High-density lipoprotein subtyping detection method, detection kit and application thereof

Abstract

The invention discloses a high-density lipoprotein subtyping detection method, a detection kit as well as a preparation method and application of the detection kit. According to the method, high-density lipoprotein in serum or plasma is separated by adopting a polyacrylamide gel electrophoresis method; after high-density lipoprotein in serum or plasma is dyed with a dyeing solution, a mixed solution is taken and added to a gel column; under the action of an electric field, the high-density lipoprotein particles are sequentially separated according to the sizes of the high-density lipoprotein particles through a polyacrylamide gel molecular sieve effect; a protein electrophoretic separation result passes through a scanning system of the electrophoresis device to obtain an optical density map of a high-density lipoprotein electrophoretic band; converting the optical density spectrum into a peak pattern spectrum, and performing area proportion analysis; and obtaining the accurate content of each component of the high-density lipoprotein subtype according to the total high-density lipoprotein content and the proportion of each peak pattern area. Classification is carried out according to the particle size of the high-density lipoprotein, the high-density lipoprotein is subtyped and quantitatively detected, and the detection sensitivity is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and relates to a method for detecting high-density lipoprotein subfractions, and also relates to a kit for detecting high-density lipoprotein subfractions, a preparation method thereof and an application thereof. Background Art

[0002] At present, for the detection of lipid metabolism, most kits are used for single-item detection or four-item lipid detection at the same time. However, the content of high-density lipoprotein is mainly determined by enzyme method, clearance method, etc., resulting in the detection result of high-density lipoprotein being only an overall result, rather than accurately obtaining the result of high-density subtypes, so as to classify high-density lipoprotein; for the detection of subtypes, methods such as nuclear magnetic resonance, non-denaturing gradient gel electrophoresis, density gradient centrifugation, etc. are mainly used. These methods are time-consuming and laborious, the test process is difficult to control, and require professional personnel with extremely high skills to operate large instruments.

[0003] In addition, some use gel electrophoresis method to detect high-density lipoprotein subfractions. However, since the commonly used Sudan Black B is a diazo dye, and the main components of each subtype in high-density lipoprotein are different, the binding of Sudan Black B to each subtype of high-density lipoprotein is not uniform, which will lead to inconsistent staining efficiency and ultimately different results.

[0004] Therefore, there is currently a lack of an improved dye in the prior art that can efficiently stain the particle size of high-density lipoprotein, so as to simply and intuitively classify and quantitatively detect high-density lipoprotein subfractions by polyacrylamide gel electrophoresis method. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for detecting high-density lipoprotein subfractions that is classified according to the particle size of high-density lipoprotein, is simple and intuitive, and mainly uses gel electrophoresis method, so as to improve the sensitivity of detecting high-density lipoprotein subfractions.

[0006] Another purpose of the present invention is to provide an improved Sudan Black B dye and a corresponding kit for detecting high-density lipoprotein subfractions.

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

[0008] To achieve the above purpose, on the one hand, the present invention provides a method for detecting high-density lipoprotein subfractions, which is detected according to the following steps:

[0009] S01: Use polyacrylamide gel electrophoresis method to separate high-density lipoprotein in serum or plasma;

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

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

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

[0013] S05: converting the optical density spectrum into a peak type spectrum through analysis software, and performing area ratio analysis of each peak type in the peak type spectrum;

[0014] S06: Obtain the accurate content of each component of high-density lipoprotein subtype according to the ratio of the detected total cholesterol content and the area of ​​each peak type.

[0015] Furthermore, the usage volume of the upper gel column is 200-400 μl, and the usage volume of the lower gel column is 1.0-1.3 ml.

[0016] Furthermore, the The usage concentration of base is 0.2-2% (W / V), the usage concentration of sucrose is 0.5-2% (W / V), the usage concentration of TEMED 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 salt ion concentration is 0.01-0.2% (W / V), and the BSA concentration is 0.005%-0.5%.

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

[0018] One aspect of the present invention provides a high-density lipoprotein subtyping detection kit.

[0019] Furthermore, the kit comprises a detection kit box body, an EPE liner, a gel column bottle, a staining solution, a buffer powder, and a quality control product I and a quality control product II, wherein the gel column bottle, the staining solution, the buffer powder, the quality control product I and the quality control product II are sequentially placed in the EPE liner, the gel column bottle contains a gel column, and the gel column is composed of a gel liquid.

[0020] Furthermore, the gel column is packed into a wide-mouth plastic bottle according to 60 portions per box, and the bottle is filled with storage buffer and stored at 2-8°C.

[0021] Furthermore, the gel column is divided into an upper and lower layer, wherein the concentration of the upper gel column is 2.01-2.19wt% polyacrylamide, including acrylamide and NN methylenebisacrylamide, and the concentration of the lower gel column is 6.04-7.49wt% polyacrylamide, including acrylamide and NN methylenebisacrylamide. The main components of the prepared gel solution are: acrylamide, NN methylenebisacrylamide, base, TEMED, ammonium persulfate and sucrose, Proclin300, MgCl 2 .

[0022] Furthermore, the volume of the upper gel column is 200-400 μl, the volume of the lower gel column is 1.0-1.3 ml, and the The usage concentration of base is 0.2-2% (W / V), the usage concentration of sucrose is 0.5-2% (W / V), the usage concentration of TEMED is 0.3-2% (V / V), the usage concentration of Proclin300 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] One aspect of the present invention provides a dye Sudan Black B2 for detecting high-density lipoprotein subtyping, wherein a hydroxymethyl group is added to the R2 position of the Sudan Black B structure.

[0024] Another aspect of the present invention provides a staining solution for the above-mentioned high-density lipoprotein subtyping detection method or the above-mentioned high-density lipoprotein subtyping detection kit, comprising the above-mentioned dye Sudan Black B2.

[0025] Another aspect of the present invention provides a method for preparing the above-mentioned dyeing solution, comprising the following steps:

[0026] The concentration of Sudan Black B derivative Sudan Black B2 is 0.1-1.5% (W / V), and Sudan Black B derivative Sudan Black B2 is added to one of acetone, propylene glycol, isopropanol and ethylene glycol, and the volume percentage of the acetone, propylene glycol, isopropanol or ethylene glycol contained therein is greater than or equal to 96.5% and less than 98.0% based on the total volume of the added acetone, propylene glycol, isopropanol or ethylene glycol; preferably, the volume percentage of the acetone, propylene glycol, isopropanol or ethylene glycol contained therein 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, a surfactant such as Triton X-405 or Triton X-100, Tween, etc. is added thereto, and the volume ratio thereof is 0.05-0.5% (V / V);

[0029] After the above mixed solution is fully mixed, it is stored away from light to obtain a dyeing solution.

[0030] One aspect of the present invention provides a high-density lipoprotein subtyping detection kit, wherein the preparation method of the staining solution comprises the following steps: taking a dye Sudan Black B2 with a concentration of 0.1-1.5% (W / V), adding the Sudan Black B2 to one of the combinations of acetone, propylene glycol, isopropanol and ethylene glycol, wherein the volume percentage of the 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, 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 is 0.05-0.5% (V / V);

[0033] After the mixed solution is fully mixed, it is stored away from light to obtain the dyeing solution.

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

[0035] Step (1), preparing a liquid, acrylamide, NN methylene bisacrylamide, base, TEMED, ammonium persulfate, sucrose, Proclin300, MgCl 2BSA is added to pure water or deionized water according to the actual concentration, and after being fully stirred, the pH value is adjusted to complete the preparation process of the glue solution. Take 1.0-1.3 ml of the above glue solution and add it to the glass column, add water or ethanol to press the glue, and let it stand for no less than 30 minutes after the glue is pressed. After the gelation, a gel column with a polyacrylamide content of 6.01-7.49wt% is obtained, which is the lower gel column; step (2), liquid preparation, acrylamide, NN methylene bisacrylamide, base, TEMED, ammonium persulfate, sucrose, Proclin300, MgCl 2 According to the actual use concentration, add it to pure water or deionized water, stir it thoroughly, adjust the pH value, and complete the glue preparation process. Remove the glue water on the top of the glass tube in step (1). Take 200-400 microliters of the above glue and add it to the upper part of the lower gel column in the glass column, add water or ethanol for glue pressing, and let it stand for no less than 30 minutes after the glue pressing is completed. After gelling, a gel column with a polyacrylamide content of 2.01-2.19wt% is obtained, which is the upper gel column;

[0036] Step (3), placing the gel column obtained in steps (1) and (2) into a wide-mouth plastic bottle and filling it with storage buffer;

[0037] Step (4), preparing a solution, setting the concentration of Sudan Black B derivative Sudan Black B2 to 0.1-1.5% (W / V), adding Sudan Black B derivative Sudan Black B2 to one selected from the group consisting 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 selected acetone, propylene glycol, isopropanol or ethylene glycol; adding DMF or 1-methyl-2-pyrrolidone therein at a volume ratio of 0.5-1.5%; then adding Triton X-405 or Triton X-100 and Tween as surfactants, wherein the surfactant is used in a volume of 0.05-0.5% (V / V); fully mixing the above mixed solution, packaging it and storing 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 above two materials are stirred and mixed by a blender, and then divided into buffer powder glass bottles according to the dosage of 16.5g / bottle;

[0039] Step (6), preparation of quality control, the received samples, after passing the safety test, are inactivated at 56°C for 30 minutes, mixed, and then tested and assigned, and packaged as quality control, each aliquot is 100 μl;

[0040] Step (7), labeling the components of steps (3), (4), (5), and (6); and inspecting the semi-finished product obtained;

[0041] Step (8), packing the semi-finished product that has passed the inspection in step (7) into a box to obtain a finished high-density lipoprotein subtyping detection kit;

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

[0043] Step (9), performing finished product testing on the finished high-density lipoprotein subtyping detection kit obtained in step (8);

[0044] Step (10), the finished product that has passed the inspection in step (9) is affixed with a qualified label and put into storage.

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

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] 1. Through the technical solution of the present invention, a newly improved Sudan Black B2 dye, which is a derivative of Sudan Black B, can be provided. By modifying Sudan Black B and optimizing the upper and lower layers of glue, the lipophilicity of the dye is greatly improved, especially for high-density lipoprotein, so that the binding conditions of the dye and high-density lipoprotein are simpler and the binding time is shorter, which can be 15 minutes at room temperature;

[0048] 2. Through the technical solution of the present invention, the detection of high-density lipoprotein is closer to the results of high-density lipoprotein content detected by other reagents;

[0049] 3. The technical solution of the present invention effectively reduces the reduction in resolution caused by the heterogeneity of high-density lipoprotein components, and can accurately distinguish each high-density lipoprotein component;

[0050] 4. The technical solution of the present invention can quantitatively detect high-density lipoprotein subtypes and analyze high-density lipoprotein components, which can be used to assess the risk of cardiovascular and cerebrovascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0052] Figure 2 This is an example of the high-density electrophoresis scanning diagram of Example 2-4.

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

[0054] Figure 4 This is the result of adding 80 μl of 3.5% glue solution to the lower layer of glue when sample #1 is added.

[0055] Figure 5 This is the result of adding 100 μl of 3.5% glue solution to the lower layer of glue when sample #1 was added.

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

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

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

[0059] Fig. 9 The result of adding 120 μl of 3.5% glue solution to the lower layer of glue for sample #2.

[0060] Fig.10 These are the test results of three samples of Sudan Black B2 and Sudan Black B. Among them, lanes 1, 3, and 5 are Sudan Black B2, and lanes 2, 4, and 6 are Sudan Black B; 1 and 2 are different dyes for the same sample, 3 and 4 are different dyes for the same sample, and 5 and 6 are different dyes for the same sample.

[0061] Fig.11 : are the structural formulas of Sudan Black B before and after transformation. From left to right are Sudan Black B, Sudan Black B1, Sudan Black B2 and Sudan Black B3. Among them, Sudan Black B derivative Sudan Black B2 is the dye used in the present invention.

[0062] In the figure, 1 is a large HDL particle, 2 is a medium HDL particle, and 3 is a small HDL particle. 11 is a very low density lipoprotein and a low density lipoprotein, 12 is a high density lipoprotein, and 13 is an albumin. 21 is a reaction of Sudan Black B derivative B2 with sample #1 at room temperature for 15 minutes; 22 is a reaction of Sudan Black B with sample #1 at 37°C for 30 minutes; 23 is a reaction of Sudan Black B derivative B2 with sample #2 at room temperature for 15 minutes; 24 is a reaction of Sudan Black B with sample #2 at 37°C for 30 minutes; 25 is a reaction of Sudan Black B derivative B2 with sample #3 at room temperature for 15 minutes; 26 is a reaction of Sudan Black B with sample #3 at 37°C for 30 minutes. DETAILED DESCRIPTION

[0063] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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 subtyping is provided, and the detection is performed according to the following steps:

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

[0066] S02: After staining the high-density lipoprotein in the serum or plasma with a staining solution, 30 μl of the mixed solution is added to a gel column;

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

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

[0069] S05: converting the optical density spectrum into a peak type spectrum through analysis software, and performing area ratio analysis of each peak type in the peak type spectrum;

[0070] S06: Obtain the accurate content of each component of high-density lipoprotein subtype according to the ratio of the detected total cholesterol content and the area of ​​each peak type.

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

[0072] Preferably, the kit comprises a detection kit box body, an EPE liner, a gel column bottle, a staining solution, a buffer powder, a quality control product I and a quality control product II, wherein the gel column bottle, the staining solution, the buffer powder, the quality control product I and the quality control product II are sequentially placed in the EPE liner, and the gel column bottle contains a gel column.

[0073] Preferably, the gel column is packed into a wide-mouth plastic bottle in a box of 60 portions, and the bottle is filled with storage buffer and stored at 2-8°C.

[0074] Preferably, the gel column is divided into two layers, wherein the concentration of the upper gel column is 2.01-2.19wt% polyacrylamide, including acrylamide and NN methylenebisacrylamide, and the concentration of the lower gel column is 6.01-7.49wt% polyacrylamide, including acrylamide and NN methylenebisacrylamide. The main components of the prepared gel solution are: acrylamide, NN methylenebisacrylamide, base, TEMED, ammonium persulfate and sucrose, Proclin300, MgCl 2 , BSA (bovine serum albumin).

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

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

[0077] The structural formula of Sudan Black B before and after transformation is shown in Figure 1 , a derivative of Sudan Black B formed by adding a specific chemical group to the structure of Sudan Black B, wherein the chemical structures of Sudan Black B, Sudan Black B1, Sudan Black B2 and Sudan Black B3 are shown from left to right. Adding a hydroxyethyl group to the R1 position of the Sudan Black B structure turns into Sudan Black B1, adding a hydroxymethyl group to the R2 position of the Sudan Black B structure turns into Sudan Black B2, and adding a hydroxyl group to the R2 position of the Sudan Black B structure turns into Sudan Black B3; preferably, adding a hydroxymethyl group to the R2 position of the Sudan Black B structure turns into Sudan Black B2. The test results of the present invention show that although Sudan Black B, Sudan Black B1, Sudan Black B2 and Sudan Black B3 can all stain high-density lipoprotein, the staining effect of Sudan Black B2 on high-density lipoprotein, especially in terms of the resolution and quantitative results of the staining bands, is significantly better than 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-mentioned high-density lipoprotein subtyping detection method or the above-mentioned high-density lipoprotein subtyping detection kit is provided, comprising the above-mentioned dye Sudan Black B2. Sudan Black B2, a derivative of Sudan Black B, is used as a staining solution to replace Sudan Black, and has a clearer staining effect on high-density lipoprotein subtyping, making the high-density lipoprotein subtyping separation effect easier and more accurate.

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

[0080] The concentration of Sudan Black B derivative Sudan Black B2 is 0.1-1.5% (W / V), and the Sudan Black B derivative Sudan Black B2 is added to one selected from the group consisting of acetone, propylene glycol, isopropanol and ethylene glycol, wherein the volume percentage of the 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, isopropanol or ethylene glycol;

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

[0082] Then Triton X-405 or Triton X-100 and Tween are added therein as surfactants, and the volume ratio thereof is 0.05-0.5% (V / V);

[0083] After the above mixed solution is fully mixed, it is stored away from light to obtain a dyeing solution.

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

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

[0086] Step (1), preparing a liquid, acrylamide, NN methylene bisacrylamide, base, TEMED, ammonium persulfate, sucrose, Proclin300, MgCl 2 , add bovine serum albumin to pure water or deionized water according to the actual use concentration, stir thoroughly, adjust the pH value, complete the preparation of the gel solution, take 1.0-1.3 ml into a glass column, add water or ethanol to perform gel pressing for not less than 30 minutes, and after gelling, obtain a gel column with a polyacrylamide content of 6.01-7.49wt%, which is the lower gel column;

[0087] Step (2), preparing the solution: Add acrylamide, N-N methylene bisacrylamide, base, TEMED, ammonium persulfate, sucrose, Proclin300 and MgCl 2 into pure water or deionized water according to the actual usage concentration. After stirring well, adjust the pH value to complete the preparation of the gel solution, and remove the pressure glue in step (1). Take 200 - 400 μL of the prepared gel solution and add it to the upper glass column of the lower layer gel. Add water or ethanol for pressure gluing for no less than 30 minutes. After forming the gel, a gel column with a polyacrylamide content of 2.01 - 2.19 wt% is obtained, which is the upper layer gel column;

[0088] Step (3), put the gel columns obtained in steps (1) and (2) into a wide-mouth plastic bottle and add the storage buffer;

[0089] Step (4), preparing the solution: The concentration of Sudan Black B derivative Sudan Black B2 is 0.1 - 1.5% (W / V). Add Sudan Black B derivative Sudan Black B2 into one of acetone, propylene glycol, isopropanol and ethylene glycol. Based on the total volume of acetone, propylene glycol, isopropanol or ethylene glycol, the volume percentage range of acetone, propylene glycol, isopropanol or ethylene glycol contained is greater than or equal to 96.5% and less than or equal to 99.3%; Add DMF or 1-methyl-2-pyrrolidone into it, with a volume ratio of 0.5 - 1.5%; Then add surfactants such as Triton X-405 or Triton X-100, Tween into it, and its usage volume is 0.05 - 0.5% (V / V); After fully mixing the above mixed solution, subpackage and store in the dark;

[0090] Step (5), mixing the buffer powder. Among them, the dosage of base is 5 g / L - 15 g / L, and the dosage of boric acid is 3 g / L - 10 g / L; Stir and mix the above two materials with a blender, and subpackage the mixed mixture into glass bottles according to the dosage of 16.5 g / bottle;

[0091] Step (6), preparing the quality control: After the received samples pass the safety test, inactivate them at 56 °C for 30 minutes. After mixing, perform detection and assignment, and subpackage them as quality control, with 100 μL subpackaged in each tube;

[0092] Step (7), label the components in steps (3), (4), (5) and (6); Inspect the obtained semi-finished products;

[0093] Step (8), pack the semi-finished products that pass the inspection in step (7) into boxes to obtain the finished high-density lipoprotein subfraction detection kit;

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

[0095] Step (9), performing finished product testing on the finished high-density lipoprotein subtyping detection kit obtained in step (8);

[0096] Step (10), the finished product that has passed the inspection in step (9) is affixed with a qualified label and put into storage.

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

[0098] Example

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

[0100] A high-density lipoprotein typing test kit, comprising a test kit box body, an EPE lining, a gel column bottle, a staining solution, a buffer powder, a quality control product I and a quality control product II, wherein the gel column bottle, the staining solution, the buffer powder, the quality control product I and the quality control product II are sequentially placed in the EPE lining. Product composition and specifications:

[0101] The ingredients and specifications are as follows:

[0102] 1. Gel column, 60 pieces / bottle × 1 bottle

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

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

[0105] 4. Instruction manual, 1 copy

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

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

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

[0109] Example 2: A dyeing solution

[0110] Sudan Black B2 is added to isopropanol at a weight-to-total-volume ratio of 0.165% (W / V), wherein the volume percentage of isopropanol is 98.8% based on the total volume of isopropanol; 1-methyl-2-pyrrolidone is added at a volume ratio of 1%; Triton X-405 is then added at a volume of 0.2% (V / V); the mixed solution is fully mixed, packaged at 1.2 mL / tube, and stored in a dark place.

[0111] Example 3: A dyeing solution

[0112] The difference from Example 2 is that the weight of Sudan Black B2 added is 0.8% (W / V) to the total volume, and the volume percentage of isopropanol contained therein is 98.8% based on the total volume of isopropanol; the volume ratio of 1-methyl-2-pyrrolidone added is 1%; Triton X-405 is added thereto, and its usage volume is 0.2% (V / V); after the above mixed solution is fully mixed, it is packaged according to 1.2 mL / tube and stored in the dark.

[0113] Embodiment 4 A kind of dyeing liquid

[0114] The difference from Example 2 and Example 3 is that the weight of Sudan Black B2 added is added to the isopropanol at a ratio of 1.5% (W / V) to the total volume. Based on the total volume of isopropanol, the volume percentage of isopropanol contained therein is 98.8%; the volume ratio of 1-methyl-2-pyrrolidone added is 1%; Triton X-405 is added thereto at a volume of 0.2% (V / V); after the above mixed solution is fully mixed, it is packaged according to 1.2 mL / tube and stored in the dark.

[0115] like Figure 2 As shown in the examples 2, 3 and 4, different concentrations of Sudan Black B2 can all be used to stain high-density lipoprotein in serum / plasma, and the staining intensities are slightly different. Obviously, when staining high-density lipoprotein, the more Sudan Black B2, the better. When the concentration is 0.8% (W / V), the staining intensity is greater than 0.165% (W / V), but 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: according to the conventional TBE electrophoresis buffer, the EDTA component is removed and the concentration of TRIS and boric acid is slightly adjusted. After weighing the appropriate weight, it is divided into suitable volumetric bottles, and each bottle weighs 16.5g. When the kit is used, each 16.5g of powder is added to 1.2L of deionized water or purified water (ion-containing mineral water is not suitable).

[0118] Weigh 1.1 kg of tris and 0.55 kg of boric acid, mix the two materials thoroughly in a blender, and then package them into 16.5 g / bottle for later use.

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

[0120] The quality control products I and II mentioned in Example 1 are: samples collected from clinical practice that meet the ideal concentration of high-density lipoprotein cholesterol detection are used as raw materials for the preparation of quality control product I; abnormal samples collected from clinical practice in high-density lipoprotein cholesterol detection are used as raw materials for the preparation of quality control product II. First, the above samples are mixed separately; then the mixed sample is inactivated at 56°C for 30 minutes; then the sample is centrifuged at 10,000 rpm for 15 minutes, filtered with a 0.45 μm filter membrane; then the diluent is added and set aside. The diluted mixed sample was tested 3 times, and the results were still consistent. At the same time, three different batches of the test kit in Example 1 were used for 20 times each batch, and the mean ± 2 times the standard deviation of each subcomponent was calculated as the detection range of the quality control product. Store at -20°C for later use.

[0121] Example 7: Gel Column A

[0122] The gel column mentioned in Example 1 is divided into two layers, the concentration of the upper gel column is 2.1wt% polyacrylamide (including acrylamide and NN methylenebisacrylamide), the concentration of the lower gel column is 7.28wt% polyacrylamide (including acrylamide and NN methylenebisacrylamide), and the main components of the prepared gel solution are: acrylamide, NN methylenebisacrylamide, base, TEMED, ammonium persulfate and MgCl 2 The concentrations of acrylamide and NN methylenebisacrylamide in the upper gel column were 1.89wt% (W / V) and 0.21wt% (W / V), respectively. The usage concentration of base is 1% (W / V), the usage concentration of sucrose is 2% (W / V), the usage concentration of TEMED is 0.15% (V / V), the concentration of Proclin300 is 0.3‰, the amount of magnesium chloride is 0.067%, the concentration of BSA is 0.013%, and the pH value is 6.8±0.2. The usage volume of the upper layer of gel is 350 microliters, and the usage volume of the lower layer of gel column is 1.2 ml.

[0123] According to the preparation of 100ml lower gel column, weigh 6.9g acrylamide, 0.38g NN methylenebisacrylamide, 1g tris, and 2% (W / V) sucrose. After fully dissolved, adjust the pH value to 6.8±0.2, add 1330μl 5% magnesium chloride, 667μl 2% BSA, and 100μl TEMED. Take 1.2ml and add it to the glass tube, add 100μl pure water for glue pressing, let it stand for 30 minutes, and use a vacuum pump to remove the glue water on the lower glue.

[0124] According to the preparation of 100ml upper gel column, weigh 2.5g acrylamide, 0.2g NN methylene bisacrylamide, 1g tris, 1g sucrose, add 30μl Proclin300, after fully dissolving, adjust the pH value to 6.8±0.2, add 1333μl magnesium chloride, add 150μl TEMED, mix well. Take 160μl and add it to the glass tube, add 100μl pure water for glue pressing, let it stand for 30 minutes, and use a vacuum pump to remove the glue water on the upper glue.

[0125] The obtained gel column was placed in a wide-mouth plastic bottle and a storage buffer was added, 60 portions per bottle.

[0126] Example 8: Gel Column B

[0127] The difference from Example 7 is mainly that the amount of acrylamide added is adjusted so that the concentration of the lower layer of gel is 6.4wt% and the concentration of the upper layer of gel is 2.1wt%. The results of Examples 7 and 8 show that different upper and lower layer gels can effectively separate the various subtypes of high-density lipoprotein.

[0128] Example 9: Electrophoresis experiment

[0129] The test kit and the matching electrophoresis device of Example 1 were used for detection.

[0130] Preparation

[0131] 1. Blood Sample Preparation and Plasma Separation

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

[0133] 1.2 Cover 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 upper plasma into a 1.5 mL clean centrifuge tube;

[0135] 1.4 Plasma samples that are tested within 7 days can be stored in a refrigerator at 2-8°C for future use. Plasma samples that are tested more than 7 days ago can be stored at -70°C or below for future use.

[0136] 2. High-density lipoprotein content detection: Use a high-density lipoprotein detection kit approved by NMPA to detect the high-density lipoprotein content (mol / L or mg / dL).

[0137] Experimental procedures

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

[0139] 2. Take out a clean 1.5 mL centrifuge tube, use a pipette to transfer 50 μL of serum or plasma sample into the centrifuge tube, and then add 15 μL of high-density lipoprotein staining solution. Vortex on a vortex shaker for 30 s or pipette 10 times to fully mix the sample and the staining solution.

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

[0141] 4. Take out 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 up into the rubber socket of the electrophoresis tank (upper part).

[0142] 5. Add an appropriate amount of electrophoresis buffer to the electrophoresis tank (lower part) to submerge the electrode column. Cover the upper electrophoresis tank with the gel column installed on the electrophoresis tank (lower part), 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 upper cover are submerged in the buffer.

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

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

[0145] 8. Remove the electrophoresis buffer from the electrophoresis tank (upper part), take out the gel column, wipe off the electrophoresis buffer on the surface of the gel column, and perform scanning analysis within 15 min.

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

[0147] For the kit mentioned in Example 1, use the detection kit of the present invention and a commercial detection kit (using the lipid multi-test card (dry chemistry) produced by Changsha Zhongsheng Zhongjie Biotechnology Co., Ltd. as the commercial detection kit) 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 It shows that under different polyacrylamide concentrations, within the concentration range of the lower layer gel of the present invention, high-density lipoprotein can be separated. Among them, as Figure 3As shown, 14-18 are the electrophoresis results of HDL in Quality Control Product I when using the Changsha kit and when the concentration of the lower layer gel of the present invention is 6.0%, 6.5%, 7.0%, and 7.5%, respectively. The staining intensity of the Changsha kit is slightly weaker, and it is not the case that the greater the concentration of the lower layer gel of the present invention, the better. When the concentration of the lower layer gel reaches 7.5%, the staining intensity is significantly inferior to that of lanes 15-17.

[0149] Example 11: High-Density Lipoprotein Subtype Detection Method

[0150] The detection is carried out according to the following steps.

[0151] S01: Use polyacrylamide gel electrophoresis to separate high-density lipoproteins in serum or plasma.

[0152] S02: Stain the high-density lipoproteins in the serum or plasma with a staining solution. After reacting with the staining solution at room temperature for 15 minutes, take 20 microliters of the mixed solution and add it to the gel column.

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

[0154] S04: The results of the electrophoresis separation of the high-density lipoprotein subtypes are obtained as an optical density map of the high-density lipoprotein electrophoresis band through the scanning system of the electrophoresis device.

[0155] S05: Convert the optical density map into a peak pattern through analysis software, and perform analysis on the area ratio of each peak pattern in the peak pattern.

[0156] S06: Obtain the accurate content of each component of the high-density lipoprotein subtype according to the detected total cholesterol content and the ratio of the area of each peak pattern.

[0157] Example 12: Preparation Method of Sudan Black B Derivative Sudan Black B2 Staining Solution, including the following steps:

[0158] Take the concentration of Sudan Black B2 as 0.165% (W / V), and add it to isopropanol. Based on the total volume of the added isopropanol, the volume percentage of the isopropanol contained therein ranges from 99%.

[0159] Add 1-methyl-2-pyrrolidone with a volume ratio of 1%.

[0160] Then add Triton X-405, and its usage volume ratio is 0.2% (V / V).

[0161] After fully mixing the above mixed solution and waiting for Sudan Black B2 to be completely dissolved, store it in the dark.

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

[0163] Step (1), preparing a liquid, acrylamide, NN methylene bisacrylamide, base, TEMED, ammonium persulfate, sucrose, Proclin300, MgCl 2、 BSA was added into deionized water according to the actual concentration, and after being fully stirred, the pH value was adjusted, and 1.2 ml was added into the glass column, and purified water was added for gelling for 30 minutes. After gelling, a gel column with a polyacrylamide content of 7.28 wt% was obtained, which was the lower gel column;

[0164] Step (2), prepare gel column liquid, acrylamide, NN methylene bisacrylamide, base, TEMED, ammonium persulfate, sucrose, Proclin300, MgCl 2 According to the actual concentration, add it into purified water, stir it thoroughly, adjust the pH value, add 350 μL into the glass column, add purified water for gel pressing for 40 minutes, and after gelling, a gel column with a polyacrylamide content of 2.1 wt% is obtained, which is the upper gel column;

[0165] Step (3), placing the gel column obtained in steps (1) and (2) into a wide-mouth plastic bottle, and adding storage buffer;

[0166] Step (4), preparing a dyeing solution, taking Sudan Black B2 at a concentration of 0.165% (W / V), adding it to isopropanol, wherein 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 Triton X-405 was added thereto at a volume ratio of 0.2% (V / V);

[0169] After the above mixed solution is fully mixed and Sudan Black B2 is completely dissolved, it is stored away from light.

[0170] Step (5), mixing of buffer powder, the buffer powder containing base and boric acid. Among them, The dosage of base is 11g / 1.2L, and the dosage of boric acid is 5.5g / 1.2L; the above two materials are stirred and mixed by a blender, and the mixed mixture is divided into glass bottles according to the dosage of 16.5g / bottle;

[0171] Step (6), preparation of quality control, the received samples, after passing the safety test, are inactivated at 56°C for 30 minutes, mixed, and diluent is added as needed, and the test value is assigned, and the samples are divided into 100 μl tubes for quality control;

[0172] Step (7), labeling the components of steps (3), (4), (5), and (6); and inspecting the semi-finished product obtained;

[0173] Step (8), packing the semi-finished product that has passed the inspection in step (7) into a box to obtain a finished high-density lipoprotein subtyping detection kit;

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

[0175] Step (9), performing finished product testing on the finished high-density lipoprotein subtyping detection kit obtained in step (8);

[0176] Step (10), the finished product that has passed the inspection in step (9) is affixed with a qualified label and put into storage.

[0177] like Figure 4-9 As shown, the present invention can use the gel column with different gel concentrations in the lower layer to perform high-density lipoprotein typing. The results show that the higher the concentration, the better the effect, that is, it does not show concentration dependence.

[0178] Example 14: Comparative experiment of Sudan Black B, Sudan Black B2 dyeing solution and three samples

[0179] Attached Fig.10 It is a comparison chart of electrophoresis scans after Sudan Black B staining solution was stained with 3 samples for 30 minutes and Sudan Black B2 was stained with 3 samples for 15 minutes. The electrophoresis scan shows that: 21 is the reaction of Sudan Black B with 1# sample at 37℃ for 30 minutes; 22 is the reaction of Sudan Black B derivative B2 with 1# sample at room temperature for 15 minutes; 23 is the reaction of Sudan Black B with 2# sample at 37℃ for 30 minutes; 24 is the reaction of Sudan Black B derivative B2 with 2# sample at room temperature for 15 minutes; 25 is the reaction of Sudan Black B with 3# sample at 37℃ for 30 minutes; 26 is the reaction of Sudan Black B derivative B2 with 3# sample at room temperature for 15 minutes.

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

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

Claims

1. A method for detecting high-density lipoprotein subtyping, characterized in that: Follow these steps to perform the test: S01: Obtain serum or plasma samples; S02: After staining the serum or plasma with a staining solution, the mixed solution is added to a gel column; the gel column is divided into an upper and lower layer, wherein the concentration of the upper gel column is 2.0wt%-2.2wt% polyacrylamide, and the concentration of the lower gel column is 6.0wt%-7.5wt% polyacrylamide; S03: Under the action of the electric field in the electrophoresis tank, the subtypes of high-density lipoprotein are separated in order according to the size of high-density lipoprotein particles through the molecular sieving effect of polyacrylamide gel; S04: The results of the high-density lipoprotein subtype electrophoretic separation are subjected to an analysis system of an electrophoresis device to obtain an optical density spectrum of high-density lipoprotein electrophoresis bands; S05: converting the optical density spectrum into a peak type spectrum through analysis software, and calculating the area ratio of each peak type in the peak type spectrum; S06: Obtain the accurate content of each component of high-density lipoprotein subtype according to the ratio of the detected total cholesterol content and the area of ​​each peak type.

2. The high-density lipoprotein subtyping detection method according to claim 1, characterized in that: The dyeing solution contains Sudan Black B2.

3. The high-density lipoprotein subtyping detection method according to claim 1, characterized in that: The preparation method of the dyeing solution comprises the following steps: Sudan Black B2 with a concentration of 0.1wt%-1.5% (W / V) is added to one selected from the group consisting 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 the 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%; 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 is 0.05-0.5% (V / V); After the mixed solution is fully mixed, it is stored away from light to obtain the dyeing solution.

4. The high-density lipoprotein subtyping detection method according to claim 1, characterized in that: The usage volume of the upper gel column is 200-400 μl, and the usage volume of the lower gel column is 1.0-1.3 ml; Said The usage concentration of base is 0.2-2% (W / V), the usage concentration of sucrose is 0.5-2% (W / V), the usage concentration of TEMED 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 salt ion concentration is 0.01-0.2% (W / V), and the BSA concentration is 0.005%-0.5%.

5. A high-density lipoprotein subtyping detection kit, characterized in that: The kit comprises a gel column bottle and a staining solution; The staining solution contains Sudan Black B2; The gel column bottle contains a gel column, which is divided into an upper and lower layer. The concentration of the upper gel column is 2.0-2.2wt% polyacrylamide, and the concentration of the lower gel column is 6.0-7.5wt% polyacrylamide.

6. The high-density lipoprotein subtyping detection kit according to claim 5, characterized in that: The kit comprises a test kit body, an EPE lining, a gel column bottle, a dye solution, a buffer powder, a quality control product I and a quality control product II; The concentration of the lower gel column is 6.5-7.0 wt% polyacrylamide.

7. The high-density lipoprotein subtyping detection kit according to claim 5, characterized in that: The polyacrylamide comprises acrylamide, NN methylenebisacrylamide, and NN methylenebisacrylamide. The main components of the prepared gel solution are: acrylamide, NN methylenebisacrylamide, NN methylenebisacrylamide, base, TEMED, ammonium persulfate and sucrose, Proclin300, salt ions and BSA.

8. The method for preparing the high-density lipoprotein subtyping detection kit according to any one of claims 5 to 7, characterized in that: The following steps are involved: Step (1), preparing a liquid, acrylamide, NN methylene bisacrylamide, Base, TEMED, ammonium persulfate, sucrose, Proclin300, MgCL2, BSA are added to purified water or deionized water according to the actual use concentration, and after being fully stirred, the pH value is adjusted, and 1.0-1.3 ml is added to the glass column, and water or ethanol is added for gelling for not less than 30 minutes. After gelling, a gel column with a polyacrylamide content of 6.01-7.49wt% is obtained, which is the lower gel column; Step (2), preparing a liquid, acrylamide, NN methylene bisacrylamide, Base, TEMED, ammonium persulfate, sucrose, Proclin300 and MgCl2 are added to pure water or deionized water according to the actual use concentration, and after being fully stirred, the pH value is adjusted to complete the preparation of the glue solution; remove the glue pressing water on the upper part of the glass tube in step (1); take 200-400 microliters into the upper glass column of the lower layer glue, add water or ethanol to press the glue for not less than 30 minutes, and after gelation, a gel column with a polyacrylamide content of 2.01-2.19wt% is obtained, which is the upper layer gel column; Step (3), placing the gel column obtained in steps (1) and (2) into a wide-mouth plastic bottle, and adding storage buffer; Step (4), preparing a solution, setting the concentration of Sudan Black B derivative Sudan Black B2 to 0.1-1.5% (W / V), adding Sudan Black B derivative Sudan Black B2 to one selected from the group consisting of acetone, propylene glycol, isopropanol and ethylene glycol, wherein the volume percentage of the 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, isopropanol or ethylene glycol; adding DMF or 1-methyl-2-pyrrolidone therein at a volume ratio of 0.5-1.5%; further adding Triton X-405 or Triton X-100 and Tween as surfactants, wherein the volume of the surfactant used is 0.05-0.5% (V / V); fully mixing the above mixed solution, packaging it and storing it in the dark; 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 above two materials are stirred and mixed by a blender, and the mixed mixture is divided into glass bottles according to the dosage of 16.5g / bottle; Step (6), preparation of quality control, the received samples, after passing the safety test, are inactivated at 56°C for 30 minutes, mixed, and then tested and assigned, and packaged as quality control, each aliquot is 100 μl; Step (7), labeling the components of steps (3), (4), (5), and (6); and inspecting the semi-finished product obtained; Step (8), packing the semi-finished product that has passed the inspection in step (7) into a box to obtain a finished high-density lipoprotein subtyping detection kit.

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

10. Use of the detection method according to claim 1 or the detection kit according to claim 5 in the quantitative detection of high-density lipoprotein subtypes.

Images