JPWO2006030866A1 - Method for quantitative determination of uric acid - Google Patents
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Abstract
ペルオキシダーゼを用いた尿酸の定量方法において、低値での正確性に優れた定量方法と試薬組成物を提供することを目的とする。被検試料中の尿酸をウリカーゼ酵素によって過酸化水素とアラントインに分解し、その過酸化水素をペルオキシダーゼを用いて定量することによって被検試料中の尿酸を定量する方法であって、尿酸のウリカーゼ酵素による分解前に尿酸をペルオキシダーゼと接触させないことにより尿酸低値を正確に定量し得ることを特徴とする尿酸の定量方法。An object of the present invention is to provide a quantification method and reagent composition excellent in accuracy at a low value in a uric acid quantification method using peroxidase. A method for quantifying uric acid in a test sample by decomposing uric acid in a test sample into hydrogen peroxide and allantoin using a uricase enzyme and quantifying the hydrogen peroxide using peroxidase, the uric acid uricase enzyme A method for quantifying uric acid, characterized in that a low value of uric acid can be accurately quantified by preventing uric acid from contacting with peroxidase before decomposition by the method.
Description
本発明は、尿酸の定量において正確に定量できる試薬およびその試薬を用いた尿酸の定量方法に関する。より詳しくは、本発明は尿酸の定量において低値を正確に定量できる試薬およびその試薬を用いた尿酸の低値の正確な定量方法に関する。 The present invention relates to a reagent capable of accurately quantifying uric acid and a method for quantifying uric acid using the reagent. More specifically, the present invention relates to a reagent capable of accurately quantifying a low value in uric acid quantification and a method for accurately quantifying a low uric acid value using the reagent.
尿酸は、デオキシリボ核酸やリボ核酸などの核酸やエネルギーの担体であるATPおよび他のヌクレオチドの構成成分であるプリン体の最終代謝産物である。プリン体はヒト体内で主にアミノ酸および関連化合物を原料として合成され、プリン代謝は細胞の増殖にかかわり、また、生命の維持に中心的な役割を果たしている。 Uric acid is a final metabolite of purines, which are constituents of nucleic acids such as deoxyribonucleic acid and ribonucleic acid, and ATP, which is an energy carrier, and other nucleotides. Purines are synthesized mainly in the human body using amino acids and related compounds as raw materials. Purine metabolism is involved in cell growth and plays a central role in maintaining life.
尿酸は生体内において一定の割合で生産され、体内に一時貯留した後、主として腎臓から尿中へ他の一部は腎外性処理として消化管や汗などに排泄される。生産と排泄はほぼ同量で、従って産生、貯留および排泄が平衡状態を成している。正常人では一日の産生・排泄量はほぼ700mgで、そのうち500mgが腎から尿中に、他の約200mgが腎外性処理により排泄される。腎外性処理は血清尿酸値の変動に並行して増減する。 Uric acid is produced at a certain rate in the living body, and after being temporarily stored in the body, the other part is excreted mainly in the digestive tract and sweat as an extrarenal treatment from the kidney to the urine. Production and excretion are approximately the same, so production, storage and excretion are in equilibrium. In normal individuals, daily production and excretion is approximately 700 mg, of which 500 mg is excreted from the kidneys into the urine and the other approximately 200 mg is excreted by extrarenal treatment. Extrarenal treatment increases or decreases in parallel with changes in serum uric acid levels.
生体内尿酸の代謝異常や排泄異常によって高尿酸血症や低尿酸血症となり、いずれも疾患の原因となる。 Abnormal metabolism and excretion of uric acid in the body cause hyperuricemia and hypouricemia, both of which cause disease.
高尿酸血症は血液中に飽和になって尿酸塩が存在した状態であり、尿酸代謝異常や尿酸の排泄の低下が主な原因である。血清尿酸値が増加する代表的疾患として痛風があるが、癌、白血病、骨髄腫などでも増加する。 Hyperuricemia is a state in which blood is saturated and urate is present, and is mainly caused by abnormal uric acid metabolism and decreased excretion of uric acid. Gout is a typical disease in which the serum uric acid level increases, but it also increases in cancer, leukemia, myeloma and the like.
低尿酸血症の原因としては、尿酸の原料となるプリン体の摂取不足、全身の消耗性疾患による尿酸の産生低下、代謝疾患による尿酸の産生低下や尿酸排泄増加などがあげられる。代謝疾患としてはキサンチンオキシダーゼ欠損症、プリンヌクレオシドホスホリラーゼ欠損症、5-ホスホリボシル-1-ピロリン酸(PRPP)合成酵素欠損症などがある。 Causes of hypouricemia include insufficient intake of purine as a raw material for uric acid, decreased production of uric acid due to systemic debilitating diseases, decreased production of uric acid due to metabolic diseases, and increased uric acid excretion. Metabolic diseases include xanthine oxidase deficiency, purine nucleoside phosphorylase deficiency, and 5-phosphoribosyl-1-pyrophosphate (PRPP) synthase deficiency.
血中および尿中の尿酸を正確に定量することは、これら疾患を診断あるいは予防する目的で重要な意義を持つ。 Accurate quantification of uric acid in blood and urine is important for the purpose of diagnosing or preventing these diseases.
尿酸測定法を原理的に分類すると、アルカリ性下での尿酸の還元性を利用した還元法と、尿酸分解酵素ウリカーゼを用いた酵素的測定法に大別される。 In principle, uric acid measurement methods can be broadly classified into a reduction method using the uric acid reducing property under alkaline conditions and an enzymatic measurement method using uric acid-degrading enzyme uricase.
還元法にはリンタングステン酸除蛋白法やリンタングステン直接法がある。ウリカーゼを用いた酵素的測定法にはウリカーゼ・カタラーゼ法、ウリカーゼ・ペルオキシダーゼ法およびウリカーゼ紫外部法がある。 The reduction method includes a phosphotungstic acid deproteinization method and a phosphotungsten direct method. Enzymatic measurement methods using uricase include uricase catalase method, uricase peroxidase method and uricase UV method.
かつてはリンタングステン酸除蛋白法が主流であったが、現在はウリカーゼ・ペルオキシダーゼ法が主流である。これは、還元法が試料中の非尿酸還元性物質によって大きな正の誤差を生じたり、除蛋白等の前処理が必要となるため、簡便性、迅速性に欠けていたことによる。ウリカーゼ・ペルオキシダーゼ法が自動分析装置への適用に優れ、簡便性、迅速性にも優れていることから、現在、普及してきている。 In the past, the phosphotungstic acid deproteinization method was the mainstream, but now the uricase peroxidase method is the mainstream. This is because the reduction method causes a large positive error due to the non-uric acid reducing substance in the sample or requires pretreatment such as deproteinization, and thus lacks convenience and speed. The uricase peroxidase method is now widely used because it is excellent in application to automatic analyzers and is also simple and quick.
ウリカーゼ・ペルオキシダーゼ法の具体的な原理としては、尿酸はウリカーゼで分解されると、アラントインになる。このときに生じた過酸化水素（H202）をペルオキシダーゼで呈色反応に導き、比色定量して尿酸量を求める。この方法は除蛋白や検体盲検が不要で、短時間に発色が終わる簡便な方法であり、各種の自動分析装置に適用が容易である。As a specific principle of the uricase peroxidase method, uric acid becomes allantoin when it is decomposed by uricase. The hydrogen peroxide (H 2 0 2 ) generated at this time is led to a color reaction with peroxidase and colorimetrically determined to determine the amount of uric acid. This method does not require deproteinization or blind test, and is a simple method that completes color development in a short time, and can be easily applied to various automatic analyzers.
前述の血清尿酸値の低下による疾患を診断するために、尿酸低値を正確に定量することが求められている。 In order to diagnose a disease caused by the aforementioned decrease in serum uric acid level, it is required to accurately quantify the low uric acid level.
ウリカーゼ・ペルオキシダーゼ法により尿酸を測定する試薬として、試薬調製の容易化の面から使用直前に希釈するための濃縮試薬が報告されている（特許文献１参照）。 As a reagent for measuring uric acid by the uricase peroxidase method, a concentrated reagent for dilution immediately before use has been reported from the viewpoint of easy preparation of the reagent (see Patent Document 1).
また、低濃度の物質の測定において、ビリルビンの影響を軽減するための測定法が報告されている（特許文献２〜４参照）。
本発明の目的は、ペルオキシダーゼを用いた尿酸の定量方法において、低値での正確性に優れた定量方法と試薬組成物を提供することであり、また、広い測定域において正確性に優れた尿酸の定量方法と試薬組成物を提供することである。 An object of the present invention is to provide a quantification method and reagent composition excellent in accuracy at a low value in a uric acid quantification method using peroxidase, and uric acid excellent in accuracy in a wide measurement range. And providing a reagent composition.
本願発明者らは、ウリカーゼ・ペルオキシダーゼ法を用いた尿酸の定量において低値の正確性の問題について鋭意研究を行った結果、試薬中のペルオキシダーゼが尿酸と反応することにより尿酸とウリカーゼとの反応を阻害し、そのため低濃度域での尿酸が正確に定量できないことを見出した。 The inventors of the present invention have conducted intensive research on the problem of accuracy of low values in uric acid quantification using the uricase peroxidase method. It was found that uric acid in the low concentration range could not be accurately quantified.
ペルオキシダーゼはキノン色素の生成に関わる試薬組成物であり通常ウリカーゼ酵素反応後に用いられる。通常ウリカーゼ酵素が第２試薬に添加されていることから、試薬全体の安定性や成分バランスより、第１試薬に添加されている場合が多い。本願発明者等が、上記問題点を見出すまでは、従来の試薬組成が有する問題点は見逃されていた。 Peroxidase is a reagent composition involved in the production of quinone dyes and is usually used after uricase enzyme reaction. Since the uricase enzyme is usually added to the second reagent, it is often added to the first reagent due to the stability and component balance of the whole reagent. Until the inventors of the present application found the above problems, the problems of the conventional reagent composition were overlooked.
本願発明者等は、上記問題点に鑑み、尿酸とウリカーゼが反応する前に尿酸とペルオキシダーゼが接触しないように、第１試薬からペルオキシダーゼを除き第２試薬へ移すことで、低濃度域での尿酸を正確に定量できることを見出し、本発明を完成した。 In view of the above problems, the inventors of the present application removed uric acid and peroxidase from the first reagent before removing uric acid and uricase so that uric acid in a low concentration range was transferred to the second reagent by removing peroxidase. Was found to be accurately quantified, and the present invention was completed.
すなわち、本発明は以下の通りである。 That is, the present invention is as follows.
[１] 被検試料中の尿酸をウリカーゼ酵素によって過酸化水素とアラントインに分解し、その過酸化水素をペルオキシダーゼを用いて定量することによって被検試料中の尿酸を定量する方法であって、尿酸のウリカーゼ酵素による分解前に尿酸をペルオキシダーゼと接触させないことにより尿酸低値を正確に定量し得ることを特徴とする尿酸の定量方法、
[２] 被検試料に水素供与化合物を含む第１試薬を添加し、次いでウリカーゼ、４−アミノアンチピリン、およびペルオキシダーゼを含む第２試薬を添加することを特徴とする[１]の尿酸の定量方法、
[３] 第１試薬添加後、1〜１０分後に第２試薬を添加することを特徴とする[１]または[２]の尿酸の定量方法、
[４] 被検試料に４−アミノアンチピリンを含む第１試薬を添加し、次いで水素供与体、ウリカーゼ、およびペルオキシダーゼを含む第２試薬を添加することを特徴とする[１]の尿酸の定量方法、
[５] 被検試料に水素供与化合物を含む第１試薬を添加し、次いで４−アミノアンチピリンを含む第２試薬を添加し、次いでウリカーゼおよびペルオキシダーゼを含む第３試薬を添加することを特徴とする[１]の尿酸の定量方法、
[６] 被検試料に4-アミノアンチピリンを含む第１試薬を添加し、次いで水素供与化合物を含む第２試薬を添加し、次いでウリカーゼおよびペルオキシダーゼを含む第３試薬を添加することを特徴とする[１]の尿酸の定量方法、
[７] 第１試薬にさらにアジ化ナトリウムを含む[２]から[６]のいずれかの尿酸の定量方法、
[８] 水素供与体を含む第１試薬と、ペルオキシダーゼおよびウリカーゼを含む第２試薬を組合せてなる尿酸定量試薬であって、第１試薬にペルオキシダーゼを含まない尿酸定量試薬、ならびに
[９] 第１試薬にさらにアジ化ナトリウムを含む[８]の尿酸定量試薬。[1] A method for quantifying uric acid in a test sample by decomposing uric acid in a test sample into hydrogen peroxide and allantoin using a uricase enzyme and quantifying the hydrogen peroxide using peroxidase. A method for quantifying uric acid, characterized in that low uric acid level can be accurately quantified by not contacting uric acid with peroxidase before degradation by uricase enzyme of
[2] The method for determining uric acid according to [1], wherein a first reagent containing a hydrogen donor compound is added to a test sample, and then a second reagent containing uricase, 4-aminoantipyrine, and peroxidase is added. ,
[3] The method for quantifying uric acid according to [1] or [2], wherein the second reagent is added 1 to 10 minutes after the addition of the first reagent,
[4] The method for quantifying uric acid according to [1], wherein a first reagent containing 4-aminoantipyrine is added to a test sample, and then a second reagent containing a hydrogen donor, uricase, and peroxidase is added. ,
[5] A first reagent containing a hydrogen donor compound is added to a test sample, then a second reagent containing 4-aminoantipyrine is added, and then a third reagent containing uricase and peroxidase is added. [1] uric acid quantitative method,
[6] A first reagent containing 4-aminoantipyrine is added to a test sample, then a second reagent containing a hydrogen donor compound is added, and then a third reagent containing uricase and peroxidase is added. [1] uric acid quantitative method,
[7] The method for quantifying uric acid according to any one of [2] to [6], wherein the first reagent further contains sodium azide,
[8] A uric acid quantitative reagent comprising a first reagent containing a hydrogen donor and a second reagent containing peroxidase and uricase, wherein the first reagent does not contain peroxidase, and
[9] The uric acid quantitative reagent according to [8], wherein the first reagent further contains sodium azide.
本発明の方法によって、低値での正確性に優れた尿酸の定量が可能となる。また、広い測定域において正確に尿酸を定量することができる。 By the method of the present invention, it is possible to quantify uric acid with excellent accuracy at a low value. In addition, uric acid can be accurately quantified in a wide measurement range.
本明細書は本願の優先権の基礎である日本国特許出願2004-270366号の明細書に記載される内容を包含する。 This specification includes the contents described in the specification of Japanese Patent Application No. 2004-270366, which is the basis of the priority of the present application.
本発明の原理は下記式に示される。 The principle of the present invention is shown in the following formula.
また、前記第一反応で生成された過酸化水素が、ペルオキシダーゼおよびロイコ色素の存在下でロイコ色素を酸化し青色色素を生成する反応系においても、本発明の方法は適用できる。臨床検査における自動分析装置では一般に、１つまたは複数の試薬を順に被検試料に所定量投入し、試薬投入一定時間後の一定波長の吸光度や散乱光等を測定し、所定の時間内の変化量や変化の速度等を求めることで、被検試料中の検査対象物質の存在量を定量することができる。 The method of the present invention can also be applied to a reaction system in which the hydrogen peroxide produced in the first reaction oxidizes leuco dye in the presence of peroxidase and leuco dye to produce a blue dye. In an automatic analyzer for clinical examination, generally, a predetermined amount of one or a plurality of reagents are sequentially put into a test sample, and the absorbance or scattered light at a certain wavelength after a certain time after the reagent is charged is measured. By determining the amount, the rate of change, and the like, the amount of the test target substance in the test sample can be quantified.
各試薬は反応順序や反応速度の調整が必要な場合、定量反応に必要な試薬組成物は複数試薬に任意に振り分けて構成されるが、各試薬は数ヶ月から数年の長期保存性を要求されるため、臨床検査に供される前に化学反応が進み、定量反応に必要な試薬組成物が分解され、あるいは定量反応を阻害するような組成物が生成されるような試薬組成物の組み合わせによる試薬構成とすることはできない。 When each reagent needs to be adjusted in reaction sequence and reaction rate, the reagent composition required for quantitative reaction is arbitrarily distributed among multiple reagents. Each reagent requires long-term storage for several months to several years. Therefore, the combination of the reagent composition is such that the chemical reaction proceeds before being subjected to a clinical test, the reagent composition necessary for the quantitative reaction is decomposed, or a composition that inhibits the quantitative reaction is generated. It is not possible to make the reagent composition by.
また、被検試料中に定量反応を阻害する物質または定量反応における生成物と同じ物質が存在する場合は、これらの物質が正の誤差要因となるため、これらの物質の影響を回避するための仕組みを準備しておかなければならない。 In addition, if a substance that inhibits the quantitative reaction or the same product as the product in the quantitative reaction is present in the test sample, these substances cause a positive error, so that the influence of these substances can be avoided. A mechanism must be prepared.
本発明の方法は、尿酸とウリカーゼが反応する前に尿酸とペルオキシダーゼが接触しないように最初に被検試料に添加する試薬からペルオキシダーゼを除いた試薬を用いる尿酸の定量方法である。 The method of the present invention is a method for quantifying uric acid using a reagent obtained by removing peroxidase from the reagent first added to a test sample so that uric acid and peroxidase do not come into contact before uric acid and uricase react.
最初に被検試料に添加する試薬からペルオキシダーゼを除いた場合の弊害として、被検試料中のヘモグロビンによる負の影響がある。鋭意検討の結果、最初に被検試料に添加する試薬にアジ化ナトリウムを加えることで、ペルオキシダーゼの弊害を回避することができる。従って、本発明の方法において、最初に被検試料に添加する試薬にアジ化ナトリウムが含まれていてもよい。 As an adverse effect of removing peroxidase from the reagent initially added to the test sample, there is a negative effect due to hemoglobin in the test sample. As a result of intensive studies, the adverse effects of peroxidase can be avoided by adding sodium azide to the reagent initially added to the test sample. Therefore, in the method of the present invention, sodium azide may be contained in the reagent initially added to the test sample.
本発明の尿酸の定量法に用いる試薬の組成物は以下の要件(a)〜(d)を満たすように選択すればよい。 What is necessary is just to select the composition of the reagent used for the determination method of uric acid of this invention so that the following requirements (a)-(d) may be satisfied.
(a) 尿酸をウリカーゼによる分解の前にペルオキシダーゼと接触させない。 (a) Do not contact uric acid with peroxidase prior to degradation by uricase.
(b) ウリカーゼによって尿酸が分解されたときに生成される過酸化水素は時間の経過と共に分解されるので、ウリカーゼとペルオキシダーゼは同一試薬に含めることが好ましい。 (b) Since hydrogen peroxide produced when uric acid is decomposed by uricase is decomposed over time, uricase and peroxidase are preferably included in the same reagent.
(c) 水素供与化合物と４AAは、保存安定性確保のため同一試薬に含めない。 (c) Hydrogen donor compounds and 4AA are not included in the same reagent to ensure storage stability.
(d) アジ化ナトリウムを最初に被検試料に添加する第１試薬に含むこともできる。 (d) Sodium azide can also be included in the first reagent first added to the test sample.
以下、具体的な試薬の構成例を各試薬の試薬組成物により示し、説明する。本発明において、上記式の反応が起こる反応系に添加する試薬を、添加する順番に第１試薬、第２試薬、第３試薬という。また、第１試薬と被検試料を混合し反応させる工程を第１工程といい、さらに第２試薬を添加し反応させる工程を第２工程という。第３試薬を用いる場合、第３試薬を添加し反応させる工程を第３工程という。 Hereinafter, specific examples of the composition of the reagents will be described with reference to the reagent composition of each reagent. In the present invention, reagents added to a reaction system in which the reaction of the above formula occurs are referred to as a first reagent, a second reagent, and a third reagent in the order of addition. Moreover, the process which mixes and reacts a 1st reagent and a test sample is called 1st process, and also the process of adding a 2nd reagent and making it react is called 2nd process. When the third reagent is used, the step of adding the third reagent and reacting is referred to as the third step.
以下の試薬の構成例において、TOOSの代わりに他の水素供与化合物を用いることができ、また第一試薬にアジ化ナトリウムが含まれていなくてもよく、第２試薬および第３試薬の一方または両方にアジ化ナトリウムが含まれていても良い。ただし、アジ化ナトリウムはペルオキシダーゼの作用を阻害する作用があり、これらを同一試薬に含めておくとペルオキシダーゼの安定性が悪くなるので、ペルオキシダーゼを含む試薬にはアジ化ナトリウムを含まないことが好ましい。ウリカーゼによって尿酸が分解されたときに生成される過酸化水素は時間の経過と共に分解されるので、ウリカーゼとペルオキシダーゼは同一試薬に含めることが好ましいが、ウリカーゼとペルオキシダーゼを同一試薬に含めずに先の試薬でウリカーゼを添加し、後の試薬でペルオキシダーゼを添加することもできる。また、第１試薬、第２試薬および第３試薬のいずれか１つ、いずれか２つまたは３つに界面活性剤が含まれていてもよい。また、第１試薬、第２試薬および第３試薬のいずれか１つ、いずれか２つまたは３つにアスコルビン酸オキシダーゼが含まれても良い。 In the following reagent configuration examples, other hydrogen donor compounds can be used instead of TOOS, and the first reagent may not contain sodium azide, and either one of the second reagent and the third reagent or Both may contain sodium azide. However, sodium azide has an action of inhibiting the action of peroxidase, and if these are included in the same reagent, the stability of peroxidase deteriorates. Therefore, it is preferable that the reagent containing peroxidase does not contain sodium azide. Since hydrogen peroxide produced when uric acid is decomposed by uricase is decomposed over time, it is preferable to include uricase and peroxidase in the same reagent, but without including uricase and peroxidase in the same reagent. Uricase can be added as a reagent, and peroxidase can be added as a later reagent. Further, any one, any two, or three of the first reagent, the second reagent, and the third reagent may contain a surfactant. Further, ascorbate oxidase may be contained in any one, any two, or three of the first reagent, the second reagent, and the third reagent.
（１）
第１試薬：TOOS、アジ化ナトリウム
第２試薬：ウリカーゼ、4-アミノアンチピリン、ペルオキシダーゼ
（２）
第１試薬：4-アミノアンチピリン、アジ化ナトリウム
第２試薬：ウリカーゼ、TOOS、ペルオキシダーゼ
（３）
第１試薬：TOOS、アジ化ナトリウム
第２試薬：4-アミノアンチピリン
第３試薬：ウリカーゼ、ペルオキシダーゼ
（４）
第１試薬：4-アミノアンチピリン、アジ化ナトリウム
第２試薬：TOOS
第３試薬：ウリカーゼ、ペルオキシダーゼ
第１試薬に含まれるTOOSは水素供与化合物である。水素供与体化合物のうちアニリン系水素供与体化合物として、N‐（2‐ヒドロキシ‐3‐スルホプロピル）‐3，5‐ジメトキシアニリン（HDAOS）、N-エチル-N-スルホプロピル-3‐メトキシアニリン（ADPS）、N-エチル-N-スルホプロピルアニリン（ALPS）、N-エチル-N-スルホプロピル-3，5‐ジメトキシアニリン（DAPS）、N‐スルホプロピル‐3，5‐ジメトキシアニリン（HDAPS）、N-エチル-N-スルホプロピル‐3，5‐ジメチルアニリン（MAPS）、N-エチル-N-スルホプロピル-3‐メチルアニリン（TOPS）、N-エチル-N‐（2‐ヒドロキシ‐3‐スルホプロピル）‐3‐メトキシアニリン（ADOS）、N-エチル-N‐（2‐ヒドロキシ‐3‐スルホプロピル）アニリン（ALOS）、N-エチル-N‐（2‐ヒドロキシ‐3‐スルホプロピル）-3，5‐ジメトキシアニリン（DAOS）、N-エチル-N‐（2‐ヒドロキシ‐3‐スルホプロピル）-3，5‐ジメチルアニリン（MAOS）、N-エチル-N‐（2‐ヒドロキシ‐3‐スルホプロピル）‐3‐メチルアニリン（TOOS）およびN‐スルホプロピルアニリン（HALPS）等があり、いずれも本発明の水素供与体として使用可能である。また、フェノール系水素供与化合物の使用も可能である。(1)
First reagent: TOOS, sodium azide Second reagent: uricase, 4-aminoantipyrine, peroxidase (2)
First reagent: 4-aminoantipyrine, sodium azide Second reagent: uricase, TOOS, peroxidase
(3)
First reagent: TOOS, sodium azide Second reagent: 4-aminoantipyrine Third reagent: uricase, peroxidase (4)
First reagent: 4-aminoantipyrine, sodium azide Second reagent: TOOS
Third reagent: uricase, peroxidase TOOS contained in the first reagent is a hydrogen donor compound. Among the hydrogen donor compounds, N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N-ethyl-N-sulfopropyl-3-methoxyaniline are used as aniline-based hydrogen donor compounds. (ADPS), N-ethyl-N-sulfopropylaniline (ALPS), N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline (DAPS), N-sulfopropyl-3,5-dimethoxyaniline (HDAPS) N-ethyl-N-sulfopropyl-3,5-dimethylaniline (MAPS), N-ethyl-N-sulfopropyl-3-methylaniline (TOPS), N-ethyl-N- (2-hydroxy-3- Sulfopropyl) -3-methoxyaniline (ADOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline (ALOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl)- 3,5-dimethoxyaniline (DAOS), N-ethyl-N- (2- Roxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS) and N-sulfopropylaniline (HALPS) Any of these can be used as the hydrogen donor of the present invention. It is also possible to use a phenolic hydrogen donor compound.
第１試薬に含めることができるアジ化ナトリウムは、被検試料中のヘモグロビンによる影響を回避するために添加するものである。第１試薬を検体試料に添加後、ヘモグロビンによる影響を回避するためには、一定時間反応させることが望ましい。 Sodium azide that can be included in the first reagent is added to avoid the influence of hemoglobin in the test sample. In order to avoid the influence of hemoglobin after adding the first reagent to the specimen sample, it is desirable to react for a certain period of time.
本発明の測定方法で用いられる被検試料は、血清、血漿、尿である。自動分析装置（日立製作所製、7150型）の場合、被検試料３〜20μLに対し第１試薬を270μL添加すればよい。用いる被検試料および試薬の量は用いる分析装置により適宜変更することができる。 The test samples used in the measurement method of the present invention are serum, plasma, and urine. In the case of an automatic analyzer (Hitachi, Model 7150), 270 μL of the first reagent may be added to 3 to 20 μL of the test sample. The amount of test sample and reagent to be used can be appropriately changed depending on the analyzer to be used.
第１工程の反応液中のアジ化ナトリウムの濃度は、0.1〜2g/L（0.01％〜0.2％(w/v)）が望ましい。 The concentration of sodium azide in the reaction solution in the first step is preferably 0.1 to 2 g / L (0.01% to 0.2% (w / v)).
第１工程は、ｐH6.0〜ｐH8.0の緩衝液中で行うことが好ましく、緩衝液はリン酸緩衝液やグッド緩衝液が好ましい。特にリン酸緩衝液やグッド緩衝液のN-（2-アセトアミド）-2-アミノエタンスルホン酸（ACES）、N,N−ビス（2-ヒドロキシエチル）-2-アミノエタンスルホン酸（BES）、2-[4-（2-ヒドロキシエチル）-1-ピペラジニル]エタンスルホン酸（HEPES）、3-モルホリノプロパンスルホン酸（MOPS）、2-ヒドロキシ-3-モルホリノプロパンスルホン酸（MOPSO）、ピペラジン-1,4-ビス（2-エタンスルホン酸）（PIPES）、及びN-トリス（ヒドロキシメチル）メチル-2-アミノエタンスルホン酸（TES）が好ましく、緩衝液の濃度は10〜200mM程度が好ましい。また、第１工程には界面活性剤が含まれていることが好ましい。第１工程に用いられる界面活性剤は非イオン系界面活性剤のポリオキシエチレンアルキルエーテル、ポリオキシエチレンアルキルフェニルエーテルが挙げられる。第１工程の反応液中の上記界面活性剤の濃度は、0.1〜10g/L（0.01〜１％（w/v））程度が好ましく、さらに好ましくは0.5〜5g/L（0.05〜0.5％（w/v））程度である。第１工程に用いる緩衝液および界面活性剤は第１試薬に含ませておいてもよいし、被検体を上記緩衝液および／または界面活性剤を含む溶液と混合した後に第１試薬を添加してもよい。 The first step is preferably performed in a pH 6.0 to pH 8.0 buffer, and the buffer is preferably a phosphate buffer or a Good buffer. Especially N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES) of phosphate buffer and Good buffer, 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES), 3-morpholinopropanesulfonic acid (MOPS), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), piperazine-1 , 4-bis (2-ethanesulfonic acid) (PIPES) and N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) are preferable, and the concentration of the buffer is preferably about 10 to 200 mM. Moreover, it is preferable that surfactant is contained in the 1st process. Examples of the surfactant used in the first step include nonionic surfactant polyoxyethylene alkyl ether and polyoxyethylene alkylphenyl ether. The concentration of the surfactant in the reaction solution in the first step is preferably about 0.1 to 10 g / L (0.01 to 1% (w / v)), more preferably 0.5 to 5 g / L (0.05 to 0.5% ( w / v)). The buffer solution and the surfactant used in the first step may be contained in the first reagent, or the first reagent is added after the sample is mixed with the solution containing the buffer solution and / or the surfactant. May be.
試薬構成が２試薬の場合、第１試薬を添加後、一定時間反応させた後、第2試薬を添加して第２工程の反応を行う。この際、反応時間（第１試薬を添加してから、第２試薬を添加するまでの時間）は１分から10分間程度でよく、５分間が好ましい。 When the reagent configuration is two reagents, after the first reagent is added, the reaction is performed for a certain time, and then the second reagent is added to perform the reaction in the second step. At this time, the reaction time (the time from the addition of the first reagent to the addition of the second reagent) may be about 1 to 10 minutes, and preferably 5 minutes.
試薬構成が３試薬の場合、第１試薬を添加後、上記時間反応させた後、第２試薬および第３試薬を順番に添加して、第２工程および第３工程の反応を行えばよい。第２試薬および第３試薬は同時に添加してもよいし、第２試薬添加後1〜10分後に第３試薬を添加してもよい。 When the reagent configuration is three reagents, after the first reagent is added and reacted for the above time, the second reagent and the third reagent are added in order, and the reactions of the second and third steps are performed. The second reagent and the third reagent may be added simultaneously, or the third reagent may be added 1 to 10 minutes after the addition of the second reagent.
第２試薬または第３試薬に含まれるウリカーゼによって上記式の第１反応に示すように、被検試料中の尿酸はウリカーゼの作用で、アラントイン、二酸化炭素および過酸化水素に分解される。 As shown in the first reaction of the above formula by uricase contained in the second reagent or the third reagent, uric acid in the test sample is decomposed into allantoin, carbon dioxide and hydrogen peroxide by the action of uricase.
尿酸の定量は、第１反応で生成した過酸化水素をペルオキシダーゼによって４−アミノアンチピリンとアニリン系水素供与体化合物との酸化縮合反応により、有色キノンに転化し、波長400〜700nmで測定する方法によって行う。 The uric acid is quantified by a method in which hydrogen peroxide generated in the first reaction is converted to colored quinone by peroxidase by oxidation condensation reaction of 4-aminoantipyrine and aniline-based hydrogen donor compound, and measured at a wavelength of 400 to 700 nm. Do.
第２工程および第３工程には界面活性剤が含まれていることが好ましい。第２工程および第３工程に用いられる界面活性剤は非イオン系界面活性剤のポリオキシエチレンアルキルエーテル、ポリオキシエチレンアルキルフェニルエーテルが挙げられる。第２工程および第３工程の反応液中の上記界面活性剤の濃度は、0.1〜10g/L（0.01〜１％（w/v））程度が好ましく、さらに好ましくは0.5〜5g/L（0.05〜0.5％（w/v））程度である。 It is preferable that a surfactant is contained in the second step and the third step. Examples of the surfactant used in the second step and the third step include nonionic surfactant polyoxyethylene alkyl ether and polyoxyethylene alkylphenyl ether. The concentration of the surfactant in the reaction solution in the second and third steps is preferably about 0.1 to 10 g / L (0.01 to 1% (w / v)), more preferably 0.5 to 5 g / L (0.05). ~ 0.5% (w / v)).
第２工程または第３工程の反応液中において過酸化水素をキノン色素に転化する場合のペルオキシダーゼの濃度は0.5〜10IU/mLが好ましく、４-アミノアンチピリンの濃度は0.1〜2.5mmol/Lが好ましく、アニリン系水素供与体化合物等の水素供与化合物の濃度は0.1〜5mmol/Lが好ましい。 The concentration of peroxidase when converting hydrogen peroxide to a quinone dye in the reaction solution of the second step or the third step is preferably 0.5 to 10 IU / mL, and the concentration of 4-aminoantipyrine is preferably 0.1 to 2.5 mmol / L. The concentration of a hydrogen donor compound such as an aniline hydrogen donor compound is preferably 0.1 to 5 mmol / L.
また、ウリカーゼの濃度は50〜500IU/Lが望ましい。第２工程および第３工程のその他の好ましい反応条件は、第１工程における好ましい反応条件と同様である。 The uricase concentration is preferably 50 to 500 IU / L. Other preferable reaction conditions in the second step and the third step are the same as the preferable reaction conditions in the first step.
試薬構成が２試薬の場合は、第２工程終了後、試薬構成が３試薬の場合は、第３工程終了後、生成したキノン色素の量を試料中の550〜650nmにおける吸光度を測定することにより測定する。吸光度の測定は、吸光度計を用いて行えばよい。 When the reagent configuration is 2 reagents, after the second step, and when the reagent configuration is 3 reagents, after the third step, the amount of quinone dye produced is measured by measuring the absorbance at 550 to 650 nm in the sample. taking measurement. Absorbance may be measured using an absorptiometer.
本発明において、「試薬」とは、試薬組成物が含まれているものをいい、「試薬組成物」とは、試薬を構成している酵素や界面活性剤などの物質を言う。 In the present invention, “reagent” refers to a substance containing a reagent composition, and “reagent composition” refers to a substance such as an enzyme or a surfactant constituting the reagent.
本発明の説明においては便宜上、「第1工程」、「第２工程」、「第３工程」や「第１試薬」、「第２試薬」、「第３試薬」等、工程名および試薬名について算用数字１、２、３を用いて説明するが、これは工程や試薬の順番を示すものであり、工程数および試薬数を２または３に限定するものではなく、任意に工程数および試薬数を増やすことができる。 In the description of the present invention, for convenience, the names of steps and reagents such as “first step”, “second step”, “third step”, “first reagent”, “second reagent”, “third reagent”, etc. The number of steps and reagents is not limited to 2 or 3, and the number of steps and the number of steps are arbitrarily determined. The number of reagents can be increased.
以下、本発明の実施例に基づき具体的に説明する。もっとも本発明は下記実施例に限定されるものではない。 The present invention will be specifically described below based on examples of the present invention. However, the present invention is not limited to the following examples.
第１工程および第２工程に用いる試薬組成物（それぞれ、第１試薬組成物および第２試薬組成物）を以下の組成になるように調製し、第１試薬および第２試薬とした。 Reagent compositions used in the first step and the second step (first reagent composition and second reagent composition, respectively) were prepared so as to have the following compositions, and used as the first reagent and the second reagent.
〔実施例１〕
測定には日立製作所製、7150型自動分析装置を使用した。 A Hitachi 7150 type automatic analyzer was used for the measurement.
試料6μLに第１試薬組成物270μLを加え、37℃、5分間加温した後、第２試薬組成物90μLを加え37℃で5分間反応させ、試薬ブランクを対照に主波長600nm、副波長700nmで吸光度を測定し、試料由来の吸光度や電源変動に対する吸光度への影響を補正するために主波長から副波長を差し引いた吸光度を求め、あらかじめ作成した検量線より、試料中の尿酸濃度を求めた。 Add 270 μL of the first reagent composition to 6 μL of the sample, heat at 37 ° C. for 5 minutes, add 90 μL of the second reagent composition, react at 37 ° C. for 5 minutes, and use the reagent blank as a control with a main wavelength of 600 nm and a sub wavelength of 700 nm. In order to correct the absorbance of the sample and the power supply fluctuation, the absorbance obtained by subtracting the sub-wavelength from the main wavelength was determined, and the uric acid concentration in the sample was determined from the calibration curve prepared in advance. .
この際、対照として従来より用いられていた、ペルオキシダーゼを含む第１試薬を用いる方法で試料中の尿酸濃度を求めた。用いた第１試薬および第２試薬の試薬組成物は以下の通りであった。 Under the present circumstances, the uric acid concentration in a sample was calculated | required by the method of using the 1st reagent containing peroxidase conventionally used as a control. The reagent compositions of the first reagent and the second reagent used were as follows.
表1に示すように、本法は従来法に比べ、低値域での正確性に優れている。 As shown in Table 1, this method is superior to the conventional method in accuracy in the low value range.
本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。 All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
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| JP2013081458A (en) * | 2011-09-30 | 2013-05-09 | Shino Test Corp | Stabilization method of measuring reagent |
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| CN110082418B (en) * | 2019-05-27 | 2021-10-15 | 三诺生物传感股份有限公司 | Uric acid electrochemical measurement method |
| JPWO2022172343A1 (en) | 2021-02-10 | 2022-08-18 | ||
| CN114441516A (en) * | 2021-12-20 | 2022-05-06 | 苏州百源基因技术有限公司 | A kind of uric acid detection kit and preparation method thereof |
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| JPH0838195A (en) * | 1994-08-02 | 1996-02-13 | Hitachi Chem Co Ltd | Chemoluminescent determining method |
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| JP2003043035A (en) * | 2001-08-01 | 2003-02-13 | Toyobo Co Ltd | Method for manufacturing liquid-like reagent raw material |
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| JP2794426B2 (en) * | 1988-11-18 | 1998-09-03 | 中外製薬株式会社 | Peroxidase-containing reagent composition and method for preventing peroxidase enzyme activity inhibition |
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| JP2013081458A (en) * | 2011-09-30 | 2013-05-09 | Shino Test Corp | Stabilization method of measuring reagent |
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