JPH0549276B2 - - Google Patents
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- Publication number
- JPH0549276B2 JPH0549276B2 JP59242797A JP24279784A JPH0549276B2 JP H0549276 B2 JPH0549276 B2 JP H0549276B2 JP 59242797 A JP59242797 A JP 59242797A JP 24279784 A JP24279784 A JP 24279784A JP H0549276 B2 JPH0549276 B2 JP H0549276B2
- Authority
- JP
- Japan
- Prior art keywords
- reagent
- lysozyme
- activity
- measuring
- present
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明はリゾチーム活性測定用試薬に関するも
のである。体液中にリゾチーム活性の測定は、急
性単球性白血病や非典型的結核症の診断及び腎疾
患のスクリーニングに有用な情報を与えるものと
して臨床的意義が高い。DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a reagent for measuring lysozyme activity. Measuring lysozyme activity in body fluids has high clinical significance as it provides useful information for diagnosing acute monocytic leukemia and atypical tuberculosis and screening for renal disease.
（従来の技術）
従来、リゾチーム活性は生菌体、例えばミクロ
コツカス・リゾデイクテイカス（Micrococcus
lysodeiktcus）等を基質として、その溶菌速度に
より活性を求めていたが、定量性に乏しく感度が
低く、測定時間も半日以上と長く、また基質が生
菌体であるから調製毎のバラツキが大きく、その
結果として測定値のバラツキが大きなものとなつ
ており、実際には臨床診断法として実用化される
ことがなかつた。(Prior art) Lysozyme activity has conventionally been measured in living bacterial cells, such as Micrococcus lysodeichteicus.
lysodeiktcus) as a substrate, and its bacteriolysis rate was used to determine the activity, but it was not quantitative and had low sensitivity, the measurement time was long (more than half a day), and since the substrate was live bacterial cells, there was a large variation between preparations. As a result, the variation in measured values was large, and it was never put to practical use as a clinical diagnostic method.
一方、最近Ｎ−アセチルグルコサミンのオリゴ
マーにアグリコンとして発色物質、例えばｐ−ニ
トロフエノールを結合した化合物を基質とするリ
ゾチームの測定法が提案されている（特開昭55−
38504号公報）。この方法は上記生菌体を用いる方
法に比べて定量性に優れるが、リゾチームの作用
PH（約6.0付近）と加水分解されて生じるｐ−ニ
トロフエノールが発色するPH（8.5以上）とが異
なるため、酸素反応と発色反応を同じ条件で行う
ことができないために、試薬数および操作ステツ
プが多く、酸素活性を求める場合に一番適当であ
るといわれている速度分析（レートアツセイ）が
出来ない欠点がある。 On the other hand, a method for measuring lysozyme has recently been proposed using as a substrate a compound in which a color-forming substance, such as p-nitrophenol, is bound as an aglycone to an oligomer of N-acetylglucosamine (Japanese Patent Laid-Open No. 1983-1983-1).
Publication No. 38504). This method has superior quantitative performance compared to the above method using live bacteria, but the effect of lysozyme
Since the pH (around 6.0) is different from the pH (8.5 or higher) at which the p-nitrophenol produced by hydrolysis develops color, the oxygen reaction and the color reaction cannot be performed under the same conditions, so the number of reagents and operating steps are different. However, it has the disadvantage that it cannot perform rate analysis, which is said to be the most appropriate method for determining oxygen activity.
（発明の解決しようとする問題点）
本発明の目的は、定量性に優れたリゾチームの
レートアツセイが可能となるリゾチーム活性測定
試薬を提供することである。(Problems to be Solved by the Invention) An object of the present invention is to provide a reagent for measuring lysozyme activity that enables rate assay of lysozyme with excellent quantitative properties.
（問題点を解決するための手段）
本発明者らは、上記目的を達成するために、
種々鋭意検討したところ、基質としてキトペンタ
オースを用い、Ｎ−アセチルヘキソサミンオキシ
ダーゼを共役酵素として用いることにより、体液
中のリゾチーム活性を短時間に正確簡単にレート
アツセイ出来ることを見出し、本発明に到達し
た。すなわち本発明はキトペンタオースおよびＮ
−アセチルヘキサソミンオキシダーゼを含有する
ことを特徴とするリゾチーム活性測定用試薬であ
る。(Means for solving the problem) In order to achieve the above object, the present inventors
After extensive research, we discovered that by using chitopentaose as a substrate and N-acetylhexosamine oxidase as a coupled enzyme, it is possible to accurately and easily rate assay lysozyme activity in body fluids in a short time, and have arrived at the present invention. . That is, the present invention provides chitopentaose and N
- A reagent for measuring lysozyme activity characterized by containing acetyl hexasomine oxidase.
本発明に使用するキトペンタオースとは、下記
一般式で示される化合物である。 Chitopentaose used in the present invention is a compound represented by the following general formula.
本発明に用いるＮ−アセチルヘキサミンオキシ
ダーゼ（以下NAHODと略記する）は、Ｎ−ア
セチルグルコサミンを基質として過酸化水素を生
成するものであればいかなる起源のものでもよい
が、例えばシユードモナス（Pseudomonas）属
に属する微生物が産生するNAHODがある。
NAHODの精製ならびに性質については日本農
芸化学会 昭和58年度大会において発表されてい
る。 The N-acetylhexamine oxidase (hereinafter abbreviated as NAHOD) used in the present invention may be of any origin as long as it produces hydrogen peroxide using N-acetylglucosamine as a substrate. There is a NAHOD produced by the microorganisms that belong to this group.
The purification and properties of NAHOD were presented at the 1988 Annual Meeting of the Japanese Society of Agricultural Chemistry.
本発明のリゾチーム活性測定試薬は上記基質、
酸素を含有する。該試薬はPH4.0〜9.0、好ましく
は5.0〜7.0を示す緩衝液、例えばリン酸緩衝液、
あるいは有機酸緩衝液として、クエン酸、フタル
酸緩衝液やグツド緩衝液を含むことが望ましい。
基質濃度としては特に制限はないが、好ましくは
最大のリゾチームの酸素活性を示す濃度が適当で
あり、例えば0.05M以上である。次に共役酵素で
あるNAHODの量としては特に制限はないが、
好ましくは最大リゾチーム活性を示す濃度、つま
りリゾチーム酸素反応を100％追随出来る量以上
添加することが適当であり、例えば0.05U／ml以
上である。本発明の試薬にはATPが１ｍＭ以上
含まれることが好ましい。 The reagent for measuring lysozyme activity of the present invention has the above-mentioned substrate,
Contains oxygen. The reagent is a buffer having a pH of 4.0 to 9.0, preferably 5.0 to 7.0, such as a phosphate buffer,
Alternatively, it is desirable to include a citric acid buffer, a phthalate buffer, or a phthalic acid buffer as the organic acid buffer.
Although there are no particular limitations on the substrate concentration, it is preferably a concentration that exhibits the maximum oxygen activity of lysozyme, for example, 0.05M or more. Next, there is no particular limit to the amount of NAHOD, which is a coupling enzyme, but
Preferably, it is appropriate to add at least a concentration that exhibits the maximum lysozyme activity, that is, an amount that can follow 100% of the lysozyme oxygen reaction, for example, 0.05 U/ml or more. The reagent of the present invention preferably contains 1 mM or more of ATP.
本発明のリゾチーム活性測定試薬を用いてリゾ
チーム活性を測定する方法としては、試料を該試
薬と反応させて、生成する過酸化水素を直接過酸
化水素電極で測定するか、あるいはペルオキシダ
ーゼあるいはカタラーゼ等の酸素を用いて過酸化
水素を発色系に導いて比色定量するか、あるいは
NAHOD作用時に消費される酸素を酸素電極等
で直接測定する方法がある。 Methods for measuring lysozyme activity using the reagent for measuring lysozyme activity of the present invention include reacting a sample with the reagent and measuring the generated hydrogen peroxide directly with a hydrogen peroxide electrode, or using peroxidase, catalase, etc. Either oxygen is used to introduce hydrogen peroxide into a color system for colorimetric determination, or
There is a method of directly measuring the oxygen consumed during NAHOD action using an oxygen electrode, etc.
（作用）
本発明のリゾチーム活性測定試薬において、基
質としてキトペンタオースを用いることにより、
体液中のリゾチーム活性を短時間に正確簡単にレ
ートアツセイすることができる。(Action) In the reagent for measuring lysozyme activity of the present invention, by using chitopentaose as a substrate,
Lysozyme activity in body fluids can be accurately and easily rate assayed in a short time.
（実施例） 以下、本発明を実施例により詳細に説明する。(Example) Hereinafter, the present invention will be explained in detail with reference to Examples.
実施例 １
被検液中のリゾチーム活性量を下記試薬を用い
て、下記方法により測定した。Example 1 The amount of lysozyme activity in the test solution was measured using the following reagent and the following method.
１ 試薬
試薬R1：
NAHOD ２単位
ペルオキシダーゼ 100単位
発色剤（TOOS） ２mg
４−アミノアンチピリン 0.5mg
ATP 1.0ｍMole
0.1Mクエン酸リン酸緩衝液（PH6.0）
全量10ml
TOOS：
Ｎ−エチル−Ｎ−（２−ヒドロキシ−３−ス
ルホプロピル）−ｍ−トルイジン−ナトリウム
試薬R2：
キトペンタオース ５ｍＭ
0.1Mクエン酸−リン酸緩衝液（PH6.0）
２ 測定方法
被検液又は既知濃度のリゾチームを含有する
サンプル50μに、試薬R12mlおよび試薬
R2500μを加えて37℃で反応させ、その吸光
度を波長550nｍで測定して発色速度を求め、
既知濃度との発色速度の比較により、被検体中
リゾチーム活性を求めた。1 Reagent Reagent R 1 : NAHOD 2 units Peroxidase 100 units Color forming agent (TOOS) 2 mg 4-aminoantipyrine 0.5 mg ATP 1.0mMole 0.1M citrate phosphate buffer (PH6.0)
Total volume 10ml TOOS: N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine-sodium reagent R 2 : Chitopentaose 5mM 0.1M citric acid-phosphate buffer (PH6.0) 2 Measurement Method Add 2 ml of Reagent R 1 and the reagent to 50μ of the test solution or sample containing lysozyme at a known concentration.
Add 500μ of R 2 and react at 37℃, measure the absorbance at a wavelength of 550nm to determine the color development rate,
The lysozyme activity in the sample was determined by comparing the color development rate with known concentrations.
反応曲線を第１図に示し、検量線を第２図に示
す。 The reaction curve is shown in FIG. 1, and the calibration curve is shown in FIG.
実施例 ２
実施例１の試薬を用い、キトペンタオースとキ
トヘキサオースの比較を示したのが、第３図であ
る。図から明らかなように約1.6倍キトペンタオ
ースを基準として用いた方が活性高く示す。Example 2 FIG. 3 shows a comparison of chitopentaose and chitohexaose using the reagent of Example 1. As is clear from the figure, the activity is approximately 1.6 times higher when chitopentaose is used as a standard.
第１図は実施例１における反応曲線を示し、第
２図は実施例１における検量線を示す。第３図は
実施例２における検量線を示す。
FIG. 1 shows the reaction curve in Example 1, and FIG. 2 shows the calibration curve in Example 1. FIG. 3 shows a calibration curve in Example 2.
Claims (1)
サミンオキシダーゼを含有することを特徴とする
リゾチーム活性測定用試薬。1. A reagent for measuring lysozyme activity characterized by containing chitopentaose and N-acetylhexosamine oxidase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP24279784A JPS61124394A (en) | 1984-11-16 | 1984-11-16 | Reagent for determination of lysozyme activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP24279784A JPS61124394A (en) | 1984-11-16 | 1984-11-16 | Reagent for determination of lysozyme activity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS61124394A JPS61124394A (en) | 1986-06-12 |
| JPH0549276B2 true JPH0549276B2 (en) | 1993-07-23 |
Family
ID=17094429
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP24279784A Granted JPS61124394A (en) | 1984-11-16 | 1984-11-16 | Reagent for determination of lysozyme activity |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS61124394A (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59156299A (en) * | 1983-02-28 | 1984-09-05 | Noda Sangyo Kagaku Kenkyusho | Determination of n-acetylhexosamine and reagent for determining it |
| JPS59156281A (en) * | 1983-02-28 | 1984-09-05 | Noda Sangyo Kagaku Kenkyusho | N-acetylhexosamine oxidase and its preparation |
-
1984
- 1984-11-16 JP JP24279784A patent/JPS61124394A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS61124394A (en) | 1986-06-12 |
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