JP3654956B2 - Residue detection method and residue detection kit - Google Patents

Description

ここで、ＮＡＤ（Ｐ）⁺は酸化型ニコチンアミドアデニンジヌクレオチドリン酸、ＮＡＤ（Ｐ）Ｈは還元型ニコチンアミドアデニンジヌクレオチドリン酸、ＤＩＡはジアホラーゼである。
【００６３】
本発明において、アルコールデヒドロゲナーゼの活性によって生成した還元型捕酵素は本発明に係る還元型捕酵素検出組成物によって定量的に表示される。
【００６４】
即ち還元型捕酵素のＮＡＤＨ或いはＮＡＤＰＨの定量は、適当な物質を用いるＮＡＤＨ或いはＮＡＤＰＨを発色色原体、蛍光前駆体、発光体等と作用させ、比色、蛍光、蛍光等で定量することができる。ここでの適当な物質とはＮＡＤＨ或いはＮＡＤＰＨを酸化し、発色色原体、蛍光前駆体、発光体等を還元する反応を触媒する物質をさし、具体的には、５−メチルフエナジムニウムメチルサルフェート、（１−メトキシ）−５−メチルメチルフエナジムニウムメチルサルフェート（以下、ＭｅＯ−ＰＭＳという）、メルドラブルー（即ち、９−ジメチルアミノベンゾ−α−フェナゾキソニウムクロライド）等の化合物や、ジヒドロリボアミドレダクターゼ（ＮＡＤ⁺）（別名ジアホラーゼ）、ＮＡＤＨデヒドロゲナーゼ（キノン）等の酵素を用いることができる。
【００６５】
この場合、好ましい発色色原体の例として３−（ｐ−ヨ−ドフェニル）−２−（ｐ−ニトロフェニル）−５−フェニル−２水素テトラゾリウムクロライド）（Ｎｅｏ−ＴＢ）、３−３′−ジメトキシ−４，４−ビフェニリレン）−ビス〔２−（ｐ−ニトロフェニル）−５−フェニル−２−水素テトテゾリヴムクロライド〕（Ｎｉｔｏｒｏ−ＴＢ）、３，３′−（３，３′−ジメトキシ−４，４′−ビフェニリレン）−ビス（２，５−ジフェニル−２水素テトラゾリウムクロライド）（ＴＢ）、３，３′−（３，３′−ジメトキシ−４，４′−ビフェニリレン）−ビス〔２，５−ビス（ｐ−ニトロフェニル）−２水素テトラゾリウムクロライド）（ＴＮＴＢ）等が挙げられる。
【００６６】
更に特異的結合反応、酵素反応、発色反応等に適したｐＨにするためには緩衝剤を含有させることが好ましい。具体的には体液試料適用時にｐＨ＝６．０〜１０．０の範囲に緩街しうるに、十分な量含有する事が好ましい。用いることができる緩衝剤としては日本化学会編「化学便覧基礎編」（東京、丸善（株）１９６６）ｐ１３１２〜１３２０、Ｎ，Ｅ，ＧＯＯｄ等；バイオケミストリ（Ｂｉｏｃｈｅｍｉｓｔｒｙ）ｖｏｌ．５，ｐ４６７（１９６６）、今村、斎藤；化学の領域．ｖｏｌ．３０（２），ｐ７９（１９７６）、Ｗ，Ｊファーギュソン（Ｆｅｒｇｕｓｏｎ）等；Ａｎａｌ．Ｂｉｏｃｈｅｍ．，ｖｏｌ．１０４，ｐ３００（１９８０）等の文献に記載されているものを挙げることができる。具体的な例としては、くえん酸塩、硼酸塩、燐酸塩、炭酸塩、トリス、バルビツール、グリシン、グッド緩衝剤等があげられる。これらの緩衝剤は必要に応じて発色反応試薬に含有させてもよい。
【００６７】
更に本発明では試薬の保存性、定量再現性の向上のため保恒剤として多価アルコール、非還元糖の少なくとも１つを含有させられる。これら保恒剤の添加量は試薬層重量に対し０．１％〜１０ｗｔ％が好ましく、更に好ましくは１〜１５％ｗｔ％である。
【００６８】
多価アルコールとしては糖アルコール、非還元糖としては還元基が消尽された多糖類が好ましい。
【００６９】
次にこれらの具体例を挙げる。
【００７０】
（糖アルコール）
エリトリトール、トレイトール、アラビニトール、リビトール、キシリトール、ソルビトール、ガラクチトール、マンニトール、アリトール、ポレミトール、プレセイトール、ｍｙｏ−イノシトール、
ｃｈｉｒｏ−イノシトール、ｓｃｙｌｌｏ−イノシトール、クエルシトール、ビブニトール、シクロヘキサンテトロール、コンズリトール。
【００７１】
（多糖類）
トレハロース、蔗糖、イソトレハロース、ラフィノース、ゲンチアノース、メソチトース、プランテオース、スタキオース、ペルバスコース、リクノース、α−デキストリン、β−デキストリン、γ−デキストリン。
【００７２】
これら発色反応試薬保恒剤、緩衝剤は、水或いは有機溶媒に溶解し検出試薬として用いられる。
【００７３】
更にアルコールの酵素分析法には、アルコールオキシダーゼを用いる方法がある。アルコールオキシダーゼ（以下ＡＯＤと略す）を用いる検出方法の反応は下記のとおりである。生成したH₂O₂を適当な比色反応系に導いて検出する。例えば、ペルオキシダーゼを触媒として、検出可能な色素を形成させて検出する方法が好ましく用いられる。
【００７４】

発色反応試薬としては、トリンダー試薬の変法として知られる４−アミノアンチピリン又はその塩、ｌ，７−ジヒドロキシナフタレン；３−Ｎ−スルホプロピルアニリン誘導体、例えばＮ−エチル−Ｎ−スルホプロピルアニリン、Ｎ−エチル−Ｎ−スルホプロピル−３、５−ジメチロキシアニリンスルホン酸及びその塩、Ｎ−スルホプロピルアニリン、Ｎ−エチルーＮ−スルホプロピル−３，５−ジメチルアニリン、Ｎ−エチル−Ｎ−スルホプロピル−ｍ−トルイジン；Ｎ−２−ヒドロキシー３−スルホプロピルアニリン誘導体、例えばＮ−エチル−Ｎ−（２−ヒドロキシ−３−スルホプロピル）−ｍ−アニシジン、Ｎ−エチル−Ｎ−（２−ヒドロキシ−３−スルホプロピル）アニリン、Ｎ−エチル−Ｎ−（２−ヒドロキシー３−スルホプロピル）−３、５−ジメチロキシアニリンスルホン酸及びその塩、Ｎ−（２−ヒドロキシ−３−スルホプロピル）−３，５−ジメチロキシアニリン、Ｎ−エチルーＮ−（２−ヒドロキシー３−スルホプロピル）−３，５−ジメチルアニリン、Ｎ−エチル−Ｎ−（２−ヒドロキシ−３−スルホプロピル）−ｍ−トルイジン等との組合せ等が挙げられる。
【００７５】
更に化学発光する化合物、例えばノレミノール（５−アミノ−２，３−ジヒドロ−１，４−フタラジンジオン）、イソルミノール（６−アミノ−２，３−ジヒドロ−ｌ，４−フタラジンジオン）、ＡＢＥＩ−Ｈ（Ｎ−（４−アミノブチル）−Ｎ−エチルイソルミノーノレヘミサクシンアミド、ＡＨＥＩ（Ｎ−（６−アミノヘキシル）−Ｎ−エチノレイソルミノール）ＡＢＥＩ（Ｎ−（４−アミノブチル）−Ｎ−エチルイソルミノール）等が挙げられる。
【００７６】
更に特開昭５７−９４６５３号、同５７−９４６５４号、同５７−９４６５５号、同５７−９４６５６号に記載された芳香族第一級アミン化合物とカップリング反応によって発色する拡散性フェノール化合物、或いはピラゾロン化合物、その他の発色試薬が用いられる。
【００７７】
前記アルコールオキシダーゼに代えて、グルコースオキシダーゼ（以下ＧＯＤと称す）を用いればグルコースを検出することが可能である。即ち、生成したＨ₂Ｏ₂を例えば、ペルオキシダーゼを触媒として、検出可能な色素を形成させて検出する方法が好ましく用いられる。
【００７８】
グルコース

また、これにα−グルコシダーゼ、β−グルコシダーゼ、β−フルクトシダーゼなどの二糖ないしオリゴ糖を加水分解する酵素を併用することによって、二糖ないしオリゴ糖を構成するグルコースとして、それらの存在を一括して検出することができる。例えば、下記の酵素を併用すればマルトース／スクロース／グルコース何れかの存在を一度に検出することが可能である。
【００７９】

中性脂肪
中性脂肪はグリセロールと脂肪酸のエステルである。検出方法は、リポプロテインリパーゼ（以下ＬＰＬと称す）及びグリセロールデヒドロゲナーゼ（以下ＧＤＨと称す）によって生成するＮＡＤＨを例えば、ジアホラーゼを触媒としてテトラゾリウム塩をホルマザン色素に変換して検出することが好ましい。検出試薬は適当な緩衝液に溶解していることが望ましく、１０ｍＭ〜１Ｍ、ｐＨ７〜１０．５の範囲で例えば、リン酸緩衝液、炭酸緩衝液、トリス−トリス塩酸緩衝液、その他グッドバッファーが適宜選択され用いられる。酸化型ニコチン酸アミドアデニンジヌクレオチド（ＮＡＤ＋）は好ましくは１〜１００ｍＭの濃度で含有させることができる。ＬＰＬ、ＧＤＨ、ＤＩＡなどの酵素は好ましくは０．１〜５０Ｕｎｉｔ／ｍｌを含有させることができる。
【００８０】
テトラゾリウム塩としては、２−（４−Ｉｏｄｏｐｈｅｎｙｌ）−３−（４−ｎｉｔｒｏｐｈｅｎｙｌ）−５−ｐｈｅｎｙｌ−２Ｈ ｔｅｔｒａｚｏｌｉｕｍｃｈｌｏｒｉｄｅ（ＩＮＴ），３−（４，５−Ｄｉｍｅｔｈｙｌ−２−ｔｈｉａｚｏｌｙｌ）−２，５−ｄｉｐｈｅｎｙｌ−２Ｈ ｔｅｔｒａｚｏｌｉｕｍｂｒｏｍｉｄｅ（ＭＴＴ），３，３′−〔３，３′−Ｄｉｍｅｔｈｏｘｙ−（１，１′−ｂｉｐｈｅｎｙｌ）−４，４′−ｄｉｙｌ〕−ｂｉｓ〔２−（４−ｎｉｔｒｏｐｈｅｎｙｌ）−５−ｐｈｅｎｙｌ−２Ｈ ｔｅｔｒａｚｏｌｉｕｍ ｃｈｌｏｒｉｄｅ〕（Ｎｉｔｒｏ−ＴＢ）などが好ましく用いられる。
【００８１】

又は、ＬＰＬによって生成するグリセロールをＡＴＰの存在下にグリセロキナーゼ（ＧＫ）でグリセリン３−リン酸（Ｇ ３−Ｐ）とし、これをグリセリン３−リン酸酸化酵素（ＧＰＯ）によって酸化し、生成したＨ₂Ｏ₂を例えば、ペルオキシダーゼを触媒として、検出可能な色素を形成させて検出する方法が好ましく用いられる。
【００８２】

脂肪酸
脂肪酸としては例えば、オレイン酸、パルミチン酸、ステアリン酸などであり、単体として或いは混合物の形で存在するものが測定対象である。これを下記に示すようにアシルＣｏＡシンセターゼ（ＡＣＳ）、アシルＣｏＡオキシダーゼ（ＡＣＯ）といった酵素を作用させることによって生成した過酸化水素を適当な比色反応系に導いて検出する。比色反応系としてペルオキシダーゼを用いた場合、ＣｏＡのＳＨ基が次の反応で色素形成を阻害することから、Ｎ−エチルマレイミドにより、未反応ＣｏＡをブロックする必要がある。これらの測定方法については、北村元仕（編）：遊離脂肪酸、実践臨床化学ｐ５３０−５４４，医歯薬出版，１９８２に記載されている。
【００８３】

中性脂肪或いは脂肪酸などのような脂質を測定する場合、検出媒体の吸水部を予め界面活性剤を含有する水溶液で湿らせておくとサンプリングの精度及び検出感度があがる。このような界面活性剤としては非イオン性界面活性剤が好ましく用いられる。具体的には、Ｔｗｅｅｎ２０やトリトンＸ−１００などのポリオキシエチレンアルキルフェニルエーテル類が好ましく用いられる。使用する界面活性剤の濃度は０．０５〜５％が好ましい。
【００８４】
これらの試薬は、好ましくは開口部を有する可撓性部材からなる１つ或いは複数の容器に収納されており、使用時に容器の一部を押圧して開口部から液滴を形成し、これを試料を採取済みの検出媒体の吸水部に点着して反応させる。或いは、別の容器（試験管など）に必要量を分注した試薬に検出媒体の吸水部を接触させて反応させる。必要に応じドライヤー又はヒーターなどで加熱させて反応を促進させるとよい。酵素を用いている反応を利用している場合は、室温（２５℃）若しくは３７℃に保温して行なわれる。必要があれば試薬ブランクをとり、発色度の差を確認することもできる。
【００８５】
標準型キット
本発明の一例を示す残留物検出用キット構成（図１）は、次の構成である。すなわち、図１（ａ）中の５の試薬Ｂ（点眼ビン形状の容器に充填）、検出媒体１（形状：綿棒状のもの、柄はプラスチック製φ２ｍｍ）、キャップ２がついた３のガラスチューブφ１０×７０ｍｍから構成されている。保存性の問題で２種以上の試薬を測定直前に混合して使用する場合は、図１の３のガラスチューブに４の試薬Ａとして、５の容器に試薬Ｂ（好ましくは点眼ビン形状の容器に充填）として分割して入れておくことができる。さらに第三の試薬Ｃが必要であれば、５の容器をもう１つ追加してキットに入れることができる。
【００８６】
また、測定対象物の表面が乾燥している場合は、検出媒体吸水部１４を水性媒体で湿らせた方が、効率的に試料を採取できる。６はそのための水性媒体で、必要に応じ蒸留水、生理食塩水、界面活性剤水溶液、緩衝溶液、含水アルコールなどが用いられる。その他に、必要に応じ、反応促進・反応速度を一定に保つために恒温槽（ブロックヒーター等）、或いは単純に加熱して反応を促進するためのドライヤー、試験管立てなどを用意する。
【００８７】
精密に測定する場合は、検量線をたて反応液中の生成色素を分光高度計によって測定する。或いは、カラーメーター（図１（ｂ））、標準カラースケール（図１（ｃ））を用いて、目視により判定することができる。図１（ｂ）、図１（ｃ）において、１ａは標準カラースケール、１ｂはライト、１ｃは比色窓、１ｄは標準カラースケールホルダー、１ｅは試験管ホルダー、１ｆは物質濃度（レベル）、１ｇは発色の度合を示す色見本である。キット操作方法測定の一例をあげれば、測定の際にガラスチューブの試薬Ａに、試薬Ｂを数滴滴下して混合し発色試薬を調製する。
【００８８】
次いで、測定対象物の表面の一定面積を検出媒体１でふき取る。このとき、試料表面が乾燥している場合は、検出媒体１の吸水部を適当な水性媒体（蒸留水、界面活性剤水溶液、生理食塩水、含水アルコール、など）で湿らせてふき取ると効率的である。ふき取った検出媒体１を発色試薬内に浸漬して反応させる。
【００８９】
反応中は検出媒体１は反応液内に浸漬し続けることが望まれる。特に、検出試薬にオキシダーゼを使用している場合は、これによって空気中の酸素が効率的に供給され反応が促進される。必要に応じ、恒温槽で加温し反応を促進させる。残留物による発色を分光光度計で測定するか、或いは目視により確認する。このとき必要に応じて標準カラースケールを用意しておくと半定量が可能である。
【００９０】
標準改良型キット
本発明の他の例を図２に示す。即ち、適当な透明チューブ９に水性媒体１０が１００〜１０００μｌ程度入っており、キャップ７の下段には８、１２の試薬Ａ，Ｂを封入した可撓性ケース１１が装着してある。キャップ７の底には、検出媒体１３が固定されている。キャップ７を強く握ることにより可撓性ケース１１が潰れ、中の試薬Ａ，Ｂが透明チューブ９内へ落ち、検出媒体１３と接触する仕組みとなっている。その他に、必要に応じ、恒温槽ブロックヒーター等）、分光光度計、カラーメーター、標準カラースケール、試験管たてなどを用意する。
【００９１】
キット操作方法
図２においてキャップ７を外し、検出媒体１３の先端に対し、上記標準型キット操作法に示した要領で測定対象物表面から試料をサンプリングする。検出媒体１３を透明チューブへ戻し、キャップ７を閉める。８，１２の試薬Ａ，Ｂの入った可撓製ケース１１を潰して、該試薬Ａ，Ｂを透明チューブ内へ落す。該透明チューブを攪拌した後、必要に応じ該透明チューブを加温或いは一定の温度に保ち、反応を進行させる。一定時間後、目視、或いは分光光度計にて発色程度を確認する。目視により確認する場合は、予め容易していた標準カラースケールと比較することによって、簡便に残留物の検出（半定量）が可能となる。発色試薬が試薬Ａ，Ｂの混合によって調製される場合を例示しているが、発色試薬が単一の溶液として供給される場合は試薬Ａ、Ｂのかわりに１種の発色試薬液を用いればよい。図２の場合、１４が吸水部であり、検出部である。
【００９２】
簡易型キット
本発明の他の例を図３に示す。この例では、全ての発色試薬を容器（点眼ビン形状の容器が好ましい）に分注保管してある。標準型キットの操作方法と同じ要領で測定対象物の表面の一定面積を検出媒体１でふき取る。この検出媒体の吸水部（試料採取部）に発色試薬を容器から直接数滴ずつ添加して、吸水部上で発色反応を行なわせる。採取した残留物量に応じ試料採取部で発色反応が進行するので、これを目視で判定することができる。必要に応じ標準カラースケールで発色の程度を比較することにより、半定量が可能である。試薬Ａ，Ｂを予め混合して使用したい場合は、容器１７で両液を混合したものを用意し、ここから試料採取部に試薬を添加すればよい。
【００９３】
キットのタイプに係わらず、分光光度計により測定する場合は発色した反応試薬液を測定することになる。一方、目視による確認を行なう場合は、発色した反応試薬液の発色によっても確認することは可能であるが、検出媒体の吸水部がより明瞭に染色されるため、この部分で判定することによって高感度な判定が可能となる。
【００９４】
【実施例】
以下、実施例を挙げて本発明を詳細に説明するが、本発明の態様はこれに限定されない。以下に本発明の残留物検出キットの形態及び測定手順を示す。キットの操作時において、何れもサンプリング時には指、手などが試薬、サンプリングの媒体に、直接触れないように十分注意する。
【００９５】
実施例１ ブドウ糖の検出
標準型キットを用いて、ブドウ糖の検出をおこなった。下記組成のＧＯＤ−ＰＯＤ発色試薬Ａ及びＢを調製し、ＧＯＤ−ＰＯＤ発色試薬Ａはガラスチューブ各１ｍｌずつ、ＧＯＤ−ＰＯＤ発色試薬Ｂは点眼ビンに充填した。
【００９６】
ＧＯＤ−ＰＯＤ発色試薬Ａ
０．１Ｍリン酸緩衝液ｐＨ６．９
０．５ｍＭ ４−アミノアンチピリン
２０ｍＭフェノール
ＧＯＤ−ＰＯＤ発色試薬Ｂ
０．１Ｍリン酸緩衝液ｐＨ６．９
５００Ｕ／ｍｌグルコースオキシダーゼ：ＧＯＤ（ベーリンガーマンハイム製）
３００Ｕ／ｍｌペルオキシダーゼ：ＰＯＤ（ベーリンガーマンハイム製）
シャーレに０．１％ブドウ糖を含む水溶液を０．１ｍｌまき、乾燥させて測定試料とした。
【００９７】
測定：ＧＯＤ−ＰＯＤ発色試薬Ａはガラスチューブ各１ｍｌに点眼ビンからＧＯＤ−ＰＯＤ発色試薬Ｂを１滴滴下し、軽く振盪して混合する。検出媒体（綿棒）に、蒸留水２〜３滴（１００μｌ程度）を含ませ、前述のシャーレをふきとり、これを検出試薬に浸漬し、３７℃で１０分間反応させた。その結果、ブドウ糖の存在を示す発色が確認された。同様に調製した検出試薬に直接０．１％ブドウ糖を含む水溶液を０．１ｍｌを入れて反応させたものと比較して、本発明の方法で反応させたものはより短時間で発色反応が進行することが確認された。又、分光光度計を用いて発色液の吸光度を測定することによって、予め作成していた検量線から検出媒体で採取したブドウ糖の量を知ることができた。
【００９８】
実施例２ ブドウ糖の検出
検出媒体の吸水部の材質として下記ものを用いてブドウ糖の検出を行なった。検出試薬として点眼ビンに入れておいた下記のＧＯＤ−ＰＯＤ発色試薬Ｃを用いた。
【００９９】
１．検出媒体として吸水部の材質：ａ．ポリエステル ｂ．ポリエチレン ｃ．ポリウレタンを用いた。
【０１００】
ＧＯＤ−ＰＯＤ発色試薬Ｃ
０．１Ｍリン酸緩衝液 ｐＨ６．５
０．５ｍＭ ４−アミノアンチピリン
１５ｍＭフェノール
５Ｕ／ｍｌグルコースオキシダーゼ：ＧＯＤ（ベーリンガーマンハイム製）
３Ｕ／ｍｌペルオキシダーゼ：ＰＯＤ（ベーリンガーマンハイム製）
２．試料のサンプリング
１０μｌの１％ブドウ糖水溶液をシャーレ底部に点着し、一旦乾燥させた。これを予め蒸留水で湿らせた各検出媒体吸水部にてふき取り、各々ＧＯＤ−ＰＯＤ発色試薬Ｃを数滴点着し、反応させた。３０分後、目視によって発色濃度別に５段階（発色なし−、薄い＋ 〜濃い＋＋＋＋）で評価した。
【０１０１】
３．結果 何れの場合もブドウ糖の存在によって、発色し検出できた。特に吸水部の材質がポリエステル又はポリエチレン繊維の場合に、強く発色し感度が高いことが確認された。
【０１０２】
材質 蒸留水のみ ブドウ糖水溶液
ａ． − ＋＋＋
ｂ． − ＋＋＋
ｃ． − ＋＋
実施例３ アミノ酸の検出
材質の吸水部からなる検出媒体を用いて、試料に付着したアミノ酸の検出を行なった。検出には下記ニンヒドリン試液を用いた。
【０１０３】
ニンヒドリン試液の調製：蒸留水５０ｍｌにニンヒドリン２ｇを溶解した液に８０ｍｇのＳｎＣｌ₂を加え、沈殿を濾過して除き、１昼夜以上暗所に放置後、蒸留水を加えて１００ｍｌとした。この溶液１ｍｌに水２ｍｌを加え、さらにイソプロパノール１８ｍｌを加えて混合した。
【０１０４】
１．検出媒体 吸水部の材質：ａ．ポリエステル ｂ．ポリウレタン ｃ．天然綿糸
２．試料のサンプリング：シャーレ底部に点着した１０μｌのグリシン水溶液（１０ｍｇ／ｍｌ）を各検出媒体吸水部にて吸い取り、各々ニンヒドリン試液を数滴点着し、これをドライヤーにて加熱させて反応させた。目視によって発色濃度別に５段階（発色なし−、薄い＋〜濃い＋＋＋＋）で評価した。
【０１０５】
３．結果：すべての例で吸水部が紫色に発色し、グリシンの存在が確認できた。ｎ＝５の測定結果を以下に示した。結晶性高分子繊維からなる吸水部を持つ検出媒体（ａ，ｂ）は天然綿糸と比べると明らかに発色のばらつきは少なかった。
【０１０６】
再現性の比較
材質 発色濃度（ｎ＝５）
ａ． ＋＋＋ ＋＋＋ ＋＋＋ ＋＋＋ ＋＋＋
ｂ． ＋＋ ＋＋ ＋＋ ＋＋ ＋＋
ｃ． ＋＋＋ ＋＋ ＋＋＋ ＋＋ ＋＋
実施例４ アルデヒドの検出実施例２と同様の手順で、異なる材質の吸水部からなる検出媒体を用いて、試料に付着したアルデヒドの検出を行なった。検出にはシッフ試薬を用いた。シッフ試薬の塩基調整フクシン２００ｍｇを水１２０ｍｌに溶解し、これに亜硫酸水素ナトリウム ２ｇを水２０ｍｌに溶かした液を加えて２００ｍｌとする。活性炭０．０３ｇを加え濾過し、使用時まで暗所に保存した。
【０１０７】
１．検出媒体 吸水部の材質：ａ．ポリエステル ｂ．ポリエチレン c.天然綿糸
２．試料のサンプリング：シャーレ底部に点着した１０μｌの０．１％ホルムアルデヒド水溶液を各検出媒体吸水部にて吸い取り、各々シッフ試薬を数滴点着し、反応させた。３０分後、目視によって発色濃度別に５段階（発色なし−、薄い＋〜濃い＋＋＋＋）で評価した。又、０．１％ホルムアルデヒド水溶液のかわりに蒸留水を用いて同様に測定しバックグラウンドを比較した。
【０１０８】
３．結果 吸水部がポリエステル、ポリエチレン等の結晶性高分子繊維からなる吸水部を有する検出媒体を用いた場合、アルデヒドを含有していない蒸留水ではまったく発色しなかったのに対し、吸水部が天然綿糸などからなる場合は、蒸留水でも発色してしまうことが判明した。
【０１０９】
材質 蒸留水 ０．１％ ホルムアルデヒド水溶液
ａ． − ＋＋
ｂ． − ＋＋
ｃ． ＋＋＋ ＋＋＋
実施例５ アルコールの検出
実施例３と同様の手順で、異なる材質の吸水部からなる検出媒体を用いて、試料に付着したエタノールの検出を行なった。検出には下記組成のＡＯＤ−ＰＯＤ発色試薬を調製し用いた。
【０１１０】
ＡＯＤ−ＰＯＤ発色試薬
５０ｍＭリン酸緩衝液ｐＨ７．５
０．０５％ ＴｒｉｔｏｎＸ−１００
２ｍＭ ＥＤＴＡ−３Ｎａ
０．５ｍＭ ４−アミノアンチピリン
２．０ｍＭ Ｓｏｄｉｕｍ−Ｎ−ｅｔｈｙｌ−Ｎ−（３−ｓｕｌｆｏｐｒｏｐｙｌ）−ｍ−ａｎｉｓｉｄｉｎｅ
５Ｕ／ｍｌペルオキシダーゼ（ベーリンガーマンハイム製）
１０Ｕ／ｍｌアルコールオキシダーゼ（東洋紡製）
１．検出媒体 吸水部の材質：ａ．ポリエステル繊維 ｂ．ポリウレタンフォーム ｃ．天然綿糸
試料のサンプリング：１０μｌの０．１％エタノール水溶液をシャーレ底部に点着し、これを各検出媒体吸水部にて吸い取り、各々ＡＯＤ−ＰＯＤ発色試薬を数滴点着し、室温で反応させた。３０分後、目視によって発色濃度別に５段階（発色なし−、薄い＋〜濃い＋＋＋＋）で評価した。
【０１１１】
２．結果 いずれの場合もエタノールの存在によって、発色し検出可能であることが確認された。ｎ＝５の測定結果を以下に示した。このように疎水性繊維からなる吸水部を持つ検出媒体（ａ，ｂ）は発色のばらつきが少なかった。
【０１１２】
再現性の比較
材質 発色濃度（ｎ＝５）
ａ． ＋＋＋ ＋＋＋ ＋＋＋ ＋＋＋ ＋＋＋
ｂ． ＋＋ ＋＋ ＋＋ ＋＋ ＋＋＋
ｃ． ＋＋ ＋＋＋ ＋＋ ＋＋＋ ＋＋
実施例６ 脂肪酸の検出
実施例１と同様の手順で、試料に付着した脂肪酸の検出を行なった。検出媒体は天然綿糸の吸水部を有するもの（綿棒）を用いた。検出には下記脂肪酸発色試薬１〜３を用いた。
【０１１３】
脂肪酸発色試薬１
５Ｕ アシルＣｏＡシンセターゼ（東洋紡製）
４Ｕ ミオキナーゼ（ベーリンガーマンハイム製）
１０ｍｇ ＡＴＰ・２Ｎａ
３ｍｇ ＣｏＡ
５ｍｇ ＭｇＣｌ₂．６Ｈ₂Ｏ
１９ｍｇ ＫＣｌ
１００ｍｇ ｐ−トルエンスルホン酸ナトリウム
３ｍｇ Ｔｒｉｔｏｎ Ｘ−１００
これらを０．１Ｍ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．８５）５ｍｌに溶解した。
【０１１４】
脂肪酸発色試薬２（反応停止液）
Ｎ−エチルマレイミド５０ｍｇを０．０１Ｎ ＨＣｌ５ｍｌに溶解した。
【０１１５】
脂肪酸発色試薬３
３Ｕ アシルＣｏＡオキシダーゼ（東洋紡製）
５Ｕ ペルオキシダーゼ（ベーリンガーマンハイム製）
１．５ｍｇ ４−アミノアンチピリン塩酸塩
２．５ｍｇ ジエチル−ｍ−トルイジン
６００ｍｇ ＫＣｌ
これらを２０ｍＭ Ｎ，Ｎ−ビス（２−ヒドロキシメチル）−２−アミノエタンスルホン酸緩衝液（ｐＨ７．５）２０ｍｌに溶解した。
【０１１６】
試料の検出：５０μｌの１０００μＥｑ／ｌのオレイン酸カリウムを含む水溶液をシャーレ底部に点着した。これを検出媒体吸水部にて吸い取り、予め試験管内に入れておいた脂肪酸発色試薬１（０．５ｍｌ）に漬けて、室温で３０分間反応させた。次いで脂肪酸発色試薬２を数滴滴下して混合し、数分後、脂肪酸発色試薬３を２ｍｌ加えて発色させた。３０分後、目視によって発色が確認され、脂肪酸が検出できることが確認された。
【０１１７】
【発明の効果】
本発明による残留物の検出方法及び残留物検出キットは、バックグラウンドが低く、再現性の高い良好な効果を有する。
【図面の簡単な説明】
【図１】本発明の一例を示す残留物検出キット（標準型）構成図である。
【図２】本発明の他の例を示す残留物検出キット（改良型）構成図である。
【図３】本発明の他の例を示す残留物検出キット（簡易型）構成図である。
【図４】本発明の好ましい一例を示す検出媒体の構成図である。
【符号の説明】
１ 検出媒体
２ キャップ
３ ガラスチューブ
４ 試薬Ａ
５ 試薬Ｂ
６ 水性媒体
７ キャップ
８ 試薬Ａ
９ 透明チューブ
１０ 水性媒体
１１ 可撓性ケース
１２ 試薬Ｂ
１３ 検出媒体
１４ 吸水部（試料採取部、検出部）
１５ 試薬Ａ
１７ 試薬混合容器[0001]
[Industrial application fields]
The present invention relates to a method for detecting residues (amino acids, carbohydrates, lipids, aldehydes and alcohols) and a residue detection kit.
[0002]
[Prior art]
Of clothing, food, and dwelling, which are the three major elements of material conditions for living a human life, clothing and dwelling can be moderated to some extent under the environment and other conditions, but food is essential in daily life.
[0003]
Therefore, food should always contain moderate nutrients as a life sustaining source and should not harm health. Although Japan's public health status has improved remarkably, it cannot be said that it is completely hygienic from a qualitative viewpoint in terms of food hygiene.
[0004]
It is necessary to hygienically control the quality and properties of food and drink-related additives, containers, and packaging environments so that hygiene hazards caused by food and drink do not occur.
[0005]
Especially in facilities such as food processing plants or pharmaceutical manufacturing plants, where different types of products are often manufactured using the same facility, the line is switched after the facility once used is cleaned. . At this time, until now, the degree of cleaning has not been particularly determined by checking the residue. For this reason, cleaning operations have been performed more than necessary, and work efficiency has been reduced. On the contrary, there is a concern that the product may be contaminated or contaminated by an insufficient cleaning operation.
[0006]
[Problems to be solved by the invention]
An object of the present invention is to provide a simple and accurate residue detection method and residue detection kit. In particular, a residue detection method for easily and accurately detecting residual substances (such as carbohydrates, sugars, amino acids, or aldehydes, alcohols, lipids (neutral fats, fatty acids)) in equipment and equipment of food processing factories and pharmaceutical manufacturing factories, and It is an object of the present invention to provide a residue detection kit, and to provide a residue detection method and a residue detection kit that can be used for determination of cleaning degree and confirmation of contamination status. The object of the present invention is not limited to the above utilization method, but is to provide a detection method and a residue detection kit for simply and quickly detecting a minute amount of residue adhering to a solid object.
[0007]
[Means for Solving the Problems]
The above object of the present invention is achieved by the following configurations.
[0008]
In the invention of claim 1, a flexible case in which a reagent that develops color upon contact with an amino acid is enclosed, a detection medium having a water absorbing part for transferring the amino acid at the tip of a support rod, and the reagent and the detection medium are brought into contact with each other. In the amino acid detection method using the amino acid detection kit, which is combined with the cap-capped tube to be stored in one,
The amino acid detection kit fixes the upper end of the support rod of the detection medium stored in the tube to the bottom of the cap, and incorporates a flexible case in which the reagent is enclosed in the cap.
By filling the tube with an aqueous medium and covering the cap, the water absorption part of the detection medium can be moistened before transferring the amino acid from the sample. After the amino acid is transferred to the detection medium, the detection medium is returned to the tube. The cap is closed, the flexible case is crushed, the reagent is dropped into the tube and brought into contact with the water-absorbing part, and amino acids attached to the surface of the sample are detected by the degree of color development.
[0009]
According to the second aspect of the present invention, a flexible case in which a reagent that develops color upon contact with a carbohydrate is enclosed, a detection medium having a water absorbing part for transferring the carbohydrate at the tip of a support rod, and the reagent and the detection medium are brought into contact with each other. In the carbohydrate detection method using the carbohydrate detection kit which is combined with the tube with the cap to be stored and stored in one,
The carbohydrate detection kit fixes the upper end of the support rod of the detection medium stored in the tube to the bottom of the cap, and incorporates a flexible case in which the reagent is enclosed in the cap.
By filling the tube with an aqueous medium and covering the cap, the water absorption part of the detection medium can be moistened before the carbohydrate is transferred from the sample, and after the carbohydrate is transferred to the detection medium, the detection medium is returned to the tube. Then, the cap is closed, the flexible case is crushed, the reagent is dropped into the tube and brought into contact with the water absorption part, and the carbohydrate adhering to the surface of the sample is detected by the degree of color development.
[0010]
According to a third aspect of the present invention, a flexible case in which a reagent that develops color upon contact with an aldehyde is sealed, a detection medium having a water absorbing portion for transferring the aldehyde at the tip of a support rod, and the reagent and the detection medium are brought into contact with each other. In an aldehyde detection method using an aldehyde detection kit that is combined with a tube with a cap to be stored in one,
The aldehyde detection kit fixes the upper end of the support rod of the detection medium stored in the tube to the bottom of the cap, and incorporates a flexible case in which the reagent is enclosed in the cap,
By filling the tube with an aqueous medium and covering the cap, the water absorption part of the detection medium can be moistened before the aldehyde is transferred from the sample. After the aldehyde is transferred to the detection medium, the detection medium is returned to the tube. The cap is closed, the flexible case is crushed, the reagent is dropped into the tube and brought into contact with the water-absorbing part, and aldehyde adhering to the surface of the sample is detected by the degree of color development.
[0011]
According to a fourth aspect of the present invention, a flexible case in which a reagent that develops color upon contact with alcohol is enclosed, a detection medium having a water-absorbing portion for transferring the alcohol at the tip of the support rod, and the reagent and the detection medium are brought into contact with each other. In the alcohol detection method using the alcohol detection kit, which is combined with a tube with a cap to be stored in one,
The alcohol detection kit fixes the upper end of the support rod of the detection medium stored in the tube to the bottom of the cap, and incorporates a flexible case in which the reagent is enclosed in the cap.
By filling the tube with an aqueous medium and covering the cap, the water absorption part of the detection medium can be moistened before the alcohol is transferred from the sample. After the alcohol is transferred to the detection medium, the detection medium is returned to the tube. Then, the cap is closed, the flexible case is crushed, the reagent is dropped into the tube and brought into contact with the water absorption part, and alcohol adhering to the surface of the sample is detected by the degree of color development.
[0012]
In the invention of claim 5, a flexible case in which a reagent that develops color upon contact with lipid is enclosed, a detection medium having a water absorbing part for transferring lipid at the tip of a support rod, and the reagent and the detection medium are brought into contact with each other. In a lipid detection method using a lipid detection kit comprising a tube with a cap to be combined and housed in one,
The lipid detection kit fixes the upper end of the support rod of the detection medium stored in the tube to the bottom of the cap, and incorporates a flexible case in which the reagent is enclosed in the cap.
By filling the tube with an aqueous medium and covering the cap, the water absorption part of the detection medium can be moistened before transferring the lipid from the sample. After the lipid is transferred to the detection medium, the detection medium is returned to the tube. The cap is closed, the flexible case is crushed, the reagent is dropped into the tube and brought into contact with the water-absorbing part, and the lipid adhering to the surface of the sample is detected by the degree of color development.
[0013]
The invention according to claim 6 is characterized in that the reagent is a coloring reagent solution containing oxidase.
[0014]
According to the seventh aspect of the present invention, a flexible case in which a reagent that develops color upon contact with an amino acid is enclosed, a detection medium having a water absorbing part for transferring the amino acid at the tip of a support rod, and the reagent and the detection medium are brought into contact with each other. In the amino acid detection kit in which the tube with a cap to be combined is stored in one,
The upper end of the support rod of the detection medium housed in the tube is fixed to the bottom of the cap, and a flexible case in which the reagent is sealed is built in the cap, and the tube is filled with an aqueous medium and the cap is covered. The water-absorbing part of the detection medium can be moistened before transferring the amino acid from the sample, and after the amino acid has been transferred to the detection medium, the detection medium is returned to the tube, the cap is closed, and the flexible case is crushed. The reagent is dropped into a tube and brought into contact with a water absorption part.
[0015]
In the invention of claim 8, a flexible case in which a reagent that develops color upon contact with a carbohydrate is sealed, a detection medium having a water absorbing part for transferring the carbohydrate at the tip of a support rod, and the reagent and the detection medium are brought into contact with each other. In the carbohydrate detection kit that is combined with the tube with the cap to be stored in one,
The upper end of the support rod of the detection medium housed in the tube is fixed to the bottom of the cap, and a flexible case in which the reagent is sealed is built in the cap, and the tube is filled with an aqueous medium and the cap is covered. The water absorption part of the detection medium can be moistened before transferring the carbohydrate from the sample in step 1. After the carbohydrate is transferred to the detection medium, the detection medium is returned to the tube, the cap is closed, and the flexible case is crushed. The reagent is dropped into a tube and brought into contact with a water absorption part.
[0016]
According to the ninth aspect of the present invention, a flexible case in which a reagent that develops color upon contact with an aldehyde is sealed, a detection medium having a water absorbing part for transferring the aldehyde at the tip of the support rod, and the reagent and the detection medium are brought into contact with each other. In the carbohydrate detection kit that is combined with the tube with the cap to be stored in one,
The upper end of the support rod of the detection medium housed in the tube is fixed to the bottom of the cap, and a flexible case in which the reagent is sealed is built in the cap, and the tube is filled with an aqueous medium and the cap is covered. The water absorption part of the detection medium can be moistened before transferring the aldehyde from the sample, and after the aldehyde has been transferred to the detection medium, the detection medium is returned to the tube, the cap is closed, and the flexible case is crushed. The reagent is dropped into a tube and brought into contact with a water absorption part.
[0017]
In the invention of claim 10, a flexible case in which a reagent that develops color upon contact with alcohol is enclosed, a detection medium having a water absorption part for transferring the alcohol at the tip of the support rod, and the reagent and the detection medium are brought into contact with each other. In the alcohol detection kit that is combined with the tube with the cap to be stored in one,
The upper end of the support rod of the detection medium housed in the tube is fixed to the bottom of the cap, and a flexible case in which the reagent is sealed is built in the cap, and the tube is filled with an aqueous medium and the cap is covered. The water absorption part of the detection medium can be moistened before transferring the alcohol from the sample in step 1. After the alcohol is transferred to the detection medium, the detection medium is returned to the tube, the cap is closed, and the flexible case is crushed. The reagent is dropped into a tube and brought into contact with a water absorption part.
[0018]
In the eleventh aspect of the invention, a flexible case in which a reagent that develops color upon contact with lipid is enclosed, a detection medium having a water absorbing part for transferring the lipid at the tip of the support rod, and the reagent and the detection medium are brought into contact with each other. In a lipid detection kit that is combined with a tube with a cap to be stored in one,
The upper end of the support rod of the detection medium housed in the tube is fixed to the bottom of the cap, and a flexible case in which the reagent is sealed is built in the cap, and the tube is filled with an aqueous medium and the cap is covered. The water absorption part of the detection medium can be moistened before transferring the lipid from the sample in step 1. After the lipid is transferred to the detection medium, the detection medium is returned to the tube, the cap is closed, and the flexible case is crushed. The reagent is dropped into a tube and brought into contact with a water absorption part.
[0019]
The invention of claim 12 is characterized in that the reagent comprises a coloring reagent solution containing oxidase.
[0020]
In a thirteenth aspect of the present invention, the water absorbing portion of the detection medium is made of a crystalline polymer fiber.
[0021]
The invention according to claim 14 is characterized in that the water absorption portion of the detection medium is made of a hydrophobic fiber.
[0022]
The invention of claim 15 is characterized in that the reagent comprises a coloring reagent solution containing oxidase.
[0023]
The invention according to claim 16 is characterized in that the flexible case is formed by individually sealing two kinds of reagents.
[0026]
As in the configuration of the present invention described above
As a result of intensive studies, the present inventors have found that in a method for detecting a residue adhering as a solid and / or a solution to a sample surface, the target portion of the sample surface is transferred to a detection medium, and this is detected as a detection reagent. A residue detection method and a residue kit were developed in a simple manner by detecting the residue attached to the surface of the sample according to the degree of color development.
[0027]
Specifically, a target portion such as equipment is wiped with a detection medium, and the adhering components are transferred to the detection medium. Next, the detection medium is reacted with the detection reagent by contact, and the presence or concentration of the residue is measured. By selecting detection reagents, it is possible to detect amino acids, carbohydrates (starch, sugar, etc.), alcohols, aldehydes, lipids (neutral fats / fatty acids), and can be used for a wider range of applications. is there.
[0028]
Specific examples of use of the present invention include, in addition to the cleaning degree inspection of equipment such as a food processing factory or a pharmaceutical manufacturing factory, for example, saliva in the oral cavity is collected using a detection medium, and this is detected with ethanol. By reacting with a reagent, it can also be used to check the presence or absence of alcohol consumption by a car driver.
[0029]
The present invention is described in detail below.
[0030]
In the present invention, the residue is transferred from the target portion of the sample surface to the detection medium, and the detection medium to which the residue has transferred is brought into contact with the reaction coloring reagent solution prepared in advance, and then heated as necessary to develop the color. Quantify the amount of residue adhering to the sample with a color sample, calibration curve (residue amount-coloring concentration), spectrophotometer, color meter, etc. in a short time of 1 to 60 minutes. This is a method for detecting residual residues.
[0031]
The sample used in the present invention is to examine whether or not residues are attached to the surface.
[0032]
The detection medium is preferably composed of a water-absorbing part and a support rod, and the water-absorbing part preferably has a structure in which fibers are gathered together, and preferably has a swab-like form. Since the detection reaction is mainly performed in the water absorption part of the detection medium, the fibers in the water absorption part that are white are dyed, and visual confirmation becomes extremely easy. The water absorbing portion is preferably made of a crystalline polymer fiber.
[0033]
As a result, there is little variation due to the product lot, and highly accurate measurement can be performed. Furthermore, non-specific color development is prevented by using a detection medium in which the water absorbing portion is made of a hydrophobic fiber, and more accurate and highly sensitive detection is possible.
[0034]
Specifically, fibers are entangled at the tip of the support like a cotton swab. A commercially available cotton swab can also be used as the detection medium of the present invention, but preferably has a water-absorbing portion made of crystalline polymer fibers or hydrophobic fibers.
[0035]
In the present invention, it is preferable that the water absorbing portion of the detection medium is wetted with an aqueous medium before wiping the sample.
[0036]
Examples of crystalline polymer fibers include:
・ Nylon type:-(NH (CH ₂ ) xNH-CO (CH ₂ ) y-CO) -n
・ Polyester: polycondensate of -CO-O-polyhydric alcohol and polybasic acid
・ Acrylic: Acrylonitrile CH ₂ = CHCN polymer
・ Polyvinyl alcohol system:-(CH ₂ C (OH) H) -n
・ Polyurethane system: -NHCOO-polymer with so-called urethane bond
・ Polyvinylidene chloride :-( CH ₂ CHCl ₂ ) -n radical polymer of vinylidene chloride
・ Polyvinyl chloride:-(CH ₂ CHCl) -n vinyl chloride polymer
・ Polyfluoroethylene type:
・ Polypropylene type :-( CH _Three CHCHCH ₂ ) -n Propylene polymer
・ Polyethylene:-(CH ₂ CH ₂ ) -n
Etc.
[0037]
Particularly preferred among these are hydrophobic fibers, for example, fibers made of a polymer that does not contain a hydrophilic group (hydroxyl group, amino group, carboxyl group, amide group) in the repeating unit of the polymer. For example, polyester fiber does not contain a hydrophilic group (hydroxyl group, amino group, carboxyl group, amide group) in the polymer repeating unit, and only contains a hydroxyl group or a carboxyl group at the end of the molecule, and is hydrophilic. Is extremely small.
[0038]
Specific examples include polyester, polyacryl, polypropylene, polyethylene, polystyrene, polyvinyl chloride, polyurethane and the like. In particular, when polyester, polyethylene, or polypropylene is used, non-specific color development is small and particularly preferable.
[0039]
In addition, as for the shape, it is only necessary that the detection medium water absorption part has water absorption retention. For example, in the case of a film-like material, its function can be sufficiently secured by processing such as providing fine holes or slits. Preferably, fibrous materials are gathered together and shaped like a cotton swab.
[0040]
Examples of a method for transferring the residue on the sample surface to the detection medium include a method of wiping, pressing, and sucking. As a wiping method, for example, a swab such as a cotton swab or a membrane filter is used. As a method of pressure bonding, for example, there is a method of pressure bonding using a tape, a stamp, a strip base, or the like with an adsorbing material, or a roller with an adsorbing material. Examples of the sucking method include a method of sucking with a water-absorbing cotton swab, a filter, and directly sucking with a dropper.
[0041]
In the case of the sucking method, sampling can be performed more efficiently, for example, by dropping water or a surfactant aqueous solution on the surface of the sample for a while and then sucking the residue on the surface.
[0042]
Examples of the aqueous medium include physiological saline, purified water (eg, distilled water, deionized water), a surfactant aqueous solution, a water-soluble organic solvent (eg, acetone, ethanol, propyl alcohol, methyl ethyl ketone, etc.) 95% solution). In the case of alcohol detection, naturally it should not contain alcohol. In particular, when detecting a lipid such as neutral fat or fatty acid, it is preferable to use an aqueous surfactant solution because the sampling accuracy and detection sensitivity are improved.
[0043]
Specific examples of the surfactant in the surfactant aqueous solution include the following.
[0044]
Nonionic activator
1. Sorbitan fatty acid ester
2. Glycerin fatty acid ester
3. Decaglycerin fatty acid ester
4). Polyglycerin fatty acid ester
5. Propylene glycol pentaerythritol fatty acid ester
6). Polyoxyethylene sorbitan fatty acid ester
7. Polyoxyethylene sorbitan fatty acid ester
8). Polyoxyethylene glycerin fatty acid ester
9. Polyethylene glycol fatty acid ester
10. Polyoxyethylene alkyl ether
11. Polyoxyethylene phytosterol / phytostanol
12 Polyoxyethylene polyoxypropylene alkyl ether
13. Polyoxyethylene alkyl phenyl ether
14 Polyoxyethylene castor oil / hardened castor oil
15. Polyoxyethylene lanolin, lanolin alcohol, beeswax derivatives
16. Polyoxyethylene alkylamine / fatty acid amide
17. Polyoxyethylene alkylphenyl formaldehyde condensate
18. Single chain length polyoxyethylene alkyl ether
As said alkyl, linear and branched C1-C18 are represented.
[0045]
Moreover, the following are mentioned as a commercial item of the said surfactant, for example.
[0046]
Nonionic surfactant: Triton X-100 (Wako Pure Chemical Industries, Ltd., Tween-20 (Wako Pure Chemical Industries, Ltd., Nonidet P-40 (Boehringer Mannheim Yamanouchi Co., Ltd.))
The residue detection kit of the present invention includes, for example, a detection medium, a reaction coloring reagent, a reaction coloring reagent preparation container, a container (for example, a glass with a cap) for reacting the detection medium from which the residue has been transferred and the reaction coloring reagent solution. (Or plastic tube), reactive color reagent titration plate, color sample, and a frame with a fixed rubbing area to transfer residue on the surface of the reagent to the detection medium. The details will be specifically described in Examples.
[0047]
Moreover, the reaction coloring reagent known in the art can be used, and these reagents can be appropriately prepared and used as a reaction coloring reagent solution for detecting the residue of the present invention.
[0048]
The detection medium of the present invention is preferably a medium including a water-absorbing part capable of transferring the residue on the surface of the sample, and the water-absorbing part is preferably composed of a fiber structure, and the water-absorbing part is at the tip of the support. What is being held is good. FIG. 4 shows an example of a preferable detection medium of the present invention. The water absorption part 14 in FIG. 4 (a) is a combination of fibers, the water absorption part 14 in FIG. 4 (b) is a porous resin (sponge-like), and the water absorption part 14 in FIG. 4 (c). The part uses cloth. Reference numeral 16 denotes a support rod.
[0049]
The water-absorbing part of the detection medium also has a function of collecting a certain amount of sample from the surface of the object to be measured, bringing the detection reagent and sample into contact with each other, reacting, and promoting the reaction.
[0050]
For example, in the case of a measurement system that uses an oxidase enzyme as a detection reagent, the reagent soaks into the fiber assembly of the water-absorbing part, so that contact with air is facilitated, and oxygen necessary for the enzyme reaction is sufficiently supplied. There is also.
[0051]
That is, the color developing reagent is usually prepared in advance and stored in an appropriate container such as an appropriate bottle or eye drop bottle. Therefore, oxygen required for the enzyme reaction of oxidase may not be sufficient, and by performing a color reaction in the presence of a detection medium as in the method of the present invention, oxygen in the air is efficiently taken up, In particular, a measurement system using an oxidase enzyme enables highly sensitive measurement in a shorter time. Furthermore, visual confirmation is facilitated by dyeing the water absorbing part itself. In particular, it is possible to confirm the color development more clearly by adsorbing the coloring material generated by the detection reaction to the fibers of the water absorbing portion.
[0052]
Examples of what can be measured by the detection method of the present invention include those shown below. Carbohydrates (eg starch, sugars (eg glucose, sucrose, maltose etc.)), amino acids (eg glycine, alanine, valine, serine, leucine, glutamic acid, proline, hydroxyproline etc.), lipids (eg neutral fat, fatty acids (eg For example, saturated fatty acids such as caproic acid, caprylic acid, lauric acid, palmitic acid, and stearic acid, and unsaturated fatty acids such as myristoleic acid, oleic acid, erucic acid, linoleic acid, and linolenic acid))], and alcohol.
[0053]
Aldehyde detection method
Aldehydes can be detected by a red-purple dye produced by reaction with a Schiff reagent. Schiff reagents are commercially available per se and are readily available. This reagent is obtained by dissolving 200 mg of basic fuchsin in 120 ml of water, adding a solution obtained by dissolving 2 g of sodium bisulfite in 20 ml of water to make 200 ml, and adding 0.03 g of activated carbon and filtering. It is desirable to store the Schiff reagent in a brown bottle or in a shaded condition.
[0054]
In order to prevent non-specific color development, it is particularly desirable that the water absorbing portion of the detection medium used for aldehyde detection is made of hydrophobic fibers. Of these, polyester fiber, polyethylene fiber and the like are preferably used. Further, it is desirable that the support rod in contact with the water absorbing portion is also made of a hydrophobic polymer.
[0055]
Amino acid detection method
Amino acids can be detected by the purple dye produced by the ninhydrin reaction. The ninhydrin is preferably included in the kit as a 0.2 to 2% aqueous solution and / or alcohol solution. For example, a 70-95% ethanol solution can be incorporated into the kit as a coloring reagent. The method using ninhydrin is excellent in sensitivity without causing nonspecific color development in the fibers used in the water absorbing portion of the detection medium.
[0056]
Carbohydrate (starch / sugar) detection method
Starch can be detected by, for example, iodine-starch reaction. As a method for measuring saccharides, the following known methods can be used. Add several solutions of Benedict into the reducing sugar Benedict reaction test tube, immerse the detection medium with the sample on it, heat it strongly for 1 to 2 minutes, and then cool it. Depending on the reducing power of the reducing sugars present, it is colored red, yellow or green. Benedict reagent can be prepared by the following procedure.
[0057]
17.3 g of purified crystalline copper sulfate is dissolved in heated distilled water to make a total volume of 60 ml. Dissolve 173 g of sodium citrate and 100 g of anhydrous sodium carbonate in heated distilled water to make a total volume of 60 ml. After cooling, dilute with distilled water to make 850 ml, add solution A to this and mix to make Benedict reagent.
[0058]
Color test with 3,6 dinitrophthalic acid
In a test tube, several drops of an aqueous solution of 0.3% 3,6-dinitrophthalic acid (pyridine salt) and an alkaline solution (K ₂ CO _Three 125g and Na ₂ S ₂ O _Three ・ 5H ₂ A solution in which 25 g of O was dissolved in water to make the total amount 500 ml. ) Add a few drops, put the detection medium with the sample in the test tube, bring it into contact, and heat it for about 10 minutes (approximately 90 to 100 ° C.) to react. If reducing sugar is present, coloration can be confirmed visually. Alternatively, it can be more accurately quantified by diluting with water after cooling and measuring the absorbance at 450 nm.
[0059]
Method for Measuring Fluorescence of Reducing Sugar Using 4-Methoxybenzamidine or Benzamidine The reducing sugar is alkaline and immediately emits strong fluorescence when heated with 4-methoxybenzamidine or benzamidine. By this reaction, uronic acid, amino acid, and sialic acid also fluoresce. In the measurement, 1 drop of 30 mM 4-methoxybenzamidine or benzamidine aqueous solution and 1 drop of 1M KOH aqueous solution are added to a test tube, heated at 100 ° C. for 2 to 3 minutes, and immediately cooled with ice water to stop the reaction.
[0060]
When 4-methoxybenzamidine is used, the fluorescence intensity can be measured at an excitation wavelength of 365 nm and an emission wavelength of 470 nm. When benzamidine is used, the fluorescence intensity can be measured at an excitation wavelength of 310 nm and an emission wavelength of 470 nm.
[0061]
Alcohol detection method
A case where ethanol is measured will be described as an example of the alcohol detection method of the present invention. As a method for measuring ethanol, a method using an enzyme such as alcohol dehydrogenase or alcohol oxidase is preferable. The reaction of the method using alcohol dehydrogenase (hereinafter abbreviated as ADH) is as follows.
[0062]

Where NAD (P) ⁺ Is oxidized nicotinamide adenine dinucleotide phosphate, NAD (P) H is reduced nicotinamide adenine dinucleotide phosphate, and DIA is diaphorase.
[0063]
In the present invention, the reduced capture enzyme produced by the activity of alcohol dehydrogenase is quantitatively displayed by the reduced capture enzyme detection composition according to the present invention.
[0064]
That is, NADH or NADPH, which is a reductive capture enzyme, can be quantified by colorimetry, fluorescence, fluorescence, etc. by reacting NADH or NADPH with a suitable substance with a chromogenic chromogen, fluorescent precursor, illuminant, etc. it can. An appropriate substance here refers to a substance that catalyzes a reaction that oxidizes NADH or NADPH to reduce a chromogenic chromogen, a fluorescent precursor, a phosphor, etc., and specifically, 5-methylphenazinium. Compounds such as methyl sulfate, (1-methoxy) -5-methylmethylphenazinium methyl sulfate (hereinafter referred to as MeO-PMS), Meldola Blue (ie, 9-dimethylaminobenzo-α-phenazoxonium chloride) Dihydroriboamide reductase (NAD) ⁺ ) (Also known as diaphorase), NADH dehydrogenase (quinone) and the like.
[0065]
In this case, 3- (p-iodophenyl) -2- (p-nitrophenyl) -5-phenyl-2hydrogentetrazolium chloride) (Neo-TB), 3-3′- Dimethoxy-4,4-biphenylylene) -bis [2- (p-nitrophenyl) -5-phenyl-2-hydrogentetotezoribum chloride] (Nitro-TB), 3,3 '-(3,3'- Dimethoxy-4,4'-biphenylylene) -bis (2,5-diphenyl-2hydrogentetrazolium chloride) (TB), 3,3 '-(3,3'-dimethoxy-4,4'-biphenylylene) -bis [ 2,5-bis (p-nitrophenyl) -2hydrogen tetrazolium chloride) (TNTB) and the like.
[0066]
Furthermore, it is preferable to contain a buffering agent in order to obtain a pH suitable for specific binding reaction, enzyme reaction, color reaction and the like. Specifically, it is preferable to contain a sufficient amount so that it can relax in the range of pH = 6.0 to 10.0 when the body fluid sample is applied. Examples of buffers that can be used include “Chemical Handbook Basics” edited by the Chemical Society of Japan (Tokyo, Maruzen Co., Ltd. 1966) p1312-1320, N, E, GOOD, etc .; Biochemistry vol. 5, p467 (1966), Imamura, Saito; Chemistry. vol. 30 (2), p79 (1976), W, J Ferguson et al .; Anal. Biochem. , Vol. 104, p300 (1980), and the like. Specific examples include citrate, borate, phosphate, carbonate, Tris, barbitur, glycine, Good buffer, and the like. These buffering agents may be contained in the coloring reaction reagent as necessary.
[0067]
Furthermore, in the present invention, at least one of a polyhydric alcohol and a non-reducing sugar can be contained as a preservative for improving the storage stability and quantitative reproducibility of the reagent. The amount of these preservatives added is preferably 0.1% to 10 wt%, more preferably 1 to 15% wt%, based on the weight of the reagent layer.
[0068]
As the polyhydric alcohol, a sugar alcohol is preferable, and as the non-reducing sugar, a polysaccharide in which a reducing group is exhausted is preferable.
[0069]
Next, specific examples will be given.
[0070]
(Sugar alcohol)
Erythritol, threitol, arabinitol, ribitol, xylitol, sorbitol, galactitol, mannitol, allitol, poremitol, preseitol, myo-inositol,
chiro-inositol, scyllo-inositol, quercitol, bibunitol, cyclohexanetetrol, conditol.
[0071]
(Polysaccharide)
Trehalose, sucrose, isotrehalose, raffinose, gentianose, mesocytose, planteose, stachyose, pervasose, lycnose, α-dextrin, β-dextrin, γ-dextrin.
[0072]
These coloring reaction reagent preservatives and buffering agents are dissolved in water or an organic solvent and used as detection reagents.
[0073]
Further, as an enzyme analysis method for alcohol, there is a method using alcohol oxidase. The reaction of the detection method using alcohol oxidase (hereinafter abbreviated as AOD) is as follows. Generated H ₂ O ₂ Is detected by introducing it into an appropriate colorimetric reaction system. For example, a method of detecting by forming a detectable dye using peroxidase as a catalyst is preferably used.
[0074]

As the coloring reaction reagent, 4-aminoantipyrine or a salt thereof known as a modification of the Trinder reagent, l, 7-dihydroxynaphthalene; 3-N-sulfopropylaniline derivatives such as N-ethyl-N-sulfopropylaniline, N -Ethyl-N-sulfopropyl-3,5-dimethyloxyanilinesulfonic acid and its salts, N-sulfopropylaniline, N-ethyl-N-sulfopropyl-3,5-dimethylaniline, N-ethyl-N-sulfopropyl -M-toluidine; N-2-hydroxy-3-sulfopropylaniline derivatives such as N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-anisidine, N-ethyl-N- (2-hydroxy- 3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopro ) -3,5-dimethyloxyanilinesulfonic acid and salts thereof, N- (2-hydroxy-3-sulfopropyl) -3,5-dimethyloxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) ) -3,5-dimethylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine, and the like.
[0075]
Further chemiluminescent compounds such as noreminol (5-amino-2,3-dihydro-1,4-phthalazinedione), isoluminol (6-amino-2,3-dihydro-1,4-phthalazinedione), ABEI-H (N- (4-aminobutyl) -N-ethylisoluminololehemisuccinamide, AHEI (N- (6-aminohexyl) -N-ethinoreisolminol) ABEI (N- (4-aminobutyl)) -N-ethylisoluminol) and the like.
[0076]
Further, a diffusible phenol compound that develops color by a coupling reaction with an aromatic primary amine compound described in JP-A-57-94653, 57-94654, 57-94655, and 57-94656, or Pyrazolone compounds and other coloring reagents are used.
[0077]
If glucose oxidase (hereinafter referred to as GOD) is used in place of the alcohol oxidase, glucose can be detected. That is, the generated H ₂ O ₂ For example, a method of detecting peroxidase as a catalyst by forming a detectable dye is preferably used.
[0078]
glucose

In addition, by using an enzyme that hydrolyzes a disaccharide or oligosaccharide such as α-glucosidase, β-glucosidase, β-fructosidase, etc., the presence of them as glucose constituting the disaccharide or oligosaccharide. Can be detected in a batch. For example, if the following enzymes are used in combination, the presence of either maltose / sucrose / glucose can be detected at a time.
[0079]

Neutral fat
Neutral fat is an ester of glycerol and fatty acids. As a detection method, NADH produced by lipoprotein lipase (hereinafter referred to as LPL) and glycerol dehydrogenase (hereinafter referred to as GDH) is preferably detected by converting a tetrazolium salt into a formazan dye using diaphorase as a catalyst, for example. It is desirable that the detection reagent is dissolved in an appropriate buffer solution. For example, phosphate buffer solution, carbonate buffer solution, Tris-Tris hydrochloride buffer solution, and other good buffers are used in the range of 10 mM to 1 M and pH 7 to 10.5. It is appropriately selected and used. Oxidized nicotinamide adenine dinucleotide (NAD +) can be contained preferably at a concentration of 1 to 100 mM. Enzymes such as LPL, GDH and DIA can preferably contain 0.1 to 50 Unit / ml.
[0080]
Examples of tetrazolium salts include 2- (4-Iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H tetrazole chloride (INT), 3- (4,5-Dimethyl-2-thiazolyl) -2,5-diphenyl. -2H tetrazolebromide (MTT), 3,3 '-[3,3'-Dimethoxy- (1,1'-biphenyl) -4,4'-diyl] -bis [2- (4-nitrophenyl) -5-phenyl -2H tetrazolium chloride] (Nitro-TB) is preferably used.
[0081]

Alternatively, glycerol produced by LPL was converted to glycerol 3-phosphate (G 3-P) with glycerokinase (GK) in the presence of ATP, and this was oxidized by glycerol 3-phosphate oxidase (GPO). H ₂ O ₂ For example, a method of detecting peroxidase as a catalyst by forming a detectable dye is preferably used.
[0082]

fatty acid
Examples of the fatty acid include oleic acid, palmitic acid, stearic acid, and the like, and those that exist as a simple substance or in the form of a mixture are measurement targets. As shown below, hydrogen peroxide produced by the action of an enzyme such as acyl CoA synthetase (ACS) or acyl CoA oxidase (ACO) is introduced into an appropriate colorimetric reaction system and detected. When peroxidase is used as a colorimetric reaction system, since the SH group of CoA inhibits dye formation in the next reaction, it is necessary to block unreacted CoA with N-ethylmaleimide. These measurement methods are described in Kitamura Motoi (ed.): Free fatty acids, Practical clinical chemistry p530-544, Ishiyaku Shuppan Publishing, 1982.
[0083]

When measuring lipids such as neutral fat or fatty acid, the accuracy of sampling and the detection sensitivity are improved if the water-absorbing part of the detection medium is previously moistened with an aqueous solution containing a surfactant. As such a surfactant, a nonionic surfactant is preferably used. Specifically, polyoxyethylene alkylphenyl ethers such as Tween 20 and Triton X-100 are preferably used. The concentration of the surfactant used is preferably 0.05 to 5%.
[0084]
These reagents are preferably housed in one or more containers made of a flexible member having an opening, and when used, a part of the container is pressed to form a droplet from the opening. The sample is spotted on the water absorption part of the collected detection medium and reacted. Alternatively, the water absorption part of the detection medium is brought into contact with a reagent dispensed in a necessary amount in another container (such as a test tube) and reacted. If necessary, the reaction may be promoted by heating with a dryer or heater. When a reaction using an enzyme is used, the reaction is performed at room temperature (25 ° C.) or 37 ° C. If necessary, a reagent blank can be taken to check the difference in color development.
[0085]
Standard kit
The residue detection kit configuration (FIG. 1) showing an example of the present invention is the following configuration. That is, 5 reagent Bs in FIG. 1A (filled in an eyeglass bottle-shaped container), detection medium 1 (shape: cotton swab-shaped, handle is plastic φ2 mm), 3 glass tubes with cap 2 It consists of φ10 × 70mm. When two or more kinds of reagents are mixed and used immediately before the measurement due to storage problems, the reagent B (preferably an ophthalmic bottle shaped container) is used as the reagent 5 as the reagent 4 in the glass tube 3 in FIG. Can be divided and put as a filling). If a third reagent C is required, another 5 container can be added to the kit.
[0086]
In addition, when the surface of the measurement object is dry, the sample can be collected more efficiently when the detection medium water-absorbing portion 14 is moistened with an aqueous medium. 6 is an aqueous medium for that purpose, and distilled water, physiological saline, surfactant aqueous solution, buffer solution, hydrous alcohol or the like is used as necessary. In addition, if necessary, a thermostat (block heater or the like) to keep the reaction acceleration / reaction rate constant, a dryer or a test tube stand for simply heating to promote the reaction is prepared.
[0087]
In the case of precise measurement, a calibration curve is established and the produced dye in the reaction solution is measured with a spectrophotometer. Alternatively, it can be visually determined using a color meter (FIG. 1B) and a standard color scale (FIG. 1C). 1 (b) and 1 (c), 1a is a standard color scale, 1b is a light, 1c is a colorimetric window, 1d is a standard color scale holder, 1e is a test tube holder, 1f is a substance concentration (level), 1 g is a color sample indicating the degree of color development. An example of the kit operation method measurement is to prepare a coloring reagent by adding a few drops of reagent B to reagent A in a glass tube and mixing them.
[0088]
Next, a certain area of the surface of the measurement object is wiped off with the detection medium 1. At this time, if the sample surface is dry, it is efficient to wipe the water absorption part of the detection medium 1 with a suitable aqueous medium (distilled water, aqueous surfactant solution, physiological saline, hydrous alcohol, etc.) and wipe it off. It is. The detection medium 1 that has been wiped off is immersed in a coloring reagent and reacted.
[0089]
It is desired that the detection medium 1 is continuously immersed in the reaction solution during the reaction. In particular, when an oxidase is used as a detection reagent, this effectively supplies oxygen in the air and promotes the reaction. If necessary, warm the reaction in a thermostatic chamber to promote the reaction. The color development due to the residue is measured with a spectrophotometer or visually confirmed. At this time, if a standard color scale is prepared as required, semi-quantification is possible.
[0090]
Standard improved kit
Another example of the present invention is shown in FIG. That is, about 100 to 1000 μl of an aqueous medium 10 is contained in a suitable transparent tube 9, and a flexible case 11 in which 8, 12 reagents A and B are enclosed is attached to the lower stage of the cap 7. A detection medium 13 is fixed to the bottom of the cap 7. By squeezing the cap 7, the flexible case 11 is crushed, and the reagents A and B fall into the transparent tube 9 and come into contact with the detection medium 13. In addition, a thermostatic bath block heater, etc.), a spectrophotometer, a color meter, a standard color scale, a fresh test tube, etc. are prepared as necessary.
[0091]
Kit operation
In FIG. 2, the cap 7 is removed, and the sample is sampled from the surface of the measurement object with respect to the tip of the detection medium 13 in the manner shown in the standard kit operation method. The detection medium 13 is returned to the transparent tube, and the cap 7 is closed. The flexible case 11 containing the 8th and 12th reagents A and B is crushed, and the reagents A and B are dropped into the transparent tube. After stirring the transparent tube, the transparent tube is heated or kept at a constant temperature as necessary to allow the reaction to proceed. After a certain time, the degree of color development is confirmed visually or with a spectrophotometer. When visually confirming, the residue can be easily detected (semi-quantitative) by comparing with a standard color scale that has been facilitated in advance. The case where the coloring reagent is prepared by mixing the reagents A and B is illustrated, but when the coloring reagent is supplied as a single solution, a single coloring reagent solution can be used instead of the reagents A and B. Good. In the case of FIG. 2, 14 is a water absorption part and is a detection part.
[0092]
Simple kit
Another example of the present invention is shown in FIG. In this example, all the coloring reagents are dispensed and stored in a container (preferably an ophthalmic bottle-shaped container). A predetermined area of the surface of the measurement object is wiped off with the detection medium 1 in the same manner as in the standard kit operation method. A coloring reagent is directly added from the container to the water absorption part (sample collection part) of this detection medium, and a color reaction is performed on the water absorption part. Since the color development reaction proceeds in the sample collection unit according to the amount of collected residue, this can be determined visually. Semi-quantification is possible by comparing the degree of color development on a standard color scale if necessary. When the reagents A and B are desired to be mixed and used in advance, a mixture of both solutions in the container 17 is prepared, and the reagent may be added from here to the sampling part.
[0093]
Regardless of the type of kit, when measuring with a spectrophotometer, the colored reagent solution is measured. On the other hand, when visual confirmation is performed, it is possible to confirm by color development of the colored reaction reagent solution, but the water absorption part of the detection medium is more clearly stained. Sensitivity can be determined.
[0094]
【Example】
EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, the aspect of this invention is not limited to this. The form and measurement procedure of the residue detection kit of the present invention are shown below. When operating the kit, be careful not to touch the reagent or sampling medium directly with your fingers or hands when sampling.
[0095]
Example 1 Detection of glucose
Glucose was detected using a standard kit. GOD-POD coloring reagents A and B having the following composition were prepared, and 1 ml each of GOD-POD coloring reagent A and a GOD-POD coloring reagent B were filled in an eye drop bottle.
[0096]
GOD-POD coloring reagent A
0.1 M phosphate buffer pH 6.9
0.5 mM 4-aminoantipyrine
20 mM phenol
GOD-POD coloring reagent B
0.1 M phosphate buffer pH 6.9
500 U / ml glucose oxidase: GOD (manufactured by Boehringer Mannheim)
300 U / ml peroxidase: POD (manufactured by Boehringer Mannheim)
An aqueous solution containing 0.1% glucose in a petri dish was seeded with 0.1 ml and dried to obtain a measurement sample.
[0097]
Measurement: GOD-POD coloring reagent A is added dropwise to 1 ml of each glass tube by dropping 1 drop of GOD-POD coloring reagent B from the eye drop bottle and mixed by shaking lightly. Two to three drops (about 100 μl) of distilled water was included in the detection medium (cotton swab), the above petri dish was wiped off, this was immersed in a detection reagent, and allowed to react at 37 ° C. for 10 minutes. As a result, a color indicating the presence of glucose was confirmed. Compared to the reaction of 0.1 ml glucose solution containing 0.1% glucose directly into the detection reagent prepared in the same way, the reaction of the method of the present invention proceeds in a shorter time. Confirmed to do. Further, by measuring the absorbance of the color developing solution using a spectrophotometer, it was possible to know the amount of glucose collected by the detection medium from a calibration curve prepared in advance.
[0098]
Example 2 Detection of glucose
Glucose was detected using the following materials as the material of the water absorption part of the detection medium. The following GOD-POD coloring reagent C, which was placed in an eye drop bottle, was used as a detection reagent.
[0099]
1. Material of water-absorbing part as detection medium: a. Polyester b. Polyethylene c. Polyurethane was used.
[0100]
GOD-POD coloring reagent C
0.1M phosphate buffer pH 6.5
0.5 mM 4-aminoantipyrine
15 mM phenol
5 U / ml glucose oxidase: GOD (manufactured by Boehringer Mannheim)
3U / ml peroxidase: POD (Boehringer Mannheim)
2. Sample sampling
10 μl of 1% aqueous glucose solution was spotted on the bottom of the petri dish and once dried. This was wiped off at each detection medium water-absorbing part previously moistened with distilled water, and a few drops of GOD-POD coloring reagent C were spotted on each, and reacted. After 30 minutes, the color density was visually evaluated on a 5-level scale (no color development-, light + to dark +++).
[0101]
3. Results In all cases, coloration was detected and detected due to the presence of glucose. In particular, when the material of the water absorbing portion is polyester or polyethylene fiber, it was confirmed that the color was strongly developed and the sensitivity was high.
[0102]
Material Distilled water only Glucose aqueous solution
a. − +++
b. − +++
c. − ++
Example 3 Detection of amino acids
The amino acid adhering to the sample was detected using a detection medium consisting of a water-absorbing part of the material. The following ninhydrin test solution was used for detection.
[0103]
Preparation of ninhydrin test solution: 80 mg of SnCl in a solution of 2 g of ninhydrin dissolved in 50 ml of distilled water ₂ The precipitate was filtered off and left in a dark place for more than one day and night, and distilled water was added to make 100 ml. 2 ml of water was added to 1 ml of this solution, and 18 ml of isopropanol was further added and mixed.
[0104]
1. Detection medium Material of water absorption part: a. Polyester b. Polyurethane c. Natural cotton yarn
2. Sampling of sample: 10 μl of glycine aqueous solution (10 mg / ml) spotted on the bottom of the petri dish was sucked by each water absorbing part of each detection medium, and several drops of each ninhydrin test solution were spotted, and this was heated with a drier to react. . Visual evaluation was made on a 5-level scale (no color development, light + to dark +++) according to the color density.
[0105]
3. Result: In all the examples, the water absorbing portion was colored purple and the presence of glycine was confirmed. The measurement results for n = 5 are shown below. The detection medium (a, b) having a water-absorbing part made of crystalline polymer fibers clearly had less variation in color compared to natural cotton yarn.
[0106]
Comparison of reproducibility
Material Color density (n = 5)
a. ++++ ++++ ++++ ++++
b. ++ ++ ++ ++ ++
c. ++++ ++ ++ ++ ++
Example 4 Detection of Aldehyde In the same procedure as in Example 2, aldehyde adhering to a sample was detected using a detection medium composed of a water absorbing portion made of a different material. A Schiff reagent was used for detection. 200 mg of base adjustment fuchsin of Schiff reagent is dissolved in 120 ml of water, and a solution of 2 g of sodium bisulfite in 20 ml of water is added to make 200 ml. 0.03 g of activated carbon was added, filtered, and stored in the dark until use.
[0107]
1. Detection medium Material of water absorption part: a. Polyester b. Polyethylene c. Natural cotton yarn
2. Sampling of sample: 10 μl of 0.1% formaldehyde aqueous solution spotted on the bottom of the petri dish was sucked up by each detection medium water-absorbing part, and several drops of each Schiff reagent were spotted and reacted. After 30 minutes, the color density was visually evaluated on a 5-level scale (no color development, light + to dark +++). Moreover, it measured similarly using distilled water instead of 0.1% formaldehyde aqueous solution, and compared the background.
[0108]
3. Result When using a detection medium having a water absorbing part made of crystalline polymer fibers such as polyester and polyethylene, the water absorbing part was not colored at all with distilled water containing no aldehyde, whereas the water absorbing part was natural cotton yarn. It has been found that even if it consists of distilled water, it will develop color even with distilled water.
[0109]
Material Distilled water 0.1% Formaldehyde aqueous solution
a. − ++
b. − ++
c. +++ +++
Example 5 Detection of alcohol
In the same procedure as in Example 3, ethanol adhering to the sample was detected using a detection medium composed of a water absorbing portion made of a different material. For detection, an AOD-POD coloring reagent having the following composition was prepared and used.
[0110]
AOD-POD coloring reagent
50 mM phosphate buffer pH 7.5
0.05% Triton X-100
2 mM EDTA-3Na
0.5 mM 4-aminoantipyrine
2.0 mM Sodium-N-ethyl-N- (3-sulfopropyl) -m-anisidine
5 U / ml peroxidase (Boehringer Mannheim)
10 U / ml alcohol oxidase (Toyobo)
1. Detection medium Material of water absorption part: a. Polyester fiber b. Polyurethane foam c. Natural cotton yarn
Sampling of sample: 10 μl of 0.1% ethanol aqueous solution was spotted on the bottom of the petri dish, and this was blotted in each water absorbing part of each detection medium, and several drops of each AOD-POD coloring reagent was spotted and reacted at room temperature. After 30 minutes, the color density was visually evaluated on a 5-level scale (no color development, light + to dark +++).
[0111]
2. Results In each case, it was confirmed that the presence of ethanol was colored and detectable. The measurement results for n = 5 are shown below. As described above, the detection medium (a, b) having a water absorbing portion made of a hydrophobic fiber had little variation in color development.
[0112]
Comparison of reproducibility
Material Color density (n = 5)
a. ++++ ++++ ++++ ++++
b. ++ ++ ++ ++ ++++
c. ++ ++++ ++ ++++ ++
Example 6 Detection of fatty acids
The fatty acid adhering to the sample was detected in the same procedure as in Example 1. A detection medium (a cotton swab) having a water absorbing portion of natural cotton yarn was used. The following fatty acid coloring reagents 1 to 3 were used for detection.
[0113]
Fatty acid coloring reagent 1
5U acyl CoA synthetase (Toyobo)
4U myokinase (Boehringer Mannheim)
10mg ATP · 2Na
3mg CoA
5mg MgCl ₂ . 6H ₂ O
19mg KCl
100 mg sodium p-toluenesulfonate
3mg Triton X-100
These were dissolved in 5 ml of 0.1 M Tris-HCl buffer (pH 7.85).
[0114]
Fatty acid coloring reagent 2 (reaction stop solution)
50 mg of N-ethylmaleimide was dissolved in 5 ml of 0.01N HCl.
[0115]
Fatty acid coloring reagent 3
3U acyl CoA oxidase (Toyobo)
5U peroxidase (Boehringer Mannheim)
1.5mg 4-aminoantipyrine hydrochloride
2.5mg diethyl-m-toluidine
600mg KCl
These were dissolved in 20 ml of 20 mM N, N-bis (2-hydroxymethyl) -2-aminoethanesulfonic acid buffer (pH 7.5).
[0116]
Sample detection: An aqueous solution containing 50 μl of 1000 μEq / l potassium oleate was spotted on the bottom of the petri dish. This was absorbed by a detection medium water absorption part, immersed in a fatty acid coloring reagent 1 (0.5 ml) previously placed in a test tube, and allowed to react at room temperature for 30 minutes. Next, a few drops of the fatty acid coloring reagent 2 were dropped and mixed, and after a few minutes, 2 ml of the fatty acid coloring reagent 3 was added to cause color development. After 30 minutes, color development was visually confirmed, and it was confirmed that fatty acids could be detected.
[0117]
【The invention's effect】
The residue detection method and residue detection kit according to the present invention have a good effect with low background and high reproducibility.
[Brief description of the drawings]
FIG. 1 is a configuration diagram of a residue detection kit (standard type) showing an example of the present invention.
FIG. 2 is a configuration diagram of a residue detection kit (improved type) showing another example of the present invention.
FIG. 3 is a configuration diagram of a residue detection kit (simple type) showing another example of the present invention.
FIG. 4 is a configuration diagram of a detection medium showing a preferred example of the present invention.
[Explanation of symbols]
1 Detection medium
2 cap
3 Glass tube
4 Reagent A
5 Reagent B
6 Aqueous media
7 Cap
8 Reagent A
9 Transparent tube
10 Aqueous medium
11 Flexible case
12 Reagent B
13 Detection medium
14 Water absorption part (sample collection part, detection part)
15 Reagent A
17 Reagent mixing container

Claims (16)

A flexible case in which a reagent that develops color upon contact with an amino acid is sealed, a detection medium having a water absorbing portion for transferring the amino acid at the tip of a support rod, and a tube with a cap that contacts the reagent and the detection medium In the amino acid detection method using the amino acid detection kit combined and stored in one,
The amino acid detection kit fixes the upper end of the support rod of the detection medium stored in the tube to the bottom of the cap, and incorporates a flexible case in which the reagent is enclosed in the cap.
By filling the tube with an aqueous medium and covering the cap, the water absorption part of the detection medium can be moistened before transferring the amino acid from the sample. After the amino acid is transferred to the detection medium, the detection medium is returned to the tube. The cap is closed, the flexible case is crushed, the reagent is dropped into the tube and brought into contact with the water-absorbing part, and the amino acid attached to the surface of the sample is detected by the degree of color development. Detection method. A flexible case in which a reagent that develops color upon contact with a carbohydrate is enclosed, a detection medium having a water absorption part for transferring the carbohydrate at the tip of a support rod, and a tube with a cap that contacts the reagent and the detection medium In the carbohydrate detection method using the carbohydrate detection kit combined and stored in one,
The carbohydrate detection kit fixes the upper end of the support rod of the detection medium stored in the tube to the bottom of the cap, and incorporates a flexible case in which the reagent is enclosed in the cap.
By filling the tube with an aqueous medium and covering the cap, the water absorption part of the detection medium can be moistened before the carbohydrate is transferred from the sample, and after the carbohydrate is transferred to the detection medium, the detection medium is returned to the tube. And closing the cap, crushing the flexible case, dropping the reagent into the tube and bringing it into contact with the water-absorbing part, and detecting the carbohydrate adhering to the surface of the sample by the degree of color development Detection method. A flexible case in which a reagent that develops color upon contact with an aldehyde is sealed, a detection medium having a water absorption part for transferring the aldehyde at the tip of a support rod, and a tube with a cap that contacts the reagent and the detection medium In the aldehyde detection method using the aldehyde detection kit combined and housed in one,
The aldehyde detection kit fixes the upper end of the support rod of the detection medium stored in the tube to the bottom of the cap, and incorporates a flexible case in which the reagent is enclosed in the cap,
By filling the tube with an aqueous medium and covering the cap, the water absorption part of the detection medium can be moistened before the aldehyde is transferred from the sample. After the aldehyde is transferred to the detection medium, the detection medium is returned to the tube. The cap is closed, the flexible case is crushed, the reagent is dropped into the tube and brought into contact with the water-absorbing part, and the aldehyde adhering to the surface of the sample is detected by the degree of color development. Detection method. A flexible case in which a reagent that develops color upon contact with alcohol is enclosed, a detection medium having a water absorbing portion for transferring alcohol at the tip of a support rod, and a tube with a cap that contacts the reagent and the detection medium In the alcohol detection method using the alcohol detection kit which is combined and stored in one,
The alcohol detection kit fixes the upper end of the support rod of the detection medium stored in the tube to the bottom of the cap, and incorporates a flexible case in which the reagent is enclosed in the cap.
By filling the tube with an aqueous medium and covering the cap, the water absorption part of the detection medium can be moistened before the alcohol is transferred from the sample. After the alcohol is transferred to the detection medium, the detection medium is returned to the tube. The cap is closed, the flexible case is crushed, the reagent is dropped into the tube and brought into contact with the water absorption part, and alcohol adhering to the surface of the sample is detected by the degree of color development. Detection method. A flexible case in which a reagent that develops color upon contact with lipid is enclosed, a detection medium having a water absorbing part for transferring lipid at the tip of a support rod, and a tube with a cap that contacts the reagent and the detection medium In a lipid detection method using a lipid detection kit that is combined and housed in one,
The lipid detection kit fixes the upper end of the support rod of the detection medium stored in the tube to the bottom of the cap, and incorporates a flexible case in which the reagent is enclosed in the cap.
By filling the tube with an aqueous medium and covering the cap, the water absorption part of the detection medium can be moistened before transferring the lipid from the sample. After the lipid is transferred to the detection medium, the detection medium is returned to the tube. The cap is closed, the flexible case is crushed, the reagent is dropped into the tube and brought into contact with the water absorption part, and the lipid adhering to the surface of the sample is detected by the degree of color development. Detection method.   6. The detection method according to claim 1, wherein the reagent is a coloring reagent solution containing oxidase. A flexible case in which a reagent that develops color upon contact with an amino acid is sealed, a detection medium having a water absorbing portion for transferring the amino acid at the tip of a support rod, and a tube with a cap that contacts the reagent and the detection medium In the amino acid detection kit that is combined and stored in one,
The upper end of the support rod of the detection medium housed in the tube is fixed to the bottom of the cap, and a flexible case in which the reagent is sealed is built in the cap, and the tube is filled with an aqueous medium and the cap is covered. The water-absorbing part of the detection medium can be moistened before transferring the amino acid from the sample, and after the amino acid has been transferred to the detection medium, the detection medium is returned to the tube, the cap is closed, and the flexible case is crushed. An amino acid detection kit, wherein the reagent is dropped into a tube and brought into contact with a water absorption part. A flexible case in which a reagent that develops color upon contact with a carbohydrate is enclosed, a detection medium having a water absorption part for transferring the carbohydrate at the tip of a support rod, and a tube with a cap that contacts the reagent and the detection medium In the carbohydrate detection kit that is combined and stored together,
The upper end of the support rod of the detection medium housed in the tube is fixed to the bottom of the cap, and a flexible case in which the reagent is sealed is built in the cap, and the tube is filled with an aqueous medium and the cap is covered. The water absorption part of the detection medium can be moistened before transferring the carbohydrate from the sample in step 1. After the carbohydrate is transferred to the detection medium, the detection medium is returned to the tube, the cap is closed, and the flexible case is crushed. A carbohydrate detection kit, wherein the reagent is dropped into a tube and brought into contact with a water absorption part. A flexible case in which a reagent that develops color upon contact with an aldehyde is sealed, a detection medium having a water absorption part for transferring the aldehyde at the tip of a support rod, and a tube with a cap that contacts the reagent and the detection medium In the carbohydrate detection kit that is combined and stored together,
The upper end of the support rod of the detection medium housed in the tube is fixed to the bottom of the cap, and a flexible case in which the reagent is sealed is built in the cap, and the tube is filled with an aqueous medium and the cap is covered. The water absorption part of the detection medium can be moistened before transferring the aldehyde from the sample, and after the aldehyde has been transferred to the detection medium, the detection medium is returned to the tube, the cap is closed, and the flexible case is crushed. An aldehyde detection kit, wherein the reagent is dropped into a tube and brought into contact with a water absorption part. A flexible case in which a reagent that develops color upon contact with alcohol is enclosed, a detection medium having a water absorbing portion for transferring alcohol at the tip of a support rod, and a tube with a cap that contacts the reagent and the detection medium In alcohol detection kits that are combined and stored together,
The upper end of the support rod of the detection medium housed in the tube is fixed to the bottom of the cap, and a flexible case in which the reagent is sealed is built in the cap, and the tube is filled with an aqueous medium and the cap is covered. The water absorption part of the detection medium can be moistened before transferring the alcohol from the sample in step 1. After the alcohol is transferred to the detection medium, the detection medium is returned to the tube, the cap is closed, and the flexible case is crushed. An alcohol detection kit, wherein the reagent is dropped into a tube and brought into contact with a water absorption part. A flexible case in which a reagent that develops color upon contact with lipid is enclosed, a detection medium having a water absorbing part for transferring lipid at the tip of a support rod, and a tube with a cap that contacts the reagent and the detection medium In the lipid detection kit that is combined and housed together,
The upper end of the support rod of the detection medium housed in the tube is fixed to the bottom of the cap, and a flexible case in which the reagent is sealed is built in the cap, and the tube is filled with an aqueous medium and the cap is covered. The water absorption part of the detection medium can be moistened before transferring the lipid from the sample in step 1. After the lipid is transferred to the detection medium, the detection medium is returned to the tube, the cap is closed, and the flexible case is crushed. A lipid detection kit, wherein the reagent is dropped into a tube and brought into contact with a water-absorbing part.   The detection kit according to claim 7, wherein the reagent is a coloring reagent solution containing oxidase.   The detection kit according to any one of claims 7 to 11, wherein the water absorbing portion of the detection medium is made of a crystalline polymer fiber.   The detection kit according to claim 13, wherein the water absorbing portion of the detection medium is made of a hydrophobic fiber.   The detection kit according to claim 7, wherein the reagent is a coloring reagent solution containing oxidase.   The detection kit according to any one of claims 7 to 11, wherein the flexible case is formed by individually sealing two kinds of reagents.

Images