JPH07300499A - Protein separator and method for removing protein using the same protein separator - Google Patents

Protein separator and method for removing protein using the same protein separator

Info

Publication number
JPH07300499A
JPH07300499A JP6113381A JP11338194A JPH07300499A JP H07300499 A JPH07300499 A JP H07300499A JP 6113381 A JP6113381 A JP 6113381A JP 11338194 A JP11338194 A JP 11338194A JP H07300499 A JPH07300499 A JP H07300499A
Authority
JP
Japan
Prior art keywords
protein
adsorbent
separator
sample
luer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
JP6113381A
Other languages
Japanese (ja)
Inventor
Norio Okuyama
典生 奥山
Kazuo Seta
和夫 瀬田
Toshihiro Ishikawa
利弘 石川
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BAIOTEKU RES KK
Original Assignee
BAIOTEKU RES KK
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BAIOTEKU RES KK filed Critical BAIOTEKU RES KK
Priority to JP6113381A priority Critical patent/JPH07300499A/en
Publication of JPH07300499A publication Critical patent/JPH07300499A/en
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G01N2030/143—Preparation by elimination of some components selective absorption
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G01N2030/146—Preparation by elimination of some components using membranes

Abstract

PURPOSE:To provide the subject separator capable of simply. rapidly and precisely separating a protein in a small amount of a sample by installing a membranous filter at the bottom on the outside of a cartridge container having a connecting part of a Luer type structure. CONSTITUTION:This protein separator 1 is obtained by installing a membranous filter 5 at the bottom on the side of a liquid outlet 6 in the interior of a diskllke or a cylindrical cartridge container, made of a plastic and having a liquid inlet 3 and the liquid outlet 6 of a Luer type structure, filling an adsorbent 4 comprising granules having 2-400mum average particle diameter such as hydroxyapatlte on the filter 5 and filling a sealing filter 7 thereonto. Furthermore, this method for simply and rapidly removing a protein in a small amount of a sample is to take the sample containing the protein in an injection syringe, connect the injection syringe to a connecting part 3 of the Luer type structure in a cartridge container 2 of the protein separator l, pass the sample containing the protein through the interior of the cartridge 2, adsorb the protein on the adsorbent 4 and recover the solution from which the protein is removed from the outlet 6 of the Luer slip type outlet 6.

Description

【発明の詳細な説明】Detailed Description of the Invention

【０００１】[0001]

【産業上の利用分野】本発明は、ディスポーザブルな蛋
白質分離器及び該蛋白質分離器を用いて蛋白質含有試料
から蛋白質を除去する方法に関し、より詳細には、円盤
カートリッジ容器に粒状吸着剤を充填した蛋白分離器及
び試料中の少量蛋白質を吸着除去する方法に関するもの
である。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a disposable protein separator and a method for removing a protein from a protein-containing sample using the protein separator, and more particularly, a disc cartridge container filled with a granular adsorbent. The present invention relates to a protein separator and a method for adsorbing and removing a small amount of protein in a sample.

【０００２】[0002]

【従来の方法】環境、医薬、農薬、化粧品、バイオテク
ノロジーの技術分野において試料の分析の際には、試料
中に混入している蛋白質を前処理として除去する必要が
生じる。従来、このような蛋白質を除去するため飽和硫
酸アンモニウム溶液、過塩素酸、トリクロロ酢酸、硫酸
亜鉛等を用いて蛋白質を沈澱させる方法、遠心限外ろ過
法を用いる方法、又は膜によるろ過方法等が採用されて
いる。しかし、従来の方法では、遠心分離器又は吸引用
のアスピレータ等の設備を必要とするなど装置面の問題
の他に、試料中に少量含有する蛋白質を効率よく確実
に、しかも簡便な操作で除去するには難点を包蔵するも
のであり、簡便な操作で少量の蛋白質を効率よく除去で
きる技術が環境、医療、農薬、及び分析分野等で要望さ
れていた。2. Description of the Related Art When analyzing a sample in the technical fields of environment, medicine, agricultural chemicals, cosmetics and biotechnology, it is necessary to remove proteins contaminated in the sample as a pretreatment. Conventionally, in order to remove such proteins, a method of precipitating a protein using a saturated ammonium sulfate solution, perchloric acid, trichloroacetic acid, zinc sulfate, etc., a method using a centrifugal ultrafiltration method, or a membrane filtration method is adopted. Has been done. However, in the conventional method, in addition to equipment problems such as the need for equipment such as a centrifuge or an aspirator for suction, the protein contained in a small amount in the sample can be removed efficiently and reliably by a simple operation. However, a technique capable of efficiently removing a small amount of protein by a simple operation has been demanded in the fields of environment, medicine, pesticides, analysis fields and the like.

【０００３】[0003]

【発明が解決しようとする課題】従って、本発明は、か
かる要望に鑑みてなされたものであり、その目的は、遠
心分離器及び吸引アスピレータ等の装置を必要とするこ
となく、簡便な操作により試料中の蛋白質を効率よく除
去することが可能な蛋白質分離器及び該分離器を用いる
蛋白質の除去方法を提供することにある。SUMMARY OF THE INVENTION Therefore, the present invention has been made in view of such a demand, and an object thereof is to provide a simple operation without requiring a device such as a centrifuge and a suction aspirator. It is an object of the present invention to provide a protein separator capable of efficiently removing proteins in a sample and a method for removing proteins using the separator.

【０００４】[0004]

【課題を解決するための手段】上記目的を達成するた
め、本発明者らは、種々検討した結果、試料注入用注射
筒が接続可能なルアー型構造の液出入口を有する円盤又
は円筒状カートリッジ容器内に吸着剤を充填させてなる
除蛋白用分離器を完成した。即ち、本発明はルアー構造
の液出入口を有する円盤カートリッジ容器内底部に膜フ
ィルターを敷設し、該膜フィルター上に平均粒径２μｍ
−４００μｍの粒状吸着剤を充填したことを特徴とする
蛋白質分離器に関する。好ましい実施の態様として
（２）試料注入用注射筒が接続可能なルアー型構造の液
出入口を有する円盤カートリッジ内底部に膜フィルター
を敷設し、該フィルター上に平均粒径２μｍ−４００μ
ｍの粒状吸着剤又は破砕状吸着剤を充填し、入口に必要
ならば封止用膜フィルターを設置してなる上記第（１）
項記載の蛋白質分離器、（３）粒状吸着剤が、水酸化ア
パタイト、フッ素アパタイト、シリカ、カーボン、活性
炭、アニオン系イオン交換樹脂、カチオン系交換樹脂の
群から選択される少なくとも一種である上記第（１）項
又は第（２）項の蛋白質分離器、（４）吸着剤の充填部
の内径が５ｍｍ−１００ｍｍであり、厚さが１ｍｍ−５
０ｍｍの範囲にある上記第（１）項又は第（２）項記載
の蛋白質分離器、（５）円盤状又は円筒状カートリッジ
が、プラスチック製である上記第（１）項ないし第
（４）項のいずれかの項記載の蛋白質分離器等を挙げる
ことが出来る。また、本発明は、（ａ）蛋白質含有試料
を注射筒に採り、該注射筒を吸着剤を充填した円盤状又
は円筒状カートリッジのルアー型構造接続部に接続し、
該試料を該カートリッジ内に通液し蛋白質を吸着剤に吸
着させ蛋白質の除去された溶液を回収することを特徴と
する蛋白質分離器による蛋白質の除去方法に関する好ま
しい実施の態様として（ｂ）吸着剤の基材が、水酸化ア
パタイト、フッ素アパタイト、シリカ、カーボン、活性
炭、アニオン系イオン交換樹脂、カチオン系交換樹脂の
群から選択される少なくとも一種である上記第（ａ）項
の蛋白質の除去方法、（ｃ）上記（ａ）項に記載の蛋白
質の除去後に、カートリッジ容器内に溶離液を通液し、
該容器内の蛋白質の吸着されている吸着剤と接触させ、
該吸着剤から蛋白質を溶離させる蛋白質の除去方法等が
挙げられる。In order to achieve the above object, the inventors of the present invention have made various studies and as a result, have a disc or cylindrical cartridge container having a liquid inlet / outlet of a luer type structure to which a sample injection syringe can be connected. A deproteinizing separator having an adsorbent filled therein was completed. That is, according to the present invention, a membrane filter is laid on the bottom of a disc cartridge container having a luer structure liquid inlet / outlet, and an average particle diameter of 2 μm is provided on the membrane filter.
The present invention relates to a protein separator characterized by being filled with a particulate adsorbent of -400 μm. As a preferred embodiment, (2) a membrane filter is laid on the inner bottom of a disk cartridge having a liquid inlet / outlet of a luer type structure to which a syringe for sample injection can be connected, and an average particle diameter of 2 μm-400 μ is provided on the filter.
The above-mentioned (1) in which the granular adsorbent or the crushed adsorbent of m is filled and a sealing membrane filter is installed at the inlet if necessary.
Item 3. The protein separator according to paragraph (3), wherein the granular adsorbent is at least one selected from the group consisting of hydroxyapatite, fluoroapatite, silica, carbon, activated carbon, anion-based ion exchange resin, and cation-based exchange resin. (1) or the protein separator according to (2), (4) the adsorbent filled portion has an inner diameter of 5 mm-100 mm and a thickness of 1 mm-5.
Item (1) to (4) above, wherein the protein separator according to item (1) or (2) above, and (5) the disk-shaped or cylindrical cartridge in the range of 0 mm, are made of plastic. The protein separator and the like described in any one of 1. The present invention also provides (a) a protein-containing sample in an injection cylinder, which is connected to a luer-type structure connecting portion of a disc-shaped or cylindrical cartridge filled with an adsorbent,
(B) an adsorbent as a preferred embodiment of a method for removing a protein by a protein separator, characterized in that the sample is passed through the cartridge to adsorb the protein on the adsorbent to recover a solution from which the protein has been removed. The base material of (a) is at least one selected from the group consisting of hydroxyapatite, fluoroapatite, silica, carbon, activated carbon, anion-based ion exchange resins, and cation-based exchange resins, and the method for removing proteins according to item (a) above, (C) After removing the protein described in (a) above, an eluent is passed through the cartridge container,
Contacting the adsorbent to which the protein in the container is adsorbed,
Examples thereof include a method for removing a protein by eluting the protein from the adsorbent.

【０００５】以下に、本発明について更に詳細に説明す
る。本発明の特徴とするところは、第一に、本発明に係
わる蛋白質分離器は、ディスポーザブルな容器であっ
て、注射筒の接続可能なルアー型の接続部を有する円盤
状又は円筒状カートリッジ容器であること、第二に、吸
着剤を用いることにより蛋白質の除去を行う方法である
ことにあり、かかる着想に基いて本発明を完成したもの
である。The present invention will be described in more detail below. The feature of the present invention is, firstly, that the protein separator according to the present invention is a disposable container, which is a disc-shaped or cylindrical cartridge container having a connectable luer-type connecting portion of a syringe. Secondly, it is a method of removing proteins by using an adsorbent, and the present invention has been completed based on such an idea.

【０００６】本発明に係わる円盤状又は円筒状カートリ
ッジ容器は、図１，図２、図３及び図４に例示する如き
形状及び構造をしたものであり、液の出入口がルアー型
構造であり、試料注入用の注射筒の接続を可能としたも
のである。図１及び図３は、入口がメスのルアー型であ
り、出口がルアースリップであることを示している。カ
ートリッジ容器の大きさは、特に限定されるものではな
いが、吸着剤の充填部の内径が５ｍｍ−１００ｍｍであ
り、厚さが１ｍｍ−５０ｍｍであるものが使用されてい
る。The disc-shaped or cylindrical cartridge container according to the present invention has a shape and structure as illustrated in FIGS. 1, 2, 3 and 4, and the liquid inlet / outlet has a luer structure. It is possible to connect an injection cylinder for sample injection. 1 and 3 show that the inlet is a female luer type and the outlet is a luer slip. The size of the cartridge container is not particularly limited, but a container having an adsorbent filled portion having an inner diameter of 5 mm-100 mm and a thickness of 1 mm-50 mm is used.

【０００７】カートリッジ容器は、ディスポーザブルな
ものであることから、生分解性の良いプラスティック等
の素材が好適である。カートリッジ容器内部の吸着剤の
充填部底部に敷設する膜フィルターの孔径は、約０．１
μｍ−３０μｍであることが好ましく吸着剤の流失を防
止すると共に、大きな破片、例えば細胞の破片等の除去
の作用も果たす。吸着剤と膜フィルターを併用すること
により、蛋白質除去効果が相乗的に増加することが見い
だされた。膜フィルターは、水溶性蛋白質を除去すると
きは親水性の材料を使用し、疎水性蛋白質を除去すると
きには疎水性材料を使用することが好ましい。好ましい
材質は、水系、イオン系についてはオレフィン系ポリマ
ー、ポリフッ化ビニリデン、セルロースアセテート等で
あり、非水系についてはポリプロピレン又はポリテトラ
フルオロエチレン等が好ましい。Since the cartridge container is disposable, a material such as plastic having good biodegradability is suitable. The pore size of the membrane filter installed at the bottom of the adsorbent filling section inside the cartridge container is about 0.1.
The thickness is preferably 30 μm to 30 μm to prevent the adsorbent from being washed away and also to remove large debris such as cell debris. It has been found that the combined use of the adsorbent and the membrane filter synergistically increases the protein removal effect. For the membrane filter, it is preferable to use a hydrophilic material when removing the water-soluble protein and a hydrophobic material when removing the hydrophobic protein. Preferred materials are olefinic polymers, polyvinylidene fluoride, cellulose acetate and the like for water-based and ionic-based materials, and polypropylene, polytetrafluoroethylene and the like for non-aqueous-based materials.

【０００８】吸着剤は蛋白質を吸着するものであれば、
制限されるものではないが、水酸化アパタイト、フッ素
アパタイト、Ｃ４シリカ、Ｃ８シリカ、Ｃ１８シリカ、
Ｃ４ポリマー、Ｃ８ポリマー、Ｃ１８ポリマー、グラフ
ァイト・カーボン、活性炭、アニオン系イオン交換樹
脂、カチオン系イオン交換樹脂を使用することができ
る。吸着剤は平均粒径２μｍ−３００μｍの顆粒又は破
砕状のものが用いられ、好ましい平均粒径は２μｍ−３
００μｍである。特に好ましい平均粒径は１０μｍ−１
００μｍである。水酸化アパタイトは、化学式Ｃａ１０
（ＰＯ４）６・（ＯＨ）２で表され、フッ素アパタイト
は、化学式Ｃａ１０（ＰＯ４）６・Ｆ２で表される。カ
ートリッジ容器内への吸着剤の充填率（容器内の全容積
に対し、吸着剤の占める容積）は、５０％以上であり、
好ましい充填率は、７０％以上であり、特に好ましい容
積率は９０％以上である。充填部の上方入口には、膜フ
ィルター又は石英綿等を設置し、吸着剤の散逸を防止す
る。If the adsorbent adsorbs a protein,
Hydroxyapatite, fluoroapatite, C 4 silica, C 8 silica, C 18 silica, but not limited to
C 4 polymer, C 8 polymer, C 18 polymer, graphite carbon, activated carbon, anionic ion exchange resins, can be used a cationic ion exchange resin. As the adsorbent, granules or crushed particles having an average particle size of 2 μm to 300 μm are used, and a preferable average particle size is 2 μm-3
It is 00 μm. Particularly preferred average particle size is 10 μm −1
It is 00 μm. Hydroxyapatite has the chemical formula Ca 10
It is represented by (PO 4 ) 6 · (OH) 2 , and fluoroapatite is represented by the chemical formula Ca 10 (PO 4 ) 6 · F 2 . The filling rate of the adsorbent in the cartridge container (the volume occupied by the adsorbent with respect to the total volume in the container) is 50% or more,
A preferable filling rate is 70% or more, and a particularly preferable volume rate is 90% or more. A membrane filter or quartz wool is installed at the upper entrance of the packing section to prevent the adsorbent from dissipating.

【０００９】図１は、本発明の円盤型の蛋白質分離器
（１）の図２のＡ−Ａ’線断面構造である。円盤（２）
の上部に注射筒が接続できるルアーロック型の入口
（３）があり。円盤内には粒径２μｍ−４００μｍの吸
着剤（４）が充填されている。円盤の底部には、吸着剤
が流出しないように０．２μｍ−２μｍの孔径の膜フィ
ルター（５）を敷設してある。円盤の下部出口（６）は
ルアースリップ型の接続部があり、注射針を接続でき
る。除蛋白処理は入口（３）から注射筒の少量試料を注
入し、円盤内部の吸着剤に試料を流動接触させ、出口か
ら除蛋白処理された試料が溶出される。図１中７は封止
用フィルターである。上記円盤２は上半部及び下半部を
融合接着その他任意の方法で一体化することにより製作
することができる。図２は、円盤型蛋白質分離器の上部
からみた平面図を示す。図３は、図４のＢ−Ｂ’線断面
図であり、本発明の円盤型の蛋白質分離器の蛋白質の吸
着を増やす場合に有効な形状である。基本的には円盤型
と変わらないが、吸着剤が円盤型よりより多く充填でき
るので円筒状試料の除蛋白処理量を増やすことができ
る。図中番号は、図１と共通である。図４は、円筒型蛋
白質分離器の上部からみた平面図である。FIG. 1 is a sectional view taken along the line AA ′ in FIG. 2 of the disc-shaped protein separator (1) of the present invention. Disk (2)
There is a luer lock type inlet (3) at the top of which a syringe can be connected. The disk is filled with an adsorbent (4) having a particle size of 2 μm to 400 μm. A membrane filter (5) having a pore size of 0.2 μm to 2 μm is laid on the bottom of the disk so that the adsorbent does not flow out. The lower outlet (6) of the disc has a luer slip type connection to which a needle can be connected. In the deproteinization treatment, a small amount of sample in a syringe is injected from the inlet (3), the sample is brought into fluid contact with the adsorbent inside the disc, and the deproteinized sample is eluted from the outlet. Reference numeral 7 in FIG. 1 denotes a sealing filter. The disc 2 can be manufactured by integrating the upper half portion and the lower half portion by fusion bonding or any other method. FIG. 2 shows a plan view of the disc type protein separator seen from above. FIG. 3 is a cross-sectional view taken along the line BB ′ of FIG. 4, which is a shape effective for increasing the adsorption of proteins in the disc-shaped protein separator of the present invention. Basically, it is the same as the disc type, but since the adsorbent can be filled more than the disc type, the deproteinization amount of the cylindrical sample can be increased. The numbers in the figure are the same as those in FIG. FIG. 4 is a plan view of the cylindrical protein separator seen from above.

【００１０】実施例１ 図１に示す図２のＡ−Ａ’線断面構造を示すプラスティ
ック製カートリッジ容器の内部の内径２５ｍｍの底部に
孔径０．４５μｍの膜フィルターを敷設し、その上に吸
着剤として平均粒径３０μｍの水酸化アパタイト顆粒を
充填、封入した蛋白質分離器を作製した。吸収剤の充填
量は、０．２５ｍｇであり、充填率９２％であった。こ
の蛋白質分離器の性能を評価するため、酸性蛋白質とし
て牛血清アルブミン１０ｍｇ／ｍｌ、リゾチーム１．２
ｍｇ／ｍｌ及び、塩基性蛋白質のチトクロムＣ１．９ｍ
ｇ／ｍｌの混合水溶液を試料として調製した。図５に除
蛋白処理をする前の混合水溶液を水酸化アパタイト・カ
ラムによる高速液体クロマトグラフィーで分析した結果
を示す、図５のクロマトグラムには牛血清アルブミン、
リゾチーム及びチトクロムＣがピークとして測定結果が
示めされている。高速液体クロマトグラフィーの分析条
件として流量１ｍｌ／ｍｉｎ、紫外線検出波長２８０ｎ
ｍを採用した。上記の試料を注射筒で吸引し、その注射
筒を上記の蛋白質にルアー構造部分で接続し通液した。
溶出液を採取し、分析したところ表１及び図６に示す結
果を得た。図６は、溶出液を高速液体クロマトグラフィ
ーで分析して得られたクロマトグラムであり、分析には
流量１ｍｌ／ｍｉｎ，紫外線検出波長２８０ｎｍの条件
を採用し、検出器の感度を除蛋白処理前の試料の分析の
際の４倍にあげた。図６のクロマトグラムには、図５の
クロマトグラムに存在した三種の蛋白質のピークが消滅
したことからも明らかな如く蛋白質が吸着剤に吸着し試
料から全量除去された。次に、蛋白質を吸着させた該分
離器に、０．４モルのリン酸ナトリウム溶液を注射筒を
用いて通液し、蛋白質を溶出した。溶出液を高速液体ク
ロマトグラフィーで分析した結果を図７に示す。牛血清
アルブミン、リゾチーム、チトロクロムＣのピークが現
われ、蛋白質が吸着剤から溶出し、回収できることが立
証された。該カートリッジ内のアパタイト顆粒吸着剤の
牛血清アルブミンの吸着量（質荷容量）は、０．６２ｍ
ｇであり、チトロクロムＣの吸着量は２．５ｍｇであっ
た。Example 1 A membrane filter having a pore diameter of 0.45 μm was laid on the bottom of an inner diameter of 25 mm inside a plastic cartridge container showing the sectional structure taken along the line AA ′ of FIG. As the above, a protein separator was prepared in which hydroxyapatite granules having an average particle size of 30 μm were filled and enclosed. The filling amount of the absorbent was 0.25 mg, and the filling rate was 92%. To evaluate the performance of this protein separator, bovine serum albumin 10 mg / ml and lysozyme 1.2 were used as acidic proteins.
mg / ml and basic protein cytochrome C 1.9 m
A mixed aqueous solution of g / ml was prepared as a sample. Fig. 5 shows the results of high performance liquid chromatography analysis using a hydroxyapatite column on the mixed aqueous solution before deproteinization. The chromatogram in Fig. 5 shows bovine serum albumin,
The measurement results are shown as peaks for lysozyme and cytochrome C. As analysis conditions for high performance liquid chromatography, flow rate 1 ml / min, UV detection wavelength 280 n
adopted m. The above-mentioned sample was sucked with a syringe, and the syringe was connected to the above-mentioned protein at the luer structure portion and passed.
When the eluate was collected and analyzed, the results shown in Table 1 and FIG. 6 were obtained. FIG. 6 is a chromatogram obtained by analyzing the eluate by high performance liquid chromatography. The conditions of a flow rate of 1 ml / min and an ultraviolet ray detection wavelength of 280 nm were adopted for the analysis, and the sensitivity of the detector was set before deproteinization. This is four times higher than that of the sample analyzed. In the chromatogram of FIG. 6, the protein was adsorbed to the adsorbent and the entire amount was removed from the sample, as is clear from the disappearance of the peaks of the three proteins present in the chromatogram of FIG. Next, 0.4 mol of sodium phosphate solution was passed through the separator having adsorbed the protein using a syringe to elute the protein. The result of analyzing the eluate by high performance liquid chromatography is shown in FIG. The peaks of bovine serum albumin, lysozyme, and cytotrochrome C appeared, which proved that the protein was eluted from the adsorbent and could be recovered. The adsorption amount (mass capacity) of bovine serum albumin on the apatite granule adsorbent in the cartridge is 0.62 m.
and the amount of adsorbed Titrochrome C was 2.5 mg.

【００１１】実施例２−６ 平均粒径５μｍ、１０μｍ、１００μｍ、２００μｍ及
び３５０μｍの水酸化アパタイト顆粒をカートリッジに
表１に示す充填率で充填したこと以外実施例１と同一構
成の蛋白質分離器を調製した。これらの蛋白質分離器を
用いて実施例１と同一の条件及び操作により蛋白質の除
去効果を評価した。結果を表１に示す。Example 2-6 A protein separator having the same structure as in Example 1 was prepared except that hydroxyapatite granules having an average particle size of 5 μm, 10 μm, 100 μm, 200 μm and 350 μm were filled in a cartridge at the filling rate shown in Table 1. Prepared. Using these protein separators, the effect of removing proteins was evaluated under the same conditions and operations as in Example 1. The results are shown in Table 1.

【００１２】比較例１ 平均粒径４００μｍの水酸化アパタイト顆粒を用い、表
１に示す構成の蛋白質分離器を作製した。実施例１と同
様にして蛋白質の除去効果を評価し、結果を表１に示
す。Comparative Example 1 A protein separator having the constitution shown in Table 1 was prepared using hydroxyapatite granules having an average particle size of 400 μm. The protein removing effect was evaluated in the same manner as in Example 1, and the results are shown in Table 1.

【００１３】実施例７ 図１に断面構造を示すプラスティック製カートリジ容器
の内部の内径２５ｍｍの底部に孔径０．２μｍの膜フィ
ルターを敷設し、その上に吸着剤として平均粒径７０μ
ｍのグラファイトカーボン顆粒を充填封入した蛋白質分
離器を作製した。吸着剤の充填量は０．０３１ｍｇであ
り、充填率は８３％であった。この蛋白質分離器の性能
を評価するため、ヒト血清アルブミン４ｍｇ／ｍｌ、リ
ボヌクレアーゼＡ１ｍｇ／ｍｌ及びα―キモトリプシン
１ｍｇ／ｍｌの疎水性蛋白質を含有する混合溶液を調製
した。図８は、混合溶液高速液体クロマトグラフィーで
分析した結果である。該図には上記三種の蛋白質の存在
を示すピークが現われている。この混合溶液を試料とし
て注射筒で吸引し、上記蛋白質分離器のルアー構造部に
その注射筒を接続し通液した。溶出液を採取し分析した
ところ、表２及び図９に示す結果を得た。図９によれば
図８で蛋白質の存在を示すクロマトグラムのピークが消
滅しており、蛋白質が具体的に全量除去されたことが示
されている。更に、除蛋白処理前の試料に存在した有機
物を除去することが出来た。Example 7 A membrane filter having a pore diameter of 0.2 μm is laid at the bottom of an inner diameter of 25 mm inside a plastic cartridge container having a sectional structure shown in FIG. 1, and an average particle diameter of 70 μm is used as an adsorbent on the membrane filter.
A protein separator was prepared in which m graphite carbon granules were filled and enclosed. The filling amount of the adsorbent was 0.031 mg, and the filling rate was 83%. In order to evaluate the performance of this protein separator, a mixed solution containing 4 mg / ml of human serum albumin, 1 mg / ml of ribonuclease A and 1 mg / ml of α-chymotrypsin hydrophobic protein was prepared. FIG. 8 shows the results of analysis by mixed solution high performance liquid chromatography. In the figure, peaks showing the existence of the above-mentioned three kinds of proteins appear. The mixed solution was used as a sample and sucked by a syringe, and the syringe was connected to the luer structure of the protein separator to pass the solution. When the eluate was collected and analyzed, the results shown in Table 2 and FIG. 9 were obtained. According to FIG. 9, the peak of the chromatogram showing the presence of the protein disappears in FIG. 8, which indicates that the entire amount of the protein was specifically removed. Furthermore, it was possible to remove the organic substances existing in the sample before the deproteinization treatment.

【００１４】実施例８−１２及び比較例１ 表２に示す如く、平均粒径の異なるグラファイトカーボ
ン顆粒を用い同表に示す充填率で充填したこと以外すべ
て実施例１と同様にして蛋白質分離器を作製し、実施例
７と同一の条件及び操作により、蛋白質の除去効果を評
価し、結果を表２に示す。Examples 8-12 and Comparative Example 1 As shown in Table 2, the same procedure as in Example 1 was repeated except that graphite carbon granules having different average particle diameters were used and packed at the packing ratio shown in the table. Was prepared, and the protein removal effect was evaluated under the same conditions and operations as in Example 7, and the results are shown in Table 2.

【表１】 [Table 1]

【表２】 [Table 2]

【００１５】[0015]

【発明の効果】本発明による蛋白質分離器は、吸着剤を
充填してなる特異な構成を有するものであり、簡便な操
作で少量の蛋白質の除去が可能であり、少量試料の分析
における前処理操作として有用である。EFFECT OF THE INVENTION The protein separator according to the present invention has a unique structure filled with an adsorbent, can remove a small amount of protein by a simple operation, and can be used as a pretreatment for analysis of a small sample. It is useful as an operation.

【図面の簡単な説明】[Brief description of drawings]

【図１】本発明の一実施例に係る蛋白質分離器の図２の
Ａ−Ａ’線断面図である。1 is a cross-sectional view of the protein separator according to the embodiment of the present invention taken along the line AA ′ in FIG.

【図２】本発明の一実施例に係る蛋白質分離器の平面図
である。FIG. 2 is a plan view of a protein separator according to an embodiment of the present invention.

【図３】本発明の一実施例に係る蛋白質分離器の図４の
Ｂ−Ｂ’線断面図である。FIG. 3 is a sectional view taken along line BB ′ of FIG. 4 of the protein separator according to the embodiment of the present invention.

【図４】本発明の一実施例に係る蛋白質分離器の平面図
である。FIG. 4 is a plan view of a protein separator according to an embodiment of the present invention.

【図５】本発明の一実施例に係る蛋白質除去方法に用い
た試料の高速液体クロマトグラフィーによる分析結果を
示す図である。FIG. 5 is a diagram showing analysis results by high performance liquid chromatography of a sample used in the protein removal method according to one example of the present invention.

【図６】本発明の一実施例に係る蛋白質除去方法により
蛋白質の除去された試料の高速液体クロマトグラフィー
による分析結果を示す図である。FIG. 6 is a diagram showing the results of high-performance liquid chromatography analysis of a sample from which proteins have been removed by the protein removal method according to one embodiment of the present invention.

【図７】本発明の一実施例に係る吸着剤から溶出回収し
た蛋白質の高速液体クロマトグラフィーによる分析の結
果を示す図である。FIG. 7 is a diagram showing the results of analysis by high performance liquid chromatography of proteins eluted and collected from the adsorbent according to one example of the present invention.

【図８】本発明の一実施例に係る蛋白質除去方法に用い
た試料の高速液体クロマトグラフィーによる分析結果を
示す図である。FIG. 8 is a diagram showing analysis results by high performance liquid chromatography of a sample used in the protein removal method according to one example of the present invention.

【図９】本発明の一実施例に係る蛋白質除去方法を用い
て蛋白質を除去して得られた試料のクロマトグラムを示
す図である。FIG. 9 is a diagram showing a chromatogram of a sample obtained by removing a protein using the protein removing method according to one example of the present invention.

【符号の説明】[Explanation of symbols]

１ ・・・・蛋白質分離器 ２ ・・・・円盤 ２’・・・・円筒 ３ ・・・・ルアーロック型入口 ４ ・・・・吸着剤 ５ ・・・・膜フィルター ６ ・・・・ルアースリップ型出口 ７ ・・・・封止用フィルター 1 ・ ・ ・ ・ Protein Separator 2 ・ ・ ・ ・ Disc 2 '・ ・ ・ ・ Cylinder 3 ・ ・ ・ ・ Lure lock type inlet 4 ・ ・ ・ ・ Adsorbent 5 ・ ・ ・ ・ Membrane filter 6 ・ ・ ・ ・ Lure Slip type outlet 7 ... Sealing filter

───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｃ０７Ｋ 1/22 Ｇ０１Ｎ 30/14 Ａ 30/60 Ａ 33/48 Ａ ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 6 Identification code Internal reference number FI Technical display location C07K 1/22 G01N 30/14 A 30/60 A 33/48 A

Claims (2)

【特許請求の範囲】[Claims] 【請求項１】 ルアー型構造の接続部を有する円盤状又
は円筒状カートリッジ容器内部の出口側底部に膜フィル
ターを敷設し、該フィルター上に平均粒径２μｍ−４０
０μｍの吸着剤を充填したことを特徴とする蛋白質分離
器。1. A membrane filter is laid on the bottom of the disc-shaped or cylindrical cartridge container having a luer-type structure on the outlet side, and an average particle diameter of 2 μm-40 is provided on the filter.
A protein separator characterized by being filled with an adsorbent of 0 μm. 【請求項２】 蛋白質含有試料を注射筒に採り、該注射
筒を吸着剤を充填した円盤状又は円筒状カートリッジ容
器のルアー型構造接続部に接続し、該蛋白質含有試料を
該カートリジ内に通液し、該蛋白質を吸着剤に吸着さ
せ、蛋白質の除去された溶液を回収することを特徴とす
る蛋白質の蛋白質分離器による除去方法。2. A protein-containing sample is taken in an injection cylinder, and the injection cylinder is connected to a luer-type structural connection part of a disk-shaped or cylindrical cartridge container filled with an adsorbent, and the protein-containing sample is passed through the cartridge. A method for removing a protein by a protein separator, which comprises lysing, adsorbing the protein on an adsorbent, and collecting a solution from which the protein has been removed.
JP6113381A 1994-05-02 1994-05-02 Protein separator and method for removing protein using the same protein separator Pending JPH07300499A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP6113381A JPH07300499A (en) 1994-05-02 1994-05-02 Protein separator and method for removing protein using the same protein separator

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP6113381A JPH07300499A (en) 1994-05-02 1994-05-02 Protein separator and method for removing protein using the same protein separator

Publications (1)

Publication Number Publication Date
JPH07300499A true JPH07300499A (en) 1995-11-14

Family

ID=14610865

Family Applications (1)

Application Number Title Priority Date Filing Date
JP6113381A Pending JPH07300499A (en) 1994-05-02 1994-05-02 Protein separator and method for removing protein using the same protein separator

Country Status (1)

Country Link
JP (1) JPH07300499A (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006153488A (en) * 2004-11-25 2006-06-15 Canon Chemicals Inc Optical measurement method
JP2011036780A (en) * 2009-08-10 2011-02-24 Asahi Kasei Chemicals Corp Hollow fiber porous membrane module having high utilization efficiency of hollow fiber porous membrane
KR101026946B1 (en) * 2007-06-29 2011-04-04 도시바 메디칼 시스템즈 코포레이션 Micro chemical analysis apparatus, method for measuring thereof and sampling apparatus for obtaining target sample

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006153488A (en) * 2004-11-25 2006-06-15 Canon Chemicals Inc Optical measurement method
KR101026946B1 (en) * 2007-06-29 2011-04-04 도시바 메디칼 시스템즈 코포레이션 Micro chemical analysis apparatus, method for measuring thereof and sampling apparatus for obtaining target sample
EP2439538A1 (en) * 2007-06-29 2012-04-11 Kabushiki Kaisha Toshiba Sample collecting tool
JP2011036780A (en) * 2009-08-10 2011-02-24 Asahi Kasei Chemicals Corp Hollow fiber porous membrane module having high utilization efficiency of hollow fiber porous membrane

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