阅读说明：本技术 一种高准确度的高密度脂蛋白胆固醇检测试剂盒 (High-accuracy high-density lipoprotein cholesterol detection kit ) 是由 王莉 侯军霞 刘振国 于 2019-10-28 设计创作，主要内容包括：本发明适用于高密度脂蛋白胆固醇检测技术领域,提供了一种高准确度的高密度脂蛋白胆固醇检测试剂盒,包括包括第一试剂和第二试剂；第一试剂包括缓冲液I、凝集剂、显色剂、第一防腐剂、表面活性剂I、表面活性剂II、保护剂和酶I；第二试剂包括缓冲液、助色剂、第二防腐剂、保护剂、表面活性剂II和酶II；酶I包括胆固醇氧化酶和过氧化物酶,其中胆固醇氧化酶来源于假单胞菌属；酶II为胆固醇酯酶,来源于裂殖菌或假单胞菌属；第一试剂中,表面活性剂I和表面活性剂II的浓度比为：1:3～4。借此,本发明能够提高HDL-C检测的准确度,解决现有技术中HDL-C检测试剂检测准确度低的问题,为相关疾病的诊断及预防提供了可靠依据。(The invention is suitable for the technical field of high-density lipoprotein cholesterol detection, and provides a high-accuracy high-density lipoprotein cholesterol detection kit, which comprises a first reagent and a second reagent; the first reagent comprises a buffer solution I, a agglutinant, a color developing agent, a first preservative, a surfactant I, a surfactant II, a protective agent and an enzyme I; the second reagent comprises a buffer solution, a color assisting agent, a second preservative, a protective agent, a surfactant II and an enzyme II; enzyme I comprises cholesterol oxidase and peroxidase, wherein the cholesterol oxidase is derived from Pseudomonas; enzyme II is cholesterol esterase derived from schizomycete or Pseudomonas; in the first reagent, the concentration ratio of the surfactant I to the surfactant II is as follows: 1: 3-4. Therefore, the invention can improve the detection accuracy of HDL-C, solve the problem of low detection accuracy of HDL-C detection reagents in the prior art, and provide reliable basis for diagnosis and prevention of related diseases.)

1. A high-accuracy high-density lipoprotein cholesterol detection kit is characterized by comprising a first reagent and a second reagent;

the first reagent comprises the following components: 0.02-0.8mol/L of buffer solution I, 2-18 g/L of agglutinant, 0.5-5.0mmol/L of color developing agent, 0.5-2.0g/L of first preservative, 1.0-12.0ml/L of surfactant I, 8.0-25.0ml/L of surfactant II, 22-160 g/L of protective agent and 2-100 Ku/L of enzyme I;

the second reagent comprises the following components: 0.02-0.8mol/L of buffer solution II, 1.0-10.0mmol/L of color assistant, 0.5-2.0g/L of second preservative, 22-160 g/L of protective agent, 8.0-25.0ml/L of surfactant II and 1.0-50.0Ku/L of enzyme II;

the enzyme I comprises cholesterol oxidase and peroxidase, wherein the cholesterol oxidase is derived from pseudomonas; the enzyme II is cholesterol esterase and is derived from schizomycete or pseudomonas;

in the first reagent, the concentration ratio of the surfactant I to the surfactant II is as follows: 1: 3-4.

2. The high-accuracy high-density lipoprotein cholesterol detection kit according to claim 1, wherein the buffer I and the buffer II are any one or more of 3-morpholine propanesulfonic acid, 1, 4-piperazine diethylsulfonic acid, 4-hydroxyethyl piperazine ethanesulfonic acid and morpholine ethanesulfonic acid monohydrate.

3. The high accuracy high density lipoprotein cholesterol assay kit of claim 2 in which the pH of buffer I and buffer II are both 6.5-7.5.

4. The high accuracy, high density lipoprotein cholesterol assay kit of claim 1 in which the agglutinating agent comprises divalent metal ions, α -cyclodextrin sulfate and dextran sodium sulfate salt.

5. The high-accuracy high-density lipoprotein cholesterol detection kit according to claim 4, wherein the mass concentration of the divalent metal ion is 1.0-10.0 g/L, the divalent metal ion is any one of magnesium sulfate, zinc sulfate or magnesium chloride, the mass concentration of the α -cyclodextrin sulfate is 0.4-4.0 g/L, and the mass concentration of the dextran sulfate sodium salt is 0.5-5.0 g/L.

6. The high-accuracy high-density lipoprotein cholesterol detection kit according to claim 1, characterized in that the color-developing agent is any one of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3' 5-dimethoxyaniline sodium salt.

7. The high accuracy high density lipoprotein cholesterol assay kit of claim 1 wherein the first and second preservatives are any one or more of sodium azide, gentamicin, Proclin 300.

8. The high accuracy high density lipoprotein cholesterol assay kit of claim 1 in which the surfactant I is any one or more of pluronic l127, Emuleng220, EmulengA90, emulengl ls114, NP-7, pluronic f-88.

9. The high accuracy, high density lipoprotein cholesterol assay kit of claim 8 in which the surfactant II is any one or more of emulgen b66, emulengl ls110, Emuleng709, pininn d6512, pininnye 309, noignea-157, noignea-167, noignea-177.

10. The kit for detecting high-accuracy high-density lipoprotein cholesterol as claimed in claim 1, wherein in the enzyme I, the active concentrations of the cholesterol oxidase and the peroxidase are both 1-50 Ku/L.

Technical Field

The invention relates to the technical field of high-density lipoprotein cholesterol detection, in particular to a high-density lipoprotein cholesterol detection kit with high accuracy.

Background

Cholesterol is present in lipoproteins in the blood, and its forms include high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol, and very low density lipoprotein cholesterol. The majority of cholesterol present in the blood is cholesterol esters bound to fatty acids, with less than 10% being present in the free form. Cholesterol has a wide range of physiological effects in the body, but when it is excessive, it causes hypercholesterolemia, which adversely affects the body. Modern studies have found that atherosclerosis, venous thrombosis and cholelithiasis are closely related to hypercholesterolemia. High density lipoproteins help to eliminate cholesterol from cells. Therefore, the determination of HDL-C is clinically significant.

HDL-C content determination is now routinely performed in clinical trials. The recommendations of the american cholesterol education program for HDL-C determination indicate: HDL-C is negatively correlated with coronary heart disease in a narrow concentration range, the limit of increased risk of coronary heart disease is at the low end of the HDL-C concentration range, and the accuracy of HDL-C determination is very important because small errors in determination have a relatively large effect.

The existing measurement methods include a first generation direct measurement method, a second generation magnetic dextran sulfate method and a third generation homogeneous measurement method; methods frequently used in the prior art are homogeneous assays, including PEG-modified enzymatic methods, selective inhibition methods, antibody methods, and hydrogen peroxide enzymatic methods. The PEG modification method needs to modify enzyme, so that the activity of the enzyme is reduced to a great extent, and the cost is increased; the specific antibody in the antibody method is expensive, has a plurality of reaction steps and is poor in practicability; the key to the selective inhibition method is the selection of the surfactant, but the use of the surfactant is difficult, the masking inhibition of non-high density lipoprotein is incomplete, and the release of high density lipoprotein is incomplete. The accuracy of HDL-C detection is retained by the above methods.

In view of the above, the prior art is obviously inconvenient and disadvantageous in practical use, and needs to be improved.

Disclosure of Invention

In view of the above-mentioned drawbacks, the present invention aims to provide a high-accuracy high-density lipoprotein cholesterol detection kit, which can improve the accuracy of HDL-C detection, solve the problem of low accuracy of HDL-C detection reagents in the prior art, and provide reliable basis for diagnosis and prevention of related diseases.

In order to achieve the above object, the present invention provides a high-density lipoprotein cholesterol detection kit with high accuracy, comprising a first reagent and a second reagent.

The first reagent comprises the following components: 0.02-0.8mol/L of buffer solution I, 2-18 g/L of agglutinant, 0.5-5.0mmol/L of color developing agent, 0.5-2.0g/L of first preservative, 1.0-12.0ml/L of surfactant I, 8.0-25.0ml/L of surfactant II, 22-160 g/L of protective agent and 2-100 Ku/L of enzyme I.

The second reagent comprises the following components: 0.02-0.8mol/L of buffer solution II, 1.0-10.0mmol/L of color assistant, 0.5-2.0g/L of second preservative, 22-160 g/L of protective agent, 8.0-25.0ml/L of surfactant II and 1.0-50.0Ku/L of enzyme II.

The enzyme I comprises cholesterol oxidase and peroxidase, wherein the cholesterol oxidase is derived from pseudomonas; the enzyme II is cholesterol esterase and is derived from schizomycete or pseudomonas.

In the first reagent, the concentration ratio of the surfactant I to the surfactant II is as follows: 1: 3-4.

According to the high-accuracy high-density lipoprotein cholesterol detection kit, the buffer solution I and the buffer solution II are any one or more of 3-morpholine propanesulfonic acid, 1, 4-piperazine diethylsulfonic acid, 4-hydroxyethyl piperazine ethanesulfonic acid and morpholine ethanesulfonic acid monohydrate.

According to the high-accuracy high-density lipoprotein cholesterol detection kit, the pH values of the buffer solution I and the buffer solution II are both 6.5-7.5.

According to the high-accuracy high-density lipoprotein cholesterol detection kit, the agglutinant comprises divalent metal ions, α -cyclodextrin sulfate and dextran sulfate sodium salt.

According to the high-accuracy high-density lipoprotein cholesterol detection kit, the mass concentration of the divalent metal ions is 1.0-10.0 g/L, the divalent metal ions are any one of magnesium sulfate, zinc sulfate or magnesium chloride, the mass concentration of the α -cyclodextrin sulfate is 0.4-4.0 g/L, and the mass concentration of the dextran sulfate sodium salt is 0.5-5.0 g/L.

According to the high-accuracy high-density lipoprotein cholesterol detection kit, the color developing agent is any one of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3' 5-dimethoxyaniline sodium salt.

According to the high-accuracy high-density lipoprotein cholesterol detection kit, the first preservative and the second preservative are any one or more of sodium azide, gentamicin and Proclin 300.

According to the high-accuracy high-density lipoprotein cholesterol detection kit, the surfactant I is any one or more of Pluronic L127, Emuleng220, EmulengA90, EmulengLS114, NP-7 and Pluronic F-88.

According to the high-accuracy high-density lipoprotein cholesterol detection kit, the surfactant II is any one or more of Emulgen B66, EmulengLS110, Emuleng709, PininnD 6512, PininnYE 309, NoigenEA-157, NoigenEA-167 and NoigenEA-177.

According to the high-accuracy high-density lipoprotein cholesterol detection kit, in the enzyme I, the active concentrations of the cholesterol oxidase and the peroxidase are both 1-50 Ku/L.

The invention aims to provide a high-accuracy high-density lipoprotein cholesterol detection kit, which is used for screening cholesterol oxidase from pseudomonas and cholesterol esterase from schizomycete or pseudomonas as enzymes for detecting the HDL-C content, so that the reaction of HDL-C is more accurate and thorough, the cholesterol content in high-density lipoprotein can be effectively selected and detected, the concentration ratio of a surfactant I and a surfactant II is limited on the basis of the selected enzymes, the interference of low-density lipoprotein, very-low-density lipoprotein and chylomicron on the HDL-C detection is effectively reduced, and the accuracy of determining the HDL-C content is further improved. In conclusion, the beneficial effects of the invention are as follows: improves the accuracy of HDL-C detection, solves the problem of low detection accuracy of HDL-C detection reagents in the prior art, and provides reliable basis for diagnosis and prevention of related diseases.

Drawings

FIG. 1 is a reaction curve in the detection process of example 1 of the present invention;

FIG. 2 is a graph showing the reaction curve during the test of comparative example 1 according to the present invention;

FIG. 3 is a graph showing the reaction curve during the test of comparative example 2 according to the present invention;

FIG. 4 is a graph showing the reaction curve during the test of comparative example 3 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. In the present invention, high density lipoprotein cholesterol is abbreviated as HDL-C.

The invention provides a high-accuracy high-density lipoprotein cholesterol detection kit, which comprises a first reagent and a second reagent.

The first reagent comprises the following components: 0.02-0.8mol/L of buffer solution I, 2-18 g/L of agglutinant, 0.5-5.0mmol/L of color developing agent, 0.5-2.0g/L of first preservative, 1.0-12.0ml/L of surfactant I, 8.0-25.0ml/L of surfactant II, 22-160 g/L of protective agent and 2-100 Ku/L of enzyme I;

the second reagent comprises the following components: 0.02-0.8mol/L of buffer solution II, 1.0-10.0mmol/L of color assistant, 0.5-2.0g/L of second preservative, 22-160 g/L of protective agent, 8.0-25.0ml/L of surfactant II and 1.0-50.0Ku/L of enzyme II.

The buffer solution I and the buffer solution II are any one or more of 3-morpholine propanesulfonic acid, 1, 4-piperazine diethylsulfonic acid, 4-hydroxyethyl piperazine ethanesulfonic acid and morpholine ethanesulfonic acid monohydrate; the pH values of the buffer solution I and the buffer solution II are both 6.5-7.5.

The agglomerant comprises divalent metal ions, α -cyclodextrin sulfate and dextran sulfate sodium salt, wherein the mass concentration of the divalent metal ions is 1.0-10.0 g/L, the divalent metal ions comprise magnesium sulfate, zinc sulfate and magnesium chloride, the mass concentration of α -cyclodextrin sulfate is 0.4-4.0 g/L, and the mass concentration of the dextran sulfate sodium salt is 0.5-5.0 g/L.

The color developing agent is any one of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3' 5-dimethoxyaniline sodium salt.

The first preservative and the second preservative are any one or more of sodium azide, gentamicin and Proclin 300.

The surfactant I is any one or more of Pluronic L127, Emuleng220, EmulengA90, EmulengLS114, NP-7 and Pluronic F-88. The surfactant I can preferentially dissolve the high-density lipoprotein, and in the present invention, the surfactant I is preferably an Emuleng series or a Pluronic series.

The surfactant II is any one or more of Emulgen B66, EmulengLS110, Emuleng709, PininnD 6512, PininnYE 309, NoigenEA-157, NoigenEA-167, and NoigenEA-177. The surfactant II is capable of inhibiting the solubilization of lipoproteins other than high-density lipoprotein, and is preferably Emulgen series or Pinin series.

Since the trade names of several surfactants I and II are all english, and there is no chinese trade name, the english trade name is used directly in the present invention.

The enzyme I comprises cholesterol oxidase and peroxidase, wherein the cholesterol oxidase is derived from pseudomonas, and the peroxidase is derived from horseradish; the active concentrations of the cholesterol oxidase and the peroxidase are both 1-50 Ku/L. Enzyme II is a cholesterol esterase derived from schizonts or Pseudomonas.

The protective agent comprises 2.0-15.0 g/L bovine serum albumin and 20.0-150.0 g/L sucrose.

The accuracy of the detection kit is verified, the detection experiment of the high-density lipoprotein is carried out, and in the experimental process, the sample treatment method comprises the following steps: taking a mixture (CM + VLDL) of pure high-density lipoprotein (HDL), low-density lipoprotein (LDL), chylomicron and very-low-density lipoprotein, and treating the three samples with phosphate buffer solution to make the volume concentration of cholesterol in the three samples 100 mg/dL; wherein the high-density lipoprotein is an analyte to be detected, and the mixture of the low-density lipoprotein, the chylomicron and the very low-density lipoprotein is an interferent for detecting HDL-C. The standard substance is a qualified Landau lipid calibration solution generally accepted in the market. The pH value of the phosphate buffer solution is 6-7, and the molar concentration is 0.05 mol/L. The invention also needs to set a blank control experiment.

The AU400 full-automatic biochemical analyzer is used for respectively detecting the samples, the standard products and the blanks, and the specific detection method comprises the following steps:

adding a first reagent and a sample into a measuring tube, uniformly mixing the first reagent and the sample, incubating for 3-7 min at the temperature of 34-40 ℃, and reading a light absorption value A1; and then adding a second reagent into the measuring tube, uniformly mixing the second reagent, the first reagent and the sample, preserving the heat for 3-7 min at the temperature of 34-40 ℃, reading the light absorption value A2, and calculating the delta A which is A2-A1.

Calculating the content of HDL-C:

the detection adopts a two-point end point method, the wavelength used for detection is 546nm, the reaction direction is a positive direction, the addition amount of the sample is 2-4 mu l, the addition amount of the first reagent is 220-260 mu l, and the addition amount of the second reagent is 70-90 mu l.

In order to verify the accuracy of the detection kit of the present invention, the detection kit was prepared according to the above-mentioned components, and the following examples were set up. Meanwhile, the source of the enzyme in the detection kit of the invention is changed, and a plurality of comparative examples are set as follows. The concentration of HDL-C in a sample is then measured according to the measuring method of the present invention using the measuring kits of the above examples and comparative examples.