JP6128808B2 - Enzyme activity detection and measurement method and kit using the same - Google Patents
Enzyme activity detection and measurement method and kit using the same Download PDFInfo
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Description
本発明は、被験物質に含まれる酵素、被験物質から抽出した酵素、被験物質に標識した酵素等の酵素活性を検出、測定することによって被験物質を検出、測定する際に使用する、酵素活性の検出、測定方法およびこれを利用したキットに関する。 The present invention relates to an enzyme activity used for detecting and measuring a test substance by detecting and measuring the enzyme activity of an enzyme contained in the test substance, an enzyme extracted from the test substance, an enzyme labeled with the test substance, etc. The present invention relates to a detection and measurement method and a kit using the same.
近年、医療現場において、時機を逸しない的確な診療行為を実施するために、患者検体を用いた臨床検査の結果が迅速に求められるようになった。そこで、Ｐｏｉｎｔ ｏｆ Ｃａｒｅ Ｔｅｓｔｉｎｇ（ＰＯＣＴ）と呼ばれる簡便な装置や器具を用いる臨床検査法が開発され（特許文献１、特許文献２）医療の現場に供されるようになって来た。 In recent years, the results of clinical tests using patient specimens have been rapidly required in order to carry out accurate medical care in a medical setting. Therefore, a clinical examination method using a simple device or instrument called Point of Care Testing (POCT) has been developed (Patent Document 1, Patent Document 2) and has come to be used in the medical field.
一方、被験物質に含まれる酵素、被験物質から抽出した酵素、被験物質に標識した酵素等の酵素活性（以下、酵素等の活性という。）を指標として患者検体中の被験物質を検出、測定する際には、発色基質等の酵素活性によってシグナルを生じさせる化合物を用いる。この場合、酵素活性による発色基質等のシグナルの発生は継続的に行われ、シグナル量が増加し続けることになる。しかし、忙しい医療現場においては、的確な時間において酵素活性によって生じたシグナル量を判定することは困難であると言わざるを得ない。 On the other hand, a test substance in a patient sample is detected and measured using an enzyme activity (hereinafter referred to as an enzyme activity) such as an enzyme contained in the test substance, an enzyme extracted from the test substance, or an enzyme labeled with the test substance as an index. In this case, a compound that generates a signal by an enzyme activity such as a chromogenic substrate is used. In this case, generation of a signal such as a chromogenic substrate due to the enzyme activity is continuously performed, and the amount of signal continues to increase. However, in a busy medical field, it must be said that it is difficult to determine the amount of signal generated by enzyme activity at an appropriate time.
そこで、自動分析装置を用いて、一定時間後に自動的に酵素活性を阻害する化合物を分注し、シグナルの発生を停止させることによって、その後の判定までの時間経過に影響を受けずに、精確にシグナル量を判定することが可能となる。しかしながら、たとえ小型であっても、自動分析装置を医療現場に持ち込むことは、費用の増大とともに装置の設置に嵩が張ることは否めない。 Therefore, using an automatic analyzer, a compound that automatically inhibits enzyme activity is dispensed after a certain period of time, and the generation of the signal is stopped, so that it is accurate without being affected by the time lapse until the subsequent determination. It is possible to determine the signal amount. However, even if it is small, it is undeniable that bringing the automatic analyzer into the medical field increases the cost and the installation of the apparatus.
それゆえ、酵素等の活性を指標として患者検体中の被験物質を検出、測定する際に、簡便な方法を用いて一定時間後に酵素反応を停止させ、酵素活性による発色基質等のシグナル量を精確に判定出来る技術が望まれている。 Therefore, when detecting and measuring a test substance in a patient sample using the activity of an enzyme or the like as an indicator, the enzyme reaction is stopped after a certain time using a simple method, and the amount of signal such as a chromogenic substrate due to the enzyme activity is accurately determined. The technology that can be judged is desired.
忙しい医療現場において、反応開始から一定時間後に酵素活性によるシグナル量の判定、もしくは酵素活性阻害剤の添加を行うことは困難と言わざるを得ない。それゆえ、反応開始から一定時間後に、検査者の手を煩わすことなく、酵素活性による発色基質等のシグナルの発生を停止することが重要な課題となっている。 In a busy medical field, it must be difficult to determine the signal amount due to enzyme activity or to add an enzyme activity inhibitor after a certain time from the start of the reaction. Therefore, after a certain time from the start of the reaction, it is an important issue to stop the generation of a signal such as a chromogenic substrate due to enzyme activity without bothering the examiner.
発明者は、酵素が存在する部位に、その酵素の反応に関与する溶液（反応液）と酵素の活性阻害剤を含む溶液（停止液）を用手法で順次添加することにより、反応液のみが酵素が存在する部位に浸透している間は酵素活性によるシグナルの発生が認められ、停止液が酵素が存在する部位に浸透するとシグナルの発生が停止することが認められることを見出し、発明の完成に至った。 The inventor adds a solution (reaction solution) involved in the enzyme reaction and a solution containing the enzyme activity inhibitor (stop solution) sequentially to the site where the enzyme is present, so that only the reaction solution is present. It was found that signal generation due to enzyme activity was observed while penetrating the site where the enzyme was present, and signal generation was stopped when the stop solution penetrated the site where the enzyme was present. It came to.
すなわち、反応液を添加した後に停止液を重層して添加することにより、短時間に作業を行うことが可能となる上、酵素が存在する部位に順次到達することによって、シグナルの発生が認められた後にその発生を停止することが可能となった。 In other words, by adding the stop solution after adding the reaction solution, it is possible to work in a short time, and in addition, the generation of signals is recognized by sequentially reaching the site where the enzyme is present. After that, it became possible to stop the occurrence.
さらに、増粘剤を反応液に添加してその粘性度を高めることにより、反応液を添加した後に停止液を重層して添加しても、反応液と停止液の界面は融合することなく、酵素が存在する部位に順次到達し、反応開始から一定時間後に酵素の活性阻害剤により反応を停止することが可能となった。 Furthermore, by adding a thickener to the reaction liquid to increase its viscosity, even if the stop liquid is added in layers after adding the reaction liquid, the interface between the reaction liquid and the stop liquid does not fuse, It was possible to reach the site where the enzyme was present sequentially, and to stop the reaction with an enzyme activity inhibitor after a certain time from the start of the reaction.
すなわち、本発明は以下の構成からなる。
（１）試料中の被験物質を検出、測定する際に、前記被験物質に関連した酵素の活性を前記酵素に特異的に反応する基質のシグナルの変化で捉える試験方法において、反応溶液と前記酵素の活性阻害剤を含む溶液が、前記酵素の存在部位に順次添加されることを特徴とする、被験物質の検出、測定方法。
（２）前記反応溶液と前記酵素の活性阻害剤を含む溶液が、重層されて前記酵素の存在部位に順次添加されることを特徴とする、（１）記載の被験物質の検出、測定方法。
（３）前記反応溶液が、さらに増粘剤を含むことを特徴とする（１）または（２）記載の被験物質の検出、測定方法。
（４）前記増粘剤が、アルギン酸ナトリウム、カラギーナン、ポリビニルピロリドン、ポリビニルアルコールから選ばれる少なくとも１である、（１）〜（３）記載の被験物質の検出、測定方法。
（５）前記被験物質に関連した酵素が、被験物質に含まれる酵素である、（１）〜（４）記載の被験物質の検出、測定方法。
（６）前記被験物質に関連した酵素が、被験物質から抽出した酵素である、（１）〜（４）記載の被験物質の検出、測定方法。
（７）前記被験物質に関連した酵素が、被験物質に標識した酵素である、（１）〜（４）記載の被験物質の検出、測定方法。
（８）前記基質が、発色基質である、（１）〜（７）記載の被験物質の検出、測定方法。
（９）前記基質が、蛍光基質である、（１）〜（７）記載の被験物質の検出、測定方法。
（１０）前記基質が、発光基質である、（１）〜（７）記載の被験物質の検出、測定方法。
（１１）前記基質が、前記酵素の存在部位にある、（１）〜（１０）記載の被験物質の検出、測定方法。
（１２）前記基質が、前記反応液に含まれる、（１）〜（１０）記載の被験物質の検出、測定方法。
（１３）（１）〜（１２）記載の被験物質の検出、測定方法を用いた、被験物質の検出、測定キット。
That is, the present invention has the following configuration.
(1) In a test method for detecting the activity of an enzyme related to a test substance by detecting a change in a signal of a substrate that reacts specifically with the enzyme when detecting and measuring the test substance in a sample, the reaction solution and the enzyme A method for detecting and measuring a test substance, comprising sequentially adding a solution containing the activity inhibitor of said enzyme to the site where the enzyme is present.
(2) The method for detecting and measuring a test substance according to (1), wherein the reaction solution and a solution containing an enzyme activity inhibitor are layered and sequentially added to the site where the enzyme is present.
(3) The method for detecting and measuring a test substance according to (1) or (2), wherein the reaction solution further contains a thickener.
(4) The method for detecting and measuring a test substance according to (1) to (3), wherein the thickener is at least one selected from sodium alginate, carrageenan, polyvinylpyrrolidone, and polyvinyl alcohol.
(5) The method for detecting and measuring a test substance according to (1) to (4), wherein the enzyme related to the test substance is an enzyme contained in the test substance.
(6) The method for detecting and measuring a test substance according to (1) to (4), wherein the enzyme related to the test substance is an enzyme extracted from the test substance.
(7) The method for detecting and measuring a test substance according to (1) to (4), wherein the enzyme related to the test substance is an enzyme labeled with the test substance.
(8) The test substance detection and measurement method according to any one of (1) to (7), wherein the substrate is a chromogenic substrate.
(9) The test substance detection and measurement method according to any one of (1) to (7), wherein the substrate is a fluorescent substrate.
(10) The method for detecting and measuring a test substance according to (1) to (7), wherein the substrate is a luminescent substrate.
(11) The method for detecting and measuring a test substance according to (1) to (10), wherein the substrate is present at the site where the enzyme is present.
(12) The test substance detection and measurement method according to (1) to (10), wherein the substrate is contained in the reaction solution.
(13) A test substance detection and measurement kit using the test substance detection and measurement method according to (1) to (12).
本発明を、一例として全血中の白血球数を測定する際に、簡易な測定装置を用いて白血球が有するエステラーゼ活性を測定して、末梢血中の白血球数を間接的に測定する方法で説明するが、本例に限定されることはない。 As an example, the present invention will be described with a method for indirectly measuring the number of white blood cells in peripheral blood by measuring the esterase activity of white blood cells using a simple measuring device when measuring the number of white blood cells in whole blood. However, the present invention is not limited to this example.
測定装置として、その筺体内に３−（Ｎ−トシル−Ｌ−アラニロシキ）インドールを含浸させた白血球捕捉膜を収容したものを用いる。全血検体を測定装置の上方に開口している添加部に添加する。その後、アルギン酸ナトリウムを含む反応液を測定装置の添加部に添加し、さらにエステラーゼの阻害剤であるクエン酸トリエチルを含む停止液を反応液の上に重層させる。 As the measuring device, a device in which a leukocyte capturing membrane impregnated with 3- (N-tosyl-L-alanyloxy) indole is accommodated in the housing is used. A whole blood sample is added to the addition part opened above the measuring apparatus. Thereafter, a reaction solution containing sodium alginate is added to the addition section of the measuring apparatus, and a stop solution containing triethyl citrate, which is an esterase inhibitor, is overlaid on the reaction solution.
反応液を添加した後より、赤血球は血漿成分とともに白血球捕捉膜内を移動して添加部近辺より離れる。一方、白血球は添加部近辺に留まり、白血球エステラーゼが溶出することにより、白血球捕捉膜に含まれる３−（Ｎ−トシル−Ｌ−アラニロシキ）インドールを加水分解してインドキシルを生成し、このインドキシルが空気中の酸素で酸化され、青色のインジゴを生成する。 After the reaction solution is added, the red blood cells move with the plasma components in the leukocyte capturing membrane and leave the vicinity of the addition portion. On the other hand, leukocytes remain in the vicinity of the addition part, and leukocyte esterase elutes to hydrolyze 3- (N-tosyl-L-alanyloxy) indole contained in the leukocyte capture membrane to produce indoxyl. Is oxidized by oxygen in the air to produce blue indigo.
ここで、アルギン酸ナトリウムを含む反応液と停止液は混ざらず、停止液はすぐには浸透しない。その結果、青色の発色反応は停止液が白血球捕捉膜に到達するまで続く。停止液が白血球捕捉膜に到達すると、停止液に含まれるクエン酸トリエチルによりエステラーゼによる発色はその時点で停止する。その結果、その後の青色の強さは変化せず、判定時間が多少ずれても同じ結果を得ることが出来る。 Here, the reaction solution containing sodium alginate and the stop solution are not mixed, and the stop solution does not penetrate immediately. As a result, the blue color development reaction continues until the stop solution reaches the leukocyte capture membrane. When the stop solution reaches the leukocyte capture membrane, the color development by esterase is stopped at that time by triethyl citrate contained in the stop solution. As a result, the intensity of the subsequent blue does not change, and the same result can be obtained even if the determination time is slightly shifted.
酵素活性を指標として用いる試料検体中に含まれる被験物質を検出、測定する場合、精確な結果を得るためには厳格な反応時間や試薬の添加時間が必要となる。しかしながら、本発明の増粘剤を含む反応液と停止液を重層することにより、一定時間の反応が得られた後に反応が停止するため、時間に縛られることなく、精確な結果を得ることが可能となる。 When detecting and measuring a test substance contained in a sample specimen that uses enzyme activity as an index, strict reaction time and reagent addition time are required to obtain accurate results. However, by laminating the reaction solution containing the thickener of the present invention and the stop solution, the reaction is stopped after a certain time of reaction is obtained, so that accurate results can be obtained without being bound by time. It becomes possible.
測定装置の筺体内の担体上に酵素反応の部位を設ける。この酵素反応の部位には、担体の孔径による排除、抗原抗体反応等の特異的結合等により被験物質を捕捉する機能を有しており、捕捉された被験物質の量を酵素等の活性を指標として測定する。このとき、筐体に酵素反応の部位の上方に開口している添加部を設けて、その添加部に反応液を添加し、さらに反応液の上に停止液を添加する。このことにより、酵素反応の開始後、一定時間が経過すると、後から添加させた停止液によって反応が停止する。反応の停止後は酵素反応の部位での変化が認められず、精確な判定が出来る。 A site for enzyme reaction is provided on the carrier in the housing of the measuring device. This enzyme reaction site has the function of capturing the test substance by specific binding such as exclusion by the pore size of the carrier, antigen-antibody reaction, etc., and the amount of the captured test substance is an indicator of the activity of the enzyme, etc. Measure as At this time, the addition part opened above the site | part of an enzyme reaction is provided in a housing | casing, a reaction liquid is added to the addition part, and also a stop liquid is added on a reaction liquid. Thus, when a certain time has elapsed after the start of the enzyme reaction, the reaction is stopped by a stop solution added later. After the reaction is stopped, no change is observed at the site of the enzyme reaction, and an accurate determination can be made.
なお、この測定装置の様式としては、フロースルー式やラテラルフロー式等が例示される。また、試験結果は、目視による測定、検出が可能であるが、より精確な結果を得るために読取装置を用いることもある。 In addition, as a form of this measuring apparatus, a flow-through type, a lateral flow type, etc. are illustrated. The test result can be visually measured and detected, but a reader may be used to obtain a more accurate result.
１．増粘剤としてアルギン酸ナトリウムを用いたときの白血球エステラーゼの活性測定
［比較例１］停止液を用いなかったときの白血球エステラーゼの活性測定。
白血球捕捉膜としてグラスファイバー濾紙（ＧＦ／ＤＶＡ：Ｗｈａｔｍａｎ社製）を用い、３−（Ｎ−トシル−Ｌ−アラニロキシ）インドールを含浸させて、上方に開口している添加部を有するデバイス内に装着した。
1. Measurement of leukocyte esterase activity when sodium alginate was used as a thickener [Comparative Example 1] Measurement of leukocyte esterase activity when no stop solution was used.
Glass fiber filter paper (GF / DVA: manufactured by Whatman) is used as a leukocyte-capturing membrane, impregnated with 3- (N-tosyl-L-alanyloxy) indole, and mounted in a device having an addition portion opened upward did.
０．０３％ ドデシル硫酸ナトリウム、１０％ ショ糖、０．２％ エチレンジアミン四酢酸・二ナトリウム、および０．４７５％ アルギン酸ナトリウムを含む０．２Ｍ トリス緩衝液（ｐＨ７．６）作製し、反応液とした。 Prepare a 0.2 M Tris buffer (pH 7.6) containing 0.03% sodium dodecyl sulfate, 10% sucrose, 0.2% ethylenediaminetetraacetic acid / disodium, and 0.475% sodium alginate. did.
白血球捕捉膜を装着したデバイスの添加口に、白血球数が１５，０００個／μＬに相当する検体を添加した。検体が白血球捕捉膜に十分浸透した後に、反応液を３滴（約１００μＬ）添加した。 A sample corresponding to a white blood cell count of 15,000 / μL was added to the addition port of the device equipped with the leukocyte capture membrane. After the specimen sufficiently penetrated the leukocyte capturing membrane, 3 drops (about 100 μL) of the reaction solution was added.
反応液の添加後５分から２０分にかけて、デバイスの添加口から観察できる反応部位を、色差計を用いて６１０ｎｍの反射率を測定した。その結果を図１に示すが、反応液の添加後５分から２０分にかけて、６１０ｎｍの反射率は約１５％に至るまで低下し続けた。すなわち、時間の経過とともにその反射率が低下するため、判定のために反射率を測定する時期に制限が生じる。 From 5 to 20 minutes after the addition of the reaction solution, the reflectance at 610 nm was measured using a color difference meter at the reaction site that can be observed from the addition port of the device. The results are shown in FIG. 1. From 5 to 20 minutes after the addition of the reaction solution, the reflectance at 610 nm continued to decrease to about 15%. That is, since the reflectance decreases with the passage of time, the timing for measuring the reflectance for determination is limited.
［実施例１］停止液を用いた白血球エステラーゼの活性測定
比較例１のデバイスおよび反応液を用い、停止液として、クエン酸トリエチルを用いた。
Example 1 Measurement of Leukocyte Esterase Activity Using Stop Solution Using the device and reaction solution of Comparative Example 1, triethyl citrate was used as the stop solution.
白血球捕捉膜を装着したデバイスの添加口に、白血球数が５，０００個／μＬ、１０，０００個／μＬ、および１５，０００個／μＬに相当する検体を添加した。検体が白血球捕捉膜に十分浸透した後に、反応液を３滴（約１００μＬ）添加し、その直後に停止液を１滴（約３０μＬ）添加した。 Samples corresponding to white blood cell counts of 5,000 / μL, 10,000 / μL, and 15,000 / μL were added to the addition port of the device equipped with the leukocyte-capturing membrane. After the specimen sufficiently penetrated the leukocyte capturing membrane, 3 drops (about 100 μL) of the reaction solution was added, and immediately after that, 1 drop (about 30 μL) of the stop solution was added.
反応液の添加後５分から２０分にかけて、デバイスの添加口から観察できる反応部位を、色差計を用いて６１０ｎｍの反射率を測定した。その結果を図２に示すが、反応液の添加後５分から２０分にかけて、添加した白血球数が５，０００個／μＬ、１０，０００個／μＬ、および１５，０００個／μＬに相当する検体の全てにおいて、６１０ｎｍの反射率の変化は認められず、その濃度に依存して低下していた。 From 5 to 20 minutes after the addition of the reaction solution, the reflectance at 610 nm was measured using a color difference meter at the reaction site that can be observed from the addition port of the device. The results are shown in FIG. 2. From 5 to 20 minutes after the addition of the reaction solution, the number of added white blood cells is 5,000 / μL, 10,000 / μL, and 15,000 / μL. In all of the cases, no change in the reflectance at 610 nm was observed, and it decreased depending on the concentration.
２．増粘剤としてカラギーナンを用いたときの白血球エステラーゼの活性測定
［実施例２］停止液を用いた白血球エステラーゼの活性測定
白血球数が１５，０００個／μＬに相当する検体のみを用い、反応液のアルギン酸ナトリウムを０．１％ カラギーナンに替えたことを除き、実施例１と同様の試験を実施した。
2. Measurement of leukocyte esterase activity when carrageenan is used as a thickener [Example 2] Measurement of leukocyte esterase activity using a stop solution Only the specimen corresponding to the number of leukocytes of 15,000 cells / μL is used. The same test as in Example 1 was performed except that sodium alginate was replaced with 0.1% carrageenan.
反応液の添加後５分から２０分にかけて、デバイスの添加口から観察できる反応部位を、色差計を用いて６１０ｎｍの反射率を測定した。その結果を図３に示すが、反応液の添加後５分から２０分にかけて、６１０ｎｍの反射率の変化は認められなかった。 From 5 to 20 minutes after the addition of the reaction solution, the reflectance at 610 nm was measured using a color difference meter at the reaction site that can be observed from the addition port of the device. The results are shown in FIG. 3, and no change in reflectance at 610 nm was observed from 5 minutes to 20 minutes after the addition of the reaction solution.
３．増粘剤としてポリビニルピロリドンを用いたときの白血球エステラーゼの活性測定
［実施例３］停止液を用いた白血球エステラーゼの活性測定
白血球数が１５，０００個／μＬに相当する検体のみを用い、反応液のアルギン酸ナトリウムを１０％ ポリビニルピロリドンに替えたことを除き、実施例１と同様の試験を実施した。
3. Measurement of leukocyte esterase activity when polyvinylpyrrolidone is used as a thickener [Example 3] Measurement of leukocyte esterase activity using a stop solution Reaction solution using only a sample corresponding to 15,000 leukocytes / μL The same test as in Example 1 was carried out except that sodium alginate was replaced with 10% polyvinylpyrrolidone.
反応液の添加後５分から２０分にかけて、デバイスの添加口から観察できる反応部位を、色差計を用いて６１０ｎｍの反射率を測定した。その結果を図４に示すが、反応液の添加後５分から２０分にかけて、６１０ｎｍの反射率の変化は認められなかった。 From 5 to 20 minutes after the addition of the reaction solution, the reflectance at 610 nm was measured using a color difference meter at the reaction site that can be observed from the addition port of the device. The results are shown in FIG. 4, and no change in reflectance at 610 nm was observed from 5 minutes to 20 minutes after the addition of the reaction solution.
４．増粘剤としてポリビニルアルコールを用いたときの白血球エステラーゼの活性測定
［実施例４］停止液を用いた白血球エステラーゼの活性測定
白血球数が１５，０００個／μＬに相当する検体のみを用い、反応液のアルギン酸ナトリウムを１５％ ポリビニルアルコールに替えたことを除き、実施例１と同様の試験を実施した。
4). Measurement of leukocyte esterase activity when polyvinyl alcohol is used as a thickener [Example 4] Measurement of leukocyte esterase activity using a stop solution Reaction solution using only a sample corresponding to 15,000 leukocytes / μL The same test as in Example 1 was performed except that 15% polyvinyl alcohol was replaced with 15% polyvinyl alcohol.
反応液の添加後５分から２０分にかけて、デバイスの添加口から観察できる反応部位を、色差計を用いて６１０ｎｍの反射率を測定した。その結果を図５に示すが、反応液の添加後５分から２０分にかけて、６１０ｎｍの反射率の変化は認められなかった。 From 5 to 20 minutes after the addition of the reaction solution, the reflectance at 610 nm was measured using a color difference meter at the reaction site that can be observed from the addition port of the device. The results are shown in FIG. 5, and no change in reflectance at 610 nm was observed from 5 minutes to 20 minutes after the addition of the reaction solution.
以上の結果より、反応液に含まれる増粘剤としてアルギン酸ナトリウム、カラギーナン、ポリビニルピロリドン、およびポリビニルアルコールの何れを用いても、酵素反応の部位に反応液を添加した後に酵素活性阻害剤を含む停止液を添加することにより、一定時間後に酵素反応が停止し、その後の酵素活性によって得られるシグナル量の増加は認められず、判定を行える期間が比較的長いものとなった。 Based on the above results, even if sodium alginate, carrageenan, polyvinylpyrrolidone, or polyvinyl alcohol is used as a thickener contained in the reaction solution, the reaction solution is stopped after the reaction solution is added to the site of the enzyme reaction. By adding the solution, the enzyme reaction was stopped after a certain time, and the increase in the amount of signal obtained by the subsequent enzyme activity was not recognized, and the period during which the determination could be made was relatively long.
５．増粘剤としてポリビニルピロリドンを用いたときのヘモグロビンＰＯＤ様活性測定
［実施例５］停止液を用いたヘモグロビンＰＯＤ様活性測定
幅４ｍｍ、長さ２５ｍｍのニトロセルロース膜（ミリポア社製）の下端から１５ｍｍの位置に１ｍｇ／ｍＬの抗ヒトヘモグロビンモノクローナル抗体１μＬを点着し、乾燥させ、テストストリップとした。
5. Measurement of hemoglobin POD-like activity when polyvinylpyrrolidone is used as a thickener [Example 5] Measurement of hemoglobin POD-like activity using a
１ｍｇ／ｍＬのヘモグロビンを含む１０ｍＭ リン酸緩衝液（ｐＨ５．０）５０μＬにテストストリップを挿入して展開させた。展開後に、テストストリップの抗体点着部から１ｃｍ手前の部位がデバイスの添加口に当たるように、テストストリップを装着させた。 A test strip was inserted into 50 μL of 10 mM phosphate buffer (pH 5.0) containing 1 mg / mL hemoglobin for development. After the development, the test strip was mounted so that the site 1 cm before the antibody spotting part of the test strip hit the addition port of the device.
１０％ クメンヒドロペルオキシド−β−シクロデキストリン包接体、１％ ＳＬＳ、０．３％ ２−アミノベンゾチアゾール硫酸塩、０．５％ テトラメチルベンチジン、０．１％ ポリビニルピロリドンを含む０．１Ｍ クエン酸緩衝液（ｐＨ５．０）を作製し、反応液とした。また、１％ アジ化ナトリウムを作製し、停止液とした。 10% cumene hydroperoxide-β-cyclodextrin inclusion complex, 0.1% containing 1% SLS, 0.3% 2-aminobenzothiazole sulfate, 0.5% tetramethylbenzidine, 0.1% polyvinylpyrrolidone A citrate buffer solution (pH 5.0) was prepared and used as a reaction solution. In addition, 1% sodium azide was prepared and used as a stop solution.
テストストリップを装着したデバイスの添加口に反応液（３０μＬ）を添加し、その直後に停止液（５０μＬ）を添加した。なお、デバイスの添加口に反応液（３０μＬ）のみを添加し、対照とした。 The reaction solution (30 μL) was added to the addition port of the device equipped with the test strip, and immediately after that, the stop solution (50 μL) was added. In addition, only the reaction solution (30 μL) was added to the addition port of the device to serve as a control.
発色試薬液等を添加した後に抗体点着部の着色を確認した結果を表１に示すが、停止液を添加したものは１分後に青色のラインが現れて、３分後まで呈色は強くならなかった。一方、対照では１分後に強い青色のラインが現れ、２分以降も呈色がさらに強くなった。 The results of confirming the coloration of the antibody spot after adding the coloring reagent solution etc. are shown in Table 1. In the case of adding the stop solution, a blue line appears after 1 minute and the color is strong until 3 minutes later. did not become. On the other hand, in the control, a strong blue line appeared after 1 minute, and the color became stronger after 2 minutes.
以上の結果より、ニトロセルロース膜に固定化した抗体や抗原等の捕捉部で捕捉した酵素活性を測定する場合でも、捕捉部に増粘剤を含む反応液を添加した後に酵素活性阻害剤を含む停止液を添加することにより、一定時間後に酵素反応が停止し、その後の判定を行える期間が延びることが判った。 Based on the above results, even when measuring the enzyme activity captured by the capture part such as antibody or antigen immobilized on the nitrocellulose membrane, the enzyme activity inhibitor is included after adding the reaction solution containing the thickener to the capture part. It was found that by adding the stop solution, the enzyme reaction was stopped after a certain time, and the period during which the subsequent determination could be performed was extended.
医療現場において、患者検体中の被験物質を検出、測定する際に、被験物質に関連した酵素の活性を指標として行う場合がある。このとき、精確に被験物質を検出、測定するためには、反応時間を一定に保つ必要があるが、忙しい医療現場では困難となっている。そこで、本発明の技術を用いることにより酵素反応が一定時間後に停止され、それ以降であれば精確な検出、測定が可能となる。 In a medical field, when detecting and measuring a test substance in a patient sample, an enzyme activity related to the test substance may be used as an index. At this time, in order to accurately detect and measure the test substance, it is necessary to keep the reaction time constant, but this is difficult in a busy medical field. Therefore, by using the technique of the present invention, the enzyme reaction is stopped after a certain time, and after that, accurate detection and measurement are possible.
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