A small and dense low density lipoprotein detection kit, its preparation method and application

Abstract

The application provides a small and dense low-density lipoprotein detection kit and a preparation method and application thereof, and belongs to the technical field of reagent detection. The detection kit comprises reagent R1 and reagent R2 packaged independently, the reagent R1 comprises a buffer, TOPS, a surfactant, sodium azide, a stabilizer, cholesteryl esterase, cholesteryl oxidase, and the enzyme activity concentration of peroxidase is 2-50 KU / L; the surfactant is EMULGEN A90 and EMULGEN LS-114; the stabilizer is magnesium ascorbyl phosphate; the reagent R2 comprises a buffer, sodium azide, 4-AAP and ascorbic acid oxidase. The kit has higher clinical use value in the aspects of precision, linearity and stability, and is higher than the industry standard and the commercially available control kit.

Description

Technical Field

[0001] This invention belongs to the field of detection reagent technology, specifically relating to a small and dense low-density lipoprotein detection kit, its preparation method, and its application. Background Technology

[0002] Low-density lipoprotein (LDL) can be mainly divided into two subtypes based on its particle size and density: "large and light" and "small and dense." Small and dense LDL (sdLDL) is significantly more harmful than regular LDL, primarily due to the following characteristics: Stronger penetrability: sdLDL particles are small and can more easily penetrate the vascular endothelium and enter the subendothelial layer of the arterial wall; Stronger retention: It has a stronger ability to bind to proteoglycans on the blood vessel wall, so once it enters the blood vessel wall, it is not easy to be cleared and is more likely to deposit. Slower clearance rate: sdLDL has a lower affinity for LDL receptors in the liver, resulting in a longer half-life in plasma, allowing more time to cause damage; More susceptible to oxidation: sdLDL has an unstable structure and is easily modified by oxidation; oxidized sdLDL is easily engulfed by immune cells (macrophages) and eventually forms foam cells, which is the starting point of atherosclerotic plaques. Compared to other indicators, sdLDL can identify "hidden" risks: many patients still experience cardiovascular events even though their LDL-C (commonly known as "bad cholesterol") levels are normal in routine blood lipid tests; sdLDL testing can identify these "residual risks," making up for the shortcomings of traditional blood lipid tests; one study showed that higher sdLDL levels were significantly associated with a greater risk of atherosclerotic cardiovascular disease (ASCVD), and this association remained even after adjusting for other risk factors. Enhanced risk prediction capabilities: Multiple studies have shown that sdLDL is an independent risk factor for cardiovascular disease, and its predictive value is even better than that of traditional LDL-C; Elevated sdLDL levels are commonly seen in individuals with insulin resistance, metabolic syndrome, type 2 diabetes, and obesity.

[0003] However, currently available reagents have poor long-term stability, with measured values ​​often being higher than or even exceeding LDL values, leading to inversions that affect diagnostic results. There is currently a lack of a small, dense low-density lipoprotein (LDL) detection kit that offers high stability and greater accuracy. Summary of the Invention

[0004] The purpose of this invention is to provide a small and dense low-density lipoprotein (LDL) detection kit, its preparation method, and its application. The kit described in this invention avoids the inversion of measured values ​​exceeding LDL, exhibiting high stability and accuracy. Specifically, the kit of this invention outperforms industry standards and commercially available control kits in terms of precision, linearity, and stability, and has high clinical value.

[0005] This invention provides a small, dense low-density lipoprotein (LDL) detection kit, comprising individually packaged reagents R1 and R2. Reagent R1 includes a buffer solution with a pH of 6.5–7.5 and a molar concentration of 20–100 mmol / L, TOPS with a mass concentration of 0.2–1 g / L, a surfactant with a mass percentage of 0.1%–4%, sodium azide with a mass concentration of 0.1–2 g / L, a stabilizer with a mass concentration of 0.03–0.1 g / L, cholesterol esterase with an enzyme activity concentration of 0.5–10 KU / L, cholesterol oxidase with an enzyme activity concentration of 0.5–10 KU / L, and peroxidase with an enzyme activity concentration of 2–50 KU / L. The surfactants are EMULGEN A90 and EMULGEN LS-114; the stabilizer is magnesium ascorbate phosphate. The reagent R2 comprises a buffer solution with a pH of 6.5–7.5 and a molar concentration of 60–180 mmol / L, sodium azide with a mass concentration of 0.1–2 g / L, 4-AAP with a mass concentration of 0.1–2 g / L, and ascorbic acid oxidase with an enzyme activity concentration of 0.5–10 KU / L.

[0006] Preferably, the reagent R1 comprises a buffer solution with a pH of 6.8-7.2 and a molar concentration of 40-60 mmol / L, TOPS with a mass concentration of 0.4-0.7 g / L, a surfactant with a mass percentage of 0.5%-2%, sodium azide with a mass concentration of 0.3-1 g / L, a stabilizer with a mass concentration of 0.03-0.06 g / L, cholesterol esterase with an enzyme activity concentration of 1-4 KU / L, cholesterol oxidase with an enzyme activity concentration of 2-6 KU / L, and peroxidase with an enzyme activity concentration of 2-18 KU / L. The reagent R2 comprises a buffer solution with a pH of 6.8–7.2 and a molar concentration of 80–130 mmol / L, sodium azide with a mass concentration of 0.3–1 g / L, 4-AAP with a mass concentration of 0.2–1 g / L, and ascorbic acid oxidase with an enzyme activity concentration of 2–5 KU / L.

[0007] Preferably, the mass ratio of EMULGEN A90 to EMULGEN LS-114 in the surfactant is (5~8):(2~4).

[0008] Preferably, the mass ratio of EMULGEN A90 to EMULGEN LS-114 in the surfactant is (5~7):(3~4).

[0009] Preferably, the buffer solution includes HEPES buffer, MOPS buffer, MES buffer, or TRIS buffer.

[0010] Preferably, in the kit, the volume ratio of reagent R1 to reagent R2 is 3:1.

[0011] The present invention also provides a method for preparing the detection kit, comprising the following steps: HEPES buffer, TOPS, surfactant, sodium azide, magnesium ascorbate phosphate, and water were mixed and the pH was adjusted to 6.5-7.5 to obtain the first mixture. The first mixture, cholesterol esterase, cholesterol oxidase, and peroxidase were mixed and the pH was adjusted to 6.5-7.5 to obtain reagent R1. HEPES buffer, sodium azide, 4-AAP, and water were mixed and the pH was adjusted to 6.5-7.5 to obtain a second mixture. The second mixture was then mixed with ascorbic acid oxidase and the pH was adjusted to 6.5-7.5 to obtain reagent R2.

[0012] The present invention also provides the application of the detection kit described in the above technical solution or the detection kit prepared by the preparation method described in the above technical solution in the preparation of products for detecting small and dense low-density lipoprotein.

[0013] The present invention also provides the application of the detection kit described in the above technical solution or the detection kit prepared by the preparation method described in the above technical solution in the preparation of products that improve the stability and / or accuracy of small and dense low-density lipoprotein detection.

[0014] The present invention also provides a method for detecting small dense low-density lipoprotein cholesterol for non-diagnostic and non-therapeutic purposes, comprising the following steps: using the detection kit described in the above technical solution or the detection kit prepared by the preparation method described in the above technical solution, and using a fully automated biochemical analysis device to detect small dense low-density lipoprotein cholesterol.

[0015] This invention provides a small, dense low-density lipoprotein (LDL) assay kit. The kit contains cholesterol esterase, cholesterol oxidase, peroxidase, and the chromogen substrate TOPS. These are susceptible to oxidation during long-term storage, leading to absorbance drift. Magnesium ascorbate phosphate exhibits excellent antioxidant capacity and, among ascorbic acid and its derivatives, shows superior stability. This invention, by adding EMULGEN A90, EMULGEN LS-114, and magnesium ascorbate phosphate to the small, dense LDL assay kit, effectively protects the enzymes and chromogen substrates, maximizing their activity and extending the kit's shelf life. The kit is stable for at least 12 months at 2–8°C. Furthermore, using magnesium ascorbate phosphate as a kit component, combined with EMULGEN A90, EMULGEN LS-114, and other components at appropriate concentrations, the resulting kit demonstrates superior precision, linearity, and stability compared to industry standards and commercially available control kits, exhibiting high clinical value. Specifically, this invention utilizes a kit containing EMULGEN A90 and EMULGEN LS-114, which can effectively remove lipoproteins other than sdLDL, thereby avoiding the inversion of measured values ​​higher than LDL, improving the accuracy of results, and when used together with magnesium ascorbate phosphate, it can effectively protect the enzymes in the reagent, greatly improving the stability of the reagent. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The correlation detection results of the kit prepared for Example 1 of this invention and the control kit 1 for detecting ordinary samples are shown in the figure. Figure 2 The correlation detection results of the kit prepared for Example 2 of this invention and the control kit 1 for detecting ordinary samples are shown in the figure. Figure 3 The correlation detection results of the kit prepared for Example 3 of this invention and the control kit 1 for detecting ordinary samples are shown in the figure. Figure 4 A graph showing the correlation detection results of the kit prepared for Comparative Example 1 and Control Kit 1 for detecting ordinary samples provided by the present invention; Figure 5 The correlation detection results of the kit prepared for Comparative Example 2 and the control kit 1 for detecting ordinary samples are shown in the figure. Figure 6 The correlation detection results of the kit prepared for Comparative Example 3 and the control kit 1 for detecting ordinary samples are shown in the figure. Detailed Implementation

[0018] This invention provides a small, dense low-density lipoprotein (LDL) detection kit, comprising individually packaged reagents R1 and R2. Reagent R1 includes a buffer solution with a pH of 6.5–7.5 and a molar concentration of 20–100 mmol / L, TOPS with a mass concentration of 0.2–1 g / L, a surfactant with a mass percentage of 0.1%–4%, sodium azide with a mass concentration of 0.1–2 g / L, a stabilizer with a mass concentration of 0.03–0.1 g / L, cholesterol esterase with an enzyme activity concentration of 0.5–10 KU / L, cholesterol oxidase with an enzyme activity concentration of 0.5–10 KU / L, and peroxidase with an enzyme activity concentration of 2–50 KU / L. The surfactants are EMULGEN A90 and EMULGEN LS-114; the stabilizer is magnesium ascorbate phosphate. The reagent R2 comprises a buffer solution with a pH of 6.5–7.5 and a molar concentration of 60–180 mmol / L, sodium azide with a mass concentration of 0.1–2 g / L, 4-AAP with a mass concentration of 0.1–2 g / L, and ascorbic acid oxidase with an enzyme activity concentration of 0.5–10 KU / L.

[0019] Unless otherwise specified, the present invention does not have special requirements for the source of each component in the reagent kit, and commercially available products well known to those skilled in the art can be used.

[0020] The R1 reagent of the small, dense low-density lipoprotein detection kit of the present invention comprises a buffer solution with a pH of 6.5-7.5 and a molar concentration of 20-100 mmol / L. In a specific embodiment, the molar concentration of the buffer solution can be 40-60 mmol / L, more specifically 45-55 mmol / L, and more specifically 50 mmol / L. In a specific embodiment, the pH of the buffer solution can be 6.8-7.2, and more specifically 7.0. The present invention improves the sensitivity of the reagent by adjusting the pH of the buffer solution. In a specific embodiment, the buffer solution includes HEPES buffer, MOPS buffer, MES buffer, or TRIS buffer.

[0021] The R1 reagent in the small, dense low-density lipoprotein detection kit of this invention includes TOPS at a mass concentration of 0.2~1 g / L, which can be 0.4~0.7 g / L, more specifically 0.4~0.6 g / L, and more specifically 0.5 g / L. The TOPS described in this invention is a chromogenic substrate and an important component of the colorimetric reaction.

[0022] The R1 reagent of the small and dense low-density lipoprotein detection kit of the present invention includes a surfactant with a mass percentage of 0.1% to 4%, which can be 0.5% to 2%, more specifically 0.8% to 1.5%, and more specifically 1%. In a specific embodiment, the surfactant is EMULGEN A90 and EMULGEN LS-114. The surfactant of the present invention has the function of removing excess lipoproteins (i.e., removing lipoprotein cholesterol other than sdLDL) and improving the accuracy of the reaction results. EMULGEN A90 and EMULGEN LS-114 at a certain ratio have the best effect and can obtain more accurate reaction results. In a specific embodiment, the mass ratio of EMULGEN A90 to EMULGEN LS-114 in the surfactant is (5~8):(2~4). In a specific embodiment, the mass ratio of EMULGEN A90 to EMULGEN LS-114 in the surfactant is (5~7):(3~4). In a specific embodiment, the EMULGEN A90 and EMULGEN LS-114 are manufactured by Kao Corporation, with product numbers 2545 and 0059, respectively.

[0023] The R1 reagent in the small, dense low-density lipoprotein detection kit of the present invention includes sodium azide at a mass concentration of 0.1~2 g / L, which can be 0.3~1 g / L, more specifically 0.6~0.9 g / L, and more specifically 0.8 g / L. In this invention, sodium azide acts as a preservative, inhibiting the growth of contaminating bacteria and extending the shelf life of the kit.

[0024] The R1 reagent of the small and dense low-density lipoprotein detection kit of the present invention includes a stabilizer with a mass concentration of 0.03~0.1 g / L, which can be 0.03~0.06 g / L, more specifically 0.04~0.05 g / L, and more specifically 0.04 g / L. In a specific embodiment, the stabilizer is magnesium ascorbate phosphate. In a specific embodiment, magnesium ascorbate phosphate is a stable vitamin C (L-ascorbic acid) derivative with antioxidant capacity, which can effectively scavenge free radicals, prevent enzyme oxidative damage, protect the enzyme and chromogen substrate in the reagent, and has chelating ability to remove interference from metal ions. Its molecular formula is C6H6Mg. 1.5 O9P·xH2O, manufactured by Sigma-Aldrich, part number A8950, structural formula is shown in Formula I; , Formula I.

[0025] The R1 reagent of the small, dense low-density lipoprotein detection kit of this invention includes cholesterol esterase with an enzyme activity concentration of 0.5~10 KU / L, which can be 1~4 KU / L, more specifically 2~3 KU / L, and more specifically 2.5 KU / L. The cholesterol esterase of this invention catalyzes the hydrolysis of cholesterol esters to release free cholesterol. All enzymes used in this invention were purchased from Toyobo, Japan.

[0026] The R1 reagent in the small, dense low-density lipoprotein detection kit of this invention includes cholesterol oxidase with an enzyme activity concentration of 0.5-10 KU / L, which can be 2-6 KU / L, more specifically 3-5 KU / L, and more specifically 4 KU / L. The cholesterol oxidase of this invention has the function of oxidizing free cholesterol to generate hydrogen peroxide.

[0027] The R1 reagent in the small, dense low-density lipoprotein detection kit of this invention comprises peroxidase with an enzyme activity concentration of 2-50 KU / L, which can be 2-18 KU / L, more specifically 2-6 KU / L, and even more specifically 3-5 KU / L, and more specifically 4 KU / L. The peroxidase of this invention catalyzes the reaction of hydrogen peroxide with a chromogenic substrate to generate a chromogenic quinone imine, which absorbs at a set wavelength.

[0028] The R2 reagent in the small, dense low-density lipoprotein detection kit of this invention comprises a buffer solution with a pH of 6.5-7.5 and a molar concentration of 60-180 mmol / L. In a specific embodiment, the molar concentration of the buffer solution can be 80-130 mmol / L, specifically 100 mmol / L. In a specific embodiment, the pH value can be 6.8-7.2, specifically 7.0. This invention improves the sensitivity of the reagent by adjusting the pH of the buffer solution. In a specific embodiment, the buffer solution includes HEPES buffer.

[0029] The R2 reagent in the small, dense low-density lipoprotein detection kit of the present invention comprises sodium azide at a mass concentration of 0.1~2 g / L, which can be 0.3~1 g / L, more specifically 0.6~0.9 g / L, and more specifically 0.8 g / L. In this invention, sodium azide acts as a preservative, inhibiting the growth of contaminating bacteria and extending the shelf life of the kit.

[0030] The R2 reagent in the small, dense low-density lipoprotein detection kit of this invention includes 4-AAP at a mass concentration of 0.1~2 g / L, specifically 0.2~1 g / L, or 0.6 g / L. In this invention, 4-AAP is a key raw material for the colorimetric reaction.

[0031] The R2 reagent in the small, dense low-density lipoprotein detection kit of this invention includes ascorbic acid oxidase with an enzyme activity concentration of 0.5~10 KU / L, specifically 2~5 KU / L, or 3 KU / L. The ascorbic acid oxidase of this invention has the function of scavenging ascorbic acid and its derivatives, thus preventing ascorbic acid and its derivatives from affecting the main Trinder reaction of the reagent.

[0032] In a specific embodiment, the volume ratio of reagent R1 to reagent R2 in the kit is 3:1.

[0033] The present invention also provides a method for preparing the detection kit, comprising the following steps: HEPES buffer, TOPS, surfactant, sodium azide, magnesium ascorbate phosphate, and water were mixed and the pH was adjusted to 6.5-7.5 to obtain the first mixture. The first mixture, cholesterol esterase, cholesterol oxidase, and peroxidase were mixed and the pH was adjusted to 6.5-7.5 to obtain reagent R1. HEPES buffer, sodium azide, 4-AAP, and water were mixed and the pH was adjusted to 6.5-7.5 to obtain a second mixture. The second mixture was then mixed with ascorbic acid oxidase and the pH was adjusted to 6.5-7.5 to obtain reagent R2.

[0034] This invention avoids the negative impact of large amounts of pH adjusters on enzyme activity by adjusting the pH value before adding the enzyme and then adjusting the pH value again. In a specific embodiment, the pH adjuster can be hydrochloric acid or NaOH.

[0035] This invention also provides the application of the detection kit described in the above-described technical solutions or the detection kit prepared by the preparation method described in the above-described technical solutions in the preparation of products for detecting small dense low-density lipoprotein (SDL). The detection kit of this invention can detect SDL cholesterol and can obtain SDL cholesterol values ​​for non-diagnostic purposes. Applications include: ① Nutritional research: assessing the effects of different dietary patterns or specific foods (such as avocados, nuts, dairy products, corn oil, etc.) on human metabolism as biomarkers; specifically, by observing changes in sdLDL-C, one can understand how dietary structure affects the subtype distribution of SDL; ② Exercise physiology research: serving as a sensitive indicator for assessing the effects of exercise; specifically, studying the impact of regular exercise on sdLDL-C levels in healthy individuals; ③ Environmental health research: a tool to explore the effects of environmental pollutants (such as dioxins, PM2.5 fine particulate matter) on organisms, specifically quantifying the degree of interference of environmental exposure on lipid metabolism by detecting changes in indicators such as sdLDL-C. ④ sdLDL is a phenotypic marker of insulin resistance and lipid transport disorders, used to assess overall metabolic status. Elevated sdLDL is a characteristic lipid phenotype of insulin resistance (high TG, low HDL, sdLDL-dominant phenotype B), reflecting systemic glucose and lipid metabolism imbalance more stably than triglycerides alone; it is suitable for grading the metabolic status of obese individuals, those with polycystic ovary syndrome, and those with impaired glucose tolerance. Abnormal hepatic lipid metabolism drives the excessive production of sdLDL, and sdLDL levels are positively correlated with hepatic steatosis and the progression of liver fibrosis; it is used to differentiate the severity of liver metabolic damage at the same BMI / transaminase levels, but not for diagnosing fatty liver. In specific embodiments, the product can be a tool for nutritional research, exercise physiology research, or environmental health research.

[0036] This invention also provides the application of the detection kit described in the above-described technical solutions or the detection kit prepared by the preparation method described in the above-described technical solutions in the preparation of products that improve the stability and / or accuracy of small and dense low-density lipoprotein (LDL) detection. By adding EMULGEN A90, EMULGEN LS-114, and magnesium ascorbate phosphate to the small and dense LDL detection kit, this invention effectively protects the enzymes and chromogen substrates in the reagent, maximizing their activity and extending the shelf life of the reagent. The reagent is stable for at least 12 months at 2–8°C. Furthermore, using magnesium ascorbate phosphate as a kit component, combined with EMULGEN A90, EMULGEN LS-114, and other components at appropriate concentrations, the resulting kit exhibits higher precision, linearity, and stability than industry standards and commercially available control kits, demonstrating high clinical value.

[0037] This invention also provides a method for detecting small dense low-density lipoprotein cholesterol (SDLC) for non-diagnostic and non-therapeutic purposes, comprising the following steps: using the detection kit described in the above technical solution or the detection kit prepared by the preparation method described in the above technical solution, and using a fully automated biochemical analysis device to detect SLC. In a specific embodiment, when using a fully automated biochemical analysis device for detection, the main detection wavelength can be 546 nm, the secondary wavelength can be 700 nm, and a two-point endpoint method is used for detection. In a specific embodiment, the mixing volume ratio of the test sample and reagent R1 can be 1:75. In a specific embodiment, during the detection process, the mixing volume ratio of reagent R1 and reagent R2 can be 3:1.

[0038] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a small and dense low-density lipoprotein detection kit, its preparation method, and its application, should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1 A stable, small and dense low-density lipoprotein cholesterol detection kit, consisting of individually packaged reagents R1 and R2, wherein the volume ratio of reagent R1 to reagent R2 is 3:1; The reagent R1 consists of the following components: 50 mmol / L HEPES buffer at pH 7.0, 0.5 g / L TOPS, 0.6% (w / w) EMULGEN A90, 0.4% (w / w) EMULGEN LS-114, 0.8 g / L sodium azide, 0.04 g / L magnesium ascorbate phosphate, 2.5 KU / L cholesterol esterase, 4 KU / L cholesterol oxidase, and 4 KU / L peroxidase. The specific preparation method is as follows: Take 800 mL of purified water, add HEPES according to the above components, dissolve fully, then add TOPS, EMULGEN A90, EMULGEN LS-114, sodium azide and magnesium ascorbate phosphate in sequence, adjust the pH to 7.0 with hydrochloric acid and sodium hydroxide, then add cholesterol esterase, cholesterol oxidase and peroxidase in sequence, and finally add purified water to make up to 900 mL, and adjust the pH to 7.0 with hydrochloric acid and sodium hydroxide.

[0040] The reagent R2 consists of the following components: 100 mmol / L HEPES buffer at pH 7.0, 0.8 g / L sodium azide, 0.6 g / L 4-AAP, and 3 KU / L ascorbic acid oxidase.

[0041] The specific preparation method is as follows: Take 250 mL of purified water, add HEPES according to the above components, dissolve it completely, then add sodium azide and 4-AAP in sequence, adjust the pH to 7.0 with hydrochloric acid and sodium hydroxide, then add ascorbic acid oxidase, and finally add purified water to make up to 300 mL, and adjust the pH to 7.0 with hydrochloric acid and sodium hydroxide.

[0042] Examples 2-3 and Comparative Examples 1-5 The small and dense low-density lipoprotein cholesterol detection kit was prepared according to Example 1. The components of the formulations in Examples 2 and 3 were the same as those in Example 1, except that the concentrations of the components in the formulations were different. The components or concentrations of the formulations in Comparative Examples 1 to 5 were different from those in Example 1. The specific parameters are listed in Table 1.

[0043] Table 1. Components of the reagent kits for Examples 1-2 and Comparative Examples 1-5

[0044] Application Example 1 Sample Correlation: Fifty fresh serum samples (numbered 1-50) were collected from the hospital. The serum levels in samples 1-50 were compared with those obtained using Examples 1-3, Comparative Examples 1-3, and commercially recognized small-dense low-density lipoprotein cholesterol (SDL) kits (as control kit 1, purchased from Beijing Jiuqiang Technology Co., Ltd., catalog number GSSD), commercially available SDL kits (as control kit 2, purchased from manufacturer A), and Zhicheng LDL cholesterol kits (catalog number CH5038). The instrument used was a Hitachi 7180 fully automated biochemical analyzer (manufacturer: Hitachi, Japan). The detection parameters were set as follows: main wavelength 546nm, secondary wavelength 700nm, sample volume 2μl, reagent R1 volume 150μl, and reagent R2 volume 50μl. The results are shown in Table 2 and... Figures 1-6 .

[0045] Table 2. Results of sample correlation detection (unit: mmol / L)

[0046] Note: Comparative Example 4 and Comparative Example 5 differ only in stability; their other basic performance characteristics are identical.

[0047] The results show that the correlation between Example 1 and Control Kit 1 is 0.9931, Example 2 is 0.9862, Example 3 is 0.9789, Comparative Example 1 is 0.9518, Comparative Example 2 is 0.9569, and Comparative Example 3 is 0.9457. Examples 1-3 indicate that the surfactant ratio in Example 1 is optimal, with the measured values ​​closest to Control Kit 1 and the best correlation. Control Kit 2 shows a poor correlation with Control Kit 1, and three samples showed higher measured values ​​than the low-density lipoprotein (LDL) sample, indicating an inversion. Examples 1 and Comparative Examples 1-3 show that samples without both surfactants or with only one surfactant have significantly higher measured values, some even exceeding the LDL value, indicating an inversion. The two surfactants in Example 1 effectively remove lipoproteins other than sdLDL.

[0048] Application Example 2 Precision Experiment: Serum samples were collected from two patients at the hospital. One patient had a high level of small dense low-density lipoprotein cholesterol (SDL-C) (approximately 2.00 mmol / L), while the other patient had a low level (approximately 0.60 mmol / L). These samples were designated as high- and low-value samples, respectively. The high- and low-value serum samples were tested using Example 1, Example 2, Comparative Example 1, Comparative Example 3, and a commercially recognized, high-dense LDL-C kit 1 (used as a control kit, purchased from Beijing Jiuqiang Technology Co., Ltd., catalog number GSSD). Each kit was tested 10 times, yielding 10 values. The mean (X), standard deviation (S), and coefficient of variation (CV) of the results were calculated to determine the precision. The instrument used was a Hitachi 7180 fully automated biochemical analyzer (manufacturer: Hitachi, Japan). The detection parameters were set as follows: main wavelength 546 nm, secondary wavelength 700 nm, sample volume 2 μl, reagent R1 volume 150 μl, and reagent R2 volume 50 μl. The results are shown in Table 3.

[0049] Table 3. Precision test results (unit: mmol / L)

[0050] As shown in Table 3, both the reagent kit of the present invention and the control reagent kit meet the requirement of a coefficient of variation of less than 5%, and the reagent kit prepared in Example 1 performs the best.

[0051] Application Example 3 A standard solution was prepared using small, dense low-density lipoprotein cholesterol (CDL cholesterol, catalog number DGZ0538B) and purified water. The 3.00 mmol / L standard solution was serially diluted with purified water to obtain six test samples with different concentrations (3.00 mmol / L, 1.50 mmol / L, 0.75 mmol / L, 0.38 mmol / L, 0.19 mmol / L, and 0.09 mmol / L). These samples were tested using Example 1, Example 3, Comparative Example 5, and a commercially recognized good control kit 1 (Beijing Jiuqiang Technology Co., Ltd., catalog number GSSD, detection method is the same as in Application Example 1). Each concentration of test sample was measured three times, and the mean (yi) of the test results was calculated. A linear regression equation was obtained with the dilution concentration (xi) as the independent variable and the mean (yi) of the test results as the dependent variable. The correlation coefficient (r) of the linear regression was calculated, and the results are shown in Table 4.

[0052] Table 4. Linearity results for different groups (unit: mmol / L)

[0053] The results above show that the kit of the present invention, the comparative kit, and the control kit all meet the linearity requirements specified in the industry standard; it is evident that the addition of magnesium ascorbate phosphate does not affect the detection results.

[0054] Application Example 4 Accelerated stability upon opening: In Examples 1, 4, and 5, and using the commercially recognized small and dense low-density lipoprotein cholesterol kit 1 (as a control kit, purchased from Beijing Jiuqiang Technology Co., Ltd., catalog number GSGR), reagents were placed in the instrument with the cap opened and then incubated at 37°C for acceleration. At 4°C, the reagents were calibrated using Jiuqiang sdLDL calibrator, catalog number GC-SD (target value 1.00 mmol / L). Subsequently, the reagents were removed 1-2 times per week and tested in the 4°C calibration channel using Jiuqiang GR quality control samples at levels 1 and 2, catalog number GQ-SD (target values ​​0.60 mmol / L and 1.20 mmol / L, respectively). The detection method was the same as in the application examples, and the results are shown in Table 5.

[0055] Table 5. Stability results for different groups (unit: mmol / L)

[0056] Table 5 shows that the reagent kit of the present invention maintains stability for one month after opening and for 21 days under accelerated conditions. The comparative samples without magnesium ascorbate phosphate and with half the amount of magnesium ascorbate phosphate exhibited poor stability after both opening and accelerated conditions, indicating that magnesium ascorbate phosphate effectively improves reagent stability. This experiment demonstrates that the reagent kit of the present invention is superior in stability to the control reagent and the comparative samples, and the reagent kit prepared in Example 1 exhibits even better stability.

[0057] Application Example 5 Long-term stability: Since accelerated stability cannot completely replace long-term stability, the following reagents were tested: Example 1, Example 2, comparative examples without magnesium ascorbate phosphate (Comparative Example 1 and Comparative Example 2), a commercially recognized small and dense low-density lipoprotein cholesterol kit (as control kit 1, purchased from Beijing Jiuqiang Technology Co., Ltd., catalog number GSSD), and a commercially available small and dense low-density lipoprotein cholesterol kit (as control kit 2, purchased from manufacturer A). Reagents were stored in a refrigerator at 2–8°C. At 4°C, the reagents were calibrated using Jiuqiang GR calibrator, catalog number GC-SD (target value 1.00 mmol / L). Every 3 months thereafter, the reagents were taken out and tested at 4°C in the calibration channel using Jiuqiang sdLDL quality control samples at levels 1 and 2, catalog number GQ-SD (target values ​​0.60 mmol / L and 1.20 mmol / L, respectively), as well as blank absorbance. The detection method was the same as in Application Example 1. The results are shown in Tables 6 and 7.

[0058] Table 6. Long-term stability results of different groups (unit: mmol / L)

[0059] Table 7. Blank absorbance results for different groups

[0060] As shown in Tables 6 and 7, the long-term stability of the reagent kit of the present invention in Example 1 can be maintained for 12 months, and its stability after 12 months is better than that of commercially recognized good reagents, and significantly better than commercially available reagents. In contrast, the long-term stability of Comparative Examples 4 and 5, which contained less or no magnesium ascorbate phosphate, was poor, indicating that a certain concentration of magnesium ascorbate phosphate has a significant effect on improving the long-term stability of the reagent.

[0061] In summary, the reagent kit of this invention outperforms industry standards and commercially available control kits in terms of precision, linearity, and stability, and has high clinical value.

[0062] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A kit for detecting small and dense low-density lipoprotein, characterized in that, The reagent comprises individually packaged reagents R1 and R2. Reagent R1 includes a buffer solution with a pH of 6.5–7.5 and a molar concentration of 20–100 mmol / L, TOPS with a mass concentration of 0.2–1 g / L, a surfactant with a mass percentage of 0.1%–4%, sodium azide with a mass concentration of 0.1–2 g / L, a stabilizer with a mass concentration of 0.03–0.1 g / L, cholesterol esterase with an enzyme activity concentration of 0.5–10 KU / L, cholesterol oxidase with an enzyme activity concentration of 0.5–10 KU / L, and peroxidase with an enzyme activity concentration of 2–50 KU / L. The surfactants are EMULGEN A90 and EMULGEN LS-114, and the stabilizer is magnesium ascorbate phosphate. The reagent R2 comprises a buffer solution with a pH of 6.5–7.5 and a molar concentration of 60–180 mmol / L, sodium azide with a mass concentration of 0.1–2 g / L, 4-AAP with a mass concentration of 0.1–2 g / L, and ascorbic acid oxidase with an enzyme activity concentration of 0.5–10 KU / L.

2. The detection kit according to claim 1, characterized in that, The reagent R1 includes a buffer solution with a pH of 6.8-7.2 and a molar concentration of 40-60 mmol / L, TOPS with a mass concentration of 0.4-0.7 g / L, a surfactant with a mass percentage of 0.5%-2%, sodium azide with a mass concentration of 0.3-1 g / L, a stabilizer with a mass concentration of 0.03-0.06 g / L, cholesterol esterase with an enzyme activity concentration of 1-4 KU / L, cholesterol oxidase with an enzyme activity concentration of 2-6 KU / L, and peroxidase with an enzyme activity concentration of 2-18 KU / L. The reagent R2 comprises a buffer solution with a pH of 6.8–7.2 and a molar concentration of 80–130 mmol / L, sodium azide with a mass concentration of 0.3–1 g / L, 4-AAP with a mass concentration of 0.2–1 g / L, and ascorbic acid oxidase with an enzyme activity concentration of 2–5 KU / L.

3. The detection kit according to claim 1 or 2, characterized in that, The mass ratio of EMULGENA90 to EMULGEN LS-114 in the surfactant is (5~8):(2~4).

4. The detection kit according to claim 3, characterized in that, In the surfactant, the mass ratio of EMULGEN A90 to EMULGEN LS-114 is (5~7):(3~4).

5. The detection kit according to claim 1 or 2, characterized in that, The buffer solution includes HEPES buffer, MOPS buffer, MES buffer, or TRIS buffer.

6. The detection kit according to claim 1 or 2, characterized in that, In the kit, the volume ratio of reagent R1 to reagent R2 is 3:

1.

7. A method for preparing the detection kit according to any one of claims 1 to 6, comprising the following steps: HEPES buffer, TOPS, surfactant, sodium azide, magnesium ascorbate phosphate, and water were mixed and the pH was adjusted to 6.5-7.5 to obtain the first mixture. The first mixture, cholesterol esterase, cholesterol oxidase, and peroxidase were mixed and the pH was adjusted to 6.5-7.5 to obtain reagent R1. HEPES buffer, sodium azide, 4-AAP, and water were mixed and the pH was adjusted to 6.5-7.5 to obtain a second mixture. The second mixture was then mixed with ascorbic acid oxidase and the pH was adjusted to 6.5-7.5 to obtain reagent R2.

8. The use of the test kit according to any one of claims 1 to 6 or the test kit prepared by the preparation method according to claim 7 in the preparation of products for detecting small and dense low-density lipoprotein.

9. The use of the test kit according to any one of claims 1 to 6 or the test kit prepared by the preparation method according to claim 7 in the preparation of products that improve the stability and / or accuracy of small and dense low-density lipoprotein detection.

10. A method for detecting small, dense low-density lipoprotein cholesterol for non-diagnostic and non-therapeutic purposes, characterized in that, Includes the following steps: Using the test kit described in any one of claims 1 to 6 or the test kit prepared by the preparation method described in claim 7, small and dense low-density lipoprotein cholesterol is detected using a fully automated biochemical analysis device.

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