JP2019027936A - Method for cleaning analyzer - Google Patents
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本発明は、分析機器の洗浄方法に関するものである。 The present invention relates to a method for cleaning an analytical instrument.
分析機器はその使用試薬や測定試料によって汚染されることがあり、しばしば測定結果に影響を与える。特に、血液試料などの様々な物質を含むクルードサンプルを分析対象とする機器においては汚染が発生しやすいことが知られており、分析機器を正常な状態に維持するための洗浄剤組成物や洗浄方法に関連する技術が必要とされている。 Analytical instruments can be contaminated by the reagents used and the measurement samples, often affecting the measurement results. In particular, it is known that contamination is likely to occur in devices that analyze crude samples containing various substances such as blood samples, and cleaning compositions and cleaning materials for maintaining the analytical device in a normal state are known. Techniques related to the method are needed.
分析機器を洗浄するとき、強酸液、強アルカリ液、有機溶媒などを単独又は組み合わせて汚染を除去する方法が知られている。例えば、液体クロマトグラフの洗浄において、硝酸水溶液、水酸化ナトリウム水溶液、アセトンなどが使用されうる。しかしながら、強酸液、強アルカリ液、有機溶媒などを洗浄剤として使用する場合、人体への有害性や危険性、分析機器に対する腐食性などからその取扱いには注意を要するため、より穏やかな条件で汚染を除去することができる洗浄方法が求められている。そのような技術として、例えば液体クロマトグラフの洗浄方法（特許文献１参照）や血液分析器に対する洗浄方法（特許文献２参照）が提案されているが、洗浄効果が不十分な場合があるため、より効果的な洗浄方法が求められていた。 When washing an analytical instrument, a method for removing contamination by using a strong acid solution, a strong alkali solution, an organic solvent or the like alone or in combination is known. For example, an aqueous nitric acid solution, an aqueous sodium hydroxide solution, acetone, or the like can be used for washing a liquid chromatograph. However, when using a strong acid solution, strong alkali solution, organic solvent, etc. as a cleaning agent, it must be handled in a milder condition because it must be handled with care because of its harmfulness and danger to the human body and corrosiveness to analytical instruments. There is a need for cleaning methods that can remove contamination. As such a technique, for example, a liquid chromatograph cleaning method (see Patent Document 1) and a blood analyzer cleaning method (see Patent Document 2) have been proposed, but the cleaning effect may be insufficient. A more effective cleaning method has been demanded.
本発明の目的は、分析機器の洗浄方法を提供することにある。 An object of the present invention is to provide a method for cleaning an analytical instrument.
本発明者は上記の課題に関し鋭意研究を行なった結果、本発明を完成するに至った。すなわち本発明は以下のとおりである。
（１）少なくとも１種類以上のプロテアーゼを含む洗浄剤を分析機器に供給した後、少なくとも１種類以上の界面活性剤を含む洗浄剤を前記分析機器に供給することを特徴とする分析機器の洗浄方法。
（２）前記プロテアーゼを含む洗浄剤及び前記界面活性剤を含む洗浄剤のいずれもｐＨ３以上１１以下であることを特徴とする（１）に記載の方法。
（３）有機溶媒を含む洗浄組成物を分析機器に供給する工程を含まないことを特徴とする（１）又は（２）に記載の方法。
（４）前記分析機器が液体クロマトグラフであることを特徴とする（１）〜（３）のいずれかに記載の方法。
As a result of intensive studies on the above problems, the present inventors have completed the present invention. That is, the present invention is as follows.
(1) A cleaning method for an analytical instrument, wherein a cleaning agent containing at least one or more types of protease is supplied to the analytical instrument, and then a detergent containing at least one or more surfactants is supplied to the analytical instrument. .
(2) The method according to (1), wherein both the detergent containing the protease and the detergent containing the surfactant have a pH of 3 or more and 11 or less.
(3) The method according to (1) or (2), which does not include a step of supplying a cleaning composition containing an organic solvent to an analytical instrument.
(4) The method according to any one of (1) to (3), wherein the analytical instrument is a liquid chromatograph.
以下に本発明を詳細に説明する。 The present invention is described in detail below.
本発明は、少なくとも１種類以上のプロテアーゼを含む洗浄剤を分析機器に供給した後、少なくとも１種類以上の界面活性剤を含む洗浄剤を前記分析機器に供給することを特徴とする分析機器の洗浄方法に関する。なお、本発明において、「洗浄剤を分析機器に供給する」とは、測定サンプルが接触する分析機器の部位に、洗浄剤を接触させることを言う。接触は連続的でも断続的でよく、通液させても滞留させてもよい。例えば、液体クロマトグラフの流路を洗浄する場合においては、ポンプやシリンジを用いて流路に洗浄剤を注入することで、流路に洗浄剤を接触させることができる。分析機器の種類に応じて適当な方法で測定サンプルが接触する部位に洗浄剤を接触させればよい。 According to the present invention, after supplying a cleaning agent containing at least one or more types of protease to an analytical instrument, a cleaning agent containing at least one or more surfactants is supplied to the analytical instrument. Regarding the method. In the present invention, “supplying the cleaning agent to the analytical instrument” means bringing the cleaning agent into contact with the part of the analytical instrument that is in contact with the measurement sample. Contact may be continuous or intermittent, and may be allowed to pass or stay. For example, when cleaning the flow path of the liquid chromatograph, the cleaning agent can be brought into contact with the flow path by injecting the cleaning agent into the flow path using a pump or a syringe. What is necessary is just to make a cleaning agent contact the site | part which a measurement sample contacts by an appropriate method according to the kind of analytical instrument.
本発明は、分析機器の汚染箇所に対してプロテアーゼによる洗浄効果を発揮させた後、界面活性剤による洗浄効果を発揮させることを特徴としている。 The present invention is characterized in that after a cleaning effect by a protease is exerted on a contaminated portion of an analytical instrument, a cleaning effect by a surfactant is exhibited.
本発明において、洗浄剤とは水を主成分として、プロテアーゼ又は界面活性剤を含有するものを言う。 In the present invention, the cleaning agent refers to one containing water as a main component and containing a protease or a surfactant.
本発明におけるプロテアーゼとしては、化学的又は遺伝的に改変された変異体を含む、動物又は植物、微生物等、種々の起源のものであってよいが、その特徴、例えば至適ｐＨや共存物質との適合性などを考慮すべきであり、かつその活性を発揮するのに十分な量を含有していることが好ましい。具体的には、０．００１−１０重量％である。使用できるプロテアーゼの例として、セリンプロテアーゼ（例えばキモトリプシン、スブチリシンなど）、金属プロテアーゼ（サーモリシンなど）、システインプロテアーゼ（パパイン、カスパーゼなど）、などが挙げられる。好ましくはｐＨ３−１１の範囲でプロテアーゼ活性を有するプロテアーゼであり、基質特異性が広いプロテアーゼや基質特異性の異なる複数のプロテアーゼからなる混合物であることがより好ましい。例えば、ブタ膵臓由来のプロテアーゼ画分やウシ膵臓由来のプロテアーゼ画分が挙げられる。 The protease in the present invention may be of various origins such as animals, plants, microorganisms, etc., including chemically or genetically modified mutants, but its characteristics such as optimum pH and coexisting substances It is preferable to contain a sufficient amount to exhibit its activity. Specifically, it is 0.001 to 10% by weight. Examples of proteases that can be used include serine proteases (such as chymotrypsin and subtilisin), metalloproteases (such as thermolysin), and cysteine proteases (such as papain and caspase). A protease having protease activity in the range of pH 3-11 is preferable, and a mixture of a protease having a wide substrate specificity or a plurality of proteases having different substrate specificities is more preferable. Examples thereof include a protease fraction derived from porcine pancreas and a protease fraction derived from bovine pancreas.
本発明における界面活性剤としては、例えば陽イオン界面活性剤（塩化ベンザルコニウムなど）、陰イオン界面活性剤（モノアルキル硫酸塩など）、非イオン界面活性剤（オクチルフェノールエトキシレートなど）、両イオン界面活性剤（アルキルアミンオキシドなど）であってよく、これらの混合物であってもよい。界面活性剤の含有量としては０．０１−３０重量％が好ましい。 Examples of the surfactant in the present invention include a cationic surfactant (such as benzalkonium chloride), an anionic surfactant (such as a monoalkyl sulfate), a nonionic surfactant (such as octylphenol ethoxylate), and both ions. It may be a surfactant (such as an alkylamine oxide) or a mixture thereof. The content of the surfactant is preferably 0.01 to 30% by weight.
上述した洗浄剤のｐＨは、３以上１１以下であることが好ましい。また、洗浄剤のｐＨを維持するために緩衝剤を使用してもよい。緩衝剤としては、リン酸、クエン酸、こはく酸、炭酸ナトリウム、重炭酸ナトリウム又はこれらの組合せが挙げられるが、特に制限はない。なお、洗浄剤に緩衝剤を添加する場合、プロテアーゼ又は界面活性剤の含有量は緩衝剤の含有量を加味した上で計算される。 The pH of the above-described cleaning agent is preferably 3 or more and 11 or less. A buffer may be used to maintain the pH of the cleaning agent. Examples of the buffering agent include phosphoric acid, citric acid, succinic acid, sodium carbonate, sodium bicarbonate, and combinations thereof, but there is no particular limitation. In addition, when adding a buffering agent to a washing | cleaning agent, content of protease or surfactant is calculated after considering the content of buffering agent.
本発明の対象となる分析機器としては、液体クロマトグラフ、フローサイトメーター、生化学分析装置、免疫測定装置などが例示されるが、中でも液体クロマトグラフに好適である。
また、本発明では、プロテアーゼを含む洗浄剤を分析機器に供給する前、プロテアーゼを含む洗浄剤を分析機器に供給した後であって界面活性剤を含む洗浄剤を分析機器に供給する前、界面活性剤を含む洗浄剤を分析機器に供給した後のいずれの場合であっても、アルコール（メタノールなど）、ケトン（アセトンなど）などの有機溶媒を含む洗浄組成物は分析機器に供給しない方が好ましい。
Examples of the analytical instrument that is the subject of the present invention include a liquid chromatograph, a flow cytometer, a biochemical analyzer, an immunoassay device, etc., among which the liquid chromatograph is suitable.
Further, in the present invention, before supplying the detergent containing protease to the analytical instrument, after supplying the detergent containing protease to the analytical instrument and before supplying the detergent containing surfactant to the analytical instrument, In any case after supplying a cleaning agent containing an activator to an analytical instrument, it is better not to supply a cleaning composition containing an organic solvent such as alcohol (such as methanol) or ketone (such as acetone) to the analytical instrument. preferable.
本発明により、分析機器、特に血液試料などの様々な物質を含むクルードサンプルを分析対象とする機器に生じる汚染を安全かつ、簡便に効率よく洗浄することができる。 According to the present invention, it is possible to safely and easily and efficiently clean contamination that occurs in an analysis apparatus, particularly an apparatus that analyzes a crude sample containing various substances such as a blood sample.
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。 EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by a present Example.
分析機器の汚染状態の評価と汚染部位の特定は下記のように実施した。
分析機器として東ソー自動ヘモグロビン分析計ＨＬＣ−７２３Ｇ８ Ｖａｒｉａｎｔ Ｍｏｄｅ（東ソー株式会社）を用いた。該分析機器はその継続的な使用により、極稀にサンプルループに汚染が発生することが確認されている。測定サンプルとしてＨｂＡ１ｃコントロールセット（東ソー株式会社）のＬｅｖｅｌ−２の蒸留水希釈サンプルおよびＨＳｉ溶血・洗浄液（東ソー株式会社）希釈サンプルを用いた。これらのサンプルのＨｂＡ１ｃ％を比較した。結果を表１に示す。汚染されたサンプルループ使用時では、蒸留水希釈サンプルのＨｂＡ１ｃ％とＨＳｉ溶血・洗浄液希釈サンプルのＨｂＡ１ｃ％の差の絶対値（以下、ＨｂＡ１ｃ％差と表記する）が０．１５ポイント以上あった。次に新品のサンプルループを取り付けて同様の測定を実施すると、ＨｂＡ１ｃ％差は０．０５ポイント以下であった。
The evaluation of the contamination state of the analytical instrument and the identification of the contaminated part were carried out as follows.
A Tosoh automatic hemoglobin analyzer HLC-723G8 Variant Mode (Tosoh Corporation) was used as an analytical instrument. It has been confirmed that the analytical instrument is rarely contaminated by the continuous use of the analytical instrument. As a measurement sample, a distilled water diluted sample of Level-2 of HbA1c control set (Tosoh Corporation) and a diluted sample of HSi hemolysis / washing liquid (Tosoh Corporation) were used. The HbA1c% of these samples were compared. The results are shown in Table 1. When the contaminated sample loop was used, the absolute value of the difference between HbA1c% of the distilled water diluted sample and HbA1c% of the HSi hemolyzed / washed solution diluted sample (hereinafter referred to as HbA1c% difference) was 0.15 points or more. Next, when a new sample loop was attached and the same measurement was performed, the difference in HbA1c% was 0.05 points or less.
以上より、ＨｂＡ１ｃコントロールセットのＬｅｖｅｌ−２のＨｂＡ１ｃ％差が０．１５ポイント以上のとき該分析機器は汚染状態とし、０．０５ポイント以下であるとき該分析機器は非汚染状態と判断した。また、サンプルループの交換により該分析機器の汚染状態が解消されたとき、該分析機器の汚染部位はサンプルループであると判断した。 From the above, when the HbA1c% difference of Level-2 of the HbA1c control set was 0.15 point or more, the analytical instrument was judged to be contaminated, and when it was 0.05 point or less, the analytical instrument was judged to be non-contaminated. Further, when the contamination state of the analytical instrument was resolved by exchanging the sample loop, it was determined that the contaminated portion of the analytical instrument was a sample loop.
（実施例１） 洗浄剤組成物による汚染分析機器の洗浄
まず、以下の洗浄剤１、２を調製した。
洗浄剤１ プロテアーゼ（シグマアルドリッチ、Ｐ４６３０）を０．１重量％含む
１０ｍＭ リン酸緩衝液（ｐＨ８．５）
洗浄剤２ ポリオキシエチレン（１０）オクチルフェニルエーテル（以下、
ＴｒｉｔｏｎＸ−１００とする）（キシダ化学、０２０−８１１５５）
を０．１重量％含む１０ｍＭ リン酸緩衝液（ｐＨ８．５）
(Example 1) Cleaning of contamination analysis equipment with cleaning composition First, the following cleaning agents 1 and 2 were prepared.
Detergent 1 contains 0.1% by weight of protease (Sigma Aldrich, P4630)
10 mM phosphate buffer (pH 8.5)
Cleaning agent 2 polyoxyethylene (10) octylphenyl ether (hereinafter referred to as “cleaning agent”)
Triton X-100) (Kishida Chemical, 020-81155)
10 mM phosphate buffer (pH 8.5) containing 0.1 wt%
次に、汚染部位であることが確認されたサンプルループ４つに対してそれぞれ、以下の条件で洗浄を行った。
洗浄方法１ 洗浄剤１を汚染状態のサンプルループに１時間接触させた後、
さらに洗浄剤１を該サンプルループに１時間接触させた。
洗浄方法２ 洗浄剤２を汚染状態のサンプルループに１時間接触させた後、
さらに洗浄剤２を該サンプルループに１時間接触させた。
洗浄方法３ 洗浄剤１を汚染状態のサンプルループに１時間接触させた後、
さらに洗浄剤２を該サンプルループに１時間接触させた。
洗浄方法４ 洗浄剤２を汚染状態のサンプルループに１時間接触させた後、
さらに洗浄剤１を該サンプルループに１時間接触させた。
Next, each of the four sample loops confirmed to be contaminated sites was cleaned under the following conditions.
Cleaning method 1 After contacting cleaning agent 1 with a contaminated sample loop for 1 hour,
Further, cleaning agent 1 was brought into contact with the sample loop for 1 hour.
Cleaning method 2 After contacting cleaning agent 2 with a contaminated sample loop for 1 hour,
Further, the cleaning agent 2 was brought into contact with the sample loop for 1 hour.
Cleaning method 3 After contacting cleaning agent 1 with a contaminated sample loop for 1 hour,
Further, the cleaning agent 2 was brought into contact with the sample loop for 1 hour.
Cleaning method 4 After contacting the cleaning agent 2 with the contaminated sample loop for 1 hour,
Further, cleaning agent 1 was brought into contact with the sample loop for 1 hour.
洗浄前後のサンプルループの汚染状態を上述の基準で評価した結果を表２に示す。洗浄方法１、２及び４では洗浄実施後のＨｂＡ１ｃ％差が０．１５ポイント以上であり、汚染状態は解消しなかった。一方、洗浄方法３では洗浄実施後のＨｂＡ１ｃ％差が０．０５％ポイント以下であり、汚染状態が解消した。すなわち、分析機器の汚染箇所に対してプロテアーゼによる洗浄効果を発揮させた後、界面活性剤による洗浄効果を発揮させることが重要であることが確認された。 Table 2 shows the results of evaluating the contamination state of the sample loop before and after the cleaning based on the above-mentioned criteria. In cleaning methods 1, 2, and 4, the HbA1c% difference after the cleaning was 0.15 points or more, and the contamination state was not resolved. On the other hand, in the cleaning method 3, the HbA1c% difference after the cleaning was 0.05 points or less, and the contamination state was solved. That is, it was confirmed that it is important to exhibit the cleaning effect by the surfactant after the cleaning effect by the protease is exerted on the contaminated portion of the analytical instrument.
（実施例２） 界面活性剤の種類による洗浄効果の比較
まず、以下の洗浄剤３〜５を調製した。
洗浄剤３ ポリオキシエチレン（２０）ソルビタンモノラウレート
（以下、Ｔｗｅｅｎ２０とする）（和光純薬、１６７−１１５１５）を
０．１重量％含む１０ｍＭ リン酸緩衝液（ｐＨ８．５）
洗浄剤４ 塩化ベンザルコニウム（東京化成工業、Ｂ０４１４）を
０．１重量％含む１０ｍＭ リン酸緩衝液（ｐＨ８．５）
洗浄剤５ ドデシル硫酸ナトリウム（和光純薬、１９６−０８６７５）を
０．１重量％含む１０ｍＭ リン酸緩衝液（ｐＨ８．５）
(Example 2) Comparison of the cleaning effect by the kind of surfactant First, the following cleaning agents 3-5 were prepared.
Detergent 3 Polyoxyethylene (20) sorbitan monolaurate
(Hereinafter referred to as Tween 20) (Wako Pure Chemicals, 167-11515) 0.1 wt% 10 mM phosphate buffer (pH 8.5)
Detergent 4 Benzalkonium chloride (Tokyo Chemical Industry, B0414)
10 mM phosphate buffer (pH 8.5) containing 0.1% by weight
Detergent 5 Sodium dodecyl sulfate (Wako Pure Chemical, 196-08675)
10 mM phosphate buffer (pH 8.5) containing 0.1% by weight
次に、汚染部位であることが確認されたサンプルループ３つに対してそれぞれ、以下の条件で洗浄を行った。
洗浄方法５ 洗浄剤１を汚染状態のサンプルループに１時間接触させた後、
さらに洗浄剤３を該サンプルループに１時間接触させた。
洗浄方法６ 洗浄剤１を汚染状態のサンプルループに１時間接触させた後、
さらに洗浄剤４を該サンプルループに１時間接触させた。
洗浄方法７ 洗浄剤１を汚染状態のサンプルループに１時間接触させた後、
さらに洗浄剤５を該サンプルループに１時間接触させた。
Next, each of the three sample loops confirmed to be contaminated sites was cleaned under the following conditions.
Cleaning method 5 After contacting cleaning agent 1 with a contaminated sample loop for 1 hour,
Further, the cleaning agent 3 was brought into contact with the sample loop for 1 hour.
Cleaning method 6 After contacting cleaning agent 1 with a contaminated sample loop for 1 hour,
Further, the cleaning agent 4 was brought into contact with the sample loop for 1 hour.
Cleaning method 7 After cleaning agent 1 is brought into contact with the contaminated sample loop for 1 hour,
Further, the cleaning agent 5 was brought into contact with the sample loop for 1 hour.
洗浄前後のサンプルループの汚染状態を実施例１の上述の基準で評価した結果を表３に示す。洗浄方法５〜７のいずれにおいても、洗浄実施後のＨｂＡ１ｃ％差が０．０５％ポイント以下であり、汚染状態が解消した。すなわち、洗浄剤組成物に用いる界面活性剤としては、非イオン界面活性剤（ＴｒｉｔｏｎＸ−１００、Ｔｗｅｅｎ２０）、陽イオン界面活性剤（塩化ベンザルコニウム）、陰イオン界面活性剤（ドデシル硫酸ナトリウム）のいずれを用いてもよいことが確認された。 Table 3 shows the results of evaluating the contamination state of the sample loop before and after the cleaning according to the above-mentioned criteria of Example 1. In any of the cleaning methods 5 to 7, the HbA1c% difference after the cleaning was 0.05% point or less, and the contamination state was eliminated. That is, as the surfactant used in the detergent composition, nonionic surfactant (Triton X-100, Tween 20), cationic surfactant (benzalkonium chloride), anionic surfactant (sodium dodecyl sulfate) It was confirmed that either may be used.
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| JPH10318999A (en) * | 1997-05-14 | 1998-12-04 | Sekisui Chem Co Ltd | Channel cleaning method for liquid chromatograph |
| JPH11118779A (en) * | 1997-10-17 | 1999-04-30 | Shino Test:Kk | Method for washing liquid chromatography carrier and its washing liquid |
| JP2003318999A (en) * | 2002-04-25 | 2003-11-07 | Matsushita Electric Ind Co Ltd | Modulation circuit, demodulation circuit and radio device |
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