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METHOD FOR DETECTING PROTEIN PRESENT IN MEMBRANE OF EXOSOME

Foreign code F180009380
File No. (S2016-1032-N0)
Posted date Apr 19, 2018
Country WIPO
International application number 2017JP029064
International publication number WO 2018030511
Date of international filing Aug 10, 2017
Date of international publication Feb 15, 2018
Priority data
  • P2016-158985 (Aug 12, 2016) JP
Title METHOD FOR DETECTING PROTEIN PRESENT IN MEMBRANE OF EXOSOME
Abstract The present invention provides a method for detecting proteins present in membranes of exosomes in a biological sample, wherein the method includes a step for bringing the exosomes isolated from said biological sample into contact with antibodies for said proteins, in a dispersed state in a bovine-serum albumin solution, thus detecting complexes formed of said proteins and said antibodies.
Outline of related art and contending technology BACKGROUND ART
Exosomes is, a diameter of about 40nm - to about 150nm secreted from cells and vesicles, proteins, mRNA, miRNA molecules such as impregnated therein. Tumor cells by tumor exosomes secreted from the cell type for containing the different molecules, the molecules of the derived exosomes has attracted attention as a biomarker. In recent years, exosomes derived from cancer cells in serum tumor marker is used as the diagnosis of cancer have been reported (Non-Patent Document 1 and 2). However, present on exosomes and the measurement of the protein, CD9, CD63, CD81 are not limited to a molecule such as, cell function and is related to a family of membrane proteins such as integrin CEA, even though the number of solutions exist, their protein exosomes on the film is not a report on the measurement.
Current, is in the blood of carcinoembryonic antigen (carcinoembryonic antigen,CEA,CEACAM 5), and general clinical tumor markers, such as colon cancer recurrence are used to diagnose. A molecular weight of about 20 million of the CEA and glycoprotein, fetal tissue and human colon cancer is found in the intestinal tract, also, a plurality of proteins having a structure similar to that of the CEA was also found. These are, CEA family, a new unified classification name is determined, thereby one of the 2 family of CEA, CEACAM(CEA-related cell adhesion molecule) PSG(pregnancy-specific glycoprotein) classified and resulted in. CEA CEACAM CEACAM 1,CEACAM 3,CEACAM 4,CEACAM 6,CEACAM 7 and CEACAM 8 with belonging to the group is. However, the recurrence of cancer CEA is used as a diagnostic marker, markers for early diagnosis of cancer and is not used as a report. Then, other CEA CEA family member is used as a tumor marker is not reported.
Also, in general, is used as a tumor marker proteins such as CEA in the diagnosis of cancer, false positive or false-negative rate is found in, for a false positive does not need to be checked or, for a false negative or delayed the discovery of cancer recurrence, unnecessary to create a burden on a patient. Therefore, a method of detecting the protein with high accuracy is demanded.
Scope of claims (In Japanese)[請求項1]
生体試料中のエクソソームの膜に存在するタンパク質の検出方法であって、
ウシ血清アルブミン溶液中に分散させた、該生体試料から単離したエクソソームと、該タンパク質に対する抗体とを接触させ、該タンパク質と該抗体との複合体を検出する工程を含む、方法。
[請求項2]
前記ウシ血清アルブミン溶液の濃度が0.5%(w/v)~5%(w/v)である、請求項1に記載の方法。
[請求項3]
前記ウシ血清アルブミン溶液の濃度が1%(w/v)である、請求項2に記載の方法。
[請求項4]
前記ウシ血清アルブミン溶液中に分散させた、該生体試料から単離したエクソソームの濃度が、前記生体試料中のエクソソームの濃度の1.5倍~10倍である、請求項1~3のいずれか1項に記載の方法。
[請求項5]
前記ウシ血清アルブミン溶液中に分散させた、該生体試料から単離したエクソソームの濃度が、前記生体試料中のエクソソームの濃度の2.5倍~5倍である、請求項4に記載の方法。
[請求項6]
生体試料が凍結された試料である場合、当該試料を4℃~60℃で融解する工程をさらに含む、請求項1~5のいずれか1項に記載の方法。
[請求項7]
融解する工程を4℃~37℃で行う、請求項6に記載の方法。
[請求項8]
エクソソームの膜に存在するタンパク質が糖タンパク質である、請求項1~7のいずれか1項に記載の方法。
[請求項9]
糖タンパク質がCEAファミリーメンバーである、請求項8に記載の方法。
[請求項10]
CEAファミリーメンバーが、CEA、CEACAM 1、及びCEACAM 6からなる群から選ばれる1以上である、請求項9に記載の方法。
[請求項11]
生体試料が体液である、請求項1~10のいずれか1項に記載の方法。
[請求項12]
体液が血清又は血漿である、請求項11に記載の方法。
[請求項13]
癌の遠隔転移を検出するための、請求項1~12のいずれか1項に記載の方法。
[請求項14]
癌が大腸癌、胃癌、及び食道癌からなる群から選ばれる1以上である、請求項13に記載の方法。
[請求項15]
癌が大腸癌である、請求項14に記載の方法。
[請求項16]
癌の遠隔転移の診断のための検査方法であって、
(1)請求項1に記載の方法により、被験者に由来する生体試料中のエクソソームの膜に存在するタンパク質を検出し、該タンパク質の濃度を測定すること、
(2)(1)により得られたタンパク質の濃度と基準値とを比較することを含み、
被験者におけるタンパク質の濃度が基準値よりも高いことが癌の遠隔転移の指標となる、方法。
[請求項17]
基準値が、遠隔転移の有無が既知の患者集団におけるROC曲線に基づいて設定されるカットオフポイントである、請求項16に記載の方法。
[請求項18]
癌が大腸癌、胃癌、及び食道癌からなる群から選ばれる1以上である、請求項16又は17に記載の方法。
[請求項19]
癌が大腸癌である、請求項18に記載の方法。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • WAKAYAMA MEDICAL UNIVERSITY
  • Inventor
  • YOKOYAMA, Shozo
  • YAMAUE, Hiroki
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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