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METHOD FOR DETECTING ENZYMATIC REACTION PRODUCT

Foreign code F200010031
File No. IP14P005
Posted date Jan 31, 2020
Country WIPO
International application number 2019JP008411
International publication number WO 2019168200
Date of international filing Mar 4, 2019
Date of international publication Sep 6, 2019
Priority data
  • P2018-037085 (Mar 2, 2018) JP
Title METHOD FOR DETECTING ENZYMATIC REACTION PRODUCT
Abstract As a technique applicable to highly sensitive detection of a pathogenic microorganism such as influenza virus, provided is a method which comprises reacting an enzyme with a substance, said substance serving as a substrate in a reaction by the enzyme, in a hydrophilic solvent being in interfacial contact with a hydrophobic solvent and then detecting the reaction product formed and released directly from the substrate, wherein the hydrophilic solvent contains a buffering substance and/or trimethylamine oxide (TMAO).
Outline of related art and contending technology BACKGROUND ART
In recent years, simple immunoaffinity chromatography using a test kit is influenza virus has been developed (see Patent Document 1). The method of using immunoaffinity chromatography, from a few minutes to several tens of minutes the influenza virus can be detected, such as diagnosis and treatment of infections are utilized.
In addition, the prior art, the influenza virus and neuraminidase is an enzyme chromogenic substrate and optically based on the reaction of the detection of an influenza virus has been known a technique (Patent Document 2, reference 3). Colored substrates include, for example, 4-α-D - methylumbellerferyl (4-Methylumbelliferyl-N-acetyl- α-D-neuraminic acid neuramatic acid: 4MU-NANA, see Patent Document 2) or, 4-or 4,7-alkoxy - N-N - dialkoxy-acetylneuraminic acid (see Patent Document 3) and derivatives thereof-acetylneuraminic acid and the like is used. For example, as a method using a chromogenic substrate is 4MU-NANA, neuraminidase 4MU-NANA by the decomposition of the fluorescent substance by 4-to generate a methylumbelliferone. The generated 4-based on the amount of enzymatic activity of the neuraminidase methylumbelliferone value can be calculated, based on the enzymatic activity of the influenza virus further particle number can be quantified.
Associated with the present invention, the non-patent document 1, and covered with the oil droplets, the droplets from the outside of the femtoliter order accessible using the array of droplets, a method of an enzyme molecule has been described.
Scope of claims (In Japanese)[請求項1]
 疎水性溶媒と界面接触する親水性溶媒中において、酵素と、該酵素による反応の基質となる物質とを反応させ、該基質から直接に生成し遊離する反応生成物を検出する方法であって、
前記親水性溶媒が、緩衝物質又はトリメチルアミンオキシド(TMAO)の少なくともいずれか一方を含む、方法。

[請求項2]
 前記親水性溶媒中の緩衝物質及びトリメチルアミンオキシドの濃度が、1M以上である、請求項1記載の方法。

[請求項3]
 前記緩衝物質が、DEA(Diethanolamine)又はHEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)である、請求項1又は2記載の方法。

[請求項4]
 前記親水性溶媒が、前記反応生成物の酸解離定数(pKa)よりも大きいpH値を有する、請求項1~3のいずれか一項に記載の方法。

[請求項5]
 前記親水性溶媒が病原性微生物を含み、
前記酵素が前記病原性微生物の表面又は内部に存在する基質切断活性を有する酵素であり、
前記物質が発色基質であり、
前記酵素による前記発色基質の切断により生成する反応生成物を光学的に検出する、請求項1~4記載の方法。

[請求項6]
 前記病原性微生物がインフルエンザウイルスであり、
前記酵素がノイラミニダーゼであり、
前記発色基質と前記反応生成物が4-メチルウンベリフェリル-α-D-ノイラミン酸(4-Methylumbelliferyl-N-acetyl-α-D-neuraminic acid)と4-メチルウンベリフェロンであるか、フルオレセイン(Fluorescein)を含む誘導体とフルオレセインである、請求項5記載の方法。

[請求項7]
 前記親水性溶媒が、前記病原性微生物に感染した対象又は感染した疑いがある対象から分離された生物試料を含む、請求項5又は6記載の方法。

[請求項8]
 疎水性溶媒と界面接触する親水性溶媒中において、酵素と、該酵素による反応の基質となる物質とを反応させ、該基質から直接に生成し遊離する反応生成物を検出する方法であって、
前記親水性溶媒に、緩衝物質又はトリメチルアミンオキシド(TMAO)の少なくともいずれか一方を添加することより、前記反応生成物の前記親水性溶媒から前記疎水性溶媒への移動を抑制する手順を含む、方法。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • Inventor
  • TABATA Kazuhito
  • MINAGAWA Yoshihiro
  • NOJI Hiroyuki
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JO JP KE KG KH KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
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