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METHOD FOR DETECTING TARGET MOLECULE, AND KIT FOR USE IN SAID METHOD

Foreign code F160008786
File No. (AF19P016)
Posted date Aug 4, 2016
Country WIPO
International application number 2015JP004577
International publication number WO 2016047068
Date of international filing Sep 9, 2015
Date of international publication Mar 31, 2016
Priority data
  • P2014-192311 (Sep 22, 2014) JP
Title METHOD FOR DETECTING TARGET MOLECULE, AND KIT FOR USE IN SAID METHOD
Abstract Provided is a method for detecting a target molecule, which comprises: a complex formation procedure of reacting the target molecule with a carrier which is modified with a first antibody capable of binding to the target molecule specifically and at least two second antibodies which are capable of binding to the target molecule specifically and are respectively labeled with enzymes each having a substrate-cleaving activity and in which the substrate specificities of the enzymes are different from each other, thereby forming a complex composed of the first antibody, the target molecule and the second antibodies all provided on the carrier; and a detection procedure of reacting at least two substrates which respectively have cleavage sites for the enzymes and in each of which a fluorescent substance is bound to one end side of the cleavage site and a color-fading substance is bound to the other end side and in which the fluorescence wavelengths of the fluorescent substances are different from each other with the complex, and then detecting fluorescence emitted from each of the fluorescent substances. It becomes possible to remove noises resulted from the non-specific adsorption of the detection antibodies onto the carrier and to detect a signal coming from the target molecule with high sensitivity and high accuracy.
Scope of claims [claim1]
1.  And the target molecule, A carrier a first antibody which specifically binds to said target molecule is modified, Specifically bind to the target molecule, and a two or more antibodies enzyme labeled with substrate cleavage activity, different second substrate specificity of the enzyme one another antibody, It was reacted, and the complex formation step to form a complex consisting of the first antibody with the target molecules on the support and the second antibody,  Having a cleavage site of the enzyme, the fluorescent substance at one side of the cleavage site is a two or more substrates decoloring material on the other end side is coupled is coupled, substrate fluorescence of different wavelengths of the fluorescent substance When, Said the complex, Are reacted, a detection procedure for detecting the fluorescence emitted from the fluorescent substance,  Method of detecting a target molecule comprising an analysis procedure for processing the detection signals of two or more fluorescent with different fluorescence wavelength as a detection signal of the target molecule, a.
[2]
[claim2]
2.  Wherein during the complex formation step and said detecting step comprises encapsulating procedure one by one enclosing said carrier to each of the droplets formed on the substrate, the detection method of claim 1 wherein.
[3]
[claim3]
3.  Wherein the carrier is a microbead, according to claim 1 or 2 detection method according.
[4]
[claim4]
4.  Wherein the first antibody the second antibody binds to a different epitope of the target molecule, the detection method according to any one of claims 1 to 3.
[5]
[claim5]
5.  A digital ELISA method, detection method according to any one of claims 1-4.
[6]
[claim6]
6.  And the target molecule, A carrier a first antibody which specifically binds to said target molecule is modified, Binds specifically to said target molecule, different from the label have been two or more of the second antibody to each other, It was reacted, and the complex formation step to form a complex consisting of the first antibody with the target molecules on the support and the second antibody,  A detecting procedure for detecting a signal from said label,  Method of detecting a target molecule comprising an analysis procedure for processing the signals from different two or more of said labels as a detection signal of the target molecule, a.
[7]
[claim7]
7.  A carrier first antibody is modified to specifically bind to a target molecule, Binds specifically to said target molecule, an enzyme having a substrate cleavage activity is a labeled two or more antibodies, organic and substrate specificity different from the second one another antibody of the enzyme, the cleavage site of the enzyme and, a two or more substrates to which the fluorescent substance is bonded to the other side to one side of the cleavage site decoloring substance is bound, different from the substrate the fluorescence wavelength of the fluorescent material with each other, The comprising at enzyme-linked immunosorbent assay kit.
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • Inventor
  • 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 DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KN KP KR 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
Reference ( R and D project ) CREST Creation of Nanosystems with Novel Functions through Process Integration AREA
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