CN112697565A - Method for detecting low-density lipoprotein in serum by improved gel electrophoresis - Google Patents
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- G—PHYSICS
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- G01N1/00—Sampling; Preparing specimens for investigation
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
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Abstract
The invention provides a method for detecting low-density lipoprotein in serum by improved gel electrophoresis, which comprises four steps of sample collection, dyeing, gel tube preparation and electrophoresis, wherein the sample collection comprises the steps of collecting a venous blood sample with an empty stomach of more than 12h, separating the serum within 3h, and storing the serum sample at-80 ℃ if the measurement cannot be carried out in time. The invention optimizes the acrylamide gel concentration and the preparation method of Sudan black B staining solution, realizes the accurate separation and quantitative analysis of serum LDL, has simple and easy detection process, does not need special mixing devices, does not need radioactive substances in the electrophoresis time, has obvious superiority in performance, and is quick and convenient to use.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for detecting low-density lipoprotein in serum by improved gel electrophoresis.
Background
Plasma lipoproteins are spherical particles responsible for the transport of cholesterol, triglycerides and phospholipids. Lipoproteins can be divided into five major groups, including chylomicrons, Very Low Density Lipoproteins (VLDL), Intermediate Density Lipoproteins (IDL), Low Density Lipoproteins (LDL), and High Density Lipoproteins (HDL). Wherein LDL (Low-density lipoprotein) is a heterogeneous group of lipoprotein composed of sub-component particles with different sizes, densities and chemical compositions, the diameter of a single LDL particle is about 2.20X 103-2.75X 103nm, and the single LDL particle generally transports 3000 to 6000 fat molecules per particle, and the size of the LDL particle is changed with the number of the fat molecules in the particle. At present, LDL can be separated into 7 LDL subfractions, designated LDL-1 to LDL-7, respectively, where LDL-1 consists of the largest particles and LDL-7 consists of the smallest particles.
The sub-composition and proportion of LDL are influenced by gene, environment, genetics, etc., and it is known that age, sex and lipid status affect LDL sub-components [7 ]. The lipoprotein electrophoresis profile is composed mainly of the larger particle LDL-1 and LDL-2 subfractions, called "type A". In addition to LDL-1 and LDL-2, there are included LDL-3 to LDL-7 subfractions consisting of smaller, denser lipoproteins, known as "type B" [ Austin MA, King MC, Vranigan KM, Krauss RM.A. heterologous protein phenyl marker for coronary heart disease, Circuse 1990; 82:495-506.]. Studies have shown [ Campos H, Genest JJ Jr, Blijlevens E, McNamara JR, Jenner JL, Ordovas JM, et al.Low dense lipid protein particulate and coral area disease. Arterioscler Thromb 1992; 187-95. ], type B LDL has a high correlation with the occurrence of Coronary Artery Disease (CAD) [ Coresh J, Kwiterovich PO Jr, Smith HH, Bachorik PS. Association of plasma triglyceridentation and LDL particulate diameter, density, and chemical composition with predictive coronary artery disease in men and white. J Lipid Res 1993; 1687-97, regardless of sex, age and weight, thus low-high density lipoprotein is an index that can predict Coronary Artery Disease (CAD) [ Gardner CD, Fortmann SP, Krauss RM.Association of small low-density lipid proteins with the involvement of the scientific area diseases in men and women, JAMA1996; 276: 875-81 ], high serum concentrations of low density lipoproteins are believed to be a major contributor to coronary heart disease. When LDL particles are oxidized, the risk of cardiovascular disease increases, mainly because the oxidized form of LDL is more easily retained by proteoglycans on the vessel wall and produces a series of complex biochemical regulatory reactions, and the increasing concentration of LDL particles over time will lead to atherosclerosis [ chenhua new main edition. Jindun press, 2012: 31 ] of the formula (I).
Lipoprotein analysis was performed By Density Gradient ultracentrifugation [ Griffin BA, Caslake MJ, YIp B, Tait GW, Packard CJ, Shepherd J. Rapid Isolation Of Low Density Lipoprotein (LDL) Subfractions From Plasma By Density Gradient centrifugation. Atherosclerosis 1990; 83(1) 59-67 according to the gold standard [ Nehemias Mun-iz. measurement of plasma characteristics by electrophoresis on polyacrylamide gel. Clin. chem.1977:23/10,1826-1833 ], but requires ultra-separation equipment and has a long operation time, and thus, large-scale detection cannot be performed. Nuclear Magnetic Resonance (NMR) [ Otvos JD. measurement Of Lipoprotein subunit films by Nuclear Magnetic Resonance spectroscopy. in: Handbook Of Lipoprotein Testing, Rifai N, Warnick GR, dominicak MH, eds. AACC Press,1999,2nd Edition, Washington, DC. pages 609. 623.] has been used since 1991 to detect lipoproteins in plasma [ Otvos JD, Jeyarajah EJ, Bennett DW. quantification Of plasma proteins by Nuclear Magnetic Resonance spectroscopy. clin Chem 1991; 37: 377-86. VLDL, LDL and HDL subtypes with different sizes in plasma emit different NMR signals simultaneously, and the single amplitude of the NMR signals can be accurately and repeatedly measured; the measured subclass signal amplitude is proportional to the number of subclass particles that signal, independent of changes in the particle lipid composition [ Jeyarajah EJ1, Cromwell WC, ovovs jd. lipoproteins particulate analysis by nuclear magnetic resonance. clin Lab med.2006 dec; 26(4) 847-70), which also requires expensive instrumentation, long operating times and inability to perform high throughput assays, and high operator requirements. Gel Electrophoresis methods are currently the most commonly used detection methods [ Shaina V.Hirany, Yusa Othman, Patricia Kutscher, David L.Rainwater, Ishwarlal Jianal and Sridevi Dearaj.Compuison of Low-sensitivity Lipoprotein Size by Polyacrylamide Tube Gel Electrophoresis and Polyacrylamide grade Gel Electrophoresis. am J Clin Patholol 2003; 119: 439-. Also developed recently are immunological detection methods [ Leary ET, Wang T, Baker DJ, Cilla DD, Zhong J, Warnick GR, Nakajima K, Havel RJ.evaluation of an immunological detection method for quantitative detection of remnants-like particles-cholesterol in server and plasma. Clin chem.1998 Dec; 44(12):2490-8 ], needs reagents such as antibodies and the like, and is not favorable for clinical rapid detection.
Disclosure of Invention
The present invention is to solve the above-mentioned disadvantages and to provide a method for detecting low density lipoprotein in serum by improved gel electrophoresis.
In order to achieve the purpose, the technical solution of the invention is as follows: the method for detecting low-density lipoprotein in serum by using improved gel electrophoresis comprises the following steps:
step 1, collecting samples: collecting venous blood sample with fasting state of more than 12h, separating serum within 3h, and storing the serum sample at-80 deg.C if it can not be measured in time;
step 2, dyeing: taking 70-90uL of the serum obtained in the step 1, adding 15-25uL of dye solution, mixing uniformly, incubating for 20min under the condition of 37 ℃ water bath, centrifuging, wherein the centrifuging time is 8-12min, the centrifuging speed is 4000rpm, and taking supernatant after centrifuging to obtain the dyed serum;
step 3, preparing a gel tube: fixing a glass tube on a support, adding separation glue into the glass tube, wherein the glue layer height of the separation glue is 71-79mm, adding concentrated glue into the glass tube after the gel surface is polymerized to be flat, the glue layer height of the concentrated glue is 6-14mm, and after the gel surface is polymerized to be flat, irradiating an incandescent lamp to 30mim for photopolymerization to obtain a gel tube;
and 4, electrophoresis: vertically placing the gel tube prepared in the step 3 into a disc electrophoresis tank, injecting electrophoresis buffer solutions into the positive and negative electrophoresis tanks respectively, slowly adding the dyed serum prepared in the step 2 into the gel tube, covering the surface of the separation gel, and switching on a voltage-stabilized power supply, wherein the current is 2-10 mA/tube, and the time is 1.5-2 h;
further, the dye liquor used in the step 2 is Sudan black B dye liquor.
Further, the preparation method of the Sudan black B dye solution comprises the steps of mixing 0.25g of Sudan black B with 25ml of ethylene glycol, heating at 50-60 ℃ for 5min, continuously stirring during heating, heating to 100-110 ℃, heating for 5min, filtering with a 0.22um membrane while hot, cooling, filtering with the 0.22um membrane once, adding 15ml of cane sugar, uniformly mixing, and keeping out of the sun.
Further, in the step 3, after the separation gel is added, 100uL of distilled water is slowly injected to cover the surface of the separation gel, the mixture is kept stand for more than 30min, and the water layer is removed after the gel is polymerized to be flat.
Further, in the step 3, after the concentrated gel is added, 100uL of distilled water is slowly injected to cover the surface of the separation gel, the separation gel is kept stand for more than 30min, and the water layer is removed after the gel is polymerized to be flat.
Further, the preparation method of the separation gel used in the step 3 comprises taking 0.857ml of 35% N, N' -methylenebisacrylamide, 2.5ml of 1.5M Tris-HCl (pH8.8), 0.1ml of 10% APS, 0.01ml of TEMED and 6.533ml of H2O。
Further, the preparation method of the concentrated glue used in the step 3 comprises a solution A, a solution B, a solution C and a solution D in a volume ratio of 1:2:1: 4;
the preparation method of the solution A comprises the steps of taking 48mL of HCL with the concentration of 1mol/L, 5.98g of Tris and 0.46mL of TEMED, adding water to 100mL, and adjusting the pH value to 6.7;
the preparation method of the solution B comprises the steps of taking 10.0g of acrylamide and 2.5g of N, N' -methylene bisacrylamide, and adding water to 100 mL;
the preparation method of the solution C comprises the steps of taking 4mg of riboflavin, and adding water to 100 mL;
the preparation method of the solution D comprises the steps of taking 40g of sucrose and adding water to 100 mL.
Furthermore, 550-600ml of electrophoresis buffer solution is injected into the anode tank, and 400-500ml of electrophoresis buffer solution is injected into the cathode tank in the step 4.
Further, the preparation method of the electrophoresis buffer used in the step 4 comprises uniformly mixing 80-100mM Tris, 70-90mM boric acid and 2-4mM EDTA, and adjusting the pH to 8.3.
Sudan black B stain is proportionally combined with the relative content of cholesterol in each lipoprotein, and the prestained lipoproteins are then subjected to electrophoresis, and in the first stage of the electrophoresis, lipoprotein particles are accumulated into a sharp narrow band by a concentrated gel and are concentrated, and when the lipoprotein particles migrate through a separation gel matrix, due to the sieving effect of the gel, the lipoprotein particles are decomposed into lipoprotein bands from large to small according to the particle size, wherein HDL migrates farthest, and then low-density LDL, IDL, VLDL and the like.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention optimizes the electrophoresis conditions to ensure that Very Low Density Lipoprotein (VLDL), medium density lipoprotein (IDL), Low Density Lipoprotein (LDL) and High Density Lipoprotein (HDL) can be accurately separated, optimizes the acrylamide gel concentration and the preparation method of Sudan black B staining solution, and realizes accurate separation and quantitative analysis of serum LDL;
2. the detection process of the invention is simple and easy to implement, does not need a special mixing device, and the electrophoresis time end and the quantitative method do not need radioactive substances, thereby having obvious superiority in performance and being fast and convenient to use.
Drawings
FIG. 1 is a gel electrophoresis chart of the serum of a clinically confirmed cardiovascular patient according to the invention.
FIG. 2 is a gel electrophoresis pattern of human serum taken in accordance with the present invention.
Detailed Description
The present invention is further described with reference to the following examples, which are intended to illustrate, unless otherwise indicated, all conventional reagents and methods known in the art.
Among them, ethylene glycol (CAS: 107-21-1), BCgrade (Biochemical reagent).
Sudan Black B (Sudanblack B, CAS:4197-25-5), BRgrade (Biologcalreagent).
Phosphotungstic acid (phosphotungstic acid44-hydrate, CAS:12067-99-1), BCgrade (Biochemical reagent).
Tris (CAS: 77-86-1), MolecuLarBiologyGrade (Biotech engineering, Shanghai, Ltd., cat # A600194).
Boric acid (CAS:10043-35-3), MolecuLarBiologyGrade (Biotech engineering (Shanghai) Co., Ltd., Cat. No.: A610044).
Disodium ethylenediaminetetraacetate (EDTA, CAS:6381-92-6), ARGrade (national chemical group, Inc., Cat. No.: 10009717).
HCl (CAS: 7647-01-0), ARGrade (national group chemical Co., Ltd., Cat. No.: 10009717).
Ammonium persulfate (APS, CAS:7727-54-0), ACSgrade (Bio-engineering (Shanghai) Co., Ltd., Cat. No.: A100486).
Acrylamide (Acrylamide, CAS: 79-06-1), MolecuLarBiologyGrade (Biotech, Ltd., Cat. No.: A100341).
N, N '-Methylenebisacrylamide (N, N' -Methylenebisacrylamide, CAS number 110-26-9) (sigma-aldrich, cat # M7256).
N, N, N ', N' -tetramethylethylenediamine (TEMED, CAS:110-18-9) BRgrade (Biotechnology engineering (Shanghai) Co., Ltd., Cat. No.: A100761).
Riboflavin (CAS: 83-88-5), UPSgrade (Biotechnology engineering (Shanghai) Co., Ltd., Cat. No.: A600470).
Sucrose (CAS:57-50-1), ARgrade (national drug group chemical Co., Ltd., product number: 10021418).
The company of the reagent used is the recommended company, and products from other companies may be used, but the reagent grade must not be lower than the above-identified grade.
Example 1:
the method for detecting low-density lipoprotein in serum by using improved gel electrophoresis comprises the following steps:
step 1, collecting samples: collecting a venous blood sample with an empty stomach of 12h, and separating serum within 3 h;
step 2, dyeing: taking 70uL of the serum obtained in the step 1, adding 15uL of Sudan black B dye solution, mixing uniformly, incubating for 20min under the condition of 37 ℃ water bath, centrifuging, wherein the centrifuging time is 8min, the centrifuging speed is 4000rpm, and taking the supernatant after centrifuging to obtain the dyed serum;
step 3, preparing a gel tube: fixing a glass tube on a bracket, adding separation glue into the glass tube, wherein the glue layer height of the separation glue is 71mm, slowly injecting 100uL of distilled water to cover the surface of the separation glue, standing for 30min, removing a water layer after gel polymerization is leveled, adding concentrated glue into the glass tube, wherein the glue layer height of the concentrated glue is 6mm, slowly injecting 100uL of distilled water to cover the surface of the separation glue, standing for 30min, removing the water layer after gel polymerization is leveled, and performing photopolymerization by using an incandescent lamp to irradiate 30mim to obtain a gel tube;
and 4, electrophoresis: vertically placing the gel tube prepared in the step 3 into a disc electrophoresis tank, respectively injecting 550ml of electrophoresis buffer solution into an anode tank, injecting 400ml of electrophoresis buffer solution into a cathode tank, slowly adding the dyed serum prepared in the step 2 into the gel tube, covering the surface of the separation gel, and switching on a voltage-stabilized power supply, wherein the current is 2 mA/tube, and the time is 1.5 h;
the preparation method of the Sudan black B dye solution comprises the steps of mixing 0.25g of Sudan black B with 25ml of ethylene glycol, heating at 50 ℃ for 5min, continuously stirring during heating, then heating to 100 ℃, heating for 5min, filtering with a 0.22um membrane while hot, cooling, filtering with a 0.22um membrane once, adding 15ml of cane sugar, uniformly mixing, and storing in a dark place.
The preparation method of the separation gel used in the step 3 comprises taking 0.857ml of 35% N, N' -methylenebisacrylamide, 2.5ml of 1.5M Tris-HCl (pH8.8), 0.1ml of 10% APS, 0.01ml of TEMED and 6.533ml of H2O。
The preparation method of the concentrated gel used in the step 3 comprises a solution A, a solution B, a solution C and a solution D, wherein the volume ratio of the solution A to the solution B to the solution C to the solution D is 1:2:1: 4;
the preparation method of the solution A comprises the steps of taking 48mL of HCL with the concentration of 1mol/L, 5.98g of Tris and 0.46mL of TEMED, adding water to 100mL, and adjusting the pH value to 6.7;
the preparation method of the solution B comprises the steps of taking 10.0g of acrylamide and 2.5g of N, N' -methylene bisacrylamide, and adding water to 100 mL;
the preparation method of the solution C comprises the steps of taking 4mg of riboflavin, and adding water to 100 mL;
the preparation method of the solution D comprises the steps of taking 40g of sucrose and adding water to 100 mL.
The preparation method of the electrophoresis buffer used in the step 4 comprises the steps of uniformly mixing 80mM Tris, 70mM boric acid and 2mM EDTA, and adjusting the pH value to 8.3.
Example 2:
the method for detecting low-density lipoprotein in serum by using improved gel electrophoresis comprises the following steps:
step 1, collecting samples: collecting a venous blood sample with an empty stomach of 13h, and separating serum within 3 h;
step 2, dyeing: taking 80uL of the serum obtained in the step 1, adding 20uL of Sudan black B dye solution, mixing uniformly, incubating for 20min under the condition of 37 ℃ water bath, centrifuging, wherein the centrifuging time is 10min, the centrifuging speed is 4000rpm, and taking the supernatant after centrifuging to obtain the dyed serum;
step 3, preparing a gel tube: fixing a glass tube on a bracket, adding separation glue into the glass tube, wherein the glue layer height of the separation glue is 75mm, slowly injecting 100uL of distilled water to cover the surface of the separation glue, standing for 35min, removing a water layer after gel polymerization is leveled, adding concentrated glue into the glass tube, wherein the glue layer height of the concentrated glue is 10mm, slowly injecting 100uL of distilled water to cover the surface of the separation glue, standing for 35min, removing the water layer after gel polymerization is leveled, and performing photopolymerization by using an incandescent lamp to irradiate 30mim to obtain a gel tube;
and 4, electrophoresis: vertically placing the gel tube prepared in the step 3 into a disc electrophoresis tank, respectively injecting 575ml of electrophoresis buffer solution into an anode tank, injecting 450ml of electrophoresis buffer solution into a cathode tank, slowly adding the dyed serum prepared in the step 2 into the gel tube, covering the surface of the separation gel, and switching on a voltage-stabilized power supply, wherein the current is 8 mA/tube, and the time is 1.7 h;
the preparation method of the Sudan black B dye solution comprises the steps of mixing 0.25g of Sudan black B with 25ml of ethylene glycol, heating at 55 ℃ for 5min while continuously stirring, then heating to 105 ℃, heating for 5min, filtering with a 0.22um membrane while the Sudan black B dye solution is hot, cooling, filtering with a 0.22um membrane once, then adding 15ml of cane sugar, uniformly mixing, and storing in a dark place.
The preparation method of the separation gel used in the step 3 comprises taking 0.857ml of 35% N, N' -methylenebisacrylamide, 2.5ml of 1.5M Tris-HCl (pH8.8), 0.1ml of 10% APS, 0.01ml of TEMED and 6.533ml of H2O。
The preparation method of the concentrated gel used in the step 3 comprises a solution A, a solution B, a solution C and a solution D, wherein the volume ratio of the solution A to the solution B to the solution C to the solution D is 1:2:1: 4;
the preparation method of the solution A comprises the steps of taking 48mL of HCL with the concentration of 1mol/L, 5.98g of Tris and 0.46mL of TEMED, adding water to 100mL, and adjusting the pH value to 6.7;
the preparation method of the solution B comprises the steps of taking 10.0g of acrylamide and 2.5g of N, N' -methylene bisacrylamide, and adding water to 100 mL;
the preparation method of the solution C comprises the steps of taking 4mg of riboflavin, and adding water to 100 mL;
the preparation method of the solution D comprises the steps of taking 40g of sucrose and adding water to 100 mL.
The preparation method of the electrophoresis buffer used in the step 4 comprises the steps of uniformly mixing 90mM Tris, 80mM boric acid and 3mM EDTA, and adjusting the pH value to 8.3.
Example 3:
the method for detecting low-density lipoprotein in serum by using improved gel electrophoresis comprises the following steps:
step 1, collecting samples: collecting a 14h fasting venous blood sample, and separating serum within 3 h;
step 2, dyeing: taking 90uL of the serum obtained in the step 1, adding 25uL of Sudan black B dye solution, mixing uniformly, incubating for 20min under the condition of 37 ℃ water bath, centrifuging, wherein the centrifuging time is 12min, the centrifuging speed is 4000rpm, and taking the supernatant after centrifuging to obtain the dyed serum;
step 3, preparing a gel tube: fixing a glass tube on a support, adding separation glue into the glass tube, wherein the glue layer height of the separation glue is 79mm, slowly injecting 100uL of distilled water to cover the surface of the separation glue, standing for 40min, removing a water layer after gel polymerization is smooth, adding concentrated glue into the glass tube, wherein the glue layer height of the concentrated glue is 14mm, slowly injecting 100uL of distilled water to cover the surface of the separation glue, standing for 40min, removing the water layer after gel polymerization is smooth, and performing photopolymerization by using an incandescent lamp to irradiate 30mim to obtain a gel tube;
and 4, electrophoresis: vertically placing the gel tube prepared in the step 3 into a disc electrophoresis tank, respectively injecting 600ml of electrophoresis buffer solution into an anode tank, injecting 500ml of electrophoresis buffer solution into a cathode tank, slowly adding the dyed serum prepared in the step 2 into the gel tube, covering the surface of the separation gel, and switching on a voltage-stabilized power supply, wherein the current is 10 mA/tube, and the time is 2 hours;
the preparation method of the Sudan black B dye solution comprises the steps of mixing 0.25g of Sudan black B with 25ml of ethylene glycol, heating at 60 ℃ for 5min, continuously stirring during heating, then heating to 110 ℃, heating for 5min, filtering with a 0.22um membrane while hot, cooling, filtering with a 0.22um membrane once, adding 15ml of cane sugar, uniformly mixing, and storing in a dark place.
The preparation method of the separation gel used in the step 3 comprises taking 0.857ml of 35% N, N' -methylenebisacrylamide, 2.5ml of 1.5M Tris-HCl (pH8.8), 0.1ml of 10% APS, 0.01ml of TEMED and 6.533ml of H2O。
The preparation method of the concentrated gel used in the step 3 comprises a solution A, a solution B, a solution C and a solution D, wherein the volume ratio of the solution A to the solution B to the solution C to the solution D is 1:2:1: 4;
the preparation method of the solution A comprises the steps of taking 48mL of HCL with the concentration of 1mol/L, 5.98g of Tris and 0.46mL of TEMED, adding water to 100mL, and adjusting the pH value to 6.7;
the preparation method of the solution B comprises the steps of taking 10.0g of acrylamide and 2.5g of N, N' -methylene bisacrylamide, and adding water to 100 mL;
the preparation method of the solution C comprises the steps of taking 4mg of riboflavin, and adding water to 100 mL;
the preparation method of the solution D comprises the steps of taking 40g of sucrose and adding water to 100 mL.
The preparation method of the electrophoresis buffer used in the step 4 comprises the steps of uniformly mixing 100mM Tris, 90mM boric acid and 4mM EDTA, and adjusting the pH value to 8.3.
And after the electrophoresis is finished, the power supply is turned off, the gel tube is taken down, the optical scanner is used for scanning and imaging the gel tube, the wavelength of the scanned monochromatic light is 610nm, blank gel is used as a uniform reference, inflection points between adjacent electrophoresis peaks are used as segmentation marks, and the percentage of the area under each electrophoresis peak to the whole serum lipoprotein is calculated.
Example 4:
serum of a clinically confirmed cardiovascular patient and serum of a healthy person were taken, gel tubes were prepared using the formulation of example 2, and electrophoreses were performed, respectively, to obtain electropherograms as shown in fig. 1-2.
Wherein, fig. 1 is a gel electrophoresis chart of serum of a clinically confirmed cardiovascular patient, fig. 2 is a gel electrophoresis chart of serum of a healthy person, and the comparison between fig. 1 and fig. 2 shows that the method of the invention can separate the dyed serum, thereby clearly separating LDL subtypes, having higher accuracy, simple operation and improved detection efficiency.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (9)
1. The method for detecting low-density lipoprotein in serum by improved gel electrophoresis is characterized by comprising the following steps: the method comprises the following steps:
step 1, collecting samples: collecting venous blood sample with fasting state of more than 12h, separating serum within 3h, and storing the serum sample at-80 deg.C if it can not be measured in time;
step 2, dyeing: adding the serum obtained in the step 1 into a dye solution, mixing uniformly, incubating for 15-25min under the condition of 37 ℃ water bath, centrifuging, and taking the supernatant after centrifuging to obtain the dyed serum;
step 3, preparing a gel tube: fixing a glass tube on a support, adding separation glue into the glass tube, wherein the glue layer height of the separation glue is 71-79mm, adding concentrated glue into the glass tube after the gel surface is polymerized to be flat, the glue layer height of the concentrated glue is 6-14mm, and after the gel surface is polymerized to be flat, irradiating an incandescent lamp to 30mim for photopolymerization to obtain a gel tube;
and 4, electrophoresis: vertically placing the gel tube prepared in the step 3 into a disc electrophoresis tank, injecting electrophoresis buffer solutions into the positive and negative electrophoresis tanks respectively, slowly adding the dyed serum prepared in the step 2 into the gel tube, covering the surface of the separation gel, and switching on a voltage-stabilized power supply, wherein the current is 2-10 mA/tube, and the time is 1.5-2 h.
2. The method for detecting low density lipoproteins in serum by modified gel electrophoresis as claimed in claim 1, wherein: the dye liquor used in the step 2 is Sudan black B dye liquor.
3. The method for detecting low density lipoproteins in serum by modified gel electrophoresis as claimed in claim 2, wherein: the preparation method of the Sudan black B dye solution comprises the steps of mixing 0.25g of Sudan black B with 25ml of ethylene glycol, heating at 50-60 ℃ for 5min, continuously stirring during heating, heating to 110 ℃ for heating, heating for 5min, filtering with a 0.22um membrane while hot, cooling, filtering with the 0.22um membrane once, adding 15ml of cane sugar, uniformly mixing, and keeping out of the sun.
4. The method for detecting low density lipoproteins in serum according to the modified gel electrophoresis as described in claim 1 or 2, wherein: and 3, slowly injecting 100uL of distilled water to cover the surface of the separation gel after the separation gel is added, standing for more than 30min, and removing the water layer after the gel is polymerized to be flat.
5. The method for detecting low density lipoproteins in serum according to the modified gel electrophoresis as described in claim 1 or 2, wherein: and 3, slowly injecting 100uL of distilled water to cover the surface of the separation gel after the concentrated gel is added, standing for more than 30min, and removing the water layer after the gel is polymerized to be flat.
6. The method for detecting low density lipoproteins in serum according to the modified gel electrophoresis as described in claim 1 or 2, wherein: the preparation method of the separation gel used in the step 3 comprises taking 0.857ml of 35% N, N' -methylenebisacrylamide, 2.5ml of 1.5M Tris-HCl (pH8.8), 0.1ml of 10% APS, 0.01ml of TEMED and 6.533ml of H2O.
7. The method for detecting low density lipoproteins in serum according to the modified gel electrophoresis as described in claim 1 or 2, wherein: the preparation method of the concentrated gel used in the step 3 comprises a solution A, a solution B, a solution C and a solution D, wherein the volume ratio of the solution A to the solution B to the solution C to the solution D is 1:2:1: 4;
the preparation method of the solution A comprises the steps of taking 48mL of HCL with the concentration of 1mol/L, 5.98g of Tris and 0.46mL of TEMED, adding water to 100mL, and adjusting the pH value to 6.7;
the preparation method of the solution B comprises the steps of taking 10.0g of acrylamide and 2.5g of N, N' -methylene bisacrylamide, and adding water to 100 mL;
the preparation method of the solution C comprises the steps of taking 4mg of riboflavin, and adding water to 100 mL;
the preparation method of the solution D comprises the steps of taking 40g of sucrose and adding water to 100 mL.
8. The method for detecting low density lipoproteins in serum according to the modified gel electrophoresis as described in claim 1 or 2, wherein: in the step 4, 550-600ml of electrophoresis buffer solution is injected into the anode tank, and 400-500ml of electrophoresis buffer solution is injected into the cathode tank.
9. The method for detecting low density lipoproteins in serum according to the modified gel electrophoresis as described in claim 1 or 2, wherein: the preparation method of the electrophoresis buffer used in the step 4 comprises the steps of uniformly mixing 80-100mM Tris, 70-90mM boric acid and 2-4mM EDTA, and adjusting the pH value to 8.3.
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