JP2019078590A - Freeze-dried reagent used in isolation of vitamins - Google Patents
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本発明は、ビタミン類の遊離に使用する凍結乾燥試薬に関するものである。 The present invention relates to a lyophilization reagent used to release vitamins.
ビタミン類にはビタミンＤ代謝産物、ビタミンＢ１２及び葉酸などの小分子がある。血液中のそれらの小分子は、その大部分が特異的又は非特異的に蛋白質、脂質などと結合しているため、それらの小分子を測定するには、小分子を前記タンパク質等から遊離する必要がある。なお、ビタミン類にはそれぞれ特異的な結合タンパク質が存在し、血液中での安定性に貢献している。例えば、ビタミンＤバインディングプロテイン（ＤＢＰ）、ハプトコリンや内因子などが良く知られている。 Vitamins include vitamin D metabolites, vitamin B 12 and small molecules such as folic acid. Most of the small molecules in the blood are specifically or nonspecifically bound to proteins, lipids and the like, so to measure the small molecules, the small molecules are released from the proteins and the like. There is a need. Each of the vitamins has a specific binding protein, which contributes to its stability in blood. For example, vitamin D binding protein (DBP), haptocholine and intrinsic factor are well known.
従来知られているビタミン類の結合タンパク質の遊離方法として、アルカリ処理と還元処理が報告されており、特にアルカリ剤として水酸化ナトリウム水溶液、還元剤としてジチオトレイトール（ＤＴＴ）を用いる場合が多い。 Alkali treatment and reduction treatment have been reported as conventionally known methods for liberating bound proteins of vitamins, and in particular, sodium hydroxide aqueous solution as an alkali agent and dithiothreitol (DTT) as a reducing agent are often used.
還元剤は水溶液中、特にアルカリ水溶液中では還元力が低下するため、凍結乾燥して保存し、且つアルカリ剤とは別の容器に保存されている。そのためビタミン類と結合タンパク質の遊離を実施する前には還元剤とアルカリ剤のどちらか一方又は双方を水などで溶解した後に、それらを混合してから遊離に用いる方法が一般的である。しかし、そのような従来法では、煩雑であり、ビタミン類と結合タンパク質の遊離の再現性を低下させ、ビタミン類の測定精度を悪化させるといった問題を有している。 The reducing agent is stored by freeze-drying and stored in a container separate from the alkaline agent, because the reducing power is reduced in an aqueous solution, particularly in an alkaline aqueous solution. Therefore, a common method is to dissolve either the reducing agent or the alkaline agent or both in water before mixing the vitamins and the binding protein, and then mix them and then use it for release. However, such conventional methods are complicated, and have problems such as decreasing the reproducibility of release of vitamins and binding proteins and deteriorating the measurement accuracy of vitamins.
本発明は、同一容器内に還元剤とアルカリ剤を含有し、且つ還元剤の還元力を保持した試薬を提供することを目的としている。 An object of the present invention is to provide a reagent which contains a reducing agent and an alkaline agent in the same container and retains the reducing power of the reducing agent.
本発明者らは、前記課題を解決すべく鋭意検討を行なった結果、還元剤とアルカリ剤を同一容器内で別個に凍結した後、凍結乾燥することにより、還元剤とアルカリ剤を同一容器内に含有した凍結乾燥試薬とできることを見出した。 As a result of intensive studies to solve the above problems, the present inventors separately freeze the reducing agent and the alkaline agent in the same container and freeze-dry the same, thereby reducing the reducing agent and the alkaline agent in the same container. It was found that it can be used as a freeze-dried reagent contained in
即ち、本発明は以下のとおりである。
（１）還元剤を含有する凍結乾燥体及びアルカリ剤を含有する凍結乾燥体を同一容器内に有することを特徴とする、凍結乾燥試薬。
（２）還元剤がトリス（２−カルボキシエチル）ホスフィン（ＴＣＥＰ）、トリブチルホスフィン（ＴＢＰ）、２−メルカプトエタノール、２−メルカプトエタノールアミン、ジチオトレイトール（ＤＴＴ）又はジチオエリトリトール（ＤＴＥ）である、（１）に記載の凍結乾燥試薬。
（３）アルカリ剤が水酸化ナトリウム又は水酸化カリウムである、（１）又は（２）に記載の凍結乾燥試薬。
（４）アルカリ剤溶液又は還元剤溶液のいずれか一方を容器に分注し、凍結させた後、その凍結状態を保ったまま、他方を当該容器に分注し、凍結させ、その後、凍結乾燥する工程を含むことを特徴とする、凍結乾燥試薬の製造方法。
（５）（１）〜（３）のいずれかに記載の凍結乾燥試薬を水溶液で溶解し、血液試料と混合し、前記血液試料中に含有されるビタミン類とタンパク質との結合物から前記ビタミン類を遊離させることを特徴とする、ビタミン類の遊離方法。
（６）ビタミン類がビタミンＤ代謝産物、ビタミンＢ１２又は葉酸である、（５）に記載の遊離方法。
That is, the present invention is as follows.
(1) A lyophilization reagent comprising a lyophilizate containing a reducing agent and a lyophilizate containing an alkaline agent in the same container.
(2) The reducing agent is tris (2-carboxyethyl) phosphine (TCEP), tributylphosphine (TBP), 2-mercaptoethanol, 2-mercaptoethanolamine, dithiothreitol (DTT) or dithioerythritol (DTE), The lyophilization reagent as described in (1).
(3) The lyophilizing reagent according to (1) or (2), wherein the alkaline agent is sodium hydroxide or potassium hydroxide.
(4) Either one of the alkaline agent solution and the reducing agent solution is dispensed into a container and frozen, and then the other is dispensed into the container while keeping its frozen state, and then it is freeze-dried A process for producing a lyophilization reagent, comprising the steps of
(5) The lyophilized reagent according to any one of (1) to (3) is dissolved in an aqueous solution, mixed with a blood sample, and a combination of vitamins and a protein contained in the blood sample is used as the vitamin A method of releasing vitamins, characterized in that
(6) The method according to (5), wherein the vitamin is a vitamin D metabolite, vitamin B 12 or folic acid.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明の凍結乾燥試薬は、還元剤を含有する凍結乾燥体及びアルカリ剤を含有する凍結乾燥体を同一容器内に有することを特徴とする。還元剤及びアルカリ剤は、各成分以外に例えば糖類やｐＨを調製するための緩衝剤等を含んでいても良い。 The freeze-dried reagent of the present invention is characterized in that the freeze-dried body containing the reducing agent and the freeze-dried body containing the alkali agent are contained in the same container. The reducing agent and the alkaline agent may contain, for example, saccharides, buffers for adjusting pH, and the like in addition to the respective components.
また、還元剤としては、トリス（２−カルボキシエチル）ホスフィン（ＴＣＥＰ）、トリブチルホスフィン（ＴＢＰ）、２−メルカプトエタノール、２−メルカプトエタノールアミン、ジチオトレイトール（ＤＴＴ）又はジチオエリトリトール（ＤＴＥ）が好ましく、アルカリ剤としては水酸化ナトリウム又は水酸化カリウムが好ましい。 Also, as the reducing agent, tris (2-carboxyethyl) phosphine (TCEP), tributylphosphine (TBP), 2-mercaptoethanol, 2-mercaptoethanolamine, dithiothreitol (DTT) or dithioerythritol (DTE) is preferable. As the alkali agent, sodium hydroxide or potassium hydroxide is preferable.
次に、本発明の製造方法について説明する。 Next, the manufacturing method of the present invention will be described.
まず、還元剤成分又はアルカリ水溶液のいずれかを容器に分注し、凍結する。使用する容器に特に制限はないが、プラスチックやガラス容器等を用いることができる。分注方法としては室温条件下、凍結方法としては−２０〜−４０℃条件下で３０分以上置くことが好ましい。次に、分注した水溶液の凍結状態を保ったまま、他方成分を分注して凍結する。容器は十分に冷却しておき、分注と同時に容器内壁に接触した部分から分注された成分が凍結されるように−２０℃程度まで冷却しておくことが好ましい。分注などの各操作は、−２０〜−４０℃条件下で実施することが、分注した各成分の溶解を防止するうえで好ましい。 First, either the reducing agent component or the alkaline aqueous solution is dispensed into a container and frozen. Although there is no restriction | limiting in particular in the container to be used, A plastic, a glass container, etc. can be used. It is preferable to set it for 30 minutes or more under room temperature conditions as a dispensing method, and as -20--40 degreeC conditions as a freezing method. Next, the other component is dispensed and frozen while maintaining the frozen state of the dispensed aqueous solution. The container is preferably sufficiently cooled and cooled to about -20 ° C. so that the components dispensed from the portion in contact with the inner wall of the container may be frozen simultaneously with the dispensing. It is preferable that each operation such as dispensing be performed under conditions of -20 to -40 ° C to prevent dissolution of each of the dispensed components.
以上のようにして各成分を接触することなく凍結した後、凍結状態にて乾燥操作を行う。この操作は通常の凍結乾燥操作で良く、具体的な条件としては、真空度１００ｍＴｏａｒ以下、−２０℃の条件下に２時間置いた後、２５℃条件下に５時間置くこと等が例示できる。乾燥操作を終了した後は、酸化による各成分の劣化等を防止する目的で不活性ガス等を注入して密閉し、保管することが好ましい。 After freezing without contacting each component as described above, the drying operation is performed in the frozen state. This operation may be a normal lyophilization operation, and specific conditions may be, for example, 2 hours under a degree of vacuum of 100 mToar or less, -20 ° C., and 5 hours under 25 ° C., and the like. After the drying operation is completed, it is preferable to inject, seal, and store inert gas or the like for the purpose of preventing deterioration of each component due to oxidation.
本発明の凍結乾燥試薬は、還元剤とアルカリ剤が乾燥状態で同一容器に入れられていることから、ビタミン類を含む血液試料を投入するだけでビタミン類と結合タンパク質との遊離を簡便に行うことができる。ビタミン類としては、ビタミンＤ代謝産物、ビタミンＢ１２又は葉酸が例示できるが、これに限定されるものではない。 Since the lyophilization reagent of the present invention contains the reducing agent and the alkali agent in a dry state in the same container, the liberation of the vitamins and the binding protein can be carried out simply by simply inputting a blood sample containing vitamins. be able to. Examples of vitamins include, but are not limited to, vitamin D metabolites, vitamin B 12 or folic acid.
本発明によれば、還元剤とアルカリ剤を同一容器内に入れておくことが可能となり、ビタミン類を含む血液試料を投入するだけで、ビタミン類と結合タンパク質との遊離を簡便に行うことができる。 According to the present invention, it becomes possible to put the reducing agent and the alkaline agent in the same container, and simply releasing the vitamins and the binding protein simply by charging the blood sample containing the vitamins. it can.
以下、実施例により本発明をさらに詳細に説明するが、本発明は本実施例により限定されるものではない。 Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited by the examples.
免疫測定装置及びビタミン類の前処理装置として全自動エンザイムイムノアッセイ装置（ＡＩＡ−ＣＬ２４００、東ソー社製）と当該装置用葉酸免疫反応試薬と葉酸前処理用試薬を用い、前処理とＤｅｌａｙ１ステップ競合法により各測定を行った。なお、葉酸免疫反応試薬と葉酸前処理用試薬は後述したようにして調製した。 Using a fully automatic enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corp.) as an immunoassay device and a pretreatment device for vitamins, a folate immunoreactant for the device and a reagent for folate pretreatment, the pretreatment and the Delay1 step competition method Each measurement was performed. The folate immune reaction reagent and the folate pretreatment reagent were prepared as described later.
葉酸免疫反応試薬
（１）固相懸濁液の調製
葉酸結合タンパク質固定化磁性微粒子をコラーゲンペプチド、糖、塩類等を含むＴｒｉｓ緩衝液で希釈し、固相懸濁液を作製した。
（２）検出用標識葉酸溶液の調製
アルカリホスファターゼ標識葉酸をコラーゲンペプチド、糖、塩類等を含むＴｒｉｓ緩衝液で希釈し、検出用標識葉酸溶液を作製した。
（３）葉酸免疫反応試薬の調製
固相懸濁液と検出用標識葉酸溶液をそれぞれ凍結乾燥し、葉酸免疫反応試薬を調製した。
Folate Immunoreactant (1) Preparation of Solid Phase Suspension The folate binding protein-immobilized magnetic fine particles were diluted with Tris buffer containing collagen peptide, sugar, salts and the like to prepare a solid phase suspension.
(2) Preparation of Labeled Folate Solution for Detection The alkaline phosphatase labeled folate was diluted with a Tris buffer solution containing collagen peptide, sugar, salts and the like to prepare a labeled folate solution for detection.
(3) Preparation of folate immune reaction reagent The solid phase suspension and the labeled folate solution for detection were lyophilized to prepare folate immune reaction reagent.
葉酸前処理用試薬
（４）アルカリ剤溶液
糖類等と水酸化ナトリウム水溶液を混合し、アルカリ剤溶液を作製した。
（５）還元剤溶液
糖類等とトリス（２−カルボキシエチル）ホスフィン（ＴＣＥＰ）を水で溶解し、還元剤溶液を作製した。
（６）中和剤溶液
糖類等とリン酸緩衝液を混合し、中和剤溶液を作製した。
（７）葉酸前処理用試薬の調製
アルカリ剤溶液を容器に分注し、凍結した。次に、分注したアルカリ剤溶液の凍結状態を保ったまま、還元剤溶液を分注して凍結した。中和剤溶液は別の容器に分注して凍結し、それぞれを凍結乾燥し、葉酸前処理用試薬として調製したものを試薬Ａとした。また、アルカリ剤溶液と還元剤溶液を混合し、２５℃で３日間保存した後に、その混合液と中和剤溶液それぞれを凍結乾燥し、葉酸前処理用試薬を調製したものを試薬Ｂ、２５℃で７日間保存した後に同様に調製したものを試薬Ｃとした。
Reagent for Folate Pretreatment (4) Alkaline Agent Solution A saccharide etc. and a sodium hydroxide aqueous solution were mixed to prepare an alkali agent solution.
(5) Reducing Agent Solution A saccharide etc. and tris (2-carboxyethyl) phosphine (TCEP) were dissolved in water to prepare a reducing agent solution.
(6) Neutralizer Solution A saccharide etc. and a phosphate buffer were mixed to prepare a neutralizer solution.
(7) Preparation of folic acid pretreatment reagent The alkaline agent solution was dispensed into a container and frozen. Next, the reducing agent solution was aliquoted and frozen while maintaining the frozen state of the aliquoted alkaline agent solution. The neutralizer solution was aliquoted into separate containers and frozen, and each was lyophilized to prepare a reagent A prepared as a folate pretreatment agent. In addition, after the alkaline agent solution and the reducing agent solution are mixed and stored at 25 ° C. for 3 days, the mixed solution and the neutralizing agent solution are each lyophilized to prepare a folic acid pretreatment reagent as reagent B, A reagent C was prepared similarly after being stored at 7 ° C. for 7 days.
次に、上記のように調製した葉酸免疫反応試薬と葉酸前処理用試薬Ａ、Ｂ、Ｃに対して前記自動免疫測定装置で各試薬に対して血清サンプル２種類を測定し、アルカリホスファターゼの基質である化学発光基質の発光強度を測定した。各血清サンプルは４回ずつ測定し、その平均値を測定値とした。結果を表１に示す。 Next, two types of serum samples are measured for each reagent using the above-mentioned automatic immunoassay apparatus for the folate immune reaction reagent and folate pre-treatment reagents A, B and C prepared as described above, and a substrate for alkaline phosphatase The luminescence intensity of the chemiluminescent substrate was measured. Each serum sample was measured four times, and the average value was taken as the measured value. The results are shown in Table 1.
葉酸前処理用試薬の試薬Ｂと試薬Ｃの２種類の血清サンプルの発光強度が試薬Ａと比較して、試薬Ｂで１０５．８％、１０６．７％、試薬Ｃで１０７．９％、１１５．３％であった。発光強度が高いことは、前処理時にサンプル中の結合タンパク質からの遊離が少なく、遊離している葉酸が少ないことを意味する。これはアルカリ剤溶液と還元剤溶液を混合してからの経過日数と関連して発光強度が高くなることから還元剤の還元力がアルカリ剤溶液中で徐々に低下していたことを示している。 The luminescence intensities of the two serum samples, reagent B and reagent C, for the pre-treatment for folic acid are 105.8%, 106.7% for reagent B, 107.9% for reagent C, 115 .3%. High luminescence intensity means less release from bound protein in the sample during pretreatment and less folate released. This indicates that the reducing power of the reducing agent gradually decreased in the alkali agent solution because the luminescence intensity increases in relation to the number of days elapsed after mixing the alkali agent solution and the reducing agent solution .
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02233700A (en) * | 1989-01-11 | 1990-09-17 | Boehringer Mannheim Gmbh | Separation of substance for analysis bound with combined protein and preparative reagent for sample for measuring vitamin b12 in serum |
| JPH08507603A (en) * | 1993-02-24 | 1996-08-13 | アボツト・ラボラトリーズ | Competitive immunoassay with binding protein in multiclonal antibody format |
| JP2010187604A (en) * | 2009-02-19 | 2010-09-02 | Sekisui Medical Co Ltd | Method for measuring sample for measuring glycosylated hemoglobin |
-
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02233700A (en) * | 1989-01-11 | 1990-09-17 | Boehringer Mannheim Gmbh | Separation of substance for analysis bound with combined protein and preparative reagent for sample for measuring vitamin b12 in serum |
| US5332678A (en) * | 1989-01-11 | 1994-07-26 | Boehringer Mannheim Gmbh | Process for liberating an analyte from its binding protein |
| JPH08507603A (en) * | 1993-02-24 | 1996-08-13 | アボツト・ラボラトリーズ | Competitive immunoassay with binding protein in multiclonal antibody format |
| JP2010187604A (en) * | 2009-02-19 | 2010-09-02 | Sekisui Medical Co Ltd | Method for measuring sample for measuring glycosylated hemoglobin |
Non-Patent Citations (2)
| Title |
|---|
| SAKURA, T. ET AL.: "One-pot preparation of mono-dispersed and physiologically stabilized gold colloid", COLLOID POLYM SCI, vol. 284, JPN6021030252, 2005, pages 97 - 101, XP019340472, ISSN: 0004570961, DOI: 10.1007/s00396-005-1339-9 * |
| 美濃 眞: "日本ビタミン学会が推奨する臨床検査検体試料のビタミン測定法", ビタミン, vol. 74, no. 10, JPN6021030255, 2000, pages 501 - 515, ISSN: 0004570962 * |
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