Apolipoprotein purification and analysis method

Abstract

To develop technology that realizes highly accurate analysis of apolipoprotein.SOLUTION: An apolipoprotein purification method includes: (1) providing a sample containing apolipoprotein, and a solid phase having an anti-apolipoprotein antibody immobilized; (2) causing, in a solution, the apolipoprotein in the sample containing the apolipoprotein to immunologically react with the anti-apolipoprotein antibody immobilized in the solid phase, and generate a solid phase in which the anti-apolipoprotein antibody immunologically reacted with the apolipoprotein is immobilized; (3) separating the solid phase having the anti-apolipoprotein antibody immunologically reacting with the apolipoprotein immobilized; (4) cleaning the separated solid phase by a detergent; and (5) causing the apolipoprotein to be eluted from the cleaned solid phase, in which one of the (2) and (4), or both of the (2) and (4) is / are performed under presence of at least one kind of a surface acting agent.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for purifying and analyzing apolipoproteins. [Background technology]

[0002] Apolipoproteins are protein components that make up lipoproteins. Apolipoproteins play various roles in disease and normal conditions. It has been reported that apolipoproteins include ApoA (e.g., ApoAI, ApoAII, ApoAIV, and ApoAV), ApoB (e.g., ApoB100 and ApoB48), Apo(a), ApoC (e.g., ApoCI, ApoCII, ApoCIII, and ApoCIV), ApoD, ApoE, ApoF, ApoH, ApoJ, ApoL (e.g., ApoLI, ApoLII, ApoLIII, ApoLIV, ApoLV, and ApoLVI), ApoM, ApoO, and their isotypes (Patent Document 1).

[0003] Apolipoprotein CIII (ApoCIII) is a 79 amino acid protein that exists in humans as multiple isotypes (e.g., CIII-0, CIII-1, and CIII-2 isotypes) with different glycosylation at threonine-74. These isotypes have different patterns of modification with glycans (sialic acid, galactose, and N-acetylgalactosamine) and different binding affinities with very low density lipoproteins (VLDL). Analysis of CIII isotypes is clinically important. For example, patients with hypertriglyceridemia have a high proportion of ApoCIII-2 isotype in VLDL. Therefore, a method for analyzing apolipoproteins such as ApoCIII has been reported (Patent Document 1).

[0004] By the way, in immunological measurement of an antigen using an antibody, a surfactant may be used. For example, a sample may be treated with a surfactant before immunological measurement in order to expose an epitope of the antigen (Patent Document 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2009-531712 A [Patent Document 2] JP 2022-174540 A Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to develop a technique for achieving high-precision analysis of apolipoproteins. [Means for solving the problem]

[0007] As a result of intensive research, the inventors discovered that by using a surfactant in a specific step in the purification of apolipoproteins by immunological methods, apolipoproteins can be highly purified, and thus high-precision analysis of apolipoproteins can be achieved, thereby completing the present invention.

[0008] That is, the present invention is as follows. [1] A method for purifying an apolipoprotein, comprising the steps of: (1) providing an apolipoprotein-containing sample and a solid phase having an anti-apolipoprotein antibody immobilized thereon; (2) immunologically reacting the apolipoprotein in the apolipoprotein-containing sample in solution with an anti-apolipoprotein antibody immobilized on a solid phase to produce a solid phase on which is immobilized the anti-apolipoprotein antibody that has immunologically reacted with the apolipoprotein; (3) isolating the solid phase on which the anti-apolipoprotein antibodies that have immunologically reacted with the apolipoprotein are immobilized; (4) washing the separated solid phase with a wash solution; and (5) Eluting the apolipoprotein from the washed solid phase. Including, The method, wherein either (2) or (4), or both (2) and (4), are carried out in the presence of at least one surfactant. [2] The method according to [1], wherein the apolipoprotein is at least one selected from the group consisting of ApoAI, ApoAII, ApoAIV, ApoAV, ApoCI, ApoCII, ApoCIII, ApoCIV, ApoB48, ApoB100, Apo(a), ApoD, ApoE, ApoF, ApoH, ApoJ, ApoLI, ApoLII, ApoLIII, ApoLIV, ApoLV, ApoLVI, ApoM, ApoO, and their isotypes. [3] The method according to [1] or [2], wherein the apolipoprotein is ApoAI or ApoCIII. [4] The method according to any one of [1] to [3], wherein the surfactant is at least one selected from the group consisting of C12-16 alkyltrimethylammonium halides, CHAPS, Tween 80, and alcohol ethoxylates. [5] The method according to [4], wherein the C12-16 alkyltrimethylammonium halide is dodecyltrimethylammonium chloride (C12TAC), tetradecyltrimethylammonium chloride (C14TAC), or hexadecyltrimethylammonium chloride (C16TAC). [6] The method according to [4], wherein the alcohol ethoxylate is Softanol 70, Tergitol 15-S-30, or Tergitol 15-S-40. [7] Any of the methods according to [1] to [6], wherein the apolipoprotein-containing sample is an animal-derived sample. [8] The method according to [7], wherein the animal-derived sample is a blood sample. [9] Any of the methods according to [1] to [8], wherein the solid phase is a bead.

[10] The method according to any one of [1] to [9], wherein the concentration of the surfactant in the solution and the cleaning solution is 0.01 w / v % or more.

[11] The method according to any one of [1] to

[10] , wherein the concentration of the surfactant in the solution and the cleaning solution is 0.01 to 5 w / v % or more.

[12] Any of the methods [1] to

[11] , wherein washing is performed two or more times.

[13] Any of the methods according to [1] to

[12] , wherein the immunological reaction time between the apolipoprotein and the anti-apolipoprotein antibody is 5 minutes or longer.

[14] A method for analyzing an apolipoprotein, comprising the steps of: (1) Obtaining an eluted apolipoprotein by carrying out any one of the methods described in [1] to

[13] ; and (2) subjecting the eluted apolipoproteins to mass spectrometry to analyze the glycosylation of apolipoproteins; A method comprising: Effect of the Invention

[0009] According to the purification method of the present invention, a target apolipoprotein contained in an apolipoprotein-containing sample can be highly purified. Furthermore, according to the analytical method of the present invention, the pattern and / or level of glycosylation of apolipoproteins can be analyzed with high accuracy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention provides a method for purifying an apolipoprotein, comprising the steps of: (1) providing an apolipoprotein-containing sample and a solid phase having an anti-apolipoprotein antibody immobilized thereon; (2) immunologically reacting the apolipoprotein in the apolipoprotein-containing sample in solution with an anti-apolipoprotein antibody immobilized on a solid phase to produce a solid phase on which is immobilized the anti-apolipoprotein antibody that has immunologically reacted with the apolipoprotein; (3) isolating the solid phase on which the anti-apolipoprotein antibodies that have immunologically reacted with the apolipoprotein are immobilized; (4) washing the separated solid phase with a detergent; and (5) Eluting the apolipoprotein from the washed solid phase. Including, Either (2) or (4), or both (2) and (4) are carried out in the presence of at least one surfactant.

[0011] Apolipoproteins are the main constituent proteins of lipoproteins. Lipids in blood are mainly composed of cholesterol, neutral fats, phospholipids, etc., but since these are insoluble in water alone, they are bound to apolipoproteins in blood and dissolved in the form of lipoproteins (e.g., chylomicrons, VLDL, IDL, LDL, HDL). Examples of apolipoproteins include ApoAI, ApoAII, ApoAIV, ApoAV, ApoCI, ApoCII, ApoCIII, ApoCIV, ApoB48, ApoB100, Apo(a), ApoD, ApoE, ApoF, ApoH, ApoJ, ApoLI, ApoLII, ApoLIII, ApoLIV, ApoLV, ApoLVI, ApoM, ApoO, and their isotypes. Therefore, the apolipoprotein purified in the present invention may be at least one (e.g., one, two, or three) selected from the group consisting of ApoAI, ApoAII, ApoB, ApoCII, ApoCIII, and ApoE. Preferably, the apolipoprotein may be ApoCIII. More preferably, the apolipoprotein may be human ApoCIII.

[0012] Regarding (1), the apolipoprotein-containing sample is not particularly limited as long as it is a sample containing apolipoprotein, and the apolipoprotein in the apolipoprotein-containing sample may be in a form bound to a binding factor (e.g., lipid) (e.g., in the form of lipoprotein), in a free form not bound to a binding factor, or in a form containing a mixture of both forms. Preferably, the apolipoprotein-containing sample may be a sample containing apolipoprotein in a form bound to a binding factor (e.g., lipoprotein). The apolipoprotein-containing sample is preferably a liquid sample derived from an animal. Examples of animals include mammals (e.g., primates such as humans and monkeys; rodents such as mice, rats, and rabbits; ungulates such as cows, pigs, goats, horses, and sheep), and birds (e.g., chickens). Examples of liquid samples derived from animals include body fluids collected from animals, extracts of tissues collected from animals, extracts of cells derived from animals, and cultures of cells derived from animals (e.g., culture supernatants). Examples of such body fluids include blood (e.g., whole blood, plasma, serum), urine, saliva, lymph, tissue fluid, cerebrospinal fluid, ascites, sweat, semen, tears, mucus, milk, pleural fluid, bronchoalveolar lavage fluid, and amniotic fluid. Examples of such tissues include animal tissues such as respiratory system tissues (e.g., lung, trachea, bronchus, pharynx, nasal cavity, paranasal cavity), digestive system tissues (e.g., stomach, small intestine, large intestine, colon, rectum), pancreas, kidney, liver, thymus, spleen, heart, thyroid, adrenal gland, prostate, ovary, uterus, brain, skin, and bone marrow. Cells derived from animals include cells derived from the above-mentioned tissues of the above-mentioned animals, and include, for example, primary culture cells and cell lines. Preferably, the animal is a mammal, and preferably a human.

[0013] The anti-apolipoprotein antibody is an antibody against the apolipoprotein as described above. Examples of the antibody include full-length antibodies (e.g., monoclonal antibodies, polyclonal antibodies) and antigen-binding fragments thereof. The antigen-binding fragment may be any antibody fragment that maintains the binding ability to the target apolipoprotein, such as Fab, Fab', F(ab') 2, scFv. In the present invention, a single antibody or multiple (e.g., two, three, four) types of antibodies can be used. Preferably, the anti-apolipoprotein antibody may be an anti-ApoCIII antibody. More preferably, the anti-apolipoprotein antibody may be an anti-human ApoCIII antibody.

[0014] Examples of the solid phase include beads, supports (e.g., membranes), and containers (e.g., plates such as plastic plates, tubes, and microchannels). Preferably, the solid phase may be beads. The anti-apolipoprotein antibody can be immobilized on the solid phase by covalent or non-covalent bonding. Immobilization of antibodies on the solid phase is well known.

[0015] Preferably, the solid phase may be beads. The solid phase is not particularly limited as long as it can directly immobilize the anti-apolipoprotein antibody or indirectly immobilize it via a linker and can be recovered from the liquid phase (e.g., by magnetic manipulation or centrifugation). Examples of beads include microbeads, nanobeads, microspheres, and nanospheres. Examples of beads include inorganic beads (e.g., metal beads, silica beads), organic beads (e.g., polymeric beads such as sepharose beads and agarose beads), and organic-inorganic composite beads. The shape of the beads may be spherical or non-spherical (e.g., elliptical).

[0016] The average primary particle size of the beads is not particularly limited. From the viewpoint of ease of recovery, it may be, for example, 0.001 to 1000 μm, preferably 0.01 to 100 μm, more preferably 0.1 to 10 μm, even more preferably 0.5 to 5 μm, and particularly preferably 1 to 3 μm. The average primary particle size of the beads can be measured by a laser diffraction / scattering method, etc.

[0017] In a specific embodiment, the beads may be magnetic beads from the viewpoint of ease of recovery by magnetic manipulation, etc. Magnetic beads are particles containing a magnetic material such as iron, nickel, cobalt, or an alloy thereof (e.g., ferrite).

[0018] In (2), apolipoproteins in an apolipoprotein-containing sample are immunologically reacted with anti-apolipoprotein antibodies immobilized on a solid phase. Such immunological reaction produces a solid phase on which anti-apolipoprotein antibodies that have immunologically reacted with apolipoproteins are immobilized. To achieve such an immunological reaction, the apolipoprotein-containing sample and the solid phase on which the anti-apolipoprotein antibodies are immobilized can be combined (e.g., mixed, immersed, added). For example, when a solid phase (e.g., beads) that can be dispersed in a liquid phase is used, the immunological reaction can proceed in a mixture of the apolipoprotein-containing sample and the beads. When a solid phase (e.g., a support) that can be immersed in the apolipoprotein-containing sample is used, the immunological reaction can proceed in the immersion liquid. When a solid phase (e.g., a container) that can accommodate the apolipoprotein-containing sample is used, the immunological reaction can proceed in the solid phase to which the apolipoprotein-containing sample has been added.

[0019] The immunological reaction time of the apolipoprotein and the anti-apolipoprotein antibody is not particularly limited as long as it is sufficient for the so-called antigen-antibody reaction, and may be, for example, 1 minute or more. The reaction time may be preferably 2 minutes or more, 3 minutes or more, 4 minutes or more, or 5 minutes or more. The reaction time may also be 4 hours or less, 2 hours or less, 1 hour or less, 45 minutes or less, 30 minutes or less, or 20 minutes or less. More specifically, the reaction time may be 1 minute to 2 hours, 2 minutes to 1 hour, 3 to 45 minutes, 4 to 30 minutes, or 5 to 20 minutes. In order to achieve such a reaction time, the time of the operation (e.g., mixing) for combining the apolipoprotein-containing sample and the solid phase on which the anti-apolipoprotein antibody is immobilized and the time of standing thereafter can be appropriately set.

[0020] The immunological reaction temperature of the apolipoprotein and the anti-apolipoprotein antibody is not particularly limited as long as it is an appropriate temperature that enables the so-called antigen-antibody reaction, and may be, for example, 4 to 40° C. (eg, room temperature).

[0021] In (3), the solid phase on which anti-apolipoprotein antibodies that have immunologically reacted with apolipoproteins are immobilized is separated from the apolipoprotein-containing sample (liquid phase). The separation of the solid phase can be performed by any method (e.g., removal of the liquid phase or fractionation of the solid phase). This separation can realize so-called B (bound) / F (free) separation, which separates factors immobilized on the solid phase from factors not immobilized on the solid phase.

[0022] When the solid phase is beads, separation can be performed appropriately depending on the type of beads. For example, separation can be performed by centrifuging the mixture of the apolipoprotein-containing sample and beads, and then removing the supernatant. In this case, the beads can be collected as a precipitate. When the solid phase is magnetic beads, separation can be performed by applying a magnetic force to the mixture of the apolipoprotein-containing sample and magnetic beads, and then collecting the magnetic beads.

[0023] In (4), the solid phase separated as described above is washed. The washing can be carried out using an aqueous solution. Examples of the aqueous solution include water (e.g., distilled water, sterile water, sterile distilled water, and pure water) and buffer solutions, with buffer solutions being preferred. Examples of the buffer solution include phosphate buffer, phosphate buffered saline (PBS), tartrate buffer, citrate buffer, acetate buffer, glycine buffer, carbonate buffer, 2-morpholinoethanesulfonic acid (MES) buffer, trishydroxymethylaminomethane (Tris) buffer, borate buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer, N,N-bis(2-hydroxyethyl)glycine (Bicine) buffer, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris) buffer, 2-[4-(2-hydroxyethyl)1-piperazinylethanesulfonic acid (HEPES) buffer, and imidazole buffer. The pH of the buffer solution is preferably neutral. More specifically, such pH may be preferably 5.0 or more, more preferably 5.5 or more, and even more preferably 6.0 or more. The pH may also be preferably 9.0 or less, more preferably 8.5 or less, and even more preferably 8.0 or less. The pH can be measured using a method known in the art. Preferably, the pH can be measured at 25°C using a pH meter with a glass electrode.

[0024] The number of times of washing is not particularly limited, and may be, for example, once, or may be twice or more (for example, twice, three times, four times, or five times). The washing temperature and time can be appropriately set.

[0025] In (5), the apolipoprotein is eluted from the solid phase washed as described above. The elution of the apolipoprotein from the solid phase on which the anti-apolipoprotein antibody that has immunologically reacted with the apolipoprotein is immobilized can be achieved by using an eluent that interferes with antigen-antibody binding, or under conditions that can cause denaturation of the antibody (e.g., heating conditions, acidic conditions, alkaline conditions). As such an eluent, for example, an acidic aqueous solution (e.g., formic acid, trifluoroacetic acid, hydrochloric acid) having a pH of about 1 to 4 is used. The eluent may contain an organic solvent such as acetonitrile, acetone, methanol, ethanol, isopropanol, or chloroform. The concentration of the eluent, as well as the elution temperature and time, can be appropriately set.

[0026] In the purification method of the present invention, either (2) or (4), or both (2) and (4) are carried out in the presence of at least one type of surfactant (e.g., 1, 2, 3, 4, or 5 types).

[0027] Examples of the surfactant include cationic surfactants, anionic surfactants, zwitterionic surfactants, and nonionic surfactants. The surfactant may be in a free form or in a salt form. Examples of the salt include metal salts (e.g., monovalent metal salts such as sodium salts and potassium salts, and divalent metal salts such as calcium salts and magnesium salts), inorganic salts (e.g., halide salts such as fluorides, chlorides, bromides, and iodides, and ammonium salts), organic salts (e.g., ammonium salts substituted with an alkyl group), and acid addition salts (e.g., salts with inorganic acids such as sulfuric acid, hydrochloric acid, hydrobromic acid, nitric acid, and phosphoric acid, and salts with organic acids such as acetic acid, oxalic acid, lactic acid, citric acid, trifluoromethanesulfonic acid, and trifluoroacetic acid).

[0028] In one embodiment, the surfactant may be a surfactant other than a negatively charged anionic surfactant, in which case the surfactant may be a cationic surfactant, a zwitterionic surfactant, or a non-ionic surfactant.

[0029] A cationic surfactant is a surfactant that contains a cationic portion (e.g., ammonium, phosphonium, sulfonium) and a hydrophobic group, but does not contain an anionic portion. Examples of cationic surfactants include quaternary ammonium surfactants, quaternary phosphonium surfactants, and salts thereof. Cationic surfactants typically contain a linear or branched (preferably linear) carbon chain (preferably an alkyl chain) that may have a cyclic structural portion (e.g., aryl, heteroaryl, cycloalkyl, non-aromatic heterocycle) as a hydrophobic group. The number of carbon atoms constituting such a carbon chain may be, for example, 6 or more, preferably 8 or more, more preferably 9 or more, even more preferably an integer of 10 or more, and particularly preferably an integer of 12 or more. The number of carbon atoms may be an integer of 30 or less, preferably an integer of 20 or less, more preferably an integer of 18 or less, and even more preferably an integer of 16 or less. More specifically, the number of carbon atoms may be, for example, 6 to 30, preferably an integer of 8 to 20, more preferably an integer of 9 to 18, even more preferably an integer of 10 to 16, and particularly preferably an integer of 12 to 16.

[0030] Specific examples of cationic surfactants include Cn alkyl trimethyl ammonium halides and Cn alkyl trimethyl phosphonium halides.

[0031] As used herein, Cn alkyl refers to an alkyl chain (preferably a straight chain) having the number of carbon atoms as described above for the cationic surfactants.

[0032] Examples of Cn alkyl trimethyl ammonium halides include octyl trimethyl ammonium halides, nonyl trimethyl ammonium halides, decyl trimethyl ammonium halides, undecyl trimethyl ammonium halides, dodecyl trimethyl ammonium halides, tridecyl trimethyl ammonium halides, tetradecyl trimethyl ammonium halides, pentadecyl trimethyl ammonium halides, and hexadecyl trimethyl ammonium halides. Examples of Cn alkyl trimethyl ammonium halides include Cn alkyl trimethyl ammonium chloride (CnTAC) and Cn alkyl trimethyl ammonium bromide (CnTAB).

[0033] Preferably, the Cn alkyl trimethyl ammonium halide may be a C12-16 alkyl trimethyl ammonium halide. Preferred examples of the C12-16 alkyl trimethyl ammonium halide include dodecyl trimethyl ammonium chloride (C12TAC), tetradecyl trimethyl ammonium chloride (C14TAC), hexadecyl trimethyl ammonium chloride (C16TAC), dodecyl trimethyl ammonium bromide (C12TAB), tetradecyl trimethyl ammonium bromide (C14TAB), or hexadecyl trimethyl ammonium bromide (C16TAB).

[0034] More preferably, the Cn alkyl trimethyl ammonium halide may be dodecyl trimethyl ammonium chloride (C12TAC), tetradecyl trimethyl ammonium chloride (C14TAC), or hexadecyl trimethyl ammonium chloride (C16TAC).

[0035] Examples of the Cn alkyl trimethylphosphonium halide include octyl trimethylphosphonium halide, nonyl trimethylphosphonium halide, decyl trimethylphosphonium halide, undecyl trimethylphosphonium halide, dodecyl trimethylphosphonium halide, tridecyl trimethylphosphonium halide, tetradecyl trimethylphosphonium halide, pentadecyl trimethylphosphonium halide, and hexadecyl trimethylphosphonium halide. Examples of the Cn alkyl trimethylphosphonium halide include Cn alkyl trimethylphosphonium chloride and Cn alkyl trimethylphosphonium bromide.

[0036] Preferably, the Cn alkyl trimethylphosphonium halide may be dodecyl trimethylphosphonium halide, tetradecyl trimethylphosphonium halide, hexadecyl trimethylphosphonium halide.

[0037] The amphoteric surfactant is a surfactant that contains both a cationic portion (e.g., ammonium, phosphonium, sulfonium) and an anionic portion (e.g., sulfate group, sulfonic acid group, carboxylate group, phosphate group), as well as a hydrophobic group. From the viewpoint of availability, for example, amphoteric surfactants include quaternary ammonium-sulfonic acid type surfactants and quaternary ammonium-carboxylate type surfactants, with quaternary ammonium-sulfonic acid type surfactants being preferred. The amphoteric surfactant may be in a free form or in the form of a salt as described above.

[0038] Examples of quaternary ammonium-sulfonic acid surfactants include 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) being preferred.

[0039] As the nonionic surfactant, any nonionic surfactant can be used, but preferably, a nonionic surfactant having a polyoxyethylene structure is used.

[0040] An example of a nonionic surfactant having a polyoxyethylene structure is polyoxyethylene sorbitan fatty acid ester. Examples of polyoxyethylene sorbitan fatty acid ester include compounds of the Tween (registered trademark) series, such as Tween 80 represented by the following formula (1). [ka] (wherein x is an integer of 1 or more; y is an integer equal to or greater than 1; z is an integer equal to or greater than 1; w is an integer equal to or greater than 1. In this specification, the values ​​of x, y, z and w indicate average values.

[0041] x, y, z, and w can be defined based on their sum. The sum of x, y, z, and w may be an integer of preferably 10 or more, more preferably 15 or more, and even more preferably 18 or more. Such a sum may also be an integer of preferably 40 or more, more preferably 30 or more, and even more preferably 25 or more. More specifically, such a sum may be an integer of preferably 10 to 40, more preferably 15 to 30, and even more preferably 18 to 25. Particularly preferably, such a sum is 20.

[0042] [Table A]

[0043] Another example of a nonionic surfactant having a polyoxyethylene structure is alcohol ethoxylate. Alcohol ethoxylate is also called poly(oxyethylene) alkyl ether, and is a compound in which a hydrophilic polyoxyethylene (POE) chain and a hydrophobic alkyl group are bonded by an ether bond. As the alcohol ethoxylate, a compound represented by the following formula (2) is preferable.

[0044] [ka] (wherein x1 is an integer of 1 or more; y1 is an integer equal to or greater than 1; z1 is an integer equal to or greater than 1; y1 ≧ z1) In this specification, the values ​​of x1, y1 and z1 indicate average values.

[0045] x1 may be an integer of 1 or more, preferably an integer of 10 or more, more preferably an integer of 20 or more, even more preferably an integer of 30 or more, and particularly preferably an integer of 40 or more. x1 may also be an integer of 300 or less, preferably an integer of 250 or less, more preferably an integer of 200 or less, even more preferably an integer of 150 or less, and particularly preferably an integer of 100 or less, 80 or less, 60 or less, or 50 or less. More specifically, x1 may be an integer of 1 to 300, preferably an integer of 10 to 250, more preferably an integer of 20 to 200, even more preferably an integer of 30 to 150, and particularly preferably an integer of 40 to 100, 40 to 80, 40 to 60, or 40 to 50.

[0046] y1 is an integer of 1 or more, and more preferably an integer of 5 or more. y1 may also be an integer of 30 or less, preferably an integer of 20 or less, and more preferably an integer of 13 or less. More specifically, y1 may be an integer of 1 to 30, preferably an integer of 1 to 20, and even more preferably an integer of 5 to 13.

[0047] z1 is an integer equal to or greater than 1. z1 may also be an integer equal to or less than 15, preferably equal to or less than 10, or 7. More specifically, z1 may be an integer from 1 to 15, preferably an integer from 1 to 10, and even more preferably an integer from 1 to 7.

[0048] y1 and z1 satisfy the relationship y1≧z1. y1 and z1 may also satisfy the relationship that y1+z1 is an integer of 5 to 30. Preferably, y1+z1 may satisfy the relationship that y1+z1 is an integer of 10 to 14.

[0049] Specific examples of such alcohol ethoxylates include Softanol (registered trademark) series compounds such as Softanol 70, and Tergitol (registered trademark) series compounds such as Tergitol 15-S-30 and Tergitol 15-S-40.

[0050] [Table B]

[0051] The surfactant is not particularly limited as long as it is a concentration sufficient for treating the apolipoprotein-containing sample, although it varies depending on factors such as the apolipoprotein-containing sample and the type of surfactant. For example, the concentration of the surfactant in the solution used in (2) above or in the washing solution used in (4) above may be 0.01 w / v% or more. Such a concentration may preferably be 0.05 w / v% or more, 0.1 w / v% or more, 0.5 w / v% or more, or 1 w / v% or more. Such a concentration may also be 10 w / v% or less, 8 w / v% or less, 7 w / v% or less, 6 w / v% or less, or 5 w / v% or less. More specifically, such a concentration may be 0.01 to 10 w / v%, 0.05 to 8 w / v%, 0.1 to 7 w / v%, 0.5 to 6 w / v%, or 1 to 5 w / v%. When a combination of two or more surfactants is used as the surfactant, the total concentration of the two or more ionic surfactants can be set to the above concentration. Alternatively, in such a case, the concentration of each ionic surfactant may be appropriately set as described above.

[0052] According to the purification method of the present invention, a target apolipoprotein contained in an apolipoprotein-containing sample can be highly purified.

[0053] The present invention also provides a method for analyzing an apolipoprotein. The method for analyzing an apolipoprotein of the present invention comprises the steps of: (1) carrying out the above purification method to obtain eluted apolipoprotein; and (2) subjecting the eluted apolipoproteins to mass spectrometry to analyze glycosylation of the apolipoproteins.

[0054] The apolipoprotein is the same as that described above. Mass spectrometry can be appropriately set (see, for example, Examples).

[0055] It is known that apolipoproteins are modified by various glycosylation processes. By subjecting the apolipoprotein purified by the purification method of the present invention to mass spectrometry, the glycosylation pattern and / or level of the apolipoprotein can be analyzed with high accuracy. EXAMPLES

[0056] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Note that the percentages attached to the components in the examples described below refer to weight percent (w / v%) unless otherwise specified.

[0057] Materials and Methods of Example 1 (1) Sample Human serum samples purchased from Golden West BioSolutions, LLC were used.

[0058] [Table C]

[0059] (2) Preparation of anti-ApoCIII antibody-bound magnetic beads Anti-human ApoCIII antibody-bound magnetic beads were prepared using the streptavidin-biotin system. Streptavidin (Fujifilm Wako Pure Chemical Industries, Osaka, Japan) was covalently immobilized on carboxylated magnetic beads by carbodiimide activation. After removing the azide in the solution using NAP Columns (Cytiva / Global Life Sciences Solutions, Marlborough, MA, USA), the antibody was biotinylated using NHS-PEG4-Biotin (Thermo Fisher Scientific, Waltham, MA, USA), and unreacted biotin in the antibody solution was removed again using NAP Columns.

[0060] (3) Immunoprecipitation of ApoCIII using magnetic beads (3-1) Equipment Microlab STARlet(Hamilton Company) KingFisfer Presto(Thermo Fisher Scientific)

[0061] (3-2) Consumables CO-RE Tips 50 μL filter tips (Hamilton Company, part number 235948) KingFisher 96 tip comb for DW Magnets (Thermo Fisher Scientific, part number 97002534) KingFisher 96 deep-well plate, V-bottom, polypropylene (Thermo Fisher Scientific, part number 95040450)

[0062] (4) Experimental procedure (4-1) Bead washing Solution: 50 μL of anti-ApoCIII antibody-bound magnetic beads added to 500 μL of PBS Mixing time: 10 sec Stirring strength: Fast Magnetism collection: 10sec×3

[0063] (4-2) Antigen-antibody reaction Solution: 10 μL of serum sample added to 500 μL of surfactant-added PBS Mixing time: 2h Stirring intensity: Medium Magnetism collection: 10sec×3

[0064] (4-3) First and second washes Solution: PBS or 5w / v% C16TAC Solution volume: 500μL Mixing time: 1 min Stirring intensity: Medium Magnetism collection: 10sec×3

[0065] (4-4) 3rd wash Solution: PBS Solution volume: 500μL Mixing time: 1 min Stirring intensity: Medium Magnetism collection: 10sec×3

[0066] (4-5) Elution Solution: 0.1v / v% formic acid in Milli-Q water Solution volume: 50μL Mixing time: 10 sec Stirring strength: Fast Magnetism collection: 10sec×3

[0067] (5) LC-MS conditions (5-1) Equipment Vanquish(Thermo Fisher Scientific) TSQ Vantage EMR (Thermo Fisher Scientific)

[0068] (5-2)LC conditions Injection volume: 10 μL Column: Inertsil C4 Cartridge Guard Column E, 5 μm, 1.5 mm, 10 mm (GL Sciences Inc., part number 5020-19338) Solvent A: 0.1v / v% formic acid in Milli-Q water Solvent B: 0.1v / v% formic acid in acetonitrile

[0069] [Table D]

[0070] (5-3) MS conditions Spray Voltage: 3000V Vaporizer Temperature: 0(Readback) Sheath Gas Pressure: 8 Ion Sweep Gas Pressure: 0 Aux Gas Pressure: 0 Capillary Temperature: 200

[0071] Scan Type: Full Scan MS Mode: Q3MS Scan Range: 750-1650m / z Scan Time: 0.5s Q3 Peak Width: 0.7FWHM Polarity: Positive Declustering Voltage: 20V Micro Scans: 1

[0072] (6) Analysis The MS spectrum with retention times from 4.5 to 5.3 min was smoothed with boxcar 7. Peak detection was performed using the MassSpecWavelet method with a signal-to-noise ratio of 10. Among the detected peaks, those detected at m / z values ​​±0.5 of the theoretical values ​​of 6-9 charge ApoCIII isoform ions (table below) were regarded as ApoCIII-derived signals, and the rest were regarded as impurity component signals. Max intensity indicates the most intense peak among each signal, and does not necessarily represent the same ion. Max intensity ratio indicates the ratio of Max intensity of each signal to PBS.

[0073] [Table E]

[0074] Example 1: Purification of ApoCIII in the presence of detergent Antigen-antibody reactions in immunoprecipitation of serum apolipoprotein CIII (ApoCIII) were performed in the presence of detergents, and the effects of detergents on ApoCIII-derived signals (peak numbers) and contaminant signal (peak numbers) were examined. The use of detergents reduced the signal intensity and signal number of contaminant components compared to PBS, and increased the number of detectable ApoCIII-derived signals. Trimethyltetradecylammonium Chloride (C14TAC), Hexadecyltrimethylammonium Chloride (C16TAC), and Tween 80 increased the ApoCIII-derived signal intensity (Table 1).

[0075] [Table 1]

[0076] Since C16TAC showed the highest detection number and intensity of ApoCIII-derived signals, the optimal concentration was examined in the range of 0.01 to 5 w / v%. The higher the C16TAC concentration, the higher the ApoCIII-derived signal intensity and number of signals, while the signal intensity of impurities decreased with increasing C16TAC concentration up to 1 w / v% (Table 2).

[0077] [Table 2]

[0078] The effect of 5 w / v% C16TAC on enhancing ApoCIII-derived signals and reducing signals from contaminants in other serum samples was examined. In both cases, the signal intensity and number of contaminant signals were reduced compared to PBS, and the number and signal intensity of ApoCIII-derived signals were increased (Table 3).

[0079] [Table 3]

[0080] The effect of using a surfactant (5 w / v% C16TAC) on the ApoCIII-derived signal and the impurity component signal in each step of the experimental procedure (antigen-antibody reaction and washing) was examined. The number of ApoCIII-derived signals and impurity component signals detected when a surfactant was used in the washing step or in both the antigen-antibody reaction and washing steps was similar to that when a surfactant was added only in the antigen-antibody reaction step (Table 4). Therefore, the addition of a surfactant is effective in either the antigen-antibody reaction step or the washing step, or in both steps. When a surfactant was used in the first and second washing steps, the peak intensities of both the ApoCIII-derived signal and the impurity component signal were maximized. When a surfactant was used in the antigen-antibody reaction step and the first and second washing steps, the number of ApoCIII-derived signals was maximized.

[0081] [Table 4]

[0082] Materials and Methods for Example 2 (1) Sample The same human serum samples as in Example 1 were used.

[0083] (2) Preparation of anti-ApoAI antibody-bound Sepharose beads Anti-human ApoAI goat polyclonal antibody (Nittobo Medical Co., Ltd., Tokyo, Japan) was concentrated using an Amicon Ultra 30K centrifugal filter (Merck KgaA, Darmstadt, Germany), and azide in the antibody solution was removed using NAP Columns (Cytiva / Global Life Sciences Solutions, Marlborough, MA, USA). An affinity column was prepared using the azide-removed antibody solution and a ligand-coupled Sepharose column (HiTrap NHS-activated HP, Cytiva / Global Life Sciences Solutions, Marlborough, MA, USA). The antibody-bound Sepharose beads were suspended in an amount of 20 v / v% ethanol equal to the column volume, and then collected from the column.

[0084] (3) Immunoprecipitation of ApoAI using Sepharose beads 10 μL of antibody-bound Sepharose beads were added to 500 μL of PBS, and the solvent was removed. 500 μL of surfactant-added PBS was added to the antibody-bound Sepharose beads from which the solvent had been removed, and 10 μL of serum sample was added and stirred at room temperature for 2 hours. After washing with PBS, ApoAI was eluted from the Sepharose with 50 μL of 0.1 v / v% formic acid.

[0085] (4) LC-MS conditions The apparatus and LC conditions were the same as in Example 1. The MS conditions are as follows: Scan Type: Full Scan MS Mode: Q3MS Scan Range: 500-2000m / z Scan Time: 0.5s Q3 Peak Width: 0.7FWHM Polarity: Positive Declustering Voltage: 20V Micro Scans: 1

[0086] (5) Analysis The MS spectrum from retention time 3.2 to 5.6 min was smoothed with boxcar 7. Peak detection was performed using the MassSpecWavelet method with a signal-to-noise ratio of 10. Among the detected peaks, those detected at a theoretical m / z value of ±1.0 of the ion derived from the ApoAI canonical isoform (average mass 28078.62 Da) were regarded as ApoAI-derived signals, and the rest were regarded as impurity component signals. Max intensity indicates the most intense peak among each signal, and does not necessarily represent the same ion. Max intensity ratio indicates the ratio of Max intensity of each signal to PBS.

[0087] Example 2: Purification of ApoAI in the presence of detergent The antigen-antibody reaction in immunoprecipitation of ApoAI in serum was performed in the presence of 5 w / v% C16TAC and 0.05 w / v% Softanol 70, which have been shown to be effective in purifying ApoCIII, to examine the effects of surfactants on the ApoAI-derived signal and the impurity component signals. In the two cases examined, the number of peaks derived from impurities was reduced compared to PBS, without decreasing the number of ApoAI-derived peaks (Table 5).

[0088] [Table 5]

Claims

1. 1. A method for purifying an apolipoprotein, comprising: (1) providing an apolipoprotein-containing sample and a solid phase to which an anti-apolipoprotein antibody is immobilized; (2) immunologically reacting the apolipoprotein in the apolipoprotein-containing sample in solution with an anti-apolipoprotein antibody immobilized on a solid phase to produce a solid phase on which the anti-apolipoprotein antibody immunologically reacted with the apolipoprotein is immobilized; (3) separating the solid phase on which the anti-apolipoprotein antibodies immunologically reacted with the apolipoprotein are immobilized; (4) washing the separated solid phase with a washing solution; and (5) Eluting the apolipoprotein from the washed solid phase. Including, The method, wherein either (2) or (4), or both (2) and (4), are carried out in the presence of at least one surfactant.

2. The method of claim 1, wherein the apolipoprotein is at least one selected from the group consisting of ApoAI, ApoAII, ApoAIV, ApoAV, ApoCI, ApoCII, ApoCIII, ApoCIV, ApoB48, ApoB100, Apo(a), ApoD, ApoE, ApoF, ApoH, ApoJ, ApoLI, ApoLII, ApoLIII, ApoLIV, ApoLV, ApoLVI, ApoM, ApoO, and isotypes thereof.

3. 2. The method of claim 1, wherein the apolipoprotein is ApoAI or ApoCIII.

4. 2. The method of claim 1, wherein the surfactant is at least one selected from C12-16 alkyltrimethylammonium halides, CHAPS, Tween 80, and alcohol ethoxylates.

5. 5. The method of claim 4, wherein the C12-16 alkyltrimethylammonium halide is dodecyltrimethylammonium chloride (C12TAC), tetradecyltrimethylammonium chloride (C14TAC), or hexadecyltrimethylammonium chloride (C16TAC).

6. 5. The method of claim 4, wherein the alcohol ethoxylate is Softanol 70, Tergitol 15-S-30, or Tergitol 15-S-40.

7. The method of claim 1, wherein the apolipoprotein-containing sample is an animal-derived sample.

8. The method of claim 7, wherein the sample from the animal is a blood sample.

9. The method of claim 1 , wherein the solid phase is a bead.

10. 2. The method according to claim 1, wherein the concentration of the surfactant in the solution and the washing solution is 0.01 w / v % or more.

11. 2. The method according to claim 1, wherein the concentration of the surfactant in the solution and the washing solution is 0.01 to 5 w / v % or more.

12. The method of claim 1 , wherein washing is performed two or more times.

13. The method according to claim 1, wherein the immunological reaction time between the apolipoprotein and the anti-apolipoprotein antibody is 5 minutes or longer.

14. A method for analyzing an apolipoprotein, comprising the steps of: (1) carrying out the method according to any one of claims 1 to 13 to obtain eluted apolipoproteins; and (2) subjecting the eluted apolipoproteins to mass spectrometry to analyze the glycosylation of the apolipoproteins; A method comprising:

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