Lipoprotein sample separation method based on instant staining

Abstract

The invention relates to the technical field of biological detection, and discloses an instant staining lipoprotein sample separation method, which comprises the following steps: S1, lipoprotein staining: taking 10 [mu] L of a sample loading staining solution, adding 50 [mu] L of a plasma lipoprotein sample, and completing staining without waiting and heating incubation; s2, sample loading of dyed lipoprotein: assembling a sample density separation liquid pre-loading column in a disc electrophoresis tank, adding an electrophoresis buffer solution, and adding the lipoprotein sample dyed in the step S1 to the upper end of the sample density separation liquid pre-loading column; and S3, electrophoretic separation of the lipoprotein subtypes: switching on a power supply of an electrophoresis apparatus, enabling the lipoprotein with negative charges to migrate to a positive electrode under the action of an electric field, and realizing separation according to different particle sizes of the lipoprotein subtypes. The lipoprotein loading staining solution is good in dispersity and uniformity, and in the preparation process of the lipoprotein loading staining solution, the dye does not need to be filtered to remove insoluble substances and does not need to be heated; the staining solution is good in stability and can be stored at normal temperature.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, in particular to a method for separating lipoprotein samples by instant staining. Background Art

[0002] The prevalence of cardiovascular disease in China is on a continuous rise. The "Guidelines for the Prevention and Treatment of Dyslipidemia in Chinese Adults (2016 Revised Edition)" points out that dyslipidemia is an important risk factor for atherosclerotic cardiovascular disease.

[0003] Among the blood lipid test items, the basic test indicators include total cholesterol (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglycerides. Among them, LDL-C is called "bad cholesterol", which is the most basic factor leading to atherosclerosis and the biggest risk factor for cardiovascular and cerebrovascular health.

[0004] Early detection of dyslipidemia and monitoring of changes in blood lipid levels are important measures to prevent atherosclerotic cardiovascular disease. However, studies have found that more than 40% of people with normal blood lipids have carotid artery plaques; and a study of 130,000 hospitalized patients diagnosed with coronary artery disease in the United States found that almost half of the cases had normal LDL-C levels; these results suggest that routine blood lipid screening in physical examinations has certain limitations, and normal levels of LDL-C do not fully represent a low risk of cardiovascular disease. The detection and analysis of blood lipid subcomponents, especially the detection and analysis of low-density lipoprotein subtypes, has received increasing attention.

[0005] Plasma lipoproteins can usually be divided into four categories: chylomicrons (CM), very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL). Among them, low density lipoproteins are heterogeneous and can be divided into up to 7 subtypes based on particle diameter and density, named LDL-1 to LDL-7. Among them, type 1-2 LDL (large buoyant LDL, 1 bLDL) has a particle diameter of >27nm and a density of <1.03g / mL, which is responsible for normal cholesterol transport; type 3-7 LDL (small dense LDL) has a particle diameter of <26nm and a density of >1.04g / mL, which is easy to form oxidized LDL, promoting the occurrence and development of atherosclerosis; it is slowly cleared and easily deposited on the blood vessel wall to form atherosclerotic plaques.

[0006] Clinical laboratories have a variety of methods for separating plasma lipoproteins, the most classic of which is ultracentrifugation. However, it is difficult to promote and is not widely used because it is cumbersome and time-consuming to operate and requires an ultracentrifugation device; the most commonly used method is electrophoresis analysis. Lipoprotein electrophoresis is a useful tool for clinical diagnosis of hyperlipoproteinemia, helps to understand the blood lipid status of coronary heart disease, and provides a good research method for clinical observation of atherosclerosis, coronary heart disease (CHD) and the efficacy of lipid-lowering drugs.

[0007] The methods of plasma lipoprotein electrophoresis can be divided into two common methods: agarose gel lipoprotein electrophoresis and polyacrylamide gel lipoprotein electrophoresis according to the different supporting media.

[0008] Lipoprotein subtypes play an important role in lipoprotein metabolism disorders. However, agarose gel is difficult to meet the clinical demand for rapid, simple and accurate determination of lipoprotein subtypes due to its low resolution. Polyacrylamide gel electrophoresis, as the highest resolution analysis technology for biological macromolecules, is increasingly widely used in lipoprotein subtype analysis.

[0009] Polyacrylamide gel electrophoresis usually uses pre-staining technology, using lipophilic dyes to color lipids in plasma before electrophoresis. Due to the limitations of Sudan Black B and other lipid dyes, such as poor water solubility, easy precipitation, instability, and uneven dispersion in the solute, traditional lipoprotein electrophoresis staining solutions are difficult to store, have poor accuracy, and need to be heated at 37°C for staining for 30 minutes. The Lipoprotein system kit of Quantimetrix in the United States uses a method of mixing the dye with the loading gel and then photopolymerizing it to pre-stain lipoproteins. However, this method requires 30 minutes to several hours to wait for the gel to be photopolymerized, and requires tools such as a light source and a fixed bracket. In addition, the process of mixing the sample and loading the gel will come into contact with highly toxic substances such as acrylamide, which is complicated and unsafe to operate, restricting the detection efficiency.

[0010] To this end, those skilled in the art provide a method for separating lipoprotein samples by instant staining to solve the problems raised in the above-mentioned background technology. Summary of the invention

[0011] In view of the shortcomings of the prior art, the present invention provides a method for separating lipoprotein samples by instant staining, which solves the problems of poor detection efficiency and complicated operation in the prior art.

[0012] To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for separating lipoprotein samples by instant staining, characterized in that it comprises the following steps:

[0013] S1: Lipoprotein staining: Take 10 μL of loading staining solution and add 50 μL of plasma lipoprotein sample. The staining is completed without waiting or heating incubation.

[0014] S2: Loading of stained lipoproteins: Assemble the sample density separation liquid preloaded column in the disc electrophoresis tank, add electrophoresis buffer, and load the lipoprotein sample stained in step S1 onto the upper end of the sample density separation liquid preloaded column;

[0015] S3: Electrophoretic separation of lipoprotein subtypes: Turn on the power of the electrophoresis instrument to allow the negatively charged lipoproteins to migrate to the positive electrode under the action of the electric field, and separate the lipoprotein subtypes according to their different particle sizes.

[0016] Preferably, the loading staining solution is prepared by mixing an organic solution and an aqueous solution;

[0017] The organic solution includes a lipid dye, a general solvent, a penetrant and a surfactant;

[0018] The aqueous solution includes a stabilizer, a buffer substance and a densifier.

[0019] Preferably, the lipid dye is one or more of Sudan Black III, Sudan Black IV, Sudan Black B, and Oil Red O.

[0020] Preferably, the general solvent is one or more of ethanol, methanol, ethylene glycol, propylene glycol, glycerol and isopropanol;

[0021] The penetrant is one or more of dimethyl sulfoxide, N,N-dimethylformamide and N,N-dimethylacetamide;

[0022] The surfactant is one or more of polysorbate, polyoxyethylene-8-octylphenyl ether, and propylene oxide-ethylene oxide-vinyl diamine copolymer.

[0023] Preferably, the stabilizer in the aqueous solution is one or more of polyethylene glycol, polypropylene glycol, ascorbic acid, sodium hydroxymethyl cellulose, and sodium alginate;

[0024] The buffer substance is selected from one or more of tris(hydroxymethyl)aminomethane, 4-hydroxyethylpiperazineethanesulfonic acid, bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane, glycine, boric acid and taurine;

[0025] The densifier is one or more of sugars or salts.

[0026] Preferably, the final concentration of the lipid dye in the staining solution is 0.05 w / v% to 0.5 w / v%;

[0027] The final concentration of the general solvent in the staining solution is 30 v / v% to 90 v / v%;

[0028] The final concentration of the penetrant in the staining solution is 5 v / v% to 50 v / v%;

[0029] The final concentration of the surfactant in the dyeing solution is 0.1 v / v% to 2 v / v%.

[0030] Preferably, the final concentration of the stabilizer in the dyeing solution is 0.5 w / v% to 2 w / v%;

[0031] The final concentration of the buffer substance in the staining solution is 50mM to 200mM;

[0032] The final concentration of the densifier in the dyeing solution is 0.5 w / v% to 8 w / v%.

[0033] Preferably, the method for preparing the loading staining solution comprises the following steps:

[0034] Preparation of organic solution: dissolve lipid dye in general solvent, add penetrant and surfactant and stir evenly;

[0035] Preparation of aqueous solution: dissolve the buffer substance in water and adjust to alkaline pH, add densifier and stabilizer, stir evenly and filter;

[0036] The organic solution and the aqueous solution are mixed in a ratio of 1:1 to 8:1, and stirred evenly to obtain a loading staining solution.

[0037] The present invention provides a method for separating lipoprotein samples by instant staining, which has the following beneficial effects:

[0038] 1. Provide a lipoprotein loading staining solution with good dispersibility and uniformity. The dye does not need to be filtered to remove insoluble matter or heated during the preparation process. The staining solution has good stability and can be stored at room temperature.

[0039] 2. It realizes instant staining of plasma lipoprotein samples without heating or incubation, and the operation is simple and fast;

[0040] 3. Due to the good penetration and uniformity of the dye, a good staining effect can be achieved, which improves the resolution of the electrophoresis bands for separation of lipoprotein subtypes;

[0041] 4. Make the lipoprotein subtype separation method simpler, promote the development of blood lipid typing methods, and provide a basis for the diagnosis and treatment of cardiovascular and cerebrovascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is a schematic diagram of the method steps of the present invention;

[0043] Figure 2 The electrophoresis pattern of lipoprotein after staining in Example 1 of the present invention;

[0044] Figure 3This is the electrophoresis pattern of lipoprotein after staining in Example 2 of the present invention;

[0045] Figure 4 The electrophoresis pattern of lipoprotein after staining in Example 3 of the present invention;

[0046] Figure 5 This is the electrophoresis pattern of lipoprotein after staining in Example 4 of the present invention;

[0047] Figure 6 This is the electrophoresis pattern of lipoprotein after staining in Example 5 of the present invention;

[0048] Figure 7 This is the electrophoresis pattern of the lipoprotein after staining in Example 6 of the present invention. DETAILED DESCRIPTION

[0049] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings 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.

[0050] Please refer to the attached Figure 1 -Attached Figure 2 Example 1: A method for preparing a plasma lipoprotein loading staining solution

[0051] S1: Preparation of organic solution:

[0052] ① Weigh 0.15g Sudan Black B dye and dissolve it in 45mL 1,2-propylene glycol, stirring to dissolve evenly;

[0053] ② Take 20 mL of N,N-dimethylformamide and add it to the solution obtained in step ①;

[0054] ③ Add 0.5 mL of Tween-20 to the solution obtained in step ②;

[0055] ④Stir well and set aside.

[0056] S2: Preparation of aqueous solution:

[0057] ① Weigh 36.34 g of tris(hydroxymethyl)aminomethane and dissolve it in 160 mL of ultrapure water, adjust the pH to 8.8 with HCl, and make up to 200 mL with ultrapure water;

[0058] ② Weigh 40g of sodium chloride and dissolve it in the buffer solution in step ①, and make up to 200mL;

[0059] ③ Weigh 2g of polyethylene glycol 10000, add it to the solution in step ②, and stir evenly;

[0060] ④ Filter using a 0.45μm filter membrane and set aside.

[0061] S3: Mixing of organic solution and aqueous solution: Mix the above organic solution and aqueous solution in a ratio of 6:4, stir evenly; store at room temperature and away from light to obtain a lipoprotein loading staining solution.

[0062] Please refer to the attached Figure 3 Example 2: A method for preparing a plasma lipoprotein loading staining solution

[0063] S1: Preparation of organic solution:

[0064] ① Weigh 0.2g Sudan Black B dye and dissolve it in 40mL 1,2-propylene glycol, stir and dissolve evenly;

[0065] ② Measure 20 mL of dimethyl sulfoxide and add it to the solution obtained in step ①;

[0066] ③ Add 2 mL of Triton X-45 to the solution obtained in step ②;

[0067] ④Stir well and set aside.

[0068] S2: Preparation of aqueous solution:

[0069] ① Weigh 36.34 g of tris(hydroxymethyl)aminomethane and dissolve it in 160 mL of ultrapure water, adjust the pH to 8.8 with HCl, and make up to 200 mL with ultrapure water;

[0070] ② Weigh 40g of sodium chloride and dissolve it in the buffer solution in step ①, and make up to 200mL;

[0071] ③ Weigh 2g of polyethylene glycol 10000, add it to the solution in step ②, and stir evenly.

[0072] ④ Filter using a 0.45μm filter membrane and set aside.

[0073] S3: Mixing of organic solution and aqueous solution: Mix the above organic solution and aqueous solution in a ratio of 6:4, stir evenly; store at room temperature and away from light to obtain a lipoprotein loading staining solution.

[0074] Please refer to the attached Figure 4 Example 3: A method for preparing a plasma lipoprotein loading staining solution

[0075] The difference from Example 1 is that in this example, the densifier is replaced by sucrose instead of sodium chloride.

[0076] Please refer to the attached Figure 5 Example 4: A method for preparing a plasma lipoprotein loading staining solution

[0077] The difference from Example 1 is that the preparation method of the aqueous solution in this example is adjusted as follows:

[0078] S2: Preparation of aqueous solution:

[0079] ① Weigh 21.81g of tris(hydroxymethyl)aminomethane) and 11.13g of boric acid and dissolve them in 160mL of ultrapure water. Stir to dissolve and then adjust the volume to 200mL.

[0080] ② Weigh 40g of sodium chloride and dissolve it in the buffer solution in step ①, and make up to 200mL;

[0081] ③ Weigh 2g of polyethylene glycol 10000, add it to the solution in step ②, and stir evenly;

[0082] ④ Filter using a 0.45μm filter membrane and set aside.

[0083] Other preparation methods are the same as in Example 1.

[0084] Please refer to the attached Figure 6 Example 5: A method for preparing a plasma lipoprotein loading staining solution

[0085] The difference from Example 1 is that in this example, the mixing ratio of the organic solution and the aqueous solution is replaced with 9:1.

[0086] Please refer to the attached Figure 7 Example 6: A method for preparing a plasma lipoprotein loading staining solution

[0087] The difference from Example 1 is that in this example, the mixing ratio of the organic solution and the aqueous solution is replaced with 1:2.

[0088] Example 7: Method for separation of lipoprotein subtypes using instant lipoprotein loading staining solution:

[0089] S1: Lipoprotein staining: Take 10 μl of the loading staining solution of Example 1 of the present invention, add 50 μl of plasma lipoprotein sample, mix well and staining is completed;

[0090] S2: Loading of stained lipoproteins: Take a sample density separation liquid preloaded column for lipoprotein subtype separation, assemble it in a disc electrophoresis tank, pour in the electrophoresis buffer, and pipette the stained lipoprotein sample in S1 and load it on the upper end of the sample density separation liquid preloaded column;

[0091] S3: Electrophoretic separation of lipoprotein subtypes: Turn on the electrophoresis instrument and perform constant current electrophoresis at 3 mA / tube for about 70 min.

[0092] S4: After the electrophoresis is completed, take out the sample density separation liquid pre-loaded column and place it under a scanner for scanning and observation of the lipoprotein subtype electrophoresis spectrum.

[0093] Example 8: Observation on the stability of lipoprotein staining solution

[0094] The stability of the lipoprotein staining solution was observed by:

[0095] ① Store the lipoprotein staining solution at room temperature and away from light for half a year, take it out and observe whether there is any residual dye precipitation. If there is no residual dye precipitation, it means that the stability is good;

[0096] ② Take out the lipoprotein staining solution to stain the plasma lipoprotein and observe whether the plasma lipoprotein is stained blue-black. If the plasma lipoprotein cannot be stained blue-black but the dye itself is gray-black, it means that the lipoprotein staining solution is not stable.

[0097] ③ Use lipoprotein staining solution to separate and detect lipoprotein subtypes. If the resolution of the electrophoresis bands decreases, it means that the stability has decreased.

[0098] Comparative Example 1: Sudan Black B dyeing solution was prepared according to the method described in CN102181173A

[0099] Weigh 0.125 g of Sudan Black B, add 25 mL of organic solvent (isopropanol: glycerol = 4:1), dissolve in the dark, place in a 37°C water bath for 24 h, centrifuge to remove the residue, and store in a sealed brown bottle at room temperature.

[0100] The volume ratio of blood lipids and Sudan Black B staining solution was 10:1. 100 μl of serum and 10 μl of staining solution were oscillated and mixed, and then incubated in a 37°C water bath for 45 min.

[0101] from Figure 2 , Figure 3 , Figure 4 , Figure 5 It can be found that the lipoprotein staining solution provided by the present invention can not only be used for rapid and instant staining, but also has an excellent staining effect, and the bands of the lipoprotein electrophoresis spectrum after staining are sharp and clear.

[0102] Figure 6 This is the electrophoresis spectrum of lipoprotein staining with a staining solution in which the organic solution and the aqueous solution are mixed in a ratio of 9:1 in Example 5. It can be seen that since the ratio of the organic solution to the aqueous solution exceeds the range described in the present invention, the proportion of the organic solution is too high, causing partial denaturation of the protein of the lipoprotein in the sample, resulting in the inability of some lipoproteins at the loading origin to migrate normally and aggregate at the loading origin, causing errors in the electrophoresis results.

[0103] Figure 7 The electrophoresis spectrum of lipoprotein staining with the staining solution in Example 6 of the present invention, in which the organic solution and the aqueous solution are mixed in a ratio of 1:2, shows that the staining effect is poor and the lipoprotein bands are dim due to the high proportion of the aqueous solution.

[0104] The stability test results of the dyeing solution of the embodiment of the present invention are shown in Table 1.

[0105]

[0106]

[0107] The stability observation results show that the high proportion of aqueous solution in the staining solution of Example 6 leads to poor dissolution of the dye, and there is a residue of dye precipitated at the bottom of the staining solution, which further leads to poor lipoprotein staining effect and low electrophoresis band resolution; it means that when the proportion of aqueous solution is higher than the above range, the stability of the staining solution is poor. In Example 5, due to the excessive organic solution part, the protein is affected by the organic solvent and denatured, and the electrophoresis band resolution is also low.

[0108] The dyeing solution of the control example needs to be heated in a water bath for 24 hours during preparation, and centrifugation is required to remove the residue, which is time-consuming and cumbersome to operate. It is also necessary to heat the water bath for 45 minutes during dyeing, and the dyeing time is long and cumbersome. In comparison, the preparation of the dyeing solution provided by the present invention is simple and fast, does not require heating and centrifugation, and dyeing is rapid and only requires mixing, does not require redundant operations, and greatly saves operating steps; at the same time, good dyeing solution stability and uniformity also ensure excellent dyeing effect and resolution.

[0109] 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 separating lipoprotein samples by instant staining, characterized in that: The following steps are involved: S1: Lipoprotein staining: Take 10 μL of loading staining solution and add 50 μL of plasma lipoprotein sample. The staining is completed without waiting or heating incubation. S2: Loading of stained lipoproteins: Assemble the sample density separation liquid preloaded column in the disc electrophoresis tank, add electrophoresis buffer, and load the lipoprotein sample stained in step S1 onto the upper end of the sample density separation liquid preloaded column; S3: Electrophoretic separation of lipoprotein subtypes: Turn on the power of the electrophoresis instrument to allow the negatively charged lipoproteins to migrate to the positive electrode under the action of the electric field, and separate the lipoprotein subtypes according to their different particle sizes.

2. The method for separating lipoprotein samples by instant staining according to claim 1, characterized in that: The loading staining solution is prepared by mixing an organic solution and an aqueous solution; The organic solution includes a lipid dye, a general solvent, a penetrant and a surfactant; The aqueous solution includes a stabilizer, a buffer substance and a densifier.

3. The method for separating lipoprotein samples by instant staining according to claim 1, characterized in that: The lipid dye is one or more of Sudan Black III, Sudan Black IV, Sudan Black B and Oil Red O.

4. The method for separating lipoprotein samples by instant staining according to claim 2, characterized in that: The general solvent is one or more of ethanol, methanol, ethylene glycol, propylene glycol, glycerol and isopropanol; The penetrant is one or more of dimethyl sulfoxide, N,N-dimethylformamide and N,N-dimethylacetamide; The surfactant is one or more of polysorbate, polyoxyethylene-8-octylphenyl ether, and propylene oxide-ethylene oxide-vinyl diamine copolymer.

5. The method for separating lipoprotein samples by instant staining according to claim 2, characterized in that: The stabilizer in the aqueous solution is one or more of polyethylene glycol, polypropylene glycol, ascorbic acid, sodium hydroxymethyl cellulose, and sodium alginate; The buffer substance is selected from one or more of tris(hydroxymethyl)aminomethane, 4-hydroxyethylpiperazineethanesulfonic acid, bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane, glycine, boric acid and taurine; The densifier is one or more of sugars or salts.

6. The method for separating lipoprotein samples by instant staining according to claim 2, characterized in that: The final concentration of the lipid dye in the staining solution is 0.05 w / v% to 0.5 w / v%; The final concentration of the general solvent in the staining solution is 30 v / v% to 90 v / v%; The final concentration of the penetrant in the staining solution is 5 v / v% to 50 v / v%; The final concentration of the surfactant in the dyeing solution is 0.1 v / v% to 2 v / v%.

7. The method for separating lipoprotein samples by instant staining according to claim 2, characterized in that: The final concentration of the stabilizer in the dyeing solution is 0.5 w / v% to 2 w / v%; The final concentration of the buffer substance in the staining solution is 50mM to 200mM; The final concentration of the densifier in the dyeing solution is 0.5 w / v% to 8 w / v%.

8. The method for separating lipoprotein samples by instant staining according to claim 2, characterized in that: The method for preparing the loading staining solution comprises the following steps: Preparation of organic solution: dissolve lipid dye in general solvent, add penetrant and surfactant and stir evenly; Preparation of aqueous solution: dissolve the buffer substance in water and adjust to alkaline pH, add densifier and stabilizer, stir evenly and filter; The organic solution and the aqueous solution are mixed in a ratio of 1:1 to 8:1, and stirred evenly to obtain a loading staining solution.

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