WO2017020830A1 - METHOD FOR USING IMPROVED GEL ELECTROPHORESIS TO DETECT PREβ1 HIGH DENSITY LIPOPROTEIN IN SERUM, AND APPLICATION - Google Patents

METHOD FOR USING IMPROVED GEL ELECTROPHORESIS TO DETECT PREβ1 HIGH DENSITY LIPOPROTEIN IN SERUM, AND APPLICATION Download PDF

Info

Publication number
WO2017020830A1
WO2017020830A1 PCT/CN2016/093019 CN2016093019W WO2017020830A1 WO 2017020830 A1 WO2017020830 A1 WO 2017020830A1 CN 2016093019 W CN2016093019 W CN 2016093019W WO 2017020830 A1 WO2017020830 A1 WO 2017020830A1
Authority
WO
WIPO (PCT)
Prior art keywords
hdl
preβ1
gel
serum
electrophoresis
Prior art date
Application number
PCT/CN2016/093019
Other languages
French (fr)
Chinese (zh)
Inventor
陈允钦
张晓金
Original Assignee
复旦大学附属中山医院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 复旦大学附属中山医院 filed Critical 复旦大学附属中山医院
Publication of WO2017020830A1 publication Critical patent/WO2017020830A1/en

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14Extraction; Separation; Purification
    • C07K1/24Extraction; Separation; Purification by electrochemical means
    • C07K1/26Electrophoresis
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416Systems
    • G01N27/447Systems using electrophoresis

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a method and an application for improved gel electrophoresis for detecting pre-beta1 high-density lipoprotein.
  • Pre-serum ⁇ 1 high-density lipoprotein is a nascent HDL that plays an important role in the reverse trans-transport of cholesterol and has anti-atherosclerotic effects.
  • pre ⁇ 1-HDL high-density lipoprotein
  • GGE gradient gel electrophoresis
  • the object of the present invention is to provide a non-linear gradient gel and/or a Sudan Black B staining solution for the separation and quantification of serum pre ⁇ 1-HDL for the deficiencies in the prior art.
  • a second object of the present invention is to provide a method for separating and quantifying serum pre ⁇ 1-HDL.
  • the technical solution provided by the present invention is: a nonlinear gradient gel and/or a Sudan
  • the concentration of Sudan Black B in the Sudan Black B staining solution was 0.5-1.0 w/v%, and the solvent was isopropanol and ethylene glycol in a volume ratio of 4:1.
  • the nonlinear gradient gel is used as a one-dimensional nonlinear gradient tubular gel system and a two-dimensional nonlinear gradient slab gel system to separate serum pre ⁇ 1-HDL electrophoresis medium, and the one-dimensional nonlinear gradient tubular gel system Sudan black B prestained serum pre ⁇ 1-HDL was isolated.
  • the technical solution provided by the present invention is: a method for separating and quantitatively analyzing serum pre ⁇ 1-HDL, and using a nonlinear gradient gel to accurately separate and quantitatively analyze serum pre ⁇ 1-HDL, wherein nonlinear gradient condensation
  • the gum consisted of a polyacrylamide gel with three concentrations of 3.0%, 3.6% and 7.0%.
  • the nonlinear gradient gel is used as a one-dimensional nonlinear gradient tubular gel system and a two-dimensional nonlinear gradient slab gel system to separate serum pre ⁇ 1-HDL electrophoresis medium.
  • the concentration of Sudan black B in the Sudan black B staining solution is 0.5-1.0 w/v%, solvent isopropanol and B The volume ratio of the diol was 4:1.
  • the three-stage concentration is 3.0%, 3.6% and 7.0% of polyacrylamide gel as separating glue, the corresponding glue layer height is 7mm, 40mm and 45mm, respectively, the error range is ⁇ 3mm, three-layer separation rubber The total height is 82mm and the error range is ⁇ 5mm.
  • the non-linear gradient gel has a specification of an inner diameter of 6-8 mm and an outer diameter of 8-10 mm in a one-dimensional tubular gel system, and the nonlinear gradient gel has a thickness of 2 in a two-dimensional slab gel system. 3mm, width 60-80mm.
  • the one-dimensional nonlinear gradient tubular gel system separates pre-stained serum pre ⁇ 1-HDL from Sudan black B.
  • the electrophoresis buffer contains 5 mM Tris and 38.4 mM glycine.
  • the electrophoresis conditions are 100 V, 2.5 h, and the migration distance is 65 after electrophoresis.
  • the isolated staining zone at -75 mm is defined as pre ⁇ 1-HDL; the two-dimensional nonlinear gradient plate gel system separates serum pre ⁇ 1-HDL, the running buffer is 5 mM Tris and 38.4 mM glycine, and the electrophoresis conditions are 100 V, 3.5 h.
  • the imprinted zone at the origin of 65°-75 mm from the origin of the pre ⁇ lane was defined as pre ⁇ 1-HDL and free Apo A1 multimer, and the imprinted band at the origin 80-90 mm was defined as free Apo A1 monomer.
  • the two-dimensional non-linear gradient slab gel system separates serum pre ⁇ 1-HDL, and a phenol red solution is added as an electrophoresis indicator in the sample application area before electrophoresis.
  • the one-dimensional nonlinear gradient tubular gel system was used to separate the pre-stained serum pre ⁇ 1-HDL from Sudan black B, and the density was scanned by optical density.
  • the blank gel scan value was used as a uniform reference to calculate the pre ⁇ 1-HDL accounted for the total blood lipids. Percentage; two-dimensional nonlinear gradient slab gel system for separation of serum pre ⁇ 1-HDL, free Apo A1 monomer and multimer, qualitatively by immunoblotting, chemiluminescence and film imaging techniques, comparing their relatives in the same batch content.
  • the method for establishing one-dimensional nonlinear GGE of the present invention uses a polyacrylamide gel having three concentrations of 3.0%, 3.6% and 7.0% as an electrophoresis medium to clearly separate the serum stained with Sudan black B. a specific HDL subclass, pre ⁇ 1-HDL;
  • the present invention also establishes a two-dimensional nonlinear GGE method capable of separating pre ⁇ 1-HDL and free Apo A1 very clearly;
  • the preparation process of the nonlinear concentration gradient independent layering of the invention is simple and easy, and no special mixing device is needed; the electrophoresis is only required to be ⁇ 3.5 h; the quantitative method does not require radioactive materials.
  • the nonlinear GGE established by the invention can accurately detect pre ⁇ 1-HDL, and has obvious superiority in performance.
  • FIG. 1 One-dimensional nonlinear GGE-lipophilic SBB staining map.
  • A Schematic representation of a one-dimensional nonlinear gradient tubular gel.
  • B Repeatability test results. The electrophoretic migration of pre ⁇ 1-HDL is 70 mm from the origin, showing a clear SBB staining zone. The coefficient of variation of pre ⁇ 1-HDL in the same blood sample was 2.31%, indicating that the method has good repeatability.
  • C One-dimensional nonlinear GGE-lipophilic SBB staining of clinical blood samples. pre ⁇ 1-HDL is abundant in neonatal cord blood, but lower in acute myocardial infarction patients and CETP variants than in healthy adults.
  • D Electrophoresis scan map.
  • FIG. 1 Two-dimensional nonlinear GGE-immunoblotting map.
  • A Schematic diagram of the preparation of a two-dimensional nonlinear gradient slab gel.
  • B Immunoblot map containing Apo A1 particles in serum after two-dimensional electrophoresis. The band of about 70 mm electrophoretically migrated on the pre ⁇ lane was pre ⁇ 1-HDL (including Apo A1 multimer), and the band of electrophoretic migration of about 85 mm was free Apo A1 monomer, and the isolated blot band was clearly visible. The spectrum of neonatal blood samples is more complex than that of adults.
  • C Schematic representation of two-dimensional nonlinear GGE-immunoblotting. The arrows indicate the direction of electrophoresis in one direction (1D) and two directions (2D).
  • FIG. 3 Experimental demonstration of pre ⁇ 1-HDL: particle density and in vitro degradation.
  • A Ultrasonic centrifugation separates serum HDL.
  • B Samples were taken from different density ranges after ultracentrifugation, and one-dimensional nonlinear GGE was performed after SBB staining.
  • pre ⁇ 1-HDL appeared in a density of 1.210 g/m1 and electrophoretically migrated to 70 mm.
  • C Two-dimensional nonlinear GGE-immunoblotting map, pre ⁇ 1-HDL also appeared in the density liquid of 1.210g/ml, migration The position is also at 70mm.
  • D DTNB in vitro inhibition assay.
  • pre ⁇ 1-HDL After normal serum in vitro at 37 ° C for 6 h, the content of pre ⁇ 1-HDL gradually decreased, and the degradation of this granule could be completely inhibited by DTNB. Because DTNB is an inhibitor of lecithin cholesterol acyltransferase, it can effectively inhibit the nascent pre ⁇ 1-HDL to the large particle HDL, so the results further demonstrate that this electrophoretic migration is 70mm and can be stained by lipophilic SBB. Above is pre ⁇ 1-HDL.
  • FIG. 4 Experimental demonstration of pre ⁇ 1-HDL: particle size and chemical composition.
  • A Electron micrograph of serum HDL. The arrow is referred to as pre ⁇ 1-HDL.
  • B Relative weight ratio of Apo A1 to cholesterol of serum HDL, wherein the ratio of pre ⁇ 1-HDL is the highest.
  • FIG. 5 Experimental demonstration of pre ⁇ 1-HDL: clinical case-control map.
  • A-B A typical two-dimensional nonlinear GGE-immunoblotting map of acute myocardial infarction patients and healthy control sera. The content of pre ⁇ 1-HDL in patients with acute myocardial infarction was significantly lower than that in healthy controls. Since pre ⁇ 1-HDL has an anti-atherosclerotic protective effect, such a result is logical.
  • C One-dimensional nonlinear GGE-lipophilic SBB staining of sera from patients with Tangier disease and healthy controls. Due to a defect in the ABCA1 gene, patients with Tangier disease are unable to produce pre ⁇ 1-HDL, resulting in complete loss in the map.
  • D Two-dimensional nonlinear GGE-immunoblotting map of serum of patients with Tangier disease. The free Apo A1 monomer was clearly visible at the migration position of 85 mm, while the micro-imprinted band was still visible at the migration position of 70 mm. Although the position was the same as that of pre ⁇ 1-HDL, it was not pre- ⁇ 1-HDL, but free Apo A1 poly-polymerization. body.
  • D Two-dimensional nonlinear GGE-immunoblotting map of free Apo A1 standard (0.2 ⁇ g, Sigma).
  • the blotting zone was mainly concentrated at the migration site of 85 mm, which was a free Apo A1 monomer, but a small number of blotting bands were still visible at the migration site of 70 mm, which is the Apo A1 multimer.
  • the Apo A1 multimer of serum from patients with Tangier disease can appear in the two-dimensional GGE-immunoblotting map, but because this part contains no lipid, the one-dimensional nonlinear GGE-lipophilic SBB staining map cannot be detected. Therefore, only one-dimensional nonlinear GGE can be used to accurately detect serum pre ⁇ 1-HDL.
  • FIG. 1 Two-dimensional linear GGE-immunoblotting map applied in China.
  • A One-dimensional agarose electrophoresis;
  • B Two-dimensional linear GGE.
  • Figure 7 Traditional two-dimensional linear GGE-immunoblotting map.
  • Initiator 1.0 g of ammonium persulfate (APS) was dissolved in dH 2 O to 10 ml, and stored at -80 ° C after dispensing.
  • APS ammonium persulfate
  • Staining solution Sultan black (SBB) 0.125 g dissolved in 20 ml of isopropanol and 5 ml of ethylene glycol, and left at 37 ° C in a water bath overnight, and stored in a sealed brown bottle at room temperature, valid for 6 months.
  • SBB Sultan black
  • Buffer H Tris 6.0 g and glycine 28.8 g were dissolved in dH 2 O to 1000 ml, stored at 4 ° C, and diluted 10 times with dH 2 O when used.
  • Tubular gel The specifications of the glass tube are 6 mm inner diameter, 8 mm outer diameter and 10 cm length. Seal one end of the glass tube and fix it vertically on the glue holder. Three different concentrations (7.0%, 3.6%, and 3.0%) of gels having a height of 45 mm, 40 mm, and 7 mm, respectively, were prepared according to Table 1. Each layer of gel was gently infused with 100 ⁇ l of dH 2 O to cover the surface immediately after filling, so that the surface of the gel was polymerized and flattened. After standing for 30 minutes, the gel layer was removed, and the aqueous layer was removed, and then the next layer of gel solution was poured.
  • Tube gel electrophoresis The prepared gel tube was placed vertically into a disk electrophoresis tank (DYY-III27A, Beijing Liuyi Electrophoresis Instrument Factory).
  • the electrophoresis buffer 600 ml of the positive electrode tank and 400 ml of the negative electrode tank
  • Pre-stained serum 50 ⁇ l/tube
  • the gel tube was removed, and the gel tube was scanned and imaged by a dual-band flying spot scanner (CS-9000, Shimadzu) and an optical scanner, respectively.
  • the flying spot scanning monochromatic light has a wavelength of 604 nm.
  • the scanning proceeds along the center line of the gel tube from the blank area to the origin.
  • the blank gel was used as the uniform reference, and the inflection point between the adjacent electrophoresis peaks was used as the segmentation marker, and the percentage of the area under each electrophoresis peak to the whole serum lipoprotein was calculated.
  • T concentration
  • C degree of crosslinking.
  • dH 2 o deionized distilled water
  • TEMED tetramethylethylenediamine
  • APS ammonium persulfate solution.
  • the glue preparation reagent is the same as above.
  • Agarose gel running buffer was used with 25 mM Tris-Tricine (pH 8.6).
  • the two-dimensional running buffer was 5 mM Tris and 38.4 mM glycine (pH 8.4). 0.3% glutaraldehyde fixative.
  • 1 Anti-Apo A1 antibody was purchased from Abcam.
  • 2 Anti-IgG was purchased from Novus Biologicals.
  • Electrophoresis of plate gel 4 ⁇ l of serum was separated by 0.75% agarose gel electrophoresis (100V, 1.5h), and 4 ⁇ l of SBB stained serum was used as a synchronization marker.
  • Agarose gel electrophoresis tank comes from GT Cell apparatus (Bio-Rad). After the electrophoresis was completed, a 40 mm long strip (thickness 2 mm) was cut along the lane from ⁇ to ⁇ , and the strip was transferred to the top of the slab gel, and two strips were simultaneously added.
  • the lipoprotein on the vinegar membrane was fixed with 0.3% glutaraldehyde solution for 10 min, rinsed with water and immunoblotted.
  • 5% de-fat milk-TBST and 5% calf serum albumin-TBST solution were added in the cold chamber for about 8 hours.
  • 1 anti-Apo A1 antibody was added (cool overnight). Unbound 1 antibody was washed away with a TBST solution on a shaker at room temperature.
  • 2 anti-IgG was added (1 h at room temperature). The unbound 2 antibody was washed away again with the TBST solution.
  • Finalization The luminescence reaction was studied and film imaging was performed in a dark room.
  • Human serum 6 ml was collected and adjusted to a density of 1.30 g/ml by using a formula (addition of potassium bromide 2.93 g). It was added to the bottom layer of a 28 ml ultracentrifuge tube, and the upper layer was covered with a density liquid of 1.063 g/ml.
  • the ultracentrifuge model Beckman L8-M has a rotor of 50.2 Ti and is centrifuged at 40K rpm, 12h, 10°C. After the end of the ultracentrifugation, a long needle was used to slowly suck out an equal volume of 1 ml of the separation solution of different density ranges from the bottom layer.
  • the densities were calculated by weighing 800 ⁇ l of the different density liquids after separation and weighing them directly with a precision balance. Finally, the volume of the separation solution of different density ranges was concentrated to 500 ⁇ l using an ultrafiltration tube (30 KD, Millipore). Samples were then taken for one-dimensional and two-dimensional GGE, respectively.
  • the electrophoretic bands of the serum HDL subclass are separately cut from the separation gel and chopped, and placed in a PBS solution containing antibiotics to freely diffuse in the cold chamber for 3-5 days.
  • the HDL solution was collected for dialysis and concentration through an ultrafiltration tube.
  • the dialysate was treated with a 0.4% sodium bicarbonate solution.
  • 10 ⁇ l of the treated HDL solution was mixed with an equal volume of 2% phosphotungstic acid solution, and a little was taken for negative staining transmission electron microscopy.
  • the transmission electron microscope is Nissan JEM-1200EX with an electron intensity of 80 kV and a magnification of 100K.
  • the HDL particles were subjected to particle size analysis using Image Analysis Software Image-Pro Plus 6.0 (Media Cybernetics, Inc. USA) to measure the particle size.
  • the experimental chart shows that the one-dimensional nonlinear GGE established by the present invention can clearly separate a special HDL subclass, namely pre ⁇ 1-HDL, also known as nascent HDL, from Sudan Black B prestained serum.
  • pre ⁇ 1-HDL also known as nascent HDL
  • the present invention applied the newly established two-dimensional nonlinear GGE-immunoblotting technique and found that this HDL subclass has the same characteristics as pre ⁇ 1-HDL originally defined by the Fielding laboratory.
  • pre ⁇ 1-HDL has a very high particle density ( ⁇ 1.20g/ml), and the apoprotein Apo A1 and cholesterol content ratio is the highest.
  • Figure 7 shows the application of linear GGE to two-dimensional gel electrophoresis, combined with immunoblotting to detect pre ⁇ 1-HDL.
  • the law was published in 1993 by Asztalos of Tuffs University in Boston, USA, and was widely used.
  • Developed by Boston Heart Diagnostics for the Boston Heart HDL The core technology.
  • the application of this technology for more than 20 years has brought economic benefits and influenced the academic community's understanding of pre ⁇ 1-HDL, which has become a recognized classic in the industry.
  • Miyazaki reported that the pre ⁇ 1-HDL detected by two-dimensional electrophoresis was essentially a free Apo A1 monomer.
  • the present invention establishes a one-dimensional nonlinear GGE method (Fig. 1).
  • the method uses three polyacrylamide gels with concentrations of 3.0%, 3.6% and 7.0% as electrophoresis media, and clearly separates the serum of Sudan black B staining into a special HDL subclass, namely pre ⁇ 1-HDL. Also known as the newborn HDL.
  • a special HDL subclass namely pre ⁇ 1-HDL.
  • pre ⁇ 1-HDL also known as the newborn HDL.
  • the HDL was subjected to two-dimensional electrophoresis and re-isolation.
  • nonlinear concentration gradient independent layering is simple and easy, and no special mixing device is needed; the electrophoresis only needs to be ⁇ 3.5h; the quantitative method does not need radioactive materials. Therefore, the nonlinear GGE we have established can accurately detect pre ⁇ 1-HDL, which has obvious superiority in performance and is the only feasible method at present.

Abstract

A method for separation and quantitative analysis of pre-β1-HDL in serum by nonlinear gradient gel electrophoresis. The invention has the advantages that the preparation process of the nonlinear concentration gradient independent layering is simple and easy, wherein no special mixing device is needed; the electrophoresis is implemented for less than or equal to 3.5 hours, and the quantitative method does not require any radioactive substance. The nonlinear gradient gel electrophoresis can accurately detect the preβ1-HDL, demonstrating obvious superiority in performance.

Description

改良凝胶电泳检测血清前β1高密度脂蛋白的方法和应用Method and application of improved gel electrophoresis for detecting pre-beta1 high-density lipoprotein 技术领域Technical field
本发明涉及生物技术领域,具体地说,涉及改良凝胶电泳检测血清前β1高密度脂蛋白的方法和应用。The present invention relates to the field of biotechnology, and in particular to a method and an application for improved gel electrophoresis for detecting pre-beta1 high-density lipoprotein.
背景技术Background technique
血清前β1高密度脂蛋白(preβ1-HDL)是一种新生的HDL,在胆固醇逆转运过程中发挥着重要功能,具有抗动脉粥样硬化的作用。但是令人费解的是,临床资料显示preβ1-HDL在冠心病患者中的含量显著升高,而不是负相关的关系。问题的关键在于方法学存在缺陷,以往应用的梯度凝胶电泳(GGE)是以线性浓度梯度为基础,结果不能区分游离的载脂蛋白Apo A1,因此不能准确检测preβ1-HDL。Pre-serum β1 high-density lipoprotein (preβ1-HDL) is a nascent HDL that plays an important role in the reverse trans-transport of cholesterol and has anti-atherosclerotic effects. However, it is puzzling that clinical data show that preβ1-HDL is significantly elevated in patients with coronary heart disease, rather than a negative correlation. The key to the problem lies in the shortcomings of the methodology. Previously applied gradient gel electrophoresis (GGE) was based on a linear concentration gradient. The result was that the free apolipoprotein Apo A1 could not be distinguished, so the preβ1-HDL could not be accurately detected.
以往线性GGE应用于二维凝胶电泳,联合免疫印迹技术检测preβ1-HDL。美国波士顿Tufts大学的Asztalos建立的方法发表于1993年,后来广为所用。经Boston Heart Diagnostics公司开发用于Boston Heart HDL
Figure PCTCN2016093019-appb-000001
的核心技术。20余年来该项技术的应用带来经济效益,同时影响本领域学术界对preβ1-HDL的认识,以致成为本行业内公认的经典。然而,2014年Miyazaki报道两维电泳技术检测的preβ1-HDL实质是一种游离的Apo A1单体。显而易见,因为无法区分游离的Apo A1,该项技术存在致命缺陷,长期以来对于preβ1-HDL的认识一直存在概念上的混淆,导致错误地认为具有保护作用的preβ1-HDL的含量在冠心病患者中反而显著升高。另外,线性梯度的凝胶(如2%-36%)在制备中需要特殊的混合装置,稳定性也不如非线性梯度的设置。实施电泳时间往往要24h。定量方法应用125I,需要严格的放射性实验条件。由此可见,工序复杂,操作耗时,且无法避免放射性问题,成为该法难以克服的缺点。参考:http://www.bostonheartdiagnostics.com/science_portfolio_map_test.php。
In the past, linear GGE was applied to two-dimensional gel electrophoresis, and combined with immunoblotting to detect preβ1-HDL. The method established by Asztalos at Tufts University in Boston, USA, was published in 1993 and was widely used. Developed by Boston Heart Diagnostics for the Boston Heart HDL
Figure PCTCN2016093019-appb-000001
The core technology. The application of this technology for more than 20 years has brought economic benefits and influenced the academic community's understanding of preβ1-HDL, which has become a recognized classic in the industry. However, in 2014 Miyazaki reported that the preβ1-HDL detected by the two-dimensional electrophoresis technique is essentially a free Apo A1 monomer. Obviously, because of the inability to distinguish free Apo A1, this technique has fatal flaws, and there has been a long-standing conceptual confusion about the understanding of preβ1-HDL, leading to the erroneously considered protective content of preβ1-HDL in patients with coronary heart disease. Instead, it has risen significantly. In addition, linear gradient gels (eg, 2% to 36%) require special mixing devices in their preparation, and the stability is not as good as the setting of nonlinear gradients. The electrophoresis time is usually 24 hours. The quantitative method of applying 125 I requires strict radioactivity experimental conditions. It can be seen that the process is complicated, the operation is time consuming, and the radioactivity problem cannot be avoided, which is a disadvantage that the method is difficult to overcome. Reference: http://www.bostonheartdiagnostics.com/science_portfolio_map_test.php.
发明内容Summary of the invention
本发明的目的是针对现有技术中的不足,提供非线性梯度凝胶和/或苏丹黑B染色液在分离和定量分析血清preβ1-HDL中的应用。The object of the present invention is to provide a non-linear gradient gel and/or a Sudan Black B staining solution for the separation and quantification of serum preβ1-HDL for the deficiencies in the prior art.
本发明的第二个目的是,提供一种分离和定量分析血清preβ1-HDL的方法。A second object of the present invention is to provide a method for separating and quantifying serum preβ1-HDL.
为实现上述目的,本发明提供的技术方案是:非线性梯度凝胶和/或苏丹 黑B染色液在分离和定量分析血清preβ1-HDL中的应用,所述的非线性梯度凝胶是由三段浓度依次为3.0%、3.6%和7.0%的聚丙烯酰胺凝胶组成,所述的苏丹黑B染色液中苏丹黑B的浓度为0.5-1.0w/v%,溶剂为异丙醇和乙二醇,体积比为4∶1。In order to achieve the above object, the technical solution provided by the present invention is: a nonlinear gradient gel and/or a Sudan The use of black B staining solution for separating and quantifying serum preβ1-HDL consisting of three layers of polyacrylamide gels having a concentration of 3.0%, 3.6% and 7.0%, respectively. The concentration of Sudan Black B in the Sudan Black B staining solution was 0.5-1.0 w/v%, and the solvent was isopropanol and ethylene glycol in a volume ratio of 4:1.
所述的非线性梯度凝胶分别作为一维非线性梯度管状凝胶体系和二维非线性梯度平板凝胶体系分离血清preβ1-HDL的电泳介质,所述的一维非线性梯度管状凝胶体系分离苏丹黑B预染色的血清preβ1-HDL。The nonlinear gradient gel is used as a one-dimensional nonlinear gradient tubular gel system and a two-dimensional nonlinear gradient slab gel system to separate serum preβ1-HDL electrophoresis medium, and the one-dimensional nonlinear gradient tubular gel system Sudan black B prestained serum preβ1-HDL was isolated.
为实现上述第二个目的,本发明提供的技术方案是:一种分离和定量分析血清preβ1-HDL的方法,采用非线性梯度凝胶精准分离和定量分析血清preβ1-HDL,其中非线性梯度凝胶是由三段浓度依次为3.0%、3.6%和7.0%的聚丙烯酰胺凝胶组成。In order to achieve the above second object, the technical solution provided by the present invention is: a method for separating and quantitatively analyzing serum preβ1-HDL, and using a nonlinear gradient gel to accurately separate and quantitatively analyze serum preβ1-HDL, wherein nonlinear gradient condensation The gum consisted of a polyacrylamide gel with three concentrations of 3.0%, 3.6% and 7.0%.
所述的非线性梯度凝胶分别作为一维非线性梯度管状凝胶体系和二维非线性梯度平板凝胶体系分离血清preβ1-HDL的电泳介质。The nonlinear gradient gel is used as a one-dimensional nonlinear gradient tubular gel system and a two-dimensional nonlinear gradient slab gel system to separate serum preβ1-HDL electrophoresis medium.
采用一维非线性梯度管状凝胶体系分离苏丹黑B预染色的血清preβ1-HDL,所述的苏丹黑B染色液中苏丹黑B的浓度为0.5-1.0w/v%,溶剂异丙醇和乙二醇的体积比为4∶1。Separation of Sudan black B prestained serum preβ1-HDL by a one-dimensional nonlinear gradient tubular gel system, the concentration of Sudan black B in the Sudan black B staining solution is 0.5-1.0 w/v%, solvent isopropanol and B The volume ratio of the diol was 4:1.
所述的三段浓度依次为3.0%、3.6%和7.0%的聚丙烯酰胺凝胶作为分离胶,对应的胶层高度分别为7mm、40mm和45mm,误差范围为±3mm,三层分离胶的总高度为82mm,误差范围±5mm。The three-stage concentration is 3.0%, 3.6% and 7.0% of polyacrylamide gel as separating glue, the corresponding glue layer height is 7mm, 40mm and 45mm, respectively, the error range is ±3mm, three-layer separation rubber The total height is 82mm and the error range is ±5mm.
所述的非线性梯度凝胶在一维管状凝胶体系中的规格为内径6-8mm,外径8-10mm,非线性梯度凝胶在二维平板凝胶体系中的规格为厚度为2-3mm,宽度为60-80mm。The non-linear gradient gel has a specification of an inner diameter of 6-8 mm and an outer diameter of 8-10 mm in a one-dimensional tubular gel system, and the nonlinear gradient gel has a thickness of 2 in a two-dimensional slab gel system. 3mm, width 60-80mm.
所述的一维非线性梯度管状凝胶体系分离苏丹黑B预染色的血清preβ1-HDL,电泳缓冲液包含5mM Tris和38.4mM甘氨酸,电泳条件为100V,2.5h,电泳后将迁移距离原点65-75mm处孤立的染色区带定义为preβ1-HDL;所述的二维非线性梯度平板凝胶体系分离血清preβ1-HDL,电泳缓冲液为5mM Tris和38.4mM甘氨酸,电泳条件为100V,3.5h,电泳后将preβ泳道上迁移距离原点65-75mm处印迹区带定义为preβ1-HDL和游离的Apo A1多聚体,迁移距离原点80-90mm处印迹区带定义为游离的Apo A1单体。The one-dimensional nonlinear gradient tubular gel system separates pre-stained serum preβ1-HDL from Sudan black B. The electrophoresis buffer contains 5 mM Tris and 38.4 mM glycine. The electrophoresis conditions are 100 V, 2.5 h, and the migration distance is 65 after electrophoresis. The isolated staining zone at -75 mm is defined as preβ1-HDL; the two-dimensional nonlinear gradient plate gel system separates serum preβ1-HDL, the running buffer is 5 mM Tris and 38.4 mM glycine, and the electrophoresis conditions are 100 V, 3.5 h. After electrophoresis, the imprinted zone at the origin of 65°-75 mm from the origin of the preβ lane was defined as preβ1-HDL and free Apo A1 multimer, and the imprinted band at the origin 80-90 mm was defined as free Apo A1 monomer.
所述的二维非线性梯度平板凝胶体系分离血清preβ1-HDL,电泳前在加样区加入酚红溶液作为电泳指示剂。 The two-dimensional non-linear gradient slab gel system separates serum preβ1-HDL, and a phenol red solution is added as an electrophoresis indicator in the sample application area before electrophoresis.
所述的一维非线性梯度管状凝胶体系分离苏丹黑B预染色的血清preβ1-HDL,采用光密度扫描定量,以空白凝胶扫描值为统一参比,计算出preβ1-HDL占血脂总体的百分含量;二维非线性梯度平板凝胶体系分离血清preβ1-HDL、游离的Apo A1单体和多聚体,采用免疫印迹、化学发光和胶片成像技术进行定性,在同一批内比较其相对含量。The one-dimensional nonlinear gradient tubular gel system was used to separate the pre-stained serum preβ1-HDL from Sudan black B, and the density was scanned by optical density. The blank gel scan value was used as a uniform reference to calculate the preβ1-HDL accounted for the total blood lipids. Percentage; two-dimensional nonlinear gradient slab gel system for separation of serum preβ1-HDL, free Apo A1 monomer and multimer, qualitatively by immunoblotting, chemiluminescence and film imaging techniques, comparing their relatives in the same batch content.
本发明优点在于:The advantages of the invention are:
1、本发明建立了一维非线性GGE的方法,采用三段浓度分别为3.0%、3.6%和7.0%的聚丙烯酰胺凝胶作为电泳介质,清晰地将苏丹黑B染色的血清分离出一种特定的HDL亚类,即preβ1-HDL;1. The method for establishing one-dimensional nonlinear GGE of the present invention uses a polyacrylamide gel having three concentrations of 3.0%, 3.6% and 7.0% as an electrophoresis medium to clearly separate the serum stained with Sudan black B. a specific HDL subclass, preβ1-HDL;
2、本发明还建立了二维非线性GGE的方法,能够非常清晰地将preβ1-HDL和游离的Apo A1分离开;2. The present invention also establishes a two-dimensional nonlinear GGE method capable of separating preβ1-HDL and free Apo A1 very clearly;
3、本发明的非线性浓度梯度独立分层的制备过程简单易行,不需要特殊混合装置;实施电泳仅需≤3.5h;定量方法无需放射性物质。本发明建立的非线性GGE能够精准检测preβ1-HDL,在性能上具有明显的优越性。3. The preparation process of the nonlinear concentration gradient independent layering of the invention is simple and easy, and no special mixing device is needed; the electrophoresis is only required to be ≤ 3.5 h; the quantitative method does not require radioactive materials. The nonlinear GGE established by the invention can accurately detect preβ1-HDL, and has obvious superiority in performance.
附图说明DRAWINGS
图1.一维非线性GGE-亲脂性SBB染色图谱。A:一维非线性梯度管状凝胶的示意图。B:重复性实验结果。preβ1-HDL的电泳迁移距离原点70mm,呈现清晰的SBB染色区带。相同血样检测preβ1-HDL的变异系数是2.31%,说明该法有很好的重复性。C:临床血样一维非线性GGE-亲脂性SBB染色图谱。preβ1-HDL在新生儿脐带血中含量丰富,而在急性心梗患者和CETP变异者中的含量低于健康成人。D:电泳扫描图谱。Figure 1. One-dimensional nonlinear GGE-lipophilic SBB staining map. A: Schematic representation of a one-dimensional nonlinear gradient tubular gel. B: Repeatability test results. The electrophoretic migration of preβ1-HDL is 70 mm from the origin, showing a clear SBB staining zone. The coefficient of variation of preβ1-HDL in the same blood sample was 2.31%, indicating that the method has good repeatability. C: One-dimensional nonlinear GGE-lipophilic SBB staining of clinical blood samples. preβ1-HDL is abundant in neonatal cord blood, but lower in acute myocardial infarction patients and CETP variants than in healthy adults. D: Electrophoresis scan map.
图2.二维非线性GGE-免疫印迹图谱。A:二维非线性梯度平板凝胶的制备示意图。B:二维电泳后血清中含有Apo A1颗粒的免疫印迹图谱。在preβ泳道上电泳迁移大约70mm的区带为preβ1-HDL(包含Apo A1多聚体),电泳迁移大约85mm的区带为游离的Apo A1单体,清晰可见孤立的印迹区带。新生儿血样的图谱表现较成人复杂。C:二维非线性GGE-免疫印迹示意图。箭头表示一向(1D)和二向(2D)的电泳方向。Figure 2. Two-dimensional nonlinear GGE-immunoblotting map. A: Schematic diagram of the preparation of a two-dimensional nonlinear gradient slab gel. B: Immunoblot map containing Apo A1 particles in serum after two-dimensional electrophoresis. The band of about 70 mm electrophoretically migrated on the preβ lane was preβ1-HDL (including Apo A1 multimer), and the band of electrophoretic migration of about 85 mm was free Apo A1 monomer, and the isolated blot band was clearly visible. The spectrum of neonatal blood samples is more complex than that of adults. C: Schematic representation of two-dimensional nonlinear GGE-immunoblotting. The arrows indicate the direction of electrophoresis in one direction (1D) and two directions (2D).
图3.preβ1-HDL的实验论证:颗粒密度和体外衍变。A:超速离心分离血清HDL。B:从超速离心后不同密度范围取样,经SBB染色后进行一维非线性GGE。preβ1-HDL出现在1.210g/m1的密度液中,电泳迁移70mm。C:二维非线性GGE-免疫印迹图谱,preβ1-HDL同样出现在1.210g/ml的密度液中,迁移 位置同样在70mm处。D:DTNB体外抑制实验。正常血清体外37℃水浴6h后,preβ1-HDL的含量逐渐减少,这种颗粒的衍变能够被DTNB完全抑制。因为DTNB是卵磷脂胆固醇酰基转移酶的抑制剂,能够有效抑制新生的preβ1-HDL向大颗粒HDL衍变,所以结果进一步说明,这种电泳迁移70mm且能够被亲脂性SBB染色的孤立电泳区带实质上是preβ1-HDL。Figure 3. Experimental demonstration of preβ1-HDL: particle density and in vitro degradation. A: Ultrasonic centrifugation separates serum HDL. B: Samples were taken from different density ranges after ultracentrifugation, and one-dimensional nonlinear GGE was performed after SBB staining. preβ1-HDL appeared in a density of 1.210 g/m1 and electrophoretically migrated to 70 mm. C: Two-dimensional nonlinear GGE-immunoblotting map, preβ1-HDL also appeared in the density liquid of 1.210g/ml, migration The position is also at 70mm. D: DTNB in vitro inhibition assay. After normal serum in vitro at 37 ° C for 6 h, the content of preβ1-HDL gradually decreased, and the degradation of this granule could be completely inhibited by DTNB. Because DTNB is an inhibitor of lecithin cholesterol acyltransferase, it can effectively inhibit the nascent preβ1-HDL to the large particle HDL, so the results further demonstrate that this electrophoretic migration is 70mm and can be stained by lipophilic SBB. Above is preβ1-HDL.
图4.preβ1-HDL的实验论证:颗粒大小和化学构成。A:血清HDL的电子显微图谱。箭头所指为preβ1-HDL。B:血清HDL的Apo A1和胆固醇的相对重量比,其中在preβ1-HDL的比值最高。Figure 4. Experimental demonstration of preβ1-HDL: particle size and chemical composition. A: Electron micrograph of serum HDL. The arrow is referred to as preβ1-HDL. B: Relative weight ratio of Apo A1 to cholesterol of serum HDL, wherein the ratio of preβ1-HDL is the highest.
图5.preβ1-HDL的实验论证:临床病例对照图谱。A-B:急性心梗患者和健康对照血清的典型二维非线性GGE-免疫印迹图谱。急性心梗患者preβ1-HDL的含量较健康对照明显减少。因为preβ1-HDL具有抗动脉粥样硬化保护作用,所以这样的结果顺理成章。C:Tangier病患者和健康对照血清的一维非线性GGE-亲脂性SBB染色图谱。由于ABCA1基因缺陷,Tangier病患者不能生成preβ1-HDL,导致在图谱中完全缺失。D:Tangier病患者血清二维非线性GGE-免疫印迹图谱。在迁移位置85mm处清晰可见游离Apo A1单体,同时在迁移位置70mm处仍然可见微量印迹区带,虽然位置和preβ1-HDL相同,但实质上不是preβ1-HDL,而是游离的Apo A1多聚体。D:游离Apo A1标准品(0.2μg,Sigma)二维非线性GGE-免疫印迹图谱。印迹区带主要集中在迁移位置85mm处,此为游离的Apo A1单体,但是在迁移位置70mm处仍然可见少量印迹区带,此为Apo A1多聚体。如此可以解释,Tangier病患者血清的Apo A1多聚体能够出现在二维GGE-免疫印迹图谱中,但是因为这部分不含脂质,在一维非线性GGE-亲脂性SBB染色图谱则无法被检测到。因此,只有一维非线性GGE才能用于精准检测血清preβ1-HDL。Figure 5. Experimental demonstration of preβ1-HDL: clinical case-control map. A-B: A typical two-dimensional nonlinear GGE-immunoblotting map of acute myocardial infarction patients and healthy control sera. The content of preβ1-HDL in patients with acute myocardial infarction was significantly lower than that in healthy controls. Since preβ1-HDL has an anti-atherosclerotic protective effect, such a result is logical. C: One-dimensional nonlinear GGE-lipophilic SBB staining of sera from patients with Tangier disease and healthy controls. Due to a defect in the ABCA1 gene, patients with Tangier disease are unable to produce preβ1-HDL, resulting in complete loss in the map. D: Two-dimensional nonlinear GGE-immunoblotting map of serum of patients with Tangier disease. The free Apo A1 monomer was clearly visible at the migration position of 85 mm, while the micro-imprinted band was still visible at the migration position of 70 mm. Although the position was the same as that of preβ1-HDL, it was not pre-β1-HDL, but free Apo A1 poly-polymerization. body. D: Two-dimensional nonlinear GGE-immunoblotting map of free Apo A1 standard (0.2 μg, Sigma). The blotting zone was mainly concentrated at the migration site of 85 mm, which was a free Apo A1 monomer, but a small number of blotting bands were still visible at the migration site of 70 mm, which is the Apo A1 multimer. This may explain that the Apo A1 multimer of serum from patients with Tangier disease can appear in the two-dimensional GGE-immunoblotting map, but because this part contains no lipid, the one-dimensional nonlinear GGE-lipophilic SBB staining map cannot be detected. Therefore, only one-dimensional nonlinear GGE can be used to accurately detect serum preβ1-HDL.
图6.我国应用的二维线性GGE-免疫印迹图谱。A:一维琼脂糖电泳;B:二维线性GGE。Figure 6. Two-dimensional linear GGE-immunoblotting map applied in China. A: One-dimensional agarose electrophoresis; B: Two-dimensional linear GGE.
图7.传统二维线性GGE-免疫印迹图谱。Figure 7. Traditional two-dimensional linear GGE-immunoblotting map.
具体实施方式detailed description
下面结合附图对本发明提供的具体实施方式作详细说明。The specific embodiments provided by the present invention are described in detail below with reference to the accompanying drawings.
实施例1Example 1
一、一维非线性GGEOne-dimensional one-dimensional nonlinear GGE
1、试剂 1, reagent
(1)贮存液A:丙烯酰胺(Acr)9.60g和甲叉双丙烯酰胺(Bis)0.25g,溶于去离子蒸馏水(dH2O)至100ml,用棕色瓶存于4℃,有效期3月。(1) Stock solution A: 9.60 g of acrylamide (Acr) and 0.25 g of bis-acrylamide (Bis), dissolved in deionized distilled water (dH 2 O) to 100 ml, stored in a brown bottle at 4 ° C, valid for 3 months .
(2)贮存液B:三羟甲基氨基甲烷(Tris)18.3g和1N盐酸24ml,溶于dH2O至100ml,用棕色瓶存于4℃,有效期3月。(2) Stock solution B: 13.3 g of Tris and 24 ml of 1N hydrochloric acid, dissolved in dH 2 O to 100 ml, stored in a brown bottle at 4 ° C, valid for 3 months.
(3)贮存液C:Acr 19.6g和Bis 0.4g,溶于dH2O至100ml,用棕色瓶存于4℃,有效期3月。(3) Stock solution C: Acr 19.6 g and Bis 0.4 g, dissolved in dH 2 O to 100 ml, stored in a brown bottle at 4 ° C, valid for 3 months.
(4)贮存液D:Tris 6.06g和乙二胺四乙酸二钠(EDTA-Na2)1.17g,溶于dH2O至100ml,用棕色瓶存于4℃,有效期3月。(4) Stock solution D: Tris 6.06 g and disodium edetate (EDTA-Na 2 ) 1.17 g, dissolved in dH 2 O to 100 ml, stored in a brown bottle at 4 ° C, valid for 3 months.
(5)引发剂:过硫酸铵(APS)1.0g溶于dH2O至10ml,分装后存于-80℃。(5) Initiator: 1.0 g of ammonium persulfate (APS) was dissolved in dH 2 O to 10 ml, and stored at -80 ° C after dispensing.
(6)染色液:苏丹黑(SBB)0.125g溶于异丙醇20ml和乙二醇5ml,37℃水浴过夜,室温下存于密封的棕色瓶内,有效期6月。(6) Staining solution: Sultan black (SBB) 0.125 g dissolved in 20 ml of isopropanol and 5 ml of ethylene glycol, and left at 37 ° C in a water bath overnight, and stored in a sealed brown bottle at room temperature, valid for 6 months.
(7)缓冲液H:Tris 6.0g和甘氨酸28.8g溶于dH2O至1000ml,贮存于4℃,使用时用dH2O稀释10倍。(7) Buffer H: Tris 6.0 g and glycine 28.8 g were dissolved in dH 2 O to 1000 ml, stored at 4 ° C, and diluted 10 times with dH 2 O when used.
2、样品  采集空腹静脉血,3小时内分离出血清。取血清100μl加SBB染色液10μl,振荡混匀后置37℃水浴45min,然后离心3000rpm,10min。2. Samples Fasting venous blood was collected and serum was separated within 3 hours. Take 100 μl of serum and add 10 μl of SBB staining solution, mix by shaking, place in a 37 ° C water bath for 45 min, then centrifuge 3000 rpm for 10 min.
3、管状凝胶  玻璃管的规格为内径6mm,外径8mm,长度10cm。密封玻璃管一端,将其垂直固定在制胶支架上。按照表1逐步制备高度分别为45mm、40mm和7mm的三层不同浓度(7.0%、3.6%和3.0%)的凝胶。每层凝胶在灌胶后立即轻轻注入100μl dH2O覆盖其表面,使凝胶表面聚合平整。静置30min凝胶聚合后去掉水层,再灌注下一层凝胶液。3. Tubular gel The specifications of the glass tube are 6 mm inner diameter, 8 mm outer diameter and 10 cm length. Seal one end of the glass tube and fix it vertically on the glue holder. Three different concentrations (7.0%, 3.6%, and 3.0%) of gels having a height of 45 mm, 40 mm, and 7 mm, respectively, were prepared according to Table 1. Each layer of gel was gently infused with 100 μl of dH 2 O to cover the surface immediately after filling, so that the surface of the gel was polymerized and flattened. After standing for 30 minutes, the gel layer was removed, and the aqueous layer was removed, and then the next layer of gel solution was poured.
4、管胶电泳  将制备好的凝胶管垂直置入圆盘电泳槽(DYY-III27A,北京六一电泳仪器厂)。分别向正负极电泳槽内注入电泳缓冲液(正极槽600ml,负极槽400ml)。将预染血清(50μl/管)缓慢地加入凝胶管内,覆盖在分离胶的表面。接通稳压电源(Model 1000/500,Bio-Rad),电泳100V,2.5h。4. Tube gel electrophoresis The prepared gel tube was placed vertically into a disk electrophoresis tank (DYY-III27A, Beijing Liuyi Electrophoresis Instrument Factory). The electrophoresis buffer (600 ml of the positive electrode tank and 400 ml of the negative electrode tank) was injected into the positive and negative electrode electrophoresis tanks, respectively. Pre-stained serum (50 μl/tube) was slowly added to the gel tube to cover the surface of the separation gel. Turn on the regulated power supply (Model 1000/500, Bio-Rad), electrophoresis 100V, 2.5h.
4、扫描和量化  电泳结束立即取下凝胶管,用双波段飞点扫描仪(CS-9000,Shimadzu)和光学扫描仪分别对凝胶管进行扫描和成像。飞点扫描单色光波长为604nm。扫描时沿凝胶管的中线,从空白区向原点的方向进行。分析时以空白凝胶为统一参比,以相邻电泳峰之间的拐点作为分段标志,计算出各电泳峰下面积占血清脂蛋白整体的百分含量。 4. Scanning and quantification Immediately after electrophoresis, the gel tube was removed, and the gel tube was scanned and imaged by a dual-band flying spot scanner (CS-9000, Shimadzu) and an optical scanner, respectively. The flying spot scanning monochromatic light has a wavelength of 604 nm. The scanning proceeds along the center line of the gel tube from the blank area to the origin. In the analysis, the blank gel was used as the uniform reference, and the inflection point between the adjacent electrophoresis peaks was used as the segmentation marker, and the percentage of the area under each electrophoresis peak to the whole serum lipoprotein was calculated.
表1.一维非线性GGE的凝胶溶液配置Table 1. Gel solution configuration for one-dimensional nonlinear GGE
Figure PCTCN2016093019-appb-000002
Figure PCTCN2016093019-appb-000002
T:浓度;C:交联度。dH2o:去离子蒸馏水;TEMED:四甲基乙二胺;APS:过硫酸铵溶液。T: concentration; C: degree of crosslinking. dH 2 o: deionized distilled water; TEMED: tetramethylethylenediamine; APS: ammonium persulfate solution.
二、二维非线性GGESecond, two-dimensional nonlinear GGE
1、制胶试剂同上。琼脂糖凝胶电泳缓冲液用25mM Tris-Tricine(pH 8.6)。二维电泳缓冲液为5mM Tris和38.4mM甘氨酸(pH 8.4)。0.3%戊二醛固定液。1抗Apo A1抗体购自Abcam。2抗IgG购自Novus Biologicals。1. The glue preparation reagent is the same as above. Agarose gel running buffer was used with 25 mM Tris-Tricine (pH 8.6). The two-dimensional running buffer was 5 mM Tris and 38.4 mM glycine (pH 8.4). 0.3% glutaraldehyde fixative. 1 Anti-Apo A1 antibody was purchased from Abcam. 2 Anti-IgG was purchased from Novus Biologicals.
2、平板凝胶玻璃板规格10cm×10cm,厚度2mm,垫片规格10cm×1cm,厚度2mm。垫片放在两块玻璃板两侧之间,垂直固定在制胶支架上,底端密封。按照一维非线性梯度凝胶的灌胶步骤逐层制备平板凝胶。2, flat gel glass plate specifications 10cm × 10cm, thickness 2mm, gasket specifications 10cm × 1cm, thickness 2mm. The gasket is placed between the two sides of the glass plate, vertically fixed on the glue holder, and the bottom end is sealed. The slab gel was prepared layer by layer according to the filling step of the one-dimensional nonlinear gradient gel.
3、板胶电泳将4μl血清进行0.75%琼脂糖凝胶电泳分离(100V,1.5h),SBB染色的血清4μl作为同步标记。琼脂糖凝胶电泳槽来自
Figure PCTCN2016093019-appb-000003
GT Cell apparatus(Bio-Rad)。电泳结束后沿泳道从β到α迁移的位置切出40mm长的胶条(厚度2mm),将胶条移入平板凝胶的上面,可同时加入两块胶条。将平板凝胶移入二维电泳槽内(XCell SureLockTM Mini-Cell apparatus,Life Technologies),加入二维电泳缓冲液,接通电源,电泳100V,3.5h。在胶条之间加入少许酚红溶液作为电泳指示剂,当酚红全部泳出凝胶后终止电泳,也可根据脂蛋白迁移位置调整电泳时间。为防止缓冲液溢出正极电泳槽,可在接触的玻璃板上涂少许凡士林隔离水层。二维电泳结束后取出平板凝胶,进行转膜电泳40V,1.5h,脂蛋白转移到醋纤膜上。
3. Electrophoresis of plate gel 4 μl of serum was separated by 0.75% agarose gel electrophoresis (100V, 1.5h), and 4μl of SBB stained serum was used as a synchronization marker. Agarose gel electrophoresis tank comes from
Figure PCTCN2016093019-appb-000003
GT Cell apparatus (Bio-Rad). After the electrophoresis was completed, a 40 mm long strip (thickness 2 mm) was cut along the lane from β to α, and the strip was transferred to the top of the slab gel, and two strips were simultaneously added. The slab gel electrophoresis tank into a two-dimensional (XCell SureLock TM Mini-Cell apparatus , Life Technologies), was added a two-dimensional electrophoresis buffer, power, electrophoresis 100V, 3.5h. A little phenol red solution is added between the strips as an electrophoretic indicator. When the phenol red is completely gelled, the electrophoresis is terminated, and the electrophoresis time can also be adjusted according to the lipoprotein migration position. To prevent the buffer from overflowing the positive electrode electrophoresis tank, apply a little Vaseline water layer to the contact glass plate. After the completion of the two-dimensional electrophoresis, the slab gel was taken out and subjected to 40 V for 1.5 h, and the lipoprotein was transferred to the vinegar membrane.
三、免疫印迹Third, immunoblotting
二维非线性GGE和转膜电泳后,用0.3%戊二醛溶液对醋纤膜上的脂蛋白固定10min,清水漂洗后进行免疫印迹。先后加入5%去脂牛奶-TBST和5%小牛血清白蛋白-TBST溶液在冷室内分别封闭8h左右。然后加入1抗Apo A1抗体(冷室内过夜)。在室温摇床上用TBST溶液洗掉未结合的1抗。接着加入2抗IgG(室温下1h)。再次用TBST溶液洗掉未结合的2抗。最后进行化 学发光反应,并在暗室内进行胶片成像。After two-dimensional nonlinear GGE and transmembrane electrophoresis, the lipoprotein on the vinegar membrane was fixed with 0.3% glutaraldehyde solution for 10 min, rinsed with water and immunoblotted. 5% de-fat milk-TBST and 5% calf serum albumin-TBST solution were added in the cold chamber for about 8 hours. Then 1 anti-Apo A1 antibody was added (cool overnight). Unbound 1 antibody was washed away with a TBST solution on a shaker at room temperature. Then 2 anti-IgG was added (1 h at room temperature). The unbound 2 antibody was washed away again with the TBST solution. Finalization The luminescence reaction was studied and film imaging was performed in a dark room.
四、超速离心Fourth, ultracentrifugation
采集人血清6ml,利用公式调密度为1.30g/ml(加入溴化钾2.93g)。加入到28ml超速离心管的底层,上层用1.063g/ml的密度液覆盖。超速离心机型号Beckman L8-M,转子为50.2Ti,离心条件为40K rpm,12h,10℃。超速离心结束后用长针头从底层开始依次缓慢吸出等体积1ml不同密度范围的分离液。针对分离后的不同密度液定量800μl,直接用精密天秤称其重量,计算出密度。最后用超滤管(30KD,Millipore)将不同密度范围分离液的体积浓缩到500μl。然后取样分别进行一维和二维GGE。 Human serum 6 ml was collected and adjusted to a density of 1.30 g/ml by using a formula (addition of potassium bromide 2.93 g). It was added to the bottom layer of a 28 ml ultracentrifuge tube, and the upper layer was covered with a density liquid of 1.063 g/ml. The ultracentrifuge model Beckman L8-M has a rotor of 50.2 Ti and is centrifuged at 40K rpm, 12h, 10°C. After the end of the ultracentrifugation, a long needle was used to slowly suck out an equal volume of 1 ml of the separation solution of different density ranges from the bottom layer. The densities were calculated by weighing 800 μl of the different density liquids after separation and weighing them directly with a precision balance. Finally, the volume of the separation solution of different density ranges was concentrated to 500 μl using an ultrafiltration tube (30 KD, Millipore). Samples were then taken for one-dimensional and two-dimensional GGE, respectively.
五、电子显微Five, electron microscopy
一维GGE结束后,将血清HDL亚类的电泳区带分别从分离凝胶中切开并切碎,放入含有抗生素的PBS溶液在冷室内自由扩散3-5天。收集HDL溶液经过超滤管透析和浓缩处理。透析液用0.4%碳酸氢钠溶液。处理后的HDL溶液10μl和等体积的2%磷钨酸溶液混合,吸取少许进行负染色透射电子显微观察。透射电子显微镜为日产JEM-1200EX,电子强度80KV,放大倍数100K。电镜成像后用图像分析软件Image-Pro Plus 6.0(Media Cybernetics,Inc.USA)对HDL颗粒进行粒度分析,测量其颗粒大小。After the one-dimensional GGE is completed, the electrophoretic bands of the serum HDL subclass are separately cut from the separation gel and chopped, and placed in a PBS solution containing antibiotics to freely diffuse in the cold chamber for 3-5 days. The HDL solution was collected for dialysis and concentration through an ultrafiltration tube. The dialysate was treated with a 0.4% sodium bicarbonate solution. 10 μl of the treated HDL solution was mixed with an equal volume of 2% phosphotungstic acid solution, and a little was taken for negative staining transmission electron microscopy. The transmission electron microscope is Nissan JEM-1200EX with an electron intensity of 80 kV and a magnification of 100K. After electron microscopy imaging, the HDL particles were subjected to particle size analysis using Image Analysis Software Image-Pro Plus 6.0 (Media Cybernetics, Inc. USA) to measure the particle size.
六、生化检测Six, biochemical testing
收集各种HDL溶液,用日产Hitachi 911全自动生化分析仪检测其中胆固醇和Apo A1的含量,计算出它们的含量比。胆固醇的检测方法采用酶法,Apo A1的检测方法采用免疫比浊法,检测试剂来自Roche Diagnostics。Various HDL solutions were collected and the contents of cholesterol and Apo A1 were measured using a Nissan Hitachi 911 automatic biochemical analyzer to calculate their content ratio. The method for detecting cholesterol is enzymatic method, the method for detecting Apo A1 is immunoturbidimetry, and the detection reagent is from Roche Diagnostics.
七、结果和结论VII. Results and conclusions
表2.健康新生儿、成人和急性心梗患者的血脂水平和脂蛋白谱Table 2. Lipid levels and lipoprotein profiles in healthy neonates, adults, and acute myocardial infarction
Figure PCTCN2016093019-appb-000004
Figure PCTCN2016093019-appb-000004
Figure PCTCN2016093019-appb-000005
Figure PCTCN2016093019-appb-000005
和<60岁健康成人比较,
Figure PCTCN2016093019-appb-000006
P<0.01,
Figure PCTCN2016093019-appb-000007
P<0.001.和>60岁健康成人比较,#P<0.05,*P<0.01,ΔP<0.001.TG:甘油三酯;TC:总胆固醇;LDL-C:低密度脂蛋白胆固醇;HDL-C:高密度脂蛋白胆固醇;IDL:中间密度脂蛋白;Lp(a):脂蛋白(a)。
Compared with healthy adults <60 years old,
Figure PCTCN2016093019-appb-000006
P<0.01,
Figure PCTCN2016093019-appb-000007
P <0.001 and> relatively healthy adults 60 years of age, # P <0.05, * P <0.01, Δ P <0.001.TG: triglycerides; TC: Total cholesterol; LDL-C: low density lipoprotein cholesterol; HDL- C: high density lipoprotein cholesterol; IDL: intermediate density lipoprotein; Lp (a): lipoprotein (a).
实验图表显示,本发明建立的一维非线性GGE能够清晰地从苏丹黑B预染色的血清中分离出一种特殊的HDL亚类,即preβ1-HDL,又称为新生的HDL。同时,本发明应用新建立的二维非线性GGE-免疫印迹技术,发现这种HDL亚类和最初由Fielding实验室定义的preβ1-HDL具有一致的特征。和其他HDL亚类比较,preβ1-HDL具有极高的颗粒密度(≥1.20g/ml),且其中载脂蛋白Apo A1和胆固醇的含量比最高。体外实验显示,2-硝基苯甲酸(DTNB)能够完全抑制preβ1-HDL向大颗粒HDL衍变。preβ1-HDL在新生儿脐带血中含量丰富,而在急性心梗患者和CETP变异者中的含量低于健康成人,在Tangier病患者中则无法检测到。研究结果表明,两维凝胶电泳-免疫印迹法不能区分preβ1-HDL和游离的Apo A1,因此该法检测preβ1-HDL并不准确。只有我们建立的非线性GGE技术才能够精准检测preβ1-HDL,具有良好的应用前景。The experimental chart shows that the one-dimensional nonlinear GGE established by the present invention can clearly separate a special HDL subclass, namely preβ1-HDL, also known as nascent HDL, from Sudan Black B prestained serum. At the same time, the present invention applied the newly established two-dimensional nonlinear GGE-immunoblotting technique and found that this HDL subclass has the same characteristics as preβ1-HDL originally defined by the Fielding laboratory. Compared with other HDL subclasses, preβ1-HDL has a very high particle density (≥1.20g/ml), and the apoprotein Apo A1 and cholesterol content ratio is the highest. In vitro experiments showed that 2-nitrobenzoic acid (DTNB) completely inhibited the degradation of preβ1-HDL to large particle HDL. preβ1-HDL is abundant in neonatal cord blood, but lower in acute myocardial infarction patients and CETP variants than in healthy adults, but not in Tangier disease patients. The results show that two-dimensional gel electrophoresis-immunoblotting cannot distinguish preβ1-HDL from free Apo A1, so the detection of preβ1-HDL by this method is not accurate. Only the nonlinear GGE technology we have established can accurately detect preβ1-HDL, which has a good application prospect.
我国于1999年由当时华西医科大学载脂蛋白研究室吴新伟和傅明德等采用琼脂糖及梯度聚丙烯酰胺凝胶双向电泳、免疫印迹试验和斑点扫描分析,建立了血清HDL亚类的检测方法,图6。这是国内较早开展检测preβ1-HDL的研究,后来陆续有很多应用文献报道。该法采用2%-30%线性GGE作为二维电泳,与Asztalos法大同小异,两者对preβ1-HDL的分辨效果相似。虽然该法在定量方法上有所改进,但仍然应用的是线性GGE,同样存在技术缺陷,无法区分preβ1-HDL和游离的Apo A1。遗憾的是,这个问题长期没有被认识到。In 1999, Wu Xinwei and Fu Mingde from the Apolipoprotein Research Laboratory of Huaxi Medical University in China used agarose and gradient polyacrylamide gel two-dimensional electrophoresis, immunoblotting and speckle scanning analysis to establish a method for detecting serum HDL subclasses. 6. This is an early study on the detection of preβ1-HDL in China, and there have been many reports in the literature. The method uses 2%-30% linear GGE as two-dimensional electrophoresis, which is similar to the Asztalos method, and the discrimination effect of preβ1-HDL is similar. Although this method has been improved in quantitative methods, linear GGE is still applied, and there are also technical defects in which it is impossible to distinguish between preβ1-HDL and free Apo A1. Unfortunately, this issue has not been recognized for a long time.
图7为线性GGE应用于二维凝胶电泳,联合免疫印迹技术检测preβ1-HDL。该法由美国波士顿Tuffs大学的Asztalos发表于1993年,后来广为所用。经Boston Heart Diagnostics公司开发用于Boston Heart HDL
Figure PCTCN2016093019-appb-000008
的核心技术。20余年来该项技术的应用带来经济效益,同时影响本领域学术界对preβ1-HDL的认识,以致成为本行业内公认的经典。然而,2014年Miyazaki 报道两维电泳技术检测的preβ1-HDL实质是一种游离的Apo A1单体。显而易见,因为无法区分游离的Apo A1,该项技术存在致命缺陷,长期以来对于preβ1-HDL的认识一直存在概念上的混淆,导致错误地认为具有保护作用的preβ1-HDL的含量在冠心病患者中反而显著升高。另外,线性梯度的凝胶(如2%-36%)在制备中需要特殊的混合装置,稳定性也不如非线性梯度的设置。实施电泳时间往往要24h。定量方法应用125I,需要严格的放射性实验条件。由此可见,工序复杂,操作耗时,且无法避免放射性问题,成为该项被视为经典的方法难以克服的技术限制。
Figure 7 shows the application of linear GGE to two-dimensional gel electrophoresis, combined with immunoblotting to detect preβ1-HDL. The law was published in 1993 by Asztalos of Tuffs University in Boston, USA, and was widely used. Developed by Boston Heart Diagnostics for the Boston Heart HDL
Figure PCTCN2016093019-appb-000008
The core technology. The application of this technology for more than 20 years has brought economic benefits and influenced the academic community's understanding of preβ1-HDL, which has become a recognized classic in the industry. However, in 2014 Miyazaki reported that the preβ1-HDL detected by two-dimensional electrophoresis was essentially a free Apo A1 monomer. Obviously, because of the inability to distinguish free Apo A1, this technique has fatal flaws, and there has been a long-standing conceptual confusion about the understanding of preβ1-HDL, leading to the erroneously considered protective content of preβ1-HDL in patients with coronary heart disease. Instead, it has risen significantly. In addition, linear gradient gels (eg, 2% to 36%) require special mixing devices in their preparation, and the stability is not as good as the setting of nonlinear gradients. The electrophoresis time is usually 24 hours. The quantitative method of applying 125 I requires strict radioactivity experimental conditions. It can be seen that the process is complicated, the operation is time consuming, and the radioactivity problem cannot be avoided, which becomes a technical limitation that is considered to be difficult to overcome by the classical method.
相比之下,我们建立的非线性GGE技术(图1-2)则能够清晰地分离开preβ1-HDL和游离的Apo A1。临床资料显示,冠心病急性心梗患者血清preβ1-HDL的含量显著降低,这使preβ1-HDL的保护作用与其临床相关性更容易理解。且非线性浓度梯度独立分层的制备过程简单易行,不需要特殊混合装置;实施电泳仅需≤3.5h;定量方法无需放射性物质。因此,我们建立的非线性GGE具有明显的技术优势,并且是目前精准检测血清preβ1-HDL的唯一可行的方法。In contrast, the nonlinear GGE technique we established (Figure 1-2) clearly separates preβ1-HDL and free Apo A1. Clinical data show that serum preβ1-HDL levels in patients with acute myocardial infarction are significantly reduced, which makes the protection of preβ1-HDL more clinically relevant. The preparation process of the independent concentration layer of the nonlinear concentration gradient is simple and easy, and no special mixing device is needed; the electrophoresis only needs to be ≤3.5h; the quantitative method does not need radioactive materials. Therefore, the nonlinear GGE we have established has obvious technical advantages and is the only feasible method for accurately detecting serum preβ1-HDL.
表3.本发明建立的非线性GGE与经典线性GGE的技术比较Table 3. Comparison of the technique of nonlinear GGE and classical linear GGE established by the present invention
Figure PCTCN2016093019-appb-000009
Figure PCTCN2016093019-appb-000009
综上所述,本发明建立了一维非线性GGE的方法(图1)。该法采用三段浓度分别为3.0%、3.6%和7.0%的聚丙烯酰胺凝胶作为电泳介质,清晰地将苏丹黑B染色的血清分离出一种特殊的HDL亚类,即preβ1-HDL,又称为新生的HDL。进而我们采用同样的三种浓度设置,将原来的管状凝胶改为平板凝胶(图2),并将这种板胶应用到两维凝胶电泳系统中,对琼脂糖凝胶电泳分离 的HDL进行二维电泳再分离,结合免疫印迹技术,结果发现这种HDL亚类的电泳特征和最初定义的preβ1-HDL(CastroG.R.and Fielding C.J.,1988)完全一致。于是我们按照惯例将这种特殊的HDL亚类命名为preβ1-HDL。同时,我们建立的二维非线性GGE技术能够非常清晰地将preβ1-HDL和游离的Apo A1分离开。临床资料显示,冠心病急性心梗患者血清preβ1-HDL的含量显著降低,这使preβ1-HDL的保护作用与其临床相关性更容易理解。非线性浓度梯度独立分层的制备过程简单易行,不需要特殊混合装置;实施电泳仅需≤3.5h;定量方法无需放射性物质。因此,我们建立的非线性GGE能够精准检测preβ1-HDL,在性能上具有明显的优越性,并且是目前唯一可行的方法。In summary, the present invention establishes a one-dimensional nonlinear GGE method (Fig. 1). The method uses three polyacrylamide gels with concentrations of 3.0%, 3.6% and 7.0% as electrophoresis media, and clearly separates the serum of Sudan black B staining into a special HDL subclass, namely preβ1-HDL. Also known as the newborn HDL. In turn, we used the same three concentration settings to change the original tubular gel to a slab gel (Fig. 2), and applied the slab to a two-dimensional gel electrophoresis system for separation by agarose gel electrophoresis. The HDL was subjected to two-dimensional electrophoresis and re-isolation. Combined with immunoblotting, it was found that the electrophoretic characteristics of this HDL subclass were completely consistent with the originally defined preβ1-HDL (CastroG.R. and Fielding C.J., 1988). So we routinely named this particular HDL subclass as preβ1-HDL. At the same time, our two-dimensional nonlinear GGE technology is able to separate preβ1-HDL from free Apo A1 very clearly. Clinical data show that serum preβ1-HDL levels in patients with acute myocardial infarction are significantly reduced, which makes the protection of preβ1-HDL more clinically relevant. The preparation process of nonlinear concentration gradient independent layering is simple and easy, and no special mixing device is needed; the electrophoresis only needs to be ≤3.5h; the quantitative method does not need radioactive materials. Therefore, the nonlinear GGE we have established can accurately detect preβ1-HDL, which has obvious superiority in performance and is the only feasible method at present.
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。 The above description is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention. These improvements and additions should also be considered. It is the scope of protection of the present invention.

Claims (10)

  1. 非线性梯度凝胶和/或苏丹黑B染色液在分离和定量分析血清preβ1-HDL中的应用,所述的非线性梯度凝胶是由三段浓度依次为3.0%、3.6%和7.0%的聚丙烯酰胺凝胶组成,所述的苏丹黑B染色液中苏丹黑B的浓度为0.5-1.0w/v%,溶剂为异丙醇和乙二醇,体积比为4∶1。The use of a non-linear gradient gel and/or Sudan Black B staining solution for the separation and quantification of serum preβ1-HDL, which is 3.0%, 3.6% and 7.0% in three stages. The polyacrylamide gel composition has a concentration of Sudan Black B in the Sudan Black B staining solution of 0.5-1.0 w/v%, and the solvent is isopropanol and ethylene glycol, and the volume ratio is 4:1.
  2. 根据权利要求1所述的应用,其特征在于,所述的非线性梯度凝胶分别作为一维非线性梯度管状凝胶体系和二维非线性梯度平板凝胶体系分离血清preβ1-HDL的电泳介质,所述的一维非线性梯度管状凝胶体系分离苏丹黑B预染色的血清preβ1-HDL。The application according to claim 1, wherein the nonlinear gradient gel is used as a one-dimensional nonlinear gradient tubular gel system and a two-dimensional nonlinear gradient slab gel system for separating serum preβ1-HDL electrophoretic medium. The one-dimensional nonlinear gradient tubular gel system is used to separate the serum preβ1-HDL pre-stained by Sudan black B.
  3. 一种分离和定量分析血清preβ1-HDL的方法,其特征在于,采用非线性梯度凝胶分离和定量分析血清preβ1-HDL,其中非线性梯度凝胶是由三段浓度依次为3.0%、3.6%和7.0%的聚丙烯酰胺凝胶组成。A method for separating and quantifying serum preβ1-HDL, characterized in that serum preβ1-HDL is separated and quantitatively analyzed by nonlinear gradient gel, wherein the nonlinear gradient gel is 3.0% and 3.6% in three stages. And 7.0% polyacrylamide gel.
  4. 根据权利要求3所述的方法,其特征在于,所述的非线性梯度凝胶分别作为一维非线性梯度管状凝胶体系和二维非线性梯度平板凝胶体系分离血清preβ1-HDL的电泳介质。The method according to claim 3, wherein the nonlinear gradient gel is used as a one-dimensional nonlinear gradient tubular gel system and a two-dimensional nonlinear gradient slab gel system to separate serum preβ1-HDL electrophoretic medium. .
  5. 根据权利要求4所述的方法,其特征在于,采用一维非线性梯度管状凝胶体系分离苏丹黑B预染色的血清preβ1-HDL,所述的苏丹黑B染色液中苏丹黑B的浓度为0.5-1.0w/v%,溶剂为异丙醇和乙二醇,体积比为4∶1。The method according to claim 4, wherein the Sudan black B prestained serum preβ1-HDL is separated by a one-dimensional nonlinear gradient tubular gel system, and the concentration of Sudan black B in the Sudan black B staining solution is 0.5-1.0 w/v%, the solvent is isopropanol and ethylene glycol, and the volume ratio is 4:1.
  6. 根据权利要求3所述的方法,其特征在于,所述的三段浓度依次为3.0%、3.6%和7.0%的聚丙烯酰胺凝胶作为分离胶,对应的胶层高度分别为7mm、40mm和45mm,误差范围为±3mm,三层分离胶的总高度为82mm,误差范围±5mm。The method according to claim 3, wherein the three-stage concentration is 3.0%, 3.6%, and 7.0% of polyacrylamide gel as a separation gel, and the corresponding glue layer heights are 7 mm, 40 mm, respectively. 45mm, the error range is ±3mm, the total height of the three-layer separation glue is 82mm, and the error range is ±5mm.
  7. 根据权利要求4所述的方法,其特征在于,所述的非线性梯度凝胶在一维管状凝胶体系中的规格为内径6-8mm,外径8-10mm,非线性梯度凝胶在二维平板凝胶体系中的规格为厚度为2-3mm,宽度为60-80mm。The method according to claim 4, wherein said non-linear gradient gel has a size of 6-8 mm in inner diameter, 8-10 mm in outer diameter, and a nonlinear gradient gel in a one-dimensional tubular gel system. The dimensions in the slab gel system are 2-3 mm in thickness and 60-80 mm in width.
  8. 根据权利要求4所述的方法,其特征在于,所述的一维非线性梯度管状凝胶体系分离苏丹黑B预染色的血清preβ1-HDL,电泳缓冲液包含5mM Tris和38.4mM甘氨酸,电泳条件为100V,2.5h,电泳后将迁移距离原点65-75mm处孤立的染色区带定义为preβ1-HDL;所述的二维非线性梯度平板凝胶体系分离血清preβ1-HDL,电泳缓冲液为5mM Tris和38.4mM甘氨酸,电泳条件为 100V,3.5h,电泳后将preβ泳道上迁移距离原点65-75mm处印迹区带定义为preβ1-HDL和游离的Apo A1多聚体,迁移距离原点80-90mm处印迹区带定义为游离的Apo A1单体。The method according to claim 4, wherein the one-dimensional nonlinear gradient tubular gel system separates pre-stained serum preβ1-HDL from Sudan black B, and the electrophoresis buffer comprises 5 mM Tris and 38.4 mM glycine, and electrophoresis conditions. 100V, 2.5h, after electrophoresis, the isolated staining zone at 65-75mm origin is defined as preβ1-HDL; the two-dimensional nonlinear gradient plate gel system separates serum preβ1-HDL, and the running buffer is 5mM. Tris and 38.4 mM glycine, electrophoresis conditions are 100V, 3.5h, after electrophoresis, the imprinted zone at the origin of 65-75mm in the preβ lane was defined as preβ1-HDL and free Apo A1 multimer, and the imprinted band at the origin 80-90mm was defined as free Apo. A1 monomer.
  9. 根据权利要求8所述的方法,其特征在于,所述的二维非线性梯度平板凝胶体系分离血清preβ1-HDL,电泳前在加样区加入酚红溶液作为电泳指示剂。The method according to claim 8, wherein the two-dimensional non-linear gradient slab gel system separates serum preβ1-HDL, and a phenol red solution is added as an electrophoresis indicator in the sample application area before electrophoresis.
  10. 根据权利要求8所述的方法,其特征在于,所述的一维非线性梯度管状凝胶体系分离苏丹黑B预染色的血清preβ1-HDL,采用光密度扫描定量,以空白凝胶扫描值为统一参比,计算出preβ1-HDL占血脂总体的百分含量;二维非线性梯度平板凝胶体系分离血清preβ1-HDL、游离的Apo A1单体和多聚体,采用免疫印迹、化学发光和胶片成像技术进行定性,在同一批内比较其相对含量。 The method according to claim 8, wherein said one-dimensional nonlinear gradient tubular gel system separates serum preβ1-HDL pre-stained by Sudan black B, and is quantified by optical density scanning, and the blank gel scan value is used. The total reference was used to calculate the percentage of preβ1-HDL in the total blood lipids; the two-dimensional nonlinear gradient slab gel system was used to separate serum preβ1-HDL, free Apo A1 monomers and multimers, using immunoblotting, chemiluminescence and Film imaging techniques were characterized and their relative contents were compared in the same batch.
PCT/CN2016/093019 2015-08-03 2016-08-03 METHOD FOR USING IMPROVED GEL ELECTROPHORESIS TO DETECT PREβ1 HIGH DENSITY LIPOPROTEIN IN SERUM, AND APPLICATION WO2017020830A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201510466708.0A CN105137070B (en) 2015-08-03 2015-08-03 The methods and applications of 1 high-density lipoprotein of β before improvement detected through gel electrophoresis serum
CN201510466708.0 2015-08-03

Publications (1)

Publication Number Publication Date
WO2017020830A1 true WO2017020830A1 (en) 2017-02-09

Family

ID=54722494

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/093019 WO2017020830A1 (en) 2015-08-03 2016-08-03 METHOD FOR USING IMPROVED GEL ELECTROPHORESIS TO DETECT PREβ1 HIGH DENSITY LIPOPROTEIN IN SERUM, AND APPLICATION

Country Status (2)

Country Link
CN (1) CN105137070B (en)
WO (1) WO2017020830A1 (en)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105137070B (en) * 2015-08-03 2018-07-24 复旦大学附属中山医院 The methods and applications of 1 high-density lipoprotein of β before improvement detected through gel electrophoresis serum
CN105527449A (en) * 2015-12-24 2016-04-27 山东博科生物产业有限公司 Apolipoprotein A1 immunoturbidimetry detection kit
CN108387630B (en) * 2018-01-29 2019-09-03 温州科技职业学院 A kind of improvement SDS- polyacrylamide gel electrophoresis technique
CN108530514B (en) * 2018-06-28 2022-05-31 中科瑞泰(北京)生物科技有限公司 Separation gel for electrophoresis and method for separating polypeptide by using separation gel
CN110031555B (en) * 2019-04-19 2022-04-05 河北大学 Method for separating and detecting serum lipoprotein
CN112305045A (en) * 2020-10-19 2021-02-02 上海宝藤生物医药科技股份有限公司 High-density lipoprotein subtyping detection kit and preparation method and application thereof
CN112798481B (en) * 2020-10-27 2022-02-18 美康生物科技股份有限公司 Reagent for detecting concentration of lipoprotein particles and using method thereof
CN112665950A (en) * 2021-01-28 2021-04-16 安徽佳创生物科技有限公司 Method for detecting high-density lipoprotein in serum
CN113462191B (en) * 2021-09-03 2021-12-07 上海淘源生物科技有限公司 Sudan black B2 dye and staining solution, detection method using the same, gel electrophoresis system containing the same, kit and preparation method

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992010277A1 (en) * 1990-12-13 1992-06-25 Opplt Jan J Discontinuous and nonsequential polymeric gel systems for separation of macromolecules
CN1699406A (en) * 2005-06-03 2005-11-23 陈允钦 Method for gel electrophoresis separation of serum lipoprotein and quantization detection thereof
CN102181173A (en) * 2011-02-14 2011-09-14 复旦大学附属妇产科医院 Sudan black B staining fluid as well as preparation method and applications of the sudan black B staining fluid
US20140243233A1 (en) * 2011-08-04 2014-08-28 Hdl Apomics Llc Methods for measuring hdl subpopulations
CN104297327A (en) * 2014-10-29 2015-01-21 邓杏飞 Method for analyzing fine sub-fractions of serum lipoprotein subtype by adopting micro-fluidic chip
CN105137070A (en) * 2015-08-03 2015-12-09 复旦大学附属中山医院 Method using improved gel electrophoresis to detect pre[beta]1 high density lipoprotein (HDL) in serum and application thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992010277A1 (en) * 1990-12-13 1992-06-25 Opplt Jan J Discontinuous and nonsequential polymeric gel systems for separation of macromolecules
CN1699406A (en) * 2005-06-03 2005-11-23 陈允钦 Method for gel electrophoresis separation of serum lipoprotein and quantization detection thereof
CN102181173A (en) * 2011-02-14 2011-09-14 复旦大学附属妇产科医院 Sudan black B staining fluid as well as preparation method and applications of the sudan black B staining fluid
US20140243233A1 (en) * 2011-08-04 2014-08-28 Hdl Apomics Llc Methods for measuring hdl subpopulations
CN104297327A (en) * 2014-10-29 2015-01-21 邓杏飞 Method for analyzing fine sub-fractions of serum lipoprotein subtype by adopting micro-fluidic chip
CN105137070A (en) * 2015-08-03 2015-12-09 复旦大学附属中山医院 Method using improved gel electrophoresis to detect pre[beta]1 high density lipoprotein (HDL) in serum and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEN, YUNQIN ET AL.: "A New Method for Detecting Serum beta-Lipoprotein Electrophoretogram", JOURNAL OF FIRST MILITARY MEDICAL UNIVERSITY, vol. 23, no. 2, 31 December 2003 (2003-12-31), pages 153 - 155 *
OSAMU, M. ET AL.: "Pre beta 1-HDL, a Key Element of Reverse Cholesterol Transport: its Potential as A Biomarker", CLINICAL LIPIDOLOGY, vol. 5, no. 3, 30 June 2010 (2010-06-30), pages 358, ISSN: 1758-4299 *
TAKASHI, M. ET AL.: "Serum Amyloid a Protein Generates Preâl High-Density Lipoprotein from a-Migrating High-Density Lipoprotein", BIOCHEMISTRY, vol. 38, 2 December 1999 (1999-12-02), pages 16958 - 16962, XP055362757 *

Also Published As

Publication number Publication date
CN105137070A (en) 2015-12-09
CN105137070B (en) 2018-07-24

Similar Documents

Publication Publication Date Title
WO2017020830A1 (en) METHOD FOR USING IMPROVED GEL ELECTROPHORESIS TO DETECT PREβ1 HIGH DENSITY LIPOPROTEIN IN SERUM, AND APPLICATION
US20170299588A1 (en) Methods for separation and immuno-detection of biomolecules, and apparatus related thereto
EP2609432B1 (en) Assay for determination of levels of lipoprotein particles in bodily fluids
AU2013267450B2 (en) Composition and method for gel electrophoresis with in-situ calibration
EP2443458A1 (en) Method for measuring lipoprotein-specific apolipoproteins
WO2006128388A1 (en) Method for gel electrophoresis separation of serum lipoproteins and quantization dection thereof
Bottenus et al. Preconcentration and detection of the phosphorylated forms of cardiac troponin I in a cascade microchip by cationic isotachophoresis
Grönwall On paper electrophoresis in the clinical laboratory
US20160109471A1 (en) Lipoprotein particle number from measurements of lipoprotein particle phospholipid concentration in lipoprotein particle membrane bilayer
EP3210029A1 (en) Quantitative molar concentration detection of specific apolipoprotein-containing particles present in bodily fluids by using capillary electrophoresis
US20180143211A1 (en) Ultra-small apob-containing particles and methods of use thereof
US20120052593A1 (en) Assay for determination of levels of lipoprotein particle number in bodily fluids
JP2009031092A (en) Lipoprotein separation method by capillary isotachophoresis and capillary tube used therein
JP5953897B2 (en) Determination method of lipoprotein particle size and lipoprotein particle marker
Rabilloud et al. Two-dimensional SDS-PAGE fractionation of biological samples for biomarker discovery
JP4171810B2 (en) Neutral fat quantification method
Jones et al. Multiplexed protein biomarker detection with microfluidic electrochemical immunoarrays
JP2024034246A (en) Test method for idiopathic nephrotic syndrome
US10520471B2 (en) Modified electrode buffers for stain-free protein detection in electrophoresis
Ranica Apllication of capillary electrophoresis to analyze human transferrin focusing on the detection and quantification of the asialo-Tf isoform
Rerabek Two-dimensional immunoelectrophrophoretic pattern of low-and very-low-density lipoproteins, with particular reference to Fredrickson's type III.
JPS58105053A (en) Method of two-dimentional electrophoresis
Hossan et al. Preconcentration and detection of the phosphorylated forms of cardiac troponin I in a cascade microchip by...
Rabilloud Two-dimensional SDS-PAGE Fractionation of Biological Samples for Biomarker Discovery Thierry Rabilloud, Sarah Triboulet 3 1: CEA Grenoble, iRTSV/LCBM, Chemistry and Biology of Metals Grenoble, France 2: CNRS UMR5249, Chemistry and Biology of Metals, UMR CNRS CEA UJF, Grenoble, France 3: Université Joseph Fourier, Chemistry and Biology of Metals, Grenoble, France
JP2009222637A (en) Immunological analysis chip and immunological analytical method using same

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16832318

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16832318

Country of ref document: EP

Kind code of ref document: A1