A method for detecting low density lipoprotein in serum by gel electrophoresis

Abstract

The application discloses a method for detecting low-density lipoprotein in serum by improved gel electrophoresis, and belongs to the technical field of biological detection. The method comprises the following steps: (1) serum staining; (2) gel tube preparation; (3) electrophoresis; and (4) quantitative detection. The gel electrophoresis system has good separation effect, high detection accuracy and high detection efficiency. By adjusting the ratio of separation gel and concentrated gel, the content of polyacrylamide in the gel liquid, and the configuration of the staining liquid, the total amount of low-density lipoprotein and each subtype can be accurately separated and quantitatively analyzed, the real level of lipoprotein can be comprehensively and accurately reflected, the detection process is simple and easy to implement, the detection time is short, and the method has important significance for better judging the risk of cardiovascular and cerebrovascular diseases.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to an improved method for detecting low-density lipoprotein in serum by gel electrophoresis. Background Technology

[0002] Cardiovascular disease (CVD) is a general term for diseases of the circulatory system, mainly including diseases of the heart, arteries, veins, and capillaries, and is generally closely related to atherosclerosis. Currently, the prevalence and mortality rates of CVD are still on the rise, ranking first among various disease mortality rates. Therefore, intervention in high-risk groups for CVD can reduce its incidence. Dyslipidemia is one of the important risk factors for CVD; therefore, standardizing blood lipid testing and measurement results plays a crucial role in the prevention and treatment of CVD.

[0003] Lipids in human blood primarily bind with proteins to form lipoproteins. Lipoproteins are classified according to their density into very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), intermediate-density lipoprotein (IDL), high-density lipoprotein (HDL), and chylomicrons (CM). Low-density lipoprotein (LDL) is heterogeneous, composed of a series of particles of varying sizes, densities, and chemical compositions. Based on the diameter and density of LDL particles, it can be divided into up to seven subtypes, named LDL-1, LDL-2, LDL-3, LDL-4, LDL-5, LDL-6, and LDL-7. Large and lightweight LDL (lbLDL), composed of LDL-1 and LDL-2 with particle diameters >27 nm and densities <1.03 g / mL, is defined as type A. Small and dense LDL (sdLDL), composed of LDL-3 to LDL-7 with particle diameters <26 nm and densities >1.04 g / mL, is defined as type B. Experiments have shown that sdLDL levels can serve as an indicator for predicting and assessing cardiovascular disease risk. LDL and its subtypes can comprehensively and accurately reflect the true levels of lipoproteins in patients with cardiovascular disease, providing a possibility for early intervention and reducing the incidence of cardiovascular disease. Therefore, lipoprotein typing analysis is of great significance in the clinical diagnosis of diseases. However, the heterogeneity of low-density lipoprotein subtypes presents many challenges in their separation and detection.

[0004] Currently, there are various methods for detecting plasma lipoproteins. Among them, electrophoresis has the advantages of high efficiency and practicality and is widely used in the separation and detection analysis of lipoproteins. Polyacrylamide gel electrophoresis, in particular, contains polyacrylamide gel, which has the dual effects of molecular sieving and charge separation, resulting in high resolution and the ability to separate serum lipoproteins into multiple bands. It also boasts advantages such as high transparency, fast separation time, and simple operation. However, traditional gel systems have lower resolution and poorer separation effects for plasma lipoproteins. This is because the polyacrylamide gel is easily hydrolyzed and has a short shelf life, causing the gel system to easily lose its separation effect. Hydrolysis shortens the migration distance of proteins, reducing the resolution of protein bands. To ensure experimental results, freshly prepared gels are often used. Although the detection effect is better, the work efficiency is low, and the time and labor costs are high.

[0005] Therefore, it is necessary to develop a gel electrophoresis system with good separation effect, high detection accuracy and efficiency, so as to accurately separate and detect various subtypes of low-density lipoprotein and comprehensively and accurately reflect the true level of lipoprotein. Summary of the Invention

[0006] To address the above problems, this invention provides an improved gel electrophoresis system with good separation effect, high detection accuracy and efficiency, which can accurately separate and detect various subtypes of low-density lipoprotein in serum.

[0007] This invention is achieved through the following technical solution:

[0008] A modified gel electrophoresis method for detecting low-density lipoprotein in serum includes the following steps:

[0009] (1) Serum staining: Take a serum sample, add staining solution, mix well, then place in a constant temperature water bath, and take the supernatant after centrifugation to obtain the stained serum;

[0010] (2) Gel tube preparation: Fix the glass tube on the support, and then add the second layer of separating gel, the first layer of separating gel and the stacking gel into the glass tube in sequence. Each time a gel solution is added, wait until the surface of the gel solution is polymerized and flat before adding the next gel solution. Finally, immerse the whole tube in the preservation solution to obtain the gel tube; The second layer of separating gel, the first layer of separating gel and the stacking gel all include: polyacrylamide, H2O, Tris-HCl, sucrose, glycerol, tetramethylethylenediamine and ammonium persulfate;

[0011] (3) Electrophoresis: After removing the surface and preservation solution from the gel tube, fix it vertically in the electrophoresis tank. The first separating gel and stacking gel are located at the negative end of the electrophoresis tank, and the second separating gel is located at the positive end of the electrophoresis tank. Then, add electrophoresis buffer to the electrophoresis tank and slowly add the stained serum into the gel tube to cover the surface of the stacking gel. After the solution in the system is stable, turn on the regulated power supply to perform electrophoresis.

[0012] (4) Quantitative detection: After electrophoresis, the gel tube was removed and the absorbance changes at different positions in the first and second separating gels of the gel tube were scanned with a scanner. Based on the curves obtained by the absorbance changes of different subtypes of low-density lipoprotein with position, the percentage of low-density lipoprotein in the total serum lipoprotein was quantitatively detected.

[0013] As a preferred technical solution, in step (2), the volume ratio of the second separating gel, the first separating gel, and the stacking gel in the gel tube is 5-7:1-2:1.

[0014] As a preferred technical solution, in step (2), the volume ratio of polyacrylamide, H2O, Tris-HCl, sucrose, glycerol, tetramethylethylenediamine and ammonium persulfate in the second separating gel is 28-35:150-300:7-9:9-11:1-2:0.1-0.2:1; the gel concentration T of the second separating gel is 3-4% (gel concentration T is the sum of the mass of acrylamide and methylenebisacrylamide divided by the total volume), and the pH value is 8.5-9.

[0015] As a preferred technical solution, in step (2), the volume ratio of polyacrylamide, H2O, Tris-HCl, sucrose, glycerol, tetramethylethylenediamine and ammonium persulfate in the first separating gel is 45-50:200-300:7-9:9-11:1-2:0.1-0.2:1; the gel concentration of the first separating gel is T = 4.5-6% and the pH value is 8.5-9.

[0016] As a preferred technical solution, in step (2), the volume ratio of polyacrylamide, H2O, Tris-HCl, sucrose, glycerol, tetramethylethylenediamine and ammonium persulfate in the concentrated gel is 20-25:150-250:4.5-6:55-65:1-2:0.1-0.2:1; the gel concentration T of the concentrated gel is 2-3%, and the pH value is 6.5-7.

[0017] As a preferred technical solution, in step (2), the preservation solution is prepared by mixing sucrose, Tris-HCl, glycerol and Proclin 300 in a mass-to-volume ratio of 9-11g:38-45mL:1mL:0.1-0.2mL.

[0018] As a preferred technical solution, in step (2), distilled water is added to the surface of the liquid immediately after each addition of adhesive, and the liquid is left to stand for 30 to 40 minutes. After the surface of the adhesive has polymerized and become smooth, the water layer is removed, and then the next adhesive is added.

[0019] As a preferred technical solution, in step (1), the volume ratio of the serum sample to the staining solution is 8 to 10:1.

[0020] As a preferred technical solution, in step (1), the staining solution is prepared from Sudan Black B, anhydrous ethanol, dimethyl sulfoxide and ethylene glycol. The concentration of Sudan Black B in the staining solution is 2-3 g / L, and the volume ratio of anhydrous ethanol, dimethyl sulfoxide and ethylene glycol is 1-2:1:1-2.

[0021] As a preferred technical solution, in step (3), the buffer solution is prepared by mixing tris(hydroxymethyl)aminomethane and boric acid in a mass ratio of 2 to 3:1.

[0022] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0023] 1. The gel electrophoresis system of the present invention has good separation effect, high detection accuracy and efficiency. By adjusting the ratio of separating gel to stacking gel, the content of polyacrylamide in the gel solution, and the preparation of staining solution, the total amount of low-density lipoprotein and its various subtypes can be accurately separated and quantitatively analyzed. It can comprehensively and accurately reflect the true level of lipoprotein. Moreover, the detection process is simple and easy to perform, and the detection time is short, only about 75 minutes. It is of great significance for better judging the risk of cardiovascular and cerebrovascular diseases.

[0024] 2. The gel separation column of this invention consists of a second separating gel, a first separating gel, and a stacking gel. Each gel contains a different amount of polyacrylamide. Using three different amounts of polyacrylamide as electrophoresis carriers allows for matching with lipoproteins of different particle sizes and densities, thereby improving the accuracy of quantitative and qualitative analysis of various lipoprotein subtypes. The main functions of the second and first separating gels are to separate the various lipoprotein subtypes, while the stacking gel acts as a packing agent, compressing all lipoprotein subtypes into narrow bands, thus improving the sensitivity of electrophoresis.

[0025] 3. The principle for setting the concentration of the second separating gel in this invention is based on the pore size of the gel. Since the diameter of the separated lipoprotein particles is relatively large (above 15nm), the gel concentration T needs to be set below 4%. A low gel concentration can make the pore size of the gel larger, so as to better separate lipoprotein particles and thus improve the detection effect.

[0026] 4. The staining solution of this invention is formulated with Sudan Black B, anhydrous ethanol, dimethyl sulfoxide (DMSO), and ethylene glycol. It exhibits good solubility, dispersibility, and stability, and has a shelf life of up to one year. DMSO acts as a solvent, primarily serving a dissolving function; anhydrous ethanol acts as a dispersant, ensuring good dispersibility of lipoproteins in serum and improving the resolution of electrophoretic detection; ethylene glycol acts as a densifying agent, increasing the density of the staining solution and causing the stained serum samples to aggregate and settle during electrophoresis, preventing sample dispersion into the buffer solution. Through the combined action of the components in the staining solution, Sudan Black B binds uniformly to various lipoprotein subtypes, resulting in uniform staining results and improving the effectiveness and efficiency of electrophoretic detection. This allows for the simultaneous qualitative detection of various lipoprotein subtypes. Attached Figure Description

[0027] Figure 1 The image shows a gel electrophoresis pattern obtained by electrophoresis of serum from a confirmed cardiovascular patient according to the method in Example 1.

[0028] Figure 2 The image shows a gel electrophoresis pattern obtained by electrophoresis of serum from healthy individuals according to the method in Example 1.

[0029] Figure 3 The image shows a gel electrophoresis pattern obtained from the serum of a confirmed cardiovascular patient, following the method described in Comparative Example 1.

[0030] Figure 4 The image shows a gel electrophoresis pattern obtained by electrophoresis of serum from healthy individuals using the method described in Comparative Example 1. Detailed Implementation

[0031] The present invention will be further described in detail below through embodiments. These embodiments are only used to illustrate the present invention and do not limit the scope of protection of the present invention.

[0032] Example 1

[0033] A modified gel electrophoresis method for detecting low-density lipoprotein in serum includes the following steps:

[0034] (1) Serum staining: Venous blood samples were collected after fasting for 12 hours. Serum was separated within 2 hours. 120 μL of serum was taken, 15 μL of staining solution was added, and the mixture was mixed evenly. The mixture was then placed in a constant temperature water bath at 37°C for 30 min. After that, it was centrifuged at 3000 r / min for 10 min. The supernatant was taken after centrifugation to obtain the stained serum.

[0035] The staining solution was prepared as follows: Sudan Black B was prepared to a concentration of 3 g / L, and then 10 mL of anhydrous ethanol, 10 mL of dimethyl sulfoxide and 10 mL of ethylene glycol were added and mixed evenly. The mixture was then heated at 40 °C for 10 min with constant stirring during heating. After that, the mixture was filtered while hot using a filter membrane with a pore size of 0.22 μm. After cooling, the mixture was filtered again to obtain the staining solution.

[0036] (2) Gel tube preparation: Fix the glass tube on the support, and then add 1050 μL of the second layer separating gel, 270 μL of the first layer separating gel and 160 μL of the stacking gel into the glass tube in sequence. After each addition of gel solution, immediately add 50 μL of distilled water to the surface of the liquid and let it stand for 30 min to isolate the air and make the surface of the gel solution smooth. After the surface of the gel solution has polymerized and become smooth, remove the water layer, and then add the next gel solution. Finally, immerse the whole tube in 500 mL of preservation solution to obtain the gel tube.

[0037] The second separating gel was prepared as follows: 1.480 mL of 30% polyacrylamide (Acr:Bis = 29:1), 12.3592 mL of H2O, 380 μL of 1 mol / L Tris-HCl (pH 8.9), 480 μL of 25% sucrose, 48 μL of 25% glycerol, 4.8 μL of tetramethylethylenediamine, and 48 μL of 10% ammonium persulfate were mixed thoroughly to prepare a second separating gel with a gel concentration T = 3.0%, pH 8.6, and a volume of 14.8 mL.

[0038] The first separating gel was prepared as follows: 3.420 mL of 30% polyacrylamide (Acr:Bis = 29:1), 16.558 mL of H2O, 550 μL of 1 mol / L Tris-HCl (pH 8.9), 700 μL of 25% sucrose, 70 μL of 25% glycerol, 7.0 μL of tetramethylethylenediamine, and 70 μL of 10% ammonium persulfate were mixed evenly to prepare the first separating gel with a gel concentration T = 4.8%, pH 8.6, and a volume of 21.375 mL.

[0039] The stacking gel was prepared as follows: 445.0 μL of 30% polyacrylamide (Acr:Bis = 29:1), 4127.2 μL of H2O, 90 μL of 1mol / L Tris-HCl (pH 6.8), 1100 μL of 25% sucrose, 18 μL of 25% glycerol, 1.8 μL of tetramethylethylenediamine, and 18 μL of 10% ammonium persulfate were mixed evenly to prepare a stacking gel with a gel concentration T = 2.3%, pH 6.9, and a volume of 5.8 mL.

[0040] The preservation solution was prepared as follows: 90 mL of 1.5 mol / L Tris-HCl (pH 6.7), 22 g of sucrose, 2.0 mL of glycerol, and 280 μL of Proclin 300 were dissolved in water, mixed thoroughly, and the volume was adjusted to 1000 mL. The solution was then filtered through a 0.22 μm pore size filter membrane to obtain the preservation solution.

[0041] (3) Electrophoresis: After removing the surface and preservation solution from the gel tube, fix it vertically in the electrophoresis tank. The first separating gel and stacking gel are located at the negative end of the electrophoresis tank, and the second separating gel is located at the positive end of the electrophoresis tank. Then, add 600 mL of electrophoresis buffer to the electrophoresis tank. Take 40 μL of stained serum and slowly add it to the gel tube to cover the surface of the stacking gel. After the solution in the system is stable, turn on the regulated power supply with a current of 2 mA / tube and an electrophoresis time of 80 min.

[0042] The electrophoresis buffer was prepared as follows: 12.025g of tris(hydroxymethyl)aminomethane and 5.8g of boric acid were dissolved in water, mixed evenly, and the volume was adjusted to 1000mL. The mixture was then filtered through a 0.22μm filter membrane to obtain the electrophoresis buffer.

[0043] (4) Quantitative detection: After electrophoresis, the gel tube was removed and the absorbance changes at different positions in the first and second separating gels of the gel tube were scanned with a scanner. Based on the curves obtained by the absorbance changes of different subtypes of low-density lipoprotein with position, the percentage of low-density lipoprotein in the total serum lipoprotein was quantitatively detected.

[0044] Example 2

[0045] A modified gel electrophoresis method for detecting low-density lipoprotein in serum includes the following steps:

[0046] (1) Serum staining: Venous blood samples were collected after fasting for 12 hours. Serum was separated within 2 hours. 150 μL of serum was taken and 18 μL of staining solution was added. The mixture was mixed evenly and then placed in a constant temperature water bath at 37°C for 30 min. After that, it was centrifuged at 3000 r / min for 10 min. The supernatant was taken after centrifugation to obtain the stained serum.

[0047] The staining solution was prepared as follows: Sudan Black B was prepared to a concentration of 2 g / L, and then 15 mL of anhydrous ethanol, 12 mL of dimethyl sulfoxide and 15 mL of ethylene glycol were added and mixed evenly. The mixture was then heated at 40 °C for 12 min with constant stirring during heating. After that, the mixture was filtered while hot using a filter membrane with a pore size of 0.22 μm. After cooling, the mixture was filtered again to obtain the staining solution.

[0048] (2) Gel tube preparation: Fix the glass tube on the support, and then add 1065 μL of the second layer separating gel, 280 μL of the first layer separating gel and 175 μL of the stacking gel into the glass tube in sequence. After each addition of gel solution, immediately add 50 μL of distilled water to the surface of the liquid and let it stand for 30 min to isolate the air and make the surface of the gel solution smooth. After the surface of the gel solution has polymerized and become smooth, remove the water layer, and then add the next gel solution. Finally, immerse the whole tube in 500 mL of preservation solution to obtain the gel tube.

[0049] The second separating gel was prepared as follows: 1.455 mL of 30% polyacrylamide (Acr:Bis = 29:1), 11.624 mL of H2O, 400 μL of 1 mol / L Tris-HCl (pH 8.9), 500 μL of 25% sucrose, 50 μL of 25% glycerol, 5 μL of tetramethylethylenediamine, and 46 μL of 10% ammonium persulfate were mixed thoroughly to prepare a second separating gel with a gel concentration T = 3.1%, pH 8.8, and a volume of 14.08 mL.

[0050] The first separating gel was prepared as follows: 3.460 mL of 30% polyacrylamide (Acr:Bis = 29:1), 17.613 mL of H2O, 580 μL of 1 mol / L Tris-HCl (pH 8.9), 750 μL of 25% sucrose, 80 μL of 25% glycerol, 7.0 μL of tetramethylethylenediamine, and 70 μL of 10% ammonium persulfate were mixed evenly to prepare the first separating gel with a gel concentration T = 4.6%, pH 8.7, and a volume of 22.56 mL.

[0051] The stacking gel was prepared as follows: 465.2 μL of 30% polyacrylamide (Acr:Bis = 29:1), 3871.8 μL of H2O, 95 μL of 1mol / L Tris-HCl (pH 6.8), 1120 μL of 25% sucrose, 25 μL of 25% glycerol, 3.0 μL of tetramethylethylenediamine, and 20 μL of 10% ammonium persulfate were mixed evenly to prepare a stacking gel with a gel concentration T = 2.5%, pH 6.7, and a volume of 5.6 mL.

[0052] The preservation solution was prepared as follows: 65 mL of 1.5 mol / L Tris-HCl (pH 6.7), 15 g of sucrose, 1.5 mL of glycerol and 250 μL of Proclin 300 were dissolved in water, mixed thoroughly, and the volume was adjusted to 1000 mL. The solution was then filtered through a 0.22 μm filter membrane to obtain the preservation solution.

[0053] (3) Electrophoresis: After removing the surface and preservation solution from the gel tube, fix it vertically in the electrophoresis tank. The first separating gel and stacking gel are located at the negative end of the electrophoresis tank, and the second separating gel is located at the positive end of the electrophoresis tank. Then, add 600 mL of electrophoresis buffer to the electrophoresis tank. Take 40 μL of stained serum and slowly add it to the gel tube to cover the surface of the stacking gel. After the solution in the system is stable, turn on the regulated power supply with a current of 2 mA / tube and an electrophoresis time of 80 min.

[0054] The electrophoresis buffer was prepared as follows: 14.120 g of tris(hydroxymethyl)aminomethane and 6.0 g of boric acid were dissolved in water, mixed evenly, and the volume was adjusted to 1000 mL. The mixture was then filtered through a filter membrane with a pore size of 0.22 μm to obtain the electrophoresis buffer.

[0055] (4) Quantitative detection: After electrophoresis, the gel tube was removed and the absorbance changes at different positions in the first and second separating gels of the gel tube were scanned with a scanner. Based on the curves obtained by the absorbance changes of different subtypes of low-density lipoprotein with position, the percentage of low-density lipoprotein in the total serum lipoprotein was quantitatively detected.

[0056] Example 3

[0057] A modified gel electrophoresis method for detecting low-density lipoprotein in serum includes the following steps:

[0058] (1) Serum staining: Venous blood samples were collected after fasting for 12 hours. Serum was separated within 2 hours. 180 μL of serum was taken, 20 μL of staining solution was added, and the mixture was mixed evenly. Then, the mixture was kept in a constant temperature water bath at 37℃ for 30 min. After that, it was centrifuged at 3000 r / min for 10 min. After centrifugation, the supernatant was taken to obtain the stained serum.

[0059] The staining solution was prepared as follows: Sudan Black B was prepared to a concentration of 3 g / L, and then 15 mL of anhydrous ethanol, 10 mL of dimethyl sulfoxide and 15 mL of ethylene glycol were added and mixed evenly. The mixture was then heated at 40 °C for 10 min with constant stirring during heating. After that, the mixture was filtered while hot using a filter membrane with a pore size of 0.22 μm. After cooling, the mixture was filtered again to obtain the staining solution.

[0060] (2) Gel tube preparation: Fix the glass tube on the support, and then add 1250 μL of the second layer separating gel, 300 μL of the first layer separating gel and 220 μL of the stacking gel into the glass tube in sequence. After each addition of gel solution, immediately add 50 μL of distilled water to the surface of the liquid and let it stand for 30 min to isolate the air and make the surface of the gel solution smooth. After the surface of the gel solution has polymerized and become smooth, remove the water layer, and then add the next gel solution. Finally, immerse the whole tube in 500 mL of preservation solution to obtain the gel tube.

[0061] The second separating gel was prepared as follows: 1.510 mL of 30% polyacrylamide (Acr:Bis = 29:1), 9.749 mL of H2O, 400 μL of 1 mol / L Tris-HCl (pH 8.9), 470 μL of 25% sucrose, 60 μL of 25% glycerol, 6.0 μL of tetramethylethylenediamine, and 45 μL of 10% ammonium persulfate were mixed thoroughly to prepare a second separating gel with a gel concentration T = 3.7%, pH 8.9, and a volume of 12.24 mL.

[0062] The first separating gel was prepared as follows: 3.230 mL of 30% polyacrylamide (Acr:Bis = 29:1), 15.646 mL of H2O, 500 μL of 1 mol / L Tris-HCl (pH 8.9), 650 μL of 25% sucrose, 80 μL of 25% glycerol, 9.0 μL of tetramethylethylenediamine, and 65 μL of 10% ammonium persulfate were mixed evenly to prepare the first separating gel with a gel concentration T = 4.8%, pH 8.5, and a volume of 20.18 mL.

[0063] The preparation of the stacking gel is as follows: Take 445.0 μL of 30% polyacrylamide (Acr:Bis = 29:1), 3977 μL of H2O, 100 μL of 1mol / L Tris-HCl (pH 6.8), 1230 μL of 25% sucrose, 25 μL of 25% glycerol, 3.0 μL of tetramethylethylenediamine and 20 μL of 10% ammonium persulfate, mix them evenly, and prepare a stacking gel with a gel concentration T = 2.3%, pH 6.9 and a system of 5.8 mL.

[0064] The preservation solution was prepared as follows: 100 mL of 1.5 mol / L Tris-HCl (pH 6.7), 25 g of sucrose, 2.3 mL of glycerol and 320 μL of Proclin 300 were dissolved in water, mixed evenly, and the volume was adjusted to 1000 mL. The solution was then filtered through a 0.22 μm filter membrane to obtain the preservation solution.

[0065] (3) Electrophoresis: After removing the surface and preservation solution from the gel tube, fix it vertically in the electrophoresis tank. The first separating gel and stacking gel are located at the negative end of the electrophoresis tank, and the second separating gel is located at the positive end of the electrophoresis tank. Then, add 600 mL of electrophoresis buffer to the electrophoresis tank. Take 40 μL of stained serum and slowly add it to the gel tube to cover the surface of the stacking gel. After the solution in the system is stable, turn on the regulated power supply with a current of 2 mA / tube and an electrophoresis time of 80 min.

[0066] The electrophoresis buffer was prepared as follows: 13.224 g of tris(hydroxymethyl)aminomethane and 5.5 g of boric acid were dissolved in water, mixed evenly, and the volume was adjusted to 1000 mL. The mixture was then filtered through a filter membrane with a pore size of 0.22 μm to obtain the electrophoresis buffer.

[0067] (4) Quantitative detection: After electrophoresis, the gel tube was removed and the absorbance changes at different positions in the first and second separating gels of the gel tube were scanned with a scanner. Based on the curves obtained by the absorbance changes of different subtypes of low-density lipoprotein with position, the percentage of low-density lipoprotein in the total serum lipoprotein was quantitatively detected.

[0068] Comparative Example 1

[0069] The difference between Comparative Example 1 and Example 1 lies in the different separating gel, stacking gel, and electrophoresis buffer used in Comparative Example 1 compared to Example 1; all other detection methods are the same as in Example 1. The preparation of the separating gel, stacking gel, and electrophoresis buffer in Comparative Example 1 is as follows:

[0070] The separating gel was prepared as follows: 0.667 mL of 30% polyacrylamide (Acr:Bis = 29:1), 4.078 mL of H2O, 200 μL of 1 mol / L Tris-HCl (pH 8.9), 5.0 μL of tetramethylethylenediamine, and 50 μL of 10% ammonium persulfate were mixed thoroughly to prepare a separating gel with a gel concentration T = 4.0%, pH 8.9, and a volume of 5.0 mL.

[0071] The stacking gel was prepared as follows: 300.0 μL of 30% polyacrylamide (Acr:Bis = 29:1), 2.567 mL of H2O, 100 μL of 1 mol / L Tris-HCl (pH 6.8), 3.0 μL of tetramethylethylenediamine, and 30 μL of 10% ammonium persulfate were mixed evenly to prepare a stacking gel with a gel concentration T = 3.0%, pH 6.9, and a volume of 3.0 mL.

[0072] The electrophoresis buffer was prepared as follows: 3.3g of tris(hydroxymethyl)aminomethane and 14.4g of glycine were dissolved in water, mixed evenly, and the volume was adjusted to 1000mL. The mixture was then filtered through a 0.22μm filter membrane to obtain the electrophoresis buffer.

[0073] Evaluation of testing results

[0074] Serum from clinically confirmed cardiovascular patients and healthy individuals was collected. Electrophoresis was performed on each serum sample according to the methods described in Example 1 and Comparative Example 1. After electrophoresis, the samples were scanned using an optical density scanner. The resulting electrophoretic patterns are shown below. Figure 1-4 As shown. Among them Figure 1 , 2 The images shown are gel electrophoresis patterns obtained by electrophoresis of serum from diagnosed cardiovascular patients and serum from healthy individuals, respectively, according to the method described in Example 1. Figure 3 , 4The images are gel electrophoresis patterns obtained by electrophoresis of serum from confirmed cardiovascular patients and healthy individuals, respectively, following the method described in Comparative Example 1.

[0075] Depend on Figure 1 and 2 It can be seen that, Figure 1 and 2 The high clarity and resolution of the gel electrophoresis patterns demonstrate that the method in Example 1 can clearly separate the various subtypes of low-density lipoprotein and has high detection accuracy. Furthermore, the gel electrophoresis patterns obtained from... Figure 3 and 4 It can be seen that, Figure 3 and Figure 4 The low clarity and resolution of the gel electrophoresis pattern indicate that the separation effect of the method in Comparative Example 1 is poor.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting low-density lipoprotein in serum by gel electrophoresis, characterized in that, Its use for non-disease diagnostic purposes includes the following steps: (1) Serum staining: Take a serum sample, add staining solution, mix well, then place in a constant temperature water bath, and take the supernatant after centrifugation to obtain the stained serum; (2) Gel tube preparation: Fix the glass tube on the support, and then add the second layer of separating gel, the first layer of separating gel and the stacking gel into the glass tube in sequence. Each time a gel solution is added, wait until the surface of the gel solution is polymerized and flat before adding the next gel solution. Finally, immerse the whole tube in the preservation solution to obtain the gel tube; The second layer of separating gel, the first layer of separating gel and the stacking gel all include: polyacrylamide, H2O, Tris-HCl, sucrose, glycerol, tetramethylethylenediamine and ammonium persulfate; The volume ratio of the second separating gel, the first separating gel, and the stacking gel in the gel tube is 5-7:1-2:1; The volume ratio of polyacrylamide, H2O, Tris-HCl, sucrose, glycerol, tetramethylethylenediamine, and ammonium persulfate in the second separating gel is 28–35:150–300:7–9:9–11:1–2:0.1–0.2:1; the gel concentration T of the second separating gel is 3–4%, and the pH value is 8.5–9. The volume ratio of polyacrylamide, H2O, Tris-HCl, sucrose, glycerol, tetramethylethylenediamine, and ammonium persulfate in the first separating gel is 45–50:200–300:7–9:9–11:1–2:0.1–0.2:1; the gel concentration T of the first separating gel is 4.5–6%, and the pH value is 8.5–9. The volume ratio of polyacrylamide, H2O, Tris-HCl, sucrose, glycerol, tetramethylethylenediamine, and ammonium persulfate in the concentrated gel is 20–25:150–250:4.5–6:55–65:1–2:0.1–0.2:1; the gel concentration T of the concentrated gel is 2–3%, and the pH value is 6.5–7. (3) Electrophoresis: After removing the surface and preservation solution from the gel tube, fix it vertically in the electrophoresis tank. The first separating gel and stacking gel are located at the negative end of the electrophoresis tank, and the second separating gel is located at the positive end of the electrophoresis tank. Then, add electrophoresis buffer to the electrophoresis tank and slowly add the stained serum into the gel tube to cover the surface of the stacking gel. After the solution in the system is stable, turn on the regulated power supply to perform electrophoresis. (4) Quantitative detection: After electrophoresis, the gel tube is removed and the absorbance changes at different positions in the first and second separating gels of the gel tube are scanned with a scanner. Based on the curves obtained by the change of absorbance of different subtypes of low-density lipoprotein with position, the percentage of low-density lipoprotein in the total serum lipoprotein is quantitatively detected.

2. The method for detecting low-density lipoprotein in serum by gel electrophoresis according to claim 1, characterized in that, In step (2), the preservation solution is prepared by mixing sucrose, Tris-HCl, glycerol and Proclin 300 in a mass-to-volume ratio of 9-11 g : 38-45 mL : 1 mL : 0.1-0.2 mL.

3. The method for detecting low-density lipoprotein in serum by gel electrophoresis according to claim 1, characterized in that, In step (2), distilled water is added to the surface of the liquid immediately after each addition of adhesive solution, and the liquid is left to stand for 30 to 40 minutes. After the surface of the adhesive solution has polymerized and become smooth, the water layer is removed, and then the next adhesive solution is added.

4. The method for detecting low-density lipoprotein in serum by gel electrophoresis according to claim 1, characterized in that, In step (1), the volume ratio of the serum sample to the staining solution is 8 to 10:

1.

5. The method for detecting low-density lipoprotein in serum by gel electrophoresis according to claim 4, characterized in that, In step (1), the staining solution is prepared from Sudan Black B, anhydrous ethanol, dimethyl sulfoxide and ethylene glycol. The concentration of Sudan Black B in the staining solution is 2-3 g / L, and the volume ratio of anhydrous ethanol, dimethyl sulfoxide and ethylene glycol is 1-2:1:1-2.

6. The method for detecting low-density lipoprotein in serum by gel electrophoresis according to claim 1, characterized in that, In step (3), the buffer solution is prepared by mixing tris(hydroxymethyl)aminomethane and boric acid in a mass ratio of 2 to 3:1.

Images