JPH11287802A - Surface protective agent - Google Patents
Surface protective agentInfo
- Publication number
- JPH11287802A JPH11287802A JP10091153A JP9115398A JPH11287802A JP H11287802 A JPH11287802 A JP H11287802A JP 10091153 A JP10091153 A JP 10091153A JP 9115398 A JP9115398 A JP 9115398A JP H11287802 A JPH11287802 A JP H11287802A
- Authority
- JP
- Japan
- Prior art keywords
- water
- polyethylene glycol
- protective agent
- surface protective
- solid phase
- 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
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- Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、免疫測定の際の固
相の表面の保護（ブロッキング）に用いる表面保護剤に
関する。TECHNICAL FIELD The present invention relates to a surface protective agent used for protecting (blocking) the surface of a solid phase during an immunoassay.
【０００２】[0002]
【従来の技術】抗原抗体反応を利用した免疫測定ではポ
リスチレンを初めとするプラスチック類からなるプレー
ト、ビーズ、ラテックス等の微粒子あるいは被覆磁性粒
子等に抗原あるいは抗体をつけた固相化抗原あるいは固
相化抗体が用いられている。しかしながら、抗原あるい
は抗体がついていない固相表面は測定の際、試料中の蛋
白質を非特異的に吸着してしまうため、正確に抗原抗体
反応を反映した免疫測定ができない。そのため固相表面
のブロッキング剤として牛血清アルブミン（ＢＳＡ）や
ゼラチン、カゼイン等の天然蛋白質が用いられている。2. Description of the Related Art In an immunoassay utilizing an antigen-antibody reaction, a solid-phased antigen or solid phase obtained by attaching an antigen or an antibody to fine particles such as plates, beads, latex, etc., or coated magnetic particles made of plastics such as polystyrene. Antibodies have been used. However, a solid phase surface without an antigen or an antibody nonspecifically adsorbs a protein in a sample during measurement, so that an immunoassay accurately reflecting an antigen-antibody reaction cannot be performed. Therefore, natural proteins such as bovine serum albumin (BSA), gelatin, and casein have been used as blocking agents on the surface of the solid phase.
【０００３】本発明に用いられる側鎖にポリエチレング
リコールを持つ水溶性共重合体、特に分子内にポリエチ
レングリコール鎖を持つビニルモノマーと該モノマーと
共重合可能な水不溶性モノマーとの共重合で得られる水
溶性共重合体は従来から増粘剤、分散剤、接着剤あるい
は生理活性ペプチドの安定剤等として知られたものであ
るが、本発明に関わる固相表面への吸着保護・ブロッキ
ングの可能性を示唆するものでは無い。[0003] The water-soluble copolymer having polyethylene glycol in the side chain used in the present invention, particularly obtained by copolymerization of a vinyl monomer having a polyethylene glycol chain in the molecule and a water-insoluble monomer copolymerizable with the monomer. The water-soluble copolymer is conventionally known as a thickener, a dispersant, an adhesive, or a stabilizer for a physiologically active peptide. It does not suggest.
【０００４】[0004]
【発明が解決しようとする課題】免疫測定の際の固相表
面の保護にＢＳＡ等の天然蛋白質を用いると、被検試料
によってはその保護層と反応する場合があり、本来の免
疫反応以外の反応によるシグナルを検出してしまう。そ
の結果陰性となるべき試料が陽性となり、判定・診断を
誤らせることがある。固相表面保護に蛋白質以外の疎水
性と親水性を合わせ持つ従来の界面活性剤を用いた場合
には、固相化した抗原あるいは抗体の蛋白質を変性ある
いは脱離してしまうため、使用量が限定され完全に固相
表面を保護するまでの量を使用できない。したがって、
固相表面に良く吸着し、免疫反応を妨害することなく固
相表面を保護する材料が望まれている。When a natural protein such as BSA is used to protect the solid phase surface during an immunoassay, depending on the test sample, it may react with the protective layer. The signal from the reaction is detected. As a result, a sample that should be negative becomes positive, which may erroneously determine or diagnose. When a conventional surfactant that combines hydrophobicity and hydrophilicity other than protein is used to protect the solid phase surface, the amount of the antigen or antibody immobilized on the solid phase is denatured or eliminated, so the amount used is limited. It cannot be used until the solid phase surface is completely protected. Therefore,
There is a need for a material that adsorbs well to the solid surface and protects the solid surface without interfering with the immune response.
【０００５】[0005]
【課題を解決するための手段】本発明者は、これら課題
を解決するため、疎水性と親水性を合わせ持つ化合物と
してポリエチレングリコール鎖を側鎖に持ち、疎水性固
相の表面に吸着させるための疎水性の官能基を主鎖に持
ち、且つ水溶性の共重合体からなる表面保護剤を見出
し、本発明を完成した。In order to solve these problems, the present inventor has proposed that a polyethylene glycol chain is provided as a compound having both hydrophobicity and hydrophilicity on a side chain and adsorbed on the surface of a hydrophobic solid phase. A surface protective agent having a hydrophobic functional group in the main chain and comprising a water-soluble copolymer has been found, and the present invention has been completed.
【０００６】即ち本発明は （１）側鎖にポリエチレングリコール鎖を有する水溶性
共重合体からなる免疫反応に使用する固相の表面保護剤 （２）分子内にポリエチレングリコール鎖を持つビニル
モノマーと該モノマーと共重合可能な水不溶性モノマー
との共重合体であって、分子量が１，０００〜１，００
０，０００の範囲にある水溶性共重合体からなる（１）
記載の表面保護剤That is, the present invention relates to (1) a solid-phase surface protective agent used for an immune reaction comprising a water-soluble copolymer having a polyethylene glycol chain in a side chain; and (2) a vinyl monomer having a polyethylene glycol chain in a molecule. A copolymer of the monomer and a copolymerizable water-insoluble monomer having a molecular weight of 1,000 to 1,000.
Consisting of a water-soluble copolymer in the range of 000 (1)
The described surface protective agent
【０００７】（３）分子内にポリエチレングリコール鎖
を持つビニルモノマーがアルコキシポリエチレングリコ
ール（メタ）アクリレートである（１）又は（２）記載
の表面保護剤 （４）分子内にポリエチレングリコール鎖を持つビニル
モノマーのポリエチレングリコール鎖の分子量が１００
〜１０，０００である（１）、（２）あるいは（３）記
載の表面保護剤 （５）（１）から（４）いずれかに記載の表面保護剤を
用いて表面処理を行った表面を用いた免疫測定方法 である。(3) The surface protective agent according to (1) or (2), wherein the vinyl monomer having a polyethylene glycol chain in the molecule is an alkoxy polyethylene glycol (meth) acrylate. (4) Vinyl having a polyethylene glycol chain in the molecule. The molecular weight of the polyethylene glycol chain of the monomer is 100
The surface protective agent according to (1), (2) or (3), which is up to 10,000. (5) The surface treated with the surface protective agent according to any one of (1) to (4) is treated. This is the immunoassay method used.
【０００８】[0008]
【発明の実施の形態】本発明において側鎖にポリエチレ
ングリコール鎖を有する水溶性共重合体は特に限定され
ず、免疫測定に使用する固相の表面を保護する能力を有
するものはいずれも使用できるが、上記（２）のものが
好ましく、より好ましくは上記（３）に記載のものであ
り、特に好ましくは上記（４）に記載のものである。本
発明に用いられるポリエチレングリコール鎖を持つビニ
ルモノマーは得られた共重合体を水溶性にし、イオン強
度あるいはｐＨの変化に対して安定にするモノマーであ
る。ポリエチレングリコールの分子量が１００〜１０，
０００のアクリレートあるいはメタクリレートが好まし
く、更に好ましくはポリエチレングリコールの分子量が
２００〜２，０００のアクリレートあるいはメタクリレ
ートであり、末端水酸基の水素がメチル基あるいはエチ
ル基等の低級アルキル基等で置換されていても良い。BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a water-soluble copolymer having a polyethylene glycol chain in a side chain is not particularly limited, and any copolymer having an ability to protect the surface of a solid phase used for immunoassay can be used. However, the above (2) is preferable, more preferably the above (3), and particularly preferably the above (4). The vinyl monomer having a polyethylene glycol chain used in the present invention is a monomer that makes the obtained copolymer water-soluble and stabilizes against a change in ionic strength or pH. Polyethylene glycol having a molecular weight of 100 to 10,
Acrylate or methacrylate is preferable, and polyethylene glycol is more preferably acrylate or methacrylate having a molecular weight of 200 to 2,000, even if the hydrogen of the terminal hydroxyl group is substituted with a lower alkyl group such as a methyl group or an ethyl group. good.
【０００９】水不溶性のモノマーは、疎水性表面に吸着
させるためのモノマーである。疎水性表面に吸着させる
ためには疎水性の高い官能基を持つモノマーが好まし
い。上記ポリエチレングリコール鎖を持つモノマーと共
重合可能ならば何でも良く、具体的には、アクリル酸又
はメタクリル酸のメチル、エチル、ブチル又はベンジル
エステルを初めとする各種アクリレートあるいはメタク
リレート、スチレン、酢酸ビニル、アクリロニトリル等
があり、好ましくはメチル（メタ）アクリレート、エチ
ル（メタ）アクリレート、ベンジル（メタ）アクリレー
ト、スチレン、酢酸ビニルが好ましい。また、これら疎
水性モノマーを組み合わせて使用することもできる。The water-insoluble monomer is a monomer for adsorbing on a hydrophobic surface. In order to adsorb on a hydrophobic surface, a monomer having a highly hydrophobic functional group is preferable. Anything is possible as long as it can be copolymerized with the monomer having a polyethylene glycol chain, and specifically, various acrylates or methacrylates including methyl, ethyl, butyl or benzyl ester of acrylic acid or methacrylic acid, styrene, vinyl acetate, acrylonitrile And the like, and preferably methyl (meth) acrylate, ethyl (meth) acrylate, benzyl (meth) acrylate, styrene, and vinyl acetate. Further, these hydrophobic monomers can be used in combination.
【００１０】分子内にポリエチレングリコール鎖を持つ
ビニルモノマーと水不溶性モノマーとの共重合割合は特
に限定されず、得られる水溶性共重合体が、免疫測定に
使用する固相の表面を保護する能力を有するものとなる
ような割合で用いれば何れの割合であっても良いが、分
子内にポリエチレングリコール鎖を持つビニルモノマー
と水不溶性モノマーとを３０〜９９．５：０．５〜７０
の重量比となるように共重合するのが好ましく、特に５
０〜９９：１〜５０の重量比となるように共重合するの
が好ましい。The copolymerization ratio of the vinyl monomer having a polyethylene glycol chain in the molecule and the water-insoluble monomer is not particularly limited, and the obtained water-soluble copolymer has an ability to protect the surface of a solid phase used for immunoassay. Any ratio may be used as long as the vinyl monomer having a polyethylene glycol chain in the molecule and the water-insoluble monomer are used in an amount of 30 to 99.5: 0.5 to 70.
It is preferable to copolymerize so as to have a weight ratio of
It is preferable to copolymerize so that the weight ratio is 0 to 99: 1 to 50.
【００１１】これらモノマーを共重合し本発明の表面保
護剤を製造するには、一般的なラジカル重合を採用する
ことで達成できる。即ち、攪拌可能な反応装置にモノマ
ーと溶媒、開始剤を加え、窒素置換の後加熱することで
重合が開始し、一定時間その温度を保つことで重合は完
結する。その後溶剤をエバポレートし、共重合体は水溶
液あるいはエタノール等の溶液として得られる。得られ
た水溶性共重合体を精製するには、共重合体不溶の溶媒
で再沈殿を行うかあるいは共重合体水溶液を透析する等
の一般的な方法で可能である。この重合の際に連鎖移動
剤を併用し分子量をコントロールすることも可能であ
る。共重合の際に用いられる溶剤としては、メタノール
やエタノールやイソプロピルアルコール等のアルコール
類、酢酸エチル、トルエン、ベンゼン、メチルエチルケ
トン等の有機溶剤、あるいは水が用いられ、これらを混
合して用いることもできる。The production of the surface protective agent of the present invention by copolymerizing these monomers can be achieved by employing general radical polymerization. That is, polymerization is started by adding a monomer, a solvent, and an initiator to a stirrable reactor and heating after nitrogen replacement, and the polymerization is completed by maintaining the temperature for a certain period of time. Thereafter, the solvent is evaporated, and the copolymer is obtained as an aqueous solution or a solution such as ethanol. The resulting water-soluble copolymer can be purified by a general method such as reprecipitation with a copolymer-insoluble solvent or dialysis of an aqueous copolymer solution. At the time of this polymerization, it is also possible to control the molecular weight by using a chain transfer agent in combination. As the solvent used in the copolymerization, alcohols such as methanol, ethanol, and isopropyl alcohol, organic solvents such as ethyl acetate, toluene, benzene, and methyl ethyl ketone, or water are used, and these may be used as a mixture. .
【００１２】重合開始剤としては、一般的にラジカル重
合で用いられる過酸化物系、アゾ系のラジカル開始剤が
用いられる。過酸化物系ラジカル開始剤としては例えば
過硫酸カリウム、過硫酸アンモニウム、過酸化水素等の
無機系過酸化物、ベンゾイルパーオキサイド、ｔ−ブチ
ルハイドロパーオキサイド、クメンパーオキサイド等の
有機系過酸化物が、アゾ系ラジカル開始剤としては例え
ば２，２’−アゾビスイソブチロニトリル、２，２’−
アゾビス（２−アミジノプロパン）ジハイドロクロライ
ド、ジメチル２，２’−アゾビスブチレート、ジメチル
２，２’−アゾビス（２−メチルプロピオネート）等が
用いられる。また、過酸化物系の開始剤に還元剤を組み
合わせたレドックス開始剤も使用できる。As the polymerization initiator, a peroxide-based or azo-based radical initiator generally used in radical polymerization is used. Examples of the peroxide-based radical initiator include inorganic peroxides such as potassium persulfate, ammonium persulfate, and hydrogen peroxide, and organic peroxides such as benzoyl peroxide, t-butyl hydroperoxide, and cumene peroxide. Examples of the azo radical initiator include 2,2′-azobisisobutyronitrile and 2,2′-
Azobis (2-amidinopropane) dihydrochloride, dimethyl 2,2′-azobisbutyrate, dimethyl 2,2′-azobis (2-methylpropionate) and the like are used. Further, a redox initiator obtained by combining a peroxide-based initiator with a reducing agent can also be used.
【００１３】重合する際の温度は、溶剤の種類、開始剤
の種類によって異なるが、開始剤の１０時間半減期温度
付近を使用するのが好ましい。重合の際に分子量を制御
するため用いられる連鎖移動剤としては、ドデシルメル
カプタン、チオリンゴ酸、チオグリコール酸等の高沸点
のチオール化合物、イソプロピルアルコール、亜リン
酸、次亜リン酸等を用いることができる。得られた共重
合体を精製するための再沈殿溶媒には、水溶性共重合体
の溶解性が低いヘキサン、ヘプタン等の脂肪族炭化水素
類が用いられ、他の溶剤と混合して用いても良い。水溶
性共重合体の分子量（ポリスチレンを標準としたＧＰＣ
よる重量平均分子量）は１，０００〜１，０００，００
０であることが好ましく、特に１０，０００〜２００，
０００であることが好ましい。The temperature for the polymerization varies depending on the type of the solvent and the type of the initiator, but it is preferable to use a temperature around the 10-hour half-life of the initiator. As the chain transfer agent used to control the molecular weight during the polymerization, a high-boiling thiol compound such as dodecyl mercaptan, thiomalic acid, thioglycolic acid, isopropyl alcohol, phosphorous acid, hypophosphorous acid, etc. may be used. it can. As the reprecipitation solvent for purifying the obtained copolymer, aliphatic hydrocarbons such as hexane and heptane having low solubility of the water-soluble copolymer are used, and used by mixing with other solvents. Is also good. Molecular weight of water-soluble copolymer (GPC using polystyrene as standard)
Weight average molecular weight) is 1,000 to 1,000,000
0, especially 10,000 to 200,
000 is preferred.
【００１４】得られた表面保護剤を一般的な放射免疫測
定法（ＲＩＡ）、酵素免疫測定法（ＥＩＡ）等の抗原抗
体反応を利用した免疫測定に利用するには、ポリスチレ
ン等からなるプラスチックの疎水性表面を前もって抗体
あるいは抗原で処理し、その後生理食塩液あるいは緩衝
液に溶解した表面保護剤を用いると良い。その濃度は水
溶性共重合体の種類や固相の材質又は免疫測定の種類や
それに用いられる試薬等によって異なるが、通常、０．
０１〜１０％程度の濃度で、好ましくは０．１〜２％の
濃度が用いられる。その場合一般的に用いられている４
℃で１晩あるいは３７℃で１時間でブロッキングするこ
とができ、その際の緩衝液は抗原あるいは抗体を変性さ
せないｐＨ条件のものが用いられる。その後必要に応じ
て緩衝液の交換を行い、目的とする免疫反応に供するこ
とができる。この際、緩衝液中に表面保護剤を含ませて
も良く、ＢＳＡ等の蛋白質を共存させても良い。In order to use the obtained surface protective agent in an immunoassay utilizing an antigen-antibody reaction such as a general radioimmunoassay (RIA) or an enzyme immunoassay (EIA), it is necessary to use a plastic such as polystyrene. It is preferable to previously treat the hydrophobic surface with an antibody or an antigen, and then use a surface protective agent dissolved in a physiological saline solution or a buffer solution. The concentration varies depending on the type of the water-soluble copolymer, the material of the solid phase, the type of the immunoassay, the reagent used therein, and the like.
A concentration of about 01 to 10%, preferably 0.1 to 2% is used. In that case, commonly used 4
Blocking can be performed overnight at 37 ° C or 1 hour at 37 ° C, and a buffer at that time is used under a pH condition that does not denature the antigen or antibody. Thereafter, the buffer solution is exchanged as necessary, and the resultant can be subjected to a target immune reaction. At this time, a surface protective agent may be included in the buffer solution, and a protein such as BSA may be coexisted.
【００１５】また、ラテックス等の微粒子の表面に抗原
あるいは抗体を付け、試料中の抗体あるいは抗原を凝集
反応で測定する一般的免疫測定法においても同様にして
感作ラテックスのブロッキングに使うことができる。本
発明の表面保護剤を用いて表面を保護する固相として
は、免疫測定に使用する抗原あるいは抗体を結合あるい
は吸着させた固相ならいずれも使用でき、例えばポリス
チレンあるいはその共重合体、ポリアクロレイン、ポリ
カーボネート、アクリル系樹脂等のプラスチック類から
なるプレート、ビーズ、ラテックス等が挙げられる。ま
た，反応セルの表面保護にも用いられる。In a general immunoassay in which an antigen or an antibody is attached to the surface of fine particles such as a latex and the antibody or the antigen in the sample is measured by agglutination, it can be similarly used for blocking the sensitized latex. . As the solid phase for protecting the surface using the surface protective agent of the present invention, any solid phase to which an antigen or an antibody used for immunoassay is bound or adsorbed can be used, for example, polystyrene or a copolymer thereof, polyacrolein. , A plate, beads, latex and the like made of plastics such as polycarbonate, acrylic resin and the like. It is also used to protect the surface of the reaction cell.
【００１６】本発明の表面保護剤を免疫測定の際の固相
表面のブロッキングに用いた場合、保護剤による免疫反
応が抑制され、偽陽性となる非特異反応の出現率が低下
する。即ち、天然蛋白質を保護剤に用いた場合に希に出
現する非特異反応による診断誤差を軽減することができ
る。When the surface protective agent of the present invention is used for blocking the surface of a solid phase during immunoassay, the immune reaction due to the protective agent is suppressed, and the incidence of false positive nonspecific reactions is reduced. That is, it is possible to reduce a diagnostic error caused by a nonspecific reaction that rarely appears when a natural protein is used as a protective agent.
【００１７】[0017]
【実施例】以下、実施例及び比較例により本発明を具体
的に説明する。 実施例１ 攪拌器、冷却器、窒素送入口を備えた５００ｍｌ反応容
器にイソプロピルアルコール２５０ｍｌ、メトキシポリ
エチレングリコール（ＭＷ＝４００）メタクリレート９
５ｇ、エチルメタクリレート５ｇを計量し、さらに２，
２’−アゾビスイソブチロニトリル１００ｍｇを添加
し、窒素気流中で７０℃にて６時間、８０℃に上げて２
時間重合した。得られた共重合表面保護剤の溶液を１，
０００ｍｌのｎ−ヘキサン中に注ぎ、再沈澱を２回繰り
返し精製した。この表面保護剤を減圧乾燥し、１０％の
濃度で水に溶解し、ｐＨを７．０に調製した。また、乾
燥した表面保護剤をテトラヒドロフランに溶解しポリス
チレンを標準としてＧＰＣにて分子量を測定したとこ
ろ、数平均分子量：２０，１４４、重量平均分子量：２
５，１１４であった。The present invention will be specifically described below with reference to examples and comparative examples. Example 1 250 ml of isopropyl alcohol and methoxypolyethylene glycol (MW = 400) methacrylate 9 were placed in a 500 ml reaction vessel equipped with a stirrer, a cooler and a nitrogen inlet.
5 g of ethyl methacrylate and 5 g of ethyl methacrylate,
100 mg of 2′-azobisisobutyronitrile was added, and the temperature was raised to 80 ° C. for 6 hours at 70 ° C. in a nitrogen stream to obtain 2
Polymerized for hours. The solution of the obtained copolymer surface protective agent was
The solution was poured into 000 ml of n-hexane, and the reprecipitation was repeated twice. This surface protective agent was dried under reduced pressure, dissolved in water at a concentration of 10%, and adjusted to pH 7.0. The dried surface protective agent was dissolved in tetrahydrofuran, and the molecular weight was measured by GPC using polystyrene as a standard. The number average molecular weight was 20,144 and the weight average molecular weight was 2
5,114.
【００１８】実施例２ 実施例１と同様にして、メトキシポリエチレングリコー
ル（ＭＷ＝１，０００）メタクリレート９５ｇとベンジ
ルメタクリレート５ｇを重合し、共重合体の１０％水溶
液を得た。 実施例３ 実施例１と同様にして、メトキシポリエチレングリコー
ル（ＭＷ＝１，０００）メタクリレート９０ｇとスチレ
ン１０ｇを重合し、共重合体の１０％水溶液を得た。Example 2 In the same manner as in Example 1, 95 g of methoxypolyethylene glycol (MW = 1,000) methacrylate and 5 g of benzyl methacrylate were polymerized to obtain a 10% aqueous solution of a copolymer. Example 3 In the same manner as in Example 1, 90 g of methoxypolyethylene glycol (MW = 1,000) methacrylate and 10 g of styrene were polymerized to obtain a 10% aqueous solution of a copolymer.
【００１９】実施例４ １０重量％の濃度の１．３μｍの平均粒子径を持つ赤色
ポリアクロレインラテックス（日本化薬（株）製）の水
懸濁液に、トレポネーマ・パリダム（ニコルス株）を１
％オクチルグルコシド水溶液にて可溶化した梅毒検査抗
原液（蛋白質濃度５０μｇ／ｍｌ）を２倍量添加し、ゆ
っくり攪拌しながら４℃にて１６時間感作した。続い
て、１，０００ｒｐｍにて遠心分離し、沈渣をリン酸緩
衝生理食塩液（ｐＨ７．２）に懸濁し、実施例１で得ら
れた１０％濃度の表面保護剤水溶液（ｐＨ７．０）をリ
ン酸緩衝生理食塩液（ｐＨ７．２）で１％濃度に希釈し
た液を等量添加混合し、４℃にて１６時間ゆっくり攪拌
し、ポリアクロレインラテックスの疎水性表面のブロッ
キングを行った。更に、遠心分離により、１％濃度の表
面保護剤を含有するリン酸緩衝生理食塩液（ｐＨ７．
２）にて２回洗浄後、０．２５％の濃度になるように同
緩衝生理食塩液に懸濁した。Example 4 Treponema pallidum (Nichols) was added to an aqueous suspension of red polyacrolein latex (manufactured by Nippon Kayaku Co., Ltd.) having an average particle diameter of 1.3 μm at a concentration of 10% by weight.
A two-fold amount of a syphilis test antigen solution (protein concentration: 50 μg / ml) solubilized with an aqueous solution of octyl glucoside was added, and sensitized at 4 ° C. for 16 hours with gentle stirring. Subsequently, the mixture was centrifuged at 1,000 rpm, the precipitate was suspended in a phosphate buffered saline (pH 7.2), and the 10% concentration aqueous solution of the surface protective agent (pH 7.0) obtained in Example 1 was added. An equal amount of a solution diluted to a concentration of 1% with a phosphate buffered saline (pH 7.2) was added and mixed, and the mixture was stirred slowly at 4 ° C. for 16 hours to block the hydrophobic surface of the polyacrolein latex. Further, by centrifugation, a phosphate buffered saline solution (pH 7.0) containing a 1% concentration of a surface protective agent was used.
After washing twice in 2), the cells were suspended in the same buffered physiological saline to a concentration of 0.25%.
【００２０】比較のため、同様にして１％の本発明のポ
リマー溶液の代わりに１％牛血清アルブミンを含有する
リン酸緩衝生理食塩液（ｐＨ７．２）を用いた条件で感
作ラテックス懸濁液を作成した。得られた各々の感作ラ
テックスを用いてマイクロタイター法にて血清試料を１
００検体比較した。以下に示す通り本発明の方が陽性判
定は少なく、ラナタイターＴＰ（日本化薬（株）製）に
よる確認のための検査結果と一致した。 実施例４ 比較 ラナタイターＴＰ ────────────────────────── 陽性 ２ ４ ２ 陰性 ９８ ９６ ９８ ────────────────────────── 従って、本発明による免疫測定法では、非特異反応によ
る偽陽性は０／１００であったが、同条件でＢＳＡを用
いた場合には２／１００の偽陽性があった。For comparison, similarly, the sensitized latex suspension was carried out under the condition that a phosphate buffered saline solution (pH 7.2) containing 1% bovine serum albumin was used instead of the 1% polymer solution of the present invention. A liquid was made. Using each of the obtained sensitized latexes, one serum sample was obtained by the microtiter method.
00 samples were compared. As shown below, the positive determination of the present invention was smaller than that of the present invention, which coincided with the test result for confirmation by Ranatiter TP (manufactured by Nippon Kayaku Co., Ltd.). Example 4 Comparative Ranatiter TP {Positive 2 42 Negative 98 96 98 98} ──────────────── Therefore, in the immunoassay according to the present invention, false positives due to non-specific reactions were 0/100, but when BSA was used under the same conditions, Had 2/100 false positives.
【００２１】実施例５ 次いでＥＩＡプレート法の例を示す。ＮＣＣ−ＳＴ４３
９マウスモノクローナル抗体（日本化薬（株）製）を
０．１％アジ化ナトリウム含有０．１Ｍリン酸緩衝液に
溶解し、１００μｇ／ｍｌの濃度に調整した。この溶液
をポリスチレン製９６穴平底マイクロプレートの各ウェ
ル当たり１００μｌずつ加え、４℃にて２４時間静置し
た。その後、モノクローナル抗体溶液を除去し、各ウェ
ルを生理食塩液にて２回洗浄後、実施例２で得られた本
発明のポリマー溶液を１０ｍＭリン酸緩衝生理食塩液
（ｐＨ７．０）で１０倍希釈した溶液を各ウェルに３０
０μｌずつ加え、４℃にて２４時間静置し保存した。
同時に比較のため本発明の表面保護剤溶液の代わりに１
％ＢＳＡ１０ｍＭリン酸緩衝生理食塩液（ｐＨ７．０）
溶液を用い、同様の操作をして保存した。Example 5 Next, an example of the EIA plate method will be described. NCC-ST43
Nine mouse monoclonal antibodies (manufactured by Nippon Kayaku Co., Ltd.) were dissolved in 0.1 M phosphate buffer containing 0.1% sodium azide and adjusted to a concentration of 100 μg / ml. This solution was added in an amount of 100 μl per well of a 96-well flat bottom microplate made of polystyrene and allowed to stand at 4 ° C. for 24 hours. Thereafter, the monoclonal antibody solution was removed, and each well was washed twice with physiological saline, and the polymer solution of the present invention obtained in Example 2 was diluted 10 times with 10 mM phosphate buffered saline (pH 7.0). Add 30 diluted solutions to each well
Each 0 μl was added, and the mixture was allowed to stand at 4 ° C. for 24 hours and stored.
At the same time, for comparison, 1 instead of the surface protective agent solution of the present invention was used.
% BSA 10 mM phosphate buffered saline (pH 7.0)
The solution was used and stored in the same manner.
【００２２】ラナザイムＳＴ−４３９測定キット（日本
化薬（株）製）の標準液及び管理血清及びＮＣＣ−ＳＴ
−４３９陽性血清（１０検体）１０μｌをキット添付の
緩衝液にて１０倍希釈した溶液を１００μｌずつ抗体結
合マイクロプレートの各ウェルに分注し、３７℃で３０
分反応させた。次いで、生理食塩液にて４回洗浄し、キ
ット添付の標識抗体を添付の溶解液にて溶解した標識抗
体液を１００μｌずつ分注し、３７℃で１時間反応さ
せ、更に生理食塩液にて４回洗浄した。次に、キット添
付の発色剤を溶解液にて溶解した発色液を１００μｌず
つ分注し、３７℃で３０分反応させ、キット添付の停止
液にて発色反応を停止させ、マイクロプレートリーダー
にて４５０ｎｍの吸光度を測定した。標準液による検量
線により、管理血清及び検体の中のＳＴ−４３９抗原の
濃度を定量した。その結果は以下の通りであった。 以上の様に、抗体結合プレート製造の際のプラスチック
表面の保護にＢＳＡと同様に使用することができた。Standard solution and control serum of Lanazyme ST-439 assay kit (manufactured by Nippon Kayaku Co., Ltd.) and NCC-ST
A solution obtained by diluting 10 μl of -439 positive serum (10 samples) with the buffer attached to the kit by 10 times was dispensed to each well of the antibody-binding microplate in an amount of 100 μl.
Minutes. Subsequently, the plate was washed four times with a physiological saline solution, and the labeled antibody solution obtained by dissolving the labeled antibody attached to the kit with the attached dissolving solution was dispensed in 100 μl portions, and allowed to react at 37 ° C. for 1 hour. Washed 4 times. Next, 100 μl of a coloring solution obtained by dissolving the coloring agent attached to the kit with the dissolving solution was dispensed in 100 μl portions, and reacted at 37 ° C. for 30 minutes. The absorbance at 450 nm was measured. The concentration of ST-439 antigen in the control serum and the sample was quantified by a calibration curve using a standard solution. The results were as follows. As described above, it could be used in the same manner as BSA for protecting the plastic surface during the production of the antibody binding plate.
【００２３】[0023]
【発明の効果】本発明の表面保護剤は各種免疫測定に用
いられる固相の表面保護剤として使用することができ
る。The surface protective agent of the present invention can be used as a solid surface protective agent used for various immunoassays.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 ＦＩ Ｇ０１Ｎ 33/531 Ｇ０１Ｎ 33/531 Ｂ ──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 6 Identification code FI G01N 33/531 G01N 33/531 B
Claims (5)
水溶性共重合体からなる免疫反応に使用する固相の表面
保護剤。1. A solid surface protective agent comprising a water-soluble copolymer having a polyethylene glycol chain in a side chain, which is used for an immunological reaction.
ビニルモノマーと該モノマーと共重合可能な水不溶性モ
ノマーとの共重合体であって、分子量が１，０００〜
１，０００，０００の範囲にある水溶性共重合体からな
る請求項１記載の表面保護剤。2. A copolymer of a vinyl monomer having a polyethylene glycol chain in the molecule and a water-insoluble monomer copolymerizable with the monomer, having a molecular weight of 1,000 to 1,000.
The surface protective agent according to claim 1, comprising a water-soluble copolymer in the range of 1,000,000.
ビニルモノマーがアルコキシポリエチレングリコール
（メタ）アクリレートである請求項１又は請求項２記載
の表面保護剤。3. The surface protective agent according to claim 1, wherein the vinyl monomer having a polyethylene glycol chain in the molecule is an alkoxy polyethylene glycol (meth) acrylate.
ビニルモノマーのポリエチレングリコール鎖の分子量が
１００〜１０，０００である請求項１、請求項２あるい
は請求項３記載の表面保護剤。4. The surface protective agent according to claim 1, wherein the molecular weight of the polyethylene glycol chain of the vinyl monomer having a polyethylene glycol chain in the molecule is 100 to 10,000.
剤を用いて表面処理を行った表面を用いた免疫測定方
法。5. An immunoassay method using a surface that has been subjected to a surface treatment using the surface protective agent according to any one of claims 1 to 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10091153A JPH11287802A (en) | 1998-04-03 | 1998-04-03 | Surface protective agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10091153A JPH11287802A (en) | 1998-04-03 | 1998-04-03 | Surface protective agent |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH11287802A true JPH11287802A (en) | 1999-10-19 |
Family
ID=14018580
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP10091153A Pending JPH11287802A (en) | 1998-04-03 | 1998-04-03 | Surface protective agent |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH11287802A (en) |
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| WO2005010529A1 (en) * | 2003-07-28 | 2005-02-03 | Tokyo University Of Science, Educational Foundation | Surface of base material being inhibited in non-specific adsorption |
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