JPS6193958A - Quantitative determination of endotoxin - Google Patents
Quantitative determination of endotoxinInfo
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- JPS6193958A JPS6193958A JP21492884A JP21492884A JPS6193958A JP S6193958 A JPS6193958 A JP S6193958A JP 21492884 A JP21492884 A JP 21492884A JP 21492884 A JP21492884 A JP 21492884A JP S6193958 A JPS6193958 A JP S6193958A
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- endotoxin
- time
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/579—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving limulus lysate
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- Urology & Nephrology (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】 (ハ)考案の目的 (産業上の利用分野) この発明は、エンドトキシンの定量法に関する。[Detailed description of the invention] (c) Purpose of the invention (Industrial application field) The present invention relates to a method for quantifying endotoxin.
さらに詳しくは発熱性物質(パイロジエン)の原因物質
の一つであるエンドトキシンの定量法に関する。More specifically, the present invention relates to a method for quantifying endotoxin, which is one of the causative substances of pyrogens.
(従来の技術)
注射剤及び医療用具の型造工程等で予期せずして混入す
る発熱性物質(パイロジエン)は、多くの場合エンドト
キシン(リボ多糖類)であると考えられており、臨床面
でもエンドトキシン血症の問題、エンドトキシン汚染を
原因とする注射事故、手術事故の問題も比較的多く、エ
ンドトキシン検出の必要性が増してきている。(Prior art) Pyrogenic substances (pyrogienes) that are unexpectedly mixed in during the molding process of injections and medical devices are thought to be endotoxins (ribopolysaccharides) in most cases, and they are of no clinical importance. However, problems with endotoxemia, injection accidents, and surgical accidents caused by endotoxin contamination are relatively common, and the need for endotoxin detection is increasing.
現在、エンドトキシンの検出法として、リムラステスト
・ゲル化法と発色性合成基質法が使われており、両方法
ともカブトガニの血球成分(す゛ムラス・アメーボサイ
ト・ライセード)を利用したものである。これらの方法
は、カブトガニの血球中に存在する前凝固性酵素(Pr
oclotting Enzyme)をエンドトキシン
が活性化し、活性化酵素(C1ot−ting Enz
yme)とするもので、ゲル化法ではカブトガニ血球中
に存在している凝固性蛋白(Coagu Iogen)
に前記活性化酵素が作用して、3種類のペプチドA、B
、Cを生成する。ペプチドAとBはS−8結合するが、
この際に肉眼で見える“ゲル”を形成するので、これを
指標としてエンドトキシンの陽性・陰性を判定するとい
う半定量的方法である。Currently, the Limulus test gelation method and the chromogenic synthetic substrate method are used to detect endotoxin, and both methods utilize horseshoe crab blood cell components (Limulus amoebocyte lysate). These methods utilize the procoagulant enzyme (Pr) present in horseshoe crab blood cells.
endotoxin activates the activating enzyme (C1ot-ting Enzyme).
yme), and the gelation method uses coagulation proteins (Coagu Iogen) present in horseshoe crab blood cells.
The activation enzyme acts on the three types of peptides A and B.
,C. Peptides A and B have an S-8 bond, but
At this time, a gel that is visible to the naked eye is formed, and this is a semi-quantitative method that uses this as an indicator to determine whether endotoxin is positive or negative.
もう一方の発色性合成基質法は、ゲル化法と原理は同じ
であるが、上記凝固性蛋白に代って発色性の合成基質を
用いる方法である。この合成基質には、種々の物質が検
討されているが、例えば有効な基質部分オリゴペプチド
にバラニトロアニリン(PNA)を結合させたものが現
在市販されている。この基質にエンドトキシンにより活
性化された酵素が作用するとバラニトロアニリンを遊離
して、反応液は黄色となる。遊離パラニトロアニリンと
添加エンドトキシン量は比例するので、黄色を吸光度(
OD 405 nm)で測定してエンドトキシン量を定
量することが出来る。The other chromogenic synthetic substrate method has the same principle as the gelation method, but uses a chromogenic synthetic substrate instead of the coagulant protein. Various substances have been investigated as this synthetic substrate, and for example, one in which valanitroaniline (PNA) is bound to an effective substrate moiety oligopeptide is currently commercially available. When an enzyme activated by endotoxin acts on this substrate, valanitroaniline is released, and the reaction solution turns yellow. Since the amount of free paranitroaniline and added endotoxin are proportional, the yellow color is expressed as absorbance (
The amount of endotoxin can be quantified by measuring at OD 405 nm).
(発明が解決しようとする問題点)
しかしながら、以上2つの検出法において、ゲル化法は
簡便で検出感度が高いが、定量性に欠けるという問題点
があり、発色性合成基質法ではエンドトキシンの定量が
可能であり、検出感度も高いが、手間がかかりかつダイ
ナミックレンジが狭いという問題点があった。(Problems to be Solved by the Invention) However, among the above two detection methods, the gelation method is simple and has high detection sensitivity, but there is a problem in that it lacks quantitative performance, and the chromogenic synthetic substrate method is used to quantify endotoxin. Although this method is possible and has high detection sensitivity, it is time-consuming and has a narrow dynamic range.
この発明は、上記問題点を解決すべくなされたものであ
り、簡便で検出感度が高くかつダイナミックレンジの広
いエンドトキシンの定量法を提供しようとするものであ
る。The present invention was made to solve the above-mentioned problems, and aims to provide a method for quantifying endotoxin that is simple, has high detection sensitivity, and has a wide dynamic range.
本発明者らは、鋭意研究の結果、まず前記ゲル化法にお
けるゲル成長過程の状態が、ゲル層の光散乱光量−を電
気的に検出して得られる電気信号の変化から簡便に知り
うろことを見出した。そしてさらに研究の結果、この光
散乱光量の変化には第1図に示すごとき一定のパターン
が認められる点を見出し、これに基づいてエンドトキシ
ンの定量を行なう点に想着し、この発明に到達した。As a result of intensive research, the present inventors have found that the state of the gel growth process in the gelation method can be easily known from the change in the electrical signal obtained by electrically detecting the amount of light scattered by the gel layer. I found out. As a result of further research, they discovered that the change in the amount of scattered light had a certain pattern as shown in Figure 1. Based on this, they came up with the idea of quantifying endotoxin, and arrived at this invention. .
(ロ)発明の構成
かくしてこの発明によれば、検体にリムラス・アメーボ
サイト・ライセート試薬を反応させてゲル化を起こさせ
、その光散乱光量を測定してゲル開始時間を求め、既知
濃度のエンドトキシンのゲル開始時間と比較して、検体
中のエンドトキシン濃度を定量することを特徴とするエ
ンドトキシンの定量法が提供される。(B) Structure of the Invention Thus, according to the present invention, a sample is reacted with a Limulus amoebosite lysate reagent to cause gelation, the amount of light scattered is measured to determine the gel initiation time, and endotoxin of a known concentration is detected. A method for quantifying endotoxin is provided, the method comprising quantifying the endotoxin concentration in a sample by comparing the gel initiation time.
この発明における「ゲル開始時間」とは、ゲル化反応に
おける光散乱光量の変化を示す第1図のごときグラフに
おける、テスト開始点(A;リムラス・アメーボサイト
・ライセート試薬添加時)から横軸に平行に引いた外挿
線と、ゲル化開始点(B)とゲル化終了点(D)の間の
最大傾斜線との交点(C)を設定した際の(A)−(C
)間の時間を意味する。In this invention, "gel start time" refers to a graph parallel to the horizontal axis from the test start point (A; when Limulus amoebosite lysate reagent is added) in a graph such as that shown in Figure 1 showing changes in the amount of light scattered during the gelation reaction. (A) - (C) when setting the intersection (C) between the extrapolation line drawn on the line and the maximum slope line between the gelation start point (B) and the gelation end point (D).
) means the time between.
この発明の最も特徴とする点は、上記で定義される「ゲ
ル開始時間」が、検体中のエンドトキシン濃度に最も相
関関係があることを見出した点にある。The most distinctive feature of this invention is that it has been found that the "gel start time" defined above has the highest correlation with the endotoxin concentration in the specimen.
このようにして設定したゲル開始時間を、まず種々の既
知濃度のエンドトキシンについて求め、それを指標とし
、それと未知b=度のエンドトキシンを含む検体につい
て求めたケル開始時間とを比較すれば、検体中の未知濃
度のエンドトキシン濃度を定量することができる。If the gel start time set in this way is first determined for various known concentrations of endotoxin, and then used as an index, and compared with the gel start time determined for a sample containing endotoxin at an unknown concentration of b, The endotoxin concentration of unknown concentrations can be quantified.
この発明に用いるリムラス・アメーボサイト・ライセー
ト試薬としては、従来のエンドトキシン検出用のリムラ
ス・アメーボサイト・ライセートをエンドトキシンフリ
ーの蒸留水に熔解したちのをそのまま用いることができ
る。またこの発明の対象とする検体は、液状のものはそ
のまま又は希釈した被検液として反応に供せられ、液状
以外のものは、適宜エンドトキシンフリーの蒸留水に溶
解又は抽出した被検液として反応に供せられる。As the Limulus amoebosite lysate reagent used in this invention, a Limulus amoebosite lysate for conventional endotoxin detection can be dissolved in endotoxin-free distilled water and used as it is. In addition, the specimens targeted by this invention are subjected to the reaction as they are or as a diluted test solution, and those in a non-liquid state are reacted as a test solution dissolved or extracted in endotoxin-free distilled water as appropriate. It is offered to
かかる検体又は被検液とリムラス・アメーボサイト・ラ
イセート試薬の反応は、エンドトキシンフリーのガラス
製反応容器中、これらを混合することにより行なわれる
。通常、37℃前後ゐ温度下で放置して反応させるのが
適しており、また混合液のpHは6〜8となるように設
定するのが適している。The reaction between the specimen or test solution and Limulus amebocyte lysate reagent is carried out by mixing them in an endotoxin-free glass reaction vessel. Generally, it is suitable to leave the mixture to react at a temperature of around 37°C, and it is suitable to set the pH of the mixed solution to 6 to 8.
この発明における光散乱光量の変化は、適当な光散乱光
量測定装置を用いて測定でき、この装置は専用機を用い
てもよく、また汎用フォトメーターを用いてその場で構
成してもよい。ただし、少なくとも前記反応容器をその
まま測定セルとして測光位置に設定でき、かつ測定部が
37℃前後に恒温化され更に光散乱光量を連続的に出力
できるよう構成されていることを要する。更に反応容器
としでは、試薬量ができるだけ少なくてすむようになる
べ(歩容量のものが好ましい。このような観点から、最
も好ましい光散乱光量測定装置として光散乱光検知式の
いわゆる血液凝固分析装置を用いることができ、その例
としては、コアグ・スタット(国際試薬株式会社製)が
挙げられる。Changes in the amount of scattered light in the present invention can be measured using an appropriate amount of light scattered light measurement device, and this device may be a dedicated device or may be constructed on the spot using a general-purpose photometer. However, it is required that at least the reaction vessel can be set as a measurement cell at a photometry position, and that the measurement section is kept at a constant temperature of around 37° C., and that the amount of scattered light can be continuously output. Furthermore, the amount of reagent used as a reaction container should be as small as possible (preferably one with walking capacity).From this point of view, the most preferable light scattering light amount measuring device is a light scattering light detection type so-called blood coagulation analyzer. An example thereof is Coag Stat (manufactured by Kokusai Reagent Co., Ltd.).
なお、かような光lt&乱光量測定装置に、マイクロプ
ロセッサを用いたゲル開始時間自動演算器及びエンドト
キシン濃度換算器とリンクすれば、自動測定が可能とな
る。かような自動演算は、出力変化の最大変化率を求め
る比較アルゴリズムと計算式とから容易にプログラム設
定することができ、濃度換算は検量線からのファクター
計算及びデータ入力により容易にプログラム設定するこ
とができる。Note that automatic measurement becomes possible by linking such a light lt & scattered light amount measuring device with an automatic gel start time calculator and an endotoxin concentration converter using a microprocessor. Such automatic calculations can be easily programmed using a comparison algorithm and calculation formula for determining the maximum rate of change in output, and concentration conversion can be easily programmed using factor calculations from calibration curves and data input. I can do it.
また第5図に示した様に、総ゲル生成量は反応液中の凝
固性蛋白濃度に比例することも見出した。Furthermore, as shown in FIG. 5, it has been found that the total amount of gel produced is proportional to the coagulant protein concentration in the reaction solution.
このことから1回の試薬に要する試薬の量を減らすこと
も可能であり、コスト的にも有効な方法であることがオ
フかる。From this, it is possible to reduce the amount of reagent required for one reagent, and it can be seen that this is a cost-effective method.
(実施例)
以下、実施例で本発明を詳説するが、これによってこの
発明は限定されるものではない。(Examples) Hereinafter, the present invention will be explained in detail using Examples, but the present invention is not limited thereby.
実施例1
(エンドトキシン濃度とゲル開始時間との相関)ゲル開
始時間とエンドトキシン濃度との相関関係を求めるため
に、エンドトキシンに標準リポポリサッカライド(E、
coli UKT−B)を使用して各エンドトキシン濃
度におけるゲル開始時間を測定した。Example 1 (Correlation between endotoxin concentration and gel initiation time) In order to determine the correlation between gel initiation time and endotoxin concentration, standard lipopolysaccharide (E,
The gel initiation time at each endotoxin concentration was measured using E.coli UKT-B).
まず、リムラス・アメーボサイト・ライセート(カブト
ガニ血球抽出物凍結乾燥品:和光純薬玉業KKM)にエ
ンドトキシンフリーの水5 mll’c無菌的に加え静
かに振り混ぜ完全に溶解し、リムラス・アメーボサイト
・ライセート試薬とした。First, add 5 ml of endotoxin-free water to Limulus amoebosite lysate (Lyophilized Limulus hemocyte extract: Wako Pure Chemical Industries KKM) under aseptic conditions, shake gently to dissolve completely, and prepare Limulus amebocyte lysate reagent. And so.
次いで光散乱光量測定装置(コアグスタット)用試験管
を250℃、2時間で乾燥熱滅菌しておき、該試験管に
上記試薬を0.2mj!注入し、その中へ各既知濃度の
エンドトキシン含有液0.21T11をそ
1れぞれ添加し、光散乱光量測定装置(コアグ・スタッ
ト:国際試薬株式会社製)にて、ゲル化時の光1i((
乱光量の変化を測定し、記録針に出力し、出力されたデ
ータをもとにしてゲル開始時間を求めた。実験は2回行
ない、その結果を第1表に示した。Next, a test tube for a light scattering light amount measuring device (Coagstat) was sterilized by dry heat at 250°C for 2 hours, and 0.2 mj! of the above reagent was added to the test tube. Inject 0.21T11 of each endotoxin-containing solution into it.
1, respectively, and measured the light 1i (((
Changes in the amount of scattered light were measured and output to a recording needle, and the gel start time was determined based on the output data. The experiment was conducted twice and the results are shown in Table 1.
第1表 この結果をもとにして第2図に示す検量線を作成した。Table 1 Based on this result, a calibration curve shown in FIG. 2 was created.
縦軸は対数目盛で表示したゲル開始時間(分)、横軸は
同じく対数目盛で表示したエンドトキシン濃度(ng/
m/)である。なお、グラフの○は第1回目を、△は第
2回目の実験結果を示す。The vertical axis is the gel start time (min) expressed on a logarithmic scale, and the horizontal axis is the endotoxin concentration (ng/min) also expressed on a logarithmic scale.
m/). Note that ◯ in the graph indicates the first experimental result, and △ indicates the second experimental result.
また、第1表中第1回目の測定データの中でエンドトキ
シン濃度が100.1.0.02ng/ mllのもの
の散乱光量とゲル開始時間との関係を求めた記録計の干
ヤードを例として第3図(a)、(bl及び(C)に示
した(図中、縦軸は散乱光量を電圧出力mVで表示した
もの、横軸は時間を示す)。In addition, in the first measurement data in Table 1, the relationship between the amount of scattered light and the gel start time when the endotoxin concentration was 100.1. 3 (a), (bl and (C)) (in the figures, the vertical axis represents the amount of scattered light expressed in voltage output mV, and the horizontal axis represents time).
実施例2
エンドトキシン既知濃度の液の代わりに姫路市の上水道
水を用いた以外は実施例1と同じ方法で測定し、第4図
のようなチャートが得られた。このチャートを基にして
、実施例1で述べた手法でゲル開始時間を求めたところ
、8.4分という値が得られた。第2図の検量線により
この値から、この水道水のエンドトキシン濃度は4.4
ng/m6ということがわかった。これは、従来法によ
る経験的な数値と同程度のものであった。Example 2 Measurement was carried out in the same manner as in Example 1, except that tap water from Himeji City was used instead of a solution with a known concentration of endotoxin, and a chart as shown in FIG. 4 was obtained. Based on this chart, gel initiation time was determined using the method described in Example 1, and a value of 8.4 minutes was obtained. Based on this value according to the calibration curve in Figure 2, the endotoxin concentration in this tap water is 4.4.
It was found that ng/m6. This value was comparable to the empirical value obtained using the conventional method.
なお、コアグスタットでの光散乱光量測定に液量が0.
3m1以上必要であるために、今回は試液0.2mj!
、サンプル液0.2m6としたが、試液0.1mff、
サンプル液0.2mj!でもよく、また装置が変われば
それらの量を任怠に選ふことが可能である。Note that when measuring the amount of light scattering with Coagstat, the liquid volume is 0.
Since more than 3ml is required, this time we used 0.2mj of test solution!
, the sample liquid was 0.2 m6, but the test liquid was 0.1 mff,
Sample liquid 0.2mj! However, if the equipment changes, it is possible to arbitrarily choose these amounts.
このようにして未知濃度のエンドトキシンを含む投法の
ゲル開始時間を、前記で作成した検量線と比較すること
により、検体のエンドトキシン濃度を容易に求めること
ができる。In this way, by comparing the gel initiation time of a dosage containing an unknown concentration of endotoxin with the calibration curve prepared above, the endotoxin concentration of the sample can be easily determined.
(ハ)発明の効果
この発明の方法によれば、簡便で検出感度の高いゲル化
法では困難であったエンドトキシン濃度の定量を正確・
簡単に行うことができ、また発色性合成基質法に比べて
ダイナミックレンジが広い等の優れたものとなる。(c) Effects of the invention According to the method of the invention, it is possible to accurately quantify endotoxin concentration, which is difficult to do with gelation methods, which are simple and have high detection sensitivity.
It is easy to carry out, and has advantages such as a wider dynamic range than the chromogenic synthetic substrate method.
また、マイクロプロセッサを用いた自動化も容易である
という効果も備えている。It also has the advantage of being easy to automate using a microprocessor.
第1図は、散乱光量と時間との関係からゲル開始時間を
求めるためのグラフ、第2図は、ゲル開始時間とエンド
トキシン濃度との相関関係を求めた検量線、第3図(a
)、fb)及び(C)は、本発明の実施例1の第1図相
当図、第4図は実施例2の第1図相当図、第5図は、凝
固性蛋白濃度とゲル生成量との関係を示すグラフである
。
、¥二
代理人 弁理士 野 河 信 太 部改・
第4図
吊木(4,4ng/ml )
チャートスピード゛5mm/min
第5図Figure 1 is a graph for determining the gel start time from the relationship between the amount of scattered light and time, Figure 2 is a calibration curve for determining the correlation between gel start time and endotoxin concentration, and Figure 3 (a)
), fb) and (C) are diagrams corresponding to FIG. 1 of Example 1 of the present invention, FIG. 4 is a diagram equivalent to FIG. 1 of Example 2, and FIG. It is a graph showing the relationship between , ¥2 Agent Patent Attorney Shin Nogawa Kai Abe Figure 4 Hanging tree (4.4 ng/ml) Chart speed ゛5mm/min Figure 5
Claims (1)
を反応させてゲル化を起こさせ、その光散乱光量を測定
してゲル開始時間を求め、既知濃度のエンドトキシンの
ゲル開始時間と比較して、検体中のエンドトキシン濃度
を定量することを特徴とするエンドトキシンの定量法。1. React the Limulus Amoebosite Lysate reagent with the sample to cause gelation, measure the amount of light scattering to determine the gel initiation time, and compare it with the gel initiation time of endotoxin at a known concentration to determine the gelation time in the sample. A method for quantifying endotoxin, characterized by quantifying endotoxin concentration.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP21492884A JPS6193958A (en) | 1984-10-13 | 1984-10-13 | Quantitative determination of endotoxin |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP21492884A JPS6193958A (en) | 1984-10-13 | 1984-10-13 | Quantitative determination of endotoxin |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS6193958A true JPS6193958A (en) | 1986-05-12 |
JPH0414310B2 JPH0414310B2 (en) | 1992-03-12 |
Family
ID=16663893
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP21492884A Granted JPS6193958A (en) | 1984-10-13 | 1984-10-13 | Quantitative determination of endotoxin |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS6193958A (en) |
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1984
- 1984-10-13 JP JP21492884A patent/JPS6193958A/en active Granted
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