A high-density lipoprotein subtyping detection method, a detection kit and application thereof

Abstract

This invention discloses a method, reagent kit, preparation method, and application for detecting high-density lipoprotein (HDL) subtypes. The method uses polyacrylamide gel electrophoresis to separate HDL in serum or plasma. After staining the HDL in serum or plasma with a staining solution, the mixture is added to a gel column. Under an electric field, the HDL particles are separated sequentially according to their size through the polyacrylamide gel molecular sieve effect. The results of the protein electrophoresis separation are used to obtain an optical density spectrum of the HDL electrophoretic bands using a scanning system of the electrophoresis apparatus. The optical density spectrum is converted into a peak pattern, and area ratio analysis is performed. The accurate content of each component of the HDL subtype is obtained based on the total HDL content and the ratio of the peak areas. This invention classifies, subtypes, and quantifies HDL based on particle size, improving detection sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and relates to a method for detecting high-density lipoprotein subtypes, as well as a high-density lipoprotein subtype detection kit, its preparation method, and its application. Background Technology

[0002] Currently, most reagent kits for lipid metabolism testing use single-item testing or simultaneous testing of four lipid parameters. However, the content of high-density lipoprotein (HDL) is mainly determined by enzymatic methods or clearance methods, resulting in an overall HDL test result rather than a precise result of HDL subtypes for classification. For subtype detection, methods such as nuclear magnetic resonance (NMR), non-denaturing gradient gel electrophoresis, and density gradient centrifugation are mainly used. These methods are time-consuming and labor-intensive, and the experimental process is difficult to control, requiring highly skilled professionals to operate large instruments.

[0003] In addition, some methods use gel electrophoresis to detect high-density lipoprotein subtypes. However, since Sudan Black B, which is commonly used, is a diazo dye, and the main components of each subtype of high-density lipoprotein are different, the binding of Sudan Black B to each subtype of high-density lipoprotein is uneven, which leads to inconsistent staining efficiency and differences in the final results.

[0004] Therefore, there is a lack of improved dyes in the current technology that can efficiently stain high-density lipoprotein particles to enable simple and intuitive quantitative detection of high-density lipoprotein subtypes using polyacrylamide electrophoresis. Summary of the Invention

[0005] The purpose of this invention is to provide a simple and intuitive method for detecting high-density lipoprotein (HDL) subtypes based on particle size, which mainly utilizes gel electrophoresis, thereby improving the sensitivity of HDL subtype detection.

[0006] Another object of the present invention is to provide an improved Sudan Black B dye and a corresponding high-density lipoprotein subtype detection kit.

[0007] Another object of the present invention is to provide the application of the above-mentioned detection method and dye.

[0008] To achieve the above objectives, one aspect of the present invention provides a method for detecting high-density lipoprotein subtypes, comprising the following steps:

[0009] S01: High-density lipoproteins in serum or plasma are separated using polyacrylamide gel electrophoresis.

[0010] S02: After staining the high-density lipoprotein in the serum or plasma with a staining solution, the mixture is added to a gel column; the gel column is divided into upper and lower layers, wherein the concentration of the upper gel column is 2.0-2.2 wt% polyacrylamide and the concentration of the lower gel column is 6.0-7.5 wt% polyacrylamide.

[0011] S03: Under the influence of the electric field in the electrophoresis tank, and through the polyacrylamide gel molecular sieve effect, high-density lipoprotein subtypes are separated in sequence according to the size of high-density lipoprotein particles.

[0012] S04: The results of the high-density lipoprotein subtype electrophoresis separation are used to obtain the optical density spectrum of the high-density lipoprotein electrophoretic bands by the scanning system of the electrophoresis device;

[0013] S05: Convert the optical density spectrum into a peak pattern using analysis software, and analyze the area ratio of each peak in the peak pattern;

[0014] S06: The accurate content of each component of the high-density lipoprotein subtype is obtained based on the detected total cholesterol content and the ratio of the peak area.

[0015] Furthermore, the volume of the upper gel column used is 200-400 microliters, and the volume of the lower gel column used is 1.0-1.3 milliliters.

[0016] Furthermore, the aforementioned The concentration of the base used is 0.2-2% (w / v), the concentration of the sucrose used is 0.5-2% (w / v), the concentration of the TEMED used is 0.3-2% (v / v), the pH value of the gel solution is 4.0-11.0, the concentration of Proclin300 is 0.05‰-5‰, the concentration of the salt ions is 0.01-0.2% (w / v), and the concentration of BSA is 0.005%-0.5%.

[0017] Furthermore, the volume of sample added during loading is determined according to the situation. For example, 25-35 ml, preferably 28-33 ml. In a preferred embodiment of the present invention, after staining high-density lipoprotein in serum or plasma with staining solution, 30 μL of the mixture is added to the gel column.

[0018] In one aspect, the present invention provides a high-density lipoprotein subtype detection kit.

[0019] Furthermore, the kit comprises a test kit box, a pearl cotton liner, a gel column bottle, a staining solution, a buffer powder, and quality control sample I and quality control sample II. The gel column bottle, the staining solution, the buffer powder, and quality control sample I and quality control sample II are sequentially placed inside the pearl cotton liner. The gel column bottle contains a gel column, and the gel column is composed of a gel solution.

[0020] Furthermore, the gel columns are packaged into wide-mouth plastic bottles in groups of 60, filled with storage buffer, and stored at 2-8°C.

[0021] Furthermore, the gel column is divided into upper and lower layers. The upper gel column has a concentration of 2.01-2.19 wt% polyacrylamide, including acrylamide and N,N-methylenebisacrylamide, while the lower gel column has a concentration of 6.04-7.49 wt% polyacrylamide, also including acrylamide and N,N-methylenebisacrylamide. The main components of the prepared gel solution are acrylamide, N,N-methylenebisacrylamide, and... base, TEMED, ammonium persulfate and sucrose, Proclin 300, MgCl2.

[0022] Furthermore, the volume of the upper gel column used is 200-400 μL, and the volume of the lower gel column used is 1.0-1.3 mL. The concentration of the base used is 0.2-2% (w / v), the concentration of the sucrose used is 0.5-2% (w / v), the concentration of the TEMED used is 0.3-2% (v / v), the concentration of the Proclin 300 used is 0.05‰-5‰, the pH value of the gel solution is 4.0-11.0, and the salt ion concentration is 0.01-0.15%.

[0023] In one aspect, the present invention provides a dye, Sudan Black B2, for the detection of high-density lipoprotein subtypes, wherein a hydroxymethyl group is added at the R2 position of the structure of Sudan Black B.

[0024] In another aspect, the present invention provides a staining solution for the above-described high-density lipoprotein subtype detection method or the above-described high-density lipoprotein subtype detection kit, comprising the above-described dye Sudan Black B2.

[0025] In another aspect, the present invention provides a method for preparing the above-mentioned staining solution, comprising the following steps:

[0026] The concentration of Sudan Black B derivative Sudan Black B2 is 0.1-1.5% (W / V). Sudan Black B derivative Sudan Black B2 is added to one of acetone, propylene glycol, isopropanol, and ethylene glycol. Based on the total volume of the added acetone, propylene glycol, isopropanol, or ethylene glycol, the volume percentage of said acetone, propylene glycol, isopropanol, or ethylene glycol is greater than or equal to 96.5% and less than 98.0%; preferably, the volume percentage of said acetone, propylene glycol, isopropanol, or ethylene glycol is greater than or equal to 96.8% and less than 97.5%.

[0027] Add 0.5-1.5% by volume of DMF or 1-methyl-2-pyrrolidone;

[0028] Then add surfactants such as Triton X-405 or Triton X-100, Tween, etc., at a volume ratio of 0.05-0.5% (V / V);

[0029] After thoroughly mixing the above solutions, store them away from light to obtain the staining solution.

[0030] In one aspect, the present invention provides a high-density lipoprotein subtype detection kit, wherein the preparation method of the staining solution includes the following steps: taking Sudan Black B2 dye with a concentration of 0.1-1.5% (W / V), adding Sudan Black B2 to one of a combination of acetone, propylene glycol, isopropanol and ethylene glycol, wherein the volume percentage of acetone, propylene glycol, isopropanol or ethylene glycol contained therein is greater than or equal to 96.5% and less than or equal to 99.3% based on the total volume of the added acetone, propylene glycol or isopropanol or ethylene glycol;

[0031] Add 0.5-1.5% by volume of DMF or 1-methyl-2-pyrrolidone;

[0032] Then, one of Triton X-405, Triton X-100, and Tween is added as a surfactant, and the volume ratio of the surfactant used is 0.05-0.5% (V / V).

[0033] After thoroughly mixing the above solutions, store them away from light to obtain the staining solution.

[0034] In another aspect, the present invention provides a method for preparing the above-mentioned high-density lipoprotein subtype detection kit, comprising the following steps:

[0035] Step (1), prepare the solution by mixing acrylamide, NN methylenebisacrylamide, Base, TEMED, ammonium persulfate, sucrose, Proclin 300, and MgCl2BSA are added to purified water or deionized water according to the actual usage concentration. After thorough mixing, the pH value is adjusted to complete the preparation of the gel solution. Take 1.0-1.3 ml of the above gel solution and add it to a glass column. Add water or ethanol to press the gel. After pressing, let it stand for no less than 30 minutes. After gel formation, a gel column with a polyacrylamide content of 6.01-7.49 wt% is obtained, which is the lower gel column. Step (2): Prepare the solution by adding acrylamide, NN methylenebisacrylamide, Add base, TEMED, ammonium persulfate, sucrose, Proclin 300, and MgCl2 to purified or deionized water according to the actual usage concentration. Stir thoroughly and adjust the pH value to complete the preparation of the gel solution. Remove the adhesive solution from the upper part of the glass tube in step (1). Take 200-400 μL of the above gel solution and add it to the upper part of the lower gel column inside the glass column. Add water or ethanol for pressing. After pressing, let it stand for no less than 30 minutes. After gel formation, a gel column with a polyacrylamide content of 2.01-2.19 wt% is obtained, which is the upper gel column.

[0036] Step (3): Place the gel column obtained in steps (1) and (2) into a wide-mouth plastic bottle and fill it with storage buffer.

[0037] Step (4): Prepare the solution by adding Sudan Black B derivative Sudan Black B2 to a mixture of acetone, propylene glycol, isopropanol, and ethylene glycol at a concentration of 0.1-1.5% (W / V). The volume percentage of acetone, propylene glycol, isopropanol, or ethylene glycol in the mixture shall be greater than or equal to 96.5% and less than or equal to 99.3% based on the total volume of the selected acetone, propylene glycol, isopropanol, or ethylene glycol. Add DMF or 1-methyl-2-pyrrolidone at a volume ratio of 0.5-1.5%. Add Triton X-405 or Triton X-100 or Tween as a surfactant at a volume ratio of 0.05-0.5% (V / V). After thoroughly mixing the above mixture, dispense it into containers and store it in the dark.

[0038] Step (5), preparation of buffer powder, wherein, The dosage of base is 5g / L-15g / L, and the dosage of boric acid is 3g / L-10g / L. The two materials are mixed evenly using a mixer, and then dispensed into buffer powder glass bottles at a dosage of 16.5g / bottle.

[0039] Step (6), quality control preparation: After the received sample passes the safety test, it is inactivated at 56°C for 30 minutes, mixed, then tested and assigned values, and dispensed as quality control, with each sample dispensed into 100 microliters.

[0040] Step (7): Label the components from steps (3), (4), (5), and (6); inspect the resulting semi-finished product.

[0041] Step (8): Pack the semi-finished product that has passed the inspection in step (7) into a box to obtain the finished high-density lipoprotein subtype detection kit.

[0042] Preferably, the preparation method further includes the following steps:

[0043] Step (9): Perform final product testing on the high-density lipoprotein subtype detection kit obtained in step (8);

[0044] Step (10): After the finished products that have passed the inspection in step (9) are affixed with a qualified label, they are put into storage.

[0045] According to another aspect of the present invention, the application of the above-described detection method, the above-described detection kit, the above-described staining solution, or the detection kit prepared by the above-described preparation method in the quantitative detection of high-density lipoprotein subtypes is also provided.

[0046] Compared with the prior art, the beneficial effects of the present invention are:

[0047] 1. The technical solution of the present invention can provide a new improved Sudan Black B derivative Sudan Black B2 dye. By modifying Sudan Black B and optimizing the upper and lower gel layers, the lipophilicity of the dye is greatly improved, especially for high-density lipoprotein, making the binding conditions between the dye and high-density lipoprotein simpler and the binding time shorter, which can be achieved in 15 minutes at room temperature.

[0048] 2. The detection results of high-density lipoprotein (HDL) obtained through the technical solution of this invention are closer to those obtained through other reagents.

[0049] 3. Through the technical solution of the present invention, the resolution reduction caused by the heterogeneity of high-density lipoprotein components is effectively reduced, and each high-density lipoprotein component can be accurately distinguished.

[0050] 4. The technical solution of the present invention enables quantitative detection of high-density lipoprotein subtypes and analysis of high-density lipoprotein components, which can be used for the assessment of cardiovascular and cerebrovascular disease risk. Attached Figure Description

[0051] Figure 1 This is a schematic diagram of the high-density detection results in this invention.

[0052] Figure 2 Examples of high-density electrophoresis scans from Examples 2-4.

[0053] Figure 3 This is an example of a high-density electrophoresis scan.

[0054] Figure 4 The result of adding 80 μL of 3.5% adhesive solution to the lower layer of sample #1.

[0055] Figure 5 The result of adding 100 μL of 3.5% adhesive solution to the lower layer of sample #1.

[0056] Figure 6 The result of adding 120 μL of 3.5% adhesive solution to the lower layer of sample #1.

[0057] Figure 7 The result of adding 80 μL of 3.5% adhesive solution to the lower layer of sample #2.

[0058] Figure 8 The result of adding 100 μL of 3.5% adhesive solution to the lower layer of sample #2.

[0059] Figure 9 The result of adding 120 μL of 3.5% adhesive solution to the lower layer of sample #2.

[0060] Figure 10 The results show the test results for three samples of Sudan Black B2 and Sudan Black B. Lanes 1, 3, and 5 are Sudan Black B2, and lanes 2, 4, and 6 are Sudan Black B. Lanes 1 and 2 are different dyes from the same sample, lanes 3 and 4 are different dyes from the same sample, and lanes 5 and 6 are different dyes from the same sample.

[0061] Figure 11 The images show the structural formulas of Sudan Black B before and after modification. From left to right, they represent Sudan Black B, Sudan Black B1, Sudan Black B2, and Sudan Black B3. Sudan Black B2, a derivative of Sudan Black B, is the dye used in this invention.

[0062] In the attached diagram, 1 represents large high-density lipoprotein particles, 2 represents medium-sized high-density lipoprotein particles, and 3 represents small high-density lipoprotein particles. 11 represents very low-density lipoprotein and low-density lipoprotein, 12 represents high-density lipoprotein, and 13 represents albumin. 21 represents Sudan Black B derivative B2 reacted with sample #1 at room temperature for 15 min; 22 represents Sudan Black B reacted with sample #1 at 37°C for 30 min; 23 represents Sudan Black B derivative B2 reacted with sample #2 at room temperature for 15 min; 24 represents Sudan Black B reacted with sample #2 at 37°C for 30 min; 25 represents Sudan Black B derivative B2 reacted with sample #3 at room temperature for 15 min; and 26 represents Sudan Black B reacted with sample #3 at 37°C for 30 min. Detailed Implementation

[0063] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0064] According to a preferred embodiment of the present invention, a method for detecting high-density lipoprotein subtypes is provided, comprising the following steps:

[0065] S01: Polyacrylamide gel electrophoresis is used to classify high-density lipoproteins in serum or plasma;

[0066] S02: After staining the high-density lipoprotein in the serum or plasma with staining solution, take 30 μL of the mixture and add it onto the gel column;

[0067] S03: Under the influence of the electric field in the electrophoresis tank, and through the effect of polyacrylamide gel molecular sieve, high-density lipoprotein subtypes are separated in sequence according to the size of high-density lipoprotein particles.

[0068] S04: The results of the high-density lipoprotein subtype electrophoresis separation are used to obtain the optical density spectrum of the high-density lipoprotein electrophoretic bands by the scanning system of the electrophoresis device;

[0069] S05: Convert the optical density spectrum into a peak pattern using analysis software, and analyze the area ratio of each peak in the peak pattern;

[0070] S06: The accurate content of each component of the high-density lipoprotein subtype is obtained based on the detected total cholesterol content and the ratio of the peak area.

[0071] According to a preferred embodiment of the present invention, a high-density lipoprotein subtype detection kit is provided, which applies the above-described high-density lipoprotein subtype detection method.

[0072] Preferably, the kit comprises a test kit body, a pearl cotton liner, a gel column bottle, a staining solution, a buffer powder, quality control product I, and quality control product II, wherein the gel column bottle, the staining solution, the buffer powder, and quality control product I and quality control product II are sequentially placed inside the pearl cotton liner, and the gel column bottle contains a gel column.

[0073] Preferably, the gel columns are packaged into wide-mouth plastic bottles in groups of 60 doses each, and the bottles are filled with storage buffer solution and stored at 2-8°C.

[0074] Preferably, the gel column consists of two layers, an upper gel column with a concentration of 2.01-2.19 wt% polyacrylamide, including acrylamide and N,N-methylenebisacrylamide, and a lower gel column with a concentration of 6.01-7.49 wt% polyacrylamide, including acrylamide and N,N-methylenebisacrylamide. The main components of the prepared gel solution are acrylamide, N,N-methylenebisacrylamide, and... base, TEMED, ammonium persulfate and sucrose, Proclin 300, MgCl2, BSA (bovine serum albumin).

[0075] Preferably, the volume of the upper gel column used is 200-400 μL, and the volume of the lower gel column used is 1.0-1.3 mL. The base concentration is 0.2-2% (W / V), the sucrose concentration is 0.5-2% (W / V), the TEMED concentration is 0.3-2% (V / V), the pH of the gel solution is 6.8±0.2, and the Proclin300 concentration is 0.3‰.

[0076] According to a preferred embodiment of the present invention, a dye, Sudan Black B2, for high-density lipoprotein subtyping detection is provided, wherein a hydroxymethyl group is added at the R2 position of the Sudan Black B structure.

[0077] The structural formulas of Sudan Black B before and after modification are shown in the figure. Figure 1Derivatives of Sudan Black B are formed by adding specific chemical groups to the structure of Sudan Black B. From left to right, these are the chemical structures of Sudan Black B, Sudan Black B1, Sudan Black B2, and Sudan Black B3. Adding a hydroxyethyl group to the R1 position of Sudan Black B results in Sudan Black B1; adding a hydroxymethyl group to the R2 position results in Sudan Black B2; and adding a hydroxyl group to the R2 position results in Sudan Black B3. Preferably, adding a hydroxymethyl group to the R2 position of Sudan Black B results in Sudan Black B2. Test results of this invention show that although Sudan Black B, Sudan Black B1, Sudan Black B2, and Sudan Black B3 can all stain high-density lipoprotein (HDL), Sudan Black B2 exhibits significantly better staining performance, especially in terms of band resolution and quantification, compared to Sudan Black B, Sudan Black B1, and Sudan Black B3.

[0078] According to a preferred embodiment of the present invention, a staining solution for the above-described high-density lipoprotein (HDL) subtype detection method or the above-described HDL subtype detection kit is provided, comprising the dye Sudan Black B2. Sudan Black B2, a derivative of Sudan Black B, serves as a staining solution in place of Sudan Black, resulting in clearer staining of HDL subtypes and making HDL subtype separation easier and more accurate.

[0079] According to a preferred embodiment of the present invention, a method for preparing the above-mentioned staining solution is provided, comprising the following steps:

[0080] The concentration of Sudan Black B derivative Sudan Black B2 is 0.1-1.5% (W / V). Sudan Black B derivative Sudan Black B2 is added to one of the following: acetone, propylene glycol, isopropanol and ethylene glycol. The volume percentage of the added acetone, propylene glycol, isopropanol or ethylene glycol is greater than or equal to 96.5% and less than or equal to 99.3% based on the total volume of the added acetone, propylene glycol, isopropanol or ethylene glycol.

[0081] Add 0.5-1.5% by volume of DMF or 1-methyl-2-pyrrolidone;

[0082] Then add Triton X-405 or Triton X-100, or Tween as a surfactant, at a volume ratio of 0.05-0.5% (V / V);

[0083] After thoroughly mixing the above solutions, store them away from light to obtain the staining solution.

[0084] According to a preferred embodiment of the present invention, a high-density lipoprotein subtype detection kit is provided, which contains the staining solution.

[0085] According to a preferred embodiment of the present invention, a method for preparing the above-mentioned high-density lipoprotein subtype detection kit is provided, comprising the following steps:

[0086] Step (1), prepare the solution by mixing acrylamide, NN methylenebisacrylamide, Add base, TEMED, ammonium persulfate, sucrose, Proclin 300, MgCl2, and bovine serum albumin to purified or deionized water according to the actual concentration to be used. Stir thoroughly and adjust the pH value to complete the gel preparation. Take 1.0-1.3 ml into a glass column, add water or ethanol, and press the gel for at least 30 minutes. After gel formation, a gel column with a polyacrylamide content of 6.01-7.49 wt% is obtained, which is the lower gel column.

[0087] Step (2), prepare the solution by mixing acrylamide, NN methylenebisacrylamide, Add base, TEMED, ammonium persulfate, sucrose, Proclin 300, and MgCl2 to purified or deionized water according to the actual usage concentration. After thorough mixing, adjust the pH value to complete the preparation of the gel solution, and remove the gelling water from step (1). Take 200-400 μL of the prepared gel solution and add it to the upper glass column of the lower gel. Add water or ethanol and press the gel for at least 30 minutes. After gel formation, a gel column with a polyacrylamide content of 2.01-2.19 wt% is obtained, which is the upper gel column.

[0088] Step (3): Place the gel column obtained in steps (1) and (2) into a wide-mouth plastic bottle and add storage buffer.

[0089] Step (4): Prepare the solution by adding Sudan Black B2, a Sudan Black B derivative, to a concentration of 0.1-1.5% (W / V). Add Sudan Black B2 to one of acetone, propylene glycol, isopropanol, or ethylene glycol, ensuring that the volume percentage of acetone, propylene glycol, isopropanol, or ethylene glycol is greater than or equal to 96.5% and less than or equal to 99.3% based on the total volume of acetone, propylene glycol, isopropanol, or ethylene glycol. Add DMF or 1-methyl-2-pyrrolidone at a volume ratio of 0.5-1.5%. Then add surfactants such as Triton X-405 or Triton X-100, Tween, etc., at a volume ratio of 0.05-0.5% (V / V). After thoroughly mixing the above solution, dispense it and store it in the dark.

[0090] Step (5), mixing of buffer powder, wherein, The dosage of base is 5g / L-15g / L, and the dosage of boric acid is 3g / L-10g / L. The two materials are mixed evenly using a mixer, and the mixture is dispensed into glass bottles at a dosage of 16.5g / bottle.

[0091] Step (6), quality control preparation: After the received sample passes the safety test, it is inactivated at 56°C for 30 minutes, mixed, and then tested and assigned values. It is then dispensed as quality control, with each sample dispensed in 100 microliters.

[0092] Step (7): Label the components from steps (3), (4), (5), and (6); inspect the resulting semi-finished product.

[0093] Step (8): Pack the semi-finished product that has passed the inspection in step (7) into a box to obtain the finished high-density lipoprotein subtype detection kit.

[0094] Preferably, the preparation method further includes the following steps:

[0095] Step (9): Perform final product testing on the high-density lipoprotein subtype detection kit obtained in step (8);

[0096] Step (10): After the finished products that have passed the inspection in step (9) are affixed with a qualified label, they are put into storage.

[0097] According to a preferred embodiment of the present invention, the application of the above-described detection method, the above-described detection kit, the above-described staining solution, or the detection kit prepared by the above-described preparation method in the quantitative detection of high-density lipoprotein subtypes is also provided.

[0098] Example

[0099] Example 1: A high-density lipoprotein typing detection kit

[0100] A high-density lipoprotein (HDL) genotyping kit comprises a kit body, a pearl cotton liner, a gel column vial, staining solution, buffer powder, and quality control I and quality control II. The gel column vial, staining solution, buffer powder, and quality control I and quality control II are sequentially placed inside the pearl cotton liner. Product composition and specifications:

[0101] The ingredients and specifications are as follows:

[0102] 1. Gel columns, 60 columns / bottle × 1 bottle

[0103] 2. Staining solution, 1.2 mL / tube × 1 tube

[0104] 3. Buffer powder, 16.5g / bottle × 2 bottles

[0105] 4. Instruction manual, 1 copy

[0106] 5. Quality control product I, 0.1ml / vial × 1 vial

[0107] 6. Quality control product II, 0.1ml / vial × 1 vial

[0108] This kit is packaged according to the specified positions of each component.

[0109] Example 2: A staining solution

[0110] Sudan Black B2 was added to isopropanol at a weight-to-volume ratio of 0.165% (W / V), resulting in an isopropanol volume percentage of 98.8%. 1-Methyl-2-pyrrolidone was added at a volume ratio of 1%. Triton X-405 was then added at a volume of 0.2% (V / V). The mixture was thoroughly mixed and dispensed into 1.2 mL vials, then stored protected from light.

[0111] Example 3: A staining solution

[0112] The difference from Example 2 is that Sudan Black B2 was added at a weight-to-volume ratio of 0.8% (W / V) to isopropanol, resulting in an isopropanol volume percentage of 98.8%; 1-methyl-2-pyrrolidone was added at a volume ratio of 1%; and Triton X-405 was added at a volume of 0.2% (V / V). After thoroughly mixing the above mixture, it was dispensed into 1.2 mL tubes and stored away from light.

[0113] Example 4: A staining solution

[0114] The difference from Examples 2 and 3 is that Sudan Black B2 was added to isopropanol at a weight-to-volume ratio of 1.5% (w / v). The volume percentage of isopropanol contained was 98.8% of the total isopropanol volume; 1-methyl-2-pyrrolidone was added at a volume ratio of 1%; and Triton X-405 was added at a volume of 0.2% (v / v). After thoroughly mixing the above solution, it was dispensed into 1.2 mL tubes and stored protected from light.

[0115] like Figure 2 As shown in Examples 2, 3, and 4, different concentrations of Sudan Black B2 can stain high-density lipoprotein (HDL) in serum / plasma, with slightly different staining intensities. Clearly, for HDL staining, more Sudan Black B2 is not necessarily better. At a concentration of 0.8% (w / v), the staining intensity is greater than 0.165% (w / v). However, when the Sudan Black B2 concentration is further increased to 1.6% (w / v), the staining intensity decreases instead of increasing.

[0116] Example 5: Buffer Powder

[0117] The buffer powder mentioned in Example 1 was prepared using standard TBE electrophoresis buffer, but with the EDTA component removed and the concentrations of TRIS and boric acid fine-tuned. After weighing an appropriate amount, it was aliquoted into suitable volumetric flasks, each weighing 16.5g. When using the kit, each 16.5g of powder was added to 1.2L of deionized water or purified water (mineral water containing ions is not suitable).

[0118] Weigh 1.1 kg of Tris and 0.55 kg of boric acid, mix the two materials thoroughly with a mixer, and then dispense them into 16.5 g / bottles for later use.

[0119] Example 6: Quality Control Sample I and Quality Control Sample II

[0120] The quality control materials I and II mentioned in Example 1 are as follows: Samples collected from clinical settings that meet the ideal concentration for high-density lipoprotein cholesterol (HDL-C) testing are used as the raw material for preparing quality control material I; abnormal HDL-C samples collected from clinical settings are used as the raw material for preparing quality control material II. The above samples are first mixed separately; then the mixed samples are inactivated at 56°C for 30 minutes; then the samples are centrifuged at 10,000 rpm for 15 minutes and filtered through a 0.45 μm filter membrane; then diluent is added for later use. The diluted mixed samples are tested three times, and the results remain consistent. Simultaneously, three different batches of the kit from Example 1 are used, each batch tested 20 times, and the mean ± 2 standard deviation of each subcomponent is calculated as the detection range of the quality control material. Store at -20°C for later use.

[0121] Example 7: Gel column A

[0122] The gel column mentioned in Example 1. The gel column consists of two layers: the upper gel column has a concentration of 2.1 wt% polyacrylamide (containing acrylamide and NN methylenebisacrylamide), and the lower gel column has a concentration of 7.28 wt% polyacrylamide (containing acrylamide and NN methylenebisacrylamide). The main components of the prepared gel solution are: acrylamide, NN methylenebisacrylamide, ... The gel column contained base, TEMED, ammonium persulfate, and MgCl2. The concentrations of acrylamide and NN-methylenebisacrylamide in the upper gel column were 1.89 wt% (W / V) and 0.21 wt% (W / V), respectively. The concentration of base used was 1% (w / v), the concentration of sucrose was 2% (w / v), the concentration of TEMED was 0.15% (v / v), the concentration of Proclin 300 was 0.3‰, the amount of magnesium chloride was 0.067%, the concentration of BSA was 0.013%, and the pH value was 6.8±0.2. The volume of the upper gel was 350 μL, and the volume of the lower gel column was 1.2 ml.

[0123] To prepare a 100ml lower gel column, weigh out 6.9g of acrylamide, 0.38g of N,N-methylenebisacrylamide, 1g of tris, and 2% (w / v) of sucrose. After complete dissolution, adjust the pH to 6.8±0.2, add 1330µL of 5% magnesium chloride, 667µL of 2% BSA, and 100µL of TEMED. Add 1.2ml to a glass tube, add 100µL of purified water, and press the gel onto the column. After standing for 30 minutes, use a vacuum pump to remove the gel residue from the lower gel column.

[0124] To prepare a 100ml upper gel column, weigh out 2.5g of acrylamide, 0.2g of N,N-methylenebisacrylamide, 1g of tris, and 1g of sucrose. Add 30µL of Proclin 300 and dissolve completely. Adjust the pH to 6.8±0.2. Add 1333µL of magnesium chloride and 150µL of TEMED, and mix thoroughly. Transfer 160µL of the mixture to a glass tube, add 100µL of purified water, and press the gel onto the surface. After standing for 30 minutes, use a vacuum pump to remove the gel residue from the upper gel column.

[0125] The resulting gel column was placed in a wide-mouth plastic bottle, and storage buffer was added, 60 doses / bottle.

[0126] Example 8: Gel column B

[0127] The main difference from Example 7 is the adjustment of the amount of acrylamide added, resulting in a lower gel concentration of 6.4 wt% and an upper gel concentration of 2.1 wt%. The results of Examples 7 and 8 show that both the upper and lower gel solutions can effectively separate the various subtypes of high-density lipoprotein.

[0128] Example 9: Electrophoresis Experiment

[0129] The detection was performed using the kit and matching electrophoresis apparatus described in Example 1.

[0130] Preparation

[0131] 1. Blood sample preparation and plasma separation

[0132] 1.1 Subjects must fast for 12 hours before blood collection. During blood collection, 4 mL of whole blood is collected using an EDTA anticoagulant tube.

[0133] 1.2 Close the tube cap and slowly invert the anticoagulant tube about 10 times to mix the blood sample;

[0134] 1.3 Centrifuge the blood sample at 3000 rpm for 5 min, and transfer the supernatant plasma into a clean 1.5 mL centrifuge tube;

[0135] 1.4 Plasma samples tested within 7 days can be stored at 2-8℃ for later use, while samples tested after 7 days can be stored at -70℃ or below for later use.

[0136] 2. High-density lipoprotein (HDL) content detection: HDL content (mol / L or mg / dL) was detected using an NMPA-approved kit for detecting HDL.

[0137] Experimental steps

[0138] 1. Take one bottle of buffer powder from the kit and pour it into a clean beaker. Add 1200 mL of double-distilled water or deionized water to prepare the electrophoresis buffer.

[0139] 2. Take out a clean 1.5mL centrifuge tube, transfer 50μL of serum or plasma sample into the centrifuge tube with a pipette, add 15μL of high-density lipoprotein staining solution, and vortex for 30s or pipette 10 times to ensure that the sample and staining solution are thoroughly mixed.

[0140] 3. Place the centrifuge tube containing the mixture at room temperature for 15 minutes to allow the high-density lipoprotein staining solution to fully stain the high-density lipoprotein in the sample.

[0141] 4. Remove the gel column from the kit, carefully discard the preservation solution at the top of the gel column, and install the gel column with the sample loading port facing upwards into the rubber socket of the electrophoresis tank (top).

[0142] 5. Add an appropriate amount of electrophoresis buffer to the bottom of the electrophoresis tank, ensuring it covers the electrode column. Place the upper electrophoresis tank (with the gel column installed) over the bottom of the electrophoresis tank and add an appropriate amount of electrophoresis buffer to ensure that both the sample loading end of the gel column and the electrode column on the top cover are submerged in the buffer.

[0143] 6. Take out the pre-stained sample, vortex for 30 seconds or pipette 10 times, and then add 20 μL of the pre-stained mixture to the sample end of the gel column.

[0144] 7. Cover the electrophoresis tank, connect the power supply, turn on the electrophoresis apparatus, set the current to 3mA per gel column, and stop electrophoresis when the dye at the bottom of the gel column is about 1cm from the bottom of the column.

[0145] 8. 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 minutes.

[0146] Example 10: High-density lipoprotein detection experiment

[0147] In the kit mentioned in Example 1, the detection kit of this invention and a commercial detection kit (using the lipid multi-test card (dry chemistry) produced by Changsha Zhong Sheng Zhong Jie Biotechnology Co., Ltd. as the commercial detection kit) were used to detect high-density lipoprotein in blood samples. The staining intensity of the present invention is greater than that of the commercial detection kit.

[0148] Figure 3 This invention demonstrates that, under different polyacrylamide concentrations, it can separate high-density lipoproteins within the concentration range of the lower gel. For example, Figure 3 As shown, lanes 14-18 represent the HDL electrophoresis results of sample I using the Changsha kit and the lower gel concentrations of this invention at 6.0%, 6.5%, 7.0%, and 7.5%, respectively. The staining intensity of the Changsha kit is slightly weaker, and the lower gel concentration of this invention is not necessarily better the higher it is. When the lower gel concentration reaches 7.5%, the staining intensity is significantly lower than that in lanes 15-17.

[0149] Example 11: High-density lipoprotein subtype detection method

[0150] Perform the test according to the following steps.

[0151] S01: High-density lipoproteins in serum or plasma are separated using polyacrylamide gel electrophoresis.

[0152] S02: Stain the high-density lipoprotein in the serum or plasma with staining solution, react with the staining solution at room temperature for 15 minutes, and then add 20 μL of the mixture onto the gel column.

[0153] S03: Under the influence of the electric field in the electrophoresis tank, and through the effect of polyacrylamide gel molecular sieve, high-density lipoprotein subtypes are separated in sequence according to the size of high-density lipoprotein particles.

[0154] S04: The results of the high-density lipoprotein subtype electrophoresis separation are used to obtain the optical density spectrum of the high-density lipoprotein electrophoretic bands by the scanning system of the electrophoresis device;

[0155] S05: Convert the optical density spectrum into a peak pattern using analysis software, and analyze the area ratio of each peak in the peak pattern;

[0156] S06: The accurate content of each component of the high-density lipoprotein subtype is obtained based on the detected total cholesterol content and the ratio of the peak area.

[0157] Example 12: A method for preparing Sudan Black B2 staining solution, a derivative of Sudan Black B, comprising the following steps:

[0158] A concentration of 0.165% (W / V) of Sudan Black B2 was added to isopropanol, and the volume percentage of the isopropanol contained therein was 99% based on the total volume of the added isopropanol.

[0159] Add 1% by volume of 1-methyl-2-pyrrolidone;

[0160] Then add Triton X-405 at a volume ratio of 0.2% (V / V);

[0161] After thoroughly mixing the above solutions, and once Sudan Black B2 has completely dissolved, store them away from light.

[0162] Example 13: A method for preparing a high-density lipoprotein subtype detection kit, comprising the following steps:

[0163] Step (1), prepare the solution by mixing acrylamide, NN methylenebisacrylamide, base, TEMED, ammonium persulfate, sucrose, Proclin 300, MgCl 2、 Add BSA to deionized water according to the actual usage concentration, stir thoroughly, adjust the pH value, add 1.2 ml into a glass column, add purified water and press gel for 30 minutes. After gelation, a gel column with a polyacrylamide content of 7.28 wt% is obtained, which is the lower gel column.

[0164] Step (2), prepare the gel column solution, and add acrylamide, NN methylenebisacrylamide, According to the actual usage concentration, add base, TEMED, ammonium persulfate, sucrose, Proclin 300, and MgCl2 to purified water, stir thoroughly, adjust the pH value, add 350 μL to a glass column, add purified water, and press gel for 40 minutes. After gelation, a gel column with a polyacrylamide content of 2.1 wt% is obtained, which is the upper gel column.

[0165] Step (3): Place the gel column obtained in steps (1) and (2) into a wide-mouth plastic bottle and add storage buffer.

[0166] Step (4): Prepare the staining solution. Take Sudan Black B2 with a concentration of 0.165% (W / V) and add it to isopropanol. The volume percentage of the isopropanol contained therein is 99% based on the total volume of the added isopropanol.

[0167] Add 1% by volume of 1-methyl-2-pyrrolidone;

[0168] Then add Triton X-405 at a volume ratio of 0.2% (V / V);

[0169] After thoroughly mixing the above solutions, and once Sudan Black B2 has completely dissolved, store them away from light.

[0170] Step (5), mixing of buffer powder, the buffer powder contains base and boric acid. Among them, The amount of base is 11g / 1.2L, and the amount of boric acid is 5.5g / 1.2L. The two materials are mixed evenly using a mixer, and the mixture is dispensed into glass bottles at a dosage of 16.5g / bottle.

[0171] Step (6), quality control preparation: After the received sample passes the safety test, it is inactivated at 56°C for 30 minutes. After mixing, diluent is added as needed, and then the test value is assigned. It is then dispensed as quality control, with each dispensing being 100 microliters.

[0172] Step (7): Label the components from steps (3), (4), (5), and (6); inspect the resulting semi-finished product.

[0173] Step (8): Pack the semi-finished product that has passed the inspection in step (7) into a box to obtain the finished high-density lipoprotein subtype detection kit.

[0174] Preferably, the preparation method further includes the following steps:

[0175] Step (9): Perform final product testing on the high-density lipoprotein subtype detection kit obtained in step (8);

[0176] Step (10): After the finished products that have passed the inspection in step (9) are affixed with a qualified label, they are put into storage.

[0177] like Figure 4-9 As shown, this invention enables high-density lipoprotein typing using gel columns with different concentrations of the lower gel layer. The results indicate that higher concentrations do not necessarily lead to better results; that is, there is no concentration dependence.

[0178] Example 14: Comparative experiment of staining Sudan Black B, Sudan Black B2 staining solutions and 3 sample staining solutions

[0179] Appendix Figure 10The image shows a comparison of electrophoretic scans after staining three samples with Sudan Black B staining solution for 30 min and Sudan Black B2 staining three samples for 15 min. The electrophoretic scans show: 21 Sudan Black B reacted with sample #1 at 37℃ for 30 min; 22 Sudan Black B derivative B2 reacted with sample #1 at room temperature for 15 min; 23 Sudan Black B reacted with sample #2 at 37℃ for 30 min; 24 Sudan Black B derivative B2 reacted with sample #2 at room temperature for 15 min; 25 Sudan Black B reacted with sample #3 at 37℃ for 30 min; 26 Sudan Black B derivative B2 reacted with sample #3 at room temperature for 15 min.

[0180] from Figure 10 The staining intensity shows that the staining intensity of Sudan Black B2, a derivative of Sudan Black B, reacted with the staining solution samples in Example 3 for 15 minutes was significantly stronger than that of Sudan Black B reacted with the samples at 37°C for 30 minutes.

[0181] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for detecting high density lipoprotein subfraction, characterized by, Perform the test according to the following steps: S01: Obtain a serum or plasma sample; S02: After staining the serum or plasma with staining solution, add the mixture to a gel column; the gel column consists of two layers, with the upper gel column containing 2.0wt%-2.2wt% polyacrylamide and the lower gel column containing 6.0wt%-7.5wt% polyacrylamide; add acrylamide, NN-methylenebisacrylamide, Trizma®base, TEMED, ammonium persulfate, sucrose, Proclin 300, MgCl2, and BSA to purified water or deionized water according to the actual usage concentration, stir thoroughly, adjust the pH value, add 1.0-1.3 ml to the glass column, add water or ethanol, and press the gel for at least 3 minutes. After 0 minutes, a gel column with a polyacrylamide content of 6.01-7.49 wt% is obtained, which is the lower gel column. Acrylamide, NN methylenebisacrylamide, Trizma®base, TEMED, ammonium persulfate, sucrose, Proclin 300, and MgCl2 are added to purified water or deionized water according to the actual usage concentration. After thorough mixing, the pH value is adjusted to complete the gel preparation. Remove the adhesive residue from the top of the glass tube. Take 200-400 μL and put it into the upper glass column of the lower gel. Add water or ethanol and press the gel for at least 30 minutes. After gel formation, a gel column with a polyacrylamide content of 2.01-2.19 wt% is obtained, which is the upper gel column. S03: Under the influence of the electric field in the electrophoresis tank, and through the effect of polyacrylamide gel molecular sieve, high-density lipoprotein subtypes are separated in sequence according to the different sizes of high-density lipoprotein particles. S04: The results of the high-density lipoprotein subtype electrophoretic separation are analyzed by the electrophoresis apparatus to obtain the optical density spectrum of the high-density lipoprotein electrophoretic bands; S05: Convert the optical density spectrum into a peak pattern using analysis software, and calculate the area ratio of each peak in the peak pattern; S06: The accurate content of each component of the high-density lipoprotein subtype is obtained based on the detected total cholesterol content and the ratio of the peak area.

2. The high-density lipoprotein subfraction detection method according to claim 1, characterized by, The staining solution contains Sudan Black B2.

3. The high-density lipoprotein subfraction typing method according to claim 1, characterized by, The method for preparing the staining solution includes the following steps: Sudan Black B2 dye with a concentration of 0.1wt%-1.5% (W / V) is taken and added to one of the following: acetone, propylene glycol, isopropanol, and ethylene glycol; the volume percentage of acetone, propylene glycol, isopropanol, or ethylene glycol contained therein, based on the total volume of the added acetone, propylene glycol, isopropanol, or ethylene glycol, is greater than or equal to 96.5% and less than or equal to 99.3%. Add 0.5-1.5% by volume of NN methylenebisacrylamide or 1-methyl-2-pyrrolidone; Then, one of Triton X-405, Triton X-100, and Tween is added as a surfactant, and the volume ratio of the surfactant used is 0.05-0.5% (V / V). After thoroughly mixing the above solutions, store them away from light to obtain the staining solution.

4. The HDL subfraction typing method according to claim 1, wherein The volume of the upper gel column used is 200-400 microliters, and the volume of the lower gel column used is 1.0-1.3 milliliters; The concentration of Trizma®base used is 0.2-2% (w / v), the concentration of sucrose is 0.5-2% (w / v), the concentration of TEMED is 0.3-2% (v / v), the pH of the gel solution is 4.0-11.0, the concentration of Proclin300 is 0.05‰-5‰, the concentration of MgCl2 is 0.01-0.2% (w / v), and the concentration of BSA is 0.005%-0.5%.

5. A high density lipoprotein subfraction typing test kit, characterized by, The kit contains gel column flasks and staining solutions; The staining solution contains Sudan Black B2; The gel column bottle contains a gel column, which is divided into upper and lower layers. The upper gel column has a concentration of 2.0-2.2 wt% polyacrylamide, and the lower gel column has a concentration of 6.0-7.5 wt% polyacrylamide. In this process, acrylamide, NN methylenebisacrylamide, Trizma®base, TEMED, ammonium persulfate, sucrose, Proclin300, MgCl2, and BSA are added to purified water or deionized water according to the actual usage concentration. After thorough mixing, the pH value is adjusted, and 1.0-1.3 ml is added to a glass column. Water or ethanol is added for gelation, and the gel is formed in a time of not less than 30 minutes. After gelation, a gel column with a polyacrylamide content of 6.01-7.49 wt% is obtained, which is the lower gel column. Add acrylamide, NN-methylenebisacrylamide, Trizma®base, TEMED, ammonium persulfate, sucrose, Proclin 300, and MgCl2 to purified or deionized water according to the actual usage concentration. Stir thoroughly and adjust the pH value to complete the preparation of the gel solution. Remove the adhesive residue from the top of the glass tube. Take 200-400 μL and add it to the upper glass column of the lower gel. Add water or ethanol and press the gel for at least 30 minutes. After gel formation, a gel column with a polyacrylamide content of 2.01-2.19 wt% is obtained, which is the upper gel column.

6. The high-density lipoprotein subfraction typing test kit according to claim 5, characterized by The kit includes a test kit box, a pearl cotton liner, a gel column bottle, a staining solution, a buffer powder, quality control I, and quality control II; The lower gel column has a concentration of 6.5-7.0 wt% polyacrylamide.

7. The method for preparing the high-density lipoprotein subfraction typing test kit according to any one of claims 5 to 6, characterized by, Includes the following steps: Step (1): Prepare the solution by adding acrylamide, NN methylenebisacrylamide, Trizma®base, TEMED, ammonium persulfate, sucrose, Proclin300, MgCl2, and BSA to purified water or deionized water according to the actual usage concentration. After stirring thoroughly, adjust the pH value and add 1.0-1.3 ml to the glass column. Add water or ethanol and press the gel for at least 30 minutes. After gelation, a gel column with a polyacrylamide content of 6.01-7.49 wt% is obtained, which is the lower gel column. Step (2): Prepare the solution by adding acrylamide, NN methylenebisacrylamide, Trizma®base, TEMED, ammonium persulfate, sucrose, Proclin300 and MgCl2 to purified water or deionized water according to the actual concentration to be used. After stirring thoroughly, adjust the pH value to complete the preparation of the gel solution. Remove the glue solution from the upper part of the glass tube in step (1). Take 200-400 μL into the upper glass column of the lower gel, add water or ethanol and press the gel for no less than 30 minutes. After gel formation, a gel column with a polyacrylamide content of 2.01-2.19 wt% is obtained, which is the upper gel column. Step (3): Place the gel column obtained in steps (1) and (2) into a wide-mouth plastic bottle and add storage buffer; Step (4): Prepare the solution by adding Sudan Black B derivative Sudan Black B2 to a mixture selected from acetone, propylene glycol, isopropanol, and ethylene glycol at a concentration of 0.1-1.5% (W / V). The volume percentage of acetone, propylene glycol, isopropanol, or ethylene glycol in the mixture shall be greater than or equal to 96.5% and less than or equal to 99.3% based on the total volume of the added acetone, propylene glycol, isopropanol, or ethylene glycol. Add DMF or 1-methyl-2-pyrrolidone at a volume ratio of 0.5-1.5%. Add Triton X-405 or Triton X-100 or Tween as a surfactant at a volume of 0.05-0.5% (V / V). After thoroughly mixing the above mixture, dispense it into containers and store it in the dark. Step (5), mixing of buffer powder, wherein the amount of Trizmabase is 5g / L-15g / L and the amount of boric acid is 3g / L-10g / L; the two materials are mixed by stirring with a mixer, and the mixture is dispensed into glass bottles at a dosage of 16.5g / bottle. Step (6), quality control preparation: After the received sample passes the safety test, it is inactivated at 56°C for 30 minutes, mixed, and then tested and assigned values. It is then dispensed as quality control, with each sample dispensed in 100 microliters. Step (7): Label the components from steps (3), (4), (5), and (6); inspect the resulting semi-finished product. Step (8): Pack the semi-finished product that has passed the inspection in step (7) into a box to obtain the finished high-density lipoprotein subtype detection kit.

8. The preparation method according to claim 7, characterized in that, The preparation method further includes the following steps: Step (9): Perform final product testing on the high-density lipoprotein subtype detection kit obtained in step (8); Step (10): After the finished product that has passed the inspection in step (9) is affixed with a qualified label, it is put into storage.

9. The application of the detection method of claim 1 or the detection kit of claim 5 in the quantitative detection of high-density lipoprotein subtypes.

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