol esterase, cholesterol oxidase, phospholipase, catalase, ascorbate oxidase and cyclodextrin; the reagent 2 comprises: good's buffer, sodium azide, TOOS, potassium ferrocyanide, alpha-amylase and peroxidase. The kit can improve the accuracy of HDL3-c detection, and does not affect other properties, such as: precision, linearity, stability. The invention also provides a preparation method and application of the high-density lipoprotein 3 cholesterol detection kit.)

1. A high density lipoprotein 3 cholesterol detect reagent box, characterized by that, including reagent 1 and reagent 2, reagent 1 includes: good's buffer solution, 4-aminoantipyrine, Proclin300, cholesterol esterase, cholesterol oxidase, phospholipase, catalase, ascorbate oxidase and cyclodextrin;

the reagent 2 comprises: good's buffer, sodium azide, TOOS, potassium ferrocyanide, alpha-amylase and peroxidase.

2. The high-density lipoprotein 3 cholesterol detection kit as claimed in claim 1, wherein the concentration of each component in the reagent 1 is: good's buffer: 50-100mmol/L, 4-aminoantipyrine: 0.1-0.5g/L, Proclin 300: 0.5-1g/L, cholesterol esterase: 1-5KU/L, cholesterol oxidase: 1-5KU/L, phospholipase: 20-50KU/L, catalase: 200-500KU/L, ascorbate oxidase: 2-5KU/L, cyclodextrin: 0.5-1g/L, pH: 6.6-7.5.

3. The high-density lipoprotein 3 cholesterol detection kit as claimed in claim 2, wherein the concentration of each component in the reagent 1 is: good's buffer: 100mmol/L, 4-aminoantipyrine: 0.5g/L, Proclin 300: 1g/L, cholesterol esterase: 5KU/L, cholesterol oxidase: 5KU/L, phospholipase: 50KU/L, catalase: 500KU/L, ascorbic acid oxidase: 5KU/L, cyclodextrin: 1g/L, pH: 7.5.

4. the high density lipoprotein 3 cholesterol detection kit of claim 1, wherein the concentration of each component in the reagent 2 is: good's buffer: 50-100mmol/L, sodium azide: 0.5-1g/L, TOOS: 0.5-1g/L, potassium ferrocyanide: 0.05-0.1g/L, alpha-amylase: 10-20KU/L, peroxidase: 2-5KU/L, pH: 6.6-7.5.

5. The high density lipoprotein 3 cholesterol detection kit of claim 4, wherein the concentration of each component in the reagent 2 is: good's buffer: 50mmol/L, sodium azide: 1g/L, TOOS: 1g/L, potassium ferrocyanide: 0.05g/L, alpha-amylase: 10KU/L, peroxidase: 5KU/L, pH: 7.0.

6. the kit for detecting high-density lipoprotein 3 cholesterol as claimed in claim 1, wherein the cyclodextrin is alpha-cyclodextrin or beta-cyclodextrin or gamma-cyclodextrin.

7. A method for preparing the high density lipoprotein 3 cholesterol assay kit according to any one of claims 1-6, comprising:

weighing raw materials required by the reagent 1: good's buffer solution, 4-aminoantipyrine, Proclin300, cholesterol esterase, cholesterol oxidase, phospholipase, catalase, ascorbate oxidase and cyclodextrin;

preparing a solution from raw materials Good's buffer solution, 4-aminoantipyrine, Proclin300, cholesterol esterase, cholesterol oxidase, phospholipase, catalase, ascorbate oxidase and cyclodextrin, and adjusting the pH by an acid solution or an alkali solution to obtain a reagent 1 having the component concentration according to claim 2 or 3;

the reagent 2 comprises Good's buffer solution, sodium azide, TOOS, potassium ferrocyanide, alpha-amylase and peroxidase;

raw materials Good's buffer, sodium azide, toss, potassium ferrocyanide, alpha-amylase and peroxidase were formulated into a solution, and the pH was adjusted by an acid solution or an alkali solution to obtain reagent 2 having the component concentration as set forth in claim 4 or 5.

8. The method for preparing a high density lipoprotein 3 cholesterol assay kit as claimed in claim 7, wherein the acid solution is hydrochloric acid and the alkali solution is sodium hydroxide.

9. The method for preparing a high density lipoprotein 3 cholesterol assay kit of claim 7 wherein the effective concentration in the Good's buffer is 50-100 mmol/L.

10. Use of a high density lipoprotein 3 cholesterol detection kit according to any one of claims 1 to 6 in a high density lipoprotein 3 cholesterol assay.

Technical Field

The invention belongs to the field of disease detection reagents, and relates to a high-density lipoprotein 3 cholesterol detection kit, a preparation method and application.

Background

High density lipoprotein cholesterol (HDL) is a major anti-atherosclerotic lipoprotein and can reduce cardiovascular disease. HDL components can be divided into two major subtypes, depending on density: HDL 2-cholesterol (HDL2-c) and less HDL 3-cholesterol (HDL 3-c).

High density lipoproteins act physiologically as a transport vehicle for cholesterol from extrahepatic tissues to the liver, thereby preventing the deposition of free cholesterol on extrahepatic tissue cells. High density lipoprotein cholesterol is an important reference index for clinical diagnosis of coronary heart disease. It is one of the clinical coronary heart disease protection factors, and can prevent, treat and delay the development of atherosclerosis. HDL2-c has anti-arteriosclerosis effect, and HDL3-c does not change to HDL2-c due to reduction of hepatic lipase activity, so HDL3-c increases, suggesting that the incidence of coronary artery disease increases due to increase of HDL 3-c.

The value of HDL3-c in serum is increased due to arteriosclerosis, so that the detection of the content of HDL3-c in serum can be used as an important detection index of cardiovascular diseases.

The existing detection kit for high-density lipoprotein 3 cholesterol generally uses an elimination method to determine HDL3-c, and the method adopts double reagents and achieves the purpose of determining HDL3-c by eliminating non-HDL 3-c. However, the surfactant is difficult to accurately identify the small particles, and causes a problem that the HDL3-c is erroneously removed, resulting in a low detection result.

Disclosure of Invention

In order to solve the technical problem of low accuracy of the conventional HDL3-c detection kit, the invention provides a high-density lipoprotein 3 cholesterol detection kit, which can improve the accuracy of HDL3-c detection and does not influence other performances, such as: precision, linearity, stability.

The invention also provides a preparation method and application of the high-density lipoprotein 3 cholesterol detection kit.

The invention is realized by the following technical scheme:

a high density lipoprotein 3 cholesterol detect reagent box, including reagent 1 and reagent 2, reagent 1 includes: good's buffer solution, 4-aminoantipyrine, Proclin300, cholesterol esterase, cholesterol oxidase, phospholipase, catalase, ascorbate oxidase and cyclodextrin;

the reagent 2 comprises: good's buffer, sodium azide, TOOS, potassium ferrocyanide, alpha-amylase and peroxidase.

Wherein, the concentration of each component in the reagent 1 is as follows:

good's buffer: 50-100mmol/L, 4-aminoantipyrine: 0.1-0.5g/L, Proclin 300: 0.5-1g/L, cholesterol esterase: 1-5KU/L, cholesterol oxidase: 1-5KU/L, phospholipase: 20-50KU/L, catalase: 200-500KU/L, ascorbate oxidase: 2-5KU/L, cyclodextrin: 0.5-1g/L, pH: 6.6-7.5.

Preferably, the concentration of each component in the reagent 1 is as follows: good's buffer: 100mmol/L, 4-aminoantipyrine: 0.5g/L, Proclin 300: 1g/L, cholesterol esterase: 5KU/L, cholesterol oxidase: 5KU/L, phospholipase: 50KU/L, catalase: 500KU/L, ascorbic acid oxidase: 5KU/L, cyclodextrin: 1g/L, pH: 7.5.

further, the concentration of each component in the reagent 2 is as follows: good's buffer: 50-100mmol/L, sodium azide: 0.5-1g/L, TOOS: 0.5-1g/L, potassium ferrocyanide: 0.05g/L, alpha-amylase: 10KU/L, peroxidase: 2-5KU/L, pH: 6.6-7.5.

Preferably, the concentration of each component in the reagent 2 is as follows: good's buffer: 50mmol/L, sodium azide: 1g/L, TOOS: 1g/L, potassium ferrocyanide: 0.05g/L, alpha-amylase: 10KU/L, peroxidase: 5KU/L, pH: 7.0.

further, the cyclodextrin adopts alpha-cyclodextrin or beta-cyclodextrin or gamma-cyclodextrin.

A preparation method of a high density lipoprotein 3 cholesterol detection kit comprises the following steps:

weighing raw materials required by the reagent 1: good's buffer solution, 4-aminoantipyrine, Proclin300, cholesterol esterase, cholesterol oxidase, phospholipase, catalase, ascorbate oxidase and cyclodextrin;

preparing raw materials of Good's buffer solution, 4-aminoantipyrine, Proclin300, cholesterol esterase, cholesterol oxidase, phospholipase, catalase, ascorbate oxidase and cyclodextrin into solution, and adjusting the pH value by acid liquor or alkali liquor to obtain a reagent 1, wherein the concentration of each component is as follows:

good's buffer: 50-100mmol/L, 4-aminoantipyrine: 0.1-0.5g/L, Proclin 300: 0.5-1g/L, cholesterol esterase: 1-5KU/L, cholesterol oxidase: 1-5KU/L, phospholipase: 20-50KU/L, catalase: 200-500KU/L, ascorbate oxidase: 2-5KU/L, cyclodextrin: 0.5-1g/L, pH: 7.5;

the reagent 2 comprises Good's buffer solution, sodium azide, TOOS, potassium ferrocyanide, alpha-amylase and peroxidase;

preparing raw materials Good's buffer solution, sodium azide, TOOS, potassium ferrocyanide, alpha-amylase and peroxidase into solution, and adjusting the pH value by acid liquor or alkali liquor to obtain a reagent 2, wherein the concentration of each component is as follows:

good's buffer: 50-100mmol/L, sodium azide: 0.5-1g/L, TOOS: 0.5-1g/L, potassium ferrocyanide: 0.05g/L, alpha-amylase: 10KU/L, peroxidase: 2-5KU/L, pH: 7.0.

further, the acid solution is hydrochloric acid, and the alkali solution is sodium hydroxide.

Furthermore, the effective concentration of the Good's buffer solution is 50-100 mmol/L.

An application of a high density lipoprotein 3 cholesterol detection kit in high density lipoprotein 3 cholesterol determination.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

according to the high-density lipoprotein 3 cholesterol detection kit, the cyclodextrin is added into the reagent 1, so that the accuracy of reagent detection is improved, the deviation of a standard substance for measuring the high-density lipoprotein 3 cholesterol is 4% or less, the anti-interference capability is provided for common interference substances, and other performances such as precision, linear correlation coefficient and functional sensitivity are not influenced.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

In order to solve the technical problems, the embodiment of the invention provides the following general ideas:

at present, a detection kit for high-density lipoprotein 3 cholesterol generally uses a clearing method to determine HDL3-c, and the method adopts double reagents, firstly uses a surfactant to wrap HDL3-c, and achieves the purpose of determining HDL3-c by clearing non-HDL 3-c. However, the surfactant has difficulty in accurately recognizing lipoprotein cholesterol having a small particle diameter of less than 25.5nm, and causes a problem that the removal of HDL3-c by mistake leads to a low detection result.

In contrast, the high density lipoprotein 3 cholesterol assay kit of the present invention includes a reagent 1 and a reagent 2, wherein the reagent 1 includes: good's buffer solution, 4-aminoantipyrine, Proclin300, cholesterol esterase, cholesterol oxidase, phospholipase, catalase, ascorbate oxidase and cyclodextrin;

the reagent 2 comprises: good's buffer, sodium azide, TOOS, potassium ferrocyanide, alpha-amylase and peroxidase.

The detection principle of the invention is as follows:

the first step is as follows: under the action of cholesterol lipase, cholesterol oxidase, phospholipase, cyclodextrin and catalase, the non-HDL 3-c component is eliminated first. The second step is that: HDL3-c is released under the action of alpha-amylase, hydrogen peroxide is generated under the action of cholesterol esterase and cholesterol oxidase, and the hydrogen peroxide and chromogen generate purple-red quinone substances under the action of peroxidase, so that the concentration of HDL3-c is detected.

According to the invention, the reagent 1 is added with cyclodextrin to specifically wrap HDL3-c, alpha-amylase in R2 degrades the cyclodextrin, and then HDL3-c is released, and the accuracy of the cyclodextrin in identifying small particles is higher than that of a common surfactant, so that the accuracy of reagent detection is improved. The method can not only improve the detection accuracy of the reagent, but also does not influence other performances (such as precision, linearity, stability) and the like.

Hereinafter, a detailed description will be given of a high density lipoprotein 3 cholesterol assay kit according to the present application with reference to examples, comparative examples and experimental data.