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METHOD AND KIT FOR MEASUREMENT OF ENDOTOXIN LEVEL meetings

Foreign code F110002700
File No. S2008-0093-C0
Posted date Apr 8, 2011
Country WIPO
International application number 2008JP070450
International publication number WO 2009063840
Date of international filing Nov 11, 2008
Date of international publication May 22, 2009
Priority data
  • P2007-293491 (Nov 12, 2007) JP
Title METHOD AND KIT FOR MEASUREMENT OF ENDOTOXIN LEVEL meetings
Abstract Disclosed is a method which can measure an endotoxin contained at such a level that has not been detected by conventional endotoxin measurement methods, in a simple manner and rapidly without the need of using any specialized measurement apparatus. The method comprises the steps of: reacting a sample to be measured, a reagent comprising factor C that can be activated upon being bound to an endotoxin, and a luminescent synthetic substrate composed of a peptide and a luminescent substrate bound to the peptide, thereby releasing a luminescent substrate from the luminescent synthetic substrate; reacting a luminescent enzyme with the released luminescent substrate; measuring the amount of an emitted light; and quantifying the level of the endotoxin in the sample based on the measurement value obtained in the previous step.
Outline of related art and contending technology BACKGROUND ART
'Endotoxin (endotoxin: endotoxin) ' is, the outer membrane of gram-negative bacteria is one of the components constituting the, LPS(lipopolysaccharide the body of its activity: lipopolysaccharide) is. The presence of endotoxin within the living organism, the surface of gram-negative bacteria present as part of the outer membrane. In addition, gram-negative bacilli are generally of the postmortem, as free of endotoxin free blood flow is present.
In the blood and endotoxin in the presence of a predetermined amount or more, the monocytes and granulocytes such as stimulation of the endotoxin in the excessive inflammatory cytokines are produced. As a result, heat generation is referred to as endotoxin disease, sepsis, septic shock, multiple organ dysfunction or the like caused the condition. Therefore, the injectable pharmaceutical or the like very significant and the detection of endotoxin, the endotoxin test method of the United States Pharmacopeia BUTT are listed. In addition, in the clinical diagnosis, the endotoxin in blood is accurately measured, the early diagnosis and therapeutic effect is considered to be critical.
As a method for measurement of endotoxin in the prior art, the body of the rabbit as direct injection of the sample to be examined, the temperature increase from the amount of endotoxin (pyrogen test) tests such as pyrogen, limulus hemocyte extract (amebocyte lysate) of endotoxin gel by the application of the phenomenon that the limulus test has been known. Of these rabbits injected directly into the method, cost, time to obtain the results, and the sensitivity is a problem, a method of measuring endotoxin limulus test is now the mainstream.
Fig. 1 by endotoxin limulus blood cell extract in the course of the reaction of the gel. Limulus hemocyte extract that specifically reacts with the endotoxin in the presence of the C factor pathway. 'C factor pathway' is, configured by the following reaction cascade. First, in the pellet, and the C factor (Factor C) tightly bound to the activated factor C. Then, activated by the binding of endotoxin (activated Factor C) is a factor C B activating factor (Factor B). Subsequently, the activated B-factor by (the active form B factor), activated clotting enzyme more previous (proclotting enzyme) (clotting enzyme) to generate a clotting enzyme. The coagulation enzyme, its substrate koagyurogen water-decomposition part (coagulogen). As a result, the peptide from the C koagyurogen (peptide C) coagulation protein is liberated is generated coagulin. Due to the coagulation effect of the coagulin gel by (see non-patent document 1).
Endotoxin by limulus test measurement method is used, the above-mentioned limulus blood cell extracts into a gel by endotoxin is an application of the above processes. A limulus test, or measurement methods is determined from a difference in the gelling of falling down of the method (gelation method), chromogenic synthetic substrate (colorimetric method), and turbidimetric time analysis method (nephelometry) or the like has been known (see Non-Patent Document 2).
'Gelling fall method' is, in the sample to be examined in vitro in the limulus blood cell extract mixture, under certain conditions (for example for 37 °C at 30-60) after reaction, tipping or the tube is tipped, or a state of a liquid sample, or a method for determining whether a solidified. In the case of the former negative endotoxin, endotoxin is positive and in the latter case. The method, without the need for special equipment but is also relatively easy operation, the raw material and lot have a variable point and the measurement result, an optical method for determination of a person than is poor in an objective usually be not only used in a simplified manner.
'Chromogenic synthetic substrate method' is, substrates of the clotting enzyme chromogenic synthetic peptide substrates is used, the amount of the chromophore liberated by absorbance by the colorimetric method to calculate the amount of endotoxin. The substrate is a chromogenic synthetic peptide, a natural substrate for clotting enzyme koagyurogen water-decomposition of the amino acid sequence of the two-site are used. Paranitroaniline (pNA) by the clotting enzyme cleavage site such as the coupling of the chromophore, the chromophore can be liberated by enzymatic cleavage is colored by. Paranitroaniline is in the case of the chromophore, the maximum absorption wavelength 405 nm paranitroaniline the absorbance is measured over time. In addition, in the case of a diazo paranitroaniline is the chromophore, the absorbance at 545 nm is measured over time. Then, the temporal change in the amount obtained by analyzing the concentration of endotoxin. Is the coloring synthetic substrate method, the reagent is relatively expensive and the operation is complicated and since the problem of such, the quantitativity, sensitivity, and excellent in terms of the objective.
'Turbidimetric time analysis method' is, the increase in turbidity due to gel as a variation in the amount of transmitted light, the transmission light amount ratio of the reaction solution to a fixed threshold value (typically around 90%) to decrease to a time required for the gel time, gel time and the endotoxin concentration is created from the relationship between the endotoxin value is calculated using a standard curve method. Quantitativity and an objective is very good, and the measurement of a dedicated apparatus.
In addition, recombinant C using a fluorescence agent, can be measured with high sensitivity an endotoxin reagent kit (trade name: pairojin rFc, manufacturing: Lonza Walkersville, Inc. sales: (strain) of the first chemical) is commercially available.
T. Miyata, M. Hiranaga et al., Amino Acid Sequence of the Coagulogen from Limulus polyphemus Hemocytes, The Journal of Biological Chemistry, 259, 8924-8933 (1984)
Fifteenth revised Japanese Pharmacopoeia, the endotoxin test method 4.01, P70-73
Scope of claims (In Japanese)請求の範囲 [1]
 試料中に含まれるエンドトキシンの濃度測定方法であって、
 試料と、エンドトキシンとの結合により活性化されるC因子を含有する試薬と、ペプチドに発光基質が結合してなる発光合成基質とを反応させ、該発光合成基質から前記発光基質を遊離させる発光基質遊離工程と、
 前記発光基質遊離工程により遊離した発光基質に発光酵素を作用させ、発光量を測定する発光量測定工程と、
 前記発光量測定工程により得られた測定値に基づいて前記試料中のエンドトキシン濃度を定量する濃度定量工程と
を包含することを特徴とするエンドトキシンの濃度測定方法。

[2]
 前記発光合成基質は、前記C因子が活性化されることにより生成される活性型C因子、活性型B因子および凝固酵素のいずれか1種の作用により、前記発光基質と前記ペプチドとの結合が切断される構造を有することを特徴とする請求項1に記載のエンドトキシンの濃度測定方法。

[3]
 前記発光基質はアミノルシフェリンであり、前記発光酵素はルシフェラーゼであることを特徴とする請求項2に記載のエンドトキシンの濃度測定方法。

[4]
 前記ルシフェラーゼが、北米ホタル、ゲンジボタル、ヘイケボタル、ツチボタル、ヒメボタル、マドボタル、オバボタル、光コメツキムシおよび鉄道虫からなる群より選択される甲虫由来であることを特徴とする請求項3に記載のエンドトキシンの濃度測定方法。

[5]
 前記ルシフェラーゼは変異型ルシフェラーゼであり、該変異型ルシフェラーゼは野生型ルシフェラーゼと比較して発光強度が増大するように、野生型ルシフェラーゼのアミノ酸配列が改変されていることを特徴とする請求項3または4に記載のエンドトキシンの濃度測定方法。

[6]
 前記変異型ルシフェラーゼが、以下の(i)~(v)からなる群より選択された1種であることを特徴とする請求項5に記載のエンドトキシンの濃度測定方法。
(i) 配列番号13に示される野生型北米ホタルルシフェラーゼのアミノ酸配列において、423位のイソロイシンがロイシンに置換され、436位のアスパラギン酸がグリシンに置換されたアミノ酸配列からなる変異型ホタルルシフェラーゼ
(ii) 配列番号13に示される北米ホタルルシフェラーゼのアミノ酸配列において、423位のイソロイシンがロイシンに置換され、530位のロイシンがアルギニンに置換されたアミノ酸配列からなる変異型ホタルルシフェラーゼ
(iii) 配列番号13に示される北米ホタルルシフェラーゼのアミノ酸配列において、436位のアスパラギン酸がグリシンに置換され、530位のロイシンがアルギニンに置換されたアミノ酸配列からなる変異型ホタルルシフェラーゼ
(iv) 配列番号13に示される北米ホタルルシフェラーゼのアミノ酸配列において、423位のイソロイシンがロイシンに置換され、436位のアスパラギン酸がグリシンに置換され、530位のロイシンがアルギニンに置換されたアミノ酸配列からなる変異型ホタルルシフェラーゼ
(v) 配列番号13に示される北米ホタルルシフェラーゼのアミノ酸配列において、47位のイソロイシンがスレオニンに、50位のアスパラギンがセリンに、59位のメチオニンがスレオニンに、252位のスレオニンがセリンに、423位のイソロイシンがロイシンに、530位のロイシンがアルギニンにそれぞれ置換されたアミノ酸配列からなる変異型ホタルルシフェラーゼ

[7]
 前記C因子を含有する試薬がカブトガニ血球抽出成分であり、反応系の全量に対する該カブトガニ血球抽出成分のタンパク質濃度が1.5mg/mL~3.5mg/mLであることを特徴とする請求項1~6のいずれか1項に記載のエンドトキシンの濃度測定方法。

[8]
 エンドトキシンと特異的に結合して活性化されるC因子を含有する試薬と、ペプチドに発光基質が結合してなる発光合成基質と、発光酵素とを構成成分として含むことを特徴とするエンドトキシンの濃度測定用キット。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • HIROSHIMA UNIVERSITY
  • Inventor
  • KURODA, Akio
  • NODA, Kenichi
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PG PH PL PT RO RS RU SC SD SE SG SK SL SM ST SV SY TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ MD RU TJ TM
EPO: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MT NL NO PL PT RO SE SI SK TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

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