Lipoprotein subcomponent phospholipid detection kit

Abstract

The invention provides a lipoprotein sub-component phospholipid detection kit, and belongs to the field of in-vitro diagnostic reagents, and in particular, the lipoprotein sub-component phospholipid detection kit provided by the invention comprises a reaction reagent, the reaction reagent comprises a buffer solution, a surfactant, magnesium chloride, 4-aminoantipyrine, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3, 3, 4-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5- The preservative is prepared from 2, 5-dimethoxyaniline sodium salt, phospholipase D, choline oxidase, peroxidase, ascorbic acid oxidase, a protective agent and a third preservative. The lipoprotein subfraction phospholipid detection kit provided by the invention can be used for determining the content of lipoprotein subfraction phospholipid by using a VAP system, simultaneously realizes simultaneous detection of a plurality of lipoprotein subfraction phospholipid items for the first time, and has the advantages of high precision and high accuracy.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic reagent technology, and more specifically, to a lipoprotein subfraction phospholipid detection kit. Background Technology

[0002] Lipoproteins are a class of globular microparticles consisting of a hydrophobic core rich in sterols and triglycerides and an outer shell composed of proteins, phospholipids, cholesterol, etc. As important biomolecules, they are mainly responsible for transporting lipids in the human body. Lipoproteins are composed of cholesterol, triglycerides, and phospholipids. Based on their density, lipoproteins are classified into chylomicrons (CM), very low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). Clinically, cholesterol levels of lipoproteins are usually measured to assess a patient's risk of cardiovascular disease. However, recent studies have shown that lipoproteins exhibit significant heterogeneity. For example, LDL can be divided into a high-risk dense subtype and a low-risk lightweight subtype, and HDL also has sub-fragments with significantly different functions. Therefore, methods that only measure total cholesterol levels are insufficient to accurately assess cardiovascular disease risk, and new detection methods that can detect phospholipids, a sub-fragment of lipoproteins, are needed.

[0003] Lipoprotein subfraction quantitative analysis is a key technical means for cardiovascular disease risk assessment. Currently, the mainstream methods for lipoprotein subfraction detection include: (1) Electrophoresis: Lipoproteins are separated by electrophoresis and then stained (usually for cholesterol), and then scanned for analysis. This method is complicated, and the separation and staining accuracy is limited, resulting in insufficient precision and accuracy, making it difficult to meet the high-throughput clinical needs; (2) Nuclear magnetic resonance: Lipoprotein particle concentration is directly measured, but the instruments are expensive, the operation is complicated, and professional maintenance is required. It is mainly limited to scientific research scenarios and is difficult to promote in clinical practice; (3) VAP method: Lipoproteins are rapidly separated by ultracentrifugation combined with a vertical rotor, and the subfraction cholesterol content is measured by cholesterol detection reagents (such as cholesterol oxidase-peroxidase system). This method is currently only used to detect lipoprotein subfraction cholesterol.

[0004] Phospholipids play a crucial role in lipoprotein metabolism, and their subfractional distribution has unique diagnostic value for hyperlipidemia and postprandial metabolic assessment. Since phospholipids and cholesterol are both components of lipoproteins, clinical practice typically only measures total phospholipids. For the detection of phospholipids in individual lipoproteins and their subfractions, traditional ultracentrifugation (over 16 hours) is usually required to collect the separated lipoproteins separately and then determine the phospholipid content of each component. This method requires separate density-adjusting solutions, ultracentrifugation, and collection for phospholipid analysis. It generally only measures phospholipids in HDL, LDL, and VLDL, making it cumbersome, inefficient, and prone to issues with precision, thus unsuitable for clinical use. Currently, there is no method that can simultaneously and rapidly detect phospholipids in multiple lipoprotein subfractions. Summary of the Invention

[0005] The technical problem to be solved by this invention is: how to detect multiple lipoprotein subfractions and phospholipids.

[0006] To address the aforementioned technical problems, this invention provides a lipoprotein subfraction phospholipid detection kit, the kit comprising:

[0007] The reaction reagents include buffer solution, surfactant, magnesium chloride, 4-aminoantipyrine, sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, phospholipase D, choline oxidase, peroxidase, ascorbic acid oxidase, protectant, and third preservative.

[0008] In the reaction reagents of the kit provided by this invention, the surfactant plays the role of cleaving lipoproteins and releasing phospholipids from lipoproteins and their sub-components. Magnesium chloride provides magnesium ions as an enzyme activator. Sodium salts of 4-aminoantipyrine and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline participate in the generation of quinone imines as chromogenic substances. Ascorbic acid oxidase can eliminate the interference of reducing substances such as ascorbic acid on the determination of hydrogen peroxide.

[0009] Preferably, the buffer solution is selected from any one of PIPES buffer, HEPES buffer, MOPS buffer, phosphate buffer, and Tris buffer with a pH of 6.0 to 9.0 and a salt concentration of 10 to 500 mM.

[0010] Preferably, the surfactant is:

[0011] Qulaton X-100; or

[0012] A mixture of Triton X-100 and alkylphenol polyoxyethylene ether;

[0013] 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.

[0014] In the kit provided by this invention, the surfactant plays a role in accelerating the cleavage of lipoproteins. To ensure cleavage efficiency, the surfactant should include at least Triton X-100. Triton X-100 can cleave lipoproteins and release phospholipids from different lipoproteins. If the amount of Triton X-100 is too low, the lipoproteins will not be completely cleaved, affecting the accuracy of the test results. If the amount is too high, the viscosity of the reagent will increase, affecting the flowability of the reagent and the accuracy of the test results.

[0015] When Triton X-100 is used alone, the reaction rate may be limited, resulting in the reaction not being completed in a short time. Therefore, the kit provided by this invention adds alkylphenol polyoxyethylene ether to the surfactant based on Triton X-100. Alkylphenol polyoxyethylene ether can effectively improve the reaction rate. After adding alkylphenol polyoxyethylene ether, the pyrolysis reaction can be completed within 60 seconds.

[0016] Preferably, the concentration of Triton X-100 in the reaction reagent is 0.1% to 1% by mass.

[0017] The concentration of the alkylphenol polyoxyethylene ether in the reaction reagent is 0.01% to 1% by mass.

[0018] Preferably, the concentration of phospholipase D is 1~100 KU / L;

[0019] The concentration of the choline oxidase is 1~100 KU / L;

[0020] The concentration of the peroxidase is 0.2~20 KU / L;

[0021] The concentration of the ascorbic acid oxidase is 0.2~20 KU / L.

[0022] In the kit provided by this invention, all enzymes should be in excess to ensure that the reaction can proceed rapidly and efficiently. If the enzyme concentration is too low, it will cause the reaction of high-value samples to be delayed.

[0023] Preferably, the protective agent is selected from one or more of marine animal gelatin, sugar protective agent, and BSA; the marine animal gelatin is selected from one or more of cod bone gelatin, squid skin gelatin, and mackerel skin gelatin; and the sugar protective agent is selected from one or more of sucrose, mannitol, trehalose, and sorbitol.

[0024] Preferably, the concentration of the third preservative in the reaction reagent is 0.01% to 1% by mass.

[0025] The concentration of 4-aminoantipyrine in the reaction reagent is 0.1~10mM;

[0026] The concentration of the sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline salt in the reaction reagent is 0.1~10mM.

[0027] Preferably, the lipoprotein subfraction phospholipid detection kit further includes a diluent, which includes a first preservative and a first density regulator;

[0028] The density liquid includes a second preservative and a second density regulator.

[0029] Preferably, the density of the diluent is adjusted by a first density regulator, the density of which is 1.15~1.25 g / mL, and the first density regulator is sucrose or iodixanol;

[0030] The density of the density liquid is adjusted by a second density regulator, the density of which is 1.0~1.1 g / mL, and the second density regulator is sucrose or iodixanol.

[0031] Preferably, the concentration of the first preservative in the diluent is 0.01% to 0.5% by mass.

[0032] The concentration of the second preservative in the diluent is 0.01% to 0.5% by mass.

[0033] In existing technologies, the diluent / density solution used in VAP lipid subcomponent analyzers is prepared with potassium bromide. However, high concentrations of potassium bromide can severely inhibit the reaction of lipoprotein subcomponent phospholipids. This invention uses a first density regulator and a second density regulator to replace the existing potassium bromide, enabling adjustable density of the diluent and density solution. This completely eliminates the inhibition of enzyme activity of phospholipid detection reagents by potassium bromide. The kit provided by this invention is compatible with VAP detection systems, enabling continuous flow cytometry detection of lipoprotein subcomponent phospholipids on VAP lipid subcomponent analyzers.

[0034] The working principle of the reagent kit provided by this invention is as follows:

[0035] First, after the sample is diluted with the diluent, an initial high-density layer with a higher density will be formed. After the initial high-density layer is added to the bottom of the density solution using the bottom loading method, a density gradient solution with upper and lower layers will be formed. After the upper and lower density gradient solutions are ultracentrifuged, a continuous density gradient solution will be formed. At the same time, lipoproteins of different densities will be separated into layers according to density under the action of centrifugal force, forming a continuous lipoprotein gradient layer, thereby achieving the separation of lipoprotein subcomponents.

[0036] Then, the centrifuged samples were tested using a lipid subfraction analyzer. The lipoproteins in the samples reacted with the reaction reagents, and the surfactants cleaved each layer of lipoproteins. The cleavage reaction was completed within 60 seconds, releasing phospholipids.

[0037] Finally, under the action of phospholipase D, phospholipids are converted into choline and phosphatidic acid. Choline is converted into hydrogen peroxide under the action of choline oxidase. In the presence of peroxidase, 4-aminoantipyrine and sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, hydrogen peroxide will generate a blue dye. The blue dye has a maximum absorption peak at a wavelength of 600 nm. By continuously detecting the change in absorbance after the sample reaction, a phospholipid spectrum can be formed. The relevant software can automatically calculate the phospholipid content in different density lipoprotein subfractions.

[0038] The kit provided by this invention enables the simultaneous detection of total phospholipids (PLIP), high-density lipoprotein phospholipids (HDL-PLIP), low-density lipoprotein phospholipids (LDL-PLIP), intermediate-density lipoprotein phospholipids (IDL-PLIP), very low-density lipoprotein phospholipids (VLDL-PLIP), high-density lipoprotein 2 phospholipids (HDL2-PLIP), high-density lipoprotein 3 phospholipids (HDL3-PLIP), small and dense low-density lipoprotein phospholipids (sdLDL-PLIP), lipoprotein α phospholipids (Lpa-PLIP), and lipoprotein remnant phospholipids (RLP-PLIP).

[0039] The sample described in this invention can be a serum sample or a plasma sample.

[0040] Unlike existing technologies, this invention has the following advantages:

[0041] 1. This invention is the first to realize the detection of phospholipid indicators of lipoprotein subfractions on a blood lipid subfraction analyzer. The detection indicators include PLIP, HDL-PLIP, LDL-PLIP, IDL-PLIP, VLDL-PLIP, HDL2-PLIP, HDL3-PLIP, sdLDL-PLIP, Lpa-PLIP, and RLP-PLIP.

[0042] 2. The kit provided by this invention uses a sucrose or iodixanol density gradient system instead of a potassium bromide system, which overcomes the inhibitory effect of potassium bromide on enzymes and enables the detection of lipoprotein subfractions phospholipids in the VAP detection system, which is of pioneering significance;

[0043] 3. This invention uses a specific combination of protective agents, which significantly increases the stability of the enzymes in the kit, meeting clinical requirements;

[0044] 4. The density gradient in the kit provided by this invention is flexibly adjustable, and the detection precision of specific subcomponents can be significantly improved by adjusting the density and centrifugation parameters;

[0045] 5. The kit provided by this invention can be used on a VAP system for detection. It can be used in conjunction with existing cholesterol VAP detection to simultaneously output lipoprotein subfraction cholesterol and phospholipid typing maps, providing a new combination of biomarkers for atherosclerosis, diabetic lipid metabolism disorders, etc., filling a gap in clinical testing. Detailed Implementation

[0046] 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 present invention.

[0047] 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.

[0048] As described in the background art, in order to enable the VAP system to be used for the detection of lipoprotein subfraction phospholipids, this invention provides a lipoprotein subfraction phospholipid detection kit, which includes:

[0049] The reaction reagents include buffer solution, surfactant, magnesium chloride, 4-aminoantipyrine, sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, phospholipase D, choline oxidase, peroxidase, ascorbic acid oxidase, protectant, and third preservative.

[0050] In the above embodiments, the buffer solution is selected from any one of PIPES buffer, HEPES buffer, MOPS buffer, phosphate buffer, and Tris buffer with a pH of 6.0 to 9.0 and a salt concentration of 10 to 500 mM.

[0051] In the above embodiments, the surfactant used as a lipoprotein cleavage agent is:

[0052] Qulaton X-100; or

[0053] A mixture of Triton X-100 and alkylphenol polyoxyethylene ether;

[0054] 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.

[0055] More specifically, in the above embodiments, if commercial raw materials are used, BASF Lutensol AP series alkylphenol polyoxyethylene ethers are preferred.

[0056] In the above embodiments, the concentration of Triton X-100 in the reaction reagent is preferably 0.1% to 1% by mass, and most preferably 0.2% to 1% by mass.

[0057] The concentration of alkylphenol polyoxyethylene ether in the reaction reagent is preferably 0.01% to 1% by mass, and most preferably 0.05% to 0.2% by mass.

[0058] In the above embodiments, the concentration of phospholipase D as a reaction catalyst is preferably 1~100 KU / L, and most preferably 5~50 KU / L;

[0059] The concentration of choline oxidase is 1~100 KU / L, with the optimal value being 5~50 KU / L;

[0060] The concentration of peroxidase is 0.2~20 KU / L, with the optimal concentration being 1~5 KU / L;

[0061] The concentration of ascorbic acid oxidase is 0.2~20 KU / L, with the optimal concentration being 1~5 KU / L.

[0062] In the above embodiments, the protective agent is selected from any one or more of marine animal gelatin, sugar protective agent, and BSA; the marine animal gelatin is selected from any one or more of cod bone gelatin, squid skin gelatin, and mackerel skin gelatin; and the sugar protective agent is selected from any one or more of sucrose, mannitol, trehalose, and sorbitol.

[0063] In the above embodiments, the concentration of the third preservative in the reaction reagent is 0.01% to 1% by mass.

[0064] The concentration of 4-aminoantipyrine in the reaction reagent is 0.1~10mM;

[0065] The concentration of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt in the reaction reagent is 0.1~10mM.

[0066] More specifically, in the above embodiments, the third preservative is selected from any one or more of PC300, sodium azide, and PC950.

[0067] More specifically, in the above embodiments, the reaction reagent can be a single reagent or multiple reagents. When the reaction reagent is multiple reagents, all components should be reasonably distributed in different reagents. For example, the chromogenic substance 4-aminoantipyrine and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt (ADOS) should be separated as much as possible to improve the stability of the reagents.

[0068] More specifically, in the above embodiments, if a sugar-based preservative is used as a component of the preservative, the concentration of the sugar-based preservative in the reagent is preferably 1~100 g / L, and most preferably 5~50 g / L.

[0069] More specifically, in the above embodiments, marine animal gelatin is used as a component of the protective agent, and the concentration of marine animal gelatin in the reagent is preferably 0.1~20 g / L, and most preferably 0.5~10 g / L.

[0070] 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.

[0071] More specifically, in the above embodiments, the lipoprotein subfraction phospholipid detection kit further includes a diluent, the diluent comprising a first preservative and a first density regulator;

[0072] The density liquid includes a second preservative and a second density regulator.

[0073] In the above embodiments, the diluent and density solution are used to create a gradient density in the lipoprotein sample.

[0074] In the above embodiments, the first preservative is PC300 and / or sodium azide;

[0075] The second preservative is PC300 and / or sodium azide.

[0076] In the above embodiments, the density of the diluent is adjusted by a first density regulator, the density of which is 1.15~1.25 g / mL, and the first density regulator is sucrose or iodixanol;

[0077] The density of the density liquid is adjusted by a second density adjuster, the density of which is 1.0~1.1 g / mL, and the second density adjuster is sucrose or iodixanol.

[0078] A specific embodiment of the present invention also provides a method for detecting lipoprotein subfraction phospholipids using a VAP detection system and the above-mentioned kit, comprising the following steps:

[0079] S1: Dilute the serum or plasma sample with diluent and adjust the liquid density;

[0080] S2: Add density solution to centrifuge tube;

[0081] S3: Add the diluted liquid sample from step S1 to the bottom of the centrifuge tube in step S2 to form two density gradients with the density liquid.

[0082] S4: Use an ultracentrifuge and a vertical rotor to ultracentrifuge the centrifuge tubes from step S3 to form a continuous density gradient. In this step, lipoproteins will be layered according to density to form a continuous lipoprotein gradient layer.

[0083] S5: Place the centrifuge tubes that were layered in step S4 onto the lipid subfraction detection instrument. The instrument will automatically and continuously draw samples from the bottom or top. The samples will react with the reaction reagents in the instrument and generate lipoprotein phospholipid profiles.

[0084] S6: The software can automatically calculate the phospholipid content of lipoprotein subfractions based on the lipoprotein phospholipid map.

[0085] More specifically, in the above embodiments, the density of the density solution, the density of the diluent, and the centrifugation parameters can be changed to improve the separation effect on a certain type of lipoprotein, depending on the different separation effects. For example, increasing the density of the diluent and the density solution, and increasing the centrifugation time can improve the separation effect of HDL, thereby improving the detection accuracy and precision of HDL2-PLIP and HDL3-PLIP. Similarly, decreasing the density of the diluent and increasing the density of the density solution can improve the separation effect of LDL, thereby improving the detection precision and accuracy of sdLDL-PLIP.

[0086] 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.

[0087] Explanation of Terminology Abbreviations: To ensure clarity, the relevant terms in the following examples are explained as follows:

[0088] BSA: Bovine serum albumin;

[0089] PC300: Proclin 300 liquid preservative, also written as PC-300 in some references;

[0090] PIPES: Piperazine-1,4-bis(2-ethanesulfonic acid), PIPES buffer is a common biological buffer;

[0091] PC950: Proclin 950 liquid preservative, also written as PC-950 in some references;

[0092] Lutensol AP 6: A commercially available alkylphenol polyoxyethylene ether derived from BASF, which is a product in the Lutensol AP series.

[0093] Lutensol AP 7: A commercially available alkylphenol polyoxyethylene ether derived from BASF, which is a product in the Lutensol AP series.

[0094] Lutensol AP 8: A commercially available alkylphenol polyoxyethylene ether derived from BASF, which is a product in the Lutensol AP series.

[0095] Lutensol AP 9: A commercially available alkylphenol polyoxyethylene ether derived from BASF, which is a product in the Lutensol AP series.

[0096] Lutensol AP 10: A commercially available alkylphenol polyoxyethylene ether derived from BASF, which is a product in the Lutensol AP series.

[0097] Lutensol AP:12: A commercially available alkylphenol polyoxyethylene ether derived from BASF, which is a product in the Lutensol AP series.

[0098] ADOS: A biochemical colorimetric reagent with CAS number 82692-96-4;

[0099] MOPS: 3-morpholinopropanesulfonic acid; MOPS buffer is a common biological buffer.

[0100] Tris: Tris(hydroxymethyl)aminomethane; Tris buffer is a common biological buffer.

[0101] PLIP: Total phospholipids;

[0102] HDL-PLIP: High-density lipoprotein phospholipids;

[0103] LDL-PLIP: Low-density lipoprotein phospholipids;

[0104] IDL-PLIP: Intermediate density lipoprotein phospholipid;

[0105] VLDL-PLIP: Very low-density lipoprotein phospholipid;

[0106] HDL2-PLIP: High-density lipoprotein 2 phospholipid;

[0107] HDL3-PLIP: High-density lipoprotein 3 phospholipid;

[0108] sdLDL-PLIP: Small, dense low-density lipoprotein phospholipids;

[0109] Lpa-PLIP: Lipoprotein a phospholipid;

[0110] RLP-PLIP: Lipoprotein Residue Phospholipids.

[0111] Example 1

[0112] The effect of different density solutions and dilution solutions on the reaction rate was determined.

[0113] Prepare the diluent and density solution according to the formula shown in Table 1.

[0114] Table 1

[0115]

[0116] S1: Dilute the serum sample with diluent and mix in a biochemical cup, wherein the dilution volume ratio is serum: diluent = 50: 1950;

[0117] S2: Take a 5.1 mL Polyallomer quick-seal tube, add 3800 μL of density solution, and then slowly add 1200 μL of pre-diluted serum sample from the bottom. Place the tube in a VTi-65.2 rotor and centrifuge using a Beckman ultracentrifuge. Parameters: Rotation speed = 65000 rpm, ω 2 t=6.6*10 10 rad 2 / sec, temperature=23℃, acceleration=6, deceleration=6.

[0118] S3: Place the centrifuged samples from step S2 onto the lipid subfraction analyzer, add reagents, and begin testing. Record the total area of ​​the reaction curve. Calculate the reaction ratio of each group to the blank control group. The samples were routine serum, with phospholipid target values ​​of 1.82 mM and 4.26 mM, respectively. The specific dilution and density solution combinations and results are shown in Table 2. The blank group served as a 100% reaction control.

[0119] Table 2

[0120]

[0121] As shown in Table 2, the diluent and density solution of the present invention have no effect on the reaction between serum and reagents. Therefore, the density solution and diluent provided by the present invention can be used in detection systems for detecting phospholipid profiles in lipoproteins without affecting the reagent reaction efficiency.

[0122] Example 2

[0123] Determination of the effect of reaction reagents on reaction rate.

[0124] Prepare different reaction reagents according to the formulas in Table 3.

[0125] Table 3

[0126]

[0127] Formula A3 from Example 1 was used as the diluent, and formula B3 from Example 1 was used as the density solution. The same detection steps as in Example 1 were employed, and the 16 reaction reagents prepared above were used for detection. The detection instrument was a lipid subfraction analyzer, and the samples were routine serum and high-phospholipid serum. The phospholipid value of the routine serum was 2.15 mM, and the phospholipid value of the high-phospholipid serum was 4.87 mM.

[0128] The reaction of this reagent is a final reaction, and the reaction time is relatively short, 1 minute. Therefore, the final total reaction area of ​​the same sample is only related to whether each reagent completely reacts with the target substance in the sample. Theoretically, if the total reaction area of ​​the 2.15 mM sample with reagent C1 is 100%, the relative total area under the reaction curve of the 4.88 mM sample should be 227%.

[0129] The specific results are shown in Table 4.

[0130] Table 4

[0131]

[0132] As shown in Table 4, the reaction reagents provided by this invention, with the combination of Triton X-100 and Lutensol AP 8 dual surfactants, exhibited a total reaction area that was largely consistent with expectations when detecting high-phospholipid samples; while the reaction curve of the single surfactant reagent containing Triton X-100 showed only 90% of the expected total area. Therefore, the single surfactant reaction reagents provided by this invention can be used for the detection of high-value samples, while the dual surfactant combination reaction reagents can meet all application scenarios and have the advantage of faster reaction speed.

[0133] Example 3

[0134] Precision study.

[0135] Prepare the reagents according to the formula in Table 5, for a total of 8 groups.

[0136] Table 5

[0137]

[0138] Based on the substances in formulation D3 except for PIPES and pH, reagents were prepared according to the buffer and pH combinations shown in Table 6, for a total of 12 groups.

[0139] Table 6

[0140]

[0141] Based on the substances in formula D4 excluding BSA, reagents were prepared according to the concentrations shown in Table 7, resulting in a total of 21 groups.

[0142] Table 7

[0143]

[0144] Based on the substances in formulation D5 except for Lutensol AP 8, reagents were prepared according to the concentrations shown in Table 8, for a total of 15 groups.

[0145] Table 8

[0146]

[0147] Based on the substances in formula D6 except for sodium azide, reagents were prepared according to the concentrations shown in Table 9, resulting in a total of 6 groups.

[0148] Table 9

[0149]

[0150] Because the reagents of this invention can react more quickly, the reaction can be performed on a lipid subcomponent analyzer.

[0151] Take 50 μl of serum and add 1950 μl of Formula A3 diluent. Dilute and mix well. Take a 5.1 mL Polyallomer quick-seal tube, add 3800 μl of Formula B3 density solution, and then slowly add 1200 μl of the pre-diluted serum sample from the bottom. Place the centrifuge tube in a VTi-65.2 rotor and centrifuge using a Beckman ultracentrifuge. Parameters: Rotation speed = 65000 rpm, ω 2 t=6.6*10 10 rad 2 / sec, temperature=23℃, acceleration=6, deceleration=6.

[0152] The centrifuged samples were carefully transferred to a lipid subfraction analyzer for testing. The testing parameters were: sample flow rate 1.0 mL / min, reagent flow rate 1.0 mL / min. The results were compared using 63 reagent formulations (C1, D1-D62) of this invention. The results are shown in Tables 10-14.

[0153] The samples were routine serum. Each sample was tested 10 times with each reagent. The mean X, standard deviation S, and coefficient of variation CV of each reagent PLIP were calculated.

[0154] Table 10

[0155]

[0156] Table 11

[0157]

[0158] Table 12

[0159]

[0160] Table 13

[0161]

[0162] Table 14

[0163]

[0164] As shown in Tables 10-14, the detection results of the 62 dual-surfactant combination reaction reagents of this invention are relatively consistent, and the coefficient of variation (CV) is good. However, the detection results of each item of the single-surfactant reaction reagents are about 10% lower. The possible reason is that the single-surfactant reaction reagents do not have sufficient reaction time on the lipid typing analyzer, resulting in incomplete reaction and thus lower detection results. This tendency to lower the detection results increases with the increase of sample values. Therefore, using the reaction reagents provided by this invention on a lipid subfraction analyzer can obtain more accurate lipoprotein subfraction phospholipid detection results.

[0165] Example 4

[0166] Linear range study.

[0167] Since the lipid subfraction analyzer can detect phospholipids of different lipoprotein subfractions, among which PLIP, HDL-PLIP, and LDL-PLIP have relatively high measured values ​​and are representative of the main items, the linear range of PLIP, HDL-PLIP, and LDL-PLIP is analyzed to illustrate the detectable linear range of the reagent detection system of the present invention. A high PLIP sample (PLIP measured value of about 8mM) was taken, diluted to different concentrations, and tested using the formula of the present invention.

[0168] Take 50 μl of each serum gradient and add 1950 μl of formulation A3 diluent. Dilute and mix well. Take a 5.1 mL Polyallomer quick-seal tube, add 3800 μl of B3 density solution, and then slowly add 1200 μl of the pre-diluted serum sample from the bottom. Place the centrifuge tube in a VTi-65.2 rotor and centrifuge using a Beckman ultracentrifuge. Parameters: speed = 65000 rpm, ω 2 t=6.6*10 10 rad 2 / sec, temperature=23℃, acceleration=6, deceleration=6.

[0169] The centrifuged samples were carefully transferred to a lipid subfraction analyzer for testing. The testing parameters were: sample flow rate 1.0 mL / min, reagent flow rate 1.0 mL / min. The results were compared using 24 different reagent formulations from this invention. The results are shown in Table 15.

[0170] Table 15

[0171]

[0172] As shown in Table 15, the multi-surfactant combination reagent of the kit provided by this invention exhibits significantly better linearity than the single-surfactant reagent, with an upper limit of its linear range reaching 8 mM. In contrast, the single-surfactant reagent generally yields lower results when detecting phospholipid samples from 0 to 8 mM. Because this invention uses a special combination of surfactants, the reaction rate between the reagent and different lipoprotein subfractions of phospholipids is significantly increased. Even when detecting high-value samples, the reaction can be completed in a short time. Therefore, the multi-surfactant reaction reagent of this invention has a wider linear range.

[0173] Example 5

[0174] Study on the detection conditions of HDL subcomponents.

[0175] The results of the precision test of the detection system show that the precision of several HDL-related items, such as HDL-PLIP, HDL2-PLIP and HDL3-PLIP, is generally low. Based on the experimental principle of this invention, we adjusted (increased) the density of the diluent, the density of the density solution, and the centrifugation time to try to find better detection conditions for HDL subcomponents.

[0176] Take 50 μl of serum and add 1950 μl of the diluent from Table 16, dilute, and mix well. Take a 5.1 mL Polyallomer quick-seal tube, add 3800 μl of the density solution from Table 16, and then slowly add 1200 μl of the pre-diluted serum sample from the bottom. Place the centrifuge tube in a VTi-65.2 rotor and centrifuge using a Beckman ultracentrifuge. Parameters: speed = 65000 rpm, centrifugation time according to Table 16, temperature = 23℃, acceleration = 6, deceleration = 6.

[0177] After centrifugation, the sample was carefully transferred to a lipid subfraction analyzer for testing. The testing parameters were: sample flow rate 1.0 mL / min and reagent flow rate 1.0 mL / min.

[0178] Using reagent D4 as the reaction reagent, the experiment was conducted according to the combination scheme shown in Table 16.

[0179] Table 16

[0180]

[0181] The experimental results are shown in Table 17.

[0182] Table 17

[0183]

[0184] As shown in Table 17, the HDL-related phospholipids in group E8 exhibited better precision, with HDL-PLIP less than 2.50%, HDL2-PLIP less than 4%, and HDL3-PLIP less than 3%. This represents a significant advantage compared to the previous precision of 3%–4% for HDL-PLIP, 5%–6% for HDL2-PLIP, and 3.5%–5% for HDL3-PLIP. Therefore, this invention can achieve even better HDL subcomponent detection results by adjusting (increasing) the density of the diluent, the density of the density solution, and the centrifugation time.

[0185] Example 6

[0186] Study on LDL subcomponent detection conditions.

[0187] The results of the precision test of the detection system show that the precision of several LDL-related items of LDL-PLIP and sdLDL-PLIP is generally poor. Based on the experimental principle of this invention, we can adjust (reduce) the density of the diluent and adjust (increase) the density of the density solution to try to find better detection conditions for LDL subcomponents.

[0188] Take 50 μl of serum and add 1950 μl of the diluent from Table 18, dilute, and mix well. Seal a 5.1 mL Polyallomer tube, add 3800 μl of the density solution from Table 18, and then slowly add 1200 μl of the pre-diluted serum sample from the bottom. Place the tube in a VTi-65.2 rotor and centrifuge using a Beckman ultracentrifuge. Parameters: Rotation speed = 65000 rpm, ω²t = 6.6 × 10⁻⁶ 10 rad 2 / sec, temperature=23℃, acceleration=6, deceleration=6.

[0189] After centrifugation, the sample was carefully transferred to a lipid subfraction analyzer for testing. The testing parameters were: sample flow rate 1.0 mL / min and reagent flow rate 1.0 mL / min.

[0190] Using reagent D4 as the reaction reagent, the experiment was conducted according to the combination scheme shown in Table 18.

[0191] Table 18

[0192]

[0193] The experimental results are shown in Table 19.

[0194] Table 19

[0195]

[0196] As shown in Table 19, the precision of LDL-related phospholipid measurements in group F12 is better, with LDL-PLIP less than 3% and sdLDL-PLIP approximately 4%. This is a significant advantage compared to the previous precision of 4%–5% for LDL-PLIP and 6%–8% for sdLDL-PLIP. Therefore, this invention can obtain better LDL subcomponent detection results by adjusting (reducing) the density of the diluent and adjusting (increasing) the density of the density solution.

[0197] 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 phospholipid detection kit, characterized in that, The lipoprotein subfraction phospholipid detection kit includes: The reaction reagents include buffer solution, surfactant, magnesium chloride, 4-aminoantipyrine, sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, phospholipase D, choline oxidase, peroxidase, ascorbic acid oxidase, protectant, and third preservative.

2. The lipoprotein subfraction phospholipid detection kit as described in claim 1, characterized in that, The buffer solution is selected from any one of PIPES buffer, HEPES buffer, MOPS buffer, phosphate buffer, and Tris buffer with a pH of 6.0 to 9.0 and a salt concentration of 10 to 500 mM.

3. The lipoprotein subfraction phospholipid detection kit as described in claim 1, characterized in that, The surfactant is Qulaton X-100; or A mixture of Triton X-100 and alkylphenol polyoxyethylene ether; 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.

4. The lipoprotein subfraction phospholipid detection kit as described in claim 3, characterized in that, The concentration of Triton X-100 in the reaction reagent is 0.1%~1% by mass. The concentration of the alkylphenol polyoxyethylene ether in the reaction reagent is 0.01% to 1% by mass.

5. The lipoprotein subfraction phospholipid detection kit as described in claim 1, characterized in that, The concentration of phospholipase D is 1~100 KU / L; The concentration of the choline oxidase is 1~100 KU / L; The concentration of the peroxidase is 0.2~20 KU / L; The concentration of the ascorbic acid oxidase is 0.2~20 KU / L.

6. The lipoprotein subfraction phospholipid detection kit as described in claim 1, characterized in that, The protective agent is selected from any one or more of marine animal gelatin, sugar protective agent, and BSA. The marine animal gelatin is selected from any one or more of cod bone gelatin, squid skin gelatin, and mackerel skin gelatin. The sugar protective agent is selected from any one or more of sucrose, mannitol, trehalose, and sorbitol.

7. The lipoprotein subfraction phospholipid detection kit as described in claim 1, characterized in that, The concentration of the third preservative in the reaction reagent is 0.01% to 1% by mass. The concentration of 4-aminoantipyrine in the reaction reagent is 0.1~10mM; The concentration of the sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline salt in the reaction reagent is 0.1~10mM.

8. The lipoprotein subfraction phospholipid detection kit as described in claim 1, characterized in that, Also includes The diluent includes a first preservative and a first density modifier; The density liquid includes a second preservative and a second density regulator.

9. The lipoprotein subfraction phospholipid detection kit as described in claim 8, characterized in that, The density of the diluent is adjusted by a first density adjuster, the density of which is 1.15~1.25 g / mL, and the first density adjuster is sucrose or iodixanol; The density of the density liquid is adjusted by a second density regulator, the density of which is 1.0~1.1 g / mL, and the second density regulator is sucrose or iodixanol.

10. The lipoprotein subfraction phospholipid detection kit as described in claim 8, characterized in that, The concentration of the first preservative in the diluent is 0.01%~0.5% by mass. The concentration of the second preservative in the diluent is 0.01% to 0.5% by mass.

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