JP2007248131A - Separating method of lipoprotein using cellulose acetate film - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new separating method of lipoprotein using an electrophoresis method, and to provide a method of detecting a disease always resulting from lipid metabolic abnormality represented by hyperlipidemia using an existence form of a subclass of the lipoprotein as an index. <P>SOLUTION: The method of separating the lipoprotein in a specimen by the electrophoresis method using cellulose acetate film containing dextran with a molecular weight of 40,000-2,000,000 is provided. A method of detecting a disease resulting from lipid metabolic abnormality by recognizing the existence form of the subclass of the separated lipoprotein VLDL, LDL, and HDL is provided. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for separating lipoproteins by electrophoresis using a cellulose acetate membrane. The present invention also relates to a method for detecting a disease caused by abnormal lipid metabolism by immunostaining lipoproteins separated by electrophoresis and confirming the presence of subclasses of VLDL, LDL, and HDL.

  By detecting quantitative and qualitative abnormalities of each lipoprotein in the specimen, abnormal lipid metabolism can be estimated. Therefore, lipoprotein separation analysis is an important clinical method for diagnosis and treatment of diseases caused by abnormal lipid metabolism such as hyperlipidemia, coronary sclerosis, hypothyroidism, obstructive liver disease, diabetes, and renal failure. One of the inspections.

  Lipoproteins are particles in which lipids and proteins are combined, solubilized in blood, and play a role in transporting lipids. As for the types of lipoproteins, from the lighter specific gravity, chylomicron (CHYL), very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), high density lipoprotein ( HDL) and the like. The difference in the specific gravity, electrical mobility, and particle size of lipoprotein is derived from the difference in the ratio of lipid and protein (apoprotein) constituting lipoprotein and the type of protein (apoprotein). The lipids constituting the lipoprotein include triglycerides, cholesterol esters, free cholesterol, and phospholipids. ApoAI, apoAII, apoB-100, apoB-48, the main apoproteins constituting the lipoprotein, There are Apo CI, Apo CII, Apo CIII, Apo E and the like.

  Hyperlipidemia, the main disease caused by abnormal lipid metabolism, is caused by an increase in the amount of one or two types of lipoproteins. The classification of various hyperlipidemias is important for the analysis of the cause and pathophysiology of primary and secondary hyperlipidemia, and the determination of treatment methods. Type I hyperlipidemia is caused by congenital lipoprotein lipase (LPL) deficiency or congenital apo CII deficiency. Type IIa hyperlipidemia increases LDL due to abnormal LDL receptors and develops hereditary familial hypercholesterolemia. There are two types of familial hypercholesterolemia, heterozygous and homozygous. Heterotypic is prevalent, about 1 in 500, and homozygous is rare, about 1 in 1 million. However, it is easy to develop arteriosclerosis from a young age, and treatment is necessary. Hyperlipidemia type IIb, in which LDL and VLDL increase, and type IV, in which VLDL increases, often have secondary diabetes as secondary hyperlipidemia. Type III hyperlipidemia increases IDL, and type V hyperlipidemia increases CHYL and VLDL. Since the most common onset is type IIa hyperlipidemia in Japan, observing an increase in lipoprotein LDL is important for the diagnosis and treatment of type IIa hyperlipidemia. .

  Lipoprotein fractionation methods include ultracentrifugation, polyacrylamide gel (PAG), agarose gel, electrophoresis using a cellulose acetate membrane as a support, and high-performance liquid chromatography (HPLC).

  In ultracentrifugation, lipoproteins in serum are separated into CHYL, VLDL, LDL, and HDL from the upper liquid based on the specific gravity of the lipoproteins. However, the ultracentrifugation method requires time and labor to prepare and process samples for ultracentrifuges, so in clinical laboratories, detection of fractions specific to specific diseases, increase and decrease of those fractions, etc. It is difficult to perform analysis with detailed analysis.

  On the other hand, since the electrophoresis method can visually grasp the separation state of lipoproteins, the separation and analysis of lipoproteins is often carried out using the electrophoresis method in daily examinations.

  The PAG electrophoresis method is an electrophoresis method using a polyacrylamide gel (PAG) having a predetermined pore size and a high molecular sieve effect. In the lipoprotein, HDL having the smallest particle size is separated first. Are separated in the order of LDL, VLDL, and CHYL. That is, this method is based on the difference in the size of each particle in the lipoprotein, not the difference in isoelectric point of each particle in the lipoprotein. However, since the PAG electrophoresis method lowers the gel concentration, the gel is soft, and development to secondary analysis such as blotting and staining is difficult, and it is difficult to perform analysis that requires detailed analysis.

  Agarose gel electrophoresis is not only a difference in the size of each particle in lipoprotein by PAG electrophoresis, but also a qualitative or quantitative abnormality in the apoprotein of particles with different types and numbers of apoproteins. It is known as a method of analyzing. For example, a method for capturing an abnormality in an apoprotein directly involved in lipid metabolism by comparing a fraction of LDL in which apoprotein B-100 is denatured with a fraction of normal LDL in which LDL is not denatured ( Patent Document 1) and the like.

An electrophoresis method using a cellulose acetate membrane of lipoprotein is made by adding a polyoxyethylene sorbitol fatty acid ester as a additive to a microporous membrane mainly composed of cellulose acetate in a certain weight ratio. The literature which provided the cellulose acetate membrane which eliminated the influence of the low density lipoprotein which influences isolation | separation of (alpha) ₂ -globulin and (beta) -globulin, and measured the fraction fraction of serum protein correctly by the made membrane (patent document 2) However, it does not clarify how to separate each lipoprotein. In addition, as a method for detecting a disease using cellulose acetate membrane electrophoresis, an early diagnostic method for diabetic nephropathy has been clarified by detecting a urinary albumin degradation product of a diabetic patient (Patent Document 3). However, it does not clarify how to estimate abnormal lipid metabolism.

  In the separation of lipoproteins using high performance liquid chromatography (HPLC), LDL and HDL subclasses are analyzed using gel filtration chromatography packed with a gel having a molecular sieving effect. It is made clear that the diagnosis of (Patent Document 4)

As immunoassay methods, LDL, HDL, VLDL, IDL and other lipoproteins present in blood have been denatured. Denatured lipoprotein detection methods include enzyme immunization, latex agglutination, immunoluminescence analysis, immunochromatography, etc. Using the immunological method described above, measurement is performed by a color development method on a microplate (Patent Document 5), and LDL and denatured LDL in blood are pretreated by ultracentrifugation or dextran sulfate / Ca precipitation. In addition, a method for measuring a labeling substance in a solution by a coloring method (Patent Document 6) is known, but an immunoassay method for lipoprotein using cellulose acetate membrane electrophoresis is not known.
JP 2000-356541 A JP-A-6-66768 JP-A-6-160384 JP 2002-139501 A JP 2001-66307 A JP 2003-227837 A

  Diseases caused by abnormal lipid metabolism, such as hyperlipidemia, are due to qualitative or quantitative abnormalities of each lipoprotein, so it is easy to increase or decrease each lipoprotein or a specific lipoprotein subclass. Measuring is important for detecting diseases caused by abnormal lipid metabolism. The present invention provides a novel method for separating lipoproteins using electrophoresis, and detects diseases caused by abnormal lipid metabolism such as hyperlipidemia using the presence of lipoprotein subclasses as indicators. The main problem is to provide a method for doing this.

  As a result of intensive studies to solve the above-mentioned problems, it was found that a good separation of lipoproteins can be obtained by incorporating a water-soluble polymer into a cellulose acetate membrane used in electrophoresis, and the present invention has been completed. It was. That is, the present invention has the following configuration.

  (1) A cellulose acetate membrane for electrophoresis, which contains a water-soluble polymer used for separating lipoproteins in a specimen.

  (2) The cellulose acetate membrane, wherein the content of the water-soluble polymer adjusts the viscosity of the buffer solution infiltrating the cellulose acetate membrane to a concentration of 10 to 50 cP.

  (3) A cellulose acetate membrane, wherein the water-soluble polymer according to (1) or (2) is dextran.

  (4) A cellulose acetate membrane, wherein the molecular weight of dextran according to (3) is 40,000 to 2,000,000.

  (5) A lipoprotein separation method, wherein lipoproteins contained in a specimen are separated by electrophoresis using the cellulose acetate membrane according to any one of (1) to (4).

  (6) Lipoprotein contained in the sample is separated by electrophoresis using the cellulose acetate membrane according to any one of (1) to (4), and then the lipoprotein on the separated membrane is Transferring to a transfer membrane, reacting the transfer membrane with an anti-lipoprotein antibody as a primary antibody, then reacting with a secondary antibody labeled with an enzyme, and detecting using a chromogenic reagent Separation method.

  (7) Lipoprotein contained in the specimen is separated by electrophoresis using the cellulose acetate membrane according to any one of (1) to (4), and then the lipoprotein on the separated membrane is It is transferred to a transfer membrane, reacted with an anti-lipoprotein sheep antibody as a primary antibody on the transfer membrane, then reacted with a peroxidase-labeled anti-sheep IgG antibody as a secondary antibody, and detected using a coloring reagent. To separate lipoproteins.

  (8) The presence of subclasses of lipoproteins VLDL, LDL, and HDL contained in the specimen is confirmed by using the separation method according to any of (5) to (7), resulting in abnormal lipid metabolism. A method of detecting a disease.

(9) The method for detecting a disease caused by abnormal lipid metabolism according to (8), wherein the disease caused by abnormal lipid metabolism is hyperlipidemia, hypercholesterolemia and hypocholesterolemia .
(10) A kit used for carrying out the separation method by electrophoresis according to any one of (5) to (7).

  In the electrophoresis method using the cellulose acetate membrane of the present invention, it is possible to easily separate and analyze the lipoprotein subclass in the specimen. The pathological condition can be detected, and appropriate treatment can be performed for each disease.

  The method for separating and analyzing lipoproteins of the present invention is based on electrophoresis using a cellulose acetate membrane. Lipoprotein separation by electrophoresis is generally based on the difference in isoelectric point, but fractionation based on the difference in the size of each particle in the lipoprotein is also important when observing subclasses. .

  Cellulose acetate membrane electrophoresis was performed in 1957 by Kohn. Since J's announcement as a method for analyzing serum proteins, it has been used in many research and clinical fields. Furthermore, as for cellulose acetate membranes, membranes excellent in protein separation have been developed. As the cellulose acetate membrane used in the present invention, various commercially available membranes can be used as long as they are used in normal screening for plasma protein abnormalities. For example, a porous membrane Separax-S (manufactured by Fuji Film Co., Ltd.), Separax-EF (manufactured by Fuji Film Co., Ltd.), Separax-SP (manufactured by Fuji Film Co., Ltd.), etc. For example, Separax-SP is preferably used for the separation of lipoproteins.

<Preparation of water-soluble polymer-containing cellulose acetate membrane>
As a method for containing a water-soluble polymer in a cellulose acetate membrane, a medium in which a buffer solution for electrophoresis can be infiltrated is placed on the cellulose acetate membrane, and the buffer solution containing the water-soluble polymer is passed through the medium through the cellulose. Infiltrate the acetate membrane. Examples of the medium that can be infiltrated with the buffer solution for electrophoresis include cellulose, nitrocellulose, and polytetrafluoroethylene (PTFE) filter paper. In general, cellulose is inexpensive and has excellent water-soluble polymer retention ability. A filter paper is preferred.

  The amount of water-soluble polymer contained in the cellulose acetate membrane varies depending on the type of water-soluble polymer or the target lipoprotein separation method, but the viscosity of the buffer solution infiltrating the cellulose acetate membrane is 1-1000 cP. Adjust the density to A more preferable concentration is a concentration at which the viscosity of the buffer solution is 1 to 100 cP, and a most preferable concentration is a concentration at which the viscosity of the buffer solution is 10 to 50 cP. For example, when dextran is used as the water-soluble polymer, when 4% dextran T2000 is dissolved in the buffer, the viscosity of the buffer is about 15 cP, and when 8% dextran T2000 is dissolved in the buffer, The viscosity of the buffer is about 30 cP.

  When water-soluble polymer is infiltrated into the cellulose acetate membrane, it can be infiltrated entirely, but partially infiltrate in the direction of electrophoresis from the vicinity of the sample application point to make a discontinuous buffer solution film of cellulose acetate membrane Is desirable.

  Examples of the water-soluble polymer include polyacrylamide, dextran, polyethylene glycol, polyethylene oxide, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, and agarose. Preferably, polyacrylamide, dextran are used. , Polyethylene glycol, polyvinyl alcohol, methylcellulose and hydroxypropylmethylcellulose, most preferably dextran.

  Dextran has the property that in a solution of the same concentration, the viscosity increases as the molecular weight increases. As the dextran to be contained in the cellulose acetate film, those having an average molecular weight of 40,000, 70,000, 100,000, 200,000, 500,000, 1,000,000 or 2,000,000 can be used. Preferred is dextran having an average molecular weight of 500,000, 1 million or 2 million, and most preferred is dextran having an average molecular weight of 2 million. As the dextran having an average molecular weight of 2 million, it is preferable to use dextran T2000 (manufactured by Amersham).

<Lipoprotein separation by cellulose acetate membrane electrophoresis>
Electrophoresis using cellulose acetate membranes can be performed by either “basic experimental method of protein / enzyme” (edited by Takeichi Horio, Nanedo, 1996), or “Shinsei Chemistry Laboratory 1, Protein I, Separation / Purification / Property”. (Tokyo Chemical Doujin, 1990).

  As the electrophoresis apparatus used in the present invention, 238 type electrophoresis apparatus (manufactured by Joko), EC-100 (manufactured by Toyo Roshi Kaisha), AE-6170AS type (manufactured by Ato), and FastSystem (manufactured by GE Healthcare Bioscience) Is suitable. As the electrophoresis buffer, veronal buffer, Tris buffer (Tris buffer) or the like is used. A Tris buffer (pH 8.6 to pH 8.8) is preferable for more clear separation. The sample coating amount is generally 0.8 to 2.4 μL per 1 cm width, but is appropriately adjusted depending on the concentration of the sample. The energization conditions are not particularly limited as long as they are normally performed, but it is desirable to pass a current of about 0.6 to 0.8 mA per 1 cm width of the film. In this case, the temperature of the cellulose acetate membrane during electrophoresis is generally constant in the range of 10 ° C to 25 ° C.

  Lipoproteins obtained by the cellulose acetate membrane electrophoresis method of the present invention are HDL, LDL, and VLDL in the order of higher electrophoretic mobility. Furthermore, the presence of a continuous subclass of lipoprotein can be confirmed by adjusting the content of the water-soluble polymer. For example, LDL subclass 1, LDL subclass 2, and LDL subclass 3 can be confirmed by performing electrophoresis using a cellulose acetate membrane infiltrated with 8% dextran T2000.

  When separating and analyzing lipoprotein subclasses, ultracentrifugation can be performed before electrophoresis to remove certain lipoproteins and effectively separate the desired lipoprotein subclass. . For example, when ultracentrifugation is performed by adding a mixed solution of dextran sulfate and magnesium chloride to a sample, VLDL and LDL of the lipoprotein are precipitated and removed, so that the HDL subclass can be analyzed.

<Lipoprotein immunostaining method>
In the immunostaining method of the present invention, lipoproteins separated by cellulose acetate membrane electrophoresis are transferred to a transfer membrane, reacted with an anti-lipoprotein antibody added to the transfer membrane, washed, and then labeled with an enzyme-labeled second antibody. In this method, the reaction is carried out and then the lipoprotein is confirmed with a coloring reagent.

  As the membrane for transferring the separated lipoprotein, a conventionally known membrane can be used, and a nitrocellulose membrane, a polyvinylidene difluoride membrane and the like are preferable. The transfer means is also performed based on a conventionally known method. Blotting buffer such as Tris buffer can be used to transfer by energization. However, when a cellulose acetate membrane is used for separation, a filter paper moistened with an aqueous solution is placed under the cellulose acetate membrane, resulting in capillary action. A transfer method using is preferable. The transferred film is blocked using skim milk solution, BSA solution, BlockAce (Dainippon Pharmaceutical Co., Ltd.), or the like.

  The transferred lipoprotein is reacted with an anti-lipoprotein antibody as a primary antibody. The reaction can be performed by a conventional method. For example, the reaction is performed by shaking the transfer membrane in a solution of anti-lipoprotein diluted with skim milk solution, BSA solution, or BlockAce solution. Unreacted primary antibody is removed by washing with, for example, a TBS solution. As the anti-human lipoprotein antibody, commercially available antibodies such as a mouse antibody, a rabbit antibody, and a sheep antibody can be used. Sigma Aldrich).

  Next, a secondary antibody labeled with an enzyme is reacted. The reaction between the primary antibody and the labeled secondary antibody is performed, for example, by shaking in a skim milk solution, a BSA solution, or a TBS solution. Unreacted labeled secondary antibody is removed by washing with, for example, a TBS solution. As the labeled secondary antibody, a peroxidase (POD) labeled antibody, an alkaline phosphatase (ALP) labeled antibody, a horseradish peroxidase (HRP) labeled antibody, a β-galactosidase (GluOx) labeled antibody, or the like can be used depending on the type of enzyme. . The enzyme-labeled antibody is used by selecting whether the final detection method is an enzyme coloring method or a fluorescence method. For example, when a POD-labeled antibody or an ALP-labeled antibody is used, both a coloring method and a fluorescence method are possible. In the color development method or the fluorescence method, the color development or fluorescence expression degree of the product produced by the reaction between the reagent and the enzyme labeled with the secondary antibody is measured.

  In the present invention, the detection of the lipoprotein bound to the secondary antibody is detected by the color development method by the reaction of the enzyme labeled with the secondary antibody and the coloring reagent. As the coloring reagent, o-phenyldiamine (OPD), tetramethylbenzidine (TMBZ), diaminobenzidine (DBA), 4-chloro-1-naphthol and the like can be used. For example, when a POD-labeled antibody is reacted with a 4-chloro-1-naphthol solution, a blue band derived from lipoprotein can be observed on the transfer membrane.

<Method for detecting diseases caused by abnormal lipid metabolism>
“Abnormal lipid metabolism” in the present invention refers to a state in which the amount of lipid in blood is abnormally large or small as compared with the case where the amount of lipid in blood is normal. Usually, the amount of lipid in the blood is constantly adjusted so as to be almost constant, but for some reason, the balance of the amount of lipid in the blood is lost, and the amount of lipid in the blood may be abnormal. If the amount of lipid in the blood is too high, it is usually judged as hyperlipidemia. Hyperlipidemia is one of the risk factors for arteriosclerotic diseases, and hyperlipidemia patients have a high risk of developing diseases such as myocardial infarction, cerebral infarction, and angina pectoris. On the other hand, when the amount of lipids in the blood is extremely small, nutrients are insufficient in the liver, brain, blood vessels, etc., leading to cerebral hemorrhage and stroke. Hypocholesterolemia, especially in young people, is considered a risk factor for cerebral hemorrhage. In blood, lipids exist as lipoproteins, so diseases caused by abnormal lipid metabolism can be detected by analyzing lipoprotein particles. For example, separation of VLDL and subclasses of LDL from patients whose cholesterol level and / or triglyceride level is higher than normal is possible by selecting the viscosity of the water-soluble polymer contained in the cellulose acetate membrane. Further, the separation form of the HDL or LDL subclass can be obtained by immunostaining after transferring the lipoprotein to the membrane after performing electrophoresis using the cellulose acetate membrane of the present invention.

  The measurement specimen containing lipoprotein for detecting a disease caused by abnormal lipid metabolism is not particularly limited as long as it is a specimen derived from human or animal. For example, biological samples that may contain measurement objects such as blood, urine, plasma, serum, spinal fluid, saliva, ascites or amniotic fluid, sweat, tears, semen, stool, tissue / cell extracts Good. Preferred as a measurement sample is serum or plasma obtained by separating components from a blood sample obtained by collecting blood from a human in accordance with a conventional method such as centrifugation. In order to obtain a more accurate measurement, the serum or plasma can be further used as a lipoprotein fraction separated by ultracentrifugation.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples.

Electrophoresis of serum lipoproteins using cellulose acetate membranes.

<Electrophoresis conditions>
Cellulose acetate membrane: Separax SP (manufactured by Fuji Film), 60 × 44 mm
Electrophoresis apparatus: PhasSystem (manufactured by Amersham), 20 ° C., 250 V, 2 mA
Membrane buffer: 47 mM Tris-HCl buffer (pH 8.8, Tris 4.54 g adjusted to pH with 1N hydrochloric acid to 100 ml), diluted 8 times when used.
Electrode buffer: 365 mM Tris borate buffer (44.2 g of Tris and 8.4 g of boric acid dissolved in 100 ml)
Scanner: Reflected light densitometry (Canon)
Analysis software: After background correction with Scion Image software, analysis is performed with width 20 using the profile plot function.

<Preparation of water-soluble polymer-containing cellulose acetate membrane>
On the cellulose acetate membrane Separax SP, a filter paper is placed 5 mm above the sample application point in the direction of electrophoresis, and a membrane buffer containing 4% or 8% dextran T2000 (Amersham, average molecular weight 2 million) is filtered. So as to impregnate the cellulose acetate membrane. In addition, a membrane buffer solution was soaked under the cellulose acetate membrane not soaked with Dextran T2000 to create a sample concentration zone. On both ends of the prepared electrophoresis filter paper, an isoelectric focusing electrode filter paper (Amersham) impregnated with an electrode buffer was placed.

<Sample serum>
1 volume of Sudan Black B staining solution was mixed with 10 volumes of serum of a healthy adult male (age 49 years old, height 183 cm, weight 86 kg), allowed to stand for 1 hour, centrifuged, and 1.6 μL of the supernatant was obtained. The electrophoretic membrane was applied to a length of 0.8 cm.

<Running time>
The energization was continued until albumin in the BPB (bromophenol blue) serum in the parallel lane moved 2.5 cm. It took about 8 minutes and was about 30 AVh.

<Results of migration of serum lipoprotein>
As shown in FIG. 1, in the film added with 4% dextran T2000, VLDL precedes LDL, and in the film added with 8% dextran T2000, VLDL migrates near the buffer boundary after LDL. HDL always precedes other lipoproteins. That is, in the presence of 8% dextran, it was found that lipoproteins migrate in order of particle size.

Separation of lipoprotein LDL subclass and VLDL in serum by cellulose acetate membrane electrophoresis

<Electrophoresis conditions>
The same cellulose acetate membrane, apparatus, buffer solution and the like as in Example 1 were used.
<Preparation of water-soluble polymer-containing cellulose acetate membrane>
A cellulose acetate membrane Separax SP was prepared in the same manner as in Example 1 using a membrane buffer containing 8% dextran T2000.

<Sample serum>
Serums obtained from 6 adults and 1 healthy person of hyperlipidemic patients were used. Serum cholesterol and triglyceride levels were measured in advance. The serum was treated in the same manner as in Example 1, and 1.6 μL of the supernatant was applied to the electrophoresis membrane to a length of 0.8 cm.

<Results of electrophoresis of lipoprotein LDL subclass and VLDL in serum>
Bands indicating VLDL and LDL subclasses were observed above the application point. This film was measured by reflected light densitometry. The results are shown in FIG. The electrophoretic distance between VLDL and LDL subclass is shown in Table 1. From these results, it was speculated that an increase in LDL subclass 2 and LDL subclass 3 had some influence on hyperlipidemia patients.

Immunostaining of lipoprotein LDL and HDL in serum Electrophoresis was performed in the same manner as in Example 1 using serum obtained from 6 hyperlipidemic patients and 1 healthy person. Lipoproteins VLDL, LDL and HDL were separated on the cellulose acetate membrane.

<Blotting>
The cellulose acetate membrane after electrophoresis was transferred to a nitrocellulose membrane (manufactured by Schleichachler). That is, the cellulose acetate membrane after migration was placed on a filter paper moistened with water, then a nitrocellulose transfer membrane was overlaid, and a dry filter paper was further placed on the top, and left for 3 minutes until moisture permeated. Lipoprotein was blotted onto the nitrocellulose membrane by capillary action.

<Immunostaining method>
After transferring to a nitrocellulose membrane, the membrane was blocked with a blocking solution of 5% skim milk-dissolved 50 mM Tris-HCl buffered saline (TBS, pH 7.4) by shaking for 30 minutes at room temperature. Next, an anti-LDL sheep antibody (manufactured by Sigma-Aldrich) or an anti-HDL sheep antibody (manufactured by Sigma-Aldrich) was diluted 1: 1000 with the above-described blocking reagent, and reacted by shaking overnight at room temperature. After the reaction, washing with shaking with 0.05% Tween20-TBS for 5 minutes was performed 3 times, and then a POD-labeled anti-sheep IgG antibody (Sigma Aldrich) diluted 1: 1000 with the blocking reagent was added at room temperature. The reaction was performed for 3 hours. After the reaction, washing with shaking with 0.05% Tween20-TBS for 5 minutes was performed 3 times. After washing, 4-chloro-1-naphthol in PBS (20% methanol) and hydrogen peroxide solution were added and shaken for 5 minutes. The color was developed. The reaction was stopped with distilled water, and the presence or absence of LDL subclass or HDL subclass was compared with the pattern of healthy subjects.

<Results of electrophoresis of lipoprotein LDL subclass or HDL subclass in serum>
As shown in FIG. 3, it was found from the LDL and HDL patterns that the presence of LDL subclass or HDL subclass was different between hyperlipidemic patients and healthy individuals. Therefore, this method is considered effective for detecting hyperlipidemic patients.

  In the electrophoresis method using the cellulose acetate membrane of the present invention, each lipoprotein can be clearly separated, and a subclass of each lipoprotein can be analyzed. There is a possibility that it can be used as a simple test method capable of detecting diseases caused by metabolic abnormalities.

It is a photograph replacing a drawing in which a buffer containing 4% or 8% dextran T2000 is contained in a membrane and electrophoresis is performed using serum of a healthy adult male. A buffer solution containing 8% dextran T2000 is contained in the membrane, and a photograph replacing the drawing of electrophoresis using serum with hyperlipidemia and normal cholesterol concentration and the electrophoretic pattern of the photograph are reflected light densit It is the chart measured by the measurement. HDL and LDL subclasses containing a buffer containing 8% dextran T2000 in the membrane, subjected to electrophoresis using serum with hyperlipidemia and normal cholesterol concentration, and immunostained the blotted ones. It is a photograph replacing a drawing that can be observed.

Claims (10)

  A cellulose acetate membrane for electrophoresis, comprising a water-soluble polymer used for separating lipoproteins in a specimen.   The cellulose acetate membrane according to claim 1, wherein the content of the water-soluble polymer adjusts the viscosity of the buffer solution infiltrating the cellulose acetate membrane to a concentration of 10 to 50 cP.   The cellulose acetate membrane according to claim 1 or 2, wherein the water-soluble polymer is dextran.   The cellulose acetate membrane according to claim 3, wherein the molecular weight of dextran is 40,000 to 2,000,000.   A method for separating lipoproteins, comprising separating lipoproteins contained in a specimen by electrophoresis using the cellulose acetate membrane according to any one of claims 1 to 4.   Lipoproteins contained in the specimen are separated by electrophoresis using the cellulose acetate membrane according to any one of claims 1 to 4, and then the lipoproteins on the separated membrane are transferred to a transfer membrane. A method for separating a lipoprotein, comprising: reacting an anti-lipoprotein antibody as a primary antibody with the transfer membrane, then reacting a secondary antibody labeled with an enzyme, and detecting using a coloring reagent.   Lipoproteins contained in the specimen are separated by electrophoresis using the cellulose acetate membrane according to any one of claims 1 to 4, and then the lipoproteins on the separated membrane are transferred to a transfer membrane. Separating the lipoprotein, wherein the transfer membrane is reacted with an anti-lipoprotein sheep antibody as a primary antibody and then reacted with a peroxidase-labeled anti-sheep IgG antibody as a secondary antibody and detected using a coloring reagent. Method.   By confirming the presence of the subclasses of lipoproteins VLDL, LDL, and HDL contained in the specimen using the separation method according to any one of claims 5 to 7, diseases caused by abnormal lipid metabolism can be confirmed. How to detect. 9. The method for detecting a disease caused by abnormal lipid metabolism according to claim 8, wherein the disease caused by abnormal lipid metabolism is hyperlipidemia, hypercholesterolemia and hypocholesterolemia. The kit used in order to implement the separation method by the electrophoresis method in any one of Claims 5-7.

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