A lipoprotein subtype detection kit, its preparation method and application

Abstract

The present invention provides a lipoprotein subtype detection kit, a preparation method and an application thereof, belonging to the technical field of biological detection. In the separating gel of the kit, BODIPY 505 / 515 is mixed, so that staining is not required during detection, and direct sample loading and electrophoresis can be carried out, reducing the operation steps and shortening the operation time, and improving the detection sensitivity. In addition, N,N'-bisacryloylpiperazine is mixed in both the separating gel and the stacking gel. The rigidity of the N,N'-bisacryloylpiperazine gel is used to reduce gel deformation and trailing phenomena during migration, significantly improving the resolution, and enhancing the stability and repeatability of detection. The detection kit provided by the present invention has the advantages of short detection operation time, high detection sensitivity, high resolution, good stability and repeatability.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a lipoprotein subtype detection kit, a preparation method thereof, and an application thereof. Background Art

[0002] Lipoproteins mainly play a transport role in the human body, transferring cholesterol to various tissues and organs of the body to exert their corresponding physiological functions. Generally, lipoproteins can be divided into five categories according to their density: chylomicrons (CM), very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). Different types of lipoprotein cholesterol have different abilities to cause atherosclerosis and different degrees of risk of causing cardiovascular diseases.

[0003] Polyacrylamide gel electrophoresis is a commonly used method for lipoprotein detection. For example, the invention patent with the patent number CN202311835108.8 discloses a lipoprotein subtype detection kit, a preparation method thereof, and an application thereof. By optimizing the separating gel and the stacking gel, this kit can separate lipoproteins into 12 subtypes within 75 minutes, enabling the content of the 12 subtypes in lipoproteins to be comprehensively, truly, accurately, and objectively detected. However, when using this kit for detection, the sample needs to be stained for 30 minutes before electrophoresis, which still takes a long time, and the stability and detection sensitivity are still insufficient. Summary of the Invention

[0004] Based on the above problems in the prior art, the present invention provides a lipoprotein subtype detection kit, including a separating gel, and fluoroboron dipyrrole 505 / 515 and N,N - bisacryloylpiperazine are mixed in the separating gel.

[0005] Among them, the content of fluoroboron dipyrrole 505 / 515 in the separating gel is 0.05 - 0.1 g / L; the content of N,N - bisacryloylpiperazine in the separating gel is 0.7 - 1.8 g / L.

[0006] Among them, the components and contents of the separating gel are as follows:

[0007] Acrylamide is 25 - 40 g / L, N,N - bisacryloylpiperazine is 0.7 - 1.8 g / L, tris buffer is 2.5 - 5 g / L, tetramethylethylenediamine is 0.5 - 1.5 ml / L, ammonium persulfate is 0.5 - 1.5 g / L, sucrose is 20 - 30 g / L, fluoroboron dipyrrole is 505 / 515 0.05 - 0.1 g / L; the pH value of the tris buffer is 7.9.

[0008] Among them, the lipoprotein subtype detection kit further includes a stacking gel, and the stacking gel contains N,N-bisacryloylpiperazine (PDA), and the content of N,N-bisacryloylpiperazine (PDA) in the stacking gel is 0.5-1 g / L.

[0009] Among them, the components and contents of the stacking gel are as follows:

[0010] Acrylamide is 20-25 g / L, N,N-bisacryloylpiperazine is 0.5-1 g / L, tris buffer is 4-8 g / L, tetramethylethylenediamine is 0.5-1.5 ml / L, ammonium persulfate is 0.5-1.5 g / L; the pH value of the tris buffer is 6.8.

[0011] The present invention also provides a preparation method of a lipoprotein subtype detection kit, which mixes boron dipyrromethene 505 / 515 and N,N-bisacryloylpiperazine into a separating gel to make a separating gel with boron dipyrromethene 505 / 515 and N,N-bisacryloylpiperazine.

[0012] Among them, the preparation method also adds N,N-bisacryloylpiperazine to the stacking gel to make a stacking gel containing N,N-bisacryloylpiperazine.

[0013] Among them, the components and contents of the separating gel are: acrylamide is 25-40 g / L, N,N-bisacryloylpiperazine is 0.7-1.8 g / L, tris buffer is 2.5-5 g / L, tetramethylethylenediamine is 0.5-1.5 ml / L, ammonium persulfate is 0.5-1.5 g / L, sucrose is 20-30 g / L, boron dipyrromethene is 505 / 515 0.05-0.1 g / L; the pH value of the tris buffer is 7.9;

[0014] The components and contents of the stacking gel are: acrylamide is 20-25 g / L, N,N-bisacryloylpiperazine is 0.5-1 g / L, tris buffer is 4-8 g / L, tetramethylethylenediamine is 0.5-1.5 ml / L, ammonium persulfate is 0.5-1.5 g / L; the pH value of the tris buffer is 6.8;

[0015] First, weigh the corresponding amounts of various components according to the components and contents of the separating gel, mix the components to make a mixed solution 1, pour the mixed solution 1 into a glass tube, and the filling volume is 1-1.5 ml, and let it stand. After the mixed solution 1 solidifies, the first-stage separating gel is obtained;

[0016] Then, weigh the corresponding amounts of various components according to the components and contents of the stacking gel, mix the components to make a mixed solution 2, pour the mixed solution 2 into a glass tube, and the filling volume is 150 ul-200 ul, and let it stand. After the mixed solution 2 solidifies, the stacking gel is obtained;

[0017] Finally, the prepared first-segment separating gel and stacking gel are placed in a preservation solution for preservation, and the lipoprotein subtype detection kit is obtained.

[0018] Among them, the preparation method further includes preparing a second-segment separating gel. First, corresponding amounts of various components are weighed according to the components and contents of the separating gel. The amounts of acrylamide and N,N-bisacryloylpiperazine are less than those of the first-segment separating gel. The components are mixed to form a mixed solution 1-1. The mixed solution 1-1 is poured into a glass tube, and the filling volume is 1-1.5 ml. It is left standing. After the mixed solution 1-1 solidifies, the second-segment separating gel is obtained.

[0019] The present invention also provides an application of the lipoprotein subtype detection kit as described above in lipoprotein subtype detection. The detection includes separately or simultaneously detecting one or more of very low density lipoprotein VLDL, intermediate density lipoprotein IDL-A, IDL-B, and IDL-C, low density lipoprotein LDL-1, LDL-2, LDL-3, LDL-4, LDL-5, LDL-6, and LDL-7, and high density lipoprotein cholesterol.

[0020] The beneficial effects of the present invention are as follows:

[0021] 1. In the kit provided by the present invention, the fluorescent probe BODIPY 505 / 515 is mixed with the separating gel. When performing the detection operation, it is not necessary to stain the sample, and it can be directly loaded for electrophoresis, which can reduce the operation steps and shorten the operation time.

[0022] 2. By using BODIPY 505 / 515 to specifically bind to lipoproteins, the detection sensitivity can be significantly improved, enabling low-concentration samples to be detected.

[0023] 3. Using N,N-bisacryloylpiperazine (PDA) as a crosslinking agent, compared with the conventional N,N'-methylenebisacrylamide (Bis), the rigid structure of the PDA gel is more suitable for the separation of macromolecular complexes (such as LDL), reducing gel deformation and trailing during migration, and significantly improving the resolution. Moreover, if the experiment needs to be carried out in an acidic or alkaline buffer solution (such as analyzing the charge heterogeneity of lipoproteins), the chemical stability of N,N-bisacryloylpiperazine (PDA) is better than that of the traditional crosslinking agent (Bis), improving the stability and repeatability of the detection. Description of the Drawings

[0024] Figure 1 It is the electrophoresis result diagram of the detection using the lipoprotein subtype detection kit provided by the present invention in the embodiment.

[0025] Figure 2 It is the electrophoresis diagram of the detection using Sudan black electrophoresis in the control example.

[0026] Figure 3 It is the verification result diagram of the functional sensitivity LOQ of Example 1.

[0027] Figure 4 It is the verification result diagram of the functional sensitivity LOQ of Example 2.

[0028] Figure 5 It is the verification result diagram of the functional sensitivity LOQ of Example 3.

[0029] Figure 6 It is the verification result diagram of the functional sensitivity LOQ of Example 4.

[0030] Figure 7 It is the verification result diagram of the functional sensitivity LOQ of Example 5. Detailed implementation manners

[0031] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] Example 1: A lipoprotein subtype detection kit.

[0033] A lipoprotein subtype detection kit includes a separating gel and a stacking gel. The separating gel is mixed with BODIPY 505 / 515 and N,N - bisacryloylpiperazine, and the stacking gel is mixed with N,N - bisacryloylpiperazine. In practical applications, other fluorescent dyes with similar properties (especially specificity) can be used to replace BODIPY 505 / 515 as the fluorescent probe. The specific components and contents of the separating gel and the stacking gel are as follows.

[0034] Separating gel: acrylamide is 32.5 g / L, N,N - bisacryloylpiperazine is 1.25 g / L, tris buffer is 3.75 g / L, N,N,N',N'-tetramethylethylenediamine is 1 ml / L, ammonium persulfate is 1 g / L, sucrose is 25 g / L, BODIPY 505 / 515 is 0.075 g / L; the pH value of the tris buffer is 7.9;

[0035] Stacking gel: acrylamide is 22.5 g / L, N,N - bisacryloylpiperazine is 0.75 g / L, tris buffer is 6 g / L, N,N,N',N'-tetramethylethylenediamine is 1 ml / L, ammonium persulfate is 1 g / L; the pH value of the tris buffer is 6.8.

[0036] The functional sensitivity LOQ of the kit provided in this example is verified.

[0037] The functional sensitivity LOQ in detection technology specifically refers to the lowest concentration or minimum amount at which a target substance can be stably and reliably detected in an experiment or by an instrument, and it is one of the core indicators for measuring the performance of a detection system. This parameter not only reflects the detection limit of the method but also emphasizes the ability to maintain the consistency of results in repeated experiments. In this test, low-concentration samples of low-density lipoprotein cholesterol (LDL-C) were used for verification. For each LDL-C concentration, 10 repeated experiments were conducted, and the results are shown in Table 1 and Figure 3 as follows:

[0038] Table 1: Data table for LOQ verification in Example 1.

[0039]

[0040] The results showed that the functional sensitivity of the detection (with 10% as the criterion), the limit of quantification LOQ = 0.047 mmol / L.

[0041] Example 2: A lipoprotein subtype detection kit.

[0042] The difference between this example and Example 1 lies in the different contents of the components of the separating gel and the stacking gel, which are specifically as follows:

[0043] Separating gel: Acrylamide is 25 g / L, N,N - bisacryloylpiperazine is 0.7 g / L, tris buffer is 2.5 g / L, tetramethylethylenediamine is 0.5 ml / L, ammonium persulfate is 0.5 g / L, sucrose is 20 g / L, fluoroboron dipyrrole 505 / 515 is 0.05 g / L; the pH value of the tris buffer is 7.9;

[0044] Stacking gel: Acrylamide is 20 g / L, N,N - bisacryloylpiperazine is 0.5 g / L, tris buffer is 4 g / L, tetramethylethylenediamine is 0.5 ml / L, ammonium persulfate is 0.5 g / L; the pH value of the tris buffer is 6.8.

[0045] Similarly, the functional sensitivity LOQ of the kit provided in this example was verified, and the results are shown in Table 2 and Figure 4 as follows:

[0046] Table 2: Data table for LOQ verification in Example 2.

[0047]

[0048] The results showed that the functional sensitivity of the detection (with 10% as the criterion), the limit of quantification LOQ = 0.074 mmol / L.

[0049] Example 3: A lipoprotein subtype detection kit.

[0050] The difference between this example and Example 1 lies in the different contents of the components of the separating gel and the stacking gel, which are specifically as follows:

[0051] Separating gel: acrylamide is 40 g / L, N,N-bisacryloylpiperazine is 1.8 g / L, tris buffer is 5 g / L, tetramethylethylenediamine is 1.5 ml / L, ammonium persulfate is 1.5 g / L, sucrose is 30 g / L, fluoroboron dipyrrole 505 / 515 is 0.1 g / L; the pH value of the tris buffer is 7.9;

[0052] Stacking gel: acrylamide is 25 g / L, N,N-bisacryloylpiperazine is 1 g / L, tris buffer is 8 g / L, tetramethylethylenediamine is 1.5 ml / L, ammonium persulfate is 1.5 g / L; the pH value of the tris buffer is 6.8.

[0053] Similarly, the functional sensitivity LOQ of the kit provided in this example was verified, and the results are shown in Table 3 and Figure 5 as follows:

[0054] Table 3: LOQ verification data table of Example 3.

[0055]

[0056] The results show that the functional sensitivity (based on 10%) of the detection, and the limit of quantification LOQ = 0.063 mmol / L.

[0057] Example 4: A lipoprotein subtype detection kit.

[0058] The difference between this example and Example 1 lies in the different contents of the components of the separating gel and the stacking gel, which are specifically as follows:

[0059] Separating gel: acrylamide is 36.25 g / L, N,N-bisacryloylpiperazine is 1.528 g / L, tris buffer is 4.375 g / L, tetramethylethylenediamine is 1.25 ml / L, ammonium persulfate is 1.25 g / L, sucrose is 27.5 g / L, fluoroboron dipyrrole 505 / 515 is 0.0875 g / L; the pH value of the tris buffer is 7.9;

[0060] Stacking gel: acrylamide is 23.75 g / L, N,N-bisacryloylpiperazine is 0.875 g / L, tris buffer is 7 g / L, tetramethylethylenediamine is 1.25 ml / L, ammonium persulfate is 1.25 g / L; the pH value of the tris buffer is 6.8.

[0061] Similarly, the functional sensitivity LOQ of the kit provided in this example was verified, and the results are shown in Table 4 and Figure 6 as follows:

[0062] Table 4: LOQ verification data table of Example 4.

[0063]

[0064] The results show that the functional sensitivity of the detection (based on 10%) and the limit of quantification LOQ = 0.068 mmol / L.

[0065] Example 5: A lipoprotein subtype detection kit.

[0066] The difference between this example and Example 1 lies in the different contents of the components of the separating gel and the stacking gel, which are specifically as follows:

[0067] Separating gel: acrylamide is 28.75 g / L, N,N-bisacryloylpiperazine is 0.975 g / L, tris buffer is 3.125 g / L, tetramethylethylenediamine is 0.75 ml / L, ammonium persulfate is 0.75 g / L, sucrose is 22.5 g / L, boron dipyrromethene 505 / 515 is 0.0625 g / L; the pH value of the tris buffer is 7.9;

[0068] Stacking gel: acrylamide is 21.25 g / L, N,N-bisacryloylpiperazine is 0.625 g / L, tris buffer is 5 g / L, tetramethylethylenediamine is 0.75 ml / L, ammonium persulfate is 0.75 g / L; the pH value of the tris buffer is 6.8.

[0069] Similarly, the functional sensitivity LOQ of the kit provided in this example was verified, and the results are shown in Table 5 and Figure 7 as follows:

[0070] Table 5: LOQ verification data table of Example 5.

[0071]

[0072] The results show that the functional sensitivity of the detection (based on 8%) and the limit of quantification LOQ = 0.02 mmol / L.

[0073] Example 6: A preparation method of a lipoprotein subtype detection kit.

[0074] A preparation method of a lipoprotein subtype detection kit, which mixes boron dipyrromethene 505 / 515 into the separating gel to form a separating gel with boron dipyrromethene 505 / 515, and adds N,N-bisacryloylpiperazine to the separating gel and the stacking gel to form a separating gel and a stacking gel containing N,N-bisacryloylpiperazine.

[0075] The components and contents of the separation gel are as follows: acrylamide is 25-40 g / L, N,N-bisacryloylpiperazine is 0.7-1.8 g / L, tris buffer is 2.5-5 g / L, tetramethylethylenediamine is 0.5-1.5 ml / L, ammonium persulfate is 0.5-1.5 g / L, sucrose is 20-30 g / L, and fluoroboron dipyrrole 505 / 515 is 0.05-0.1 g / L; the pH value of the tris buffer is 7.9;

[0076] The components and contents of the concentrated gel are: 20-25 g / L acrylamide, 0.5-1 g / L N,N-bisacryloylpiperazine, 4-8 g / L tris buffer, 0.5-1.5 ml / L tetramethylethylenediamine, and 0.5-1.5 g / L ammonium persulfate; the pH value of the tris buffer is 6.8. The detailed preparation steps are as follows:

[0077] Step S1: preparing the first stage separation gel: weighing the corresponding amounts of various components according to the components and contents of the separation gel, which are: 30 g / L acrylamide, 1.5 g / L N,N-bisacryloylpiperazine, 2.5 g / L tris buffer, 0.5 ml / L tetramethylethylenediamine, 0.5 g / L ammonium persulfate, 20 g / L sucrose, and 0.05 g / L fluoroborane dipyrrole 505 / 515; the pH value of the tris buffer is 7.9, mixing the components and water to prepare a mixed solution 1, taking the mixed solution 1 and pouring it into a glass tube, the filling volume is 1-1.5 ml, standing at 32° C. for 40 min, and after the mixed solution 1 is condensed, a gel column with a polyacrylamide content of 4.5-6.5 wt% is obtained, which is the first stage separation gel; the first stage separation gel is a high-concentration separation gel, which can separate bilirubin in serum and remove the interference of bilirubin;

[0078] Step S2: preparing the second stage separation gel: weighing the corresponding amounts of various components according to the components and contents of the separation gel, which are: 25 g / L acrylamide, 0.7 g / L N,N-bisacryloylpiperazine, 2.5 g / L tris buffer, 0.5 ml / L tetramethylethylenediamine, 0.5 g / L ammonium persulfate, 20 g / L sucrose, and 0.05 g / L fluoroborane dipyrrole 505 / 515; the pH value of the tris buffer is 7.9, mixing the components and water to prepare a mixed solution 1-1, taking the mixed solution 1-1 and filling it onto the first stage separation gel, the filling volume is 1-1. After the filling is completed, water is added to the liquid surface to isolate the air and make the gel surface flat, and the gel is allowed to stand for a while. After the gel solution is coagulated, a gel column with a polyacrylamide content of 3.0-4.5 wt% is obtained, the surface water is poured out and the water on the surface of the gel column is dried, which is the second stage separation gel;

[0079] Step S3: Preparation of stacking gel: Weigh corresponding amounts of various components according to the composition and content of the stacking gel, which are: acrylamide is 20 g / L, N,N - bisacryloylpiperazine is 0.5 g / L, tris buffer is 4 g / L, tetramethylethylenediamine is 0.5 ml / L, ammonium persulfate is 0.5 g / L; the pH value of the tris buffer is 6.8. Mix the components to form a mixed solution 2. Pour the mixed solution 2 on top of the second - stage separating gel. After pouring, add water on the liquid surface to isolate air and make the gel surface flat. Let it stand. After the gel solution solidifies, a gel column with a polyacrylamide content of 2.5 - 3.5 wt% is obtained, which is the stacking gel.

[0080] Step S4: Preparation of the kit: Immerse the structures of the first - stage separating gel, the second - stage separating gel, and the stacking gel in the preservation solution for storage, then the lipoprotein subtype detection kit is obtained. The application of the lipoprotein subtype detection kit in the detection of lipoprotein subtypes, and the detection includes separately or simultaneously detecting one or more of very - low - density lipoprotein VLDL, intermediate - density lipoprotein IDL - A, IDL - B, IDL - C (three subtypes), low - density lipoprotein LDL - 1, LDL - 2, LDL - 3, LDL - 4, LDL - 5, LDL - 6, and LDL - 7 (seven subtypes), and high - density lipoprotein cholesterol.

[0081] Example 7: Precision verification of the lipoprotein subtype detection kit in Examples 1 - 5.

[0082] Precision refers to the degree of consistency or dispersion between the measurement results when measuring the same target multiple times under the same conditions, reflecting the repeatability and stability of the experiment or instrument.

[0083] In this verification experiment, the repeatability of detecting the LDL - C concentration of two levels of quality control products (high and low) was used as the verification:

[0084] Table 6: Detection result table of low - level quality control product.

[0085]

[0086] Table 7: Detection result table of high - level quality control product.

[0087]

[0088] The results show that in Examples 1 - 5, the coefficient of variation of detecting two levels of quality control products is < 3%.

[0089] Example 8: Control experiment on the detection application of the kit provided in Example 1 to blood samples.

[0090] Use the detection kit provided in Example 1 to detect blood samples. The specific steps are as follows:

[0091] Step S1: Dissolve 12.375 g of tris(hydroxymethyl)aminomethane and 5.5 g of boric acid in distilled water, make up the volume to 1000 mL. After mixing evenly, obtain the electrophoresis buffer solution for standby.

[0092] Step S2: Fix the gel column prepared in Example 1 in the electrophoresis tank, where the first-stage separating gel is located at the positive electrode end of the electrophoresis tank, and the second-stage separating gel and the stacking gel are located at the negative electrode end of the electrophoresis tank. Add the electrophoresis buffer solution to the electrophoresis tank, add the serum sample into the gel column. After the solution in the system is stable, turn on the power supply and perform electrophoresis for 70 - 75 min.

[0093] Step S3: After electrophoresis, remove the gel column, and scan and analyze the gel column within 10 - 15 min. The results are as shown in the appendix Figure 1 The sample separation liquid in the gel column from top to bottom is very low density lipoprotein, intermediate density lipoprotein, low density lipoprotein 1 - 7 subtypes, and high density lipoprotein in sequence, and the concentration percentage of each lipoprotein subtype is obtained.

[0094] Control example: Use the conventional Sudan black staining gel electrophoresis as a control experiment. The specific steps are as follows:

[0095] Step S1: Dissolve 0.003 g of Sudan black B in 0.334 mL of dimethyl sulfoxide, stir for 5 min. While stirring, add 0.333 mL of ethylene glycol, stir for 3 min. Finally, add 0.333 mL of absolute ethanol. After mixing evenly, obtain the staining solution. Take 50 μL of serum sample, mix it thoroughly with 10 μL of the staining solution, and then let it stand in the dark at room temperature for 25 min to obtain the stained serum.

[0096] Step S2: Dissolve 12.375 g of tris(hydroxymethyl)aminomethane and 5.5 g of boric acid in distilled water, make up the volume to 1000 mL. After mixing evenly, obtain the electrophoresis buffer solution. Fix the gel column in Example 1 in the electrophoresis tank, where the first-stage separating gel is located at the positive electrode end of the electrophoresis tank, and the second-stage separating gel and the stacking gel are located at the negative electrode end of the electrophoresis tank. Add the electrophoresis buffer solution to the electrophoresis tank, add the stained serum into the gel column. After the solution in the system is stable, turn on the power supply and perform electrophoresis for 70 min.

[0097] Step S3: After electrophoresis, remove the gel column, and scan and analyze the gel column within 10 min. The results are as Figure 2 The sample separation liquid in the gel column from top to bottom is very low density lipoprotein, intermediate density lipoprotein, low density lipoprotein 1 - 7 subtypes, and high density lipoprotein in sequence, and the concentration percentage of each lipoprotein subtype is obtained.

[0098] Lipoproteins in the blood are spherical particles responsible for the transport of cholesterol, triglycerides, and phospholipids. Lipoproteins are composed of multiple subtypes, and these subcomponents differ in particle size, density, and chemical composition. Various analytical methods have demonstrated the heterogeneity of lipoproteins. This kit separates each lipoprotein subtype by polyacrylamide gel electrophoresis. Since the particle sizes and charges of each subtype are different, their mobilities in polyacrylamide gel electrophoresis are also different. The lipoprotein subtypes are separated by the molecular sieve effect of polyacrylamide gel. After protein separation, the optical density map of the electrophoresis band is obtained through a gel imaging scanning system, and then the optical density map is converted into a peak pattern through a software analysis system, and at the same time, the area percentage of each subcomponent is obtained, which is the concentration percentage of each lipoprotein subtype.

[0099] The above-described embodiments merely represent several implementation manners of the present invention, and the description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent shall be subject to the appended claims.

Claims

1. A lipoprotein subtype detection kit, comprising a separating gel and a stacking gel, characterized in that, The components and contents of the separating gel are as follows: acrylamide is 25 - 40 g / L, N,N - bisacryloylpiperazine is 0.7 - 1.8 g / L, tris buffer is 2.5 - 5 g / L, tetramethylethylenediamine is 0.5 - 1.5 ml / L, ammonium persulfate is 0.5 - 1.5 g / L, sucrose is 20 - 30 g / L, fluoroboron dipyrrole 505 / 515 is 0.05 - 0.1 g / L; the pH value of the tris buffer is 7.9; The components and contents of the stacking gel are as follows: acrylamide is 20 - 25 g / L, N,N - bisacryloylpiperazine is 0.5 - 1 g / L, tris buffer is 4 - 8 g / L, tetramethylethylenediamine is 0.5 - 1.5 ml / L, ammonium persulfate is 0.5 - 1.5 g / L; the pH value of the tris buffer is 6.

8.

2. A method for preparing a lipoprotein subtype detection kit as described in claim 1, characterized in that, It mixes fluoroboron dipyrrole 505 / 515 and N,N - bisacryloylpiperazine into the separating gel to make a separating gel with fluoroboron dipyrrole 505 / 515 and N,N - bisacryloylpiperazine; adds N,N - bisacryloylpiperazine to the stacking gel to make a stacking gel containing N,N - bisacryloylpiperazine.

3. The preparation method according to claim 2, wherein First, weigh the corresponding amounts of various components according to the components and contents of the separating gel, mix the components to make a mixed solution 1, take the mixed solution 1 and pour it into a glass tube, the filling volume is 1 - 1.5 ml, let it stand, and after the mixed solution 1 solidifies, obtain the first - stage separating gel; Then, weigh the corresponding amounts of various components according to the components and contents of the stacking gel, mix the components to make a mixed solution 2, take the mixed solution 2 and pour it into a glass tube, the filling volume is 150 ul - 200 ul, let it stand, and after the mixed solution 2 solidifies, obtain the stacking gel; Finally, place the prepared first - stage separating gel and stacking gel in the preservation solution for preservation, and thus obtain the lipoprotein subtype detection kit described above.

4. The preparation method according to claim 3, wherein, It also includes preparing the second - stage separating gel. First, weigh the corresponding amounts of various components according to the components and contents of the separating gel, and the amounts of acrylamide and N,N - bisacryloylpiperazine used are less than those of the first - stage separating gel. Mix the components to make a mixed solution 1 - 1, take the mixed solution 1 - 1 and pour it into a glass tube, the filling volume is 1 - 1.5 ml, let it stand, and after the mixed solution 1 - 1 solidifies, obtain the second - stage separating gel.

5. Use of a lipoprotein subtype detection kit according to any one of claims 1 - 4 in the detection of lipoprotein subtypes.

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