Low-density lipoprotein electrophoretic typing method and gel electrophoretic separation column for electrophoretic typing

Abstract

The invention provides a low density lipoprotein electrophoresis typing method and a gel electrophoresis separation column for electrophoresis typing, the gel electrophoresis separation column comprises a concentrated gel and a separation gel, the concentrated gel and the separation gel both comprise acrylamide, N, N-dimethacrylamide, cane sugar, glycerin and a Tris-HCl solution, the content of acrylamide in the separation gel is 1.42-1.53 times that of the concentrated gel, the content of the N, N-dimethacrylamide in the separation gel is 1.42-1.53 times that of the Tris-HCl solution, and the content of the N, N-dimethacrylamide in the separation gel is 1.42-1.53 times that of the Tris-HCl solution in the separation gel. And the content of N, N-bis (methacrylamide) in the separation gel is 1.41-1.52 times that in the concentrated gel. The electrophoresis typing method provided by the invention has the advantages of simplicity and convenience in operation, rapidness, high efficiency, high sensitivity, good resolution ratio and low cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of low-density lipoprotein typing, in particular to a low-density lipoprotein electrophoresis typing method and a gel electrophoresis separation column for electrophoresis typing. BACKGROUND

[0002] Cardiovascular and cerebrovascular diseases are one of the main causes of death and disability worldwide, including coronary heart disease, stroke, hypertension, etc. According to the statistics of the World Health Organization, cardiovascular and cerebrovascular diseases cause about 18 million deaths each year, accounting for 31% of the total number of deaths worldwide. The harm is not only in high mortality, but also in high disability rate. Survivors often face sequelae such as limb dysfunction and cognitive decline, which seriously affect the quality of life. Cardiovascular and cerebrovascular diseases are closely related to lipoprotein levels, especially low-density lipoprotein (LDL) and high-density lipoprotein (HDL). Different types of lipoproteins play different roles in the transport and metabolism of cholesterol and triglycerides, and thus have important effects on cardiovascular and cerebrovascular health. Lipoproteins mainly include low-density lipoprotein (LDL), high-density lipoprotein (HDL), very low-density lipoprotein (VLDL), chylomicrons and lipoprotein (a) [Lp(a)], and their levels directly relate to the occurrence and development of atherosclerosis and cardiovascular and cerebrovascular diseases. Low-density lipoprotein (LDL) is known as "bad cholesterol", and its main function is to transport cholesterol from the liver to tissues throughout the body. When LDL levels are too high, cholesterol is easily deposited in the arterial wall, forming plaques, leading to atherosclerosis, and thus increasing the risk of coronary heart disease, myocardial infarction and stroke. Therefore, controlling LDL levels is one of the key targets for preventing cardiovascular and cerebrovascular diseases. High-density lipoprotein (HDL) is known as "good cholesterol", which can transport cholesterol from peripheral tissues back to the liver for metabolism and excretion. Higher HDL levels help remove cholesterol from the arteries, slowing the progression of atherosclerosis, and thus reducing the risk of cardiovascular and cerebrovascular diseases. Very low-density lipoprotein (VLDL) is mainly responsible for transporting triglycerides. Elevated VLDL levels are usually associated with hypertriglyceridemia, which may indirectly promote the occurrence of atherosclerosis and increase the risk of cardiovascular and cerebrovascular diseases. In addition, although chylomicrons are mainly involved in the transport of dietary fat, their metabolic abnormalities can also lead to diseases such as pancreatitis, which indirectly affects cardiovascular health.

[0003] Low-density lipoprotein (LDL) is not a single substance, but is composed of subtypes with different particle sizes, densities, and chemical compositions. Studies have shown that there are significant differences in the ability of different LDL subtypes to cause atherosclerosis, with the small and dense LDL (sdLDL) subtype having a stronger atherosclerotic effect. Traditional LDL detection methods, such as ultracentrifugation and chemical precipitation, can only measure the total LDL level and cannot distinguish between different LDL subtypes. In recent years, LDL typing techniques based on electrophoresis principles have gradually developed, such as gradient gel electrophoresis (GGE) and capillary electrophoresis (CE). These methods can separate LDL into different subtypes and quantitatively analyze them, providing more accurate information for assessing cardiovascular disease risk. However, existing LDL electrophoresis typing techniques still have some shortcomings: (1) complex operation, time-consuming, GGE requires multiple steps such as gel preparation, sample loading, electrophoresis, staining, and destaining, and the entire process takes several hours or even longer; (2) low sensitivity, limited resolution, CE has high separation efficiency, but the detection sensitivity for low concentration LDL subtypes is low, and it is difficult to distinguish between similar LDL subtypes; (3) high cost, difficult to popularize, some advanced LDL electrophoresis typing instruments are expensive, limiting their widespread use in clinical settings.

[0004] Therefore, there is an urgent need to develop a low-density lipoprotein electrophoresis typing method that is simple, fast, efficient, sensitive, and has good resolution and low cost to overcome the shortcomings of existing technologies. SUMMARY

[0005] To solve the above problems, the present application provides a gel electrophoresis separation column for low-density lipoprotein electrophoresis typing, comprising: concentrated gel and separation gel, both the concentrated gel and the separation gel contain acrylamide, N,N dimethyl acrylamide, sucrose, glycerol, and Tris-HCl solution, and the content of acrylamide in the separation gel is 1.42-1.53 times that in the concentrated gel, and the content of N,N dimethyl acrylamide in the separation gel is 1.41-1.52 times that in the concentrated gel.

[0006] In one example of the present application, the content of acrylamide in the separation gel is 1.48 times that in the concentrated gel.

[0007] In one example of the present application, the content of acrylamide in the separation gel is 1.49 times that in the concentrated gel.

[0008] In one example of the present application, the content of acrylamide in the separation gel is 1.46 times that in the concentrated gel.

[0009] In one example of the present application, the content of acrylamide in the separation gel is 1.44 times that in the concentrated gel.

[0010] In one example of the present application, the content of acrylamide in the separation gel is 1.5 times that in the concentrated gel.

[0011] In one example of the present application, the content of acrylamide in the separation gel is 1.52 times that in the concentrated gel.

[0012] In one example of the present application, the content of N,N dimethyl acrylamide in the separation gel is 1.48 times that in the concentrated gel.

[0013] In one example of the present application, the content of N,N dimethyl acrylamide in the separation gel is 1.46 times that in the concentrated gel.

[0014] In one example of the present application, the content of N,N dimethyl acrylamide in the separation gel is 1.44 times that in the concentrated gel.

[0015] In one example of the present application, the content of N,N dimethyl acrylamide in the separation gel is 1.42 times that in the concentrated gel.

[0016] In one example of the present application, the content of N,N dimethyl acrylamide in the separation gel is 1.49 times that in the concentrated gel.

[0017] In one example of the present application, the content of N,N dimethyl acrylamide in the separation gel is 1.5 times that in the concentrated gel.

[0018] In one example of the present application, the content of N,N dimethyl acrylamide in the separation gel is 1.51 times that in the concentrated gel.

[0019] In one example of the present application, the N,N dimethyl acrylamide is N,N'- methylenebisacrylamide.

[0020] In one example of the present application, the concentrated gel contains Tris-HCl solution, acrylamide solution, N,N dimethyl acrylamide solution, sucrose solution, glycerol solution, water in a volume ratio of (98-102):(65-70):(130-140):(98-102):(9-11):(577-578).

[0021] In one example of the present application, the concentrated gel contains Tris-HCl solution, acrylamide solution, N,N dimethyl acrylamide solution, sucrose solution, glycerol solution, distilled water, tetramethylethylenediamine, ammonium persulfate solution in a volume ratio of (98-102):(65-70):(130-140):(98-102):(9-11):(577-578):(0.3-0.5):(9-11).

[0022] In one example of the present application, the concentrated gel contains Tris-HCl solution, acrylamide solution, N,N dimethyl acrylamide solution, sucrose solution, glycerol solution, distilled water, tetramethylethylenediamine, ammonium persulfate solution in a volume ratio of 100:67.5:135:100:10:577.1:0.4:10.

[0023] In one example of the present application, the concentrated gel contains Tris-HCl solution, acrylamide solution, N,N dimethyl acrylamide solution, sucrose solution, glycerol solution, distilled water, tetramethylethylenediamine, ammonium persulfate solution in a volume ratio of 100:67:135:100:10:577.6:0.4:10.

[0024] In one example of the present application, the concentrated gel contains Tris-HCl solution, acrylamide solution, N,N dimethyl acrylamide solution, sucrose solution, glycerol solution, distilled water, tetramethylethylenediamine, ammonium persulfate solution in a volume ratio of 100:67.5:134.5:100:10:577.6:0.4:10.

[0025] In one example of the present application, the separation gel contains Tris-HCl solution, acrylamide solution, N,N dimethyl acrylamide solution, sucrose solution, glycerol solution, water in a volume ratio of (98-102):(95-105):(195-205):(98-102):(9-11):(479-480).

[0026] In one example of the present application, the separation gel contains Tris-HCl solution, acrylamide solution, N,N dimethyl acrylamide solution, sucrose solution, glycerol solution, distilled water, tetramethylethylenediamine, ammonium persulfate solution in a volume ratio of (98-102):(95-105):(195-205):(98-102):(9-11):(479-480):(0.3-0.5):(9-11).

[0027] In one example of the present application, the separation gel contains Tris-HCl solution, acrylamide solution, N,N dimethyl acrylamide solution, sucrose solution, glycerol solution, distilled water, tetramethylethylenediamine, ammonium persulfate solution in a volume ratio of 100:100:200:100:10:479.6:0.4:10.

[0028] In one example of the present application, the separation gel contains Tris-HCl solution, acrylamide solution, N,N dimethyl acrylamide solution, sucrose solution, glycerol solution, distilled water, tetramethylethylenediamine, ammonium persulfate solution in a volume ratio of 100:100.5:200:100:10:479.1:0.4:10.

[0029] In one example of the present application, the separating gel contains Tris-HCl solution, acrylamide solution, N,N dimethyl acrylamide solution, sucrose solution, glycerol solution, distilled water, tetramethylethylenediamine, ammonium persulfate solution in a volume ratio of 100:99.6:200:100:10:480:0.4:10.

[0030] In one example of the present application, acrylamide solution with a mass fraction of 28%-32% and N,N dimethyl acrylamide solution with a mass fraction of 14%-16% are used in the concentrating gel and / or the separating gel.

[0031] In one example of the present application, acrylamide solution with a mass fraction of 30% and N,N dimethyl acrylamide solution with a mass fraction of 15% are used in both the concentrating gel and the separating gel.

[0032] In one example of the present application, acrylamide solution with a mass fraction of 30% and N,N dimethyl acrylamide solution with a mass fraction of 15% are used in the concentrating gel, and acrylamide solution with a mass fraction of 30% and N,N dimethyl acrylamide solution with a mass fraction of 15.5% are used in the separating gel.

[0033] In one example of the present application, acrylamide solution with a mass fraction of 29% and N,N dimethyl acrylamide solution with a mass fraction of 14.5% are used in the concentrating gel, and acrylamide solution with a mass fraction of 31% and N,N dimethyl acrylamide solution with a mass fraction of 14% are used in the separating gel.

[0034] In one example of the present application, sucrose solution with a mass fraction of 20%-30% and glycerol solution with a mass fraction of 20%-30% and Tris-HCl solution with a mass fraction of 16%-20% are used in the concentrating gel and / or the separating gel, and Tris-HCl solution with a pH of 6.6-7 is used in the concentrating gel and / or the separating gel.

[0035] In one example of the present application, sucrose solution with a mass fraction of 25% is used in both the concentrating gel and the separating gel.

[0036] In one example of the present application, sucrose solution with a mass fraction of 26% is used in both the concentrating gel and the separating gel.

[0037] In one example of the present application, sucrose solution with a mass fraction of 24% is used in both the concentrating gel and the separating gel.

[0038] In one example of the present application, sucrose solution with a mass fraction of 23% is used in the concentrating gel, and sucrose solution with a mass fraction of 27% is used in the separating gel.

[0039] In one example of the present application, sucrose solution with a mass fraction of 26% is used in the concentrating gel, and sucrose solution with a mass fraction of 24% is used in the separating gel.

[0040] In one example of the present application, a glycerol solution with a mass fraction of 25% is used in both the concentrated gel and the separation gel.

[0041] In one example of the present application, a glycerol solution with a mass fraction of 24% is used in both the concentrated gel and the separation gel.

[0042] In one example of the present application, a glycerol solution with a mass fraction of 26% is used in both the concentrated gel and the separation gel.

[0043] In one example of the present application, a glycerol solution with a mass fraction of 28% is used in the concentrated gel, and a glycerol solution with a mass fraction of 23% is used in the separation gel.

[0044] In one example of the present application, a glycerol solution with a mass fraction of 25% is used in the concentrated gel, and a glycerol solution with a mass fraction of 26% is used in the separation gel.

[0045] In one example of the present application, a Tris-HCl solution with a mass fraction of 18% and a pH of 6.8 is used in both the concentrated gel and the separation gel.

[0046] In one example of the present application, a Tris-HCl solution with a pH of 6.7 is used in the concentrated gel, and a Tris-HCl solution with a pH of 6.8 is used in the separation gel.

[0047] In one example of the present application, a Tris-HCl solution with a pH of 6.9 is used in the concentrated gel, and a Tris-HCl solution with a pH of 6.8 is used in the separation gel.

[0048] In one example of the present application, tetramethyl ethylenediamine and ammonium persulfate solution are further included in both the concentrated gel and the separation gel.

[0049] In one example of the present application, an ammonium persulfate solution with a mass fraction of 10% is used in both the concentrated gel and the separation gel.

[0050] In one example of the present application, the specific preparation steps of the gel electrophoresis separation column are as follows: S1: uniformly mix the Tris-HCl solution, the acrylamide solution, the N,N dimethyl acrylamide solution, the sucrose solution, the glycerol solution, and the distilled water according to the proportion, and then add tetramethyl ethylenediamine to obtain a first mixture; S2: add the ammonium persulfate solution to the first mixture to obtain a second mixture; S3: divide the second mixture, discharge the air bubbles, then seal the gel with distilled water, and after standing for 40-90 min, remove the gel sealing distilled water to obtain the separation gel; S4: according to the proportion, the Tris-HCl solution, the acrylamide solution, the N, N dimethyl acrylamide solution, the sucrose solution, the glycerol solution and the distilled water are mixed uniformly, then tetramethyl ethylenediamine is added, and a third mixture is obtained; S5: the ammonium persulfate solution is added to the third mixture, and a fourth mixture is obtained; S6: the fourth mixture is added above the separation gel, the bubbles are discharged, then the separation gel is sealed with distilled water, and after standing for 45min-90min, a gel electrophoresis separation column is obtained.

[0051] The application further provides a low-density lipoprotein electrophoresis typing method, and the specific steps are as follows: S100: buffer powder and water are mixed in a mass ratio of (35-36):2000 to obtain an electrophoresis buffer; S200: the to-be-tested liquid and the staining liquid are mixed in a volume ratio of (4.5-5.5):1, and then after standing for 18min-22min, a staining mixed liquid is obtained; S300: the above gel electrophoresis separation column is installed into an electrophoresis device, and the electrophoresis buffer is added to the electrophoresis tank; S400: the staining mixed liquid is vortexed and oscillated for 25s-35s, and then a mini vortex centrifuge is used for centrifugation for 25s-35s, and the staining mixed liquid in the upper part after centrifugation is added to the sample end of the gel electrophoresis separation column; S500: the electrophoresis device is operated, the current is set to 2.8mA-3.2mA, and the electrophoresis time is set to 75min-85min, then the gel electrophoresis separation column is taken out from the electrophoresis device, scanning analysis is performed, and typing data are obtained.

[0052] In one example of the application, in S100, the buffer powder comprises sodium borate decahydrate and boric acid.

[0053] In one example of the application, in S200, the staining liquid comprises Sudan black and propylene glycol.

[0054] Compared with the prior art, the application has the following advantages: (1) The electrophoresis typing method of the application is simple, rapid and efficient, simplifies the experimental steps, shortens the detection time, improves the detection efficiency, has high sensitivity and good resolution, can effectively separate and detect low-concentration LDL subtypes and distinguish similar LDL subtypes, is low in cost, easy to popularize, uses conventional experimental instruments and reagents, reduces the detection cost, and is conducive to the popularization and application in the clinic; (2) The gel electrophoresis separation column of the present application comprises two parts, a concentration gel and a separation gel. The concentration gel can concentrate the sample. The pore size of the concentration gel is small, and the lipoprotein in the sample can be concentrated into a narrow band, thereby improving the resolution of subsequent separation. At the same time, the concentration gel can also form a pH gradient. The pH difference between the concentration gel and the separation gel forms a discontinuous electrophoresis system, so that the sample is concentrated before entering the separation gel, ensuring that the sample enters the separation gel in the form of a uniform narrow band. Moreover, the concentration gel can also improve the separation effect. The concentration effect of the concentration gel reduces the diffusion of the sample, so that the lipoprotein can be more clearly separated in the separation gel, thereby improving the accuracy and sensitivity of detection. (3) The separation gel in the gel electrophoresis separation column of the present application has a molecular sieve effect. The pore size of the separation gel is small, and the separation gel can separate the lipoprotein according to the molecular size. Smaller molecules (such as HDL) migrate faster, while larger molecules (such as LDL and VLDL) migrate slower, thereby achieving separation. The separation gel can also achieve high-resolution separation. The uniform pore size and appropriate concentration of the separation gel can provide high-resolution separation effect, so that different lipoproteins (such as LDL, HDL and VLDL) form clear bands. At the same time, the separation gel also has the effect of charge separation. Under the action of an electric field, the lipoprotein is further separated according to the charge difference, thereby enhancing the separation effect. Moreover, the separation gel also supports quantitative analysis. The clear bands of the separation gel facilitate subsequent staining and quantitative analysis, thereby providing reliable data for lipoprotein typing. (4) The electrophoresis typing method of the present application can be applied to cardiovascular disease risk assessment. By detecting the level of sdLDL and other atherogenic LDL subtypes, the risk of an individual suffering from cardiovascular disease can be assessed. The electrophoresis typing method of the present application can also be applied to the evaluation of the efficacy of lipid-lowering drugs. By monitoring the changes in the levels of different LDL subtypes, the efficacy and safety of lipid-lowering drugs can be evaluated. The electrophoresis typing method of the present application can also be used for the study of LDL metabolism mechanism, thereby providing a new technical means for the study of the relationship between LDL metabolism mechanism and diseases. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 Figure 1 is a gel electrophoresis separation column typing data graph of sample 1 in Example 2 of the present application. Figure 2 Figure 2 is a gel electrophoresis separation column typing data graph of sample 2 in Example 2 of the present application. Figure 3 Figure 3 is a gel electrophoresis separation column typing data graph of sample 3 in Example 2 of the present application. Figure 4 Figure 4 is a gel electrophoresis separation column typing data graph of sample 4 in Example 2 of the present application. Figure 5 Figure 5 is a gel electrophoresis separation column typing data graph of sample 5 in Example 2 of the present application. Figure 6Figure for typing data of gel electrophoresis separation column of sample 6 in embodiment two of the present application; Figure 7 Figure for typing data of gel electrophoresis separation column of sample 7 in embodiment two of the present application; Figure 8 Figure for typing data of gel electrophoresis separation column of sample 8 in embodiment two of the present application; Figure 9 Figure for typing data of gel electrophoresis separation column of sample 9 in embodiment two of the present application; Figure 10 Figure for typing data of gel electrophoresis separation column of sample 10 in embodiment two of the present application; Figure 11 Figure for typing data of gel electrophoresis separation column of sample 11 in embodiment two of the present application; Figure 12 Figure for typing data of gel electrophoresis separation column of sample 12 in embodiment two of the present application; Figure 13 Figure for gel electrophoresis separation column of sample 1 to sample 12 in embodiment two of the present application. DETAILED DESCRIPTION

[0056] In order to make the above objectives, characteristics and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application are described in detail below.

[0057] Embodiment one: The embodiment provides a gel electrophoresis separation column for low density lipoprotein electrophoresis typing, which comprises two parts of concentrated gel and separation gel, wherein the concentrated gel contains the following components: acrylamide, N,N dimethyl acrylamide, sucrose, glycerol, Tris-HCl solution, tetramethyl ethylenediamine (TEMED) and ammonium persulfate, and the specific concentration is shown in Table 1: Table 1 Concentrated gel component Content 1 Tris-HCl solution (18%, PH=6.8) 100 mL / L 2 Acrylamide solution (30%) 67.5 mL / L 3 N,N dimethyl acrylamide solution (15%) 135 mL / L 4 Sucrose solution (25%) 100 mL / L 5 Glycerol solution (25%) 10 mL / L 6 Distilled water 577.1 mL / L 7 TEMED 400 μL / L 8 Ammonium persulfate solution (10%) 10 mL / L The separation gel contains the following components: acrylamide, N,N dimethyl acrylamide, sucrose, glycerol, Tris-HCl solution, TEMED and ammonium persulfate, and the specific concentration is shown in Table 2: Table 2 Concentrated gel component Content 1 Tris-HCl solution (18%, PH=6.8) 100 mL / L 2 Acrylamide solution (30%) 100 mL / L 3 N,N dimethyl acrylamide solution (15%) 200 mL / L 4 Sucrose solution (25%) 100 mL / L 5 Glycerol solution (25%) 10 mL / L 6 Distilled water 479.6 mL / L 7 TEMED 400 μL / L 8 Ammonium persulfate solution (10%) 10 mL / L The gel electrophoresis separation column is prepared by the following steps: S0: Wear disposable gloves, first insert one end of a clean glass tube (75mm*7mm*0.7mm) into the bottom of a customized silicone pad hole with force, and fix it with a plastic hole plate of 8*12 from above, the fixed position is the lower quarter of the top of the glass column, press the top of the glass column with force after inserting a plate to make it more firm combined with the silicone plate, prevent liquid leakage during pouring glue, and place it vertically; S1: according to the proportion, mix Tris-HCl solution, acrylamide solution, N, N bis-methyl acrylamide solution, sucrose solution, glycerol solution and distilled water uniformly, and then divide into bottles; use a pipette to add 400 μL of TEMED to the separation gel mixture; after adding TEMED, mix slowly for 5 times to prevent air bubbles; and obtain a first mixture; S2: use a 5 mL pipette to add 10 mL of 10% ammonium persulfate solution to the first mixture; immediately mix uniformly (not too vigorously to prevent air bubbles); mix thoroughly; and obtain a second mixture; S3: immediately adjust the peristaltic pump dispensing amount to 2000 μL; divide the second mixture into each glass tube; after filling, gently shake the table top to make the gel surface level and the air bubbles in the glass column discharge; then add 13.5 uL of ddH2O to seal the gel in each glass tube; when adding ddH2O, suspend it in the center of the glass tube to prevent edge effects and cause uneven liquid surface; use a daylight lamp to irradiate the gel column for not less than 40 min and not more than 90 min to achieve a good solidification state (obviously stratified and flat); if stratification is not flat with jagged or inclined, directly discard; after the time arrives, remove the upper ddH2O; if there is residual water, use a water absorption paper to absorb it; and obtain a separation gel; S4: according to the proportion, mix Tris-HCl solution, acrylamide solution, N, N bis-methyl acrylamide solution, sucrose solution, glycerol solution and distilled water uniformly, and then divide into bottles; use a pipette to add 400 μL of TEMED to the concentrated gel mixture; after adding TEMED, mix slowly for 5 times to prevent air bubbles; and obtain a third mixture; S5: use a 5 mL pipette to add 10 mL of 10% ammonium persulfate solution to the third mixture; immediately mix uniformly (not too vigorously to prevent air bubbles); mix thoroughly; and obtain a fourth mixture; S6: immediately adjust the peristaltic pump dispensing amount to 1350 μL; divide the fourth mixture into each glass tube containing the separation gel; after filling, add 15 μL of ddH2O to seal the gel in each glass tube; use a daylight lamp to irradiate the gel column for not less than 45 min and not more than 90 min; then turn off the daylight lamp; carefully remove the gel column from the silica gel plate; observe the obvious height difference, visible air bubbles and uneven interface between the concentrated gel and the separation gel; discard the column; place the prepared gel column into a clean small white bottle; add ddH2O to seal the gel column; and obtain a gel electrophoresis separation column.

[0058] Example Two The embodiment provides a low-density lipoprotein electrophoresis typing method, and the specific steps are as follows: S100: Take 35.5 g of buffer powder into a clean beaker, add 2000 mL of double distilled water or deionized water to prepare an electrophoresis buffer; S200: Take a clean 1.5 mL centrifuge tube, use a pipette to transfer 50 μL of the sample into the centrifuge tube, then add 10 μL of the staining solution, shake for 30 s on a vortex shaker (or blow 10 times with a pipette) to mix the sample and the staining solution thoroughly, then stand for 20 min to obtain a staining mixture; S300: Take the gel electrophoresis separation column prepared in Example 1, carefully discard the preservative liquid at the top of the gel column, install the gel column with the sample loading end upward into the rubber jack of the upper part of the electrophoresis tank, add an appropriate amount of electrophoresis buffer to the lower part of the electrophoresis tank, which should cover the electrode column; cover the upper part of the electrophoresis tank with the gel column on the lower part of the electrophoresis tank, and add an appropriate amount of electrophoresis buffer to ensure that the sample loading end of the gel column and the electrode column of the upper cover can be immersed in the buffer; S400: Take the staining mixture, vortex for 30 s, and then centrifuge for 30 s with a mini vortex machine, take 30 μL of the upper part of the dye mixture after centrifugation and add it to the sample loading end of the gel column; S500: Cover the electrophoresis tank cover, turn on the power supply, turn on the electrophoresis device (power) switch, set the current to 3 mA, and the electrophoresis time to 80 min (the electrophoresis can be ended early when the bottom dye in the gel column is about 1 cm away from the bottom end of the column), remove the electrophoresis buffer from the upper part of the electrophoresis tank, take out the gel column, wipe off the electrophoresis buffer on the surface of the gel column, and perform scanning analysis within 15 min to obtain the typing data.

[0059] The samples 1-12 were tested by the electrophoresis typing method of the present example, and the test results are shown in Table 1. Figure 1-12 Among them, samples 1-12 are serum or plasma samples collected.

[0060] From the test results, it can be seen that sample 1 is of low-density lipoprotein type 4, having a high risk of causing cardiovascular diseases; sample 2 is of low-density lipoprotein type 2, having a low risk of causing cardiovascular diseases; sample 3 is of low-density lipoprotein type 3, having a low risk of causing cardiovascular diseases; sample 4 is of low-density lipoprotein type 2, having a low risk of causing cardiovascular diseases; sample 5 is of low-density lipoprotein type 5, having a high risk of causing cardiovascular diseases; sample 6 is of low-density lipoprotein type 4, having a high risk of causing cardiovascular diseases; sample 7 is of low-density lipoprotein type 3, having a low risk of causing cardiovascular diseases; sample 8 is of low-density lipoprotein type 5, having a high risk of causing cardiovascular diseases; sample 9 is of low-density lipoprotein type 4, having a high risk of causing cardiovascular diseases; sample 10 is of low-density lipoprotein type 4, having a high risk of causing cardiovascular diseases; sample 11 is of low-density lipoprotein type 4, having a high risk of causing cardiovascular diseases; and sample 12 is of low-density lipoprotein type 3, having a low risk of causing cardiovascular diseases.

[0061] Figure 13 From left to right are sample 1 to sample 12 using the low-density lipoprotein electrophoresis typing method of the present application, and after the low-density lipoprotein in serum or plasma is separated by electrophoresis, the photo of the gel electrophoresis separation column.

[0062] It can be proved by the above steps that the low-density lipoprotein electrophoresis typing method of the present application has the advantages of simple operation, rapid and efficient, high sensitivity, good resolution, and low cost.

[0063] Although the present application is disclosed as above, the present application is not limited thereto. Any person skilled in the art, without departing from the spirit and scope of the present application, can make various changes and modifications, and therefore the protection scope of the present application should be defined by the scope defined in the claims.

Claims

1. A gel electrophoresis separation column for low-density lipoprotein electrophoresis typing, characterized in that, include: The mixture comprises a stacking gel and a separating gel, both of which contain acrylamide, N,N-dimethylacrylamide, sucrose, glycerol, and Tris-HCl solution. The acrylamide content in the separating gel is 1.42-1.53 ​​times that in the stacking gel, and the N,N-dimethylacrylamide content in the separating gel is 1.41-1.52 times that in the stacking gel.

2. The gel electrophoresis separation column according to claim 1, characterized in that, The concentrated gel contains Tris-HCl solution, acrylamide solution, N,N-dimethylacrylamide solution, sucrose solution, glycerol solution, and water in a volume ratio of (98-102):(65-70):(130-140):(98-102):(9-11):(577-578).

3. The gel electrophoresis separation column according to claim 1, characterized in that, The separating gel contains Tris-HCl solution, acrylamide solution, N,N-dimethylacrylamide solution, sucrose solution, glycerol solution, and water in a volume ratio of (98-102):(95-105):(195-205):(98-102):(9-11):(479-480).

4. The gel electrophoresis separation column according to any one of claims 1-3, characterized in that, The concentrating gel and / or the separating gel use an acrylamide solution with a mass fraction of 28%-32% and an N,N-bismethylacrylamide solution of 14%-16%.

5. The gel electrophoresis separation column according to claim 4, characterized in that, The stacking gel and / or the separating gel use a sucrose solution with a mass fraction of 20%-30%, a glycerol solution with a mass fraction of 20%-30%, and a Tris-HCl solution with a mass fraction of 16%-20%, and the stacking gel and / or the separating gel use a Tris-HCl solution with a pH of 6.6-7.

6. The gel electrophoresis separation column according to claim 1, characterized in that, The concentrating gel and the separating gel also contain tetramethylethylenediamine and ammonium persulfate solution.

7. The gel electrophoresis separation column according to claim 6, characterized in that, The specific preparation steps are as follows: S1: Mix Tris-HCl solution, acrylamide solution, N,N-dimethylacrylamide solution, sucrose solution, glycerol solution, and distilled water evenly according to the formula, and then add tetramethylethylenediamine to obtain the first mixture; S2: Add ammonium persulfate solution to the first mixture to obtain a second mixture; S3: Dispense the second mixture, remove air bubbles, seal with distilled water, let stand for 40-90 minutes, remove the distilled water used for sealing, and obtain the separating adhesive; S4: Mix Tris-HCl solution, acrylamide solution, N,N-dimethylacrylamide solution, sucrose solution, glycerol solution, and distilled water evenly according to the formula, and then add tetramethylethylenediamine to obtain the third mixture; S5: Add ammonium persulfate solution to the third mixture to obtain a fourth mixture; S6: Add the fourth mixture on top of the separating gel, remove air bubbles, seal the gel with distilled water, and let it stand for 45-90 minutes to obtain the gel electrophoresis separation column.

8. A method for electrophoretic typing of low-density lipoproteins, characterized in that, The specific steps are as follows: S100: Mix buffer powder with water in a mass ratio of (35-36):2000 to obtain electrophoresis buffer; S200: Mix the test solution and staining solution in a volume ratio of (4.5-5.5):1 until homogeneous, and then let stand for 18-22 minutes to obtain the staining mixture; S300: Install the gel electrophoresis separation column according to any one of claims 1-7 into the electrophoresis apparatus, and add the electrophoresis buffer to the electrophoresis tank; S400: Take the staining mixture, vortex for 25s-35s, then centrifuge for 25s-35s using a mini vortex mixer, and add the upper part of the staining mixture after centrifugation to the loading end of the gel electrophoresis separation column. S500: Run the electrophoresis apparatus, set the current to 2.8mA-3.2mA and the electrophoresis time to 75min-85min, then remove the gel electrophoresis separation column from the electrophoresis apparatus, scan and analyze it to obtain the typing data.

9. The electrophoretic typing method according to claim 8, characterized in that, In S100, the buffer powder contains sodium borate decahydrate and boric acid.

10. The electrophoretic typing method according to claim 8, characterized in that, In S200, the staining solution contains Sudan Black and propylene glycol.

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