JP6059546B2 - Reagent composition for measuring glycated protein and method for measuring glycated protein - Google Patents
Reagent composition for measuring glycated protein and method for measuring glycated protein Download PDFInfo
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- JP6059546B2 JP6059546B2 JP2013024571A JP2013024571A JP6059546B2 JP 6059546 B2 JP6059546 B2 JP 6059546B2 JP 2013024571 A JP2013024571 A JP 2013024571A JP 2013024571 A JP2013024571 A JP 2013024571A JP 6059546 B2 JP6059546 B2 JP 6059546B2
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Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
本開示は、糖化タンパク質測定用試薬組成物、糖化タンパク質測定用試薬キット、及び、糖化タンパク質の測定方法に関する。 The present disclosure relates to a reagent composition for measuring glycated protein, a reagent kit for measuring glycated protein, and a method for measuring glycated protein.
アゾ色素は、フェノチアジン誘導体色素の吸収スペクトルをシフトさせるシフト剤、及び発色剤などの光により分解する化合物に対する保護剤として使用されることがある。引用文献１は、フェノチアジン誘導体色素を用いて糖化タンパク質（ＨｂＡ１ｃ）を測定する方法を開示するが、この中で、アゾ色素であるタートラジンを使用してフェノチアジン誘導体色素の吸収スペクトルを長波長側にシフトさせることを開示する。 An azo dye is sometimes used as a shift agent that shifts the absorption spectrum of a phenothiazine derivative dye and a protective agent for a compound that decomposes by light, such as a color former. Cited Document 1 discloses a method for measuring a glycated protein (HbA1c) using a phenothiazine derivative dye. Among them, the absorption spectrum of a phenothiazine derivative dye is shifted to a longer wavelength side using tartrazine, which is an azo dye. To disclose.
糖化タンパク質やＨｂＡ１ｃの酵素法による測定は、自動分析装置で行われることが多い。 Measurement of glycated protein and HbA1c by an enzyme method is often performed by an automatic analyzer.
酵素法で糖化タンパク質等の測定を行う自動分析装置は、一般に、複数の反応セルを備え、これらの反応セル内で反応を行い、光学的測定を行う。したがって、反応セルが汚れた場合には反応セルの洗浄や交換といったメンテナンスの手間や費用が必要となる。 An automatic analyzer that measures a glycated protein or the like by an enzyme method generally includes a plurality of reaction cells, performs a reaction in these reaction cells, and performs optical measurement. Therefore, when the reaction cell becomes dirty, maintenance work such as cleaning or replacement of the reaction cell is required.
しかしながら、糖化タンパク質測定の試薬には、自動分析装置の反応セル上面で析出物が生じるものがあり、この析出物による反応セルの汚れが自動分析装置のメンテナンスの回数や費用の増加をもたらすという問題点が見出された。 However, some reagents for measuring glycated protein cause precipitates on the upper surface of the reaction cell of the automatic analyzer, and the contamination of the reaction cell due to the precipitates increases the number of maintenance and costs of the automatic analyzer. A point was found.
そこで、本開示は、一態様において、自動分析装置の反応セル上面での析出物の発生を抑制できる糖化タンパク質の測定に用いる試薬組成物を提供する。 Thus, in one aspect, the present disclosure provides a reagent composition used for measurement of a glycated protein that can suppress the generation of precipitates on the upper surface of a reaction cell of an automatic analyzer.
本開示は、一態様において、糖化タンパク質測定用試薬組成物であって、フルクトシルアミノ酸オキシダーゼ（ＦＡＯＤ）又はプロテアーゼ、水、タール色素、及び多価アルコールを含む試薬組成物に関する。 In one aspect, the present disclosure relates to a reagent composition for measuring a glycated protein, which includes fructosyl amino acid oxidase (FAOD) or protease, water, a tar dye, and a polyhydric alcohol.
本開示は、その他の態様において、糖化タンパク質測定用試薬キットであって、フルクトシルアミノ酸オキシダーゼ（ＦＡＯＤ）又はプロテアーゼ、水、タール色素、及び多価アルコールを含む試薬組成物である第一試薬と、水を含む第二試薬を含む糖化タンパク質測定用試薬キットに関する。ここで、第二試薬は、さらに、第一試薬がＦＡＯＤを含む場合プロテアーゼを含み、第一試薬がプロテアーゼを含む場合ＦＡＯＤを含む。 In another aspect, the present disclosure provides a reagent kit for measuring a glycated protein, the first reagent being a reagent composition comprising fructosyl amino acid oxidase (FAOD) or protease, water, a tar dye, and a polyhydric alcohol; The present invention relates to a glycated protein measurement reagent kit containing a second reagent containing water. Here, the second reagent further includes protease when the first reagent includes FAOD, and includes FAOD when the first reagent includes protease.
本開示は、その他の態様において、下記工程（１）〜（４）を行うことを含む糖化タンパク質の測定方法に関する。
工程（１）：フルクトシルアミノ酸オキシダーゼ（ＦＡＯＤ）又はプロテアーゼ、水、タール色素、及び多価アルコールを含む試薬組成物である第一試薬と、測定対象試料とを混合する工程。
工程（２）：第一試薬と測定対象試料とを混合した混合液に、さらに、水を含む第二試薬を混合する工程。ここで、第二試薬は、さらに、第一試薬がＦＡＯＤを含む場合プロテアーゼを含み、第一試薬がプロテアーゼを含む場合ＦＡＯＤを含み、第一試薬と第二試薬の一方が発色剤を含む。
工程（３）：工程（１）の混合液を光学測定するか、或いは、工程（２）の混合後速やかに混合液を光学測定する工程。
工程（４）：工程（２）の混合液を一定時間後に光学測定する工程。
In another aspect, the present disclosure relates to a method for measuring a glycated protein comprising performing the following steps (1) to (4).
Step (1): A step of mixing a sample to be measured with a first reagent which is a reagent composition containing fructosyl amino acid oxidase (FAOD) or protease, water, tar dye, and polyhydric alcohol.
Step (2): A step of further mixing a second reagent containing water with a mixed liquid obtained by mixing the first reagent and the sample to be measured. Here, the second reagent further includes a protease when the first reagent includes FAOD, includes FAOD when the first reagent includes a protease, and one of the first reagent and the second reagent includes a color former.
Step (3): A step of optically measuring the liquid mixture in step (1) or a step of optically measuring the liquid mixture immediately after mixing in step (2).
Step (4): A step of optically measuring the mixed solution of step (2) after a certain time.
本開示によれば、一態様において、自動分析装置の反応セル上面での析出物の発生を抑制できる糖化タンパク質測定用試薬組成物、糖化タンパク質測定用試薬キット、又は、糖化タンパク質測定方法を提供できる。 According to the present disclosure, in one aspect, it is possible to provide a glycated protein measuring reagent composition, a glycated protein measuring reagent kit, or a glycated protein measuring method capable of suppressing the generation of precipitates on the upper surface of a reaction cell of an automatic analyzer. .
本開示は、一態様において、タール色素を用いる酵素反応系で糖化タンパク質を測定する自動分析装置において、自動分析装置の反応セル上面で析出物が生じてセルが汚染される場合がある、という課題を見出したことに基づく。 In one aspect of the present disclosure, in an automatic analyzer that measures glycated protein in an enzyme reaction system using a tar dye, a precipitate may be generated on the upper surface of the reaction cell of the automatic analyzer, and the cell may be contaminated. Based on the finding.
前記析出物の詳細な組成はまだ明らかではないが、前記析出物には少なくとも前記試薬中の色素が含まれる。本開示は、一態様において、前記析出物が、前記試薬に多価アルコールを含有させることで抑制できるという知見に基づく。したがって、本開示は、一態様において、フルクトシルアミノ酸オキシダーゼ（ＦＡＯＤ）又はプロテアーゼ、水、タール色素、及び多価アルコールを含む試薬組成物に関する（以下、「本開示の試薬組成物」ともいう）。本開示の試薬組成物は、一又は複数の実施形態において、ＦＡＯＤとプロテアーゼの２成分を同時に含有しない。 Although the detailed composition of the precipitate is not yet clear, the precipitate contains at least the dye in the reagent. This indication is based on the knowledge that the deposit can be controlled by making the reagent contain a polyhydric alcohol in one mode. Therefore, in one aspect, the present disclosure relates to a reagent composition containing fructosyl amino acid oxidase (FAOD) or protease, water, a tar dye, and a polyhydric alcohol (hereinafter, also referred to as “reagent composition of the present disclosure”). In one or a plurality of embodiments, the reagent composition of the present disclosure does not contain two components of FAOD and protease at the same time.
［多価アルコール］
本開示の試薬組成物は、析出物の発生を抑制する観点から、多価アルコールを含有する。同様の観点から、多価アルコールとしては、分子内ヒドロキシル基の数が２〜４であるものが好ましく、２〜３がより好ましく、２がさらに好ましい。多価アルコールは、限定されない一又は複数の実施形態において、グリセリン、エチレングリコール、ジエチレングリコール、トリエチレングリコール、プロピレングリコール、ジプロピレングリコール、ポリエチレングリコール＃２００、ポリエチレングリコール＃３００、ポリエチレングリコール＃４００、ポリエチレングリコール＃６００、又はこれらの組み合わせが挙げられる。
[Polyhydric alcohol]
The reagent composition of the present disclosure contains a polyhydric alcohol from the viewpoint of suppressing the generation of precipitates. From the same viewpoint, the polyhydric alcohol preferably has 2 to 4 hydroxyl groups in the molecule, more preferably 2 to 3, and still more preferably 2. In one or more non-limiting embodiments, the polyhydric alcohol is glycerin, ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol, polyethylene glycol # 200, polyethylene glycol # 300, polyethylene glycol # 400, polyethylene glycol. # 600, or a combination thereof.
また、測定対象試料中のヘモグロビン濃度の影響を受けにくいという観点から、多価アルコールとして、平均重量分子量が２００未満のものが好ましく、より好ましくは１９０以下、さらに好ましくは、１５０以下である。多価アルコールの分子量の下限は特に限定されないが、例えば、５０以上である。したがって、析出物の発生を抑制する観点、及び、測定対象試料中のヘモグロビン濃度の影響を受けにくいという観点から、多価アルコールは、限定されない一又は複数の実施形態において、グリセリン、エチレングリコール、ジエチレングリコール、トリエチレングリコール、プロピレングリコール、ジプロピレングリコール、又はこれらの組み合わせが挙げられる。 Further, from the viewpoint of being hardly affected by the hemoglobin concentration in the sample to be measured, the polyhydric alcohol preferably has an average weight molecular weight of less than 200, more preferably 190 or less, and even more preferably 150 or less. Although the minimum of the molecular weight of a polyhydric alcohol is not specifically limited, For example, it is 50 or more. Therefore, from the viewpoint of suppressing the generation of precipitates, and from the viewpoint of being hardly affected by the hemoglobin concentration in the sample to be measured, the polyhydric alcohol is glycerol, ethylene glycol, diethylene glycol in one or more embodiments that are not limited. , Triethylene glycol, propylene glycol, dipropylene glycol, or combinations thereof.
さらに、多価アルコールの添加の有無で測定結果が大きく変動すると測定精度が低下する恐れがある。よって、多価アルコールの添加が測定結果に与える測定値の変化は、抑制されることは好ましい。測定値に与える多価アルコールの影響を抑制する観点、及び、析出物の発生を抑制する観点から、多価アルコールは、限定されない一又は複数の実施形態において、グリセリン、エチレングリコール、プロピレングリコール、ジプロピレングリコール、ポリエチレングリコール＃２００、ポリエチレングリコール＃４００、ポリエチレングリコール＃６００又はこれらの組み合わせが挙げられる。 Furthermore, if the measurement result varies greatly depending on whether polyhydric alcohol is added or not, the measurement accuracy may be lowered. Therefore, it is preferable that the change of the measured value which the addition of a polyhydric alcohol gives to a measurement result is suppressed. From the viewpoint of suppressing the influence of the polyhydric alcohol on the measured value and the viewpoint of suppressing the generation of precipitates, the polyhydric alcohol is one or more embodiments that are not limited. In one or more embodiments, glycerin, ethylene glycol, propylene glycol, di Examples include propylene glycol, polyethylene glycol # 200, polyethylene glycol # 400, polyethylene glycol # 600, or combinations thereof.
よって、析出物の発生を抑制する観点、測定対象試料中のヘモグロビン濃度の影響を受けにくいという観点、及び、測定値に与える多価アルコールの影響を抑制する観点から、多価アルコールは、グリセリン、エチレングリコール、プロピレングリコール、ジプロピレングリコール、又はこれらの組み合わせが挙げられる。 Therefore, from the viewpoint of suppressing the generation of precipitates, from the viewpoint of being less affected by the hemoglobin concentration in the measurement target sample, and from the viewpoint of suppressing the influence of the polyhydric alcohol on the measurement value, the polyhydric alcohol is glycerin, Examples include ethylene glycol, propylene glycol, dipropylene glycol, or combinations thereof.
さらにまた、本開示の試薬組成物に含まれるフルクトシルアミノ酸オキシダーゼ（ＦＡＯＤ）が耐熱安定性を有する場合、その耐熱安定性を大きく阻害しないことが好ましい。析出物の発生を抑制する観点、測定対象試料中のヘモグロビン濃度の影響を受けにくいという観点、測定値に与える多価アルコールの影響を抑制する観点、及び、ＦＡＯＤの耐熱安定性の維持の観点から、多価アルコールは、エチレングリコール、プロピレングリコール、ジプロピレングリコール、又はこれらの組み合わせが挙げられる。 Furthermore, when the fructosyl amino acid oxidase (FAOD) contained in the reagent composition of the present disclosure has heat stability, it is preferable that the heat stability is not significantly inhibited. From the viewpoint of suppressing the generation of precipitates, from the viewpoint of being hardly affected by the hemoglobin concentration in the sample to be measured, from the viewpoint of suppressing the influence of polyhydric alcohol on the measurement value, and from the viewpoint of maintaining the heat stability of FAOD. The polyhydric alcohol includes ethylene glycol, propylene glycol, dipropylene glycol, or a combination thereof.
本開示の試薬組成物における多価アルコールの含有量は、限定されない一又は複数の実施形態において、析出物の発生を抑制する観点から、０．１重量％以上が好ましく、より好ましくは０．５重量％以上、さらに好ましくは０．８重量％以上、さらにより好ましくは０．９重量％以上である。本開示の試薬組成物における多価アルコールの含有量は、限定されない一又は複数の実施形態において、測定値に与える多価アルコールの影響を抑制する観点から、１０重量％以下、７重量％以下、５重量％以下、又は４重量％以下である。 In one or more embodiments that are not limited, the polyhydric alcohol content in the reagent composition of the present disclosure is preferably 0.1% by weight or more, more preferably 0.5%, from the viewpoint of suppressing the generation of precipitates. % By weight or more, more preferably 0.8% by weight or more, still more preferably 0.9% by weight or more. The content of the polyhydric alcohol in the reagent composition of the present disclosure is 10% by weight or less, 7% by weight or less from the viewpoint of suppressing the influence of the polyhydric alcohol on the measurement value in one or more embodiments that are not limited. 5 wt% or less, or 4 wt% or less.
［タール色素］
本開示の試薬組成物は、タール色素を含有する。糖化タンパク質測定試薬において、タール色素は一般にフェノチアジン誘導体色素の吸収スペクトルのシフト剤、及び発色剤などの光により分解する化合物に対する保護剤として使用することを目的として含有されることがある（特許文献１）。しかしながら、本開示において、タール色素を含有する目的はこれに限定されない。
[Tar dye]
The reagent composition of the present disclosure contains a tar dye. In glycated protein measurement reagents, tar dyes are generally contained for the purpose of use as a shift agent for the absorption spectrum of phenothiazine derivative dyes and as a protective agent for compounds that decompose by light, such as color formers (Patent Document 1). ). However, in the present disclosure, the purpose of containing the tar dye is not limited to this.
タール色素を用いる酵素反応系で糖化タンパク質を測定する自動分析装置の反応セル上面で生み出す析出物には、少なくともタール色素が含まれる。ただし、前記析出物の詳細な組成は未だ明らかではない。 The precipitate generated on the upper surface of the reaction cell of the automatic analyzer that measures glycated protein in an enzyme reaction system using a tar dye contains at least a tar dye. However, the detailed composition of the precipitate is not yet clear.
本開示の試薬組成物に使用されるタール色素としては、限定されない一又は複数の実施形態において、アゾ色素である。アゾ色素としては、限定されない一又は複数の実施形態において、下記式（Ｉ）で表される化合物が挙げられる。
Ｒ1−Ｎ＝Ｎ−Ｒ2 （Ｉ）
前記式（Ｉ）において、Ｒ1は、
Ｘは、水素、ハロゲン、ナトリウム又はカリウムであり、
Ｙは、水素又はＳＯ3Ｘであり、
各Ｘは同一でも異なっていても良く、各Ｙは同一でも異なっていてもよく、
Ｚは、水素、メチル基又はメトキシ基であり、各Ｚは同一でも異なっていてもよい。
The tar dye used in the reagent composition of the present disclosure is an azo dye in one or more embodiments that are not limited. Examples of the azo dye include a compound represented by the following formula (I) in one or a plurality of non-limiting embodiments.
R 1 −N = N−R 2 (I)
In the formula (I), R 1 is
X is hydrogen, halogen, sodium or potassium;
Y is hydrogen or SO 3 X;
Each X may be the same or different, each Y may be the same or different,
Z is hydrogen, a methyl group or a methoxy group, and each Z may be the same or different.
前記式（Ｉ）で表されるアゾ色素物質の限定されない一又は複数の実施形態として、５−ヒドロキシ−１−（４−スルホフェニル）−４−（４−スルホフェニルアゾ）ピラゾール−３−カルボン酸又はその塩（例えば、三ナトリウム塩）、６−ヒドロキシ−５−（４−スルホフェニルアゾ）−２−ナフタレンスルホン酸又はその塩（例えば、二ナトリウム塩）、３−ヒドロキシ−４−（４−スルホナフチルアゾ）−２，７−ナフタレンジスルホン酸（「３−ヒドロキシ−４−［（スルホナトナフタレン−１−イル）ジアゼニル］ナフタレン−２，７−ジスルホン酸」ともいう）又はその塩（例えば、三ナトリウム塩）、７−ヒドロキシ−８−（４−スルホナフチルアゾ）−１，３−ナフタレンジスルホン酸もしくはその塩（例えば、三ナトリウム塩）又は水和物（例えば、１１／２水和物）等が挙げられる。これらの塩や水和物としては、例えば、市販品であるタートラジン（黄色４号）、黄色５号、赤色２号、赤色４０号、赤色１０２号等が挙げられる。これらの色素物質は、食用であってもよいし、非食用であってもよい。 One or more non-limiting embodiments of the azo dye material represented by the formula (I) include 5-hydroxy-1- (4-sulfophenyl) -4- (4-sulfophenylazo) pyrazole-3-carbon Acid or a salt thereof (for example, trisodium salt), 6-hydroxy-5- (4-sulfophenylazo) -2-naphthalenesulfonic acid or a salt thereof (for example, disodium salt), 3-hydroxy-4- (4 -Sulfonaphthylazo) -2,7-naphthalenedisulfonic acid (also referred to as "3-hydroxy-4-[(sulfonatonaphthalen-1-yl) diazenyl] naphthalene-2,7-disulfonic acid") or a salt thereof (for example, Trisodium salt), 7-hydroxy-8- (4-sulfonaphthylazo) -1,3-naphthalenedisulfonic acid or a salt thereof (e.g. Unsalted) or hydrates (for example, 11/2 hydrate) and the like. Examples of these salts and hydrates include tartrazine (yellow No. 4), yellow No. 5, red No. 2, red No. 40, and red No. 102, which are commercially available products. These pigment substances may be edible or non-edible.
本開示の試薬組成物に含まれるタール色素は、一種類であってもよく二種類以上であってもよい。本開示の試薬組成物におけるタール色素の含有量は、限定されない一又は複数の実施形態において、反応系に含まれるフェノチアジン誘導体色素１モルに対して、１〜１０００モル、２〜２００モル、又は、４〜１００モルである。また、本開示の試薬組成物におけるタール色素の含有量は、限定されない一又は複数の実施形態において、反応液に添加する発色剤の量に応じて決定でき、発色剤１モルに対して、０．１〜１０００モル、１〜５００モル、又は４〜３００モルである。また、反応系におけるタール色素の最終濃度は、限定されない一又は複数の実施形態において、１×１０-7〜０．１ｍｏｌ／Ｌ、２×１０-7〜０．０５ｍｏｌ／Ｌ、又は、５×１０-7〜０．０３ｍｏｌ／Ｌである。 The tar dye contained in the reagent composition of the present disclosure may be one type or two or more types. The content of the tar dye in the reagent composition of the present disclosure is, in one or a plurality of non-limiting embodiments, 1 to 1000 mol, 2 to 200 mol, or 1 mol to 1 mol of the phenothiazine derivative dye contained in the reaction system. 4 to 100 moles. Further, the content of the tar dye in the reagent composition of the present disclosure can be determined according to the amount of the color former added to the reaction liquid in one or a plurality of non-limiting embodiments. .1 to 1000 mol, 1 to 500 mol, or 4 to 300 mol. In addition, the final concentration of the tar dye in the reaction system is 1 × 10 −7 to 0.1 mol / L, 2 × 10 −7 to 0.05 mol / L, or 5 × in one or more embodiments that are not limited. 10 −7 to 0.03 mol / L.
［ＦＡＯＤ］
本開示の試薬組成物に含まれうるフルクトシルアミノ酸オキシダーゼ（ＦＡＯＤ）は、糖化アミノ酸及び／又は糖化ペプチドを酸化的加水分解する酵素をいい、糖化タンパク質の検出に従来使用され或いは今後開発され使用されるものを含む。ＦＡＯＤの基質としては、限定されない一又は複数の実施形態において、フルクトシルバリン、フルクトシルリジン、及び／又はフルクトシルバリルヒスチジンが挙げられる。ＦＡＯＤとしては、限定されない一又は複数の実施形態において、商品名ＦＰＯＸ-ＣＥ（キッコーマン社製）、耐熱性が付与された商品名ＦＰＯＸ-ＣＥＴ（キッコーマン社製）、商品名ＦＰＯＸ−ＥＥ（キッコーマン社製）、商品名ＦＯＤ（旭化成社製）等が挙げられる。
[FAOD]
Fructosyl amino acid oxidase (FAOD) that can be included in the reagent composition of the present disclosure refers to an enzyme that oxidatively hydrolyzes glycated amino acids and / or glycated peptides, and has been conventionally used for detection of glycated proteins or will be developed and used in the future. Including things. FAOD substrates include, in one or more non-limiting embodiments, fructosyl valine, fructosyl lysine, and / or fructosyl valyl histidine. In one or a plurality of embodiments that are not limited as FAOD, the product name FPOX-CE (manufactured by Kikkoman), the product name FPOX-CET (manufactured by Kikkoman), and the product name FPOX-EE (Kikkoman) Product name) FOD (manufactured by Asahi Kasei Co., Ltd.) and the like.
本開示の試薬組成物におけるＦＡＯＤの含有量は、限定されない一又は複数の実施形態において、０．１〜１０．０Ｕ／ｍｌ、０．２〜８．０Ｕ／ｍｌ、０．５〜５．０Ｕ／ｍｌ、０．６〜３．０Ｕ／ｍｌである。本開示において、ＦＡＯＤの酵素単位「Ｕ」は、フルクトシルバリンを基質として１分間に１マイクロモルの過酸化水素を生成する量を１Ｕとする。 The content of FAOD in the reagent composition of the present disclosure is, in one or more non-limiting embodiments, 0.1 to 10.0 U / ml, 0.2 to 8.0 U / ml, 0.5 to 5.0 U. / Ml, 0.6 to 3.0 U / ml. In the present disclosure, the enzyme unit “U” of FAOD is defined as 1 U that generates 1 micromole of hydrogen peroxide per minute using fructosyl valine as a substrate.
［プロテアーゼ］
本開示の試薬組成物に含まれうるプロテアーゼとしては、測定対象試料中のヘモグロビン（及び糖化ヘモグロビン）のβ鎖Ｎ末端バリンや、前記Ｎ末端バリンを含むペプチド（β鎖Ｎ末端ペプチド）を切り出すことができるものであって、糖化タンパク質の検出に従来使用され或いは今後開発され使用されるものを含む。
[Protease]
As a protease that can be included in the reagent composition of the present disclosure, the β-chain N-terminal valine of hemoglobin (and glycated hemoglobin) in a sample to be measured and a peptide containing the N-terminal valine (β-chain N-terminal peptide) are cut out. Including those that are conventionally used for the detection of glycated proteins or that will be developed and used in the future.
前記プロテアーゼとしては、限定されない一又は複数の実施形態において、メタロプロテアーゼ、セリンプロテアーゼ、セリンカルボキシペプチダーゼ、プロテイナーゼＫ、ブロメライン、パパイン、ブタ膵臓由来トリプシン、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ由来プロテアーゼ、Ａｓｐｅｒｇｉｌｌｕｓ ｏｒｙｚａｅ由来プロテアーゼ等が使用できる。前記プロテアーゼは、限定されない一又は複数の実施形態において、測定感度及び精度を向上する観点から、好ましくはエンドプロテアーゼである。限定されない一又は複数の実施形態において、市販品として、メタロプロテアーゼ（商品名、アークレイ社製）、プロテアーゼＡ「アマノ」Ｇ（商品名、天野エンザイム社製）、プロテアーゼＭ「アマノ」Ｇ（商品名、天野エンザイム社製）、プロテアーゼＳ「アマノ」Ｇ（商品名、天野エンザイム社製）、ペプチダーゼＲ（商品名、天野エンザイム社製）、パパインＭ−４０（商品名、天野エンザイム社製）、プロテアーゼＮ（商品名、フルカ社製）、プロテアーゼＮ「アマノ」（商品名、天野製薬社製）、Ｂａｃｉｌｌｕｓ属由来メタロプロテイナーゼ（商品名、トヨチーム東洋紡社製）等が挙げられる。 As the protease, in one or a plurality of non-limiting embodiments, metalloprotease, serine protease, serine carboxypeptidase, proteinase K, bromelain, papain, porcine pancreatic trypsin, Bacillus subtilis-derived protease, Aspergillus oryzae-derived protease, and the like can be used. . In one or a plurality of non-limiting embodiments, the protease is preferably an endoprotease from the viewpoint of improving measurement sensitivity and accuracy. In one or a plurality of non-limiting embodiments, commercially available products include metalloprotease (trade name, manufactured by Arkray), protease A “Amano” G (trade name, manufactured by Amano Enzyme), protease M “Amano” G (trade name) , Amano Enzyme), protease S “Amano” G (trade name, Amano Enzyme), peptidase R (trade name, Amano Enzyme), papain M-40 (trade name, Amano Enzyme), protease N (trade name, manufactured by Fluka), protease N “Amano” (trade name, manufactured by Amano Pharmaceutical Co., Ltd.), Bacillus genus-derived metalloproteinase (trade name, manufactured by Toyoteam Toyobo Co., Ltd.), and the like.
前記プロテアーゼとしては、限定されない一又は複数の実施形態において、測定感度及び精度を向上する観点から、β鎖Ｎ末端に特異的に作用してＮ末端ペプチドの切断を触媒するプロテアーゼ（例えば、特開２０００−３００２９４号公報、特開２００４−３４４０５２号公報等）が好ましい。また、β鎖Ｎ末端バリンの切断を触媒するプロテアーゼとしては、例えば、国際公開第２０００／５０５７９号パンフレット（日本国特許３６６８８０１）、国際公開第２０００／６１７３２号パンフレット、特開２００２−３１５６００号公報等に開示されているプロテアーゼが挙げられる。 As the protease, in one or a plurality of non-limiting embodiments, from the viewpoint of improving measurement sensitivity and accuracy, a protease that specifically acts on the N-terminus of the β chain to catalyze the cleavage of the N-terminal peptide (for example, JP 2000-300294, JP-A-2004-344052, etc.) are preferable. Examples of proteases that catalyze the cleavage of β-chain N-terminal valine include, for example, International Publication No. 2000/50579 pamphlet (Japan Patent 3668801), International Publication No. 2000/61732 pamphlet, Japanese Patent Application Laid-Open No. 2002-315600, etc. Proteases disclosed in (1).
本開示の試薬組成物におけるプロテアーゼの含有量は、限定されない一又は複数の実施形態において、５００〜８０００Ｕ／ｍｌ、６００〜７０００Ｕ／ｍｌ、７００〜５０００Ｕ／ｍｌ、８００〜４０００Ｕ／ｍｌである。本開示において、プロテアーゼの活性「Ｕ」は、１分間に１マイクロモルのチロシンに相当する２７５ｎｍの吸光度の増加を与える酵素量を１Ｕとする。 The content of the protease in the reagent composition of the present disclosure is 500 to 8000 U / ml, 600 to 7000 U / ml, 700 to 5000 U / ml, 800 to 4000 U / ml in one or more non-limiting embodiments. In the present disclosure, the activity “U” of the protease is defined as the amount of enzyme that gives an increase in absorbance at 275 nm corresponding to 1 micromole of tyrosine per minute.
［水］
本開示の試薬組成物は、水溶液であり、水を含む。水は、蒸留水、イオン交換水、又は超純水等が使用され得る。
[water]
The reagent composition of the present disclosure is an aqueous solution and contains water. As the water, distilled water, ion-exchanged water, ultrapure water, or the like can be used.
［その他の成分］
本開示の試薬組成物は、その他の成分として、ペルオキシダーゼ（ＰＯＤ）又は発色剤を含有しうる。本開示の試薬組成物は、一又は複数の実施形態において、ＰＯＤと発色剤の２成分を同時に含有しない。したがって、本開示の試薬組成物は、限定されない一又は複数の実施形態において、水、タール色素、及び多価アルコールに加えて下記表１の組み合わせの成分を含む実施形態１〜４が挙げられる。
The reagent composition of the present disclosure may contain peroxidase (POD) or a color former as other components. In one or a plurality of embodiments, the reagent composition of the present disclosure does not contain two components of POD and color former at the same time. Therefore, the reagent composition of this indication includes Embodiment 1-4 which contains the component of the combination of following Table 1 in addition to water, a tar pigment | dye, and a polyhydric alcohol in one or some embodiment which is not limited.
本開示の試薬組成物は、一又は複数の実施形態において、さらに、亜硝酸又はその塩、緩衝液又は緩衝剤、及び界面活性剤の１つ以上を含有しうる。また、本開示の試薬組成物が発色剤を含む場合、さらに発色剤安定化剤を含有しうる。 In one or a plurality of embodiments, the reagent composition of the present disclosure may further contain one or more of nitrous acid or a salt thereof, a buffer solution or a buffering agent, and a surfactant. Further, when the reagent composition of the present disclosure includes a color former, it may further contain a color former stabilizer.
［ＰＯＤ］
本開示の試薬組成物に含まれうるペルオキシダーゼ（ＰＯＤ）は、ＦＡＯＤが反応することで生成された過酸化水素を基質として分解するとともに発色剤を酸化することで過酸化水素の測定を可能とするものが使用でき、糖化タンパク質の検出に従来使用され或いは今後開発され使用されるものを含む。
[POD]
The peroxidase (POD) that can be contained in the reagent composition of the present disclosure enables measurement of hydrogen peroxide by decomposing hydrogen peroxide produced by the reaction of FAOD as a substrate and oxidizing the color former. Can be used, including those conventionally used or later developed and used for detection of glycated proteins.
本開示の試薬組成物におけるＰＯＤの含有量は、限定されない一又は複数の実施形態において、０．５〜１００Ｕ／ｍｌ、１．０〜８０Ｕ／ｍｌ、２．０〜５０Ｕ／ｍｌ、又は３．０〜２０Ｕ／ｍｌである。本開示において、ＰＯＤの活性「Ｕ」は、２０秒間に１ミリグラムのプルプロガリンを生成できる量を１プルプロガリンＵとする。 The content of POD in the reagent composition of the present disclosure is, in one or more non-limiting embodiments, 0.5 to 100 U / ml, 1.0 to 80 U / ml, 2.0 to 50 U / ml, or 3. 0-20 U / ml. In the present disclosure, the activity “U” of POD is defined as the amount capable of producing 1 milligram of purprogalin in 20 seconds.
［発色剤］
本開示の試薬組成物に含まれうる発色剤としては、反応系の過酸化水素とＰＯＤとの反応に応じて発色、変色、消失するものであって、糖化タンパク質の検出に従来使用され或いは今後開発され使用されるものを含む。発色剤は、１つの化合物であってもよく、二種類以上の化合物の組み合わせであってもよい。本開示の試薬組成物における発色剤は、限定されない一又は複数の実施形態において、フェノチアジン骨格を有する酸化発色剤、酸化還元反応によりフェノチアジン誘導体色素を生成する発色剤、又は、フェノチアジン誘導体色素が挙げられる。
[Coloring agent]
The color former that can be included in the reagent composition of the present disclosure is one that develops color, changes color, or disappears in response to the reaction between hydrogen peroxide and POD in the reaction system, and is conventionally used for the detection of glycated protein or will be used in the future. Includes those developed and used. The color former may be a single compound or a combination of two or more compounds. Examples of the color former in the reagent composition of the present disclosure include, but are not limited to, an oxidative color former having a phenothiazine skeleton, a color former that generates a phenothiazine derivative dye by a redox reaction, or a phenothiazine derivative dye. .
フェノチアジン誘導体色素は、限定されない一又は複数の実施形態において、下記化学式で表されるメチレンブルー、アズールＡ、アズールＢ、アズールＣ、トルイジンブルーＯ、１，９−ジメチル−３，７−ビス（ジメチルアミノ）フェノチアジン塩、メチレングリーン等が挙げられる。
酸化還元反応によりフェノチアジン誘導体色素を生成する発色剤は、限定されない一又は複数の実施形態において、酸化又は還元によってフェノチアジン誘導体色素を遊離（脱離
）する発色剤（化合物）が挙げられる。
Examples of the color former that generates a phenothiazine derivative dye by an oxidation-reduction reaction include, in one or more embodiments without limitation, a color former (compound) that liberates (eliminates) the phenothiazine derivative dye by oxidation or reduction.
酸化によりフェノチアジン誘導体色素であるメチレンブルーを遊離する発色剤（化合物）としては、限定されない一又は複数の実施形態において、１０−（カルボキシメチルアミノカルボニル）−３，７−ビス（ジメチルアミノ）フェノチアジン又はその塩（例えば、商品名ＤＡ−６７、和光純薬社製）、１０−（アセチルアミノカルボニル）−３，７−ビス（ジメチルアミノ）フェノチアジン又はその塩、特公平４−２７８３９号記載の１０−（フェニルカルボニル）−３，７−ビス（ジメチルアミノ）フェノチアジン又はその塩等が挙げられる。また、特公昭６０−３３４７９号記載の化合物Ｎｏ．II−４〜９、II−１０である、１０−（３−（メチルカルボキシアミノ）−ヘキサメチル−アミノ）−フェノチアジン、１０−（３−（メチルカルボキシアミノ）−４−メチル−フェニル）−アミノ）−フェノチアジン、１０−（（３−（メチルカルボキシアミノメチル）−フェニル）−メチルアミノ）−フェノチアジン、１０−（１−ナフタレンアミノ）−フェノチアジン、１０−（メチル）−フェノチアジン、１０−（フェニルアミノ）−フェノチアジン、１０−（メチルアミノ）−フェノチアジンや、これらの塩も挙げられる。中でも、水溶性が高いという観点から、１０−（カルボキシメチルアミノカルボニル）−３，７−ビス（ジメチルアミノ）フェノチアジン塩が好ましい。 The color former (compound) that liberates methylene blue, which is a phenothiazine derivative dye, by oxidation is, in one or more non-limiting embodiments, 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine or its Salt (for example, trade name DA-67, manufactured by Wako Pure Chemical Industries, Ltd.), 10- (acetylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine or a salt thereof, 10- (described in Japanese Patent Publication No. 4-27839 Phenylcarbonyl) -3,7-bis (dimethylamino) phenothiazine or a salt thereof. In addition, the compound No. described in JP-B-60-33479 is also disclosed. II-4-9, II-10, 10- (3- (methylcarboxyamino) -hexamethyl-amino) -phenothiazine, 10- (3- (methylcarboxyamino) -4-methyl-phenyl) -amino) -Phenothiazine, 10-((3- (methylcarboxyaminomethyl) -phenyl) -methylamino) -phenothiazine, 10- (1-naphthaleneamino) -phenothiazine, 10- (methyl) -phenothiazine, 10- (phenylamino) -Phenothiazine, 10- (methylamino) -phenothiazine, and salts thereof are also included. Among these, 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine salt is preferable from the viewpoint of high water solubility.
また、フェノチアジン誘導体色素であるメチレンブルーを生成する発色剤としては、この他に、例えば、ロイコメチレンブルー等のロイコ化合物が挙げられる。ロイコメチレンブルーは、無色の化合物（還元型）であり、酸化によってメチレンブルー（酸化型）となる。このため、ロイコメチレンブルーは、例えば、酸化物質を検出するための基質として使用できる。本発明においては、例えば、前記反応系に発色剤としてロイコメチレンブルーを添加し、ロイコメチレンブルーと前記反応系における酸化物質との酸化還元反応により、メチレンブルーを生成させ、これを吸光度測定すればよい。また、メチレンブルー以外のフェノチアジン誘導体色素を生成する発色剤としては、各種フェノチアジン誘導体色素のロイコ体等が挙げられる。 Other examples of the color former that generates methylene blue, which is a phenothiazine derivative dye, include leuco compounds such as leucomethylene blue. Leucomethylene blue is a colorless compound (reduced type) and becomes methylene blue (oxidized type) by oxidation. For this reason, leucomethylene blue can be used as a substrate for detecting an oxidized substance, for example. In the present invention, for example, leucomethylene blue may be added to the reaction system as a color former, and methylene blue may be generated by an oxidation-reduction reaction between leucomethylene blue and an oxidizing substance in the reaction system, and the absorbance may be measured. Examples of color formers that produce phenothiazine derivative dyes other than methylene blue include leuco bodies of various phenothiazine derivative dyes.
本開示の試薬組成物における発色剤の含有量は、限定されない一又は複数の実施形態において、０．００１〜０．１００ｍｇ／ｍｌ、０．００３〜０．０８０ｍｇ／ｍｌ、０．００６〜０．０５０ｍｇ／ｍｌ、又は、０．００８〜０．０３０ｍｇ／ｍｌである。 The content of the color former in the reagent composition of the present disclosure is, in one or more non-limiting embodiments, 0.001 to 0.100 mg / ml, 0.003 to 0.080 mg / ml, 0.006 to 0.00. It is 050 mg / ml or 0.008-0.030 mg / ml.
［亜硝酸又はその塩］
本開示の試薬組成物に含まれうる亜硝酸又はその塩は、ヘモグロビンを変性させるために使用され得る。亜硝酸塩としては、限定されない一又は複数の実施形態において、亜硝酸カリウム、亜硝酸アミル、亜硝酸ブチル、亜硝酸ナトリウム等が挙げられる。本開示の試薬組成物における亜硝酸又は亜硝酸塩としては、０．０５〜３．００ｍｇ／ｍｌ、０．１０〜１．５０ｍｇ／ｍｌ、又は０．１５〜０．７５ｍｇ／ｍｌである。
[Nitrous acid or its salt]
Nitrous acid or a salt thereof that can be included in the reagent composition of the present disclosure can be used to denature hemoglobin. The nitrite includes, in one or more non-limiting embodiments, potassium nitrite, amyl nitrite, butyl nitrite, sodium nitrite and the like. The nitrite or nitrite in the reagent composition of the present disclosure is 0.05 to 3.00 mg / ml, 0.10 to 1.50 mg / ml, or 0.15 to 0.75 mg / ml.
［緩衝液又は緩衝剤］
本開示の試薬組成物に含まれうる緩衝液又は緩衝剤は、限定されない一又は複数の実施形態において、Ｔｒｉｓ (Tris(hydroxymethyl)amino-methane) 緩衝液、Ｔｒｉｓ−ＨＣｌ緩衝液、ＭＥＳ (2-Morpholinoehanesulfonic acid) 緩衝液、ＭＥＳ−Ｔｒｉｓ緩衝液、ＭＯＰＳ (3-Moropholinopropanesulfonic acid) 緩衝液、ＭＯＰＳ−Ｔｒｉｓ緩衝液、ＡＤＡ (N-(2-Acetamido)iminodiacetic acid) 緩衝液、Ｂｉｓ−Ｔｒｉｓ緩衝液、ＰＩＰＥＳ (Piperazine-1,4-bis(2-ethanesulfonic acid)) 緩衝液、リン酸緩衝液、クエン酸緩衝液、ＨＥＰＥＳ (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) 緩衝液、ＴＡＰＳ (N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) 緩衝液、グリシルグリシン緩衝液、グリシンアミド緩衝液等が挙げられる。上記緩衝液の濃度は、限定されない一又は複数の実施形態において、１〜５００ｍｍｏｌ／Ｌの範囲であり、又は、５〜１００ｍｍｏｌ／Ｌの範囲である。
[Buffer or buffer]
In one or more non-limiting embodiments, the buffer or buffer that can be included in the reagent composition of the present disclosure is a Tris (Tris (hydroxymethyl) amino-methane) buffer, Tris-HCl buffer, MES (2- Morpholinoehanesulfonic acid) buffer, MES-Tris buffer, MOPS (3-Moropholinopropanesulfonic acid) buffer, MOPS-Tris buffer, ADA (N- (2-Acetamido) iminodiacetic acid) buffer, Bis-Tris buffer, PIPES (Piperazine-1,4-bis (2-ethanesulfonic acid)) buffer, phosphate buffer, citrate buffer, HEPES (2- [4- (2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid) buffer TAPS (N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid) buffer, glycylglycine buffer, glycinamide buffer, and the like. The concentration of the buffer solution is in the range of 1 to 500 mmol / L or in the range of 5 to 100 mmol / L in one or more embodiments that are not limited.
［界面活性剤］
本開示の試薬組成物に含まれうる界面活性剤は、限定されない一又は複数の実施形態において、ｎ−ドデシル−β−Ｄ−マルトシド（ｎ−ドデシル−β−Ｄ−マルトピラノシド）、６−シクロヘキシルヘキシル−β−Ｄ−マルトシド、スクロースモノラウレート（β−Ｄ−フルクトピラノシル−α−Ｄ−グルコピラノシドモノドデカノエート）、ｎ−デシル−β−Ｄ−マルトシド（ｎ−デシル−β−Ｄ−マルトピラノシド）、ｎ−ノニル−β−Ｄ−チオマルトシド（ｎ−ノニル−β−Ｄ−チオマルトピラノシド）、５−シクロヘキシルペンチル−β−Ｄ−マルトシド、ウンデシル−β−Ｄ−マルトシド、ｎ−ドデシル−α−Ｄ−マルトシド、ヘキサデシル−β−Ｄ−マルトシド、及び、３−オキサトリデシル−α−Ｄ−マンノシド等が挙げられる。上記界面活性剤の濃度は、限定されない一又は複数の実施形態において、発色剤と界面活性剤のモル比が、１：１〜１：１００，０００の範囲であることが好ましく、より好ましくは、１：２〜１：５０，０００の範囲である。
[Surfactant]
In one or more non-limiting embodiments, the surfactant that can be included in the reagent composition of the present disclosure is n-dodecyl-β-D-maltoside (n-dodecyl-β-D-maltopyranoside), 6-cyclohexylhexyl. -Β-D-maltoside, sucrose monolaurate (β-D-fructopyranosyl-α-D-glucopyranoside monododecanoate), n-decyl-β-D-maltoside (n-decyl-β-D) -Maltopyranoside), n-nonyl-β-D-thiomaltoside (n-nonyl-β-D-thiomaltopyranoside), 5-cyclohexylpentyl-β-D-maltoside, undecyl-β-D-maltoside, n- Examples include dodecyl-α-D-maltoside, hexadecyl-β-D-maltoside, and 3-oxatridecyl-α-D-mannoside. The concentration of the surfactant is not limited, and in one or more embodiments, the molar ratio of the color former to the surfactant is preferably in the range of 1: 1 to 1: 100,000, more preferably It is in the range of 1: 2 to 1: 50,000.
［ｐＨ］
本開示の試薬組成物のｐＨは、酵素活性の維持及び/又は効率化の観点から、５．０〜９．０が好ましく、より好ましくは５．５〜８．５、より好ましくは６．０〜８．０である。
[PH]
The pH of the reagent composition of the present disclosure is preferably 5.0 to 9.0, more preferably 5.5 to 8.5, and more preferably 6.0 from the viewpoint of maintaining enzyme activity and / or increasing efficiency. ~ 8.0.
本開示の試薬組成物に含まれうる発色剤安定化剤は、限定されない一又は複数の実施形態において、マルトシル−β−シクロデキストリン、グルコシル−β−シクロデキストリン、カルボキシメチル−β−シクロデキストリン、ジメチル−β−シクロデキストリン、モノアミノ−β−シクロデキストリン、２−ヒドロキシプロピル−β−シクロデキストリン、メチル−β−シクロデキストリン、グルクロニルグルコシル−β−シクロデキストリン、ヘプタキス（２，６−ジ−Ｏ−メチル）−β−シクロデキストリン、トリ−Ｏ−メチル−β−シクロデキストリン、サクシニル−β−シクロデキストリン、スルホプロピル−β−シクロデキストリン、サクシニルヒドロキシプロピル−β−シクロデキストリン、トリアセチル−β−シクロデキストリン等が挙げられる。発色剤安定化剤の濃度は、特に制限はされないが、発色剤と発色剤安定化剤のモル比が例えば、１：１〜１：１００，０００の範囲であることが好ましく、より好ましくは、１：２〜１：５０，０００の範囲である。 The color former stabilizer that can be included in the reagent composition of the present disclosure includes, in one or more non-limiting embodiments, maltosyl-β-cyclodextrin, glucosyl-β-cyclodextrin, carboxymethyl-β-cyclodextrin, dimethyl -Β-cyclodextrin, monoamino-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, glucuronylglucosyl-β-cyclodextrin, heptakis (2,6-di-O- Methyl) -β-cyclodextrin, tri-O-methyl-β-cyclodextrin, succinyl-β-cyclodextrin, sulfopropyl-β-cyclodextrin, succinylhydroxypropyl-β-cyclodextrin, triacetyl-β-cyclodextrin Etc. Can be mentioned. The concentration of the color former stabilizer is not particularly limited, but the molar ratio of the color former and the color former stabilizer is, for example, preferably in the range of 1: 1 to 1: 100,000, more preferably It is in the range of 1: 2 to 1: 50,000.
［本開示の試薬組成物の調製方法］
本開示の試薬組成物の調製方法は、水又は緩衝液に上述した成分を添加混合することで調製できる。本開示の試薬組成物は、溶液の状態であってもよく、粉末、例えば、凍結乾燥粉末であってもよい。
[Method for Preparing Reagent Composition of Present Disclosure]
The method for preparing the reagent composition of the present disclosure can be prepared by adding and mixing the above-described components to water or a buffer solution. The reagent composition of the present disclosure may be in a solution or may be a powder, for example, a lyophilized powder.
［第２の試薬組成物］
本開示の試薬組成物のみでは、糖化タンパク質を測定することは難しい。糖化タンパク質を測定する場合、第２の試薬組成物として、水を含む試薬組成物を使用することが好ましい（以下、「本開示の第２の試薬組成物」ともいう）。本開示の第２の試薬組成物は、本開示の試薬組成物がＦＡＯＤを含む場合プロテアーゼを含み、本開示の試薬組成物がプロテアーゼを含む場合ＦＡＯＤを含む。
[Second reagent composition]
It is difficult to measure glycated protein only with the reagent composition of the present disclosure. When measuring glycated protein, it is preferable to use a reagent composition containing water as the second reagent composition (hereinafter, also referred to as “second reagent composition of the present disclosure”). The second reagent composition of the present disclosure includes a protease when the reagent composition of the present disclosure includes FAOD, and includes a FAOD when the reagent composition of the present disclosure includes a protease.
［第２の試薬組成物の水、ＦＡＯＤ、及びプロテアーゼ］
本開示の第２の試薬組成物は、水溶液であり、水を含む。水は、蒸留水、イオン交換水、又は超純水等が使用され得る。本開示の第２の試薬組成物に含まれうるＦＡＯＤ及びプロテアーゼは、本開示の試薬組成物に含まれ得る上述の物が使用できる。
[Second reagent composition of water, FAOD, and protease]
The second reagent composition of the present disclosure is an aqueous solution and contains water. As the water, distilled water, ion-exchanged water, ultrapure water, or the like can be used. As the FAOD and protease that can be contained in the second reagent composition of the present disclosure, those described above that can be contained in the reagent composition of the present disclosure can be used.
［第２の試薬組成物のその他の成分］
本開示の第２の試薬組成物は、その他の成分として、タール色素、ペルオキシダーゼ（ＰＯＤ）又は発色剤の少なくとも１つを含有しうる。本開示の第２の試薬組成物は、一又は複数の実施形態において、ＰＯＤと発色剤の２成分を同時に含有しない。したがって、本開示の試薬組成物と本開示の第２の試薬組成物は、限定されない一又は複数の実施形態において、下記表２の組み合わせの成分を含む実施形態１〜４が挙げられる。なお、表２において、本開示の試薬組成物の成分は、水、タール色素、及び多価アルコールに加えて含有する成分を示し、本開示の第２の試薬組成物は、水に加えて含有する成分を示す。
The second reagent composition of the present disclosure may contain at least one of a tar dye, a peroxidase (POD), or a color former as other components. In one or a plurality of embodiments, the second reagent composition of the present disclosure does not contain two components of POD and color former at the same time. Therefore, the reagent composition of this indication and the 2nd reagent composition of this indication include Embodiment 1-4 which contains the component of the combination of following Table 2 in one or some embodiment which is not limited. In Table 2, the components of the reagent composition of the present disclosure indicate components contained in addition to water, tar dyes, and polyhydric alcohols, and the second reagent composition of the present disclosure contains in addition to water. Ingredients to be shown.
本開示の第２の試薬組成物は、一又は複数の実施形態において、その他の成分として、さらに、緩衝液又は緩衝剤、界面活性剤を含有しうる。また、本開示の第２の試薬組成物が発色剤を含む場合、本開示の第２の試薬組成物はさらに発色剤安定化剤を含有しうる。 In one or a plurality of embodiments, the second reagent composition of the present disclosure may further contain a buffer solution or a buffering agent and a surfactant as other components. Moreover, when the 2nd reagent composition of this indication contains a color former, the 2nd reagent composition of this indication can contain a color former stabilizer further.
本開示の第２の試薬組成物に含まれうる緩衝液又は緩衝剤、及び、発色剤安定化剤は、限定されない一又は複数の実施形態において、本開示の試薬組成物に含まれ得る上述の物が使用できる。 The buffer or buffering agent and the color former stabilizer that may be included in the second reagent composition of the present disclosure may be included in the reagent composition of the present disclosure in one or more non-limiting embodiments. Things can be used.
本開示の第２の試薬組成物に含まれうる界面活性剤は、限定されない一又は複数の実施形態において、ベンゼトニウム、ステアリルトリメチルアンモニウム、セチルトリメチルアンモニウム、ヘキサデシルトリメチルアンモニウム、ベンザルコニウム、ベンジルジメチルテトラデシルアンモニウム、ミリスチルトリメチルアンモニウム、ラウリルトリメチルアンモニウム、ドデシルトリメチルアンモニウム、およびココナットアミンアセテート等が挙げられる。上記界面活性剤の濃度は、特に制限はされないが、前記発色剤と安定化剤のモル比が例えば、例えば、１：１〜１：１００，０００の範囲であることが好ましく、より好ましくは、１：２〜１：５０，０００の範囲である。 The surfactant that can be included in the second reagent composition of the present disclosure includes, in one or more non-limiting embodiments, benzethonium, stearyltrimethylammonium, cetyltrimethylammonium, hexadecyltrimethylammonium, benzalkonium, benzyldimethyltetra Examples include decyl ammonium, myristyl trimethyl ammonium, lauryl trimethyl ammonium, dodecyl trimethyl ammonium, and coconut amine acetate. The concentration of the surfactant is not particularly limited, but the molar ratio of the color former and the stabilizer is, for example, preferably in the range of 1: 1 to 1: 100,000, and more preferably, It is in the range of 1: 2 to 1: 50,000.
［本開示の第２の試薬組成物のｐＨ］
本開示の第２の試薬組成物のｐＨは、酵素活性の維持及び/又は効率化の観点から、５．０〜９．０が好ましく、より好ましくは５．５〜８．５、より好ましくは６．０〜８．０である。
[PH of the second reagent composition of the present disclosure]
The pH of the second reagent composition of the present disclosure is preferably 5.0 to 9.0, more preferably 5.5 to 8.5, and more preferably from the viewpoint of maintaining enzyme activity and / or increasing efficiency. 6.0 to 8.0.
［本開示の第２の試薬組成物の調製方法］
本開示の第２の試薬組成物の調製方法は、水又は緩衝液に上述した成分を添加混合することで調製できる。本開示の第２の試薬組成物は、溶液の状態であってもよく、粉末、例えば、凍結乾燥粉末であってもよい。
[Method for Preparing Second Reagent Composition of Present Disclosure]
The preparation method of the 2nd reagent composition of this indication can be prepared by adding and mixing the ingredient mentioned above to water or a buffer solution. The second reagent composition of the present disclosure may be in solution or may be a powder, such as a lyophilized powder.
［糖化タンパク質の測定方法］
本開示の試薬組成物及び本開示の第２の試薬組成物を用いて検体中の糖化タンパク質を測定する方法の限定されない一又は複数の実施形態は以下のようになる。
工程（１）：本開示の試薬組成物と、測定対象試料とを混合する工程。
工程（２）：本開示の試薬組成物と測定対象試料とを混合した混合液に、さらに、本開示の第２の試薬組成物を混合する工程。
工程（３）：工程（１）の混合液を光学測定するか、或いは、工程（２）の混合後速やかに混合液を光学測定する工程。
工程（４）：工程（２）の混合液を一定時間後に光学測定する工程。
[Method for measuring glycated protein]
One or a plurality of non-limiting embodiments of a method for measuring a glycated protein in a specimen using the reagent composition of the present disclosure and the second reagent composition of the present disclosure are as follows.
Step (1): A step of mixing the reagent composition of the present disclosure and the sample to be measured.
Step (2): a step of further mixing the second reagent composition of the present disclosure with a mixed liquid obtained by mixing the reagent composition of the present disclosure and the sample to be measured.
Step (3): A step of optically measuring the liquid mixture in step (1) or a step of optically measuring the liquid mixture immediately after mixing in step (2).
Step (4): A step of optically measuring the mixed solution of step (2) after a certain time.
より具体的には、限定されない下記実施形態１及び２が挙げられる。
実施形態１
工程（１）：本開示の試薬組成物と測定対象試料とを混合する
工程（２）：一定時間後に工程（１）の混合液を光学測定する。
工程（３）：工程（２）後にさらに本開示の第２の試薬組成物を混合する。
工程（４）：一定時間後に工程（３）後の混合液を光学測定する。
実施形態２
工程（１）：本開示の試薬組成物と測定対象試料とを混合する
工程（２）：一定時間後に工程（１）後にさらに本開示の第２の試薬組成物を混合する。
工程（３）：工程（２）の実施後に速やかに混合液を光学測定する。
工程（４）：一定時間後に工程（３）後の混合液を光学測定する。
More specifically, the following Embodiments 1 and 2 are not limited.
Embodiment 1
Step (1): Step of mixing the reagent composition of the present disclosure and the sample to be measured (2): After a certain time, the mixed solution of step (1) is optically measured.
Step (3): After the step (2), the second reagent composition of the present disclosure is further mixed.
Step (4): The liquid mixture after step (3) is optically measured after a certain time.
Embodiment 2
Step (1): Mixing the reagent composition of the present disclosure and the sample to be measured (2): After a predetermined time, the second reagent composition of the present disclosure is further mixed after the step (1).
Step (3): The liquid mixture is optically measured immediately after step (2).
Step (4): The liquid mixture after step (3) is optically measured after a certain time.
工程（１）の前に、検体から測定対象試料を調製してもよい。検体は、限定されない一又は複数の実施形態において、糖化タンパク質を含みうるものであって、全血、血漿、血清、血球等の血液試料、尿、髄液等の生体試料や、飲料水、食品類等が挙げられる。測定目的の糖化タンパク質が血球内成分の場合には、例えば、血球や血球を含む全血を溶血処理して測定対象試料を調製できる。溶血方法は、特に制限されず、例えば、浸透圧差を利用する方法、超音波による方法、界面活性剤処理方法等が使用できる。浸透圧差を利用する場合、全血（または血球）に対して、例えば、２〜１００倍体積量の精製水を添加して溶血させればよい。或いは、測定対象試料は、限定されない一又は複数の実施形態において、検体そのものであってもよい。 Prior to step (1), a sample to be measured may be prepared from the specimen. In one or a plurality of non-limiting embodiments, the specimen may contain a glycated protein, and blood samples such as whole blood, plasma, serum, blood cells, biological samples such as urine and cerebrospinal fluid, drinking water, food And the like. When the glycated protein to be measured is a blood cell component, for example, a measurement target sample can be prepared by hemolysis of blood cells or whole blood including blood cells. The hemolysis method is not particularly limited, and for example, a method using an osmotic pressure difference, an ultrasonic method, a surfactant treatment method, and the like can be used. When utilizing the osmotic pressure difference, for example, 2 to 100 times volume of purified water may be added to whole blood (or blood cells) to cause hemolysis. Alternatively, the sample to be measured may be the specimen itself in one or more embodiments that are not limited.
実施形態１の工程（２）と工程（４）で得られた光学的測定値の差、及び実施形態２の工程（３）と工程（４）で得られた光学的測定値の差から、糖化タンパク質量を算出できる。算出は、限定されない一又は複数の実施形態において、糖化量が既知の標準試料と光学的測定値との関係をプロットした検量線を用いることによって行える。標準試料は、１種類又は２種類以上である。光学的測定としては、限定されない一又は複数の実施形態において、吸光度測定、反射光測定等が挙げられる。実施形態１及び２の工程（２）における「一定時間」とは、限定されない一又は複数の実施形態において、１〜６０分、１〜１０分又は２〜５分である。実施形態１及び２の工程（４）における「一定時間」とは、限定されない一又は複数の実施形態において、１〜２０分、１〜１０分又は２〜５分である。また、実施形態１及び２の工程（１）〜（４）は、限定されない一又は複数の実施形態において、１５〜４５℃、２５〜４０℃、３０〜４０℃、又は３５〜３７℃の温度条件下で行われうる。 From the difference between the optical measurement values obtained in Step (2) and Step (4) of Embodiment 1 and the difference between the optical measurement values obtained in Step (3) and Step (4) of Embodiment 2, The amount of glycated protein can be calculated. In one or a plurality of non-limiting embodiments, the calculation can be performed by using a calibration curve in which a relationship between a standard sample with a known amount of saccharification and an optical measurement value is plotted. There are one or more standard samples. Examples of the optical measurement include, but are not limited to, absorbance measurement and reflected light measurement in one or more embodiments. The “certain time” in step (2) of Embodiments 1 and 2 is 1 to 60 minutes, 1 to 10 minutes, or 2 to 5 minutes in one or more embodiments that are not limited. The “certain time” in the step (4) of Embodiments 1 and 2 is 1 to 20 minutes, 1 to 10 minutes, or 2 to 5 minutes in one or a plurality of non-limiting embodiments. Moreover, process (1)-(4) of Embodiment 1 and 2 is 15-45 degreeC, 25-40 degreeC, 30-40 degreeC, or temperature of 35-37 degreeC in one or some embodiment which is not limited. It can be performed under conditions.
測定目的となる糖化タンパク質は、限定されない一又は複数の実施形態において、糖化ヘモグロビン、糖化アルブミン、ＨｂＡ１ｃなどが挙げられる。 The glycated protein to be measured includes, but is not limited to, glycated hemoglobin, glycated albumin, HbA1c and the like in one or more embodiments.
本開示の糖化タンパク質の測定方法は、その他の一又は複数の実施形態において、さらに測定対象試料中のタンパク質量（糖化タンパク質と非糖化タンパク質の合計量）を測定することを含みうる。このタンパク質量の測定は、本開示の第２の試薬組成物を混合する前又は後に測定することで達成できる。例えば、糖化タンパク質の測定方法の前記実施形態１においては、工程（２）と同時、工程（２）の前後、工程（４）と同時、又は工程（４）の前後に測定できる。糖化タンパク質の測定方法の前記実施形態１においては、工程（２）の前、工程（３）と同時、工程（４）と同時、又は工程（４）の前後に測定できる。 In one or a plurality of other embodiments, the method for measuring a glycated protein of the present disclosure may further include measuring the amount of protein (total amount of glycated protein and non-glycated protein) in the measurement target sample. This measurement of protein amount can be achieved by measuring before or after mixing the second reagent composition of the present disclosure. For example, in the said Embodiment 1 of the measuring method of glycated protein, it can measure simultaneously with a process (2), before and after a process (2), simultaneously with a process (4), or before and after a process (4). In the said Embodiment 1 of the measuring method of glycated protein, it can measure before a process (2), simultaneous with a process (3), simultaneous with a process (4), or before and after a process (4).
本開示の試薬組成物を用いた糖化タンパク質の測定方法であれば、限定されない一又は複数の実施形態において、自動分析装置の反応セルにおける析出物の発生を抑制できる。よって、本開示は、一態様において、下記工程（１）〜（４）を行う自動分析装置を使用する糖化タンパク質の測定方法（以下、「本開示の測定方法」ともいう）に関する。
工程（１）：本開示の試薬組成物である第一試薬と、測定対象試料とを混合する工程。
工程（２）：第一試薬と測定対象試料とを混合した混合液に、さらに、本開示の第２の試薬組成物である第二試薬を混合する工程。
工程（３）：工程（１）の混合液を光学測定するか、或いは、工程（２）の混合後速やかに混合液を光学測定する工程。
工程（４）：工程（２）の混合液を一定時間後に光学測定する工程。
If it is the measuring method of glycated protein using the reagent composition of this indication, in one or some embodiment which is not limited, generation | occurrence | production of the precipitate in the reaction cell of an automatic analyzer can be suppressed. Therefore, this indication is related with the measuring method (henceforth the "measuring method of this indication") of glycated protein which uses the automatic analyzer which performs the following process (1)-(4) in one mode.
Step (1): A step of mixing the first reagent that is the reagent composition of the present disclosure and the sample to be measured.
Step (2): A step of further mixing a second reagent, which is the second reagent composition of the present disclosure, with a mixed liquid obtained by mixing the first reagent and the sample to be measured.
Step (3): A step of optically measuring the liquid mixture in step (1) or a step of optically measuring the liquid mixture immediately after mixing in step (2).
Step (4): A step of optically measuring the mixed solution of step (2) after a certain time.
［自動分析装置］
自動分析装置としては、糖化タンパク質の検出に従来使用され或いは今後開発され使用されるものが挙げられる。限定されない一又は複数の実施形態において、自動分析装置は、下記手段１〜４を備える。
測定手段１
手段（１）：本開示の試薬組成物と測定対象試料とを混合する
手段（２）：手段（１）の混合液を光学測定する。
手段（３）：手段（２）後にさらに本開示の第２の試薬組成物を混合する。
手段（４）：手段（３）後の混合液を光学測定する。
測定手段２
手段（１）：本開示の試薬組成物と測定対象試料とを混合する
手段（２）：一定時間後に手段（１）後にさらに本開示の第２の試薬組成物を混合する。
手段（３）：手段（２）の実施後に速やかに混合液を光学測定する。
手段（４）：一定時間後に手段（３）後の混合液を光学測定する。
[Automatic analyzer]
Examples of the automatic analyzer include those that are conventionally used for the detection of glycated proteins or that are developed and used in the future. In one or some embodiment which is not limited, an automatic analyzer is provided with the following means 1-4.
Measuring means 1
Means (1): Means for mixing the reagent composition of the present disclosure and the sample to be measured (2): Optically measuring the mixed solution of means (1).
Means (3): After the means (2), the second reagent composition of the present disclosure is further mixed.
Means (4): The liquid mixture after means (3) is optically measured.
Measuring means 2
Means (1): Means for mixing the reagent composition of the present disclosure and the sample to be measured (2): The second reagent composition of the present disclosure is further mixed after the means (1) after a certain time.
Means (3): The liquid mixture is optically measured immediately after the means (2) is carried out.
Means (4): The liquid mixture after means (3) is optically measured after a certain time.
自動分析装置は、さらに、測定手段１では手段（２）と手段（４）で得られた光学的測定値の差、及び測定手段１では手段（２）と手段（４）で得られた光学的測定値の差から糖化タンパク質量を算出する手段を備えてもよい。 The automatic analyzer further includes a difference between the optical measurement values obtained by the means (2) and the means (4) in the measurement means 1, and an optical value obtained by the means (2) and the means (4) in the measurement means 1. There may be provided means for calculating the amount of glycated protein from the difference between the measured values.
［糖化タンパク質測定用試薬キット］
本開示は、その他の態様において、本開示の試薬組成物である第一試薬と、本開示の第２の試薬組成物である第二試薬とを含む、糖化タンパク質測定用試薬キット（以下、「本開示の試薬キット」ともいう）に関する。本開示の試薬キットと自動分析装置とを用いて、本開示の測定方法を行うことができる。本開示の試薬キットは、さらに、標準試料を含んでもよい。
[Reagent kit for measuring glycated protein]
In another aspect, the present disclosure provides a reagent kit for measuring a glycated protein (hereinafter referred to as “the second reagent composition which is the second reagent composition of the present disclosure”) (hereinafter referred to as “the reagent composition of the present disclosure”). Also referred to as “the reagent kit of the present disclosure”. The measurement method of the present disclosure can be performed using the reagent kit and the automatic analyzer of the present disclosure. The reagent kit of the present disclosure may further include a standard sample.
すなわち、本開示は以下の一又は複数の実施形態に関しうる；
［Ａ１］ 糖化タンパク質測定用試薬組成物であって、フルクトシルアミノ酸オキシダーゼ（ＦＡＯＤ）又はプロテアーゼ、水、タール色素、及び多価アルコールを含む試薬組成物。
［Ａ２］ タール色素が、アゾ色素である、［Ａ１］記載の試薬組成物。
［Ａ３］ アゾ色素がタートラジンである、［Ａ２］記載の試薬組成物。
［Ａ４］ 多価アルコールの分子内ヒドロキシル基数が２個である、［Ａ１］から［Ａ３］のいずれかに記載の試薬組成物。
［Ａ５］ 多価アルコールが、エチレングリコール、プロピレングリコール、ジプロピレングリコール、及びこれらの組み合わせからなる群から選択される、［Ａ１］から［Ａ４］のいずれかに記載の試薬組成物。
［Ａ６］ 試薬組成物中の多価アルコールの含有量が、０．５重量％以上１０重量％以下である、［Ａ１］から［Ａ５］のいずれかに記載の試薬組成物。
［Ａ７］ 糖化タンパク質測定自動分析装置で用いるための、［Ａ１］から［Ａ６］のいずれかに記載の試薬組成物。
［Ａ８］ さらに、ペルオキシダーゼ（ＰＯＤ）又は発色剤を含む、［Ａ１］から［Ａ７］のいずれかに記載の試薬組成物。
［Ａ９］ ［Ａ１］から［Ａ８］のいずれかに記載の試薬組成物である第一試薬と、水を含む第二試薬を含み、第二試薬は、さらに、第一試薬がＦＡＯＤを含む場合プロテアーゼを含み、第一試薬がプロテアーゼを含む場合ＦＡＯＤを含む、糖化タンパク質測定用試薬キット。
［Ａ１０］ 第二試薬が、さらに、タール色素、ペルオキシダーゼ（ＰＯＤ）及び発色剤からなる群から選択される少なくとも一つを含む、［Ａ９］に記載の糖化タンパク質測定用試薬キット。
［Ａ１１］ さらに、標準試料を含む、［Ａ９］又は［Ａ１０］に記載の糖化タンパク質測定用試薬キット。
［Ａ１２］ 下記工程（１）〜（４）を行うことを含む、糖化タンパク質の測定方法。
工程（１）：［Ａ１］から［Ａ８］のいずれかに記載の試薬組成物である第一試薬と、測定対象試料とを混合する工程。
工程（２）：第一試薬と測定対象試料とを混合した混合液に、さらに、水を含む第二試薬を混合する工程。ここで、第二試薬は、さらに、第一試薬がＦＡＯＤを含む場合プロテアーゼを含み、第一試薬がプロテアーゼを含む場合ＦＡＯＤを含み、さらに、第一試薬が発色剤を含まない場合発色剤を含む。
工程（３）：工程（１）の混合液を光学測定するか、或いは、工程（２）の混合後速やかに混合液を光学測定する工程。
工程（４）：工程（２）の混合液を一定時間後に光学測定する工程。
That is, the present disclosure may relate to one or more of the following embodiments;
[A1] A reagent composition for measuring a glycated protein, which comprises fructosyl amino acid oxidase (FAOD) or protease, water, a tar dye, and a polyhydric alcohol.
[A2] The reagent composition according to [A1], wherein the tar dye is an azo dye.
[A3] The reagent composition according to [A2], wherein the azo dye is tartrazine.
[A4] The reagent composition according to any one of [A1] to [A3], wherein the polyhydric alcohol has 2 hydroxyl groups in the molecule.
[A5] The reagent composition according to any one of [A1] to [A4], wherein the polyhydric alcohol is selected from the group consisting of ethylene glycol, propylene glycol, dipropylene glycol, and combinations thereof.
[A6] The reagent composition according to any one of [A1] to [A5], wherein the content of the polyhydric alcohol in the reagent composition is 0.5 wt% or more and 10 wt% or less.
[A7] The reagent composition according to any one of [A1] to [A6] for use in a glycated protein measurement automatic analyzer.
[A8] The reagent composition according to any one of [A1] to [A7], further comprising peroxidase (POD) or a color former.
[A9] When the first reagent which is the reagent composition according to any one of [A1] to [A8] and a second reagent containing water are included, and the second reagent further includes FAOD A reagent kit for measuring a glycated protein, which contains a protease and FAOD when the first reagent contains a protease.
[A10] The reagent kit for measuring a glycated protein according to [A9], wherein the second reagent further contains at least one selected from the group consisting of a tar dye, peroxidase (POD), and a color former.
[A11] The reagent kit for measuring a glycated protein according to [A9] or [A10], further including a standard sample.
[A12] A method for measuring a glycated protein, comprising performing the following steps (1) to (4).
Step (1): A step of mixing the first reagent that is the reagent composition according to any one of [A1] to [A8] and a sample to be measured.
Step (2): A step of further mixing a second reagent containing water with a mixed liquid obtained by mixing the first reagent and the sample to be measured. Here, the second reagent further includes a protease when the first reagent includes FAOD, includes a FAOD when the first reagent includes a protease, and further includes a color former when the first reagent does not include a color former. .
Step (3): A step of optically measuring the liquid mixture in step (1) or a step of optically measuring the liquid mixture immediately after mixing in step (2).
Step (4): A step of optically measuring the mixed solution of step (2) after a certain time.
以下、実施例により本開示をさらに詳細に説明するが、これらは例示的なものであって、本開示はこれら実施例に制限されるものではない。 Hereinafter, the present disclosure will be described in more detail by way of examples. However, these examples are illustrative, and the present disclosure is not limited to these examples.
血中のヘモグロビンＡ１ｃ濃度及び総ヘモグロビン濃度を測定するための試薬組成物の組み合わせとして、下記第１試薬及び第２試薬を準備した。
＜第１試薬＞
フルクトシルアミノ酸オキシダーゼ（ＦＡＯＤ）（商品名：ＦＰＯＸ−ＣＥ、キッコーマン社製）：０．６−３．０ Ｕ／ｍｌ
亜硝酸カリウム：０．１５−０．７５ ｍｇ／ｍｌ
ペルオキシダーゼ（東洋紡社製）：３−２０ Ｕ／ｍｌ
タートラジン：０．０５−０．３０ ｍｇ／ｍｌ
緩衝液（Ｔｒｉｓ）：２−１０ ｍｍｏｌ／Ｌ，ｐＨ ７．０
＜第２試薬＞
中性プロテアーゼ・・・８００−４０００ Ｕ／ｍｌ
１０−（カルボキシメチルアミノカルボニル）−３，７−ビス（ジメチルアミノ）フェノチアジン（和光純薬社製）・・・０．００８−０．０３ ｍｇ／ｍｌ
緩衝液（Ｔｒｉｓ）：５０−１００ ｍｍｏｌ／Ｌ，ｐＨ ６．５
界面活性剤：０．０５−０．３ ｍｇ／ｍｌ
発色剤安定化剤：１０−３０ ｍｇ／ｍｌ
The following first reagent and second reagent were prepared as a combination of reagent compositions for measuring hemoglobin A1c concentration and total hemoglobin concentration in blood.
<First reagent>
Fructosyl amino acid oxidase (FAOD) (trade name: FPOX-CE, manufactured by Kikkoman): 0.6-3.0 U / ml
Potassium nitrite: 0.15-0.75 mg / ml
Peroxidase (Toyobo): 3-20 U / ml
Tartrazine: 0.05-0.30 mg / ml
Buffer (Tris): 2-10 mmol / L, pH 7.0
<Second reagent>
Neutral protease ... 800-4000 U / ml
10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine (manufactured by Wako Pure Chemical Industries) ... 0.008-0.03 mg / ml
Buffer (Tris): 50-100 mmol / L, pH 6.5
Surfactant: 0.05-0.3 mg / ml
Color former stabilizer: 10-30 mg / ml
［析出試験］
前記第１試薬に下記表３記載の多価アルコールを２ｗ／ｖ％添加して実施例１〜１０及び比較例１の試薬組成物を作製した。これらの試薬組成物１００μＬを、自動分析装置（商品名：ＪＣＡ−ＭＢ８，日本電子社製）の反応セルに分注し、室温で５時間静置した。その結果の一例を図１に示す。その結果、比較例１の試薬組成物（第１試薬そのもの）では、自動分析装置の反応セル上面の４つのコーナーに色素を含む析出物が発生した。一方で、多価アルコールを添加した実施例１〜１０の試薬組成物では析出物が確認されず、析出物の発生が抑制された。
[Precipitation test]
The reagent compositions of Examples 1 to 10 and Comparative Example 1 were prepared by adding 2 w / v% of the polyhydric alcohol described in Table 3 below to the first reagent. 100 μL of these reagent compositions were dispensed into a reaction cell of an automatic analyzer (trade name: JCA-MB8, manufactured by JEOL Ltd.) and allowed to stand at room temperature for 5 hours. An example of the result is shown in FIG. As a result, in the reagent composition of Comparative Example 1 (first reagent itself), precipitates containing pigments were generated at the four corners on the upper surface of the reaction cell of the automatic analyzer. On the other hand, precipitates were not confirmed in the reagent compositions of Examples 1 to 10 to which polyhydric alcohol was added, and the generation of precipitates was suppressed.
実施例１〜１０及び比較例１の試薬組成物１００μＬを、自動分析装置（商品名：ＪＣＡ−ＭＢ８，日本電子社製）の反応セルに分注し、室温で５時間静置した。その結果の一例を図１に示す。図１に示すとおり、比較例１の試薬組成物（第１試薬そのもの）では、自動分析装置の反応セル上面の４つのコーナーに色素を含む析出物が発生した。一方で、多価アルコールを添加した実施例１〜１０の試薬組成物では析出物が確認されず、析出物の発生が抑制された。 100 μL of the reagent compositions of Examples 1 to 10 and Comparative Example 1 were dispensed into a reaction cell of an automatic analyzer (trade name: JCA-MB8, manufactured by JEOL Ltd.) and allowed to stand at room temperature for 5 hours. An example of the result is shown in FIG. As shown in FIG. 1, in the reagent composition of Comparative Example 1 (first reagent itself), precipitates containing pigments were generated at the four corners on the upper surface of the reaction cell of the automatic analyzer. On the other hand, precipitates were not confirmed in the reagent compositions of Examples 1 to 10 to which polyhydric alcohol was added, and the generation of precipitates was suppressed.
［試薬性能試験１］
第１試薬として実施例１〜１０及び比較例１の試薬組成物を用いてＨｂＡ１ｃ値を測定し、試薬性能を確認した。
[Reagent performance test 1]
Using the reagent compositions of Examples 1 to 10 and Comparative Example 1 as the first reagent, the HbA1c value was measured to confirm the reagent performance.
〔測定対象試料〕
下記試料を使用してＨｂＡ１ｃ （ＪＤＳ）値が４〜１４％のリファレンス試料を作成した。また、ＨｂＡ１ｃ （ＪＤＳ）値が同じ（６．５又は７．０％）でヘモグロビン濃度が３０〜１５０μｍｏｌ／Ｌの範囲で異なるリファレンス試料を準備した。
糖尿病検査コントロールＬｅｖｅｌ １−３（リクイチェック（商品名）、ＢＩＯ−ＲＡＤ社製）
−８０℃凍結保存全血Ｈｂ濃度違い３種類
−８０℃凍結保存全血ＨｂＡ１ｃ濃度違い８種類
[Samples to be measured]
A reference sample having an HbA1c (JDS) value of 4 to 14% was prepared using the following sample. Further, different reference samples having the same HbA1c (JDS) value (6.5 or 7.0%) and a hemoglobin concentration in the range of 30 to 150 μmol / L were prepared.
Diabetes test control Level 1-3 (Liqui check (trade name), manufactured by BIO-RAD)
-80 ° C cryopreserved whole blood Hb concentration difference 3 types -80 ° C cryopreserved whole blood HbA1c concentration difference 8 types
〔ＨｂＡ１ｃ濃度の測定手順〕
測定試料８μＬと実施例１〜１０及び比較例１の試薬組成物（第１試薬）９６μＬを混合し、３７℃、５分で反応する。その後、主波長６９４ｎｍ副波長７５１ｎｍで吸光度を測定し、第２試薬６３μＬを添加する。さらに３７℃、５分間反応させた後、再度、主波長６９４ｎｍ副波長７５１ｎｍで吸光度を測定する。吸光度の差からＨｂＡ１ｃ濃度（ｍｍｏｌ／ｌ）を算出する。
〔総ヘモグロビン濃度の測定手順〕
測定試料８μＬと実施例１〜１０及び比較例１の試薬組成物（第１試薬）８μＬを混合し、３７℃、５分で反応する。その後、主波長５７１ｎｍ副波長６９４ｎｍで吸光度を測定し、その吸光度から総ヘモグロビン濃度（ｍｏｌ／ｌ）を算出する。
〔ＨｂＡ１ｃ（ＪＤＳ）値の算出手順〕
ＨｂＡ１ｃ （ＪＤＳ）値は、以下の式で求めた。その結果の一例を図２及び３に示す。
ＨｂＡ１ｃ （ＮＧＳＰ）値＝０．０９１５ × ＨｂＡ１ｃ濃度（ｍｍｏｌ／ｌ）／総Ｈｂ濃度（ｍｏｌ／ｌ） ＋ ２．１５
ＨｂＡ１ｃ （ＪＤＳ）値＝０．９８０ × ＨｂＡ１ｃ （ＮＧＳＰ）値 − ０．２４５
[Measurement procedure of HbA1c concentration]
8 μL of the measurement sample and 96 μL of the reagent composition (first reagent) of Examples 1 to 10 and Comparative Example 1 are mixed and reacted at 37 ° C. for 5 minutes. Thereafter, the absorbance is measured at a main wavelength of 694 nm and a subwavelength of 751 nm, and 63 μL of the second reagent is added. Further, after reacting at 37 ° C. for 5 minutes, the absorbance is measured again at a main wavelength of 694 nm and a subwavelength of 751 nm. The HbA1c concentration (mmol / l) is calculated from the difference in absorbance.
[Measurement procedure of total hemoglobin concentration]
8 μL of the measurement sample and 8 μL of the reagent compositions (first reagent) of Examples 1 to 10 and Comparative Example 1 are mixed and reacted at 37 ° C. for 5 minutes. Thereafter, the absorbance is measured at a main wavelength of 571 nm and a subwavelength of 694 nm, and the total hemoglobin concentration (mol / l) is calculated from the absorbance.
[Calculation procedure of HbA1c (JDS) value]
The HbA1c (JDS) value was determined by the following formula. An example of the result is shown in FIGS.
HbA1c (NGSP) value = 0.0915 × HbA1c concentration (mmol / l) / total Hb concentration (mol / l) + 2.15
HbA1c (JDS) value = 0.980 × HbA1c (NGSP) value−0.245
ＨｂＡ１ｃ（ＪＤＳ）値が４〜１４％の試料を実施例１〜１０及び比較例１の試薬組成物を用いて上記手順に従ってＨｂＡ１ｃ（ＪＤＳ）値を測定した。その結果の一例を図２に示す。図２は、実施例１〜１０と比較例１とのＨｂＡ１ｃ（ＪＤＳ）値の差（ΔＪＤＳ％）を示すグラフである。図２に示すとおり、実施例３（ジエチレングリコール）、実施例４（トリエチレングリコール）、実施例８（ポリエチレングリコール＃３００）では、比較例１と比べて高値化する傾向が見られた。 The HbA1c (JDS) value of 4 to 14% of the sample was measured for the HbA1c (JDS) value according to the above procedure using the reagent compositions of Examples 1 to 10 and Comparative Example 1. An example of the result is shown in FIG. FIG. 2 is a graph showing a difference (ΔJDS%) in HbA1c (JDS) values between Examples 1 to 10 and Comparative Example 1. As shown in FIG. 2, in Example 3 (diethylene glycol), Example 4 (triethylene glycol), and Example 8 (polyethylene glycol # 300), a tendency to increase the price compared to Comparative Example 1 was observed.
ヘモグロビン（Ｈｂ）濃度が低（４５μｍｏｌ／Ｌ）、中（９０μｍｏｌ／Ｌ）、高（１３５μｍｏｌ／Ｌ）の３種類の試料（ＨｂＡ１ｃ （ＪＤＳ）値６．５％）を実施例１〜１０及び比較例１の試薬組成物を用いて上記手順に従ってＨｂＡ１ｃ（ＪＤＳ）値を測定した。その結果の一例を図３に示す。図３は、Ｈｂ濃度が中濃度（９０μｍｏｌ／Ｌ）の試料の測定値と、低又は高濃度の試料の測定値との差を示すグラフである。図３に示すとおり、実施例３（ジエチレングリコール）、実施例４（トリエチレングリコール）、実施例７（ポリエチレングリコール＃２００）、実施例８（ポリエチレングリコール＃３００）、実施例９（ポリエチレングリコール＃４００）、実施例１０（ポリエチレングリコール＃６００）では、Ｈｂ濃度の影響を強く受けた。 Three types of samples (HbA1c (JDS) value 6.5%) with low (45 μmol / L), medium (90 μmol / L), and high (135 μmol / L) hemoglobin (Hb) concentrations were compared with Examples 1 to 10. Using the reagent composition of Example 1, the HbA1c (JDS) value was measured according to the above procedure. An example of the result is shown in FIG. FIG. 3 is a graph showing a difference between a measured value of a sample having a medium Hb concentration (90 μmol / L) and a measured value of a sample having a low or high concentration. As shown in FIG. 3, Example 3 (diethylene glycol), Example 4 (triethylene glycol), Example 7 (polyethylene glycol # 200), Example 8 (polyethylene glycol # 300), and Example 9 (polyethylene glycol # 400). ), Example 10 (polyethylene glycol # 600) was strongly influenced by the Hb concentration.
［試薬性能試験２］
前記第１試薬（比較例１）のＦＡＯＤ（商品名：ＦＰＯＸ−ＣＥ、キッコーマン社製）を耐熱性ＦＡＯＤ（商品名：ＦＰＯＸ−ＣＥＴ、キッコーマン社製）に換えた試薬組成物を調製し、これを比較例２とした。比較例２の組成に、下記表４記載の多価アルコール（エチレングリコール、プロピレングリコール、ジプロピレングリコール、及びグリセリン）を１〜３ｗ／ｖ％添加して実施例１１〜１８及び比較例２の試薬組成物を作製した。
[Reagent performance test 2]
A reagent composition was prepared by replacing FAOD (trade name: FPOX-CE, manufactured by Kikkoman Corp.) of the first reagent (Comparative Example 1) with heat-resistant FAOD (trade name: FPOX-CET, manufactured by Kikkoman Corp.). Was referred to as Comparative Example 2. Reagents of Examples 11 to 18 and Comparative Example 2 by adding 1 to 3 w / v% of polyhydric alcohols (ethylene glycol, propylene glycol, dipropylene glycol, and glycerin) described in Table 4 below to the composition of Comparative Example 2 A composition was prepared.
第１試薬として、実施例１１〜１８、及び比較例１〜２の試薬組成物、及び、３７℃で７日間保存した組成物を使用して上述の手順にしたがってＨｂＡ１ｃ（ＪＤＳ）値を算出した。測定試料として、下記４種類の試料を用いた。 Using the reagent compositions of Examples 11 to 18 and Comparative Examples 1 and 2 and the composition stored at 37 ° C. for 7 days as the first reagent, the HbA1c (JDS) value was calculated according to the procedure described above. . The following four types of samples were used as measurement samples.
〔測定試料〕
酵素法ＨｂＡ１測定試薬キャリブレータＬｏｗ及びＨｉｇｈ（アークレイ社製）
（以下、「Ｃａｌ Ｌ」及び「Ｃａｌ Ｈ」とも表記する）
−８０℃凍結保存全血Ｈｂ濃度違い２種類である、ヘモグロビン（Ｈｂ）濃度中（９０μｍｏｌ／Ｌ）および高（１３５μｍｏｌ／Ｌ）検体（以下、それぞれ「ＭＮ」及び「ＭＨ」とも表記する）
[Measurement sample]
Enzyme method HbA1 measuring reagent calibrator Low and High (manufactured by ARKRAY)
(Hereinafter also referred to as “Cal L” and “Cal H”)
-80 ° C frozen stored whole blood Hb concentration difference, two types of hemoglobin (Hb) concentration (90 μmol / L) and high (135 μmol / L) samples (hereinafter also referred to as “MN” and “MH”, respectively)
図４〜８は、３７℃７日間保存の前（初期）と後（３７℃７日）の実施例１１〜１８、及び比較例１〜２の試薬組成物を用いてＨｂＡ１ｃ濃度の測定したときの反応タイムコースの吸光度（波長７００ｎｍ）を示す。また、下記表５は、試料ＭＮ及びＮＨを測定した場合における保存前（初期）と保存後（３７℃７日）におけるＨｂＡ１ｃ（ＪＤＳ）値とその変動値を示す。 4 to 8 show the measurement of HbA1c concentration using the reagent compositions of Examples 11 to 18 and Comparative Examples 1 to 2 before (initial) and after storage (37 ° C. for 7 days) at 37 ° C. for 7 days. The absorbance (wavelength 700 nm) of the reaction time course is shown. Table 5 below shows the HbA1c (JDS) value and its fluctuation value before (initial) and after storage (7 days at 37 ° C.) when samples MN and NH are measured.
図４及び表５に示すとおり、比較例１の試薬組成物は、３７℃７日間の培養後は反応活性が著しく低下していた。一方、比較例２及び実施例１１〜１７は、図４〜７に示すとおり保存前後で反応性に変化は見られず、また、表５に示すとおり反応活性にも影響は見られなかった。実施例１８のグリセリンは、図８に示すとおり反応活性に変化があり、表５に示すとおり測定値も大幅に変化した。 As shown in FIG. 4 and Table 5, the reagent composition of Comparative Example 1 had a markedly decreased reaction activity after culturing at 37 ° C. for 7 days. On the other hand, in Comparative Example 2 and Examples 11 to 17, no change was observed in the reactivity before and after storage as shown in FIGS. 4 to 7, and no influence on the reaction activity was seen as shown in Table 5. As for glycerin of Example 18, there was a change in the reaction activity as shown in FIG.
Claims (12)
フルクトシルアミノ酸オキシダーゼ（ＦＡＯＤ）、水、タール色素、及び多価アルコールを含む第一試薬と、
プロテアーゼを含む第二試薬とを含む、糖化タンパク質測定用試薬キット。 A reagent kit for measuring glycated protein, comprising:
A first reagent comprising fructosyl amino acid oxidase (FAOD), water, tar dye, and polyhydric alcohol ;
A reagent kit for measuring a glycated protein, comprising a second reagent containing a protease.
工程（１）：フルクトシルアミノ酸オキシダーゼ（ＦＡＯＤ）、水、タール色素、及び多価アルコールを含む第一試薬と、測定対象試料とを混合する工程。
工程（２）：第一試薬と測定対象試料とを混合した混合液に、さらに、プロテアーゼ、及び発色剤を含む第二試薬を混合する工程。
工程（３）：工程（１）の混合液を光学測定するか、或いは、工程（２）の混合後速やかに混合液を光学測定する工程。
工程（４）：工程（２）の混合液を一定時間後に光学測定する工程。 The measuring method of glycated protein including performing following process (1)-(4).
Step (1): A step of mixing a first reagent containing fructosyl amino acid oxidase (FAOD), water, a tar dye, and a polyhydric alcohol with a sample to be measured.
Step (2): A step of further mixing a protease and a second reagent containing a color-developing agent with a mixed liquid obtained by mixing the first reagent and the sample to be measured.
Step (3): A step of optically measuring the liquid mixture in step (1) or a step of optically measuring the liquid mixture immediately after mixing in step (2).
Step (4): A step of optically measuring the mixed solution of step (2) after a certain time.
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