JP2002350448A - Quantitative analysis method and reagent for assay of whole protein - Google Patents
Quantitative analysis method and reagent for assay of whole proteinInfo
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- JP2002350448A JP2002350448A JP2001163059A JP2001163059A JP2002350448A JP 2002350448 A JP2002350448 A JP 2002350448A JP 2001163059 A JP2001163059 A JP 2001163059A JP 2001163059 A JP2001163059 A JP 2001163059A JP 2002350448 A JP2002350448 A JP 2002350448A
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- reagent
- absorbance
- biuret
- total protein
- quantification
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、臨床検査の分野で
用いる検体中の干渉物質の影響を受けることがほとんど
無い総蛋白質の定量方法及び定量試薬に関する。TECHNICAL FIELD The present invention relates to a method and a reagent for quantitative determination of total protein which are hardly affected by interfering substances in specimens used in the field of clinical examination.
【０００２】[0002]
【従来の技術】血清及び血漿中の総蛋白質量は、脱水、
網内系疾患、慢性感染症、栄養不良、肝機能障害等の各
種疾患時に変化し、その測定は疾患の診断及び治療上重
要視されている。従来、臨床検査の分野では、血清及び
血漿中の総蛋白質の定量方法として、屈折計法、ケルダ
ール法、ビウレット法等が用いられてきた。2. Description of the Related Art Total protein levels in serum and plasma are determined by dehydration,
It changes during various diseases such as reticuloendothelial disease, chronic infection, malnutrition, and liver dysfunction, and its measurement is regarded as important in the diagnosis and treatment of diseases. Conventionally, in the field of clinical examination, refractometer method, Kjeldahl method, biuret method, etc. have been used as methods for quantifying total protein in serum and plasma.
【０００３】ケルダール法は従来から標準法とされてき
たが、操作が複雑であることや蛋白質の種類により窒素
量から蛋白質量への換算係数においてかなりの差がある
ことから日常検査法としては充分なものではない。ま
た、屈折計法は定量の精度がやや劣り、専用装置による
分析であることから日常検査には適さない。一方、ビウ
レット法は、操作が簡便で自動分析装置への適応が可能
であること、蛋白質の種類に関係なく発色感度が一定で
あり簡単に比色定量できること、測定感度が血清及び血
漿中の総蛋白質量の測定に適していることなどの理由か
ら、日常検査法として広く普及している。[0003] The Kjeldahl method has long been regarded as a standard method, but it is sufficient as a routine examination method because the procedure is complicated and the conversion factor from the amount of nitrogen to the amount of protein varies considerably depending on the type of protein. not something In addition, the refractometer method is slightly inferior in accuracy of quantification, and is not suitable for routine inspection because analysis is performed using a dedicated device. On the other hand, the biuret method is easy to operate and can be applied to automatic analyzers, has a constant color development sensitivity regardless of the type of protein and can be easily colorimetrically determined, and has a high measurement sensitivity for all serum and plasma samples. It is widely used as a routine test method because it is suitable for measuring protein levels.
【０００４】日常検査では、しばしば、乳ビ、溶血、ビ
リルビンを高濃度に含む検体や、血漿増量薬として使用
されるデキストランを含む検体について検査することが
あるが、ビウレット法では、これらの干渉物質により測
定値に誤差を生じる。このような誤差を補正するため
に、ビウレット法において、銅を含まない別の試薬で検
体盲検を取り演算する、デュマス（Doumas）らの方法が
考案されている(Doumaset.al., Clin.Chem. Vol.27、N
o.10、1642-1650 (1981)）。[0004] Routine examinations often include specimens containing high concentrations of chyle, hemolysis, bilirubin, and specimens containing dextran, which is used as a plasma expander. cause an error in the measured value. In order to correct for such errors, a method has been devised by Doumas et al., in which the biuret method is blinded and calculated with a separate copper-free reagent (Doumaset et al., Clin. Chem. Vol.27, N.
o.10, 1642-1650 (1981)).
【０００５】この方法を現在日常検査に使用されている
検体中の総蛋白質を測定するための自動分析装置におい
て実行する場合、自動分析装置では搭載できる試薬の数
が限られているため、１つの測定対象に対して２種類の
試薬を搭載することは、他の様々な検査のために搭載す
べき試薬が搭載できなくなってしまう不都合があり、実
用的ではない。また、常に盲検を取るため、１つの検体
に対して、２つの測定対象を測定するのと同じだけの時
間と試薬が必要となり、検査時間が長く、検査費用が高
くなる。[0005] When this method is carried out in an automatic analyzer for measuring total protein in a specimen currently used for daily examinations, the number of reagents that can be loaded in the automatic analyzer is limited. Loading two types of reagents on the object to be measured is not practical because it is inconvenient that the reagents that should be loaded for various other tests cannot be loaded. In addition, since a blind test is always performed, the same amount of time and reagents as measuring two objects to be measured are required for one sample, resulting in a long test time and a high test cost.
【０００６】現在臨床検査の分野で普及している自動分
析装置は、一般的には１つの測定対象に対し１種類の試
薬を搭載し、その試薬は２つに分割することができる
(第１試薬、第２試薬)。この方法は２試薬系と呼ばれ、
測定に必要な成分を第１試薬と第２試薬に分割して含有
させることにより、試薬の保存安定性の確保や、検体中
に存在する反応妨害物質の影響の解消に効果を与えてい
る。２試薬系では、検体の第１試薬での反応の吸光度測
定と、更に第２試薬を添加した後の吸光度測定を行い、
２つの吸光度差を求め目的物質の定量を行う方法が使用
することができ、盲検を行うのと同様に検体由来の色、
濁りの誤差を緩和することができる。このとき、第１試
薬の反応時間は５分、第２試薬の反応時間も５分である
(合計１０分)。試薬を２つに分割しない場合(１試薬
系)、反応時間は１０分である。[0006] An automatic analyzer, which is currently widely used in the field of clinical examination, is generally loaded with one kind of reagent for one measurement object, and the reagent can be divided into two.
(first reagent, second reagent). This method is called a two-reagent system,
By dividing the components necessary for measurement into the first reagent and the second reagent, the storage stability of the reagents can be ensured and the effects of reaction-interfering substances present in the sample can be eliminated. In the two-reagent system, the absorbance measurement of the reaction of the sample with the first reagent and the absorbance measurement after the addition of the second reagent are performed,
A method of determining the difference between two absorbances and quantifying the target substance can be used, and the color derived from the sample, as well as performing a blind test,
Turbidity errors can be mitigated. At this time, the reaction time of the first reagent is 5 minutes, and the reaction time of the second reagent is also 5 minutes.
(10 minutes total). If the reagents are not split in two (one reagent system), the reaction time is 10 minutes.
【０００７】総蛋白質量測定試薬においてデキストラン
の影響は沈殿物を生じるため、上記の２試薬系による方
法のみでは解消できない。また、検体中のデキストラン
が与える誤差の防止策として、エチレンジアミン四酢酸
ナトリウム(ＥＤＴＡ)に代表されるキレート剤の添加が
効果的であるという報告があり、市販のビウレット法を
用いた総蛋白質量測定試薬では広く使用されているが、
ＥＤＴＡに代表される強いキレート剤を使用すると反応
速度が遅くなり規定の時間内で反応が終了しないため、
２試薬系による方法が使用できず、市販品のほとんどが
１試薬系となっている。そのため、デキストランが与え
る測定値の誤差は解消されているものの、他の妨害物質
が与える測定値の誤差については解消できていない。[0007] Since the effect of dextran on the reagent for measuring the amount of total protein causes precipitates, it cannot be eliminated by the above two-reagent system alone. In addition, it has been reported that the addition of a chelating agent represented by sodium ethylenediaminetetraacetate (EDTA) is effective as a measure to prevent errors caused by dextran in the sample. Widely used in reagents,
If a strong chelating agent such as EDTA is used, the reaction rate will slow down and the reaction will not be completed within the specified time.
Most of the commercially available products are one-reagent systems, as methods using two-reagent systems cannot be used. Therefore, although the errors in the measured values caused by dextran have been eliminated, the errors in the measured values caused by other interfering substances have not been eliminated.
【０００８】さらに、２試薬系の試薬において、第２試
薬に銅イオンが含まれる場合、第２試薬の添加により、
ビリルビンがビリベルジンに変化し( 臨床化学 (２０)
補冊２号 ５１b １９９１)、測定波長に吸光度変化を
与えるため、ビリルビンの与える誤差は解消できない。
それゆえ、ビウレット試薬の成分を２試薬系にするだけ
では、デキストランとビリルビンの与える誤差は解消で
きないとされている。[0008] Furthermore, in a two-reagent system reagent, when the second reagent contains copper ions, addition of the second reagent results in
Bilirubin is converted to biliverdin (Clinical Chemistry (20)
Supplementary Volume 2, 51b, 1991), the error caused by bilirubin cannot be eliminated because the absorbance changes at the measurement wavelength.
Therefore, it is considered that the error caused by dextran and bilirubin cannot be eliminated simply by making the components of the biuret reagent into a two-reagent system.
【０００９】これらの問題に対し、２試薬系で第１試薬
に銅イオンとＥＤＴＡに代表される強いキレート剤を含
有させ、アルカリ溶液には水酸化リチウムを用いること
で、多くの妨害物質の与える誤差を解消できる方法が開
示されている(特開平１０-１９８９８)。この方法では
第１試薬に銅が含まれておりｐＨ１３以上になるとビウ
レット反応を起こしてしまうため、緩衝液を使用してｐ
Ｈを調製している。そのため、同分野で使用されている
カルシウム測定試薬、アルカリフォスファターゼ測定試
薬と同様に、アルカリ性の緩衝液は、開栓後使用中に大
気中の炭酸ガスを吸収してｐＨが低下してしまい試薬の
保存性が低く、測定値が変動し測定精度が悪くなるとい
う問題を抱えている。To solve these problems, the first reagent in the two-reagent system contains copper ions and a strong chelating agent represented by EDTA, and lithium hydroxide is used as the alkaline solution to provide many interfering substances. A method capable of eliminating the error has been disclosed (Japanese Unexamined Patent Application Publication No. 10-19898). In this method, the first reagent contains copper, and if the pH becomes 13 or higher, a biuret reaction will occur.
I am preparing H. Therefore, as with reagents for measuring calcium and alkaline phosphatase, which are used in the same field, alkaline buffers absorb carbon dioxide gas in the atmosphere during use after opening, resulting in a drop in pH. There is a problem that the storage stability is low, the measured value fluctuates, and the measurement accuracy deteriorates.
【００１０】[0010]
【発明が解決しようとする課題】従って、本発明の課題
は、上記問題を解決し、検体中の妨害物質の影響を回避
できる、開栓後保存安定性の優れた総蛋白質の定量方法
及び定量用試薬を提供することにある。SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to solve the above-mentioned problems, and to avoid the influence of interfering substances in the sample, and to provide a method and a method for quantifying total protein that are excellent in storage stability after opening the bottle. The purpose is to provide a reagent for
【００１１】[0011]
【課題を解決するための手段】本発明者らは、上記課題
を解決すべく鋭意研究を進める中で、適当なｐＨ範囲で
デキストラナーゼにより検体中のデキストランを分解で
きること、デキストラナーゼは総蛋白質量の測定値に影
響を及ぼさないことを見出し、本発明を完成するに至っ
た。DISCLOSURE OF THE INVENTION The inventors of the present invention have made intensive studies to solve the above problems, and found that dextran in a sample can be decomposed by dextranase in an appropriate pH range, and that dextranase can The inventors have found that it does not affect the measured protein amount, and have completed the present invention.
【００１２】したがって本発明は、検体中のデキストラ
ンを酵素で分解することを特徴とする、総蛋白質の定量
方法に関する。また本発明は、酵素がデキストラナーゼ
である、前記の方法に関する。さらに本発明は、総蛋白
質の定量が、酵素を含む第１試薬およびアルカリ性の第
２試薬を用いる２試薬系ビウレット反応による定量であ
る、前記の方法に関する。また本発明は、第１試薬がｐ
Ｈ３〜１０である、前記の方法に関する。さらに本発明
は、第２試薬の各成分濃度が、ビウレット反応に必要な
濃度の１倍〜５倍の濃度である、前記の方法に関する。Accordingly, the present invention relates to a method for quantifying total protein, which comprises enzymatically degrading dextran in a specimen. The present invention also relates to the above method, wherein the enzyme is dextranase. Furthermore, the present invention relates to the above method, wherein the quantification of total protein is quantification by a two-reagent biuret reaction using a first reagent containing an enzyme and a second alkaline reagent. Further, in the present invention, the first reagent is p
H3-10. Further, the present invention relates to the above method, wherein the concentration of each component of the second reagent is 1 to 5 times the concentration required for the biuret reaction.
【００１３】また本発明は、デキストラナーゼを含む総
蛋白質の定量のための試薬に関する。さらに本発明は、
総蛋白質の定量のための試薬が、酸化剤を含む第１試薬
およびアルカリ性の第２試薬からなる２試薬系ビウレッ
ト反応用試薬である、前記の試薬に関する。また本発明
は、デキストラナーゼが第１試薬に含まれる、前記の試
薬に関する。さらに本発明は、第１試薬がｐＨ３〜１０
である、前記の試薬に関する。[0013] The present invention also relates to reagents for the quantification of total protein, including dextranase. Furthermore, the present invention
The reagent for quantification of total protein is a two-reagent system biuret reaction reagent consisting of a first reagent containing an oxidizing agent and an alkaline second reagent. The present invention also relates to the above reagent, wherein dextranase is included in the first reagent. Furthermore, according to the present invention, the first reagent has a pH of 3 to 10.
It relates to the reagent described above.
【００１４】本発明のデキストランを分解する酵素は、
ビウレット反応などの総蛋白質の定量方法において、デ
キストラナーゼのようにデキストランが干渉しない程度
に分解し得る酵素であればよく、一般にデキストラナー
ゼと呼ばれるもの（デキストラン加水分解酵素）に限定
されるものではない。これら酵素は、定量方法として２
試薬系ビウレット反応を用いる場合、検体の総蛋白質量
の定量の直前に第１試薬に混ぜてもよく、既に第１試薬
に含まれていてもよい。The dextran-degrading enzyme of the present invention is
Enzymes such as dextranase, which can be degraded to the extent that dextran does not interfere in methods for quantifying total protein such as biuret reaction, are generally limited to those called dextranase (dextran hydrolase). is not. These enzymes are quantified in two ways.
When using the reagent-based biuret reaction, it may be mixed with the first reagent immediately before quantification of the total protein amount of the sample, or may already be contained in the first reagent.
【００１５】本発明に用いる蛋白質の定量方法は、呈色
反応であればよく、本発明の趣旨からデキストランによ
り、該呈色反応が干渉されるような定量方法が含まれ
る。このような定量方法には、２試薬系ビウレット法、
ビウレット法、Lowryらの方法、ビシンコニン酸法など
があげられる。とりわけ自動分析装置に適用可能で、日
常検査法として簡便である２試薬系ビウレット法が好ま
しい。ここでいうところの２試薬系ビウレット法とは、
ビウレット反応を用いて定量する際に、定量用試薬を第
１試薬と第２試薬とに分けて調製し、保存可能な試薬を
用いて行なう方法である。[0015] The method for quantifying the protein used in the present invention may be a color reaction, and from the purport of the present invention, quantification methods in which the color reaction is interfered with by dextran are included. Such quantitative methods include the two-reagent biuret method,
Biuret method, Lowry et al. method, bicinchoninic acid method and the like. In particular, the two-reagent biuret method is preferred because it is applicable to automatic analyzers and convenient as a routine test method. The two-reagent biuret method referred to here is
In this method, the reagents for quantification are prepared separately into a first reagent and a second reagent, and storable reagents are used for quantification using the biuret reaction.
【００１６】本発明の第１試薬には、クエン酸、リン
酸、コハク酸などの緩衝液を使用することができ、デキ
ストランを分解する酵素が酵素活性を阻害せず、本発明
の第２試薬を混合したのちのビウレット反応を阻害しな
い緩衝液であれば、特に限定されない。Buffers such as citric acid, phosphoric acid, and succinic acid can be used for the first reagent of the present invention, and the enzyme that decomposes dextran does not inhibit the enzymatic activity, and the second reagent of the present invention can be used. is not particularly limited as long as it does not inhibit the biuret reaction after mixing.
【００１７】好ましくは、本発明で用いる酵素はデキス
トラナーゼであって、医薬品成分であるデキストラン４
０、デキストラン７０を分解できるもので、総蛋白質量
の測定値に影響を与えないものであればよい。Preferably, the enzyme used in the present invention is dextranase and the pharmaceutical ingredient dextran 4
0, any substance that can decompose dextran 70 and does not affect the measured value of the total protein amount.
【００１８】本発明の第１試薬のｐＨはデキストラナー
ゼなどの酵素が安定であり、且つ、活性発現できる領域
である３〜１０が好ましい。ただし、第２試薬と混合し
た後のｐＨが強アルカリ性を呈すものでなければならな
い。The pH of the first reagent of the present invention is preferably in the range of 3 to 10, in which enzymes such as dextranase are stable and capable of exhibiting activity. However, the pH after mixing with the second reagent must be strongly alkaline.
【００１９】さらに、本発明の第１試薬には、ビリルビ
ンが与える誤差の回避を目的として、ビリルビンをあら
かじめビリベルジンに変換するため、微量の酸化剤を添
加していてもよく、酸化剤としてはフェリシアン化物、
フェロシアン化物、硝酸塩、亜硝酸塩、塩素酸塩、ヨウ
素酸塩などがあげられる。中でも、試薬中での保存安定
性が確保できるとの理由からフェリシアン化カリウムが
好ましい。Furthermore, in order to avoid errors caused by bilirubin, the first reagent of the present invention may contain a small amount of an oxidizing agent for converting bilirubin into biliverdin in advance. phosphide,
ferrocyanides, nitrates, nitrites, chlorates, iodates and the like. Among them, potassium ferricyanide is preferable because the storage stability in the reagent can be ensured.
【００２０】本発明のビウレット反応に必要な成分と
は、硫酸銅、ＥＤＴＡ銅、硝酸銅などの銅（II）イオン
を含む化合物と、水酸化ナトリウム、水酸化カリウム、
水酸化リチウムなどの塩基性化合物である。また、ビウ
レット反応を安定に行なうために、本発明のビウレット
反応に必要な成分に上記以外の成分を添加することも可
能である。The components necessary for the biuret reaction of the present invention are compounds containing copper (II) ions such as copper sulfate, copper EDTA and copper nitrate, sodium hydroxide, potassium hydroxide,
It is a basic compound such as lithium hydroxide. Further, in order to stably carry out the biuret reaction, it is also possible to add components other than those mentioned above to the components necessary for the biuret reaction of the present invention.
【００２１】本発明の第２試薬には、ビウレット反応に
必要な成分が含まれている。該第２試薬に含まれる各成
分濃度は、ビウレット反応に必要な濃度の１〜５倍、好
ましくは１.５〜３倍である。１倍濃度のビウレット試
薬の処方の１例を表１に示す。ここで、表１の処方は代
表的な例であって、本発明で用いられるビウレット試薬
の処方は、この処方に限るわけではない。ビウレット反
応は、強アルカリ水溶液中の蛋白質が銅（II）イオンと
錯体を形成し、赤紫色を呈する呈色反応であることか
ら、銅（II）イオンが溶液中に存在し、強アルカリ性で
あれば、その処方は任意に改変可能である。[0021] The second reagent of the present invention contains components necessary for the biuret reaction. The concentration of each component contained in the second reagent is 1 to 5 times, preferably 1.5 to 3 times the concentration required for the biuret reaction. Table 1 shows an example of a 1×biuret reagent formulation. Here, the formulations in Table 1 are representative examples, and the formulation of the biuret reagent used in the present invention is not limited to this formulation. The biuret reaction is a color reaction in which a protein in a strong alkaline aqueous solution forms a complex with copper(II) ions, resulting in a reddish purple coloration. For example, the formulation can be modified arbitrarily.
【００２２】[0022]
【表１】 [Table 1]
【００２３】本発明において、第１試薬と第２試薬の混
合比は、第２試薬のビウレット試薬の成分濃度に依存
し、両試薬混合後のビウレット試薬濃度の最終濃度が、
１倍濃度に近似することが望ましく、再現性良く測定値
を得るためには、第１試薬と第２試薬の比を１：１に近
づけることが望ましい。In the present invention, the mixing ratio of the first reagent and the second reagent depends on the component concentration of the biuret reagent of the second reagent, and the final concentration of the biuret reagent after mixing both reagents is
It is desirable to approximate a 1-fold concentration, and in order to obtain a measured value with good reproducibility, it is desirable to bring the ratio of the first reagent to the second reagent close to 1:1.
【００２４】[0024]
【実施例】〔実施例１〕表２に示す処方で試薬を調製
し、第１試薬と第２試薬の混合比を５：４として自動分
析装置を用いて反応妨害物質を含む検体の測定を行っ
た。また、デキストラナーゼ無添加の試薬は、表２に示
す処方からデキストラナーゼを除いて調製した。[Example 1] Reagents were prepared according to the formulation shown in Table 2, and the mixture ratio of the first reagent and the second reagent was 5:4. gone. Further, a dextranase-free reagent was prepared from the formulation shown in Table 2 by omitting dextranase.
【００２５】[0025]
【表２】 [Table 2]
【００２６】測定方法は以下に示すとおりである。検体
量 ６μl、第１試薬 ２００μlを混和し、３７℃で５分
間反応後、主波長５４６nm 副波長 ７００nmで吸光度を
測定(１point)し、続いて第２試薬 １６０μl添加し、
３７℃で５分間反応後に再度吸光度を測定(２point)す
る。１pointの測定吸光度の液量補正を行った後、２poi
ntの吸光度から１pointの吸光度を減じ吸光度変化量を
求める。あらかじめ、検体に生理食塩水と既知濃度
（７．０ｇ／ｄｌ）の人血清標準液を用い、上記方法に
より測定を行い検量線を作成する。次に総蛋白質量を求
めたい検体を測定して吸光度変化量を求め、作製した検
量線より総蛋白質量を求める。反応妨害物質を含む検体
は、各妨害物質をそれぞれ表３左欄に記載の１０倍濃度
で調製し、血清９容量に対し１容量の割合で添加して調
製した。各妨害物質を添加した人血清の測定値は表３に
示す。表中の「添加」、「無添加」は、それぞれデキス
トラナーゼの添加、無添加を示す。The measuring method is as follows. 6 μl of the specimen and 200 μl of the first reagent are mixed, reacted at 37° C. for 5 minutes, absorbance is measured (1 point) at a dominant wavelength of 546 nm and a sub-wavelength of 700 nm, then 160 μl of the second reagent is added,
After reacting at 37°C for 5 minutes, absorbance is measured again (2 points). After correcting the liquid volume of 1 point measurement absorbance, 2 poi
Subtract the absorbance of 1 point from the absorbance of nt to obtain the amount of change in absorbance. Physiological saline and a human serum standard solution with a known concentration (7.0 g/dl) are used as specimens in advance, and measurements are made by the above method to create a calibration curve. Next, the sample for which the total protein content is to be determined is measured to determine the amount of change in absorbance, and the total protein content is determined from the prepared calibration curve. Samples containing reaction-interfering substances were prepared by adding each interfering substance at 10 times the concentration shown in the left column of Table 3 and adding 1 volume to 9 volumes of serum. Table 3 shows the measured values of human serum to which each interfering substance was added. "Addition" and "no addition" in the table indicate addition and non-addition of dextranase, respectively.
【００２７】[0027]
【表３】 [Table 3]
【００２８】表３の結果から、デキストラナーゼを無添
加の場合、明らかにデキストランによる測定値への影響
が出ており、実際の総蛋白質量（生理食塩水を添加した
場合）よりも多くの蛋白質が存在するかのような誤差が
生じていることがわかる。したがって、本発明のデキス
トラナーゼを添加した試薬で行なうビウレット反応はデ
キストランによる干渉を受けることなく、正確な総蛋白
質量を定量することを可能としていることが明らかであ
る。[0028] From the results in Table 3, when dextranase was not added, the measurement value was clearly affected by dextran, and the actual amount of protein (when physiological saline was added) was larger than the actual amount. It can be seen that there is an error as if the protein exists. Therefore, it is clear that the biuret reaction performed with the reagent containing the dextranase of the present invention enables accurate quantification of the total protein amount without interference by dextran.
【００２９】〔実施例２〕表２に示した処方の試薬と以
下の表４に示す特開平１０-１９８９８に開示されてい
る処方の試薬を調製し、それぞれの第１試薬、第２試薬
を常時開栓した状態で自動分析装置にセットし、５日間
放置した後、標準液の測定を行い、得られた吸光度変化
量から試薬の安定性を比較した。[Example 2] Reagents having the formulation shown in Table 2 and reagents having the formulation disclosed in JP-A-10-19898 shown in Table 4 below were prepared. It was set in an automatic analyzer with the cap always open, and after being left for 5 days, the standard solution was measured, and the stability of the reagent was compared from the amount of change in absorbance obtained.
【００３０】[0030]
【表４】 [Table 4]
【００３１】表２に示した処方の試薬においては、検体
量 ６μl、第１試薬 ２００μlを混和し、３７℃で５分
間反応後、主波長 ５４６nm 副波長 ７００nmで吸光度
を測定(１point)し、続いて第２試薬 １６０μl添加
し、３７℃で５分間反応後に再度吸光度を測定(２poin
t)する。１pointの吸光度の液量補正を行った後、２poi
ntの吸光度から１pointの吸光度を減じ吸光度変化量を
求めた。表４に示した処方の試薬においては、検体量
５μl、第１試薬 ２００μlを混和し、３７℃で５分間
反応後、主波長 ５４６nm 副波長 ７００nmで吸光度を
測定(１point)し、続いて第２試薬 ５０μl添加し、３
７℃で５分間反応後に再度吸光度を測定(２point)す
る。１pointの吸光度の液量補正を行った後、２pointの
吸光度から１pointの吸光度を減じ吸光度変化量を求め
た。各処方で求めた標準液の吸光度変化量の変動を表５
に示す。In the reagent formulation shown in Table 2, 6 µl of the specimen and 200 µl of the first reagent were mixed and reacted at 37°C for 5 minutes. 160 μl of the second reagent was added using the 2nd reagent, reacted at 37° C. for 5 minutes, and then the absorbance was measured again (2 points
t) do. After performing 1 point absorbance liquid volume correction, 2 poi
The amount of change in absorbance was obtained by subtracting the absorbance at 1 point from the absorbance at nt. For the reagents with the prescriptions shown in Table 4, the amount of sample
5 μl and 200 μl of the first reagent were mixed and reacted at 37° C. for 5 minutes.
After reacting at 7°C for 5 minutes, the absorbance is measured again (2 points). After the 1-point absorbance was corrected for liquid volume, the 1-point absorbance was subtracted from the 2-point absorbance to determine the amount of change in absorbance. Table 5 shows the variation in the amount of change in absorbance of the standard solution obtained for each formulation.
shown in
【００３２】[0032]
【表５】 [Table 5]
【００３３】表５の結果から、特開平１０-１９８９８
に開示されている処方のビウレット試薬では保存期間が
短く、５日間程度の放置によって測定に大きな影響を与
え、実際の自動分析装置などでの使用において、実用的
に耐え得る保存性が無いことがわかる。一方で、本発明
の試薬においては、５日程度の放置によっても、測定値
に大きな影響は無く、充分に実用に耐え得るものである
ことが見出された。[0033] From the results in Table 5,
The storage period of the biuret reagent with the formulation disclosed in is short, and the measurement is greatly affected by leaving it for about 5 days. Recognize. On the other hand, it was found that the reagent of the present invention can be used for practical use without any significant effect on measured values even after being left for about 5 days.
【００３４】〔実施例３〕表２に示した処方の試薬と、
２試薬系ビウレット反応による総蛋白質量の定量用試薬
として用いられるTP試薬・B「コクサイ」（国際試薬株式
会社製）のそれぞれの第１試薬、第２試薬を常時開栓し
た状態で自動分析装置にセットし、１ヵ月以上継続して
測定を行い、得られた吸光度変化量から安定性を比較し
た。検体は人血清を小分け凍結保存したものを用い、使
用時に融解して使用した。表２に示した処方の試薬にお
いては、検体量 ６μl、第１試薬 ２００μlを混和し、
３７℃で５分間反応後、主波長 ５４６nm 副波長 ７０
０nmで吸光度を測定(１point)し、続いて第２試薬 １６
０μl添加し、３７℃で５分間反応後に再度吸光度を測
定(２point)する。１pointの吸光度の液量補正を行った
後、２pointの吸光度から１pointの吸光度を減じ吸光度
変化量を求めた。TP試薬・B「コクサイ」においては、検
体量 ５μl、第１試薬 ２００μlを混和し、３７℃で５
分間反応後、主波長 ５４６nm 副波長 ７００nmで吸光
度を測定(１point)し、続いて第２試薬 ５０μl添加
し、３７℃で５分間反応後に再度吸光度を測定(２poin
t)する。１pointの吸光度の液量補正を行った後、２poi
ntの吸光度から１pointの吸光度を減じ吸光度変化量を
求めた。各試薬の吸光度変化量の変動を図１に示す。[Example 3] A reagent having the formulation shown in Table 2,
TP Reagent B “Kokusai” (manufactured by International Reagent Co., Ltd.), which is used as a reagent for quantification of the total protein amount by the two-reagent biuret reaction, is used in an automatic analyzer with the first and second reagents always open. The measurement was continued for one month or more, and the stability was compared from the amount of change in absorbance obtained. Human serum was subdivided and cryopreserved as specimens, and thawed before use. In the reagent formulation shown in Table 2, 6 μl of the specimen and 200 μl of the first reagent are mixed,
After reacting at 37°C for 5 minutes, the main wavelength is 546 nm and the sub wavelength is 70.
Absorbance was measured at 0 nm (1 point) followed by the second reagent 16
0 μl is added, reacted at 37° C. for 5 minutes, and then the absorbance is measured again (2 points). After the 1-point absorbance was corrected for liquid volume, the 1-point absorbance was subtracted from the 2-point absorbance to determine the amount of change in absorbance. For TP Reagent B “Kokusai” , mix 5 μl of the sample and 200 μl of the first reagent and mix at 37°C for 5 minutes.
After reacting for 1 minute, the absorbance was measured at a dominant wavelength of 546 nm and a sub-wavelength of 700 nm (1 point), then 50 μl of the second reagent was added, and after reacting at 37° C. for 5 minutes, the absorbance was measured again (2 points).
t) do. After performing 1 point absorbance liquid volume correction, 2 poi
The amount of change in absorbance was obtained by subtracting the absorbance at 1 point from the absorbance at nt. FIG. 1 shows the change in the amount of change in absorbance for each reagent.
【００３５】図１から、明らかなように一般的な市販品
の総蛋白質量の定量用試薬が、１０日程度で既に定量の
精度に大きな影響がでるのに対し、本発明の試薬は、３
０日以上経ても試薬の劣化がほとんど無く、良好に定量
が行なえることが示された。As is clear from FIG. 1, the general commercial reagents for quantification of the amount of total protein have a large effect on the accuracy of quantification in about 10 days, whereas the reagent of the present invention has a large effect on the accuracy of quantification in 3 days.
There was almost no deterioration of the reagent even after 0 days or more, indicating that quantification could be carried out satisfactorily.
【００３６】[0036]
【発明の効果】以上のように本発明は、検体中の妨害物
質の影響、特にデキストランの影響を回避でき、開栓後
の保存安定性に優れ、長期間にわたって正確な測定結果
を得ることができる総蛋白質の定量方法及び定量用試薬
を提供することを可能とした。INDUSTRIAL APPLICABILITY As described above, the present invention can avoid the influence of interfering substances in the specimen, especially the influence of dextran, has excellent storage stability after opening, and can obtain accurate measurement results over a long period of time. It is possible to provide a method and a reagent for quantitative determination of total protein that can be obtained.
【図１】 開栓後の測定感度の変動を示したグラフであ
る。FIG. 1 is a graph showing changes in measurement sensitivity after opening.
───────────────────────────────────────────────────── フロントページの続き Ｆターム(参考） 2G045 AA25 BB51 BB60 CA25 CA26 DA36 FB01 FB11 4B063 QA01 QQ67 QR15 QS11 ──────────────────────────────────────────────────── ──── continuation of the front page F term (reference) 2G045 AA25 BB51 BB60 CA25 CA26 DA36 FB01 FB11 4B063 QA01 QQ67 QR15 QS11
Claims (9)
ことを特徴とする、総蛋白質の定量方法。1. A method for quantifying total protein, which comprises enzymatically degrading dextran in a specimen.
１に記載の方法。2. The method of claim 1, wherein the enzyme is dextranase.
およびアルカリ性の第２試薬を用いる２試薬系ビウレッ
ト反応による定量である、請求項１または２に記載の方
法。3. The method according to claim 1, wherein the quantification of total protein is quantification by a two-reagent biuret reaction using a first reagent containing an enzyme and a second alkaline reagent.
３に記載の方法。4. The method of claim 3, wherein the first reagent has a pH of 3-10.
応に必要な濃度の１倍〜５倍の濃度である、請求項３ま
たは４に記載の方法。5. The method according to claim 3, wherein the concentration of each component of the second reagent is 1 to 5 times the concentration required for the biuret reaction.
のための試薬。6. A reagent for the quantification of total protein containing dextranase.
を含む第１試薬およびアルカリ性の第２試薬からなる２
試薬系ビウレット反応用試薬である、請求項６に記載の
試薬。7. The reagent for quantification of total protein consists of a first reagent containing an oxidizing agent and an alkaline second reagent.
7. The reagent according to claim 6, which is a reagent-based biuret reaction reagent.
る、請求項７に記載の試薬。8. The reagent of claim 7, wherein dextranase is included in the first reagent.
７または８に記載の試薬。9. The reagent according to claim 7 or 8, wherein the first reagent has a pH of 3-10.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005024430A1 (en) * | 2003-09-03 | 2005-03-17 | Arkray, Inc. | Method of analyzing protein and protein analysis reagent to be used therein |
| CN105548798A (en) * | 2016-02-29 | 2016-05-04 | 珠海格力电器股份有限公司 | Grounding detection method and device for direct current bus |
| JP2020115112A (en) * | 2019-01-18 | 2020-07-30 | 株式会社シノテスト | Measurement reagent for measuring total protein in sample and stabilization method for the same |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH1019898A (en) * | 1996-07-01 | 1998-01-23 | Internatl Reagents Corp | Determination method and reagent for total protein |
| JP2000512750A (en) * | 1996-06-07 | 2000-09-26 | アボツト・ラボラトリーズ | Methods for improving the performance of immunoreagents in immunoassays |
| JP2001099826A (en) * | 2000-08-25 | 2001-04-13 | Internatl Reagents Corp | Method for developing color of complex, and reagent for developing color |
-
2001
- 2001-05-30 JP JP2001163059A patent/JP4602595B2/en not_active Expired - Lifetime
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000512750A (en) * | 1996-06-07 | 2000-09-26 | アボツト・ラボラトリーズ | Methods for improving the performance of immunoreagents in immunoassays |
| JPH1019898A (en) * | 1996-07-01 | 1998-01-23 | Internatl Reagents Corp | Determination method and reagent for total protein |
| JP2001099826A (en) * | 2000-08-25 | 2001-04-13 | Internatl Reagents Corp | Method for developing color of complex, and reagent for developing color |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005024430A1 (en) * | 2003-09-03 | 2005-03-17 | Arkray, Inc. | Method of analyzing protein and protein analysis reagent to be used therein |
| CN105548798A (en) * | 2016-02-29 | 2016-05-04 | 珠海格力电器股份有限公司 | Grounding detection method and device for direct current bus |
| JP2020115112A (en) * | 2019-01-18 | 2020-07-30 | 株式会社シノテスト | Measurement reagent for measuring total protein in sample and stabilization method for the same |
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| Publication number | Publication date |
|---|---|
| JP4602595B2 (en) | 2010-12-22 |
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