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FUSION PROTEIN AND METHOD FOR DETECTING ANTIGEN USING SAME

Foreign code F170009301
File No. S2016-0199-C0
Posted date Dec 20, 2017
Country WIPO
International application number 2016JP088061
International publication number WO 2017130610
Date of international filing Dec 21, 2016
Date of international publication Aug 3, 2017
Priority data
  • P2016-011475 (Jan 25, 2016) JP
Title FUSION PROTEIN AND METHOD FOR DETECTING ANTIGEN USING SAME
Abstract In order to establish an antigen detection means capable of simply and quickly detecting an antigen and having excellent detection sensitivity, etc., this fusion protein contains an enzyme variant which is activated by multimeric formation, a linker peptide linked to the enzyme variant, and the VH domain or VL domain of an antibody which binds to the linker peptide, and is characterized in that the enzyme variant has an introduced mutation which reduces linking affinity between monomers.
Outline of related art and contending technology BACKGROUND ART
Current, is becoming increasingly important in clinical diagnosis and immunoassay technique that has been. It is the adoption of the individual immunoassay, sensitivity, as well as improve specificity, rapid measurement, also simplified has been an important factor. In the current mainstream immunoassay, protein biomarkers in the detection of the sandwich method, competition in the detection of low molecular weight is used as means of measurement. However both of which, after washing several times the main reaction of the label using enzyme-linked immunosorbent assay measuring the enzymatic activity in many cases, the measurement is time-consuming and trouble is several hours. In contrast, the sample and assay reagents mixed with the reaction, a homogeneous immunoassay is detected, it is possible to measure in can be easily and quickly. As homogeneous immunoassay, fluorescent ellipsometry, method EMIT, CEDIA method, OS-FIA method, and the like are known method Quenchbody, competition in the detection of a low molecular weight comparable to that of the conventional practical sensitivity can be obtained in many cases may also be used. However, in the method using fluorescence detection sensitivity of the fluorescent label and the sensitivity is limited to a difficult problem for improving sensitivity while the signal amplification, the sensitivity by signal amplification in the method using enzymes capable of the enzyme stability becomes a problem in many cases. For example, a deletion mutant of β-galactosidase activity between complementary CEDIA method used, OS-ECIA method (non-patent document 1, 2) in, easy-to-substrate has the advantage that the stability of the enzyme but on the other hand, specific activity, the amount of change of a small amount of the active when a severe problem in practical use.
Scope of claims (In Japanese)[請求項1]
多量体の形成により活性化する酵素の変異体、この酵素の変異体と結合するリンカーペプチド、及びこのリンカーペプチドと結合する抗体のVH領域又はVL領域を含む融合タンパク質であって、前記酵素の変異体が、単量体間の結合親和性を低下させる変異が導入された変異体であることを特徴とする融合タンパク質。
[請求項2]
多量体の形成により活性化する酵素の変異体が、βグルクロニダーゼの変異体であることを特徴とする請求項1に記載の融合タンパク質。
[請求項3]
βグルクロニダーゼの変異体が、大腸菌βグルクロニダーゼのアミノ酸配列における516番目のメチオニン及び517番目のチロシンが他のアミノ酸に置換された変異体であることを特徴とする請求項2に記載の融合タンパク質。
[請求項4]
βグルクロニダーゼの変異体が、大腸菌βグルクロニダーゼのアミノ酸配列における516番目のメチオニンがリジンに置換され、517番目のチロシンがグルタミン酸に置換された変異体であることを特徴とする請求項2に記載の融合タンパク質。
[請求項5]
融合タンパク質中に、1又は2個のβグルクロニダーゼの変異体が含まれることを特徴とする請求項2乃至4のいずれか一項に記載の融合タンパク質。
[請求項6]
請求項1乃至5のいずれか一項に記載の融合タンパク質をコードすることを特徴とする核酸。
[請求項7]
試料中の抗原を検出する方法であって、試料を、抗体のVH領域を含む請求項1乃至5のいずれか一項に記載の融合タンパク質及び抗体のVL領域を含む請求項1乃至5のいずれか一項に記載の融合タンパク質と接触させる工程、並びに多量体の形成を酵素活性の変化により検出する工程を有することを特徴とする抗原の検出方法。
[請求項8]
抗体のVH領域を含む請求項1乃至5のいずれか一項に記載の融合タンパク質及び抗体のVL領域を含む請求項1乃至5のいずれか一項に記載の融合タンパク質を含むことを特徴とする抗原検出キット。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • TOKYO INSTITUTE OF TECHNOLOGY
  • Inventor
  • UEDA HIROSHI
  • Tung Jinhua
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 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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