Lipoprotein subfraction triglyceride detection kit

Abstract

The invention provides a lipoprotein subfraction triglyceride detection kit which is suitable for a blood fat subfraction detector. Belongs to the technical field of in vitro diagnosis, the lipoprotein subfraction triglyceride detection reagent provided by the invention comprises a diluent, and the diluent comprises a first preservative, a first density regulator and a first solvent; the density liquid comprises a second preservative, a second density regulator and a second solvent; the reaction reagent comprises a surfactant, magnesium chloride, 4-aminoantipyrine, 4-chlorophenol, lipoprotein esterase, glycerophosphate oxidase, glycerol kinase, peroxidase, ascorbic acid oxidase, a protective agent, a third preservative and a third solvent; the kit provided by the invention determines the content of the lipoprotein subfraction triglyceride through a VAP system for the first time, and has the advantages of high precision and high accuracy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of in vitro diagnosis, in particular, it relates to a lipoprotein subcomponent triglyceride detection kit, more particularly, the kit is suitable for blood lipid subcomponent detector. BACKGROUND

[0002] As an important biomolecule, lipoprotein is mainly responsible for transporting lipids in the human body. The composition of lipoprotein includes cholesterol and triglyceride. According to the density of lipoprotein, lipoprotein is classified into chylomicron (CM), very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL) and high density lipoprotein (HDL). Generally, the risk of cardiovascular disease in patients is evaluated by measuring the cholesterol content of lipoprotein in clinic. However, recent studies have shown that there is significant heterogeneity in lipoprotein, for example, LDL can be divided into high-risk dense subtype and low-risk light subtype, and HDL also has significantly different functional subcomponents. Therefore, the detection method of detecting only the total cholesterol content is insufficient to accurately evaluate the risk of cardiovascular disease, and a new detection method for detecting lipoprotein subcomponent triglyceride needs to be developed.

[0003] Lipoprotein subcomponent quantitative analysis is a key technical means for cardiovascular disease risk assessment. At present, the mainstream methods for lipoprotein subcomponent detection include: (1) electrophoresis method: after separating lipoprotein by electrophoresis and staining (usually for cholesterol), scanning analysis is performed. This method is complicated, and the separation and staining precision is limited, resulting in insufficient precision and accuracy, which is difficult to meet the clinical high-throughput demand; (2) nuclear magnetic resonance method: directly measures the concentration of lipoprotein particles, but the instrument is expensive, the operation is complex, and professional maintenance is required, which is mainly limited to scientific research scenes, and it is difficult to promote in clinic; (3) VAP method: uses ultracentrifugation combined with vertical rotor to quickly separate lipoprotein, and measures the subcomponent cholesterol content by cholesterol detection reagent (such as cholesterol oxidase-peroxidase system). This method is currently only used for detecting lipoprotein subcomponent cholesterol.

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

[0005] The technical problem to be solved by this invention is how to simultaneously detect multiple lipoprotein subfractions, including triglycerides.

[0006] To address the aforementioned technical problems, this invention provides a lipoprotein subfraction triglyceride detection kit suitable for lipid subfraction analyzers, the triglyceride detection kit comprising:

[0007] The diluent comprises a first preservative, a first density modifier, and a first solvent;

[0008] A density liquid, the density liquid comprising a second preservative, a second density modifier, and a second solvent;

[0009] The reaction reagents include surfactants, magnesium chloride, 4-aminoantipyrine, 4-chlorophenol, lipoprotein lipase, glycerol phosphate oxidase, glycerol kinase, peroxidase, ascorbic acid oxidase, a preservative, a third preservative, and a third solvent.

[0010] In existing technologies, the density solution used in VAP lipid subfraction analyzers is prepared with potassium bromide. However, high concentrations of potassium bromide can severely inhibit the reaction of lipoprotein subfraction triglycerides. This invention uses a first density regulator and a second density regulator to replace the existing potassium bromide to achieve adjustable density of the diluent and density solution, completely eliminating the inhibition of enzyme activity of the triglyceride detection reagent by potassium bromide. The kit provided by this invention is compatible with VAP detection systems, enabling continuous flow cytometry detection of lipoprotein subfraction triglycerides on VAP lipid subfraction analyzers.

[0011] In the reaction reagents of the kit provided by this invention, the surfactant plays the role of cleaving lipoproteins and releasing triglycerides from lipoproteins and their sub-components; magnesium chloride acts as an enzyme activator to provide magnesium ions; 4-aminoantipyrine and 4-chlorophenol act as chromogenic substances to participate in the generation of quinone imines; and ascorbic acid oxidase can eliminate the interference of reducing substances such as ascorbic acid on the determination of hydrogen peroxide.

[0012] 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;

[0013] 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.

[0014] Using the diluent and density solution of specific densities provided by this invention, it can be ensured that lipoprotein subfractions are effectively separated by density during ultracentrifugation, ultimately enabling the detection of lipoprotein subfraction triglyceride profiles.

[0015] Preferably, the first solvent is a phosphate buffer solution with a pH of 6.0 to 8.0 and a salt concentration of 10 to 1000 mM;

[0016] The second solvent is a phosphate buffer solution with a pH of 6.0 to 8.0 and a salt concentration of 10 to 1000 mM;

[0017] The third solvent is selected from any one of PIPES buffer, HEPES buffer, MOPS buffer, TES buffer, and Tris buffer with a pH of 6.0 to 9.0 and a salt concentration of 10 to 500 mM.

[0018] Preferably, the surfactant is

[0019] Qulaton X-100; or

[0020] A mixture of Triton X-100 and alkylphenol polyoxyethylene ether; or

[0021] A mixture of Triton X-100, alkylphenol polyoxyethylene ether, and fatty alcohol polyoxyethylene ether.

[0022] 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.

[0023] 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 triglycerides 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.

[0024] When Triton X-100 is used alone as a surfactant, 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 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.

[0025] For samples with high VLDL or chyle content, even with the simultaneous use of Triton X-100 and alkylphenol polyoxyethylene ether, it is impossible to guarantee that the lysis reaction will be completed in a short time. This will lead to lower results for high-value samples in subsequent testing. Based on this, the kit provided by this invention provides fatty alcohol polyoxyethylene ether in addition to Triton X-100 and alkylphenol polyoxyethylene ether. The fatty alcohol polyoxyethylene ether, in conjunction with Triton X-100 and alkylphenol polyoxyethylene ether, can enable high triglyceride samples such as chylous blood to complete the lysis reaction rapidly in a short time.

[0026] Preferably, the concentration of Triton X-100 in the reaction reagent is 0.1% to 1% by mass.

[0027] The concentration of the alkylphenol polyoxyethylene ether in the reaction reagent is 0.01%~1% by mass.

[0028] The concentration of the fatty alcohol polyoxyethylene ether in the reaction reagent is 0.01~1% by mass.

[0029] Preferably, the concentration of the lipoprotein lipase is 0.2~20 KU / L;

[0030] The concentration of the glycerol phosphate oxidase is 1~100 KU / L;

[0031] The concentration of the glycerol kinase is 0.2~20 KU / L;

[0032] The concentration of the peroxidase is 0.2~20 KU / L;

[0033] The concentration of the ascorbic acid oxidase is 0.2~20 KU / L.

[0034] 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.

[0035] 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.

[0036] Preferably, the first preservative is PC300 and / or sodium azide;

[0037] The second preservative is PC300 and / or sodium azide;

[0038] The third preservative is selected from any one or more of PC300, sodium azide, and PC950.

[0039] Preferably, the concentration of the first preservative in the diluent is 0.01% to 0.5% by mass.

[0040] The concentration of the second preservative in the diluent is 0.01%~0.5% by mass.

[0041] The concentration of the third preservative in the reaction reagent is 0.01% to 1% by mass.

[0042] Preferably, the concentration of 4-aminoantipyrine in the reaction reagent is 0.1~10mM;

[0043] The concentration of 4-chlorophenol in the reaction reagent is 0.1~10mM.

[0044] The working principle of the reagent kit provided by this invention is as follows:

[0045] 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. At this time, the lipoproteins with higher density will be located in the lower layer and the lipoproteins with lower density will be located in the upper layer, thus realizing the separation of lipoprotein subcomponents.

[0046] 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 60-120 seconds, releasing triglycerides.

[0047] Finally, triglycerides are converted into glycerol by lipoprotein lipase, and glycerol is converted into glycerol 3-phosphate by glycerol kinase. Glycerol 3-phosphate can be catalyzed by glycerol phosphate oxidase to generate hydrogen peroxide. In the presence of peroxidase, 4-aminoantipyrine and 4-chlorophenol, hydrogen peroxide will generate red quinone imine. Quinone imine has absorbance at λ=546nm. By continuously detecting the absorbance changes of the sample after the reaction, a triglyceride spectrum can be formed, and the relevant software can automatically calculate the triglyceride content in different density lipoprotein subfractions.

[0048] The kit provided by this invention enables the simultaneous detection of 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), chylotriglycerides (CM-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).

[0049] The sample described in this invention can be a serum sample or a plasma sample.

[0050] Unlike existing technologies, this invention has the following advantages:

[0051] 1. This invention is the first to realize the detection of lipoprotein subfraction triglyceride indexes on a blood lipid subfraction analyzer. The detection indexes include TG, HDL-TG, LDL-TG, IDL-TG, VLDL-TG, CM-TG, HDL2-TG, HDL3-TG, sdLDL-TG, Lpa-TG, and RLP-TG.

[0052] 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 subfraction triglycerides in the VAP detection system, which is of pioneering significance;

[0053] 3. This invention provides a specific combination of surfactants that enable high triglyceride samples such as chylous blood to complete the lysis reaction in a short time, which can effectively reduce detection bias and improve the accuracy and precision of the kit;

[0054] 4. This invention uses a specific combination of protective agents, which significantly increases the stability of the enzymes in the kit, meeting clinical requirements;

[0055] 5. 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;

[0056] 6. The kit provided by this invention can be used on a VAP system and can be combined with existing cholesterol VAP detection to simultaneously output lipoprotein subfraction cholesterol and triglyceride typing maps, providing a new combination of biomarkers for atherosclerosis, diabetic lipid metabolism disorders, etc., filling a gap in clinical testing. Detailed Implementation

[0057] 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.

[0058] 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.

[0059] As described in the background art, in order to enable the VAP system to be used for the detection of lipoprotein subfraction triglycerides, this invention provides a lipoprotein subfraction triglyceride detection kit, which includes:

[0060] The diluent comprises a first preservative, a first density modifier, and a first solvent;

[0061] A density liquid, the density liquid comprising a second preservative, a second density modifier, and a second solvent;

[0062] The reaction reagents include surfactants, magnesium chloride, 4-aminoantipyrine, 4-chlorophenol, lipoprotein lipase, glycerol phosphate oxidase, glycerol kinase, peroxidase, ascorbic acid oxidase, a preservative, a third preservative, and a third solvent.

[0063] In the above embodiments, the diluent and density solution serve to create a density gradient in the lipoprotein sample.

[0064] 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;

[0065] 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.

[0066] In the above embodiments, the first solvent is a phosphate buffer solution with a pH of 6.0 to 8.0 and a salt concentration of 10 to 1000 mM.

[0067] The second solvent is a phosphate buffer solution with a pH of 6.0 to 8.0 and a salt concentration of 10 to 1000 mM;

[0068] The third solvent is selected from any one of the following: PIPES buffer, HEPES buffer, MOPS buffer, TES buffer, and Tris buffer, with a pH of 6.0 to 9.0 and a salt concentration of 10 to 500 mM.

[0069] In the above embodiments, the surfactant is used as a lipoprotein cleavage agent.

[0070] Qulaton X-100; or

[0071] A mixture of Triton X-100 and alkylphenol polyoxyethylene ether; or

[0072] A mixture of Triton X-100, alkylphenol polyoxyethylene ether, and fatty alcohol polyoxyethylene ether.

[0073] 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.

[0074] More specifically, in the above embodiments, if commercial raw materials are used, BASF Lutensol AP series alkylphenol polyoxyethylene ethers are preferred.

[0075] 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.

[0076] 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.

[0077] The concentration of fatty alcohol polyoxyethylene ether (AEO-7) in the reaction reagent is 0.01%~1% by mass, and the most preferred concentration is 0.05%~0.2% by mass.

[0078] In the above embodiments, the concentration of lipoprotein lipase as a reaction catalyst is preferably 0.2~20 KU / L, and most preferably 1~5 KU / L;

[0079] The concentration of glycerol phosphate oxidase is 1~100 KU / L, with the most preferred concentration being 5~50 KU / L;

[0080] The concentration of glycerol kinase is 0.2~20 KU / L, with the most preferred concentration being 1~5 KU / L;

[0081] The concentration of peroxidase is 0.2~20 KU / L, with the optimal concentration being 1~5 KU / L;

[0082] The concentration of ascorbic acid oxidase is 0.2~20 KU / L, with the optimal concentration being 1~5 KU / L.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] In the above embodiments, the first preservative is PC300 and / or sodium azide;

[0088] The second preservative is PC300 and / or sodium azide;

[0089] The third preservative is selected from any one or more of PC300, sodium azide, and PC950.

[0090] In the above embodiments, the concentration of the first preservative in the diluent is 0.01% to 0.5% by mass.

[0091] The concentration of the second preservative in the diluent is 0.01%~0.5% by mass.

[0092] The concentration of the third preservative in the reaction reagent is 0.01% to 1% by mass.

[0093] In the above embodiments, the concentration of 4-aminoantipyrine as the chromogenic substance in the reaction reagent is 0.1~10mM;

[0094] The concentration of 4-chlorophenol in the reaction reagent is 0.1~10mM.

[0095] 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 substances 4-aminoantipyrine and 4-chlorophenol should be separated as much as possible to improve the stability of the reagents.

[0096] A specific embodiment of the present invention also provides a method for detecting lipoprotein subfraction triglycerides using a VAP detection system and the above-mentioned kit, comprising the following steps:

[0097] S1: Dilute the serum or plasma sample with diluent and adjust the liquid density;

[0098] S2: Add density solution to centrifuge tube;

[0099] 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.

[0100] S4: Use an ultracentrifuge and a vertical rotor to ultracentrifuge the centrifuge tubes in step S3 to form a continuous density gradient. In this step, lipoproteins will be stratified according to density, with the densest lipoproteins located in the lower layer and the least dense lipoproteins located in the upper layer.

[0101] S5: Place the centrifuge tubes that were layered in step S4 onto the lipid sub-fraction 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 a lipoprotein triglyceride profile.

[0102] S6: The software can automatically calculate the triglyceride content of lipoprotein subfractions based on the lipoprotein triglyceride map.

[0103] 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 density solution, and increasing the centrifugation time can improve the separation effect of HDL, thereby improving the detection accuracy and precision of HDL2-TG and HDL3-TG. Furthermore, in normal samples, the chyle content is extremely low, so there is no need to distinguish between VLDL and CM. However, some samples with high chyle content (such as postprandial samples, hyperlipidemia samples, and chylous blood) contain a certain amount of CM. In this case, reducing the density of the diluent and density solution, and reducing the centrifugation time, can improve the separation effect of CM and VLDL, thereby enabling the detection of CM-TG. 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-TG.

[0104] 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.

[0105] Example 1

[0106] The effect of different density solutions and dilution solutions on the reaction rate was determined.

[0107] Prepare the diluent and density solution according to the formula shown in Table 1.

[0108] Table 1

[0109]

[0110] S1: Dilute the serum sample with diluent and mix in a biochemical cup, wherein the dilution volume ratio is serum: diluent = 50: 1950;

[0111] 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 rad2 / sec, temperature=23℃, acceleration=6, deceleration=6.

[0112] 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 triglyceride target values ​​of 1.2 mM and 2.6 mM, respectively. The specific diluent and density solution combinations and results are shown in Table 2. The blank group served as a 100% reaction control.

[0113] Table 2

[0114]

[0115] 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 the detection system for detecting triglyceride profiles in lipoproteins without affecting the reagent reaction efficiency.

[0116] Example 2

[0117] Determination of the effect of reaction reagents on reaction rate.

[0118] Prepare different reaction reagents according to the formulations in Tables 3 and 4. Table 3 shows reaction reagents without AEO-7, and Table 4 shows reaction reagents containing different concentrations of fatty alcohol polyoxyethylene ether AEO-7.

[0119] Table 3

[0120]

[0121] Table 4

[0122]

[0123] 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 triglyceride serum. The triglyceride value of the routine serum was 1.2 mM, and the triglyceride value of the high triglyceride serum was 5.8 mM.

[0124] 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 1.2 mM sample with reagent C1 is 100%, the relative total area under the reaction curve of the 5.8 mM sample should be 483%.

[0125] The specific results are shown in Table 5.

[0126] Table 5

[0127]

[0128] As shown in Table 5, the reaction reagents provided by this invention, with the three surfactant combination reagents containing Triton X-100, Lutensol AP 8, and AEO-7, show a relative total area under the reaction curve that is basically consistent with expectations. The two surfactant combination reagents containing Triton X-100 and Lutensol AP 8 show a total reaction area of ​​approximately 90% to 95% of the expected area when detecting high triglyceride samples. In contrast, the single surfactant reagent containing Triton X-100 shows a relative total area of ​​only 84% of the expected area. Therefore, the single surfactant reaction reagents provided by this invention can be used in routine lipoprotein triglyceride profile detection systems, while the two surfactant combination reagents can be used with some difficulty for high-value samples. The three surfactant combination reagents can meet all application scenarios and have the advantage of faster reaction speed.

[0129] Example 3

[0130] Precision study.

[0131] Prepare the reagents according to the formula in Table 6, for a total of 8 groups.

[0132] Table 6

[0133]

[0134] Based on the substances in Formula E3 excluding PIPES and pH, reagents were prepared according to the buffer and pH combinations shown in Table 7, for a total of 12 groups.

[0135] Table 7

[0136]

[0137] Based on the substances in Formula E4 excluding BSA, reagents were prepared according to the concentrations shown in Table 8, resulting in a total of 21 groups.

[0138] Table 8

[0139]

[0140] Based on the substances in formulation E5 except for Lutensol AP 8, reagents were prepared according to the concentrations shown in Table 9, for a total of 15 groups.

[0141] Table 9

[0142]

[0143] Based on the substances in Formula E6 excluding sodium azide, reagents were prepared according to the concentrations shown in Table 10, resulting in a total of 6 groups.

[0144] Table 10

[0145]

[0146] Because the reagents of this invention can react more quickly, the reaction can be performed on a lipid subcomponent analyzer.

[0147] Take 50 μl of serum and add 1950 μl of Formula A3 diluent. Dilute and mix well. Take 5.1 mL of 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.

[0148] 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 66 reagent formulations (C1, E1-E65) of this invention. The results are shown in Table 11.

[0149] 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 TG were calculated for each reagent.

[0150] Table 11

[0151]

[0152] Table 12

[0153]

[0154] Table 13

[0155]

[0156] Table 14

[0157]

[0158] Table 15

[0159]

[0160] As shown in Tables 11-15, the detection results of the 65 multi-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% to 15% 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 triglyceride detection results.

[0161] Example 4

[0162] Linear range study.

[0163] Since the lipid subfraction analyzer can detect triglycerides of different lipoprotein subfractions, among which TG, HDL-TG, LDL-TG, and VLDL-TG have relatively high measured values ​​and are relatively representative of the main items, the linear range of TG, HDL-TG, LDL-TG, and VLDL-TG is analyzed to illustrate the detectable linear range of the reagent detection system of the present invention. A high TG sample (TG measured value of about 12mM) was taken, diluted to different concentrations, and tested using the formula of the present invention.

[0164] Take 50 μl of each grade of serum 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.

[0165] 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 16.

[0166] Table 16

[0167]

[0168] As shown in Table 16, 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 12 mM. In contrast, the single-surfactant reagent generally yields lower results when detecting triglyceride samples in the 0-12 mM range. Because this invention uses a special combination of surfactants, the reaction rate of the reagent with different lipoprotein triglycerides is significantly increased. Even when detecting high-value samples, the reaction can be completed in a short time. Therefore, the multi-surfactant reagent of this invention has a wider linear range.

[0169] Example 5

[0170] Study on the detection conditions of HDL subcomponents.

[0171] The results of the precision test of the detection system show that the precision of several HDL-related items, such as HDL-TG, HDL2-TG and HDL3-TG, 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.

[0172] Take 50 μl of serum and add 1950 μl of the diluent from Table 17. Dilute and mix well. Take a 5.1 mL Polyallomer quick-seal tube, add 3800 μl of the density solution from Table 17, 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 17, temperature = 23℃, acceleration = 6, deceleration = 6.

[0173] 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.

[0174] Using reagent E4 as the reaction reagent, the experiment was conducted according to the combination scheme shown in Table 17.

[0175] Table 17

[0176]

[0177] The experimental results are shown in Table 18.

[0178]

[0179] As shown in Table 18, the precision of HDL-related triglyceride measurements in groups F7 and F8 is better, with HDL-TG at 3%–4% and HDL2-TG and HDL3-TG at 4%–5%, which is significantly better than the previous precision of 7%–9% for HDL-TG and 9%–11% for HDL2-TG and HDL3-TG. Therefore, this invention can obtain better HDL subcomponent detection results by adjusting (increasing) the density of the diluent, adjusting (increasing) the density of the density solution, and adjusting (increasing) the centrifugation time.

[0180] Example 6

[0181] Study on LDL subcomponent detection conditions.

[0182] The results of the precision experiment of the detection system show that the precision of several LDL-related items, such as LDL-TG and sdLDL-TG, is generally low. 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.

[0183] Take 50 μl of serum and add 1950 μl of the diluent from Table 19, dilute, and mix well. Seal a 5.1 mL Polyallomer tube, add 3800 μl of the density solution from Table 19, 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.

[0184] 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.

[0185] Using reagent E4 as the reaction reagent, the experiment was conducted according to the combination scheme shown in Table 19.

[0186] Table 19

[0187]

[0188] The experimental results are shown in Table 20.

[0189] Table 20

[0190]

[0191] As shown in Table 20, the precision of LDL-related triglyceride measurements in groups G8 and G12 is better, with LDL-TG at 2%–3% and sdLDL-TG at 4%–5%. This is a significant advantage compared to the previous precision of 4%–6% for LDL-TG and 9%–12% for sdLDL-TG. Therefore, this invention can obtain better LDL subcomponent detection results by adjusting (decreasing) the density of the diluent and adjusting (increasing) the density of the density solution.

[0192] Example 7

[0193] Study on CM detection conditions

[0194] When a sample contains a certain amount of chylomicrons (CM), since the proportion of triglycerides in chyle is significantly higher than that of other components, it is necessary to distinguish CM from VLDL as much as possible in order to more accurately determine the concentrations of TG and VLDL-TG in the sample. Based on the experimental principle of this invention, the density of the diluent, the density of the density solution, and the centrifugation time were adjusted (reduced) in an attempt to find better CM detection conditions.

[0195] Take 50 μl of slightly chylous serum and add 1950 μl of the diluent from Table 21. Dilute and mix well. Take a 5.1 mL Polyallomer quick-seal tube, add 3800 μl of the density solution from Table 21, 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 21, temperature = 23℃, acceleration = 6, deceleration = 6.

[0196] 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.

[0197] Using reagent E4 as the reaction reagent, the experiment was conducted according to the combination scheme shown in Table 21.

[0198] Table 21

[0199]

[0200] The experimental results are shown in Table 22.

[0201] Table 22

[0202]

[0203] As shown in Table 22, the precision of VLDL-TG and CM-related triglyceride measurements in group H7 was good, with LDL-TG at 2.51% and sdLDL-TG at 4%–5%, which was close to the coefficient of variation (CV) of VLDL-TG detection in conventional serum. The CV for CM was only 3.91%. Compared to the previous CV of 7.41% for VLDL-TG, the CV for CM was 37.5%, showing a significant precision advantage and demonstrating good separation of CM and VLDL-TG components under these conditions. The poor separation in group H8 may be due to insufficient centrifugation time, leading to a poorer separation effect. Therefore, this invention can separate and detect CM and VLDL-TG by adjusting (reducing) the density of the diluent, adjusting (reducing) the density of the densitizing solution, and adjusting (reducing) the centrifugation time.

[0204] 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 triglyceride detection kit, characterized in that, The lipoprotein subfraction triglyceride detection kit includes: The diluent comprises a first preservative, a first density modifier, and a first solvent; A density liquid, the density liquid comprising a second preservative, a second density modifier, and a second solvent; The reaction reagents include surfactants, magnesium chloride, 4-aminoantipyrine, 4-chlorophenol, lipoprotein lipase, glycerol phosphate oxidase, glycerol kinase, peroxidase, ascorbic acid oxidase, a preservative, a third preservative, and a third solvent.

2. The lipoprotein subfraction triglyceride detection kit as described in claim 1, 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.

3. The lipoprotein subfraction triglyceride detection kit as described in claim 1, characterized in that, The first solvent is a phosphate buffer solution with a pH of 6.0 to 8.0 and a salt concentration of 10 to 1000 mM; The second solvent is a phosphate buffer solution with a pH of 6.0 to 8.0 and a salt concentration of 10 to 1000 mM; The third solvent is selected from any one of PIPES buffer, HEPES buffer, MOPS buffer, TES buffer, and Tris buffer with a pH of 6.0 to 9.0 and a salt concentration of 10 to 500 mM.

4. The lipoprotein subfraction triglyceride 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; or A mixture of Triton X-100, alkylphenol polyoxyethylene ether, and fatty alcohol 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.

5. The lipoprotein subfraction triglyceride detection kit as described in claim 4, 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%~1% by mass. The concentration of the fatty alcohol polyoxyethylene ether in the reaction reagent is 0.01~1% by mass.

6. The lipoprotein subfraction triglyceride detection kit as described in claim 1, characterized in that, The concentration of the lipoprotein lipase is 0.2~20 KU / L; The concentration of the glycerol phosphate oxidase is 1~100 KU / L; The concentration of the glycerol kinase is 0.2~20 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.

7. The lipoprotein subfraction triglyceride 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.

8. The lipoprotein subfraction triglyceride detection kit as described in claim 1, characterized in that, The first preservative is PC300 and / or sodium azide; The second preservative is PC300 and / or sodium azide; The third preservative is selected from any one or more of PC300, sodium azide, and PC950.

9. The lipoprotein subfraction triglyceride detection kit as described in claim 1, 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%~0.5% by mass. The concentration of the third preservative in the reaction reagent is 0.01% to 1% by mass.

10. The lipoprotein subfraction triglyceride detection kit as described in claim 1, characterized in that, The concentration of 4-aminoantipyrine in the reaction reagent is 0.1~10mM; The concentration of 4-chlorophenol in the reaction reagent is 0.1~10mM.

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