WO2016162950A1 - SEPARATION AND PURIFICATION METHOD FOR IgM ANTIBODY - Google Patents
SEPARATION AND PURIFICATION METHOD FOR IgM ANTIBODY Download PDFInfo
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- WO2016162950A1 WO2016162950A1 PCT/JP2015/060848 JP2015060848W WO2016162950A1 WO 2016162950 A1 WO2016162950 A1 WO 2016162950A1 JP 2015060848 W JP2015060848 W JP 2015060848W WO 2016162950 A1 WO2016162950 A1 WO 2016162950A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/34—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood group antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
Definitions
- the present invention belongs to the technical field of immunoglobulins.
- the present invention relates to a method for separating and purifying the immunoglobulin, particularly IgM antibody in human serum.
- Immunoglobulin is the main protein that constitutes gamma globulin and is contained in the serum and body fluids of all vertebrates.
- Each class of immunoglobulin differs in properties such as molecular weight, charge, amino acid composition, sugar content and the like.
- IgM (Immunoglobulin M) antibody is present in blood and lymph and is produced at the early stage of immune reaction against cell membrane antigens, infectious microorganisms, and lytic antigens.
- IgM antibodies have the largest molecular weight and exist mainly as pentamers consisting of 10 heavy chains (H chains) and 10 light chains (L chains). Since IgM has high specificity for polysaccharides, it is expected to be applied to cancer diagnosis by detecting cancer-related sugar chains.
- Patent Document 1 is characterized in that an IgM antibody-containing liquid is brought into contact with sodium urate crystals to selectively bind IgM antibodies to sodium urate crystals, and then IgM antibodies are dissociated from sodium urate crystals and recovered. A method for separating and purifying IgM antibodies is disclosed.
- a non-ionic polymer for example, polyethylene glycol (PEG)
- PEG polyethylene glycol
- an additive for example, a urea compound
- the main object of the present invention is to provide a novel separation and purification method capable of easily separating and purifying IgM antibodies in human serum.
- Examples of the present invention include the following.
- [1] A method for separating and purifying IgM antibodies in human serum, comprising a step of solid-phase extraction with an anion exchange resin, and a step of subsequent treatment with polyethylene glycol.
- [2] The separation and purification method according to [1], wherein the anion exchange resin is an anion exchange resin to which diethylaminoethyl is bonded.
- [3] The separation and purification method according to the above [1] or [2], wherein the anion exchange resin is based on a polymethacrylate polymer.
- [4] The separation and purification method according to any one of [1] to [3] above, wherein the anion exchange resin is of a size exclusion type.
- IgM antibodies in human serum can be easily separated and purified.
- IgM antibodies in human serum can be separated and purified at high purity and at low cost.
- FIG. 1 represents a protocol according to one embodiment of the present invention. It is a dot blot figure of a process elution fraction. It is an overlap figure of an elution curve and dot blot analysis.
- the horizontal axis represents fraction No.
- the left vertical axis represents absorbance at a wavelength of 280 nm
- the right vertical axis represents the number of pixels.
- the rhombic curve represents the change in absorbance
- the square curve represents the change in the number of pixels.
- SDS-PAGE Western blotting diagram photo
- It is an SDS-PAGE CBB staining electrophoresis diagram (photograph).
- FIG. 3 is an image analysis plot diagram of SDS-PAGE CBB staining.
- FIG. 5 is a dot blot diagram of samples treated with various concentrations of PEG. It is an SDS-PAGE immunoblotting diagram (photograph).
- the present invention is characterized by comprising a step of performing solid phase extraction with an anion exchange resin, and a step of subsequent treatment with polyethylene glycol (PEG).
- PEG polyethylene glycol
- the purification method of the present invention includes a step of performing solid phase extraction with an anion exchange resin.
- Human serum can be obtained, for example, by removing platelets and clotting factors from blood collected from humans by a conventional method. This serum sample is usually diluted several tens to several hundred times, preferably 100 to 200 times with 3 to 20 mM phosphate buffer (pH 7 to 8), 3 to 20 mM Tris-HCl buffer, and the like. Can be treated with an anion exchange resin in a conventional manner.
- the treatment with an anion exchange resin according to this step is usually performed by applying a serum sample to a column packed with an anion exchange resin and flowing a mobile phase of pH 7 to 8 (chromatography).
- a weak basic ion exchange resin that is, a resin having a weak basic ion functional group (exchange group) bonded thereto is suitable.
- the weakly basic ion functional group is not particularly limited, and examples thereof include tertiary amino groups such as diethylaminoethyl group (DEAE) and diethylaminopropyl group (DEAP), primary amino groups such as oleylamine and stearylamine, Secondary amino groups such as distearylamine can be mentioned.
- a tertiary amino group is preferable, and a diethylaminoethyl group (DEAE) is more preferable.
- the resin used as the base material of the anion exchange resin is not particularly limited, such as a polymethacrylate polymer, a polyethylene polymer, a cellulose polymer, and a polystyrene polymer, but a polymethacrylate polymer is preferable.
- the mobile phase examples include a phosphate buffer having a pH of 7 to 8, a phosphate buffer having a pH of 7 to 8 containing sodium chloride, and a tris hydrochloride buffer having a pH of 7 to 8. These may be used in appropriate combinations, but it is preferable to first carry out a mobile phase from which IgM is not eluted (washing step), and then flow through a mobile phase from which IgM is eluted (elution step). This removes many proteins and contaminants other than IgM.
- the mobile phase from which IgM is not eluted and the mobile phase from which IgM is eluted vary depending on the type of anion exchange resin, but can be determined by, for example, an experiment described in Test Example 1 described later.
- the amount of anion exchange resin, the column size, the amount of mobile phase, and the like vary depending on the type, processing amount, purpose, etc., and are appropriately selected.
- ultrafiltration membrane After performing solid phase extraction with an anion exchange resin as described above, it is preferable to concentrate with an ultrafiltration membrane in order to enhance the separation and purification of IgM antibodies.
- ultrafiltration membranes include those having a fractional molecular weight in the range of 10,000 to 120,000, and those having a molecular weight in the range of 30,000 to 100,000 are suitable. If the molecular weight cut-off is less than 10,000, it may take time for separation, and the impurities may not be sufficiently separated. If the molecular weight is more than 120,000, the separation and purification yield of IgM decreases. There is a case.
- the purification method of the present invention includes a process with polyethylene glycol (PEG). This step is performed after solid-phase extraction with the anion exchange resin and, if necessary, concentration with an ultrafiltration membrane.
- PEG polyethylene glycol
- This step is performed by adding PEG to the specimen obtained by the above steps and stirring. More specifically, for example, an aqueous PEG solution is added to the aqueous solution of the specimen obtained by the above process, both are vigorously stirred to obtain a precipitate, and after standing at a low temperature of usually 4 to 8 ° C., It is carried out by centrifuging with a refrigerated centrifuge set at 0 to 10 ° C. After centrifugation, the supernatant is discarded to obtain a separated and purified IgM antibody precipitate.
- polyethylene glycol for example, those having an average molecular weight in the range of 4,000 to 12,000 are suitable, and preferably 6,000 to 10,000.
- PEG4000, PEG6000, PEG8000, PEG10000, and PEG12000 can be mentioned, and among these, PEG8000 is preferable.
- the treatment concentration of the polyethylene glycol varies depending on the type of PEG, but is preferably within a range of 6 to 10 w / v%, more preferably 8 w / v%. In particular, when PEG 8000 is used, the treatment concentration is preferably 8 w / v%.
- the precipitate obtained in the PEG step is dissolved in a suitable solvent, methanol is added and stirred, and then chloroform is further added and stirred. Subsequently, water (eg, water through a reverse osmosis membrane (RO water), purified water, distilled water) is added and stirred, centrifuged and the supernatant discarded. An appropriate amount of methanol is added thereto and mixed, and then centrifuged again to discard the supernatant, thereby obtaining a separated and purified IgM antibody precipitate.
- RO water reverse osmosis membrane
- the chromate process as described above can also be performed before the PEG process.
- the IgM antibody precipitate obtained by the purification method of the present invention is dissolved in an appropriate solvent, and a phosphate buffer of pH 7-8 is added and stirred as necessary, to obtain a separated and purified IgM antibody solution. Can do.
- a phosphate buffer of pH 7-8 is added and stirred as necessary, to obtain a separated and purified IgM antibody solution.
- Such an IgM antibody solution can be stored at a low temperature (eg, 4 ° C.).
- an alkaline agent can be mentioned, and an alkaline agent is preferable. Although there was a concern about the denaturation of IgM antibody with an alkaline agent, it was surprisingly good in dissolving IgM antibody.
- alkali agent examples include sodium hydroxide and potassium hydroxide having a pH of 10 to 13, and a carbonate-bicarbonate buffer having a pH of 9 to 10. Of these, sodium hydroxide having a pH of 12 to 13 is preferred.
- the alkali agent preferably contains an appropriate amount of a polysorbate surfactant such as Tween20.
- IgM After dissolving the IgM antibody with an alkaline agent, it is preferable to neutralize with an appropriate acid. If neutralized with an acid, IgM may be denatured. Examples of such acids include hydrochloric acid and acetic acid. Of these, hydrochloric acid is preferred.
- the concentration of the acid varies depending on the type, amount, concentration, and the like of the alkaline agent, and may be a concentration that can be neutralized.
- Analytical method of IgM antibody provides an IgM antibody solution obtained by dissolving a precipitate of IgM antibody obtained by the purification method of the present invention with an alkaline agent and neutralizing with an acid if necessary. And an IgM antibody analysis method (hereinafter referred to as “analysis method of the present invention”).
- the analysis method of the present invention is performed by dissolving the precipitate of IgM antibody obtained by the purification method of the present invention with an alkaline agent.
- alkaline agent for dissolving the precipitate of IgM antibody examples include sodium hydroxide and potassium hydroxide at pH 10 to 13, and carbonate-bicarbonate buffer at pH 9 to 10. Of these, sodium hydroxide having a pH of 12 to 13 is preferred.
- the alkali agent preferably contains an appropriate amount of a polysorbate surfactant such as Tween20.
- examples of the acid for neutralization include hydrochloric acid and acetic acid. Of these, hydrochloric acid is preferred.
- the concentration of the acid varies depending on the type, amount, concentration, and the like of the alkaline agent, and may be a concentration that can be neutralized.
- an IgM antibody solution obtained by dissolving the IgM antibody precipitate obtained by the purification method of the present invention with an alkaline agent and neutralizing with an acid as necessary is used.
- the law can be applied.
- Examples of such known analytical methods include electrophoresis, polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate (SDS) -PAGE, Western blotting, immunoblotting, and dot blot analysis. .
- DEAE ion exchange resin TOYOPEARL DEAE 650M, manufactured by Tosoh Corporation
- Solid phase extraction column ISOLUTE (registered trademark) double fritted filtration column (25 ml, ⁇ 20 mm), manufactured by Biotage Japan
- Polyethylene glycol PEG8000 Powder (Molecular Biology Grade), Promega Corporation (Madison WI, USA)
- Ultrafiltration membrane Vivaspin6 (MWCO 50000), manufactured by Sartorius Stedim Biotech GmbH (Goettingen, Germany)
- PVDF Polyvinylidene fluoride
- MA Millipore Corporation
- Electrophoresis buffer is AE-1410 EzRun
- Blotting transfer buffer is EzFastBlot
- Blocking reagent is AE-1475 EzBlock Chemi
- HRP labeled antibody chromogenic substrate is AE-1490 Ez WestBlue (all manufactured by Ato)
- Anti-human IgM-HRP-labeled antibody (H chain): Polyclonal Rabbit Anti-Human IgM / HRP code No. P0215, manufactured by Dako Denmark A / S (Glostrup, Denmark)
- Human-derived IgM antibody H-IgM (Immunoglobulin M from (human Serum Lot No. 44004406, manufactured by Oriental Yeast Co., Ltd.)
- -Human-derived IgG antibody HUMAN IgG (Product No. 14506, manufactured by Sigma) ⁇ Bovine serum albumin solution (BSA, for biochemistry, manufactured by Wako Pure Chemical Industries, Ltd.)
- Atophoresis Rapidas AE-6530M Horizblot 2M AE-6687 ( ⁇ S) are used for SDS-polyacrylamide gel electrophoresis (SDA-PAGE), and Anatech is a constant power supply. PowerPhoreStar 3810 made by the company was used.
- PB phosphate buffer (pH 7.1)
- NaCl sodium chloride
- NaOH sodium hydroxide
- HCl hydrochloric acid
- Vortex mixer vigorously are simply called “vortex”.
- Example 1 Separation and purification of IgM antibody (1)
- Treatment with DEAE ion exchange resin column Blood was collected from 20 healthy volunteers, and serum was obtained by a conventional method. A solid phase extraction column was packed with 5 mL of DEAE650M, equilibrated with 8 mMPB, and a serum dilution (15 mL of 8 mMPB was added to 0.1 mL of serum and mixed. The same applies hereinafter) was applied thereto.
- Example 2 Separation and purification of IgM antibody (2) DEAE ion exchange resin column treatment and PEG treatment were carried out in the same manner as in Example 1, and the supernatant was discarded and 100 ⁇ L of 0.1% Tween20-0.1N NaOH was added and completely dissolved, and then 50 ⁇ L of 0.1 MPB was added and vortexed. Vortex by adding 50 ⁇ L of 0.2N hydrochloric acid, vortex by adding 100 ⁇ L of methanol, vortex by adding 100 ⁇ L of chloroform, and then vortex by adding 300 ⁇ L of water (RO water) that has passed through a reverse osmosis membrane. The supernatant was discarded after centrifugation for 2 seconds.
- RO water water
- pass is the column passing solution
- 8 mMPB is the 8 mMPB washing solution
- No. Nos. 1 to 10 are eluate fractions of 0.1M NaCl / 8mMPB.
- 11 to 20 are the results of the eluate fraction of 0.2M NaCl / 8mMPB.
- IgM antibody is not contained in the eluate of pass, 8mMPB, and 0.1M NaCl / 8mMPB, and IgM antibody is contained in the fraction eluted with the eluate of 0.2M NaCl / 8mMPB. It was confirmed that it was included.
- Test Example 2 SDS-PAGE and Western Blotting of Separated and Purified IgM Antibody The 10% hand-cast gel used had a gel size of 90 ⁇ 80 mm and a thickness of 1 mm.
- the IgM antibody solution obtained in Example 1 and a 2.7 mg / mL human-derived IgG antibody solution were mixed with a sample processing solution for SDS-PAGE (50 mM Tris-HCl buffer solution pH 6.8 (1% SDS, 20% glycerin, 1.5% Diluted 2-fold with 2-mercaptoethanol), heated at 100 ° C. for 4 minutes, and then applied to the gel with 5 to 10 ⁇ L (No. 1 and No. 2 were 10 ⁇ L, No. 3 and No. 4 were 5 ⁇ L) After electrophoresis at 20 mA and constant flow for 65 to 75 minutes, the gel was transferred to a PVDF membrane with Horizblot 2M AE-6687 (• S) at 250 mA for 35 to 40 minutes.
- SDS-PAGE 50 mM Tris-HCl buffer solution pH 6.8 (1% SDS, 20% glycerin, 1.5% Diluted 2-fold with 2-mercaptoethanol
- FIG. 1 and No. 3 shows the results of the human IgG antibody.
- 2 and No. 4 shows the results of IgM antibodies isolated from human serum by the purification method of the present invention.
- 1 and No. 2 shows the results of CBB staining.
- 3 and no. 4 shows the results of TMB chromogenic substrate staining, respectively.
- Example 3 Protein content of separated and purified IgM antibody An SDS-PAGE 10% gel was prepared, and the IgM antibody solution, 1 mg / mL bovine serum albumin solution (BSA) obtained in Example 1 was used for SDS-PAGE. Dilute 5 times with sample treatment solution (50 mM Tris-HCl buffer pH 6.8 (containing 1% SDS, 20% glycerin, 1.5% 2-mercaptoethanol), heat at 100 ° C. for 4 minutes, and dilute IgM antibody on the gel.
- sample treatment solution 50 mM Tris-HCl buffer pH 6.8 (containing 1% SDS, 20% glycerin, 1.5% 2-mercaptoethanol
- the sample treatment solution for SDS-PAGE 50 mM Tris-HCl buffer pH 6.8 (1% SDS, 20% The mixture was diluted 5 times with glycerin and 1.5% 2-mercaptoethanol), heated at 100 ° C. for 4 minutes, and 20 ⁇ L was applied to the gel, and after electrophoresis at 20 mA, constant flow for 65 to 75 minutes, the gel was subjected to Horizblot 2M AE. -6687 ( ⁇ S) at 250 mA for 35-40 minutes, transferred to a PVDF membrane, and stained with 0.01% CBB, the results of which are shown in FIG.
- FIG. 1 shows the result of 10% PEG
- 2 shows the result of 8% PEG
- 3 shows the result of 6% PEG
- 4 shows the result of 4% PEG
- 5 shows the result of no PEG treatment.
- 6 shows the results of the human-derived IgM antibody (standard product).
- 7 shows the results of human-derived IgG antibody (standard product).
- the PVDF membrane was hydrophilized by immersing it in methanol for 2 to 5 minutes, then immersed in RO water for 20 to 30 minutes, placed on a filter paper moistened with RO water, and 5 ⁇ L of each specimen was applied.
- the membrane was transferred to a tray, blocked with a blocking reagent for 40 minutes, reacted with anti-human IgM-HRP labeled antibody (H chain) for 30 minutes, and stained with a chromogenic substrate for HRP labeled antibody (TMB chromogenic substrate).
- TMB chromogenic substrate a chromogenic substrate for HRP labeled antibody
- Example 5 Examination of content by SDS-PAGE immunoblotting As in Example 1, 100 ⁇ L of human serum was subjected to DEAE column treatment and PEG treatment, and the resulting IgM antibody solution, 2.7 mg / mL human-derived IgG 5 times the antibody solution and 560 ⁇ g / mL human-derived IgM antibody solution in SDS-PAGE sample treatment solution (50 mM Tris-HCl buffer pH 6.8 (containing 1% SDS, 20% glycerin, 1.5% 2-mercaptoethanol) After heating at 100 ° C. for 4 minutes, 10 ⁇ L (No. 1 to 3) or 20 ⁇ L (No.
- the purification method of the present invention is useful for biochemical examination of IgM antibodies and the like because IgM antibodies in human serum can be separated and purified with high purity simply and inexpensively.
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Abstract
The present invention mainly addresses the problem of providing a novel separation and purification method whereby IgM antibody in human serum can be easily separated and purified. Provided is, for example, a separation and purification method for IgM antibody in human serum, said separation and purification method being characterized by comprising a step for performing solid phase extraction using an anion exchange resin (for example, DEAE anion exchange resin), and a subsequent step for treating with polyethylene glycol (for example, PEG8000). Also provided is an analysis method for IgM antibody, said analysis method being characterized by comprising analyzing an IgM antibody solution that is obtained by dissolving an IgM sediment obtained by the aforesaid separation and purification method using an alkali agent and, if necessary, neutralizing with an acid. According to the present invention, IgM antibody in human serum can be easily separated and purified. Moreover, IgM antibody in human serum can be economically separated and purified at a high purity.
Description
本発明は、免疫グロブリンの技術分野に属する。本発明は、その免疫グロブリン、特にヒト血清中のIgM抗体を分離精製する方法に関するものである。
The present invention belongs to the technical field of immunoglobulins. The present invention relates to a method for separating and purifying the immunoglobulin, particularly IgM antibody in human serum. *
免疫グロブリンは、γグロブリンを構成する主要なタンパク質であり、すべての脊椎動物の血清や体液中に含まれる。免疫グロブリンには5種類の異なったクラスのものが存在し、それぞれIgG、IgA、IgM、IgD、およびIgEと称される。各クラスの免疫グロブリンは、分子量、電荷、アミノ酸組成、糖含量等の性状が異なる。
この中、IgM(Immunoglobulin M)抗体は、血液やリンパ液中に存在し、細胞膜抗原、感染性微生物、溶解性抗原に対する免疫反応の初期に産生される。IgM抗体は最も大きな分子量を持ち、主に10個の重鎖(H鎖)と10個の軽鎖(L鎖)からなる5量体として存在する。
IgMは多糖類に対して高い特異性があるため、癌関連糖鎖の検出による癌診断への応用が期待される。 Immunoglobulin is the main protein that constitutes gamma globulin and is contained in the serum and body fluids of all vertebrates. There are five different classes of immunoglobulins, referred to as IgG, IgA, IgM, IgD, and IgE, respectively. Each class of immunoglobulin differs in properties such as molecular weight, charge, amino acid composition, sugar content and the like.
Among them, IgM (Immunoglobulin M) antibody is present in blood and lymph and is produced at the early stage of immune reaction against cell membrane antigens, infectious microorganisms, and lytic antigens. IgM antibodies have the largest molecular weight and exist mainly as pentamers consisting of 10 heavy chains (H chains) and 10 light chains (L chains).
Since IgM has high specificity for polysaccharides, it is expected to be applied to cancer diagnosis by detecting cancer-related sugar chains.
この中、IgM(Immunoglobulin M)抗体は、血液やリンパ液中に存在し、細胞膜抗原、感染性微生物、溶解性抗原に対する免疫反応の初期に産生される。IgM抗体は最も大きな分子量を持ち、主に10個の重鎖(H鎖)と10個の軽鎖(L鎖)からなる5量体として存在する。
IgMは多糖類に対して高い特異性があるため、癌関連糖鎖の検出による癌診断への応用が期待される。 Immunoglobulin is the main protein that constitutes gamma globulin and is contained in the serum and body fluids of all vertebrates. There are five different classes of immunoglobulins, referred to as IgG, IgA, IgM, IgD, and IgE, respectively. Each class of immunoglobulin differs in properties such as molecular weight, charge, amino acid composition, sugar content and the like.
Among them, IgM (Immunoglobulin M) antibody is present in blood and lymph and is produced at the early stage of immune reaction against cell membrane antigens, infectious microorganisms, and lytic antigens. IgM antibodies have the largest molecular weight and exist mainly as pentamers consisting of 10 heavy chains (H chains) and 10 light chains (L chains).
Since IgM has high specificity for polysaccharides, it is expected to be applied to cancer diagnosis by detecting cancer-related sugar chains.
一方、IgM抗体の精製には、IgMの親和性が高いリガンドである2-メルカプトピリジンを結合したアフィニティーカラムとIgG等の除去にProtein A/Gのカラムを使用し最終精製としてゲルろ過クロマトグラフィーを行うのが一般的である。
他にも様々な精製方法があるが、それらの精製方法単独での精製では不十分であり、複数の精製方法を組み合わせる必要があり、精製には煩雑で長時間の操作が必要とされる。また、プロテインAやプロテインGは菌類から産生されるものであるので大量生産が困難であり、抗体を精製する材料としては非常に高価である。 On the other hand, for purification of IgM antibody, gel filtration chromatography is used as final purification, using an affinity column bound with 2-mercaptopyridine, which is a ligand with high affinity for IgM, and a Protein A / G column for removing IgG and the like. It is common to do it.
Although there are various other purification methods, purification by these purification methods alone is insufficient, and it is necessary to combine a plurality of purification methods, and the purification requires complicated and long-time operation. In addition, protein A and protein G are produced from fungi, so that mass production is difficult, and materials for purifying antibodies are extremely expensive.
他にも様々な精製方法があるが、それらの精製方法単独での精製では不十分であり、複数の精製方法を組み合わせる必要があり、精製には煩雑で長時間の操作が必要とされる。また、プロテインAやプロテインGは菌類から産生されるものであるので大量生産が困難であり、抗体を精製する材料としては非常に高価である。 On the other hand, for purification of IgM antibody, gel filtration chromatography is used as final purification, using an affinity column bound with 2-mercaptopyridine, which is a ligand with high affinity for IgM, and a Protein A / G column for removing IgG and the like. It is common to do it.
Although there are various other purification methods, purification by these purification methods alone is insufficient, and it is necessary to combine a plurality of purification methods, and the purification requires complicated and long-time operation. In addition, protein A and protein G are produced from fungi, so that mass production is difficult, and materials for purifying antibodies are extremely expensive.
特許文献1には、IgM抗体含有液を尿酸ナトリウム結晶と接触させることによりIgM抗体を尿酸ナトリウム結晶に選択的に結合させ、その後にIgM抗体を尿酸ナトリウム結晶から解離、回収することを特徴とするIgM抗体の分離精製方法が開示されている。
Patent Document 1 is characterized in that an IgM antibody-containing liquid is brought into contact with sodium urate crystals to selectively bind IgM antibodies to sodium urate crystals, and then IgM antibodies are dissociated from sodium urate crystals and recovered. A method for separating and purifying IgM antibodies is disclosed.
特許文献2には、非イオン性ポリマー(例えば、ポリエチレングリコール(PEG))を用い、タンパク質凝集物を除去する第1のクロマトグラフィー工程と、その後に、溶解度を高める添加剤(例えば、尿素化合物、アルキレングリコール、グリシン)を用いた第2のイオン交換クロマトグラフィー工程とを含む、IgM抗体の精製方法が開示されている。
In Patent Document 2, a non-ionic polymer (for example, polyethylene glycol (PEG)) is used to remove a protein aggregate, followed by an additive (for example, a urea compound, And a second ion exchange chromatography step using (alkylene glycol, glycine).
IgM抗体を分離精製する方法として様々なものが知られているが、必ずしも十分なものではない。より簡便で高純度かつ廉価な、IgM抗体の分離精製方法の開発が望まれている。
本発明は、ヒト血清中のIgM抗体を簡便に分離精製することができる新規な分離精製法を提供することを主な課題とする。 Various methods for separating and purifying IgM antibodies are known, but not necessarily sufficient. Development of a simpler, more pure and less expensive method for separating and purifying IgM antibodies is desired.
The main object of the present invention is to provide a novel separation and purification method capable of easily separating and purifying IgM antibodies in human serum.
本発明は、ヒト血清中のIgM抗体を簡便に分離精製することができる新規な分離精製法を提供することを主な課題とする。 Various methods for separating and purifying IgM antibodies are known, but not necessarily sufficient. Development of a simpler, more pure and less expensive method for separating and purifying IgM antibodies is desired.
The main object of the present invention is to provide a novel separation and purification method capable of easily separating and purifying IgM antibodies in human serum.
本発明者らは、鋭意検討を重ねた結果、血清に陰イオン交換樹脂の固相抽出とその後にポリエチレングリコール(以下、「PEG」ともいう。)処理を施すことにより、上記課題を解決できることを見出し、本発明を完成した。
As a result of intensive studies, the present inventors have found that the above-mentioned problems can be solved by subjecting serum to solid phase extraction of an anion exchange resin and subsequent treatment with polyethylene glycol (hereinafter also referred to as “PEG”). The headline and the present invention were completed.
本発明として、例えば、下記のものを挙げることができる。
[1]陰イオン交換樹脂で固相抽出を行う工程、およびその後にポリエチレングリコールで処理する工程を含むことを特徴とする、ヒト血清中のIgM抗体の分離精製法。
[2]前記陰イオン交換樹脂が、ジエチルアミノエチルが結合する陰イオン交換樹脂である、上記[1]に記載の分離精製法。
[3]前記陰イオン交換樹脂がポリメタクリレート系高分子を基材とするものである、上記[1]または[2]に記載の分離精製法。
[4]前記陰イオン交換樹脂がサイズ排除型のものである、上記[1]~[3]のいずれか一項に記載の分離精製法。 Examples of the present invention include the following.
[1] A method for separating and purifying IgM antibodies in human serum, comprising a step of solid-phase extraction with an anion exchange resin, and a step of subsequent treatment with polyethylene glycol.
[2] The separation and purification method according to [1], wherein the anion exchange resin is an anion exchange resin to which diethylaminoethyl is bonded.
[3] The separation and purification method according to the above [1] or [2], wherein the anion exchange resin is based on a polymethacrylate polymer.
[4] The separation and purification method according to any one of [1] to [3] above, wherein the anion exchange resin is of a size exclusion type.
[1]陰イオン交換樹脂で固相抽出を行う工程、およびその後にポリエチレングリコールで処理する工程を含むことを特徴とする、ヒト血清中のIgM抗体の分離精製法。
[2]前記陰イオン交換樹脂が、ジエチルアミノエチルが結合する陰イオン交換樹脂である、上記[1]に記載の分離精製法。
[3]前記陰イオン交換樹脂がポリメタクリレート系高分子を基材とするものである、上記[1]または[2]に記載の分離精製法。
[4]前記陰イオン交換樹脂がサイズ排除型のものである、上記[1]~[3]のいずれか一項に記載の分離精製法。 Examples of the present invention include the following.
[1] A method for separating and purifying IgM antibodies in human serum, comprising a step of solid-phase extraction with an anion exchange resin, and a step of subsequent treatment with polyethylene glycol.
[2] The separation and purification method according to [1], wherein the anion exchange resin is an anion exchange resin to which diethylaminoethyl is bonded.
[3] The separation and purification method according to the above [1] or [2], wherein the anion exchange resin is based on a polymethacrylate polymer.
[4] The separation and purification method according to any one of [1] to [3] above, wherein the anion exchange resin is of a size exclusion type.
[5]前記ポリエチレングリコールがポリエチレングリコール8000である、上記[1]~[4]のいずれか一項に記載の分離精製法。
[6]前記ポリエチレングリコールの処理濃度が6~10w/v%の範囲内である、上記[1]~[5]のいずれか一項に記載の分離精製法。 [5] The separation and purification method according to any one of [1] to [4], wherein the polyethylene glycol is polyethylene glycol 8000.
[6] The separation and purification method according to any one of [1] to [5] above, wherein the treatment concentration of the polyethylene glycol is in the range of 6 to 10 w / v%.
[6]前記ポリエチレングリコールの処理濃度が6~10w/v%の範囲内である、上記[1]~[5]のいずれか一項に記載の分離精製法。 [5] The separation and purification method according to any one of [1] to [4], wherein the polyethylene glycol is polyethylene glycol 8000.
[6] The separation and purification method according to any one of [1] to [5] above, wherein the treatment concentration of the polyethylene glycol is in the range of 6 to 10 w / v%.
[7]前記固相抽出を行う工程の後、ポリエチレングリコールで処理する工程の前に、限外ろ過膜で濃縮する工程を更に含む、上記[1]~[6]のいずれか一項に記載の分離精製法。
[8]上記[1]~[7]のいずれか一項に記載の分離精製法により得られたIgM抗体の沈渣を、界面活性剤が含まれていてもよいアルカリ剤で溶解し、必要に応じて酸で中和することによって得られるIgM抗体液を分析に供することを特徴とする、IgM抗体の分析法。
[9]前記アルカリ剤が、水酸化ナトリウムまたは水酸化カリウムである、上記[8]に記載の分析法。 [7] The method according to any one of [1] to [6], further comprising a step of concentrating with an ultrafiltration membrane after the step of performing solid-phase extraction and before the step of treating with polyethylene glycol. Separation and purification method.
[8] The precipitate of IgM antibody obtained by the separation and purification method according to any one of [1] to [7] above is dissolved in an alkaline agent that may contain a surfactant, and A method for analyzing IgM antibody, characterized in that an IgM antibody solution obtained by neutralization with an acid is subjected to analysis.
[9] The analysis method according to the above [8], wherein the alkaline agent is sodium hydroxide or potassium hydroxide.
[8]上記[1]~[7]のいずれか一項に記載の分離精製法により得られたIgM抗体の沈渣を、界面活性剤が含まれていてもよいアルカリ剤で溶解し、必要に応じて酸で中和することによって得られるIgM抗体液を分析に供することを特徴とする、IgM抗体の分析法。
[9]前記アルカリ剤が、水酸化ナトリウムまたは水酸化カリウムである、上記[8]に記載の分析法。 [7] The method according to any one of [1] to [6], further comprising a step of concentrating with an ultrafiltration membrane after the step of performing solid-phase extraction and before the step of treating with polyethylene glycol. Separation and purification method.
[8] The precipitate of IgM antibody obtained by the separation and purification method according to any one of [1] to [7] above is dissolved in an alkaline agent that may contain a surfactant, and A method for analyzing IgM antibody, characterized in that an IgM antibody solution obtained by neutralization with an acid is subjected to analysis.
[9] The analysis method according to the above [8], wherein the alkaline agent is sodium hydroxide or potassium hydroxide.
本発明によれば、ヒト血清中のIgM抗体を簡便に分離精製することができる。また、高純度に廉価に、ヒト血清中のIgM抗体を分離精製することができる。
According to the present invention, IgM antibodies in human serum can be easily separated and purified. In addition, IgM antibodies in human serum can be separated and purified at high purity and at low cost.
According to the present invention, IgM antibodies in human serum can be easily separated and purified. In addition, IgM antibodies in human serum can be separated and purified at high purity and at low cost.
以下、本発明について詳述する。
1 IgM抗体の分離精製法
本発明は、陰イオン交換樹脂で固相抽出を行う工程、およびその後にポリエチレングリコール(PEG)で処理する工程を含むことを特徴とする。以下、本発明に係る、IgM抗体の分離精製法を「本発明精製法」という。 Hereinafter, the present invention will be described in detail.
1 Separation and purification method of IgM antibody The present invention is characterized by comprising a step of performing solid phase extraction with an anion exchange resin, and a step of subsequent treatment with polyethylene glycol (PEG). Hereinafter, the method for separating and purifying IgM antibodies according to the present invention is referred to as “the purification method of the present invention”.
1 IgM抗体の分離精製法
本発明は、陰イオン交換樹脂で固相抽出を行う工程、およびその後にポリエチレングリコール(PEG)で処理する工程を含むことを特徴とする。以下、本発明に係る、IgM抗体の分離精製法を「本発明精製法」という。 Hereinafter, the present invention will be described in detail.
1 Separation and purification method of IgM antibody The present invention is characterized by comprising a step of performing solid phase extraction with an anion exchange resin, and a step of subsequent treatment with polyethylene glycol (PEG). Hereinafter, the method for separating and purifying IgM antibodies according to the present invention is referred to as “the purification method of the present invention”.
1.1 陰イオン交換樹脂で固相抽出を行う工程
本発明精製法は、陰イオン交換樹脂により固相抽出を行う工程を含む。 1.1 Step of performing solid phase extraction with anion exchange resin The purification method of the present invention includes a step of performing solid phase extraction with an anion exchange resin.
本発明精製法は、陰イオン交換樹脂により固相抽出を行う工程を含む。 1.1 Step of performing solid phase extraction with anion exchange resin The purification method of the present invention includes a step of performing solid phase extraction with an anion exchange resin.
本工程を実施するに先立ち、ヒト血清を用意する。ヒト血清は、例えば、ヒトから採血された血液から、常法により血小板や凝固因子を除くことにより得ることができる。この血清検体は、通常、3~20mMリン酸緩衝液(pH7~8)や3~20mMトリス塩酸緩衝液等で数十倍~数百倍、好ましくは100~200倍に希釈し、かかる希釈液を常法により陰イオン交換樹脂で処理することができる。
* Preparation of human serum prior to carrying out this process. Human serum can be obtained, for example, by removing platelets and clotting factors from blood collected from humans by a conventional method. This serum sample is usually diluted several tens to several hundred times, preferably 100 to 200 times with 3 to 20 mM phosphate buffer (pH 7 to 8), 3 to 20 mM Tris-HCl buffer, and the like. Can be treated with an anion exchange resin in a conventional manner.
本工程に係る陰イオン交換樹脂による処理は、通常、陰イオン交換樹脂が詰められたカラムに血清検体をアプライし、pH7~8の移動相を流すことによって行われる(クロマトグラフィー)。
The treatment with an anion exchange resin according to this step is usually performed by applying a serum sample to a column packed with an anion exchange resin and flowing a mobile phase of pH 7 to 8 (chromatography).
本発明で用いる陰イオン交換樹脂としては、弱塩基性イオン交換樹脂、すなわち弱塩基性イオン官能基(交換基)が結合したものが適当である。かかる弱塩基性イオン官能基としては、特に制限されないが、例えば、ジエチルアミノエチル基(DEAE)、ジエチルアミノプロピル基(DEAP)等の第3級アミノ基、オレイルアミン、ステアリルアミン等の第1級アミノ基、ジステアリルアミン等の第2級アミノ基を挙げることができる。この中、第3級アミノ基が好ましく、ジエチルアミノエチル基(DEAE)がより好ましい。
As the anion exchange resin used in the present invention, a weak basic ion exchange resin, that is, a resin having a weak basic ion functional group (exchange group) bonded thereto is suitable. The weakly basic ion functional group is not particularly limited, and examples thereof include tertiary amino groups such as diethylaminoethyl group (DEAE) and diethylaminopropyl group (DEAP), primary amino groups such as oleylamine and stearylamine, Secondary amino groups such as distearylamine can be mentioned. Among these, a tertiary amino group is preferable, and a diethylaminoethyl group (DEAE) is more preferable.
陰イオン交換樹脂の基材となる樹脂としては、ポリメタクリレート系高分子、ポリエチレン高分子、セルロース系高分子、ポリスチレン系高分子など特に制限されないが、ポリメタクリレート系高分子が好ましい。
The resin used as the base material of the anion exchange resin is not particularly limited, such as a polymethacrylate polymer, a polyethylene polymer, a cellulose polymer, and a polystyrene polymer, but a polymethacrylate polymer is preferable.
移動相としては、例えば、pH7~8のリン酸緩衝液や、塩化ナトリウムを含んだpH7~8のリン酸緩衝液、pH7~8のトリス塩酸緩衝液を挙げることができる。これらを適宜組み合わせて用いうるが、まずIgMが溶出されない移動相を流し(洗浄工程)、続いてIgMが溶出される移動相を流して行う(溶出工程)ことが好ましい。これによりIgM以外のタンパク質や夾雑物の多くが除かれる。
IgMが溶出されない移動相、およびIgMが溶出される移動相は、陰イオン交換樹脂の種類などによって異なるが、例えば、後述する試験例1に記載の実験により決定することができる。 Examples of the mobile phase include a phosphate buffer having a pH of 7 to 8, a phosphate buffer having a pH of 7 to 8 containing sodium chloride, and a tris hydrochloride buffer having a pH of 7 to 8. These may be used in appropriate combinations, but it is preferable to first carry out a mobile phase from which IgM is not eluted (washing step), and then flow through a mobile phase from which IgM is eluted (elution step). This removes many proteins and contaminants other than IgM.
The mobile phase from which IgM is not eluted and the mobile phase from which IgM is eluted vary depending on the type of anion exchange resin, but can be determined by, for example, an experiment described in Test Example 1 described later.
IgMが溶出されない移動相、およびIgMが溶出される移動相は、陰イオン交換樹脂の種類などによって異なるが、例えば、後述する試験例1に記載の実験により決定することができる。 Examples of the mobile phase include a phosphate buffer having a pH of 7 to 8, a phosphate buffer having a pH of 7 to 8 containing sodium chloride, and a tris hydrochloride buffer having a pH of 7 to 8. These may be used in appropriate combinations, but it is preferable to first carry out a mobile phase from which IgM is not eluted (washing step), and then flow through a mobile phase from which IgM is eluted (elution step). This removes many proteins and contaminants other than IgM.
The mobile phase from which IgM is not eluted and the mobile phase from which IgM is eluted vary depending on the type of anion exchange resin, but can be determined by, for example, an experiment described in Test Example 1 described later.
なお、陰イオン交換樹脂の量、カラムの大きさ、移動相の量などは、それらの種類や処理量、目的などによって異なり、適宜選択される。
The amount of anion exchange resin, the column size, the amount of mobile phase, and the like vary depending on the type, processing amount, purpose, etc., and are appropriately selected.
上記のように陰イオン交換樹脂で固相抽出を行った後、IgM抗体の分離精製を高めるために、限外ろ過膜で濃縮することが好ましい。かかる限外ろ過膜としては、例えば、分画分子量が10,000~120,000の範囲内のものを挙げることができ、30,000~100,000の範囲内のものが適当である。分画分子量が10,000より小さいものは、分離に時間を要するおそれがあり、不純物の分離が十分に行えない場合があり、120,000より大きいものは、IgMの分離精製収率が低下する場合がある。
After performing solid phase extraction with an anion exchange resin as described above, it is preferable to concentrate with an ultrafiltration membrane in order to enhance the separation and purification of IgM antibodies. Examples of such ultrafiltration membranes include those having a fractional molecular weight in the range of 10,000 to 120,000, and those having a molecular weight in the range of 30,000 to 100,000 are suitable. If the molecular weight cut-off is less than 10,000, it may take time for separation, and the impurities may not be sufficiently separated. If the molecular weight is more than 120,000, the separation and purification yield of IgM decreases. There is a case.
1.2 ポリエチレングリコールで処理する工程
本発明精製法は、ポリエチレングリコール(PEG)で処理する工程を含む。本工程は、前記陰イオン交換樹脂で固相抽出を行い、必要に応じて限外ろ過膜で濃縮を行った後に行われる。 1.2 Process with polyethylene glycol The purification method of the present invention includes a process with polyethylene glycol (PEG). This step is performed after solid-phase extraction with the anion exchange resin and, if necessary, concentration with an ultrafiltration membrane.
本発明精製法は、ポリエチレングリコール(PEG)で処理する工程を含む。本工程は、前記陰イオン交換樹脂で固相抽出を行い、必要に応じて限外ろ過膜で濃縮を行った後に行われる。 1.2 Process with polyethylene glycol The purification method of the present invention includes a process with polyethylene glycol (PEG). This step is performed after solid-phase extraction with the anion exchange resin and, if necessary, concentration with an ultrafiltration membrane.
本工程は、前記工程までに処理され得られた検体にPEGを加え、攪拌することによって行われる。より具体的には、例えば、前記工程までに処理され得られた検体の水溶液にPEG水溶液を加え、両者を激しく攪拌して沈殿物を得、通常4~8℃の低温で静置した後、0~10℃に設定した冷却遠心分離機で遠心分離することにより行われる。そして、遠心分離の後、上清を捨てることにより、分離精製されたIgM抗体の沈渣を得る。
This step is performed by adding PEG to the specimen obtained by the above steps and stirring. More specifically, for example, an aqueous PEG solution is added to the aqueous solution of the specimen obtained by the above process, both are vigorously stirred to obtain a precipitate, and after standing at a low temperature of usually 4 to 8 ° C., It is carried out by centrifuging with a refrigerated centrifuge set at 0 to 10 ° C. After centrifugation, the supernatant is discarded to obtain a separated and purified IgM antibody precipitate.
当該ポリエチレングリコールとしては、例えば、平均分子量が4,000~12,000の範囲内のものが適当であり、好ましくは6,000~10,000のものである。具体的には、例えば、PEG4000、PEG6000、PEG8000、PEG10000、PEG12000を挙げることができ、この中、PEG8000が好ましい。
As the polyethylene glycol, for example, those having an average molecular weight in the range of 4,000 to 12,000 are suitable, and preferably 6,000 to 10,000. Specifically, for example, PEG4000, PEG6000, PEG8000, PEG10000, and PEG12000 can be mentioned, and among these, PEG8000 is preferable.
当該ポリエチレングリコールの処理濃度としては、PEGの種類などによって異なるが、6~10w/v%の範囲内が好ましく、8w/v%がより好ましい。特にPEG8000を用いる場合には、その処理濃度が8w/v%であることが好ましい。
The treatment concentration of the polyethylene glycol varies depending on the type of PEG, but is preferably within a range of 6 to 10 w / v%, more preferably 8 w / v%. In particular, when PEG 8000 is used, the treatment concentration is preferably 8 w / v%.
1.3 その他の工程
本発明精製法では、必要に応じて、いわゆるクロロホルム/メタノール処理(クロメタ沈殿法)を行うことができる。かかるクロメタ処理は常法により行うことができる。具体的には、例えば、次のようにしてクロメタ処理が行われる。 1.3 Other Steps In the purification method of the present invention, so-called chloroform / methanol treatment (chromate precipitation method) can be performed as necessary. Such chromate treatment can be performed by a conventional method. Specifically, for example, the chroma processing is performed as follows.
本発明精製法では、必要に応じて、いわゆるクロロホルム/メタノール処理(クロメタ沈殿法)を行うことができる。かかるクロメタ処理は常法により行うことができる。具体的には、例えば、次のようにしてクロメタ処理が行われる。 1.3 Other Steps In the purification method of the present invention, so-called chloroform / methanol treatment (chromate precipitation method) can be performed as necessary. Such chromate treatment can be performed by a conventional method. Specifically, for example, the chroma processing is performed as follows.
前記PEG工程で得られた沈渣を適当な溶媒に溶かし、メタノールを加えて攪拌後、更にクロロホルムを加えて攪拌する。続いて、水(例、逆浸透膜を通した水(RO水)、精製水、蒸留水)を加え攪拌し、遠心分離して上清を廃棄する。これにメタノールを適当量加え混和し、再度遠心分離して上清を廃棄し、分離精製されたIgM抗体の沈渣を得る。
上記のようなクロメタ処理は、前記PEG処理の前に行うこともできる。 The precipitate obtained in the PEG step is dissolved in a suitable solvent, methanol is added and stirred, and then chloroform is further added and stirred. Subsequently, water (eg, water through a reverse osmosis membrane (RO water), purified water, distilled water) is added and stirred, centrifuged and the supernatant discarded. An appropriate amount of methanol is added thereto and mixed, and then centrifuged again to discard the supernatant, thereby obtaining a separated and purified IgM antibody precipitate.
The chromate process as described above can also be performed before the PEG process.
上記のようなクロメタ処理は、前記PEG処理の前に行うこともできる。 The precipitate obtained in the PEG step is dissolved in a suitable solvent, methanol is added and stirred, and then chloroform is further added and stirred. Subsequently, water (eg, water through a reverse osmosis membrane (RO water), purified water, distilled water) is added and stirred, centrifuged and the supernatant discarded. An appropriate amount of methanol is added thereto and mixed, and then centrifuged again to discard the supernatant, thereby obtaining a separated and purified IgM antibody precipitate.
The chromate process as described above can also be performed before the PEG process.
また、本発明精製法により得られたIgM抗体の沈渣を適当な溶媒で溶かし、必要に応じてpH7~8のリン酸緩衝剤などを加え攪拌した後、分離精製されたIgM抗体液とすることができる。かかるIgM抗体液は、低温(例えば、4℃)で保存することができる。
In addition, the IgM antibody precipitate obtained by the purification method of the present invention is dissolved in an appropriate solvent, and a phosphate buffer of pH 7-8 is added and stirred as necessary, to obtain a separated and purified IgM antibody solution. Can do. Such an IgM antibody solution can be stored at a low temperature (eg, 4 ° C.).
上記のIgM抗体の沈渣を溶かす適当な溶媒として、例えば、アルカリ剤を挙げることができ、またアルカリ剤が好ましい。アルカリ剤では、IgM抗体の変性を起こすことが懸念されたが、意外にもIgM抗体の溶解に良好であった。
As an appropriate solvent for dissolving the above IgM antibody precipitate, for example, an alkaline agent can be mentioned, and an alkaline agent is preferable. Although there was a concern about the denaturation of IgM antibody with an alkaline agent, it was surprisingly good in dissolving IgM antibody.
上記アルカリ剤としては、例えば、pH10~13の水酸化ナトリウムや水酸化カリウム、pH9~10の炭酸-重炭酸緩衝液を挙げることができる。この中、pH12~13の水酸化ナトリウムが好ましい。また、当該アルカリ剤には、Tween20などのポリソルベート系界面活性剤が適当量含まれていることが好ましい。
Examples of the alkali agent include sodium hydroxide and potassium hydroxide having a pH of 10 to 13, and a carbonate-bicarbonate buffer having a pH of 9 to 10. Of these, sodium hydroxide having a pH of 12 to 13 is preferred. The alkali agent preferably contains an appropriate amount of a polysorbate surfactant such as Tween20.
IgM抗体をアルカリ剤で溶解した後は、適当な酸で中和することが好ましい。酸で中和しないでおくと、IgMが変性するおそれがある。かかる酸としては、例えば、塩酸、酢酸を挙げることができる。この中、塩酸が好ましい。当該酸の濃度は、アルカリ剤の種類や量、濃度などによって異なり、中和できる濃度で行えばよい。
After dissolving the IgM antibody with an alkaline agent, it is preferable to neutralize with an appropriate acid. If neutralized with an acid, IgM may be denatured. Examples of such acids include hydrochloric acid and acetic acid. Of these, hydrochloric acid is preferred. The concentration of the acid varies depending on the type, amount, concentration, and the like of the alkaline agent, and may be a concentration that can be neutralized.
1.4 本発明精製法の具体的実施態様
本発明精製法の具体的実施態様としては、例えば、図1に記載のプロトコルを挙げることができる。
1.4 Specific Embodiment of the Purification Method of the Present Invention As a specific embodiment of the purification method of the present invention, for example, the protocol shown in FIG. 1 can be mentioned.
本発明精製法の具体的実施態様としては、例えば、図1に記載のプロトコルを挙げることができる。
1.4 Specific Embodiment of the Purification Method of the Present Invention As a specific embodiment of the purification method of the present invention, for example, the protocol shown in FIG. 1 can be mentioned.
2 IgM抗体の分析法
本発明は、本発明精製法により得られたIgM抗体の沈渣をアルカリ剤で溶解し、必要に応じて酸で中和することによって得られるIgM抗体液を分析に供することを特徴とするIgM抗体の分析法(以下、「本発明分析法」という。)を含む。本発明分析法は、本発明精製法により得られたIgM抗体の沈渣をアルカリ剤で溶解することによって行われる。 2. Analytical method of IgM antibody The present invention provides an IgM antibody solution obtained by dissolving a precipitate of IgM antibody obtained by the purification method of the present invention with an alkaline agent and neutralizing with an acid if necessary. And an IgM antibody analysis method (hereinafter referred to as “analysis method of the present invention”). The analysis method of the present invention is performed by dissolving the precipitate of IgM antibody obtained by the purification method of the present invention with an alkaline agent.
本発明は、本発明精製法により得られたIgM抗体の沈渣をアルカリ剤で溶解し、必要に応じて酸で中和することによって得られるIgM抗体液を分析に供することを特徴とするIgM抗体の分析法(以下、「本発明分析法」という。)を含む。本発明分析法は、本発明精製法により得られたIgM抗体の沈渣をアルカリ剤で溶解することによって行われる。 2. Analytical method of IgM antibody The present invention provides an IgM antibody solution obtained by dissolving a precipitate of IgM antibody obtained by the purification method of the present invention with an alkaline agent and neutralizing with an acid if necessary. And an IgM antibody analysis method (hereinafter referred to as “analysis method of the present invention”). The analysis method of the present invention is performed by dissolving the precipitate of IgM antibody obtained by the purification method of the present invention with an alkaline agent.
IgM抗体の沈渣を溶解するためのアルカリ剤としては、例えば、pH10~13の水酸化ナトリウムや水酸化カリウム、pH9~10の炭酸-重炭酸緩衝液を挙げることができる。この中、pH12~13の水酸化ナトリウムが好ましい。また、当該アルカリ剤には、Tween20などのポリソルベート系界面活性剤が適当量含まれていることが好ましい。
Examples of the alkaline agent for dissolving the precipitate of IgM antibody include sodium hydroxide and potassium hydroxide at pH 10 to 13, and carbonate-bicarbonate buffer at pH 9 to 10. Of these, sodium hydroxide having a pH of 12 to 13 is preferred. The alkali agent preferably contains an appropriate amount of a polysorbate surfactant such as Tween20.
また、中和するための酸としては、例えば、塩酸、酢酸を挙げることができる。この中、塩酸が好ましい。当該酸の濃度は、アルカリ剤の種類や量、濃度などによって異なり、中和できる濃度で行えばよい。
Further, examples of the acid for neutralization include hydrochloric acid and acetic acid. Of these, hydrochloric acid is preferred. The concentration of the acid varies depending on the type, amount, concentration, and the like of the alkaline agent, and may be a concentration that can be neutralized.
本発明分析法においては、本発明精製法により得られたIgM抗体の沈渣をアルカリ剤で溶解し、必要に応じて酸で中和することによって得られるIgM抗体液を用いる以外は、公知の分析法を適用することができる。かかる公知の分析法としては、例えば、電気泳動法、ポリアクリルアミドゲル電気泳動(PAGE)法、ドデシル硫酸ナトリウム(SDS)-PAGE、ウェスタン・ブロッティング法、イムノブロッティング法、ドットブロット分析を挙げることができる。
In the analysis method of the present invention, a known analysis is used except that an IgM antibody solution obtained by dissolving the IgM antibody precipitate obtained by the purification method of the present invention with an alkaline agent and neutralizing with an acid as necessary is used. The law can be applied. Examples of such known analytical methods include electrophoresis, polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate (SDS) -PAGE, Western blotting, immunoblotting, and dot blot analysis. .
In the analysis method of the present invention, a known analysis is used except that an IgM antibody solution obtained by dissolving the IgM antibody precipitate obtained by the purification method of the present invention with an alkaline agent and neutralizing with an acid as necessary is used. The law can be applied. Examples of such known analytical methods include electrophoresis, polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate (SDS) -PAGE, Western blotting, immunoblotting, and dot blot analysis. .
以下に実施例、試験例を掲げて本発明を更に詳しく説明するが、本発明はこれら実施例等により何ら限定されるものではない。
Hereinafter, the present invention will be described in more detail with reference to examples and test examples, but the present invention is not limited to these examples and the like.
以下の実施例、試験例で用いた主な試薬および機器の一覧は、下記の通りである。
・DEAEイオン交換樹脂:TOYOPEARL DEAE 650M 、東ソー社製
・固相抽出用カラム:ISOLUTE(登録商標)double fritted filtration column(25ml,φ20mm)、バイオタージ・ジャパン社製
・ポリエチレングリコール:PEG8000 Powder(Molecular Biology Grade)、Promega Corporation (Madison WI, USA)製 The list of main reagents and instruments used in the following examples and test examples is as follows.
・ DEAE ion exchange resin: TOYOPEARL DEAE 650M, manufactured by Tosoh Corporation ・ Solid phase extraction column: ISOLUTE (registered trademark) double fritted filtration column (25 ml, φ20 mm), manufactured by Biotage Japan ・ Polyethylene glycol: PEG8000 Powder (Molecular Biology Grade), Promega Corporation (Madison WI, USA)
・DEAEイオン交換樹脂:TOYOPEARL DEAE 650M 、東ソー社製
・固相抽出用カラム:ISOLUTE(登録商標)double fritted filtration column(25ml,φ20mm)、バイオタージ・ジャパン社製
・ポリエチレングリコール:PEG8000 Powder(Molecular Biology Grade)、Promega Corporation (Madison WI, USA)製 The list of main reagents and instruments used in the following examples and test examples is as follows.
・ DEAE ion exchange resin: TOYOPEARL DEAE 650M, manufactured by Tosoh Corporation ・ Solid phase extraction column: ISOLUTE (registered trademark) double fritted filtration column (25 ml, φ20 mm), manufactured by Biotage Japan ・ Polyethylene glycol: PEG8000 Powder (Molecular Biology Grade), Promega Corporation (Madison WI, USA)
・限外ろ過膜:Vivaspin6 (MWCO 50000)、Sartorius Stedim Biotech GmbH(Goettingen, Germany)製
・ポリフッ化ビニリデン(PVDF)膜:Immobilon-P Transfer Membrane(Pore size 0.45μm)、Millipore Corporation(MA,USA)製 ・ Ultrafiltration membrane: Vivaspin6 (MWCO 50000), manufactured by Sartorius Stedim Biotech GmbH (Goettingen, Germany) ・ Polyvinylidene fluoride (PVDF) membrane: Immobilon-P Transfer Membrane (Pore size 0.45 μm), Millipore Corporation (MA, USA) Made
・ポリフッ化ビニリデン(PVDF)膜:Immobilon-P Transfer Membrane(Pore size 0.45μm)、Millipore Corporation(MA,USA)製 ・ Ultrafiltration membrane: Vivaspin6 (MWCO 50000), manufactured by Sartorius Stedim Biotech GmbH (Goettingen, Germany) ・ Polyvinylidene fluoride (PVDF) membrane: Immobilon-P Transfer Membrane (Pore size 0.45 μm), Millipore Corporation (MA, USA) Made
・電気泳動用アクリルアミド、電気泳動用NNメチレンビス(アクリルアミド)、ペルオキソ二硫酸アンモニウム電気泳動用、NNNNテトラメチルエチレンジアミン電気泳動用、Coomassie Brilliant Blue (CBB)G-250電気泳動用、ドデシル硫酸ナトリウム(生化学用)は和光純薬工業社製
・電気泳動緩衝液はAE-1410 EzRun、ブロッティング用転写バッファーはEzFastBlot、ブロッキング試薬はAE-1475 EzBlock Chemi、HRP標識抗体用発色基質(TMB発色基質)はAE-1490 Ez WestBlue(いずれもアトー社製) ・ Acrylamide for electrophoresis, NN methylene bis (acrylamide) for electrophoresis, ammonium peroxodisulfate electrophoresis, NNNN tetramethylethylenediamine electrophoresis, Coomassie Brilliant Blue (CBB) G-250 electrophoresis, sodium dodecyl sulfate (for biochemistry) ) Is manufactured by Wako Pure Chemical Industries, Ltd. Electrophoresis buffer is AE-1410 EzRun, Blotting transfer buffer is EzFastBlot, Blocking reagent is AE-1475 EzBlock Chemi, HRP labeled antibody chromogenic substrate (TMB chromogenic substrate) is AE-1490 Ez WestBlue (all manufactured by Ato)
・電気泳動緩衝液はAE-1410 EzRun、ブロッティング用転写バッファーはEzFastBlot、ブロッキング試薬はAE-1475 EzBlock Chemi、HRP標識抗体用発色基質(TMB発色基質)はAE-1490 Ez WestBlue(いずれもアトー社製) ・ Acrylamide for electrophoresis, NN methylene bis (acrylamide) for electrophoresis, ammonium peroxodisulfate electrophoresis, NNNN tetramethylethylenediamine electrophoresis, Coomassie Brilliant Blue (CBB) G-250 electrophoresis, sodium dodecyl sulfate (for biochemistry) ) Is manufactured by Wako Pure Chemical Industries, Ltd. Electrophoresis buffer is AE-1410 EzRun, Blotting transfer buffer is EzFastBlot, Blocking reagent is AE-1475 EzBlock Chemi, HRP labeled antibody chromogenic substrate (TMB chromogenic substrate) is AE-1490 Ez WestBlue (all manufactured by Ato)
・抗ヒトIgM-HRP標識抗体(H鎖):Polyclonal Rabbit Anti-Human IgM/HRP code No.P0215、Dako Denmark A/S(Glostrup, Denmark)製
・ヒト由来IgM抗体:H-IgM(Immunoglobulin M from human Serum Lot No.44004406、オリエンタル酵母工業社製)
・ヒト由来IgG抗体:HUMAN IgG(Product No.14506、シグマ社製)
・牛血清アルブミン液(BSA、生化学用、和光純薬工業社製) Anti-human IgM-HRP-labeled antibody (H chain): Polyclonal Rabbit Anti-Human IgM / HRP code No. P0215, manufactured by Dako Denmark A / S (Glostrup, Denmark) Human-derived IgM antibody: H-IgM (Immunoglobulin M from (human Serum Lot No. 44004406, manufactured by Oriental Yeast Co., Ltd.)
-Human-derived IgG antibody: HUMAN IgG (Product No. 14506, manufactured by Sigma)
・ Bovine serum albumin solution (BSA, for biochemistry, manufactured by Wako Pure Chemical Industries, Ltd.)
・ヒト由来IgM抗体:H-IgM(Immunoglobulin M from human Serum Lot No.44004406、オリエンタル酵母工業社製)
・ヒト由来IgG抗体:HUMAN IgG(Product No.14506、シグマ社製)
・牛血清アルブミン液(BSA、生化学用、和光純薬工業社製) Anti-human IgM-HRP-labeled antibody (H chain): Polyclonal Rabbit Anti-Human IgM / HRP code No. P0215, manufactured by Dako Denmark A / S (Glostrup, Denmark) Human-derived IgM antibody: H-IgM (Immunoglobulin M from (human Serum Lot No. 44004406, manufactured by Oriental Yeast Co., Ltd.)
-Human-derived IgG antibody: HUMAN IgG (Product No. 14506, manufactured by Sigma)
・ Bovine serum albumin solution (BSA, for biochemistry, manufactured by Wako Pure Chemical Industries, Ltd.)
・SDS-ポリアクリルアミドゲル電気泳動(SDA-PAGE)に必要な電気泳動槽およびブロッティング装置はアトー社製のRapidas AE-6530M、Horizblot 2M AE-6687(・S)を使用し、コンスタントパワーサプライはアナテック社製のPowerPhoreStar 3810を使用した。
・ Atophoresis Rapidas AE-6530M, Horizblot 2M AE-6687 (・ S) are used for SDS-polyacrylamide gel electrophoresis (SDA-PAGE), and Anatech is a constant power supply. PowerPhoreStar 3810 made by the company was used.
・ゲルは10%Hand-Castゲルを作成し使用した。
・メタノール(HPLC用)およびその他の試薬は和光純薬工業社製を用いた。 -A 10% Hand-Cast gel was prepared and used.
-Methanol (for HPLC) and other reagents were manufactured by Wako Pure Chemical Industries.
・メタノール(HPLC用)およびその他の試薬は和光純薬工業社製を用いた。 -A 10% Hand-Cast gel was prepared and used.
-Methanol (for HPLC) and other reagents were manufactured by Wako Pure Chemical Industries.
また、リン酸緩衝液(pH7.1)を「PB」と、塩化ナトリウムを「NaCl」と、水酸化ナトリウムを「NaOH」と、塩酸を「HCl」と、「ボルテックスミキサーで激しく攪拌」することを単に「ボルテックス」と、それぞれいう。
In addition, “PB” for phosphate buffer (pH 7.1), “NaCl” for sodium chloride, “NaOH” for sodium hydroxide, “HCl” for hydrochloric acid, and “Vortex mixer vigorously”. Are simply called “vortex”.
[実施例1]IgM抗体の分離精製(1)
(1)DEAEイオン交換樹脂カラムによる処理
ボランティア健常者20名より採血し、常法により血清を得た。
固相抽出用カラムにDEAE650Mを5mL詰め、8mMPBで平衡化し、これに血清希釈液(血清0.1mLに8mMPB15mLを加え混和したもの。以下同じ。)をアプライした。 [Example 1] Separation and purification of IgM antibody (1)
(1) Treatment with DEAE ion exchange resin column Blood was collected from 20 healthy volunteers, and serum was obtained by a conventional method.
A solid phase extraction column was packed with 5 mL of DEAE650M, equilibrated with 8 mMPB, and a serum dilution (15 mL of 8 mMPB was added to 0.1 mL of serum and mixed. The same applies hereinafter) was applied thereto.
(1)DEAEイオン交換樹脂カラムによる処理
ボランティア健常者20名より採血し、常法により血清を得た。
固相抽出用カラムにDEAE650Mを5mL詰め、8mMPBで平衡化し、これに血清希釈液(血清0.1mLに8mMPB15mLを加え混和したもの。以下同じ。)をアプライした。 [Example 1] Separation and purification of IgM antibody (1)
(1) Treatment with DEAE ion exchange resin column Blood was collected from 20 healthy volunteers, and serum was obtained by a conventional method.
A solid phase extraction column was packed with 5 mL of DEAE650M, equilibrated with 8 mMPB, and a serum dilution (15 mL of 8 mMPB was added to 0.1 mL of serum and mixed. The same applies hereinafter) was applied thereto.
8mMPB20mL、および0.1MNaCl/8mMPB20mLで洗浄後、さらに0.2MNaCl/8mMPB3.0mLで洗浄してから、0.2MNaCl/8mMPB6.0mLで溶出し、限外ろ過膜で200μLまで濃縮した。
After washing with 20 mL of 8 mMPB and 20 mL of 0.1 M NaCl / 8 mMPB, the product was further washed with 3.0 mL of 0.2 M NaCl / 8 mMPB, eluted with 6.0 mL of 0.2 M NaCl / 8 mMPB, and concentrated to 200 μL with an ultrafiltration membrane.
(2)PEG処理
2.0mLマイクロチューブ(ワトソン社製)に移し1.0MNaCl/8mMPBを100μL加えボルテックスした後、12w/v%PEG(0.5MNaCl含有/8mMPB)を600μL加えボルテックスし、4℃で2時間静置した後、12000g、4℃で10分間遠心した。 (2) PEG treatment After transferring to a 2.0 mL microtube (manufactured by Watson) and adding 100 μL of 1.0 M NaCl / 8 mMPB, vortexing, adding 600 μL of 12 w / v% PEG (containing 0.5 M NaCl / 8 mMPB), vortexing, And then allowed to stand for 2 hours at 12,000 g and centrifuged at 4 ° C. for 10 minutes.
2.0mLマイクロチューブ(ワトソン社製)に移し1.0MNaCl/8mMPBを100μL加えボルテックスした後、12w/v%PEG(0.5MNaCl含有/8mMPB)を600μL加えボルテックスし、4℃で2時間静置した後、12000g、4℃で10分間遠心した。 (2) PEG treatment After transferring to a 2.0 mL microtube (manufactured by Watson) and adding 100 μL of 1.0 M NaCl / 8 mMPB, vortexing, adding 600 μL of 12 w / v% PEG (containing 0.5 M NaCl / 8 mMPB), vortexing, And then allowed to stand for 2 hours at 12,000 g and centrifuged at 4 ° C. for 10 minutes.
(3)分離精製されたIgM抗体の保存液の調製
上清を廃棄し、沈渣に0.1%Tween20-0.1NNaOHを100μL加え完全に溶かした後、0.1MPBを50μL加えてボルテックスし、0.2NHCl50μLを加えボルテックスして、分離精製されたIgM抗体の保存液を得た。これを4℃に保存した。 (3) Preparation of stock solution of separated and purified IgM antibody Discard the supernatant, dissolve 100 μL of 0.1% Tween20-0.1N NaOH in the sediment and dissolve completely, then add 50 μL of 0.1 MPB and vortex, A stock solution of separated and purified IgM antibody was obtained by adding 50 μL of 0.2N HCl and vortexing. This was stored at 4 ° C.
上清を廃棄し、沈渣に0.1%Tween20-0.1NNaOHを100μL加え完全に溶かした後、0.1MPBを50μL加えてボルテックスし、0.2NHCl50μLを加えボルテックスして、分離精製されたIgM抗体の保存液を得た。これを4℃に保存した。 (3) Preparation of stock solution of separated and purified IgM antibody Discard the supernatant, dissolve 100 μL of 0.1% Tween20-0.1N NaOH in the sediment and dissolve completely, then add 50 μL of 0.1 MPB and vortex, A stock solution of separated and purified IgM antibody was obtained by adding 50 μL of 0.2N HCl and vortexing. This was stored at 4 ° C.
[実施例2]IgM抗体の分離精製(2)
実施例1と同様にしてDEAEイオン交換樹脂カラム処理とPEG処理を行い、上清を捨て0.1%Tween20-0.1NNaOHを100μL加えて完全に溶かした後、0.1MPBを50μL加えてボルテックスし、0.2N塩酸を50μL加えてボルテックスし、メタノール100μLを加えボルテックスした後、クロロホルム100μLを加えボルテックスし、続いて逆浸透膜を通した水(RO水)300μL加えボルテックスした後、12000g、60秒間遠心し、上清を廃棄した。 [Example 2] Separation and purification of IgM antibody (2)
DEAE ion exchange resin column treatment and PEG treatment were carried out in the same manner as in Example 1, and the supernatant was discarded and 100 μL of 0.1% Tween20-0.1N NaOH was added and completely dissolved, and then 50 μL of 0.1 MPB was added and vortexed. Vortex by adding 50 μL of 0.2N hydrochloric acid, vortex by adding 100 μL of methanol, vortex by adding 100 μL of chloroform, and then vortex by adding 300 μL of water (RO water) that has passed through a reverse osmosis membrane. The supernatant was discarded after centrifugation for 2 seconds.
実施例1と同様にしてDEAEイオン交換樹脂カラム処理とPEG処理を行い、上清を捨て0.1%Tween20-0.1NNaOHを100μL加えて完全に溶かした後、0.1MPBを50μL加えてボルテックスし、0.2N塩酸を50μL加えてボルテックスし、メタノール100μLを加えボルテックスした後、クロロホルム100μLを加えボルテックスし、続いて逆浸透膜を通した水(RO水)300μL加えボルテックスした後、12000g、60秒間遠心し、上清を廃棄した。 [Example 2] Separation and purification of IgM antibody (2)
DEAE ion exchange resin column treatment and PEG treatment were carried out in the same manner as in Example 1, and the supernatant was discarded and 100 μL of 0.1% Tween20-0.1N NaOH was added and completely dissolved, and then 50 μL of 0.1 MPB was added and vortexed. Vortex by adding 50 μL of 0.2N hydrochloric acid, vortex by adding 100 μL of methanol, vortex by adding 100 μL of chloroform, and then vortex by adding 300 μL of water (RO water) that has passed through a reverse osmosis membrane. The supernatant was discarded after centrifugation for 2 seconds.
メタノール300μLを加えゆるやかに混和し、12000g、2分間遠心して上清を廃棄し、沈渣に0.1%Tween20-0.1NNaOHを100μL加えて完全に溶かした後、0.1MPBを50μL加えてボルテックスし、0.2N塩酸50μLを加えボルテックスして、分離精製されたIgM抗体の保存液を得た。これを4℃に保存した。
Add 300 μL of methanol, mix gently, centrifuge at 12,000 g for 2 minutes, discard the supernatant, add 100 μL of 0.1% Tween20-0.1N NaOH to the sediment and dissolve completely, then add 50 μL of 0.1 MPB and vortex Then, 50 μL of 0.2N hydrochloric acid was added and vortexed to obtain a separated and purified IgM antibody stock solution. This was stored at 4 ° C.
[試験例1]溶出液と溶出液量の検討
血清希釈液をDEAEイオン交換樹脂カラムにアプライし、8mMPBの洗浄液を20mL通し、続いて0.1MNaCl/8mMPBと0.2MNaCl/8mMPBの溶出液を順に20mLずつ通した。 [Test Example 1] Examination of eluate and eluate volume The serum dilution was applied to a DEAE ion exchange resin column, and 20 mL of 8mMPB washing solution was passed through, followed by elution of 0.1M NaCl / 8mMPB and 0.2M NaCl / 8mMPB. 20 mL each was passed in order.
血清希釈液をDEAEイオン交換樹脂カラムにアプライし、8mMPBの洗浄液を20mL通し、続いて0.1MNaCl/8mMPBと0.2MNaCl/8mMPBの溶出液を順に20mLずつ通した。 [Test Example 1] Examination of eluate and eluate volume The serum dilution was applied to a DEAE ion exchange resin column, and 20 mL of 8mMPB washing solution was passed through, followed by elution of 0.1M NaCl / 8mMPB and 0.2M NaCl / 8mMPB. 20 mL each was passed in order.
当該カラムから流出してきた溶出液を2mLずつ分取し、各分取液(画分)についてドットブロット分析を行った。
2 mL each of the eluate flowing out from the column was collected, and each blotted solution (fraction) was subjected to dot blot analysis.
PVDF膜をメタノールで2~3分間浸した後、メタノールを棄てRO水に浸し、シェーカー(インビトロシェーカー Wave-SI slim、タイテック社製)で10~20分間振とうした後、PVFD膜をRO水に浸した濾紙の上に置き、各画分サンプルを5μLずつアプライした。次いでPVFD膜をブロッキング試薬でブロックし、抗ヒトIgM‐HRP標識抗体(H鎖)と反応させた後、HRP標識抗体用発色基質(TMB発色基質)で染色した。その結果を図2に示す。
After immersing the PVDF membrane in methanol for 2 to 3 minutes, discard the methanol and immerse in RO water, shake with a shaker (Invitro Shaker イ ン ビ ト ロ Wave-SI slim, manufactured by Taitec) for 10 to 20 minutes, and then immerse the PVFD membrane in RO water. Placed on soaked filter paper, 5 μL of each fraction sample was applied. Next, the PVFD membrane was blocked with a blocking reagent, reacted with an anti-human IgM-HRP labeled antibody (H chain), and then stained with a chromogenic substrate for HRP labeled antibody (TMB chromogenic substrate). The result is shown in FIG.
また、各画分の画像解析(色の濃淡)を画像処理ソフトウェアのImageJで行うと共に、分光光度計(HITACHI U-2900 Spectrophotometer 日立製作所社製)を用いて波長280nmにおける各溶出液の吸光度を測定した。その結果を図3に示す。
In addition, image analysis (color shading) of each fraction is performed using ImageJ, an image processing software, and the absorbance of each eluate at a wavelength of 280 nm is measured using a spectrophotometer (HITACHI U-2900 Spectrophotometer manufactured by Hitachi, Ltd.). did. The result is shown in FIG.
図2、図3中、passはカラム通過液、8mMPBは8mMPB洗浄液、No.1~10は0.1MNaCl/8mMPBの溶出液画分、No.11~20は0.2MNaCl/8mMPBの溶出液画分の結果である。
2 and 3, in FIG. 2 and FIG. 3, “pass” is the column passing solution, 8 mMPB is the 8 mMPB washing solution, No. Nos. 1 to 10 are eluate fractions of 0.1M NaCl / 8mMPB. 11 to 20 are the results of the eluate fraction of 0.2M NaCl / 8mMPB.
図2、図3の結果から、passや8mMPB、0.1MNaCl/8mMPBの溶出液にはIgM抗体は含まれておらず、0.2MNaCl/8mMPBの溶出液で溶出された画分にIgM抗体が含まれていることが確認された。
From the results of FIGS. 2 and 3, IgM antibody is not contained in the eluate of pass, 8mMPB, and 0.1M NaCl / 8mMPB, and IgM antibody is contained in the fraction eluted with the eluate of 0.2M NaCl / 8mMPB. It was confirmed that it was included.
[試験例2]分離精製されたIgM抗体のSDS-PAGEおよびウェスタン・ブロッティング
使用した10%hand-castゲルはゲルサイズ90×80mm、厚さ1mmとした。 Test Example 2 SDS-PAGE and Western Blotting of Separated and Purified IgM Antibody The 10% hand-cast gel used had a gel size of 90 × 80 mm and a thickness of 1 mm.
使用した10%hand-castゲルはゲルサイズ90×80mm、厚さ1mmとした。 Test Example 2 SDS-PAGE and Western Blotting of Separated and Purified IgM Antibody The 10% hand-cast gel used had a gel size of 90 × 80 mm and a thickness of 1 mm.
実施例1で得られたIgM抗体液、2.7mg/mLのヒト由来IgG抗体液をSDS-PAGE用サンプル処理液(50mMTris-HCl緩衝液 pH6.8(1%SDS,20%グリセリン,1.5% 2-メルカプトエタノール含有)で5倍に希釈し、100℃、4分間加熱後、ゲルに5~10μL(No.1とNo.2が10μL、No.3とNo.4が5μL)をアプライした。20mA、定流で65~75分間泳動した後、ゲルをHorizblot 2M AE-6687(・S)で250mA、35~40分間PVDF膜に転写した。
The IgM antibody solution obtained in Example 1 and a 2.7 mg / mL human-derived IgG antibody solution were mixed with a sample processing solution for SDS-PAGE (50 mM Tris-HCl buffer solution pH 6.8 (1% SDS, 20% glycerin, 1.5% Diluted 2-fold with 2-mercaptoethanol), heated at 100 ° C. for 4 minutes, and then applied to the gel with 5 to 10 μL (No. 1 and No. 2 were 10 μL, No. 3 and No. 4 were 5 μL) After electrophoresis at 20 mA and constant flow for 65 to 75 minutes, the gel was transferred to a PVDF membrane with Horizblot 2M AE-6687 (• S) at 250 mA for 35 to 40 minutes.
転写膜を半分に切り、一枚は0.02%CBBで染色し、残り一枚はブロッキング試薬でブロックし、抗ヒトIgM‐HRP標識抗体(H鎖)と反応させた後、HRP標識抗体用発色基質(TMB発色基質)で染色した。その結果を図4に示す。
Cut the transfer membrane in half, one is stained with 0.02% CBB, the other is blocked with a blocking reagent, reacted with anti-human IgM-HRP labeled antibody (H chain), and then for HRP labeled antibody Stained with a chromogenic substrate (TMB chromogenic substrate). The result is shown in FIG.
図4中、No.1とNo.3はヒト由来IgG抗体の結果を、No.2とNo.4は本発明精製法によりヒト血清中から分離されたIgM抗体の結果を、それぞれ示し、No.1とNo.2はCBB染色での結果を、No.3とNo.4はTMB発色基質染色での結果を、それぞれ示す。
In FIG. 1 and No. 3 shows the results of the human IgG antibody. 2 and No. 4 shows the results of IgM antibodies isolated from human serum by the purification method of the present invention. 1 and No. 2 shows the results of CBB staining. 3 and no. 4 shows the results of TMB chromogenic substrate staining, respectively.
図4の結果から、本発明精製法のNo.2におけるHバンドがIgM抗体H鎖であることが確認された。
4 From the result of FIG. It was confirmed that the H band in Fig. 2 is an IgM antibody H chain.
[試験例3]分離精製されたIgM抗体のタンパク質含有率
SDS-PAGE10%ゲルを作成し、実施例1で得られたIgM抗体液、1mg/mL牛血清アルブミン液(BSA)をSDS-PAGE用サンプル処理液(50mMTris-HCl緩衝液 pH6.8(1%SDS,20%グリセリン,1.5% 2-メルカプトエタノール含有)で5倍に希釈し、100℃、4分間加熱後、ゲルにIgM抗体希釈液を20、10、または5μLアプライし、BSA希釈液を10μLアプライした。20mA、定流で65~75分間泳動した後、0.08%CBBで約30分染色し、次いで脱色液A(50%メタノール-10%酢酸-40%RO水)で約1時間脱色し、さらに脱色液B(30%メタノール-10%酢酸-60%RO水)で一夜脱色した。その結果を図5に示す。 [Test Example 3] Protein content of separated and purified IgM antibody An SDS-PAGE 10% gel was prepared, and the IgM antibody solution, 1 mg / mL bovine serum albumin solution (BSA) obtained in Example 1 was used for SDS-PAGE. Dilute 5 times with sample treatment solution (50 mM Tris-HCl buffer pH 6.8 (containing 1% SDS, 20% glycerin, 1.5% 2-mercaptoethanol), heat at 100 ° C. for 4 minutes, and dilute IgM antibody on the gel. 20, 10 or 5 μL was applied, and 10 μL of BSA diluted solution was applied, followed by electrophoresis at 65 mA for 75 to 75 minutes at 20 mA, followed by staining with 0.08% CBB for about 30 minutes, followed by Decolorization Solution A (50% The color was decolored with methanol-10% acetic acid-40% RO water for about 1 hour, and further decolorized with Decoloring Solution B (30% methanol-10% acetic acid-60% RO water) overnight, and the results are shown in FIG.
SDS-PAGE10%ゲルを作成し、実施例1で得られたIgM抗体液、1mg/mL牛血清アルブミン液(BSA)をSDS-PAGE用サンプル処理液(50mMTris-HCl緩衝液 pH6.8(1%SDS,20%グリセリン,1.5% 2-メルカプトエタノール含有)で5倍に希釈し、100℃、4分間加熱後、ゲルにIgM抗体希釈液を20、10、または5μLアプライし、BSA希釈液を10μLアプライした。20mA、定流で65~75分間泳動した後、0.08%CBBで約30分染色し、次いで脱色液A(50%メタノール-10%酢酸-40%RO水)で約1時間脱色し、さらに脱色液B(30%メタノール-10%酢酸-60%RO水)で一夜脱色した。その結果を図5に示す。 [Test Example 3] Protein content of separated and purified IgM antibody An SDS-
図5中、No.1~3はIgM抗体希釈液を順に20、10、5μLアプライした結果を、No.4はBSA希釈液のアプライ結果を、それぞれ示す。
In FIG. 1 to 3 show the results obtained by applying 20, 10, and 5 μL of IgM antibody dilution solution in order. 4 shows the results of applying the BSA dilution.
また、図5のNo.1についてImageJ画像解析ソフトで処理し、色の濃淡をピクセル数で数値化した。その結果を図6に示す。全ピクセルは17126.972であり、H鎖とL鎖のピクセルは合計で12361.367であった。よって、本発明精製法で分離されたIgM抗体(H鎖とL鎖)のピクセルの占める割合は72%となり、本割合はIgMの含有率と推定された。
Also, No. 5 in FIG. 1 was processed with ImageJ image analysis software, and the color shading was quantified by the number of pixels. The result is shown in FIG. The total number of pixels was 17126.972, and the total of H chain and L chain pixels was 12361.367. Therefore, the ratio of the IgM antibody (H chain and L chain) separated by the purification method of the present invention to the pixel was 72%, and this ratio was estimated to be the IgM content.
[試験例4]PEG処理濃度の検討
(1)SDS-PAGE Trans-Blot法による検討
実施例1と同様にヒト血清についてDEAEカラムで処理した。続いて、かかる処理液(200μL)に1.0MNaCl/8mMPBを100μL加えボルテックスした後、PEG濃度が4w/v%、6w/v%、8w/v%、または10w/v%の濃度となるように12w/v%PEG(0.5MNaCl含有/8mMPB)を加えて実施例1と同様に処理した後、SDS-PAGE用サンプル処理液(50mMTris-HCl緩衝液pH6.8(1%SDS,20%グリセリン,1.5% 2-メルカプトエタノール含有)で5倍に希釈し、100℃、4分間加熱後、ゲルに20μLをアプライした。20mA、定流で65~75分間泳動した後、ゲルをHorizblot 2M AE-6687(・S)で250mA、35~40分間PVDF膜に転写し、0.01%CBBで染色した。その結果を図7に示す。 [Test Example 4] Examination of PEG treatment concentration (1) Examination by SDS-PAGE Trans-Blot method In the same manner as in Example 1, human serum was treated with a DEAE column. Subsequently, 100 μL of 1.0M NaCl / 8mMPB is added to the treatment solution (200 μL) and vortexed, so that the PEG concentration becomes 4 w / v%, 6 w / v%, 8 w / v%, or 10 w / v%. After adding 12 w / v% PEG (containing 0.5 M NaCl / 8 mMPB) to the sample, the sample treatment solution for SDS-PAGE (50 mM Tris-HCl buffer pH 6.8 (1% SDS, 20% The mixture was diluted 5 times with glycerin and 1.5% 2-mercaptoethanol), heated at 100 ° C. for 4 minutes, and 20 μL was applied to the gel, and after electrophoresis at 20 mA, constant flow for 65 to 75 minutes, the gel was subjected to Horizblot 2M AE. -6687 (· S) at 250 mA for 35-40 minutes, transferred to a PVDF membrane, and stained with 0.01% CBB, the results of which are shown in FIG.
(1)SDS-PAGE Trans-Blot法による検討
実施例1と同様にヒト血清についてDEAEカラムで処理した。続いて、かかる処理液(200μL)に1.0MNaCl/8mMPBを100μL加えボルテックスした後、PEG濃度が4w/v%、6w/v%、8w/v%、または10w/v%の濃度となるように12w/v%PEG(0.5MNaCl含有/8mMPB)を加えて実施例1と同様に処理した後、SDS-PAGE用サンプル処理液(50mMTris-HCl緩衝液pH6.8(1%SDS,20%グリセリン,1.5% 2-メルカプトエタノール含有)で5倍に希釈し、100℃、4分間加熱後、ゲルに20μLをアプライした。20mA、定流で65~75分間泳動した後、ゲルをHorizblot 2M AE-6687(・S)で250mA、35~40分間PVDF膜に転写し、0.01%CBBで染色した。その結果を図7に示す。 [Test Example 4] Examination of PEG treatment concentration (1) Examination by SDS-PAGE Trans-Blot method In the same manner as in Example 1, human serum was treated with a DEAE column. Subsequently, 100 μL of 1.0M NaCl / 8mMPB is added to the treatment solution (200 μL) and vortexed, so that the PEG concentration becomes 4 w / v%, 6 w / v%, 8 w / v%, or 10 w / v%. After adding 12 w / v% PEG (containing 0.5 M NaCl / 8 mMPB) to the sample, the sample treatment solution for SDS-PAGE (50 mM Tris-HCl buffer pH 6.8 (1% SDS, 20% The mixture was diluted 5 times with glycerin and 1.5% 2-mercaptoethanol), heated at 100 ° C. for 4 minutes, and 20 μL was applied to the gel, and after electrophoresis at 20 mA, constant flow for 65 to 75 minutes, the gel was subjected to Horizblot 2M AE. -6687 (· S) at 250 mA for 35-40 minutes, transferred to a PVDF membrane, and stained with 0.01% CBB, the results of which are shown in FIG.
図7中、No.1は10%PEGの結果を、No.2は8%PEGの結果を、No.3は6%PEGの結果を、No.4は4%PEGの結果を、No.5はPEG無処理の結果を、No.6はヒト由来IgM抗体(標準品)の結果を、No.7はヒト由来IgG抗体(標準品)の結果を、それぞれ示す。
In FIG. 1 shows the result of 10% PEG, 2 shows the result of 8% PEG. 3 shows the result of 6% PEG. 4 shows the result of 4% PEG, 5 shows the result of no PEG treatment. 6 shows the results of the human-derived IgM antibody (standard product). 7 shows the results of human-derived IgG antibody (standard product).
(2)ドットブロット法による検討
ヒト由来IgM抗体(標準品)は、25mMトリス(トリスヒドロキシメチルアミノメタンをRO水で溶かして25mMとしたもの)で希釈し、1.12μg/mL濃度を作成して使用した。NCは、ヒト血清0.1mLを実施例1と同様にDEAEカラム処理し、限外ろ過膜にて0.1mLまで濃縮したものを、25mMトリスで500倍に希釈して使用した(PEG処理は行わない)。各上清は上記25mMトリスで10倍に希釈して使用した。各沈殿物(ppt)はPEG処理により得た沈渣を0.1%Tween20-0.1NNaOH0.1mLで溶かした後、25mMトリスで500倍に希釈して使用した。 (2) Examination by dot blot method Human-derived IgM antibody (standard) is diluted with 25 mM Tris (trishydroxymethylaminomethane dissolved in RO water to 25 mM) to prepare a concentration of 1.12 μg / mL. Used. NC was treated with DEAE column treated with 0.1 mL of human serum in the same manner as in Example 1 and concentrated to 0.1 mL with an ultrafiltration membrane, and diluted 500-fold with 25 mM Tris and used. Not performed). Each supernatant was diluted 10-fold with the 25 mM Tris and used. Each precipitate (ppt) was used by dissolving the precipitate obtained by PEG treatment with 0.1 mL of 0.1% Tween20-0.1N NaOH and diluting it 500 times with 25 mM Tris.
ヒト由来IgM抗体(標準品)は、25mMトリス(トリスヒドロキシメチルアミノメタンをRO水で溶かして25mMとしたもの)で希釈し、1.12μg/mL濃度を作成して使用した。NCは、ヒト血清0.1mLを実施例1と同様にDEAEカラム処理し、限外ろ過膜にて0.1mLまで濃縮したものを、25mMトリスで500倍に希釈して使用した(PEG処理は行わない)。各上清は上記25mMトリスで10倍に希釈して使用した。各沈殿物(ppt)はPEG処理により得た沈渣を0.1%Tween20-0.1NNaOH0.1mLで溶かした後、25mMトリスで500倍に希釈して使用した。 (2) Examination by dot blot method Human-derived IgM antibody (standard) is diluted with 25 mM Tris (trishydroxymethylaminomethane dissolved in RO water to 25 mM) to prepare a concentration of 1.12 μg / mL. Used. NC was treated with DEAE column treated with 0.1 mL of human serum in the same manner as in Example 1 and concentrated to 0.1 mL with an ultrafiltration membrane, and diluted 500-fold with 25 mM Tris and used. Not performed). Each supernatant was diluted 10-fold with the 25 mM Tris and used. Each precipitate (ppt) was used by dissolving the precipitate obtained by PEG treatment with 0.1 mL of 0.1% Tween20-0.1N NaOH and diluting it 500 times with 25 mM Tris.
PVDF膜は、メタノールに2~5分間浸して親水化した後、RO水に20~30分間浸し、RO水で湿らせた濾紙の上に置き、各検体5μLをアプライした。膜をトレイに移しブロッキング試薬で40分間ブロックした後、抗ヒトIgM‐HRP標識抗体(H鎖)と30分間反応させ、HRP標識抗体用発色基質(TMB発色基質)で染色した。その結果を図8に示すと共に、ImageJにより画像解析を行い、PEG無処理(NC)の場合を100としたときの回収率(%)を求めた。その結果を表1に示す。
The PVDF membrane was hydrophilized by immersing it in methanol for 2 to 5 minutes, then immersed in RO water for 20 to 30 minutes, placed on a filter paper moistened with RO water, and 5 μL of each specimen was applied. The membrane was transferred to a tray, blocked with a blocking reagent for 40 minutes, reacted with anti-human IgM-HRP labeled antibody (H chain) for 30 minutes, and stained with a chromogenic substrate for HRP labeled antibody (TMB chromogenic substrate). The results are shown in FIG. 8 and image analysis was performed using ImageJ, and the recovery rate (%) was calculated when the case of no PEG treatment (NC) was set to 100. The results are shown in Table 1.
[試験例5]SDS-PAGEイムノブロッティングによる含有率の検討
実施例1と同様にヒト血清100μLについてDEAEカラム処理とPEG処理を行い、得られたIgM抗体液、2.7mg/mLのヒト由来IgG抗体液、および560μg/mLのヒト由来IgM抗体液をSDS-PAGE用サンプル処理液(50mMTris-HCl緩衝液pH6.8(1%SDS,20%グリセリン,1.5% 2-メルカプトエタノール含有)で5倍に希釈し、100℃、4分間加熱後、ゲルに10μL(No.1~3)、または20μL(No.4~6)をアプライした。20mA、定流で65~75分間泳動した後、ゲルをHorizblot 2M AE-6687(・S)で250mA、35~40分間PVDF膜に転写した。 [Test Example 5] Examination of content by SDS-PAGE immunoblotting As in Example 1, 100 μL of human serum was subjected to DEAE column treatment and PEG treatment, and the resulting IgM antibody solution, 2.7 mg / mL human-derivedIgG 5 times the antibody solution and 560 μg / mL human-derived IgM antibody solution in SDS-PAGE sample treatment solution (50 mM Tris-HCl buffer pH 6.8 (containing 1% SDS, 20% glycerin, 1.5% 2-mercaptoethanol) After heating at 100 ° C. for 4 minutes, 10 μL (No. 1 to 3) or 20 μL (No. 4 to 6) was applied to the gel, and after electrophoresis at 20 mA, constant flow for 65 to 75 minutes, the gel was applied. Was transferred to a PVDF membrane with Horizblot 2M AE-6687 (· S) at 250 mA for 35-40 minutes.
実施例1と同様にヒト血清100μLについてDEAEカラム処理とPEG処理を行い、得られたIgM抗体液、2.7mg/mLのヒト由来IgG抗体液、および560μg/mLのヒト由来IgM抗体液をSDS-PAGE用サンプル処理液(50mMTris-HCl緩衝液pH6.8(1%SDS,20%グリセリン,1.5% 2-メルカプトエタノール含有)で5倍に希釈し、100℃、4分間加熱後、ゲルに10μL(No.1~3)、または20μL(No.4~6)をアプライした。20mA、定流で65~75分間泳動した後、ゲルをHorizblot 2M AE-6687(・S)で250mA、35~40分間PVDF膜に転写した。 [Test Example 5] Examination of content by SDS-PAGE immunoblotting As in Example 1, 100 μL of human serum was subjected to DEAE column treatment and PEG treatment, and the resulting IgM antibody solution, 2.7 mg / mL human-derived
転写膜を半分に切り、一枚は0.01%CBBで染色し、残り一枚はブロッキング試薬で40分間ブロックし、抗ヒトIgM-HRP標識抗体(H鎖)と30分間反応させた後、HRP標識抗体用発色基質(TMB発色基質)で染色した。その結果を図9に示す。
Cut the transfer membrane in half, one was stained with 0.01% CBB, the other was blocked with blocking reagent for 40 minutes, reacted with anti-human IgM-HRP labeled antibody (H chain) for 30 minutes, Stained with a chromogenic substrate for HRP-labeled antibody (TMB chromogenic substrate). The result is shown in FIG.
図9中、No.1はヒト由来IgG抗体の結果を、No.2はDEAE処理-PEG処理(本発明精製法)の結果を、No.3はヒト由来IgM抗体の結果を、No.4はDEAE処理-PEG処理(本発明精製法)の結果を、No.5はヒト由来IgM抗体の結果を、No.6はヒト由来IgG抗体の結果を、それぞれ示す。また、No.1~3は、ブロッキング試薬でブロックし、抗ヒトIgM‐HRP標識抗体(H鎖)と反応させた後、HRP標識抗体用発色基質(TMB発色基質)で染色し、No.4~6は0.01%CBBで染色した。
In FIG. No. 1 shows the results of human IgG antibody. No. 2 shows the results of DEAE treatment-PEG treatment (the purification method of the present invention). 3 shows the results of the human-derived IgM antibody. 4 shows the results of DEAE treatment-PEG treatment (the purification method of the present invention). No. 5 shows the results of human-derived IgM antibody. 6 shows the results of the human-derived IgG antibody. No. Nos. 1 to 3 were blocked with a blocking reagent, reacted with an anti-human IgM-HRP labeled antibody (H chain), and then stained with a chromogenic substrate for HRP labeled antibody (TMB chromogenic substrate). 4-6 were stained with 0.01% CBB.
No.4(本発明精製法)とNo.5(ヒト由来IgM抗体(標準品))について、ImageJにより画像解析を行った結果、IgM含有率はそれぞれ88%と87%であり、本発明精製法により得られたIgM抗体の含有率は、ヒト由来IgM抗体(標準品)と遜色がなかった。
No. 4 (invention purification method) and No. 4 5 (human-derived IgM antibody (standard product)), as a result of image analysis using ImageJ, the IgM content was 88% and 87%, respectively. The IgM antibody content obtained by the purification method of the present invention was There was no inferiority to human-derived IgM antibody (standard product).
No. 4 (invention purification method) and No. 4 5 (human-derived IgM antibody (standard product)), as a result of image analysis using ImageJ, the IgM content was 88% and 87%, respectively. The IgM antibody content obtained by the purification method of the present invention was There was no inferiority to human-derived IgM antibody (standard product).
本発明精製法は、ヒト血清中のIgM抗体を高純度に簡便に、また廉価に分離精製することができるものであるから、IgM抗体の生化学検査などにおいて有用である。
The purification method of the present invention is useful for biochemical examination of IgM antibodies and the like because IgM antibodies in human serum can be separated and purified with high purity simply and inexpensively.
The purification method of the present invention is useful for biochemical examination of IgM antibodies and the like because IgM antibodies in human serum can be separated and purified with high purity simply and inexpensively.
Claims (9)
- 陰イオン交換樹脂で固相抽出を行う工程、およびその後にポリエチレングリコールで処理する工程を含むことを特徴とする、ヒト血清中のIgM抗体の分離精製法。 A method for separating and purifying IgM antibodies in human serum, comprising a step of solid-phase extraction with an anion exchange resin and a step of subsequent treatment with polyethylene glycol.
- 前記陰イオン交換樹脂が、ジエチルアミノエチルが結合する陰イオン交換樹脂である、請求項1に記載の分離精製法。 The separation and purification method according to claim 1, wherein the anion exchange resin is an anion exchange resin to which diethylaminoethyl is bonded.
- 前記陰イオン交換樹脂がポリメタクリレート系高分子を基材とするものである、請求項1または2に記載の分離精製法。 The separation and purification method according to claim 1 or 2, wherein the anion exchange resin is based on a polymethacrylate polymer.
- 前記陰イオン交換樹脂がサイズ排除型のものである、請求項1~3のいずれか一項に記載の分離精製法。 The separation and purification method according to any one of claims 1 to 3, wherein the anion exchange resin is of a size exclusion type.
- 前記ポリエチレングリコールがポリエチレングリコール8000である、請求項1~4のいずれか一項に記載の分離精製法。 The separation and purification method according to any one of claims 1 to 4, wherein the polyethylene glycol is polyethylene glycol 8000.
- 前記ポリエチレングリコールの処理濃度が6~10w/v%の範囲内である、請求項1~5のいずれか一項に記載の分離精製法。 The separation and purification method according to any one of claims 1 to 5, wherein a treatment concentration of the polyethylene glycol is in a range of 6 to 10 w / v%.
- 前記固相抽出を行う工程の後、ポリエチレングリコールで処理する工程の前に、限外ろ過膜で濃縮する工程を更に含む、請求項1~6のいずれか一項に記載の分離精製法。 The separation and purification method according to any one of claims 1 to 6, further comprising a step of concentrating with an ultrafiltration membrane after the step of performing solid phase extraction and before the step of treating with polyethylene glycol.
- 請求項1~7のいずれか一項に記載の分離精製法により得られたIgM沈渣を、界面活性剤が含まれていてもよいアルカリ剤で溶解し、必要に応じて酸で中和することによって得られるIgM抗体液を分析に供することを特徴とする、IgM抗体の分析法。 The IgM sediment obtained by the separation and purification method according to any one of claims 1 to 7 is dissolved with an alkaline agent that may contain a surfactant, and neutralized with an acid as necessary. A method for analyzing IgM antibody, comprising subjecting the IgM antibody solution obtained by the above method to analysis.
- 前記アルカリ剤が、水酸化ナトリウムまたは水酸化カリウムである、請求項8に記載の分析法。
The analysis method according to claim 8, wherein the alkaline agent is sodium hydroxide or potassium hydroxide.
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EP4335869A1 (en) | 2022-09-07 | 2024-03-13 | Bio T Biyoteknoloji Cozumleri Ve Uretim Anonim | Resin and chromatography column that purifies antibodies with protease resistant small peptides |
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EP4335869A1 (en) | 2022-09-07 | 2024-03-13 | Bio T Biyoteknoloji Cozumleri Ve Uretim Anonim | Resin and chromatography column that purifies antibodies with protease resistant small peptides |
CN116606370A (en) * | 2023-07-21 | 2023-08-18 | 南京松天盛科生物科技有限公司 | Natural IgM purification method |
CN116606370B (en) * | 2023-07-21 | 2023-09-19 | 南京松天盛科生物科技有限公司 | Natural IgM purification method |
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