A lipoprotein subfraction test kit and a lipoprotein subfraction test system

Abstract

The application provides a lipoprotein subfraction detection kit and a lipoprotein subfraction detection system, and belongs to the technical field of medical examination.The lipoprotein subfraction detection kit provided by the application comprises reagent R1, which comprises: a first buffer, a surfactant, magnesium chloride, a first protective agent, a first preservative, 4-aminoantipyrine, lipoprotein esterase, cholesteryl esterase and ascorbate oxidase; reagent R2, which comprises: a second buffer, a second protective agent, a second preservative, NAD + , glycerol dehydrogenase; and reagent R3, which comprises: a third buffer, a third protective agent, a third preservative, cholesteryl oxidase, peroxidase and a chromogenic substance.The lipoprotein subfraction detection kit provided by the application can be combined with the lipoprotein subfraction detection system provided by the application to realize the synchronous detection of cholesterols and triglycerides, and further realize the detection of 20 lipoprotein subfractions.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, and more specifically, to a lipoprotein subfraction detection kit and a lipoprotein subfraction detection system. Background Technology

[0002] Lipoproteins, as important biomolecules, are mainly responsible for transporting lipids in the human body. Lipoproteins are composed of cholesterol and triglycerides. Based on their density, lipoproteins are classified into chylomicrons (CM), very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). Clinically, cholesterol levels in lipoproteins are typically measured to assess a patient's risk of cardiovascular disease. However, recent studies have shown significant heterogeneity in lipoproteins. For example, LDL can be divided into a high-risk dense subtype and a low-risk light subtype, and HDL also has sub-fractions with significantly different functions. Therefore, methods that only measure total cholesterol levels are insufficient for accurately assessing cardiovascular disease risk. There is a need to develop new methods that can simultaneously detect both cholesterol and triglyceride sub-fractions of lipoproteins.

[0003] Currently, there are few methods for detecting lipoprotein subfractions. Clinically applicable methods include electrophoresis, nuclear magnetic resonance (NMR), and vertical density gradient centrifugation (VAP). Electrophoresis first separates lipoproteins by electrophoresis, then stains total lipoproteins and cholesterol using different methods, and finally collects data by scanning. Staining methods detect the cholesterol content of lipoproteins and their subfractions. However, this method is cumbersome and cannot detect triglycerides within the lipoprotein subfraction. Furthermore, electrophoretic separation and staining scanning have accuracy issues, making it unsuitable as the preferred clinical method. NMR can be used to detect the particle concentration of total lipoproteins and lipoprotein subfractions; however, NMR... The expensive testing instruments and high operator skill requirements make the clinical application of MRI extremely difficult. VAP, a novel lipid testing method developed in the United States in the 1990s, first uses ultracentrifugation to separate lipoproteins, then uses a VAP instrument to detect continuous lipoprotein cholesterol, obtaining lipoprotein cholesterol images. The cholesterol content of each lipoprotein component is then calculated. This technology, utilizing a vertical rotor, can accurately detect the cholesterol content of lipoproteins and lipoprotein subfractions in extremely small samples, showing broad application prospects. However, the lipoprotein subfractions detected by the VAP method are still limited to cholesterol, and it cannot simultaneously detect triglycerides, another component of lipoproteins.

[0004] Therefore, there is a need to develop a method for simultaneously detecting lipoprotein subfractions cholesterol and triglycerides on a single system to achieve the detection of lipoprotein subfractions. Summary of the Invention

[0005] The technical problem to be solved by this invention is: how to realize the detection of lipoprotein subfraction cholesterol and lipoprotein subfraction triglycerides in a single system.

[0006] To achieve the above objectives, the first aspect of the present invention provides a lipoprotein subfraction detection kit, comprising:

[0007] Reagent R1, wherein reagent R1 comprises: a first buffer solution, a surfactant, magnesium chloride, a first protectant, a first preservative, 4-aminoantipyrine, lipoprotein lipase, cholesterol lipase, and ascorbic acid oxidase;

[0008] Reagent R2, wherein reagent R2 comprises: a second buffer solution, a second protective agent, a second preservative, and NAD+. + glycerol dehydrogenase;

[0009] Reagent R3, wherein reagent R3 comprises: a third buffer solution, a third protectant, a third preservative, cholesterol oxidase, peroxidase, and chromogen;

[0010] The diluent includes a fourth preservative and potassium bromide;

[0011] The density liquid includes a fifth preservative and potassium bromide.

[0012] In the lipoprotein subfraction detection kit provided by the present invention, reagent R1 is a lysis reagent, reagent R2 is a triglyceride detection reagent, and reagent R3 is a cholesterol detection reagent.

[0013] In existing triglyceride detection methods, the GPO-PAP method is commonly used. In this method, the final detection principle is that hydrogen peroxide reacts with 4-aminoantipyrine and phenol under the action of catalase to generate red benzoquinone imine. The generated benzoquinone imine can be measured colorimetrically using a spectrophotometer. However, since the detection wavelength of the GPO-PAP method is 500~600nm, this wavelength will mask the color reaction of cholesterol, making it impossible for existing technology to simultaneously detect lipoprotein subfraction cholesterol and lipoprotein subfraction triglycerides.

[0014] This invention utilizes the glycerol dehydrogenase in reagent R2 to oxidize glycerol, while simultaneously reducing NAD+. +The NADH is reduced to NADH, and the concentration of triglycerides can be further calculated by detecting changes in the absorbance of NADH. In the above method, the detection wavelength of NADH is 340 nm, which is not in the same range as the detection wavelength of cholesterol, so the detection results of the two will not interfere with each other. Moreover, the function of glycerol dehydrogenase is not interfered with by high concentrations of potassium bromide, enabling simultaneous detection of lipoprotein subfraction triglycerides and lipoprotein subfraction cholesterol.

[0015] Cholesterol oxidase in reagent R3 can oxidize cholesterol to hydrogen peroxide. In the presence of peroxidase and chromogen, hydrogen peroxide can generate a blue-violet compound, which can be colorimetrically determined by a spectrophotometer at a wavelength of 500-600 nm.

[0016] Preferably, the first buffer is selected from any one of PIPES buffer, HEPES buffer, MOPS buffer, TES buffer, and Tris buffer, and the pH of the first buffer is 5 to 9.

[0017] The second buffer is selected from any one of PIPES buffer, HEPES buffer, MOPS buffer, TES buffer, and Tris buffer, and the pH of the second buffer is 5 to 9.

[0018] The third buffer is selected from any one of PIPES buffer, HEPES buffer, MOPS buffer, TES buffer, and Tris buffer, and the pH of the third buffer is 5 to 9.

[0019] Preferably, the surfactant is Triton X-100 or a mixture of Triton X-100 and alkylphenol polyoxyethylene ether, wherein the alkylphenol polyoxyethylene ether is selected from any one or more of alkylphenol polyoxyethylene ether, heptaphenol polyoxyethylene ether, octylphenol polyoxyethylene ether, nonylphenol polyoxyethylene ether, decylphenol polyoxyethylene ether, and dodecyl polyoxyethylene ether.

[0020] To improve the reaction rate, this invention introduces another surfactant, alkylphenol polyoxyethylene ether, into reagent R1. Alkylphenol polyoxyethylene ether can effectively improve the reaction rate. After adding alkylphenol polyoxyethylene ether, the pyrolysis reaction can be completed within 60 seconds.

[0021] Preferably, the first preservative is selected from any one or more of sodium azide, PC300, and PC950;

[0022] The second preservative is selected from any one or more of sodium azide, PC300, and PC950;

[0023] The third preservative is selected from any one or more of sodium azide, PC300, and PC950;

[0024] The fourth preservative is selected from any one or more of sodium azide, PC300, and PC950;

[0025] The fifth preservative is selected from any one or more of sodium azide, PC300, and PC950.

[0026] Preferably, the colorant is TOOS and / or TODB.

[0027] In the lipoprotein subfraction detection kit provided by this invention, reagent R1 serves as a lysis reagent, which can rapidly and effectively lyse lipoproteins through the presence of a special surfactant or combination of surfactants, enabling the lipoproteins to release their contained triglycerides and cholesterol esters. Simultaneously, the lipoprotein lipase in reagent R1 can further hydrolyze the triglycerides released by the lipoproteins into glycerol and free fatty acids; the cholesterol lipase in reagent R1 can further hydrolyze the cholesterol esters released by the lipoproteins into free cholesterol.

[0028] In the lipoprotein subfraction detection kit provided by this invention, the glycerol produced by the hydrolysis of triglycerides by lipoprotein lipase in reagent R1 can be oxidized by glycerol dehydrogenase in reagent R2, thereby reducing the NAD content in reagent R2. + It is reduced to NADH, which has absorbance at 340 nm. By detecting the change in absorbance of the sample at 340 nm, the glycerol content can be quantitatively measured, and then the triglyceride content can be calculated.

[0029] In the lipoprotein subfraction detection kit provided by this invention, free cholesterol in the sample after being reacted by reagent R1 or detected by reagent R2 can be oxidized by cholesterol oxidase in reagent R3 to generate hydrogen peroxide. Hydrogen peroxide can generate a blue-violet compound in the presence of peroxidase and chromogen. This blue-violet compound has absorbance at 500-600 nm. By detecting the change in absorbance of the sample at 500-600 nm, the cholesterol content can be quantitatively calculated.

[0030] Therefore, the content of lipoprotein subfractions can be further deduced from the content of triglycerides and cholesterol.

[0031] The second aspect of the present invention provides a lipoprotein subfraction detection system, which, in conjunction with the lipoprotein subfraction detection kit provided in the first aspect, enables rapid detection of lipoprotein subfractions.

[0032] The lipoprotein subfraction detection system includes:

[0033] Controller used to control the lipoprotein subfraction detection system;

[0034] Sample containers used to store liquid samples;

[0035] The first reagent container used to store reagent R1;

[0036] The first reaction module is used for mixing and incubating the first reagent and liquid sample;

[0037] The sample container and the first reaction module are connected by pipes, and the first reagent container and the first reaction module are connected by pipes.

[0038] The second reagent container used to store reagent R2;

[0039] The second reaction module is used for mixing and incubating the second reagent and the effluent from the first reaction module;

[0040] The first reaction module and the second reaction module are connected by pipes, and the second reagent container and the second reaction module are connected by pipes.

[0041] The third reagent container used to store reagent R3;

[0042] The third reaction module is used for mixing and incubating the third reagent and the effluent from the second reaction module;

[0043] The second reaction module and the third reaction module are connected by pipes, and the third reagent container and the third reaction module are connected by pipes.

[0044] Waste liquid container used to receive the effluent from the lipoprotein subcomponent detection system;

[0045] A controller used to control the various modules of the system, system temperature, and liquid flow rate. The controller is either communicatively connected or electrically connected to other modules.

[0046] Preferably, the lipoprotein subfraction detection system also includes

[0047] The first detector is used to detect the liquid flowing out of the first reaction module;

[0048] With a second detector used to detect the effluent from the second reaction module;

[0049] A third detector for detecting the effluent from the third reaction module.

[0050] As a preferred option, the lipoprotein subfraction detection system also includes:

[0051] A cleaning solution container for storing cleaning solution used to clean all the pipes of the lipoprotein subfraction detection system.

[0052] As a preferred option, the lipoprotein subfraction detection system also includes:

[0053] A controller used to control the various modules of the system, system temperature, and liquid flow rate.

[0054] The lipoprotein subfraction detection system and kit provided by this invention can detect total cholesterol (TCH), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), intermediate-density lipoprotein cholesterol (IDL-C), very low-density lipoprotein cholesterol (VLDL-C), high-density lipoprotein 2 cholesterol (HDL2-C), high-density lipoprotein 3 cholesterol (HDL3-C), small and dense low-density lipoprotein cholesterol (sdLDL-C), lipoprotein a cholesterol (Lpa-C), and lipoprotein remnant cholesterol (LPD). The study included the detection of RLP-C, total triglycerides (TG), high-density lipoprotein triglycerides (HDL-TG), low-density lipoprotein triglycerides (LDL-TG), intermediate-density lipoprotein triglycerides (IDL-TG), very low-density lipoprotein triglycerides (VLDL-TG), high-density lipoprotein 2 triglycerides (HDL2-TG), high-density lipoprotein 3 triglycerides (HDL3-TG), small and dense low-density lipoprotein triglycerides (sdLDL-TG), lipoprotein α triglycerides (Lpa-TG), and lipoprotein remnant triglycerides (RLP-TG).

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] 1. This invention is the first to propose a mechanism that abandons the traditional GPO-PAP method and uses the glycerol dehydrogenase method and cholesterol oxidase method to achieve wavelength separation, ultimately realizing the accurate and simultaneous detection of lipoprotein subfraction triglycerides and lipoprotein subfraction cholesterol. It completely overcomes the defect of wavelength superposition interference in the traditional GPO-PAP method and also eliminates the influence of potassium bromide on triglyceride detection, providing an accurate assessment basis for cardiovascular disease and other risks.

[0057] 2. The kit provided by this invention enables lipoproteins to be cleaved within 60 seconds through the synergistic effect of Triton X-100 and alkylphenol polyoxyethylene ether, thereby improving the detection speed and efficiency of lipoprotein subfractions while ensuring reagent fluidity.

[0058] 3. The reagent kit and detection system provided by this invention can be used together with only an instrument with colorimetric analysis function to detect lipoprotein subfractions. Its cost is much lower than that of nuclear magnetic resonance method. At the same time, the reagent kit and detection system provided by this invention do not require highly skilled operators and can be widely used.

[0059] 4. The reagent kit and detection system provided by this invention are the first to achieve simultaneous detection of 20 lipoprotein subfractions of cholesterol and lipoprotein subfractions of triglycerides, including TCH, HDL-C, LDL-C, IDL-C, VLDL-C, HDL2-C, HDL3-C, sdLDL-C, Lpa-C, RLP-C, TG, HDL-TG, LDL-TG, IDL-TG, VLDL-TG, HDL2-TG, HDL3-TG, sdLDL-TG, Lpa-TG, and RLP-TG. The detection method is simple and rapid, which can support personalized cardiovascular risk assessment and has broad application prospects in precision medicine. Attached Figure Description

[0060] Figure 1 A schematic diagram of the lipoprotein subfraction detection system provided for a specific embodiment of the present invention.

[0061] Explanation of reference numerals in the attached figures:

[0062] 1. Sample container; 2. First reagent container; 3. First reaction module; 4. Second reagent container; 5. Second reaction module; 6. Third reagent container; 7. Third reaction module; 8. Waste liquid container; 9. Controller; 10. First detector; 11. Second detector; 12. Third detector; 13. First three-way regulating valve; 14. Second three-way regulating valve; 15. Third three-way regulating valve; 16. Cleaning solution container. Detailed Implementation

[0063] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the claims of the present invention.

[0064] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0065] As described in the background section, existing electrophoresis methods for lipoprotein subfraction detection have issues with detection accuracy, nuclear magnetic resonance has extremely high costs, and the VAP method can only detect cholesterol levels in lipoprotein subfractions at a time, and cannot detect triglyceride levels in lipoprotein subfractions.

[0066] In view of this, a specific embodiment of the present invention provides a lipoprotein subfraction detection kit. The kit includes:

[0067] Reagent R1 for lysing lipoproteins includes: first buffer, surfactant, magnesium chloride, first protectant, first preservative, 4-aminoantipyrine, lipoprotein lipase, cholesterol lipase, and ascorbic acid oxidase.

[0068] Reagent R2 for detecting triglyceride levels includes: second buffer, second protectant, second preservative, and NAD+. + glycerol dehydrogenase;

[0069] Reagent R3 used to detect cholesterol levels includes: third buffer, third protectant, third preservative, cholesterol oxidase, peroxidase, and chromogen.

[0070] The diluent includes a fourth preservative and potassium bromide;

[0071] The density liquid includes a fifth preservative and potassium bromide.

[0072] In the above embodiments, the diluent and density solution are used to create a density gradient in the lipoprotein sample, wherein the density of the diluent is 1.15~1.25 g / mL and the density solution is 1.0~1.1 g / mL.

[0073] In the above embodiments, the role of reagent R1 is to cleave lipoproteins, breaking them down into triglycerides and cholesterol esters. Lipoprotein lipases hydrolyze triglycerides into glycerol and free fatty acids, and cholesterol lipases hydrolyze cholesterol esters into free cholesterol.

[0074] The role of reagent R2 is to oxidize glycerol through glycerol dehydrogenase, thereby increasing NAD+. + The glycerol is reduced to NADH. The absorbance change of the sample at 340 nm is detected by a spectrophotometer. The glycerol content can be quantitatively determined by reagent R2, and then the triglyceride content can be calculated.

[0075] The role of reagent R3 is to react cholesterol oxidase with the reaction solution of reagent R1 or with free cholesterol in the sample after being detected by reagent R2 to generate hydrogen peroxide. In the presence of peroxidase and chromogen, hydrogen peroxide generates a blue-violet compound. This blue-violet compound has absorbance at 500-600 nm. By detecting the change in absorbance of the sample at 500-600 nm, the cholesterol content can be quantitatively calculated.

[0076] In the lipoprotein subfraction detection kit provided in the specific embodiments of the present invention, all reagents should contain an excess of enzymes to ensure that the reaction proceeds smoothly.

[0077] In the above embodiments, the first buffer is selected from any one of PIPES buffer, HEPES buffer, MOPS buffer, TES buffer, and Tris buffer with a salt concentration of 10~500mM, and the pH of the buffer is 5~9.

[0078] The second buffer is selected from any one of PIPES buffer, HEPES buffer, MOPS buffer, TES buffer, and Tris buffer, and the pH of the buffer is 5 to 9;

[0079] The third buffer solution is selected from any one of PIPES buffer, HEPES buffer, MOPS buffer, TES buffer, and Tris buffer, and the pH of the buffer solution is 5 to 9.

[0080] More specifically, in the above embodiments, the salt concentration in the first buffer solution is preferably 50-250 mM; the salt concentration in the second buffer solution is preferably 50-250 mM; and the salt concentration in the third buffer solution is preferably 50-250 mM.

[0081] In the above embodiments, the surfactant is Triton X-100 or a mixture of Triton X-100 and alkylphenol polyoxyethylene ether, wherein the alkylphenol polyoxyethylene ether is selected from any one or more of alkylphenol polyoxyethylene ether, heptaphenol polyoxyethylene ether, octylphenol polyoxyethylene ether, nonylphenol polyoxyethylene ether, decylphenol polyoxyethylene ether, and dodecyl polyoxyethylene ether.

[0082] More specifically, in the above embodiments, the concentration of Triton X-100 in reagent R1 is preferably 0.01% to 5.0% by mass, and most preferably 0.1% to 1% by mass. If the amount of Triton X-100 used is too low, the lipoprotein will not be completely dissolved, affecting the accuracy of the detection results. If the amount used is too high, the viscosity of the reagent will increase, affecting the flowability of the reagent and the accuracy of the detection results.

[0083] More specifically, in the above embodiments, the concentration of alkylphenol polyoxyethylene ether in reagent R1 is preferably 0.01% to 1% by mass, and most preferably 0.05% to 0.2% by mass.

[0084] More specifically, in the above embodiments, if commercial raw materials are used, BASF Lutensol AP series alkylphenol polyoxyethylene ethers are preferred.

[0085] In the above embodiments, magnesium chloride, as an enzyme activator, is present in reagent R1 at a concentration of 1-200 mM.

[0086] In the above embodiments, the first protective agent is selected from any one or more of sugar protective agents, marine animal gelatin, and BSA;

[0087] The second preservative is selected from any one or more of carbohydrate preservatives, marine animal gelatin, and BSA;

[0088] The third protective agent is selected from any one or more of sugar protective agents, marine animal gelatin, and BSA.

[0089] More specifically, in the above embodiments, the sugar protectant is selected from any one or more of sucrose, mannitol, trehalose, and sorbitol. If a sugar protectant is used as a component of the protectant, the concentration of the sugar protectant in the reagent is preferably 1~100 g / L, and most preferably 5~50 g / L.

[0090] More specifically, in the above embodiments, the marine animal gelatin is selected from any one or more of cod bone gelatin, squid skin gelatin, and mackerel skin gelatin. When marine animal gelatin is used as a component of the preservative, the concentration of marine animal gelatin in the reagent is preferably 0.1~20 g / L, and most preferably 0.5~10 g / L.

[0091] More specifically, in the above embodiments, if BSA is used as a component of the protective agent, the concentration of BSA in the reagent is 0.1~20 g / L, and most preferably 0.5~10 g / L.

[0092] In the above embodiments, the first preservative is selected from any one or more of sodium azide, PC300, and PC950, and the concentration of the first preservative in reagent R1 is preferably 0.01% to 1%, and most preferably 0.02% to 0.2%.

[0093] The second preservative is selected from any one or more of sodium azide, PC300, and PC950. The concentration of the second preservative in reagent R2 is preferably 0.01% to 1%, and most preferably 0.02% to 0.2%.

[0094] The third preservative is selected from any one or more of sodium azide, PC300, and PC950. The concentration of the third preservative in reagent R3 is preferably 0.01% to 1%, and most preferably 0.02% to 0.2%.

[0095] The fourth preservative is selected from any one or more of sodium azide, PC300, and PC950, and the concentration of the third preservative in reagent R3 is preferably 0.01% to 1%, and most preferably 0.02% to 0.2%.

[0096] The fifth preservative is selected from any one or more of sodium azide, PC300, and PC950, and the concentration of the third preservative in reagent R3 is preferably 0.01% to 1%, and most preferably 0.02% to 0.2%.

[0097] In reagent R3 of the above embodiments, the chromogen is TOOS and / or TODB, and the concentration of the chromogen in reagent R3 is preferably 0.1~10mM, and most preferably 0.5~5mM.

[0098] In reagent R1 of the above embodiments, 4-aminoantipyrine is used as a chromogenic substance, and its concentration in reagent R1 is preferably 0.1~10mM, and most preferably 0.5~5mM.

[0099] In reagent R1 of the above embodiments, the concentration of lipoprotein lipase is preferably 0.2~20 KU / L, and most preferably 1~5 KU / L.

[0100] In reagent R1 of the above embodiments, the concentration of cholesterol esterase is preferably 0.2~20 KU / L, and most preferably 1~5 KU / L.

[0101] In reagent R1 of the above embodiments, the concentration of ascorbic acid oxidase is preferably 0.2~20 KU / L, and most preferably 1~5 KU / L.

[0102] In reagent R2 of the above embodiments, the concentration of glycerol dehydrogenase is 0.2~50 KU / L, most preferably 2~10 KU / L.

[0103] In reagent R3 of the above embodiments, the concentration of cholesterol oxidase is preferably 0.2~20 KU / L, and most preferably 1~5 KU / L.

[0104] In reagent R3 of the above embodiments, the concentration of peroxidase is preferably 0.2~20 KU / L, and most preferably 1~5 KU / L.

[0105] More specifically, the test samples for the kits provided in the above embodiments are selected from any one of whole blood, plasma, serum, or lipoprotein-containing biological buffer systems.

[0106] A specific embodiment of the present invention also provides a lipoprotein subfraction detection system.

[0107] See Figure 1 The lipoprotein subfraction detection system provided in the specific embodiments of the present invention includes:

[0108] Controller 9 for controlling the lipoprotein subfraction detection system;

[0109] Sample container 1 for storing liquid samples;

[0110] First reagent container 2 for storing reagent R1;

[0111] The first reaction module 3, used for mixing and incubating reagent R1 and liquid samples, preferably has temperature control functions such as water bath, sand bath, metal bath, and gas bath.

[0112] The sample container 1 and the first reaction module 3 are connected by a pipe, and the first reagent container 2 and the first reaction module 3 are connected by a pipe.

[0113] The second reagent container 4 is used to store reagent R2;

[0114] The second reaction module 5 is used for mixing and incubating reagent R2 and the effluent from the first reaction module 3. Preferably, the second reaction module 5 has temperature control functions such as water bath, sand bath, metal bath, and air bath.

[0115] The first reaction module 3 and the second reaction module 5 are connected by pipes, and the second reagent container 4 and the second reaction module 5 are connected by pipes.

[0116] The third reagent container 6 is used to store reagent R3;

[0117] The third reaction module 7, used for mixing and incubating reagent R3 and the effluent from the second reaction module 5, preferably has temperature control functions such as water bath, sand bath, metal bath, and gas bath.

[0118] The second reaction module 5 and the third reaction module 7 are connected by pipes, and the third reagent container 6 and the third reaction module 7 are connected by pipes.

[0119] Waste liquid container 8 for receiving the effluent from the lipoprotein subfraction detection system;

[0120] The controller 9 is communicatively or electrically connected to the first reaction module 3, the second reaction module 5, and the third reaction module 7, respectively, and is used to control the reaction temperature and the liquid inflow and outflow rate. The controller 9 is also communicatively or electrically connected to the sample container 1, the first reagent container 2, the second reagent container 4, and the third reagent container 6, respectively, and is used to control the liquid flow rate.

[0121] A first detector 10 is used to detect the liquid flowing out of the first reaction module 3. The first detector 10 is connected to the pipeline of the first reaction module 3.

[0122] The second detector 11 is used to detect the liquid flowing out of the second reaction module 5. The second detector 11 is connected to the pipeline of the second reaction module 5.

[0123] The third detector 12 is used to detect the effluent from the third reaction module 7, and the third detector 12 is connected to the pipeline of the third reaction module 7.

[0124] In the above embodiments, the first detector 10, the second detector 11, and the third detector 12 are all instruments with colorimetric analysis functions, and can be spectrophotometers, microplate readers, or microplate detectors.

[0125] In the above embodiments, the waste liquid container 8 can be connected to the first detector 10, the second detector 11, and the third detector 12 by a pipeline for receiving the detection waste liquid.

[0126] The first detector 10, the second detector 11, and the third detector 12 are all connected to the controller 9 via communication or electrical connection. The controller 9 can be used to control and adjust the absorbance of the detectors, and at the same time, receive the detection data of the relevant detectors for subsequent analysis and calculation.

[0127] In a specific embodiment of the present invention, the first reaction module 3, the first detector 10, the second reaction module 5, the second detector 11, the third reaction module 7, and the third detector 12 are arranged in series. By arranging them in series, the sample volume of the lipoprotein subcomponent detection system can be effectively reduced.

[0128] In the above embodiment, the first reagent container 2 and the sample container 1 share a pipe to enter the first reaction module 3. A first three-way regulating valve 13 is provided at the liquid junction for mixing the liquid sample and the first reagent. Similarly, a second three-way regulating valve 14 is provided at the liquid junction of the first reaction module 3, the second reagent container 4, and the second reaction module 5 for mixing the liquids, and a third three-way regulating valve 15 is provided at the liquid junction of the second reaction module 5, the third reagent container 6, and the third reaction module 7 for mixing the liquids.

[0129] In the above embodiments, the lipoprotein subcomponent detection kit also includes a cleaning solution container 16 for storing cleaning solution. The controller 9 is communicatively or electrically connected to the cleaning solution container 16 to control the flow rate of the cleaning solution, which can clean the pipelines of the lipoprotein subcomponent detection system.

[0130] The lipoprotein subfraction detection system provided in this invention may include the following steps to achieve quantitative detection of lipoprotein subfractions:

[0131] S1: The sample is first treated with diluent and density solution, and after ultracentrifugation, it is transferred to sample container 1. The liquid sample in sample container 1 is fully mixed with reagent R1 in the first reagent container 2 in the pipeline or the first reaction module 3, and then incubated in the first reaction module 3 to complete the relevant enzymatic reaction.

[0132] S2: After the sample is incubated in the first reaction module 3, the first detector 10 detects its absorbance. The detection wavelength is set to 340nm, which is used to quantitatively determine the blank absorbance of triglyceride content.

[0133] S3: After the sample has been incubated in the first reaction module 3, it is thoroughly mixed with reagent R2 in the second reagent container 4 in the pipe or the second reaction module 5. The mixture is then incubated in the second reaction module 5 to complete the relevant enzymatic reaction.

[0134] S4: After the sample is incubated in the second reaction module 5, the second detector 11 detects its absorbance. The detection wavelength is set to 340nm and 500~600nm. The 340nm wavelength is used for subsequent quantitative determination of triglyceride content, and the 500~600nm wavelength is used for subsequent quantitative determination of cholesterol and its blank absorbance.

[0135] S5: After the sample has been incubated in the second reaction module 5, it is thoroughly mixed with reagent R3 in the third reagent container 6 in the pipe or the third reaction module 7. The mixture is then incubated in the third reaction module 7 to complete the relevant enzymatic reaction.

[0136] S6: After the sample is incubated in the third reaction module 7, the third detector 12 detects its absorbance at a wavelength of 500~600nm, which is used to quantitatively determine the absorbance after the reaction of cholesterol content.

[0137] The lipoprotein triglyceride and cholesterol profiles are obtained by using the second detector 11 and the third detector 12. The triglyceride and cholesterol contents are then calculated using specialized software.

[0138] The technical solutions of the present invention are further described below through specific embodiments. Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. In some cases, terms with conventional meanings are limited herein for clarification or ease of reference, and such limitations should not be construed as indicating a significant difference from conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and have been employed by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments shall be performed according to the manufacturer's instructions and parameters.

[0139] Example 1

[0140] Feasibility Study on Lipoprotein Subfraction Detection System

[0141] In this embodiment, the structural diagram of the lipoprotein subfraction detection system is as follows: Figure 1 As shown, the first reagent container is loaded with reagent R1, the second reagent container is loaded with reagent R2, and the third actual container is loaded with reagent R3.

[0142] The components of reagent R1 are: 100mM HEPES buffer at pH 6.8, 0.05% Triton X-100, 10g / L magnesium chloride, 1g / L BSA, 2g / L squid skin gelatin, 0.05% PC300, 0.05% sodium azide, 2mM 4-aminoantipyrine, 2KU / L lipoprotein lipase, 2KU / L cholesterol lipase, and 2KU / L ascorbic acid oxidase.

[0143] The components of reagent R2 are: 250 mM MOPS buffer (pH 7.2), 1 g / L sucrose, 2 g / L BSA, 0.1% sodium azide, and 1 mM NAD. + 3 KU / L of glycerol dehydrogenase.

[0144] The components of reagent R3 are: 50 mM MTE buffer at pH 8.0, 0.1 g / L BSA, 1 g / L mackerel skin gelatin, 10 g / L trehalose, 0.02% PC950, 2 KU / L cholesterol oxidase, 2 KU / L peroxidase, 0.1 mM TOOS and 0.1 mM TODB.

[0145] The diluent consists of 0.05% PC300 by mass and 336.31 g / L potassium bromide.

[0146] The composition of the density solution is: 0.05% PC300 by mass and 36.31 g / L potassium bromide.

[0147] Take 50 μL of serum and add 1950 μL of diluent, mix well to obtain serum diluent. Then take a 5.1 mL Polyallomer quick-seal tube, add 3800 μL of density solution, and then slowly add 1200 μL of serum diluent from the bottom. Place the quick-seal tube in an ultracentrifuge and centrifuge at the following parameters: speed = 65000 rpm, ω 2 t=6.6×10 10 rad 2 / sec, temperature=23℃, acceleration=6, deceleration=6.

[0148] The centrifuged samples were placed in the sample container of the lipoprotein subfraction detection system for testing. The system parameters were: sample flow rate 1.0 ml / min, reagent R1 flow rate 0.5 ml / min, reagent R2 flow rate 0.5 ml / min, and reagent R3 flow rate 0.5 ml / min. The test results are shown in Table 1.

[0149] After centrifugation, the samples were placed on a fully automated biochemical analyzer and tested using commercially available T-CH and TG detection kits. Serum samples were assigned values ​​with T-CH and TG targets. The relative deviations between the measured and assigned values ​​of the lipoprotein subfraction system were compared. The test results are shown in Table 1.

[0150] Table 1

[0151]

[0152] As shown in Table 1, the lipoprotein subfraction detection system provided by this invention exhibits good consistency between the measured values ​​and the target values, with deviations all within ±3%. This demonstrates the feasibility of the lipoprotein subfraction detection kit and system provided by this invention.

[0153] Example 2

[0154] Precision study

[0155] Prepare different diluents and density solutions according to Table 2.

[0156] Table 2

[0157]

[0158] Prepare different reagents R1 according to Table 3.

[0159] Table 3

[0160]

[0161] Prepare different reagents R2 according to Table 4.

[0162] Table 4

[0163]

[0164] Prepare different reagents R3 according to Table 5.

[0165] Table 5

[0166]

[0167] Integrate the reagents in Tables 2-4 according to Table 6.

[0168] Table 6

[0169]

[0170] In this embodiment, conventional serum with a T-CH target value of 3.4 mM and high-value serum with a measured value of 6.6 mM were selected as the precision detection serum, respectively.

[0171] Take 50 μL of serum and add 1950 μL of diluent, mix well to obtain serum diluent. Then take a 5.1 mL Polyallomer quick-seal tube, add 3800 μL of density solution, and then slowly add 1200 μL of serum diluent from the bottom. Place the quick-seal tube in an ultracentrifuge and centrifuge at the following parameters: speed = 65000 rpm, ω 2 t=6.6×10 10 rad 2 / sec, temperature=23℃, acceleration=6, deceleration=6.

[0172] Centrifuged samples were placed in the sample container of the lipoprotein subfraction detection system for testing. The system parameters were: sample flow rate 1.0 ml / min, reagent R1 flow rate 0.5 ml / min, reagent R2 flow rate 0.5 ml / min, and reagent R3 flow rate 0.5 ml / min. Each serum sample was tested ten times per reagent group, and the mean X, standard deviation S, and coefficient of variation CV were calculated.

[0173] The precision test results are shown in Tables 7 and 8. Table 7 shows the test results for lipoprotein subfraction triglycerides, and Table 8 shows the test results for lipoprotein subfraction cholesterol.

[0174] Table 7

[0175]

[0176] Table 8

[0177]

[0178] As can be seen from the results in Tables 7 and 8, the CV values ​​of the main items under each reagent combination provided in this embodiment are generally less than 5%. The CV values ​​of the sub-items are higher than 5% due to the smaller measured values, but all CV values ​​are below 10%. This shows that the lipoprotein subfraction detection kit provided by the present invention has good precision.

[0179] Example 3

[0180] Linear range study

[0181] Because this testing system tests a large number of items, high-linearity samples need to be prepared for each item. However, for lipoprotein subfraction cholesterol testing items, except for TCH, LDL-C has the highest linearity requirement and best represents the cholesterol linearity detection range of this testing system. Therefore, samples with LDL-C values ​​of around 10mM and TCH values ​​greater than 13mM were used for the linearity range study of lipoprotein subfraction cholesterol testing items. For lipoprotein subfraction triglyceride testing items, except for TG, VLDL-TG has the highest linearity requirement and best represents the lipoprotein subfraction cholesterol linearity detection range of this testing system. Therefore, samples with VLDL-TG values ​​of around 10mM and TG values ​​greater than 12mM were used for the linearity range study of triglyceride testing items.

[0182] The high-value serum samples were diluted to different concentrations, and linear range detection experiments were conducted using the sample processing methods described below and the reagent combinations in Table 5.

[0183] Take 50 μL of serum and add 1950 μL of diluent, mix well to obtain serum diluent. Then take a 5.1 mL Polyallomer quick-seal tube, add 3800 μL of density solution, and then slowly add 1200 μL of serum diluent from the bottom. Place the quick-seal tube in an ultracentrifuge and centrifuge at the following parameters: speed = 65000 rpm, ω 2 t=6.6×10 10 rad 2 / sec, temperature=23℃, acceleration=6, deceleration=6.

[0184] The centrifuged samples were placed in the sample container of the lipoprotein subfraction detection system for testing. The system parameters were: sample flow rate 1.0 ml / min, reagent R1 flow rate 0.5 ml / min, reagent R2 flow rate 0.5 ml / min, and reagent R3 flow rate 0.5 ml / min. The linear slope, intercept, and correlation coefficient for each item and each set of reagents were calculated. The results are shown in Tables 9 and 10, where Table 9 shows the detection results for high linear values ​​in the LDL category, and Table 10 shows the detection results for high linear values ​​in the VLDL category.

[0185] Table 9

[0186]

[0187] Table 10

[0188]

[0189] As shown in Tables 9 and 10, the lipoprotein subfraction detection kit and system provided by this invention exhibit good linearity for cholesterol subfractions when LDL-C values ​​are around 0-10 mM and TCH values ​​are around 0-13 mM, with a slope between 0.96 and 1.01, a small intercept, and R > 0.99. Similarly, for triglycerides subfractions, when VLDL-TG values ​​are around 0-10 mM and TG values ​​are around 0-12 mM, the slope also shows a small intercept, with R > 0.99. Therefore, the lipoprotein subfraction detection kit and system provided by this invention demonstrate good linearity.

[0190] Comparative Example 1

[0191] Methodological Comparison

[0192] The lipoprotein subfraction detection kit and lipoprotein subfraction detection system provided by this invention can be used to compare the detection results of lipoprotein subfraction cholesterol items with the results of each lipoprotein cholesterol component measured together with VAP.

[0193] Take 50 μL of serum, add 1950 μL of diluent, mix well to obtain serum diluent, then take 5.1 mL of Polyallomer quick-seal tube, add 3800 μL of density solution, and then slowly add 1200 μL of serum diluent from the bottom. Place the quick-seal tube in an ultracentrifuge and centrifuge at the following parameters: speed = 65000 rpm, ω 2 t=6.6×10 10 rad 2 / sec, temperature=23℃, acceleration=6, deceleration=6.

[0194] The centrifuged samples were placed in the sample container of the lipoprotein subfraction detection system for detection. The system parameters were: sample flow rate 1.0 ml / min, reagent R1 flow rate 0.5 ml / min, reagent R2 flow rate 0.5 ml / min, and reagent R3 flow rate 0.5 ml / min.

[0195] Simultaneously, the same serum was processed in the same way, and the lipoprotein cholesterol results of each component were obtained by using the detection method in Chinese invention patent application with publication number CN107561297A.

[0196] Forty serum samples were randomly selected for testing. The results of each lipoprotein cholesterol component detected by the detection method in Chinese invention patent application CN107561297A were used as a control, and the results of each reagent combination in this invention were used as the comparison results. The comparison results are shown in Table 11.

[0197] Table 11

[0198]

[0199] As shown in Table 11, the slope of the comparison between the detection results of the lipoprotein subfraction detection kit and the lipoprotein subfraction detection system provided by the present invention and the detection results of the prior art is within 0.98-1.04, with a small intercept. The correlation of all items is above 0.99. Therefore, it can be seen that the lipoprotein subfraction detection kit and the lipoprotein subfraction detection system provided by the present invention have good correlation with existing detection methods.

[0200] The results of the above studies show that the lipoprotein subfraction detection kit and lipoprotein subfraction detection system provided by the present invention can simultaneously detect lipoprotein subfraction cholesterol and lipoprotein subfraction triglyceride indicators while ensuring detection precision and accuracy, and have broad application prospects.

[0201] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. A lipoprotein subfraction detection system, characterized in that, include: Controller used to control the lipoprotein subfraction detection system; Sample containers used to store liquid samples; The first reagent container used to store reagent R1; The first reaction module is used for mixing and incubating reagent R1 and liquid samples; The second reagent container used to store reagent R2; The second reaction module is used for mixing and incubating reagent R2 and the effluent from the first reaction module; The third reagent container used to store reagent R3; The third reaction module is used for mixing and incubating reagent R3 and the effluent from the second reaction module; Waste liquid container used to receive the effluent from the lipoprotein subcomponent detection system; The lipoprotein subfraction detection system also includes: The first detector is used to detect the liquid flowing out of the first reaction module; A second detector for detecting the effluent from the second reaction module; and A third detector used to detect the effluent from the third reaction module; The first reaction module, the first detector, the second reaction module, the second detector, the third reaction module, and the third detector are connected in series. The first reagent container contains reagent R1, the second reagent container contains reagent R2, and the third reagent container contains reagent R3. The components of reagent R1 are: 100mM HEPES buffer at pH 6.8, 0.05% Triton X-100, 10g / L magnesium chloride, 1g / L BSA, 2g / L squid skin gelatin, 0.05% PC300, 0.05% sodium azide, 2mM 4-aminoantipyrine, 2KU / L lipoprotein lipase, 2KU / L cholesterol lipase, and 2KU / L ascorbic acid oxidase; The components of reagent R2 are: 250 mM MOPS buffer at pH 7.2, 1 g / L sucrose, 2 g / L BSA, 0.1% sodium azide, 1 mM NAD+, and 3 KU / L glycerol dehydrogenase. The components of reagent R3 are: 50 mM TES buffer at pH 8.0, 0.1 g / L BSA, 1 g / L mackerel skin gelatin, 10 g / L trehalose, 0.02% PC950, 2 KU / L cholesterol oxidase, 2 KU / L peroxidase, 0.1 mM TOOS and 0.1 mM TODB; The system is used to achieve quantitative detection of lipoprotein subfractions according to the following process: S1: The sample is first treated with diluent and density solution, and after ultracentrifugation, it is transferred to the sample container. The liquid sample in the sample container is thoroughly mixed with reagent R1 in the first reagent container in the tubing or the first reaction module, and then incubated in the first reaction module to complete the relevant enzymatic reaction. S2: After the sample is incubated in the first reaction module, the first detector detects its absorbance. The detection wavelength is set to 340nm, which is used to quantitatively determine the blank absorbance of triglyceride content. S3: After the sample has been incubated in the first reaction module, it is thoroughly mixed with reagent R2 in the second reagent container in the pipe or the second reaction module. The mixture is then incubated in the second reaction module to complete the relevant enzymatic reaction. S4: After the sample is incubated in the second reaction module, the second detector detects its absorbance. The detection wavelength is set to 340nm and 500-600nm. The 340nm wavelength is used for subsequent quantitative determination of triglyceride content, and the 500-600nm wavelength is used for blank absorbance for subsequent quantitative determination of cholesterol content. S5: After the sample has been incubated in the second reaction module, it is thoroughly mixed with reagent R3 in the third reagent container in the pipe or the third reaction module. The mixture is then incubated in the third reaction module to complete the relevant enzymatic reaction. S6: After the sample is incubated in the third reaction module, the third detector detects its absorbance at a wavelength of 500-600 nm. This absorbance is used to quantitatively determine the cholesterol content after the reaction.

2. The lipoprotein subfraction detection system as described in claim 1, characterized in that, The liquid sample was obtained as follows: 50 μL of serum was taken, and 1950 μL of diluent was added. The mixture was stirred until homogeneous to obtain a serum diluent. Then, 3800 μL of density solution was added to a 5.1 mL Polyallomer quick-seal tube, followed by the slow addition of 1200 μL of serum diluent from the bottom. The quick-seal tube was then placed in an ultracentrifuge and centrifuged. The centrifugation parameters were set as follows: rotation speed = 65000 rpm, ω 2 t=6.6×10 10 rad 2 / sec, temperature=23℃, acceleration=6, deceleration=6.

3. The lipoprotein subfraction detection system as described in claim 1, characterized in that, The first reaction module has temperature control functions such as water bath, sand bath, metal bath, and air bath; the second reaction module has temperature control functions such as water bath, sand bath, metal bath, and air bath; the third reaction module has temperature control functions such as water bath, sand bath, metal bath, and air bath.

4. The lipoprotein subfraction detection system as described in claim 1, characterized in that, Also includes: A cleaning solution container for storing cleaning solution used to clean all the pipes of the lipoprotein subfraction detection system.

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