A kit for detecting small, low-density lipoprotein cholesterol and a method for preparing the same

Abstract

The application provides a kit for detecting small and low-density lipoprotein cholesterol and a preparation method thereof, the kit comprising reagent R1 and reagent R2; wherein the reagent R1 comprises a buffer, an eliminating agent, CHOE and the like; and the reagent R2 comprises a buffer, a reaction agent and the like. The application improves the detection accuracy, precision, linearity and specificity of the reagents by improving the formula of the reagent R1 and the reagent R2; and the kit is used in cooperation with a full-automatic biochemical analyzer, has the advantages of fast detection speed, automatic detection and simple operation, and thus improves the detection performance of small and low-density lipoprotein cholesterol in clinical treatment.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, specifically to a kit for detecting small low-density lipoprotein cholesterol and its preparation method. Background Technology

[0002] Low-density lipoprotein (LDL) is heterogeneous, consisting of a series of particles of varying sizes, densities, and chemical compositions. Generally, the LDL subunits with smaller particles and higher density are called small dense LDL (sd LDL); those with larger particles and lower density are called large light LDL; and the subunits in between are called intermediate-density LDL.

[0003] Recent studies have found that sdLDL has a stronger atherosclerotic effect than regular LDL and has been listed as one of the newly discovered important cardiovascular risk factors by the Adult Treatment Group of the National Cholesterol Education Program (NCEP) Committee.

[0004] Small-particle dense low-density lipoprotein (sd LDL) in the LDL subgroup has a strong atherosclerotic effect, while elevated plasma triglyceride levels can promote the conversion of LDL from large-particle loose LDL to sd LDL. The conversion of LDL subgroups to large-particle loose LDL can be effectively promoted by lipid-lowering drugs such as niacin, phenoxyacetic acid, or in combination with statins, thereby reducing plasma sd LDL levels to some extent.

[0005] The possible mechanisms by which sd LDL leads to atherosclerosis, coronary heart disease, etc. include:

[0006] 1. Easily oxidized and modified: High-density LDL subcomponents (sd LDL) are easily oxidized and modified into oxidized LDL in the human body. Oxidized LDL is easily phagocytosed by phagocytes indefinitely, thus developing into foam cells.

[0007] 2. Low antioxidant content: Studies have found that the content of coenzyme Q10 and vitamin E in sd LDL particles isolated from normal human blood plasma is significantly lower than that in large and light LDL and medium-density LDL particles, while the content of peroxides is very high.

[0008] 3. Slow clearance: Dejager et al. found in in vitro cell culture that high-density LDL particles in normal human plasma have a significantly lower affinity for LDL receptors than other LDL particles. This indicates that sd LDL particles, due to their slow degradation via the receptor pathway, remain in vivo for a longer time than other LDL particles. This increases their chances of oxidation in blood vessels and the likelihood of them being phagocytosed by macrophages to form foam cells.

[0009] 4. Easy Adhesion: In vitro studies have found that sd LDL easily binds to proteoglycans on the blood vessel wall, thus adhering to the vessel wall. If this occurs in vivo, the adhered sd LDL is likely to enter human vascular endothelial cells through some pathway. Under the action of various cellular oxidases, sd LDL undergoes oxidative modification within the blood vessel wall, thereby inducing the occurrence of various diseases such as atherosclerosis and coronary heart disease.

[0010] Currently known methods for detecting small low-density lipoprotein cholesterol (SDL-C) mainly include centrifugation and direct detection. Centrifugation separates LDL from plasma and then detects SDL through electrophoresis or immunoblotting. This method has relatively high accuracy, but it is not only cumbersome and time-consuming, but also has a narrow linear range, making it unsuitable for the needs of emergency and clinical patients requiring timely diagnosis. These problems have caused significant inconvenience to the clinical use of SDL-C testing. On the other hand, commercially available reagents for direct detection have issues with unstable sample values ​​and narrow linearity, leading to higher values ​​for chylous samples and lower reagent accuracy. Summary of the Invention

[0011] (a) Technical problems to be solved

[0012] To address the shortcomings of existing technologies, this invention provides a kit for detecting small low-density lipoprotein cholesterol and its preparation method, which solves the problems of slow detection speed, low automation, cumbersome operation, and inaccurate results of known small low-density lipoprotein cholesterol detection methods.

[0013] (II) Technical Solution

[0014] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution:

[0015] In a first aspect, the present invention provides a reagent composition for small low-density lipoprotein cholesterol, comprising reagent R1 and reagent R2, which are independent of each other, with a volume ratio of 3:1; wherein reagent R1 has a pH of 7.0 to 7.8 and contains a buffer, a stabilizer, a surfactant, an enzyme, and an eliminator; and reagent R2 has a pH of 7.2 to 7.8 and contains a stabilizer, a modifier, a surfactant, an enzyme, and a substrate.

[0016] In reagent R1, the buffer solution is PIPES at a concentration of 100–150 mmol / L. The stabilizer contains 0.2%–0.4% BSA and 0.1%–0.5% sodium glutamate; the surfactants B66, A90, and CH40 are 0.1%–0.3%, A90, and CH40, respectively; the enzymes are 2–5 KU / L phospholipase, 2–5 KU / L cholesterol esterase, 2–5 KU / L cholesterol oxidase, and 1–10 KU / L POD; the ions are 2–5 mmol / L Mg; the eliminator is 10 KU peroxidase and 0.1%–0.5% TOOS; and the concentration of dextran sulfate 2000 is 1–2 g / L.

[0017] In reagent R2, the stabilizer is glutathione at a concentration of 30–60 mmol / L and BSA at a concentration of 0.5%–1%; the modifier is HEPES at a concentration of 30–60 mmol / L and triethanolamine at a concentration of 30–60 mmol / L; the surfactant is 709 at a concentration of 0.7%–1.3%; the enzyme is cholesterol esterase at a concentration of 2–5 KU / L, cholesterol oxidase at a concentration of 2–5 KU / L, and POD at a concentration of 10 KU / L; and the substrate is 4AA at a concentration of 0.1%–0.3%.

[0018] Secondly, the present invention provides a kit for detecting small low-density lipoprotein cholesterol, comprising the above-mentioned reagent composition, and further comprising quality control and calibrators, wherein the quality control and calibrators each contain at least one concentration level of small low-density lipoprotein cholesterol, and the buffer solutions are all phosphate buffers containing human serum, wherein the content of human serum is not less than 5%, and the pH value of the buffer solutions is 7.35 to 7.45.

[0019] Preferably, the quality control product contains at least one concentration level of low-density lipoprotein cholesterol, with the following two concentration levels: Level 1: 20 mg / dL to 30.0 mg / dL; Level 2: 45 mg / dL to 65 mg / dL.

[0020] Preferably, the calibrator has a concentration of 40 mg / dL to 55 mg / dL.

[0021] Thirdly, the present invention provides a method for preparing a modified cholesterol esterase, comprising the following steps: S1: adding 30-60 mmol / L HEPES and 30-60 mmol / L triethanolamine, and adjusting the pH to 7.5-8.0;

[0022] S2: Add 2-5 KU / L cholesterol esterase to S1 buffer and treat for 1-3 hours to obtain modified cholesterol esterase;

[0023] In R1, the preferred composition includes PIPES at 110–130 mmol / L; BSA at 0.2%–0.7% and monosodium glutamate at 0.1%–0.2%; surfactants B66 at 0.15%–0.25%, A90 at 0.15%–0.25%, and CH40 at 0.02%–0.04%; enzymes of 3–4 KU / L phospholipase, 3–4 KU / L cholesterol esterase, 3–4 KU / L cholesterol oxidase, and 10 KU / L POD; ions at 3–4 mmol / L; eliminators of 10 KU peroxidase and 0.2%–0.4% TOOS; and dextran sulfate 2000 at a concentration of 1–2 g / L. The pH range is 6.8–7.2.

[0024] Preferably, in R2, the stabilizer is a diglucopyranoside at a concentration of 40–50 mmol / L and BSA at a concentration of 0.6%–0.9%; the modifier is 40–50 mmol / L HEPES and 40–50 mmol / L triethanolamine; the surfactant is 0.4%–0.6% 709; the enzyme is 3–4 KU / L cholesterol esterase, 3–4 KU / L cholesterol oxidase, and 10 KU / L POD enzyme; and the substrate is 0.15%–0.25% 4AA. The pH range of reagent R2 is 7.2–7.6.

[0025] Fourthly, the present invention provides a reagent composition, a kit for detecting small low-density lipoprotein cholesterol, and a method for modifying cholesterol esterase, for use in the preparation of products for detecting small low-density lipoprotein cholesterol.

[0026] (III) Beneficial Effects

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] This invention provides an optimized reagent composition for detecting small low-density lipoprotein cholesterol and its preparation method. The kit includes reagent R1, reagent R2, calibrators, and quality control reagents. Reagent R1 includes buffer, elimination agent, surfactant, etc.; reagent R2 includes buffer, enzyme, modifier, substrate, etc. By improving the formulation of reagent R1 and reagent R2, this invention significantly improves the detection accuracy, sample detection stability, sensitivity, and specificity of the reagents, and also broadens the linear range.

[0029] In the preparation of the reagent composition for detecting small low-density lipoprotein cholesterol described in this invention, a series of buffer solutions are preferred according to the preparation purpose. For example, PIPES buffer is preferred as a protective buffer, and HEPES and triethanolamine buffer is preferred as a modification solution. Furthermore, the concentration conditions of various buffers are optimized to ensure optimal modification and preservation while adjusting the buffering capacity of the solution within a certain range to provide an optimal reaction environment for the kit. In particular, the HEPES and triethanolamine buffer not only preserves the enzyme for a long time and improves its anti-interference ability, but also has a buffering capacity that adjusts the pH to 7.2–7.6. The ionic strength and pH value of this buffer will not inhibit the enzymatic reaction during the detection process.

[0030] In this invention, reagent R1 contains: specially added surfactants B66 (0.1%–0.3%), A90 (0.1%–0.3%), and CH40 (0.01%–0.15%), which interact with cholesterol esterase to not only remove high-density lipoprotein cholesterol and very low-density lipoprotein cholesterol, but also have no effect on the stability of reagent R1, effectively improving the interference removal ability of reagent R1 and improving reagent precision; the interaction between phospholipase and surfactant can remove low-density lipoprotein cholesterol other than small and small low-density lipoprotein cholesterol, with phospholipase concentrations of 3–4 KU / L, preferably 3.5 KU; the buffer solution contains 110–130 mmol of PIPES. The buffer solution, preferably 120 mmol / L, effectively protects enzyme activity over a long period. The stabilizer contains 0.2%–0.7% BSA, preferably 0.45%, and 0.1%–0.2% sodium glutamate, preferably 0.15%, significantly improving the stability of the kit at 37°C. The eliminator consists of 10 KU peroxidase and 0.2%–0.4% TOOS, effectively eliminating hydrogen peroxide generated during the R1 reaction. Cholesterol oxidase, essential for eliminating other lipoprotein cholesterol, is present at a concentration of 3–4 KU / L, preferably 3.5 KU / L, effectively eliminating other lipoprotein cholesterol without affecting the final measurement value.

[0031] In this invention, reagent R2 contains the core cholesterol esterase for the detection of small low-density lipoprotein cholesterol (SLDL-C). Different treatment methods result in different reactions to different SLDL-C. HEPES and triethanolamine modify the cholesterol esterase, enhancing its specificity for SLDL-C and further eliminating the influence of other SLDL-C levels. The addition of a surfactant (0.5%–1.5%, preferably 1.0%) further enhances the specificity of the cholesterol esterase for SLDL-C, thereby improving the accuracy of the measurement. The stabilizer contains 40–50 mmol / L of gluconate, preferably 45 mmol / L, and 0.6%–0.9% of BSA, preferably 0.75%, which significantly improves the stability of the kit at 37°C and enhances its long-term stability at 2–8°C. It also exhibits high linearity. The enzymes are 3–4 KU / L cholesterol esterase, 3–4 KU / L cholesterol oxidase, and 10 KU / L POD enzyme; the substrate is 0.1%–0.3% 4AA, providing greater sensitivity and accuracy within a linear range of 1–100 mg / dL.

[0032] Furthermore, this invention optimizes the method for modifying cholesterol esterase. Cholesterol esterase recognizes all lipoprotein cholesterol. Modifying the cholesterol esterase with a buffer solution induces it to specifically recognize small low-density lipoprotein cholesterol. This modification alters the enzyme's substrate recognition ability, improving the specificity of cholesterol esterase for small low-density lipoprotein cholesterol. The preferred modification buffer is HEPES and triethanolamine. This modified sterol esterase not only improves specificity but also effectively enhances stability and improves product linearity.

[0033] Furthermore, in the preservative, the optimal stabilizer, preservative and their optimal concentration range were screened; the optimal concentration of these components was screened, which further improved the stability and low-value sensitivity of reagent R2, not only increasing the stability of the kit of the present invention in preservation, but also improving the practicality of the related products of the present invention in auxiliary clinical applications.

[0034] In summary, the small low-density lipoprotein cholesterol reagent composition and kit disclosed in this invention, as well as the formulations of reagents R1 and R2, not only effectively reduce production costs and are simple to manufacture, but can also be used on fully automated biochemical analyzers with a high degree of automation. Moreover, compared with similar products, they have a higher detection range, accuracy, and stability, achieving unexpected technical effects. Attached Figure Description

[0035] Figure 1 This is a graph showing the linear correlation results in the performance evaluation of the kit for detecting small low-density lipoprotein cholesterol described in Example 1 of this invention.

[0036] Figure 2This is a graph showing the linear correlation results in the performance evaluation of the kit for detecting small low-density lipoprotein cholesterol described in Example 2 of this invention.

[0037] Figure 3 This is a graph showing the linear correlation results in the performance evaluation of the kit for detecting small low-density lipoprotein cholesterol described in Example 3 of this invention.

[0038] Figure 4 This is a graph showing the accuracy results in the performance evaluation of the kit for detecting small low-density lipoprotein cholesterol corresponding to Example 1 of this invention.

[0039] Figure 5 This is a graph showing the accuracy results in the performance evaluation of the kit for detecting small low-density lipoprotein cholesterol corresponding to Example 2 of this invention.

[0040] Figure 6 This is a graph showing the accuracy results in the performance evaluation of the kit for detecting small low-density lipoprotein cholesterol corresponding to Example 3 of this invention.

[0041] Figure 7 This is a graph showing the accuracy results in the performance evaluation of the kit for detecting small low-density lipoprotein cholesterol, which corresponds to the control reagent in this invention. Detailed Implementation

[0042] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available. Cholesterol esterase and cholesterol oxidation were purchased from Beijing Dacheng Biotechnology Co., Ltd.; A90 and B66 were purchased from Xiamen Yutaikang Biotechnology Co., Ltd.; PIPES (100 mmol / L) was purchased from Bailingwei Technology Co., Ltd.; bovine serum albumin (BSA) was purchased from Shanghai Xibao Biotechnology Co., Ltd.; diglucopyranoside was purchased from Sinopharm; monosodium glutamate was purchased from Suzhou Yake Biotechnology Co., Ltd.; POD was purchased from Xiamen Yutaikang Biotechnology Co., Ltd.; and the automated biochemical analyzer was a Hitachi 7180 automated biochemical analyzer. However, it is obvious that one or more examples can be performed without these specific details. Where specific conditions are not specified in the examples, they should be performed under conventional conditions or the manufacturer's recommended conditions. Molecular biology experimental methods not specifically described in the following examples were performed according to the specific methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition), or according to the kit and product instructions.

[0044] The application of this invention only pertains to biological samples taken from outside the body, specifically human bodily fluids, such as blood samples (whole blood / serum / plasma) that have been separated from living organisms. These are not living human or animal bodies, but rather inanimate biological samples separated from the body. The direct purpose of the detection is to detect the content of inanimate samples in vitro. The detection objective is to detect the adiponectin content in the sample, thereby helping doctors to indirectly understand the corresponding adiponectin content in the human body during the diagnostic process.

[0045] This invention provides a reagent composition for detecting small low-density lipoprotein cholesterol and a method for preparing the same kit. Specific embodiments of this invention are described below:

[0046] The product for detecting small low-density lipoprotein cholesterol described in this invention uses an enzymatic method, and the biochemical results can be automatically analyzed using a fully automated biochemical analyzer. The general detection process steps are as follows:

[0047] A reagent composition for detecting small low-density lipoprotein cholesterol includes reagents R1 and R2, which are independent of each other. Reagent R1 has a pH of 7.0 to 7.8 and contains a buffer, a stabilizer, an eliminator, a surfactant, and an enzyme. Reagent R2 has a pH of 7.2 to 7.8 and contains a buffer, a stabilizer, a modifier, a surfactant, an enzyme, and a substrate.

[0048] The preparation process of the cholesterol esterase modified in reagent R2 is as follows:

[0049] S1: The modification buffer consists of 40–50 mmol / L HEPES and 40–50 mmol / L triethanolamine.

[0050] S2: Added cholesterol esterase purchased from Beijing Dacheng Biotechnology Co., Ltd.;

[0051] A kit for detecting small low-density lipoprotein cholesterol (MLC) comprises reagent compositions (reagent R1 and reagent R2), quality control products, and calibrators, wherein the quality control products and the calibrators each contain at least one concentration level of MLC, and the buffer solutions are phosphate buffers containing human serum, wherein the human serum content is not less than 5%, and the pH value of the buffer solutions is 7.35 to 7.45.

[0052] Among them, the quality control product contains two concentration levels of low-density lipoprotein cholesterol: Level 1: 25 mg / dL; Level 2: 55 mg / dL.

[0053] The calibrator has a concentration of 40 mg / dL to 55 mg / dL.

[0054] The preferred concentration level for the calibrators is 47 mg / dL.

[0055] The reagents, reagent compositions, and dosages used in the detection process are set as follows: Three types of reaction tubes are set up, including blank tubes, sample tubes, and calibration tubes. 3 μL of distilled water is added to the blank tube, 3 μL of the sample to be tested is added to the sample tube, and 3 μL of standard is added to the calibration tube. 150 μL of reagent R1 solution is added to each of the three reaction tubes, and after incubation at 37°C for 5 minutes, the absorbance (A1) of each reaction tube is read using a fully automated biochemical analyzer. Then, 50 μL of reagent R2 solution is added to each reaction tube, and after incubation at 37°C for 5 minutes, the absorbance (A2) of each reaction tube is read again using the fully automated biochemical analyzer. The detection conditions of the fully automated biochemical analyzer are: main wavelength set to 600 nm, incubation temperature at 37°C, cuvette optical path 1 cm, and the detection principle is based on the absorbance difference ΔA (i.e., endpoint rise method), calculated using the following formula: ΔA = A2 ~ A1.

[0056] Example 1: Preparation of a reagent composition 1

[0057] Reagent composition 1 comprises reagents R1 and R2, which are independent of each other, wherein the specific amounts or final concentrations of each component pair in reagent R1 are as follows:

[0058] Table 1: Composition of reagent R1 in reagent composition 1

[0059] Components concentration PIPES 125mmol / l BSA 0.30％ mgCL2 3.5 mmol / L Sulfated dextran 2000 1.5g / L A90 0.20％ B66 0.20％ CH40 0.03％ phosphatase 3.5KU / L cholesterol esterase 3.5KU / L cholesterol oxidase 3.5KU / L peroxidase 10KU / L TOOS 0.30％ dichloroacetamide 0.03％ Monosodium glutamate 0.15％

[0060] Adjust the pH of reagent R1 solution to 7.3 using a pH adjuster.

[0061] The specific amounts or final concentrations of each component pair in reagent R2 are as follows:

[0062] Table 2: Composition of reagent R2 in reagent composition 1

[0063] Components concentration HEPES 40mmol / l Triethanolamine 40mmol / l 709 1.00％ dichloroacetamide 0.08％ Diglycinate 45mmol / l BSA 0.75％ 4AA 0.20％ cholesterol esterase 3.5KU / L cholesterol oxidase 3.5KU / L peroxidase 10KU / L

[0064] Example 2: Preparation of a reagent composition 2

[0065] Reagent composition 2 includes reagents R1 and R2, which are independent of each other. The specific amounts or final concentrations of each component pair in reagent R1 are as follows:

[0066] Table 3: Composition of reagent R1 in reagent composition 2

[0067]

[0068]

[0069] Adjust the pH of reagent R1 solution to 7.4 using a pH adjuster.

[0070] The specific amounts or final concentrations of each component pair in reagent R2 are as follows:

[0071] Table 4: Composition of reagent R2 in reagent composition 2

[0072] Components concentration HEPES 40mmol / l Triethanolamine 40mmol / l 709 1.00％ dichloroacetamide 0.08％ Diglycinate 50mmol / l BSA 0.75％ 4AA 0.20％ cholesterol esterase 3.5KU / L cholesterol oxidase 3.5KU / L peroxidase 10KU / L

[0073] Adjust the pH of reagent R2 solution to 7.5 using a pH adjuster.

[0074] Example 3: Preparation of a reagent composition 3

[0075] Reagent composition 3 includes reagents R1 and R2, which are independent of each other. The specific amounts or final concentrations of each component pair in reagent R1 are as follows:

[0076] Table 5: Composition of reagent R1 in reagent composition 3

[0077] Components concentration PIPES 125mmol / l BSA 0.30％ mgCL2 3.5 mmol / L Sulfated dextran 2000 1.0g / L A90 0.20％ B66 0.20％ CH40 0.04％ phosphatase 3.5KU / L cholesterol esterase 3.5KU / L cholesterol oxidase 3.5KU / L peroxidase 10KU / L TOOS 0.30％ dichloroacetamide 0.03％ Monosodium glutamate 0.10％

[0078] Adjust the pH of reagent R1 solution to 7.6 using a pH adjuster.

[0079] The specific amounts or final concentrations of each component pair in reagent R2 are as follows:

[0080] Table 6: Composition of reagent R2 in reagent composition 3

[0081]

[0082]

[0083] Adjust the pH of reagent R2 solution to 7.4.

[0084] Example 4: Preparation of a reagent composition 4

[0085] Reagent composition 4 includes reagents R1 and R2, which are independent of each other. The specific amounts or final concentrations of each component pair in reagent R1 are as follows:

[0086] Table 7: Composition of reagent R1 in reagent composition 4

[0087]

[0088]

[0089] Adjust the pH of reagent R1 solution to 7.4.

[0090] The specific amounts or final concentrations of each component pair in reagent R2 are as follows:

[0091] Table 8: Composition of reagent R2 in reagent composition 4

[0092] Components concentration HEPES 40mmol / l Triethanolamine 40mmol / l 709 1.00％ dichloroacetamide 0.08％ BSA 0.75％ 4AA 0.20％ cholesterol esterase 3.5KU / L cholesterol oxidase 3.5KU / L peroxidase 10KU / L

[0093] Adjust the pH of reagent R2 solution to 7.4.

[0094] Control reagent: Centrifugation separates LDL from plasma, and electrophoresis is used to detect the concentration.

[0095] Performance evaluation: linear correlation evaluation

[0096] Using reagent composition 1, reagent composition 2 and reagent composition 3 prepared in Examples 1 to 3 above, comparative tests were performed with control reagents. Forty clinical serum samples were randomly tested, and the test values ​​and control values ​​of Examples 1 to 3 were obtained respectively. The specific data results are shown in Table 10 below (the unit of test value and control value is ug / L).

[0097] Table 10 Results of linear correlation test

[0098]

[0099]

[0100]

[0101] Based on the detection results shown in Table 10, the linear correlation results corresponding to Examples 1 to 3 are obtained, as follows: Figure 1 , Figure 2 as well as Figure 3 As shown, the linear correlation curve for Example 1 was y = 1.0036x + 0.1504, with a correlation coefficient r = 0.9988; the linear correlation curve for Example 2 was y = 1.007x + 0.2408, with a correlation coefficient r = 0.9986; and the linear correlation curve for Example 3 was y = 1.0117x + 0.3467, with a correlation coefficient r = 0.9992. This indicates that the results obtained from Examples 1 to 3 and the control reagent were all correlated.

[0102] Performance evaluation: Intra-batch precision evaluation

[0103] Add physiological saline to the blank tube instead of distilled water. Use reagent composition 1, reagent composition 2, and reagent composition 3 prepared in Examples 1-3 of this invention to test the calibrators and quality control samples prepared at a preferred concentration level. Test each calibrator and quality control sample 10 times consecutively, and calculate the mean and standard deviation (SD). Parallel tests were performed simultaneously with the control reagent for comparison. The test results are shown in Table 11 below.

[0104] Table 11 Results of Intra-Batch Precision Comparison Test

[0105]

[0106]

[0107]

[0108] As can be seen from Table 11, the intra-batch precision of the calibrators and quality control samples tested in Examples 1 to 3 of the present invention does not exceed 3%, while the intra-batch precision of the control reagents tested for calibrators and quality control samples cannot be guaranteed to be within the range of 5%.

[0109] Performance Evaluation: Accuracy Evaluation

[0110] Using reagent compositions 1, 2, and 3 prepared in Examples 1-3 of this invention, any 10 clinical samples with known concentrations within the linear range were tested, and parallel tests were performed with control reagents to compare the results and calculate the deviation. The test data results are shown in Table 12.

[0111] Table 12. Accuracy Comparison Test Results

[0112]

[0113]

[0114] Plot the accuracy comparison test results as shown in Table 12. See details. Figure 4 , Figure 5 , Figure 6 as well as Figure 7 As shown. From Figure 4 , Figure 5 , Figure 6 as well as Figure 7 It is evident that the theoretical and measured values ​​of reagent compositions 1, 2, and 3 prepared in Examples 1-3 of this invention show little linear difference between the actual and measured values ​​for clinical sample detection, and are all significantly superior to the control reagents. This demonstrates that by optimizing the components of reagents R1 and R2, the three reagent compositions ultimately obtained by this invention exhibit higher specificity than the control reagents, thus improving the accuracy and sensitivity of the reagents.

[0115] Performance Evaluation: Anti-interference Capability Evaluation

[0116] Using reagent composition 1 and reagent composition 4 prepared in Examples 1 and 4 of this invention, five clinical samples of mild chylous blood with known concentrations within the linear range were tested twice consecutively, and the results were compared with those of the control reagent. The deviation was calculated. The test data results are shown in Table 13.

[0117] Table 13 Results of Anti-interference Capability Test

[0118]

[0119]

[0120] A graph was plotted based on the test results of the anti-interference capability shown in Table 13. (See details...) Figure 4 As shown. The only difference between reagent composition 1 prepared in Example 1 and reagent composition 4 prepared in Example 4 is the addition of 0.4 mmol / L dextran sulfate in reagent composition 1. Figure 4 As can be seen, the test results show that the linear difference between the theoretical value and the measured value of Example 1 for detecting clinical mild chylous blood samples is not significant, and it is significantly better than Example 4 and the control reagent. This shows that by optimizing the composition of reagent R1 and combining surfactant and dextran sulfate, the present invention can not only reduce the interference of chylous particles, but also has no effect on the stability of reagent R1, effectively improving the anti-interference ability of the reagent and thus improving the precision of the reagent.

[0121] Performance evaluation: Stability evaluation

[0122] Stability evaluation in clinical trials generally includes thermally accelerated stability evaluation tests at 37°C and long-term stability evaluation tests at 2–8°C.

[0123] (1) Stability at 37℃

[0124] The reagent compositions 1, 2, and 3 prepared in Examples 1-3 of this invention were placed in a 37°C water bath for 1, 3, 5, 7, and 14 days, respectively, and the preferred calibrator (47 mg / dL) was analyzed three times consecutively. The reagent compositions 1, 2, and 3 prepared in Examples 1-3 of this invention were compared in parallel with the control reagent. The detection results after thermal acceleration at 37°C are shown in Table 14.

[0125] Table 14. Stability test results at 37℃

[0126]

[0127]

[0128] As shown in Table 14, compared to the control reagent, the reagent compositions 1, 2, and 3 prepared in Examples 1-3 of this invention showed no significant changes after being stored at 37°C for 14 days (approximately equivalent to storage at 2-8°C for 1 year), and were superior to the control reagent (CV = 5.55%). This indicates that the reagent compositions prepared in Examples 1-3 can maintain stability even after storage at 37°C for 14 days. Therefore, it can be concluded that the reagent compositions prepared in Examples 1-3 of this invention have higher stability under room temperature conditions.

[0129] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A reagent composition for detecting small low-density lipoprotein cholesterol, comprising reagents R1 and R2, which are independent of each other, characterized in that: The volume ratio of reagent R1 to reagent R2 is 3:1; The reagent R1 has a pH of 7.0–7.8 and contains 100–150 mmol / L PIPES buffer, stabilizer, preservative, eliminator, 1–2 g / L dextran sulfate 2000, 0.1%–0.3% surfactant A90, 0.1%–0.3% surfactant B66, 0.01%–0.15% surfactant CH40, 2–5 KU / L phospholipase, 2–5 KU / L cholesterol esterase, and 2–5 KU / L cholesterol oxidase; the stabilizer contains 0.1%–0.5% BSA and 2–5 mmol / L magnesium ions; the preservative is a dichloroacetamide solution with a final concentration of 0.01%–0.05%; and the eliminator is 10 KU / L LOD and 0.1%–0.2% TOOS. The reagent R2 has a pH of 7.2–7.8 and contains a buffer, stabilizer, preservative, modifier, substrate, 0.5%–1.5% surfactant 709, 2–5 KU / L cholesterol esterase, 2–5 KU / L cholesterol oxidase, and 10 KU / L POD; the stabilizer is 1.0%–2% BSA; the preservative is a dichloroacetamide solution with a final concentration of 0.05%–0.1%; and the modifier is 30–60 mmol / L HEPES and 30–60 mmol / L triethanolamine, used to modify cholesterol esterase.

2. A small, low-density lipoprotein cholesterol detection kit, characterized in that, The reagent composition comprises the one described in claim 1.

3. A method for preparing a small, low-density lipoprotein cholesterol detection reagent, applied to the reagent composition of claim 1, characterized in that, The preparation of reagent R1 includes: using 100–150 mmol / L LPIPES buffer, adding 2–5 mmol / L magnesium ions, surfactants B66 / A90 / CH40, 0.1%–0.5% BSA, and 0.1%–0.2% sodium glutamate as stabilizers; adding 2–5 KU / L cholesterol esterase, 2–5 KU / L cholesterol oxidase, and 2–5 KU / L phospholipase; combined with 10 KU / L LPOD and 0.1%–0.5% TOOS as eliminators; and 1–2 g / L dextran sulfate 2000; adjusting the pH to 7.0–7.

8. Preparation of reagent R2: 30–60 mmol / L HEPES and 30–60 mmol / L triethanolamine were used as modification buffers. 30–60 mmol / L gluconate and 0.5%–1% BSA were added as stabilizers. 2–5 KU / L cholesterol esterase, 2–5 KU / L cholesterol oxidase, and 10 KU / L LPD were added, along with 0.1%–0.3% 4AA as a substrate and 0.5%–1.5% surfactant 709. The pH was adjusted to 7.2–7.

8.

4. The application of the reagent composition of claim 1, the small low-density lipoprotein cholesterol detection kit of claim 2, or the small low-density lipoprotein cholesterol detection reagent preparation method of claim 3 in the preparation of small low-density lipoprotein cholesterol detection products.

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