JP2017184730A - Methods for producing denatured antibody measuring reagent - Google Patents
Methods for producing denatured antibody measuring reagent Download PDFInfo
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- JP2017184730A JP2017184730A JP2017048921A JP2017048921A JP2017184730A JP 2017184730 A JP2017184730 A JP 2017184730A JP 2017048921 A JP2017048921 A JP 2017048921A JP 2017048921 A JP2017048921 A JP 2017048921A JP 2017184730 A JP2017184730 A JP 2017184730A
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Abstract
Description
本発明は、溶液中に含まれる変性した抗体の量を測定するための試薬を製造する方法に関する。特に本発明は、凍結乾燥形態の前記試薬を製造する方法に関する。また、本発明は当該方法によって製造される試薬にも関する。 The present invention relates to a method for producing a reagent for measuring the amount of denatured antibody contained in a solution. In particular, the present invention relates to a method for producing the reagent in lyophilized form. The present invention also relates to a reagent produced by the method.
医薬品用途で用いる抗体は、通常当該抗体を発現可能な細胞を培養後、得られた培養液を遠心分離、濾過、カラムクロマトグラフィー等の精製操作を行ない、製造する。その際、得られた抗体の中には、製造時のストレス等により変性を受けることで生じる、当該抗体の凝集体が含まれている可能性がある。抗体凝集体は免疫原性を有するおそれがある（非特許文献１）ため、特に医薬品用途で用いる場合、当該凝集体を精度よく検出する必要がある。 Antibodies used for pharmaceutical applications are usually produced by culturing cells capable of expressing the antibody, and then subjecting the obtained culture solution to purification operations such as centrifugation, filtration, and column chromatography. At this time, the obtained antibody may contain an aggregate of the antibody that is generated by being denatured due to stress during production. Since antibody aggregates may have immunogenicity (Non-patent Document 1), it is necessary to detect the aggregates with high precision, particularly when used for pharmaceutical applications.
抗体凝集体を検出する方法として、サイズ排除クロマトグラフィーやフィールドフローフラクショネーションを用いた方法が知られている。しかしながら、これらの方法はタンパク質のサイズに基づき抗体凝集体を検出する方法であり、構造・性状に基づいた方法ではない。また抗体の高次構造の精密な解析手法として、Ｘ線結晶構造解析やＮＭＲを用いた方法が知られているが、これらの方法は非常に手間のかかる方法であり、製造時の純度管理等、迅速・簡便な測定が求められる用途には不向きである
近年、ストレス等により変性を受けた抗体を、その分子量を問わず、特異的に認識する分子が報告されている（特許文献１〜３並びに非特許文献２）。しかしながら、これら文献に記載の前記分子を用いた変性抗体測定法は表面プラズモン共鳴を利用した方法であり、当該方法を実施するにはＢｉａｃｏｒｅ（ＧＥヘルスケア製）等の高価な専用装置を必要とする。
Known methods for detecting antibody aggregates include methods using size exclusion chromatography and field flow fractionation. However, these methods are methods for detecting antibody aggregates based on protein size, and are not methods based on structure and properties. In addition, X-ray crystal structure analysis and NMR-based methods are known as precise analysis methods of antibody higher-order structures, but these methods are very time-consuming methods, such as purity control during production. In recent years, molecules that specifically recognize antibodies that have been denatured by stress or the like regardless of their molecular weight have been reported (Patent Documents 1 to 3). And Non-Patent Document 2). However, the denatured antibody measurement method using the molecule described in these documents is a method using surface plasmon resonance, and an expensive dedicated device such as Biacore (manufactured by GE Healthcare) is required to perform the method. To do.
また測定試薬を製造／利用するにあたり、当該測定試薬の保存安定性は高いほうがよい。試薬を安定的に保存する方法としては、一般に、凍結保存法、冷却保存法、凍結乾燥法等が用いられている。凍結保存法は試薬を氷点下（好ましくは−２０℃以下）の温度で凍結し保存することでタンパク質の変性等による失活を防ぎ、長期保存を可能とする方法である。しかしながら凍結融解を繰り返すことでタンパク質が変性する（失活する）おそれがある。冷却保存法はグリセリン等の添加剤を試薬に加えた後、凍結保存法と同様、氷点下（好ましくは−２０℃以下）で冷却し保存する方法である。しかしながら前記添加剤が、試薬に含まれる酵素活性に影響を与える例が知られている。また凍結保存法や冷却保存法は、常に試薬を氷点下で凍結又は冷却する必要があり、試薬を搬送する際や消費地で保存する際、大きな負担となる。 Moreover, when manufacturing / utilizing a measurement reagent, the storage stability of the measurement reagent should be high. In general, a cryopreservation method, a cryopreservation method, a freeze-drying method, or the like is used as a method for stably storing a reagent. In the cryopreservation method, the reagent is frozen and stored at a temperature below freezing point (preferably −20 ° C. or lower), thereby preventing inactivation due to protein denaturation and the like, and enabling long-term storage. However, the protein may be denatured (deactivated) by repeated freezing and thawing. The cold storage method is a method in which an additive such as glycerin is added to the reagent and then cooled and stored at a temperature below freezing point (preferably −20 ° C. or lower), similarly to the cryopreservation method. However, examples in which the additive affects the enzyme activity contained in the reagent are known. In addition, the cryopreservation method and the cold storage method always require that the reagent be frozen or cooled below freezing point, and this is a heavy burden when the reagent is transported or stored at a consumption place.
一方、凍結乾燥法は、凍結乾燥後、冷蔵（４から１０℃程度）で保存することで、安定かつ長期間試薬を保存できる方法であり、試薬安定化方法として、現在広く用いられる方法である。しかしながら、凍結及び乾燥する条件によっては、試薬中に含まれるタンパク質（抗原、抗体、酵素等）並びに核酸（ＤＮＡ、ＲＮＡ等）等が変性する（失活するおそれがある。そのため、凍結乾燥法により試薬安定化を図る際は、試薬中に含まれる成分や凍結乾燥条件（温度／時間）等を最適化する必要がある。 On the other hand, the freeze-drying method is a method that can store the reagent stably and for a long period of time by refrigeration (about 4 to 10 ° C.) after lyophilization, and is currently widely used as a reagent stabilization method. . However, depending on the conditions for freezing and drying, proteins (antigens, antibodies, enzymes, etc.) and nucleic acids (DNA, RNA, etc.) contained in the reagents may be denatured (inactivated). When stabilizing the reagent, it is necessary to optimize the components contained in the reagent, lyophilization conditions (temperature / time), and the like.
本発明は、前記従来技術の有する課題に鑑みてなされたものであり、溶液中に含まれる変性した抗体を測定するための試薬を製造する方法であって、前記試薬の性能を保持したまま、冷蔵での長期保存が可能な試薬を製造する方法を提供することを目的とする。 The present invention has been made in view of the problems of the prior art, and is a method for producing a reagent for measuring a denatured antibody contained in a solution, while maintaining the performance of the reagent, It aims at providing the method of manufacturing the reagent which can be preserve | saved for a long time by refrigeration.
本発明者らは、上記課題を解決すべく、凍結乾燥させる際、試薬に含ませる安定化剤について鋭意検討を重ねた結果、本発明に到達した。 In order to solve the above-mentioned problems, the inventors of the present invention have reached the present invention as a result of intensive studies on stabilizers contained in reagents when freeze-dried.
すなわち本発明の第一の態様は、
変性した抗体を測定するための試薬を製造する方法であって、
変性した抗体を認識するオリゴペプチドを結合した担体に、安定化剤を含む溶液を添加した後、添加後の前記担体及び前記溶液を凍結乾燥する工程を含み、
前記オリゴペプチドが配列番号：１に記載のアミノ酸配列を含むオリゴペプチドであり、かつ前記安定化剤が非イオン界面活性剤及び／又は陽イオン界面活性剤である方法である。
That is, the first aspect of the present invention is:
A method for producing a reagent for measuring a denatured antibody, comprising:
A step of adding a stabilizer-containing solution to a carrier bound with an oligopeptide that recognizes a denatured antibody, and then freeze-drying the carrier and the solution after the addition,
In this method, the oligopeptide is an oligopeptide comprising the amino acid sequence shown in SEQ ID NO: 1, and the stabilizer is a nonionic surfactant and / or a cationic surfactant.
また本発明の第二の態様は、安定化剤が０．００００５〜０．０２％（ｖ／ｖ）のポリソルベート２０、０．００５〜０．１％（ｖ／ｖ）のオクチルフェノールエトキシレート及び０．００５〜０．１％（ｗ／ｖ）のセチルトリメチルアンモニウムブロミドからなる群から選択される少なくとも１つの界面活性剤である、前記第一の態様に記載の方法である。 The second aspect of the present invention is that the stabilizer is 0.00005 to 0.02% (v / v) polysorbate 20, 0.005 to 0.1% (v / v) octylphenol ethoxylate and 0 The method according to the first aspect, which is at least one surfactant selected from the group consisting of 0.005 to 0.1% (w / v) cetyltrimethylammonium bromide.
さらに本発明の第三の態様は、０．００００５〜０．０２％（ｖ／ｖ）のポリソルベート２０、０．００５〜０．１％（ｖ／ｖ）のオクチルフェノールエトキシレート及び０．００５〜０．１％（ｗ／ｖ）のセチルトリメチルアンモニウムブロミドからなる群から選択される少なくとも１つの界面活性剤を含む溶液が添加されているオリゴペプチド結合担体を凍結乾燥してなる、かつ、前記オリゴペプチド結合担体が、配列番号：１に記載のアミノ酸配列を含み、変性した抗体の量を測定するための試薬である。 The third aspect of the present invention further comprises 0.00005-0.02% (v / v) polysorbate 20, 0.005-0.1% (v / v) octylphenol ethoxylate and 0.005-0. A lyophilized oligopeptide-binding carrier to which a solution containing at least one surfactant selected from the group consisting of 1% (w / v) cetyltrimethylammonium bromide is added, and said oligopeptide The binding carrier contains the amino acid sequence shown in SEQ ID NO: 1, and is a reagent for measuring the amount of denatured antibody.
さらに本発明の第四の態様は、以下の（１）及び（２）に示す工程を含む、変性した抗体の量を測定する方法である。
（１）被検抗体を含む試料と、前記第三の態様に記載の試薬と、被検抗体を認識する抗体に標識物質が結合している標識抗体とを接触させることにより、オリゴペプチド結合担体と変性した抗体と標識抗体とを含む複合体を形成させる工程
（２）（１）で形成した複合体中の標識物質を検出することにより、前記試料中の変性した抗体の量を測定する工程。
Furthermore, the fourth aspect of the present invention is a method for measuring the amount of denatured antibody, comprising the steps shown in the following (1) and (2).
(1) An oligopeptide-binding carrier by contacting a sample containing a test antibody, the reagent according to the third aspect, and a labeled antibody in which a labeling substance is bound to an antibody that recognizes the test antibody And (2) a step of forming a complex comprising a denatured antibody and a labeled antibody, and a step of measuring the amount of denatured antibody in the sample by detecting the labeling substance in the complex formed in (1) .
本発明は、変性した抗体を認識するオリゴペプチドを結合した担体に安定化剤を含む試薬を添加後、前記溶液を凍結乾燥することで、変性抗体測定試薬を製造する際、変性した抗体を認識するオリゴペプチドとして配列番号：１に記載のアミノ酸配列を含むオリゴペプチドを用い、かつ安定化剤が非イオン界面活性剤及び／又は陽イオン界面活性剤であることを特徴としている。本発明により、凍結乾燥前（液体試薬）における試薬性能は保持しつつ、冷蔵での長期保存が可能な試薬を提供することができる。 The present invention recognizes a denatured antibody when producing a denatured antibody measurement reagent by adding a reagent containing a stabilizer to a carrier bound with an oligopeptide recognizing the denatured antibody and then freeze-drying the solution. The oligopeptide comprising the amino acid sequence shown in SEQ ID NO: 1 is used as the oligopeptide, and the stabilizer is a nonionic surfactant and / or a cationic surfactant. According to the present invention, it is possible to provide a reagent that can be stored for a long time in a refrigerator while maintaining the reagent performance before lyophilization (liquid reagent).
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
＜変性した抗体を測定するための試薬を製造する方法＞
本発明の変性した抗体の量を測定するための試薬を製造する方法は、変性した抗体を認識するオリゴペプチドを結合した担体に、安定化剤を含む溶液を添加した後、添加後の前記担体及び前記溶液を凍結乾燥する工程を含み、前記オリゴペプチドが配列番号：１に記載のアミノ酸配列を含むオリゴペプチドであり、かつ前記安定化剤が非イオン界面活性剤及び／又は陽イオン界面活性剤である、方法である。
<Method for Producing Reagent for Measuring Denatured Antibody>
The method for producing a reagent for measuring the amount of denatured antibody of the present invention comprises adding a solution containing a stabilizer to a carrier bound with an oligopeptide that recognizes a denatured antibody, and then adding the carrier after the addition. And the step of freeze-drying the solution, wherein the oligopeptide is an oligopeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, and the stabilizer is a nonionic surfactant and / or a cationic surfactant It is a method.
本発明において、変性した抗体とは、酸、アルカリ、加熱、凍結融解、撹拌等によるストレスで抗体の全構造又は一部構造が変化したものをいう。なお変性した抗体は、Ｆｃ領域を有する限り、その種類について特に限定はなく、例えば、免疫グロブリンのアイソタイプ（ＩｇＧ、ＩｇＡ、ＩｇＤ、ＩｇＥ、ＩｇＭ等）及びそれらのアイソタイプのサブクラスが挙げられ、より具体的に、ヒトの免疫グロブリンとして、ＩｇＧ１、ＩｇＧ２、ＩｇＧ３、ＩｇＧ４、ＩｇＡ１、ＩｇＡ２、ＩｇＤ、ＩｇＥ、ＩｇＭの９種類のクラスが挙げられる。また、変性した抗体の由来について特に限定はなく、例えばヒト、マウス、ラット、ウサギ、ヤギ等に由来する抗体が挙げられ、特にヒトに由来する抗体が好ましい。さらに、変性した抗体は、このような単一の動物種に由来する抗体に限らず、異なる動物種に由来する抗体の断片が結合されてなる抗体であってもよい。かかる抗体としては、例えば、キメラ抗体（ヒト抗体のＦｃ領域と、他の動物に由来する抗体（例えば、マウス抗体、ラット抗体、ウサギ抗体、ヤギ抗体）に由来する配列（可変領域、ＣＤＲ等）を含むキメラ抗体等）、ヒト化抗体であってもよい。また、本発明にかかる抗体は、モノクローナル抗体であってもよく、ポリクローナル抗体であってもよい。 In the present invention, a denatured antibody refers to an antibody whose entire structure or partial structure has been changed by stress due to acid, alkali, heating, freeze-thawing, stirring, or the like. The modified antibody is not particularly limited as long as it has an Fc region, and includes, for example, immunoglobulin isotypes (IgG, IgA, IgD, IgE, IgM, etc.) and subclasses of those isotypes. In particular, there are nine classes of human immunoglobulins: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. The origin of the modified antibody is not particularly limited, and examples thereof include antibodies derived from humans, mice, rats, rabbits, goats, and the like, and antibodies derived from humans are particularly preferable. Furthermore, the denatured antibody is not limited to an antibody derived from such a single animal species, and may be an antibody formed by binding fragments of antibodies derived from different animal species. Examples of such antibodies include chimeric antibodies (human antibody Fc region and sequences derived from other animals (eg, mouse antibody, rat antibody, rabbit antibody, goat antibody) (variable region, CDR, etc.). Or a humanized antibody. The antibody according to the present invention may be a monoclonal antibody or a polyclonal antibody.
本発明において、変性した抗体を認識するオリゴペプチドは、配列番号：１に記載のアミノ酸配列を含むオリゴペプチドである。かかるオリゴペプチドとして、配列番号：１に記載のアミノ酸配列のみからなるオリゴペプチドを用いてもよいし、前記オリゴペプチドのＮ末端側又はＣ末端側に後述の担体と結合させるためのリンカーペプチドといった数アミノ酸残基から数十アミノ残基からなるオリゴペプチドを付加したオリゴペプチドを用いてもよい。本発明にかかるオリゴペプチドの長さとしては、変性した抗体を認識する限り、特に制限はないが、通常５０〜２５アミノ酸であり、好ましくは４０〜２５アミノ酸であり、より好ましくは３０〜２５アミノ酸であり、特に好ましくは２５アミノ酸（配列番号；１に記載のアミノ酸配列のみ）である。また、オリゴペプチドは、通常天然アミノ酸がペプチド結合により連結されて構成されるものであるが、本発明において非天然型のアミノ酸を含むものであってもよく、また修飾が施されているものであってもよい。 In the present invention, an oligopeptide that recognizes a denatured antibody is an oligopeptide comprising the amino acid sequence set forth in SEQ ID NO: 1. As such oligopeptide, an oligopeptide consisting only of the amino acid sequence described in SEQ ID NO: 1 may be used, or the number of linker peptides for binding to the carrier described later on the N-terminal side or C-terminal side of the oligopeptide. An oligopeptide to which an oligopeptide consisting of amino acid residues to several tens of amino acid residues is added may be used. The length of the oligopeptide according to the present invention is not particularly limited as long as it recognizes a denatured antibody, but is usually 50 to 25 amino acids, preferably 40 to 25 amino acids, more preferably 30 to 25 amino acids. And particularly preferably 25 amino acids (SEQ ID NO: 1 only amino acid sequence). In addition, the oligopeptide is usually composed of natural amino acids linked by peptide bonds. However, in the present invention, it may contain a non-natural amino acid and is modified. There may be.
本発明において、前記オリゴペプチドを固定化させる担体に特に限定はなく、例えば、プレート（複数の凹部が形成されたプレート（ウェルプレート、マイクロプレート等）も含む）、ビーズ、ゲル等が挙げられる。また、担体の材料としては、プラスチック（例えば、ポリスチレン等のスチレン系樹脂、ポリ塩化ビニル等のビニル系重合体、ポリメチル（メタ）アクリレート等のアクリル系樹脂、ポリエチレン等のポリオレフィン系樹脂、ポリエチレンテレフタレート等のポリエステル系樹脂、ポリカーボネート樹脂）、金属（金、銀、アルミ、ステンレス等）、酸化物（ガラス、シリカ、アルミナ、チタニア、ジルコニア、インジウムスズ酸化物（ＩＴＯ）等）、寒天等が挙げられる。より具体的には、ＥＬＩＳＡ（Ｅｎｚｙｍｅ−Ｌｉｎｋｅｄ ＩｍｍｕｎｏＳｏｒｂｅｎｔ Ａｓｓａｙ）法で用いられるウェルプレートや、ガラスビーズ、磁性ビーズ、金属粒子、シリカビーズ、ジルコニアビーズ、セルロースゲル、アガロースゲル、ＴＯＹＯＰＥＡＲＬ（登録商標、東ソー株式会社製）等のポリマーゲルが挙げられる。 In the present invention, the carrier on which the oligopeptide is immobilized is not particularly limited, and examples thereof include plates (including plates (including well plates and microplates) in which a plurality of recesses are formed), beads, gels, and the like. Examples of the carrier material include plastics (for example, styrene resins such as polystyrene, vinyl polymers such as polyvinyl chloride, acrylic resins such as polymethyl (meth) acrylate, polyolefin resins such as polyethylene, polyethylene terephthalate, etc. Polyester resins, polycarbonate resins), metals (gold, silver, aluminum, stainless steel, etc.), oxides (glass, silica, alumina, titania, zirconia, indium tin oxide (ITO), etc.), agar and the like. More specifically, well plates used in ELISA (Enzyme-Linked Immunosorbent Assay) method, glass beads, magnetic beads, metal particles, silica beads, zirconia beads, cellulose gel, agarose gel, TOYOPEARRL (registered trademark, Tosoh Corporation) Polymer gels).
本発明で用いるオリゴペプチドを結合した担体（オリゴペプチド結合担体）は、前記オリゴペプチドを前述した担体に化学的又は物理的に固定化することで得られる。本発明にかかるオリゴペプチドの前記担体への固定化方法としては特に制限はなく、例えば、物理吸着、静電的相互作用、疎水的相互作用、架橋剤等を利用する方法が挙げられる。本発明にかかるオリゴペプチドの固定化時の濃度は、担体の材質、形状、固定化の方法等に合わせて適宜調整すればよく、例えば、５〜５０μｇ／ｍＬの濃度が挙げられ、好ましくは１０〜４０μｇ／ｍＬである。また、その際、オリゴペプチド固定化後の担体に対しブロッキング剤によるブロッキング操作を行なうと、非特異吸着が抑えられ、測定時のバックグラウンドも抑えられるため、好ましい。ブロッキング剤は、ＥＬＩＳＡ法等、抗原抗体反応を利用した測定の分野で通常用いられる剤の中から適宜選択すればよく、好ましいブロッキング剤の一態様として、コラーゲンペプチドが挙げられる。 The carrier to which the oligopeptide used in the present invention is bound (oligopeptide binding carrier) can be obtained by chemically or physically immobilizing the oligopeptide on the above-mentioned carrier. The method for immobilizing the oligopeptide according to the present invention on the carrier is not particularly limited, and examples thereof include a method using physical adsorption, electrostatic interaction, hydrophobic interaction, a crosslinking agent and the like. What is necessary is just to adjust the density | concentration at the time of the fixation | immobilization of the oligopeptide concerning this invention suitably according to the material of a support | carrier, a shape, the immobilization method etc., for example, the density | concentration of 5-50 microgram / mL is mentioned, Preferably it is 10 ~ 40 μg / mL. Further, at that time, it is preferable to perform a blocking operation with a blocking agent on the carrier after immobilization of the oligopeptide because nonspecific adsorption can be suppressed and the background during measurement can also be suppressed. What is necessary is just to select a blocking agent suitably from the agents normally used in the field | area of the measurement using an antigen antibody reaction, such as ELISA method, A collagen peptide is mentioned as one aspect | mode of a preferable blocking agent.
本発明において、上述のオリゴペプチド結合担体に添加する溶液に含まれる安定化剤は、非イオン界面活性剤及び／又は陽イオン界面活性剤である。非イオン界面活性剤や陽イオン界面活性剤は、抗原抗体反応を利用した測定試薬において通常用いられる試薬の中から適宜選択すればよい。非イオン界面活性剤の一例としては、ポリソルベート２０（商品名：Ｔｗｅｅｎ ２０）、ポリソルベート８０（商品名：Ｔｗｅｅｎ ８０）、Ｔｒｉｔｏｎ Ｘ−１００（商品名）やＴｒｉｔｏｎ Ｘ−１１４（商品名）等のオクチルフェノールエトキシレート、Ｂｒｉｊ ５８（商品名）、Ｂｒｉｊ ３５（商品名）、ｎ−オクチル−β−Ｄ−グルコピラノシド、ｎ−オクチル−β−Ｄ−チオグルコピラノシド、ｎ−ドデシル−β−Ｄ−マルトピラノシド、ｎ−ドデシル−α−Ｄ−マルトピラノシド、ｎ−ドデシル−Ｎ，Ｎ-ジメチルアミン−Ｎ−オキシド、スクロースモノドデカン酸、ｎ−オクチル−β−Ｄ−グルコピラノシド、ｎ−ドデシル−β−Ｄ−マルトピラノシド、ｎ−トリデシル−β−Ｄ−マルトピラノシドが挙げられる。また陽イオン界面活性剤の一例としては、セチルトリメチルアンモニウムブロミド（ＣＴＡＢ）、セチルジメチルアンモニウムブロミド、セチルトリメチルアンモニウムクロライド、ミリスチルトリメチルアンモニウムブロミドが挙げられる。界面活性剤の濃度も、臨界ミセル濃度及び抗原抗体反応を利用した測定試薬で通常用いられる濃度範囲を考慮し、適宜決定すればよい。一例として、安定化剤として非イオン界面活性剤であるポリソルベート２０を用いる場合、０．００００５％（ｗ／ｖ）から０．０２％（ｗ／ｖ）までの間とすればよく、０．０００１％（ｗ／ｖ）から０．０１％（ｗ／ｖ）までの間とすると好ましく、０．０００５％（ｗ／ｖ）から０．００８％（ｗ／ｖ）までの間とするとより好ましい。また安定化剤として非イオン界面活性剤であるオクチルフェノールエトキシレートを用いる場合、０．００５％（ｖ／ｖ）から０．１％（ｖ／ｖ）までの間とすると好ましく、０．０１％（ｖ／ｖ）から０．０５％（ｖ／ｖ）までの間とするとより好ましい。さらに安定化剤として陽イオン界面活性剤であるＣＴＡＢを用いる場合、０．００５％（ｗ／ｖ）から０．１％（ｗ／ｖ）までの間とすればよく、０．０１％（ｗ／ｖ）から０．０５％（ｗ／ｖ）までの間とすると好ましい。 In the present invention, the stabilizer contained in the solution added to the above-mentioned oligopeptide binding carrier is a nonionic surfactant and / or a cationic surfactant. What is necessary is just to select a nonionic surfactant and a cationic surfactant suitably from the reagents normally used in the measuring reagent using an antigen antibody reaction. Examples of nonionic surfactants include octylphenols such as polysorbate 20 (trade name: Tween 20), polysorbate 80 (trade name: Tween 80), Triton X-100 (trade name) and Triton X-114 (trade name). Ethoxylate, Brij 58 (trade name), Brij 35 (trade name), n-octyl-β-D-glucopyranoside, n-octyl-β-D-thioglucopyranoside, n-dodecyl-β-D-maltopyranoside, n- Dodecyl-α-D-maltopyranoside, n-dodecyl-N, N-dimethylamine-N-oxide, sucrose monododecanoic acid, n-octyl-β-D-glucopyranoside, n-dodecyl-β-D-maltopyranoside, n- And tridecyl-β-D-maltopyranoside. Examples of cationic surfactants include cetyltrimethylammonium bromide (CTAB), cetyldimethylammonium bromide, cetyltrimethylammonium chloride, and myristyltrimethylammonium bromide. The concentration of the surfactant may be appropriately determined in consideration of the critical micelle concentration and the concentration range usually used in the measurement reagent utilizing the antigen-antibody reaction. As an example, when using polysorbate 20 which is a nonionic surfactant as a stabilizer, it may be between 0.00005% (w / v) and 0.02% (w / v). % (W / v) to 0.01% (w / v) is preferable, and 0.0005% (w / v) to 0.008% (w / v) is more preferable. Moreover, when using octylphenol ethoxylate which is a nonionic surfactant as a stabilizer, it is preferable to make it between 0.005% (v / v) and 0.1% (v / v), 0.01% ( More preferably, it is between v / v) and 0.05% (v / v). Furthermore, when using CTAB which is a cationic surfactant as a stabilizer, it may be between 0.005% (w / v) and 0.1% (w / v), and 0.01% (w / V) to 0.05% (w / v).
本発明で用いる安定化剤は、非イオン界面活性剤及び陽イオン界面活性剤のうち、少なくとも１つ含んでいればよく、非イオン界面活性剤のみを１つ又は複数含んでもよいし、陽イオン界面活性剤のみを１つ又は複数含んでもよいし、１つ又は複数の非イオン界面活性剤と１つ又は複数の非イオン界面活性剤とを組み合わせてもよい。 The stabilizer used in the present invention may contain at least one of a nonionic surfactant and a cationic surfactant, may contain only one or a plurality of nonionic surfactants, Only one or more surfactants may be included, or one or more nonionic surfactants and one or more nonionic surfactants may be combined.
また、本発明で用いる安定化剤を溶解させるための液体としては、特に制限はなく、緩衝液（例えば、ｐＨ６〜８の緩衝液（リン酸緩衝食塩水（ＰＢＳ））等）が挙げられる。 Moreover, there is no restriction | limiting in particular as a liquid for dissolving the stabilizer used by this invention, For example, a buffer solution (For example, buffer solution (phosphate buffered saline (PBS)) etc. of pH 6-8) etc. are mentioned.
そして、本発明においては、かかる安定化剤を含む溶液を、上述のオリゴヌクレオチド結合担体に添加した後、当該溶液を凍結乾燥することで、変性抗体測定試薬を製造することができる。 And in this invention, after adding the solution containing this stabilizer to the above-mentioned oligonucleotide binding support | carrier, the modified | denatured antibody measuring reagent can be manufactured by freeze-drying the said solution.
安定化剤を含む溶液の添加は、用いる担体の種類、形態に応じ、適宜公知の手法を用いて行えばよい。また、凍結乾燥の条件としては、前記溶液の凍結、及びそれに続く乾燥に適した温度での水の昇華を行える条件であればよく、変性抗体測定試薬の組成、担体の種類及び大きさ等に応じて変化し得るが、通常、凍結の際の温度は、−１０℃以下の温度（例えば−５０℃〜−１０℃、好ましくは−４０℃〜−２０℃）である。凍結時間は、通常３０分以上（例えば０．５〜４８時間、好ましくは１〜２４時間、さらに好ましくは２〜１０時間）である。なお、凍結を多段階で行う場合、各段階において、凍結温度及び凍結時間は、それぞれ、同じであってもよく、異なっていてもよい。また、その後の乾燥において、乾燥温度は、凍結温度と同じ温度であってもよく、凍結温度より高めの温度（例えば、−１０℃〜４０℃、好ましくは−２０℃〜２５℃）である。また、乾燥時間は、例えば、０．５〜７２時間（好ましくは１〜７２時間、より好ましくは２〜４８時間）が挙げられる。なお、凍結後の乾燥を多段階で行う場合、各段階において、乾燥温度及び乾燥時間は、それぞれ、同じであってもよく、異なっていてもよい。また、凍結乾燥において、乾燥圧力は、通常３００ｍｍＴｏｒｒ以下であり、好ましくは２００ｍｍＴｏｒｒ以下、より好ましくは１００ｍｍＴｏｒｒ以下（例えば、５０〜１００ｍｍＴｏｒｒ）である。本発明における凍結乾燥の条件のより具体的な一例として、−４０℃で３０分以上放置することで凍結させた後、真空条件（１００ｍｍｔｏｒｒ以下）で−２０℃で２時間放置することで１次乾燥し、２５℃で２時間放置することで２次乾燥することが挙げられる。 The addition of the solution containing the stabilizer may be appropriately performed using a known method depending on the type and form of the carrier used. The freeze-drying conditions may be any conditions as long as the solution can be frozen and water sublimated at a temperature suitable for subsequent drying, and the composition of the denatured antibody measurement reagent, the type and size of the carrier, etc. The temperature during freezing is usually -10 ° C or lower (eg, -50 ° C to -10 ° C, preferably -40 ° C to -20 ° C). The freezing time is usually 30 minutes or longer (for example, 0.5 to 48 hours, preferably 1 to 24 hours, more preferably 2 to 10 hours). When freezing is performed in multiple stages, the freezing temperature and the freezing time may be the same or different in each stage. Further, in the subsequent drying, the drying temperature may be the same as the freezing temperature, or a temperature higher than the freezing temperature (for example, −10 ° C. to 40 ° C., preferably −20 ° C. to 25 ° C.). The drying time is, for example, 0.5 to 72 hours (preferably 1 to 72 hours, more preferably 2 to 48 hours). In addition, when drying after freezing is performed in multiple stages, in each stage, the drying temperature and the drying time may be the same or different. In freeze-drying, the drying pressure is usually 300 mmTorr or less, preferably 200 mmTorr or less, more preferably 100 mmTorr or less (for example, 50 to 100 mmTorr). As a more specific example of the lyophilization conditions in the present invention, after freezing for 30 minutes or longer at −40 ° C., the primary condition is left for 2 hours at −20 ° C. under vacuum conditions (100 mmtorr or less). It can be dried and left to stand at 25 ° C. for 2 hours for secondary drying.
また、担体がウェルプレート等のプレートの場合は、該プレートに設けた保持部（ウェル等）に前記オリゴペプチドを結合させた後、当該保持部に安定化剤を含む溶液を添加し、前記溶液をプレートごと凍結乾燥（条件：一例として、−４０℃で３０分以上放置することで凍結させた後、真空条件（１００ｍｍｔｏｒｒ以下）で−２０℃で２時間放置することで１次乾燥し、２５℃で２時間放置することで２次乾燥する）することで、本発明の変性抗体測定試薬を製造することができる。また担体がビーズの場合は、該ビーズに前記オリゴペプチドを結合させた後、得られたオリゴペプチド結合ビーズと安定化剤を含む溶液とを一つの容器に収容し、前記溶液を容器ごと凍結乾燥（条件：一例として、−４０℃で３０分以上放置することで凍結させた後、真空条件（１００ｍｍｔｏｒｒ以下）で−２０℃で２時間放置することで１次乾燥し、２５℃で２時間放置することで２次乾燥する）することで、本発明の変性抗体測定試薬を製造することができる。 In the case where the carrier is a plate such as a well plate, the oligopeptide is bound to a holding part (well or the like) provided on the plate, and then a solution containing a stabilizer is added to the holding part. The plate was freeze-dried (conditions: as an example, after being left to stand for 30 minutes or longer at −40 ° C., and then dried for 2 hours at −20 ° C. under a vacuum condition (100 mmtorr or less) for primary drying, 25 The denatured antibody measurement reagent of the present invention can be produced by subjecting it to secondary drying by allowing it to stand at 2 ° C. for 2 hours. When the carrier is a bead, after the oligopeptide is bound to the bead, the resulting oligopeptide-bound bead and a solution containing a stabilizer are contained in one container, and the solution is freeze-dried together with the container. (Condition: As an example, after freezing by leaving it to stand at −40 ° C. for 30 minutes or more, it was primarily dried by leaving it at −20 ° C. for 2 hours under vacuum conditions (100 mmtorr or less), and left at 25 ° C. for 2 hours. Then, the modified antibody measurement reagent of the present invention can be produced.
＜変性した抗体の量を測定する方法＞
上述の通り、本発明の方法によれば、その性能を保持したまま、冷蔵での長期保存が可能な試薬を提供することが可能となる。そして、その試薬は、変性した抗体の量を測定することに好適に用いられる。したがって、本発明の変性した抗体の量を測定する方法は、以下の（１）及び（２）に示す工程を含む方法である。
（１）被検抗体を含む試料と、本発明の変性した抗体の量を測定するための試薬と、被検抗体を認識する抗体に標識物質が結合している標識抗体とを接触させることにより、オリゴペプチド結合担体と変性した抗体と標識抗体とを含む複合体を形成させる工程
（２）（１）で形成した複合体中の標識物質を検出することにより、前記試料中の変性した抗体の量を測定する工程。
<Method for measuring the amount of denatured antibody>
As described above, according to the method of the present invention, it is possible to provide a reagent that can be stored for a long time in a refrigerator while maintaining its performance. The reagent is suitably used for measuring the amount of denatured antibody. Therefore, the method for measuring the amount of the modified antibody of the present invention is a method comprising the steps shown in the following (1) and (2).
(1) By contacting a sample containing a test antibody, a reagent for measuring the amount of the modified antibody of the present invention, and a labeled antibody in which a labeling substance is bound to an antibody that recognizes the test antibody (2) Forming a complex containing the oligopeptide-binding carrier, the denatured antibody and the labeled antibody (2) By detecting the labeling substance in the complex formed in (1), the denatured antibody in the sample is detected. The process of measuring the quantity.
本発明において、試料とは、変性、未変性の如何を問わず、上述の抗体を含むものであれば特に制限はなく、通常、抗体を含む溶液であり、特にヒト抗体を含むものが好ましい。また、溶液としても、変性した抗体と上記オリゴペプチドとの結合を阻害しない限り、特に制限はない。 In the present invention, the sample is not particularly limited as long as it contains the above-mentioned antibody regardless of whether it is denatured or undenatured, and is usually a solution containing an antibody, and particularly preferably contains a human antibody. Further, the solution is not particularly limited as long as it does not inhibit the binding between the modified antibody and the oligopeptide.
本発明において、被検抗体を認識する抗体としては、上記変性した抗体を含む被検抗体を認識し得る抗体であればよく、ＩｇＧ、ＩｇＡ、ＩｇＤ、ＩｇＥ、ＩｇＭ、ＩｇＡ、ＩｇＹ等のいずれのアイソタイプであってもよい。また、抗体の形態についても特に制限はなく、被検抗体を認識する抗体は、その機能的断片であってもよい。しかしながら、被検抗体を認識する抗体が変性していたとしても、該変性した認識抗体は前記オリゴペプチド結合担体に結合し難く、かつ、披検抗体中の変性した抗体のみが前記オリゴペプチド結合担体に結合し易くなるという観点から、被検抗体を認識する抗体は、Ｆｃ領域を含まない抗体の機能的断片が好ましい。かかるＦｃ領域を含まない抗体の機能的断片としては、例えば、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、Ｆｖ、ｓｃＦｖ、ダイアボディーが挙げられる。また、被検抗体を認識する抗体と変性した抗体を含む被検抗体とは、異なる種由来であることが好ましく、例えば、変性した抗体を含む被検抗体がヒト由来の抗体である場合には、被検抗体を認識する抗体は、非ヒト動物由来の抗体（例えば、マウス抗体、ラット抗体、ウサギ抗体、ヤギ抗体）が挙げられる。 In the present invention, the antibody that recognizes the test antibody may be any antibody that can recognize the test antibody including the modified antibody, and any of IgG, IgA, IgD, IgE, IgM, IgA, IgY, etc. It may be an isotype. The form of the antibody is not particularly limited, and the antibody that recognizes the test antibody may be a functional fragment thereof. However, even if the antibody recognizing the test antibody is denatured, the denatured recognition antibody is difficult to bind to the oligopeptide-binding carrier, and only the denatured antibody in the test antibody is the oligopeptide-binding carrier. In view of facilitating binding to the antibody, the antibody recognizing the test antibody is preferably a functional fragment of an antibody not containing the Fc region. Examples of such a functional fragment of an antibody that does not contain the Fc region include Fab, Fab ', F (ab') 2, Fv, scFv, and diabody. The antibody recognizing the test antibody and the test antibody containing the denatured antibody are preferably derived from different species. For example, when the test antibody containing the denatured antibody is a human-derived antibody, Examples of the antibody recognizing the test antibody include antibodies derived from non-human animals (eg, mouse antibody, rat antibody, rabbit antibody, goat antibody).
本発明で用いる被検抗体を認識する抗体に結合する標識物質は、抗原抗体反応を利用した測定の分野で通常用いられる物質の中から適宜選択すればよく、一例として、フルオレセイン等の蛍光物質や、アルカリホスファターゼ等の酵素、放射性物質が挙げられる。 The labeling substance that binds to the antibody that recognizes the test antibody used in the present invention may be appropriately selected from substances usually used in the field of measurement using antigen-antibody reaction. As an example, a fluorescent substance such as fluorescein, , Enzymes such as alkaline phosphatase, and radioactive substances.
また、被検抗体を認識する抗体と標識物質との結合は、後述の当該物質の検出における簡便さから、通常リンカー（例えば、ＮＨＳエステル、マレイミド）等を介した架橋反応により行われるが、標識物質を結合させた二次抗体を用いて行うこともできる。ここで、二次抗体とは、被検抗体を認識する抗体に特異的な結合性を示す抗体である。そして、この特異的な結合性を利用することにより、当該二次抗体を介し、被検抗体を認識する抗体に標識物質を間接的に結合させることができる。また、この標識物質が結合された二次抗体を用いる場合には、オリゴペプチド結合担体及び変性した抗体と複合体を形成した後に、当該二次抗体を介して抗体を認識する抗体に標識物質を結合させてもよい。 In addition, the binding between the antibody recognizing the test antibody and the labeling substance is usually performed by a cross-linking reaction via a linker (for example, NHS ester, maleimide) or the like for the convenience of detection of the substance described later. It can also be performed using a secondary antibody to which a substance is bound. Here, the secondary antibody is an antibody that exhibits specific binding to an antibody that recognizes the test antibody. Then, by utilizing this specific binding property, the labeling substance can be indirectly bound to the antibody recognizing the test antibody via the secondary antibody. When a secondary antibody to which this labeling substance is bound is used, after forming a complex with the oligopeptide binding carrier and the denatured antibody, a labeling substance is added to the antibody that recognizes the antibody via the secondary antibody. It may be combined.
本発明において、本発明の変性した抗体の量を測定するための試薬と被検抗体を含む試料と標識抗体とを接触させる際の条件としては、上述の前記試薬と当該試料中の変性した抗体とが結合できる条件であり、また被検抗体と標識抗体とが結合できる条件であればよく、通常、０〜４５℃の温度下での接触であり、好ましくは０〜４０℃の温度下での接触であり、より好ましくは４〜３７℃の温度下であり、さらに好ましくは２５℃〜３７℃の温度下での接触である。また、接触させる時間としても特に制限はなく、前記試薬中のオリゴペプチド結合担体の種類、試料中の抗体及び標識抗体の濃度、標識物質の種類等により、適宜調整され得るが、通常５分〜６時間であり、好ましくは１０分〜２時間、より好ましくは２０分〜１時間である。 In the present invention, the conditions for bringing the reagent for measuring the amount of the modified antibody of the present invention, a sample containing the test antibody, and the labeled antibody into contact with the above-mentioned reagent and the modified antibody in the sample are as follows. And can be any conditions as long as the test antibody and the labeled antibody can bind to each other. Usually, the contact is performed at a temperature of 0 to 45 ° C., preferably at a temperature of 0 to 40 ° C. More preferably, the contact is performed at a temperature of 4 to 37 ° C., and more preferably the contact is performed at a temperature of 25 to 37 ° C. Further, the contacting time is not particularly limited, and may be appropriately adjusted depending on the type of oligopeptide binding carrier in the reagent, the concentration of antibody and labeled antibody in the sample, the type of labeled substance, etc. 6 hours, preferably 10 minutes to 2 hours, more preferably 20 minutes to 1 hour.
また、この接触後、前記試薬に結合できなかった抗体、被検抗体に結合できなかった標識抗体等を除去するため、本発明の方法においては洗浄工程を設けてもよい。その洗浄に用いられる洗浄液としては、前記試薬と変性した抗体との結合及び被検抗体と標識抗体との結合を阻害せず、担体等に非特異的に吸着した抗体を洗浄できるものであればよく、例えば、緩衝液（ｐＨ６〜８）、より具体的には、トリス緩衝液、リン酸緩衝液、ＨＥＰＥＳ緩衝液が挙げられる。また、これらの緩衝液においては、塩類、界面活性化剤、タンパク質、糖、双性イオン化合物等を適宜含有していてもよい。また、市販の免疫反応用試薬キットに含まれる洗浄液（例えば、Ｅテスト「ＴＯＳＯＨ」ＩＩ洗浄液、東ソー株式会社製）も好適に用いられる。 Further, after this contact, a washing step may be provided in the method of the present invention in order to remove the antibody that could not be bound to the reagent, the labeled antibody that could not be bound to the test antibody, and the like. The washing solution used for the washing is not limited to the binding between the reagent and the denatured antibody and the binding between the test antibody and the labeled antibody, and can wash the antibody adsorbed nonspecifically on the carrier or the like. Well, for example, a buffer solution (pH 6-8), more specifically, a Tris buffer solution, a phosphate buffer solution, and a HEPES buffer solution may be mentioned. Moreover, these buffers may contain salts, surfactants, proteins, sugars, zwitterionic compounds and the like as appropriate. In addition, a cleaning liquid (for example, E test “TOSOH” II cleaning liquid, manufactured by Tosoh Corporation) included in a commercially available reagent kit for immune reaction is also preferably used.
なお、本発明にかかる工程（１）に関し、上述のとおり、本発明の変性した抗体の量を測定するための試薬と被検抗体を含む試料と標識抗体とを一遍に接触させることにより、前記試薬と変性した抗体と標識抗体とを含む複合体を形成させる態様について説明したが、本発明にかかる工程（１）は上記実施形態に限定されるものではない。例えば、前記試薬と被検抗体を含む試料とを先ず接触させることにより、前記試薬と変性した抗体とを含む複合体を形成し、次いで標識抗体を更に接触させることにより、前記試薬と変性した抗体と標識抗体とを含む複合体を形成してもよい。また、本発明にかかる工程（１）においては、被検抗体を含む試料と標識抗体とを先ず接触させることにより、変性した抗体と標識抗体とを含む複合体を形成し、次いで前記試薬を更に接触させることにより、前記試薬と変性した抗体と標識抗体とを含む複合体を形成してもよい。なお、前記各物質の接触条件は上述のとおりである。また、前記各物質の接触後においても、前記試薬に結合できなかった抗体、被検抗体に結合できなかった標識抗体等を除去するため、上述の洗浄工程を適宜設けてもよい。また、このような実施形態において、変性した抗体の検出感度がより向上し易いという観点から、本発明にかかる工程（１）は、前記試薬と被検抗体を含む試料とを先ず接触させ、次いで標識抗体と接触させる工程であることが好ましい。 In addition, regarding the step (1) according to the present invention, as described above, the reagent for measuring the amount of the denatured antibody of the present invention, the sample containing the test antibody, and the labeled antibody are contacted together, thereby Although the aspect which forms the composite_body | complex containing a reagent, the modified | denatured antibody, and a labeled antibody was demonstrated, the process (1) concerning this invention is not limited to the said embodiment. For example, the reagent and the sample containing the test antibody are first brought into contact to form a complex containing the reagent and the denatured antibody, and then the labeled antibody is further brought into contact with the reagent and the denatured antibody. And a complex containing the labeled antibody may be formed. In the step (1) according to the present invention, the sample containing the test antibody and the labeled antibody are first contacted to form a complex containing the denatured antibody and the labeled antibody, and then the reagent is further added. By contacting, a complex containing the reagent, the denatured antibody and the labeled antibody may be formed. In addition, the contact conditions of each said substance are as above-mentioned. In addition, the above-described washing step may be appropriately provided in order to remove the antibody that could not be bound to the reagent, the labeled antibody that could not be bound to the test antibody, and the like even after contact with each substance. In such an embodiment, from the viewpoint that the detection sensitivity of the denatured antibody is more easily improved, the step (1) according to the present invention first contacts the reagent and the sample containing the test antibody, and then The step of contacting with a labeled antibody is preferable.
次に、本発明の方法の工程（２）においては、上述の工程（１）における接触を経て形成された複合体中の標識物質を検出することにより、試料中の変性した抗体の量を測定する。 Next, in the step (2) of the method of the present invention, the amount of the denatured antibody in the sample is measured by detecting the labeling substance in the complex formed through the contact in the above-mentioned step (1). To do.
本発明において標識物質の検出とは、標識物質に由来した信号の検出を意味し、その検出は、当該技術分野の公知の方法にしたがって行うことができる。例えば、標識物質として蛍光物質を用いる場合には、それが発する蛍光をプレートリーダー等を用いて検出及び定量することができる。また、標識物質として酵素を用いる場合には、当該酵素の作用によって分解して発色、発光等する基質を添加することにより、その発色、発光等をプレートリーダー等を用いて検出及び定量することができる。また、標識物質として放射性物質を用いる場合には、当該物質の発する放射線量をシンチレーションカウンター等により検出及び定量することができる。 In the present invention, detection of a labeling substance means detection of a signal derived from the labeling substance, and the detection can be performed according to a known method in the art. For example, when a fluorescent substance is used as the labeling substance, the fluorescence emitted from the fluorescent substance can be detected and quantified using a plate reader or the like. In addition, when an enzyme is used as a labeling substance, it is possible to detect and quantify the coloration, luminescence, etc. using a plate reader or the like by adding a substrate that decomposes by the action of the enzyme to develop coloration, luminescence, etc. it can. When a radioactive substance is used as the labeling substance, the radiation dose emitted from the substance can be detected and quantified using a scintillation counter or the like.
本発明においては、後述の実施例に示す通り、このようにして検出される標識物質に由来した信号の強度（吸光度、蛍光強度等）と、試料中の変性した抗体の量とは、高い相関性を示す。そのため、濃度既知の変性した抗体を含む標準試料を用いて本発明の方法を実施し、被検抗体を含む試料を用いて得られる測定値との間の相関データを取得し、当該相関データに基づき当該被検試料に存在する変性した抗体の量を測定（定量）することができる。なお相関データとは、例えば検量線である。 In the present invention, as shown in Examples described later, the signal intensity (absorbance, fluorescence intensity, etc.) derived from the labeling substance thus detected and the amount of denatured antibody in the sample are highly correlated. Showing gender. For this reason, the method of the present invention is carried out using a standard sample containing a denatured antibody with a known concentration, and correlation data with a measurement value obtained using a sample containing a test antibody is obtained. Based on this, the amount of denatured antibody present in the test sample can be measured (quantified). The correlation data is, for example, a calibration curve.
＜変性した抗体の量を測定するための試薬＞
上述の通り、本発明において、変性した抗体を認識するオリゴペプチドとして配列番号：１に記載のアミノ酸配列を含むオリゴペプチドを用い、かつ安定化剤として非イオン界面活性剤及び／又は陽イオン界面活性剤を含む溶液を用いることにより、凍結乾燥前（液体試薬）における試薬性能は保持しつつ、冷蔵での長期保存が可能な凍結乾燥形態の試薬を得ることができる。
<Reagent for measuring the amount of denatured antibody>
As described above, in the present invention, an oligopeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 is used as an oligopeptide that recognizes a denatured antibody, and a nonionic surfactant and / or cationic surfactant is used as a stabilizer. By using a solution containing an agent, it is possible to obtain a lyophilized reagent that can be stored for a long period of time in a refrigerator while maintaining the reagent performance before lyophilization (liquid reagent).
したがって、本発明の変性した抗体の量を測定するための試薬は、
非イオン界面活性剤及び／又は陽イオン界面活性剤を含む溶液が添加されているオリゴペプチド結合担体を凍結乾燥してなり、かつ、前記オリゴペプチド結合担体が、配列番号：１に記載のアミノ酸配列を含み、変性した抗体を認識するオリゴペプチドが結合している担体である、変性した抗体の量を測定するための試薬である。
Therefore, the reagent for measuring the amount of the modified antibody of the present invention is:
An oligopeptide binding carrier to which a solution containing a nonionic surfactant and / or a cationic surfactant is added is lyophilized, and the oligopeptide binding carrier is an amino acid sequence set forth in SEQ ID NO: 1. Is a reagent for measuring the amount of denatured antibody, which is a carrier to which an oligopeptide that recognizes denatured antibody is bound.
また、その好適な態様として、
０．００００５〜０．０２％（ｖ／ｖ）のポリソルベート２０、０．００５〜０．１％（ｖ／ｖ）のオクチルフェノールエトキシレート及び０．００５〜０．１％（ｗ／ｖ）のセチルトリメチルアンモニウムブロミドからなる群から選択される少なくとも１つの界面活性剤を含む溶液が添加されているオリゴペプチド結合担体を凍結乾燥してなり、かつ、前記オリゴペプチド結合担体が、配列番号：１に記載のアミノ酸配列を含み、変性した抗体を認識するオリゴペプチドが結合している担体である、変性した抗体の量を測定するための試薬が、挙げられる。
Moreover, as the suitable aspect,
0.00005-0.02% (v / v) polysorbate 20, 0.005-0.1% (v / v) octylphenol ethoxylate and 0.005-0.1% (w / v) cetyl An oligopeptide binding carrier to which a solution containing at least one surfactant selected from the group consisting of trimethylammonium bromide is added is lyophilized, and the oligopeptide binding carrier is described in SEQ ID NO: 1. And a reagent for measuring the amount of denatured antibody, which is a carrier to which an oligopeptide that recognizes denatured antibody is bound.
さらに、本発明においては、当該試薬と、上記抗体を認識する抗体に標識物質が結合している標識抗体とを含む、変性した抗体の量を測定するためのキットという態様もとり得る。本発明のキットは、これら以外に、上述の、被検抗体を溶解又は希釈させるための液、ブロッキング剤、洗浄液、酵素の作用によって分解して発色、発光等する基質、その発色等を停止するための停止液、濃度既知の変性した抗体を含む標準試料等を含むものであってもよい。さらに、本発明の試薬及びキットには、本発明の方法を実施する際の、前記オリゴペプチド結合担体等の使用説明書を含めることができる。 Furthermore, in the present invention, an embodiment of a kit for measuring the amount of denatured antibody comprising the reagent and a labeled antibody in which a labeling substance is bound to the antibody recognizing the antibody can be employed. In addition to these, the kit of the present invention stops the above-described solution for dissolving or diluting the test antibody, blocking agent, washing solution, substrate that decomposes by the action of an enzyme, develops color, emits light, etc. And a standard sample containing a denatured antibody with a known concentration may be included. Furthermore, the reagent and kit of the present invention can include instructions for using the oligopeptide-binding carrier and the like when the method of the present invention is carried out.
以下、実施例に基づき、本発明を詳細に説明するが、本発明はこれら実施例により限定されるものではない。 EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited by these Examples.
（実施例１） 安定化剤の検討（その１）
変性抗体試薬の凍結乾燥体を製造する際、添加する、安定化剤の検討を以下の方法で行なった。
（１）本実施例においては、本発明にかかるヒト抗体として抗ＣＤ２０抗体を用いた。そして、１０ｍｇ／ｍＬの抗ＣＤ２０抗体溶液をリン酸緩衝液（ｐＨ７．４）で１ｍｇ／ｍＬに希釈したものを１００ｍｍｏｌ／Ｌ グリシン緩衝液（ｐＨ２．０）で一晩透析することで、変性したヒト抗体溶液を調製した。
（２）下記に示す方法で、配列番号：１に記載のアミノ酸配列を含み、変性したヒト抗体を認識するオリゴペプチド（以下、「２Ａ１」とも称する）の磁性ビーズ（フェライトを練り込んだエチレン−酢酸ビニル共重合体（ＥＶＡ）による粒子）への固定化を行った。
（２−１）４％（ｖ／ｖ）グルタルアルデヒドを磁性ビーズへ添加し、３７℃で２時間撹拌後、２Ａ１を添加し、３７℃で２時間放置することで、磁性ビーズへ固定化した。
（２−２）１％（ｗ／ｖ）コラーゲンペプチド（魚鱗又は魚皮由来のコラーゲン又はゼラチンを加水分解して得られた、重量平均分子量：３０００〜５０００のコラーゲンペプチド、製品名：イクオスＨＤＬ、新田ゼラチン株式会社製）を含む炭酸緩衝液（ｐＨ９．６）を３００μｌ／ｗｅｌｌで添加し、３７℃で２時間撹拌することで、ブロッキング処理を行なった。
（３）全自動エンザイムイムノアッセイ装置ＡＩＡ−６００ＩＩ（東ソー株式会社製）用測定容器に、配列番号：１に記載のアミノ酸配列からなるオリゴペプチド（以下、２Ａ１と表記）を結合した磁性ビーズ及び下記（Ａ）から（Ｈ）に示すいずれかの溶液を添加後、当該測定容器を凍結乾燥機（ＶｉｒＴｉｓ Ｇｅｎｅｓｉｓ ２５ＸＬ、ＳＰ Ｓｃｉｅｎｔｉｆｉｃ製）を用い、凍結乾燥させることで、凍結乾燥試薬を製造した。凍結乾燥の条件としては、−４０℃で３０分以上放置することで凍結した後、真空条件（１００ｍｍｔｏｒｒ以下）で−２０℃で２時間放置することで１次乾燥し、２５℃で２時間放置することで２次乾燥することで行った。
(Example 1) Examination of stabilizer (part 1)
The following method was used to examine the stabilizer added when the lyophilized product of the modified antibody reagent was produced.
(1) In this example, an anti-CD20 antibody was used as the human antibody according to the present invention. Then, 10 mg / mL anti-CD20 antibody solution diluted to 1 mg / mL with phosphate buffer (pH 7.4) was denatured by dialysis overnight with 100 mmol / L glycine buffer (pH 2.0). A human antibody solution was prepared.
(2) An oligopeptide (hereinafter also referred to as “2A1”) that contains the amino acid sequence of SEQ ID NO: 1 and recognizes a denatured human antibody by the method shown below (ethylene-kneaded ethylene- Immobilization to particles with a vinyl acetate copolymer (EVA).
(2-1) 4% (v / v) glutaraldehyde was added to the magnetic beads, stirred for 2 hours at 37 ° C, 2A1 was added, and the mixture was allowed to stand at 37 ° C for 2 hours to be immobilized on the magnetic beads. .
(2-2) 1% (w / v) collagen peptide (obtained by hydrolyzing fish scale or fish skin-derived collagen or gelatin, weight average molecular weight: 3000-5000 collagen peptide, product name: Ikuos HDL, A carbonate buffer solution (pH 9.6) containing Nitta Gelatin Co., Ltd.) was added at 300 μl / well and stirred at 37 ° C. for 2 hours for blocking treatment.
(3) Magnetic beads in which an oligopeptide consisting of the amino acid sequence described in SEQ ID NO: 1 (hereinafter referred to as 2A1) is bound to a measurement container for a fully automatic enzyme immunoassay device AIA-600II (manufactured by Tosoh Corporation) and the following ( After adding any solution shown in A) to (H), the measurement container was freeze-dried using a freeze-dryer (VirTis Genesis 25XL, manufactured by SP Scientific) to produce a freeze-dried reagent. As freeze-drying conditions, after freezing by standing at −40 ° C. for 30 minutes or more, it was first dried by leaving it at −20 ° C. for 2 hours under vacuum conditions (100 mmtorr or less), and left at 25 ° C. for 2 hours. This was done by secondary drying.
なお比較対象として、ＡＩＡ−６００ＩＩ用測定容器に２Ａ１を結合した磁性ビーズのみを添加した試薬（凍結乾燥なし、試薬（Ｉ））も製造した。
（Ａ）リン酸緩衝液（ｐＨ７．４）（添加剤なし）
（Ｂ）０．００５％（ｖ／ｖ）Ｔｗｅｅｎ ２０（商品名）を含むリン酸緩衝液（ｐＨ７．４）
（Ｃ）０．１ｍｏｌ／Ｌスクロースを含むリン酸緩衝液（ｐＨ７．４）
（Ｄ）０．０１ｍｏｌ／Ｌスクロースを含むリン酸緩衝液（ｐＨ７．４）
（Ｅ）１％（ｗ／ｖ）コラーゲンペプチドを含むリン酸緩衝液（ｐＨ７．４）
（Ｆ）０．１％（ｗ／ｖ）コラーゲンペプチドを含むリン酸緩衝液（ｐＨ７．４）
（Ｇ）１０ｍｍｏｌ／Ｌヒスチジンを含むリン酸緩衝液（ｐＨ７．４）
（Ｈ）１ｍｍｏｌ／Ｌヒスチジンを含むリン酸緩衝液（ｐＨ７．４）
（４）（３）で作製した試薬に（１）で調製した変性ヒト抗体溶液を添加し、３７℃で２０分間撹拌した。
（５）Ｅテスト「ＴＯＳＯＨ」ＩＩ洗浄液（東ソー株式会社製）で洗浄することで遊離成分を除去後、アルカリ性ホスファターゼ標識ヤギ由来抗ヒトＩｇＧ Ｆ（ａｂ’）２（ジャクソンイムノリサーチ製、カタログ番号：１０９−０５６−０９７）を含む溶液を添加し、３７℃で２０分間撹拌した。
（６）Ｅテスト「ＴＯＳＯＨ」ＩＩ洗浄液（東ソー株式会社製）で洗浄することで余剰の標識ヒト抗体を除去後、基質である４−メチルウンベリフェリルりん酸を添加し、酵素反応により生じる蛍光物質（４−メチルウンベリフェロン）の生成速度（レート値）を全自動エンザイムイムノアッセイ装置ＡＩＡ−６００ＩＩ（東ソー株式会社製）で測定することで、磁性ビーズに結合した変性ヒト抗体量を測定した。
As a comparison object, a reagent (without lyophilization, reagent (I)) in which only magnetic beads bound with 2A1 were added to a measurement container for AIA-600II was also produced.
(A) Phosphate buffer solution (pH 7.4) (without additives)
(B) Phosphate buffer solution (pH 7.4) containing 0.005% (v / v) Tween 20 (trade name)
(C) Phosphate buffer solution (pH 7.4) containing 0.1 mol / L sucrose
(D) Phosphate buffer solution (pH 7.4) containing 0.01 mol / L sucrose
(E) Phosphate buffer solution (pH 7.4) containing 1% (w / v) collagen peptide
(F) Phosphate buffer solution (pH 7.4) containing 0.1% (w / v) collagen peptide
(G) Phosphate buffer solution (pH 7.4) containing 10 mmol / L histidine
(H) Phosphate buffer solution (pH 7.4) containing 1 mmol / L histidine
(4) The modified human antibody solution prepared in (1) was added to the reagent prepared in (3) and stirred at 37 ° C. for 20 minutes.
(5) After removing free components by washing with E test “TOSOH” II washing solution (manufactured by Tosoh Corporation), alkaline phosphatase-labeled goat-derived anti-human IgG F (ab ′) 2 (manufactured by Jackson ImmunoResearch, catalog number: 109-056-097) was added and stirred at 37 ° C. for 20 minutes.
(6) After removing excess labeled human antibody by washing with E test “TOSOH” II washing solution (manufactured by Tosoh Corporation), the substrate is added with 4-methylumbelliferyl phosphate, and the fluorescence generated by the enzyme reaction The production rate (rate value) of the substance (4-methylumbelliferone) was measured with a fully automatic enzyme immunoassay device AIA-600II (manufactured by Tosoh Corporation), thereby measuring the amount of denatured human antibody bound to the magnetic beads.
種々の濃度の変性ヒト抗体溶液を添加したときのレート値測定結果を図１Ａ〜Ｈに示す。安定化剤としてＴｗｅｅｎ ２０を０．００５％（ｖ／ｖ）添加したとき（図１Ｂ）は、凍結乾燥処理しない試薬（図１Ｉ）と同等の検出感度を有し、検量線の直線性も良好であった（Ｒ２＝０．９９７）。一方、安定化剤を含まない、または非イオン界面活性剤以外の安定化剤を含んだ溶液を添加し、凍結乾燥させると検出感度は低下した（図１Ａ及びＣ〜Ｈ）。このことから、変性ヒト抗体試薬の凍結乾燥体を製造する際、非イオン界面活性剤を添加して凍結乾燥することで、凍結乾燥前（液体試薬）における試薬性能は保持しつつ、冷蔵での長期保存が可能な試薬を提供できることがわかる。 The rate value measurement results when various concentrations of modified human antibody solutions were added are shown in FIGS. When 0.005% (v / v) of Tween 20 is added as a stabilizer (FIG. 1B), the detection sensitivity is equivalent to that of a reagent that is not lyophilized (FIG. 1I), and the linearity of the calibration curve is also good. (R 2 = 0.997). On the other hand, when a solution containing no stabilizer or containing a stabilizer other than the nonionic surfactant was added and freeze-dried, the detection sensitivity decreased (FIGS. 1A and C to H). Therefore, when producing a lyophilized product of a modified human antibody reagent, by adding a nonionic surfactant and lyophilizing it, the reagent performance before lyophilization (liquid reagent) is maintained, while refrigerated. It can be seen that a reagent capable of long-term storage can be provided.
（実施例２） 安定化剤の検討（その２）
実施例１（２）でＡＩＡ−６００ＩＩ用容器に添加する溶液を、下記（Ａ）から（Ｋ）に示すいずれかの溶液とした他は、実施例１と同様な方法で、凍結乾燥体を製造する際、添加する、安定化剤の検討を行なった。
（Ａ）０．０００１％（ｖ／ｖ）Ｔｗｅｅｎ ２０を含むリン酸緩衝液（ｐＨ７．４）
（Ｂ）０．００１％（ｖ／ｖ）Ｔｗｅｅｎ ２０を含むリン酸緩衝液（ｐＨ７．４）
（Ｃ）０．０１％（ｖ／ｖ）Ｔｗｅｅｎ ２０を含むリン酸緩衝液（ｐＨ７．４）
（Ｄ）０．０１％（ｖ／ｖ）Ｔｒｉｔｏｎ Ｘ−１００（商品名）を含むリン酸緩衝液（ｐＨ７．４）
（Ｅ）０．０５％（ｖ／ｖ）Ｔｒｉｔｏｎ Ｘ−１００を含むリン酸緩衝液（ｐＨ７．４）
（Ｆ）０．０１％（ｗ／ｖ）セチルトリメチルアンモニウムブロミド（ＣＴＡＢ）を含むリン酸緩衝液（ｐＨ７．４）
（Ｇ）０．０５％（ｗ／ｖ）ＣＴＡＢを含むリン酸緩衝液（ｐＨ７．４）
（Ｈ）０．１％（ｗ／ｖ）ドデシル硫酸ナトリウム（ＳＤＳ）を含むリン酸緩衝液（ｐＨ７．４）
（Ｉ）０．５％（ｗ／ｖ）ＳＤＳを含むリン酸緩衝液（ｐＨ７．４）
（Ｊ）０．１％（ｗ／ｖ）ＣＨＡＰＳ（３−［（３−Ｃｈｏｌａｍｉｄｏｐｒｏｐｙｌ）ｄｉｍｅｔｈｙｌａｍｍｏｎｉｏ］ｐｒｏｐａｎｅｓｕｌｆｏｎａｔｅ）を含むリン酸緩衝液（ｐＨ７．４）
（Ｋ）０．５％（ｗ／ｖ）ＣＨＡＰＳを含むリン酸緩衝液（ｐＨ７．４）
種々の濃度の変性ヒト抗体溶液を添加したときのレート値測定結果を図２Ａ〜Ｋに示す。安定化剤としてＴｗｅｅｎ ２０を０．０００１％（ｖ／ｖ）から０．０１％（ｖ／ｖ）添加したとき（図２Ａ〜Ｃ）、Ｔｒｉｔｏｎ Ｘ−１００を０．０１％（ｖ／ｖ）から０．０５％（ｖ／ｖ）添加したとき（図２Ｄ及びＥ）、及びＣＴＡＢを０．０１％（ｗ／ｖ）から０．０５％（ｗ／ｖ）添加したとき（図２Ｆ及びＧ）は、凍結乾燥処理しない試薬（図１Ｉ）と同等の検出感度を有した。一方、ＳＤＳ等の陰イオン界面活性剤やＣＨＡＰＳ等の両性界面活性剤を含んだ溶液を添加し、凍結乾燥させると検出感度は低下した（図２Ｈ〜Ｋ）。このことから、変性抗体試薬の凍結乾燥体を製造する際、非イオン界面活性剤又は陽イオン界面活性剤を添加して凍結乾燥することで、凍結乾燥前（液体試薬）における試薬性能は保持しつつ、冷蔵での長期保存が可能な試薬を提供できることがわかる。
(Example 2) Examination of stabilizer (part 2)
In the same manner as in Example 1 except that the solution added to the container for AIA-600II in Example 1 (2) was any one of the following solutions (A) to (K), A study was made on stabilizers to be added during production.
(A) Phosphate buffer solution (pH 7.4) containing 0.0001% (v / v) Tween 20
(B) Phosphate buffer solution (pH 7.4) containing 0.001% (v / v) Tween 20
(C) Phosphate buffer solution (pH 7.4) containing 0.01% (v / v) Tween 20
(D) Phosphate buffer solution (pH 7.4) containing 0.01% (v / v) Triton X-100 (trade name)
(E) Phosphate buffer solution (pH 7.4) containing 0.05% (v / v) Triton X-100
(F) Phosphate buffer solution (pH 7.4) containing 0.01% (w / v) cetyltrimethylammonium bromide (CTAB)
(G) Phosphate buffer solution (pH 7.4) containing 0.05% (w / v) CTAB
(H) Phosphate buffer solution (pH 7.4) containing 0.1% (w / v) sodium dodecyl sulfate (SDS)
(I) Phosphate buffer solution (pH 7.4) containing 0.5% (w / v) SDS
(J) Phosphate buffer (pH 7.4) containing 0.1% (w / v) CHAPS (3-[(3-Cholamidopropyl) dimethylamino] propansulfonate)
(K) Phosphate buffer solution (pH 7.4) containing 0.5% (w / v) CHAPS
The rate value measurement results when various concentrations of modified human antibody solutions were added are shown in FIGS. When Tween 20 is added as a stabilizer from 0.0001% (v / v) to 0.01% (v / v) (FIGS. 2A-C), Triton X-100 is 0.01% (v / v). To 0.05% (v / v) (FIGS. 2D and E) and CTAB from 0.01% (w / v) to 0.05% (w / v) (FIGS. 2F and G) ) Had a detection sensitivity equivalent to that of the reagent that was not lyophilized (FIG. 1I). On the other hand, when a solution containing an anionic surfactant such as SDS or an amphoteric surfactant such as CHAPS was added and lyophilized, the detection sensitivity decreased (FIGS. 2H to K). Therefore, when producing a lyophilized product of a modified antibody reagent, the reagent performance before lyophilization (liquid reagent) is maintained by adding a nonionic surfactant or a cationic surfactant and lyophilizing. However, it can be seen that a reagent that can be stored for a long time in a refrigerator can be provided.
以上説明したように、変性した抗体を認識するオリゴペプチドを結合した担体に安定化剤を含む試薬を添加後、添加後の前記担体及び前記溶液を凍結乾燥することで、変性抗体測定試薬を製造する際、変性した抗体を認識するオリゴペプチドとして配列番号：１に記載のアミノ酸配列を含むオリゴペプチドを用い、かつ安定化剤が非イオン界面活性剤及び／又は陽イオン界面活性剤を用いることにより、凍結乾燥前（液体試薬）における試薬性能は保持しつつ、冷蔵での長期保存が可能な試薬を提供することが可能となる。 As described above, after adding a reagent containing a stabilizer to a carrier bound with an oligopeptide that recognizes a denatured antibody, the carrier after the addition and the solution are freeze-dried to produce a denatured antibody measurement reagent. In that case, an oligopeptide comprising the amino acid sequence shown in SEQ ID NO: 1 is used as an oligopeptide that recognizes a denatured antibody, and the stabilizer is a nonionic surfactant and / or a cationic surfactant. It is possible to provide a reagent that can be stored for a long period of time in a refrigerator while maintaining the reagent performance before lyophilization (liquid reagent).
そして、当該試薬を用いれば、ストレス（例えば、酸、アルカリ、加熱、凍結融解、撹拌）等により変性を受けた抗体の量を迅速・簡便に測定することできる。したがって、本発明は、抗体を含む医薬品の品質管理等において有用である。 And if the said reagent is used, the quantity of the antibody which modified | denatured by stress (for example, an acid, an alkali, a heating, freeze-thaw, stirring) etc. can be measured rapidly and simply. Therefore, the present invention is useful for quality control of pharmaceuticals containing antibodies.
Claims (4)
変性した抗体を認識するオリゴペプチドを結合した担体に、安定化剤を含む溶液を添加した後、添加後の前記担体及び前記溶液を凍結乾燥する工程を含み、
前記オリゴペプチドが配列番号：１に記載のアミノ酸配列を含むオリゴペプチドであり、かつ前記安定化剤が非イオン界面活性剤及び／又は陽イオン界面活性剤である、方法。 A method for producing a reagent for measuring a denatured antibody, comprising:
A step of adding a stabilizer-containing solution to a carrier bound with an oligopeptide that recognizes a denatured antibody, and then freeze-drying the carrier and the solution after the addition,
A method wherein the oligopeptide is an oligopeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 and the stabilizer is a nonionic surfactant and / or a cationic surfactant.
（１）被検抗体を含む試料と、請求項３に記載の試薬と、被検抗体を認識する抗体に標識物質が結合している標識抗体とを接触させることにより、オリゴペプチド結合担体と変性した抗体と標識抗体とを含む複合体を形成させる工程
（２）（１）で形成した複合体中の標識物質を検出することにより、前記試料中の変性した抗体の量を測定する工程。 A method for measuring the amount of denatured antibody comprising the following steps (1) and (2): (1) a sample containing a test antibody; the reagent according to claim 3; and recognizing the test antibody In the complex formed in the step (2) and (1), a complex containing the oligopeptide binding carrier, the denatured antibody and the labeled antibody is formed by contacting the labeled antibody with the labeled substance bound to the antibody. A step of measuring the amount of the denatured antibody in the sample by detecting the labeling substance.
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