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KIT FOR DETECTING OR MEASURING ANTIGEN

Foreign code F190009774
File No. S2016-1193-C0
Posted date May 7, 2019
Country WIPO
International application number 2018JP001186
International publication number WO 2018147018
Date of international filing Jan 17, 2018
Date of international publication Aug 16, 2018
Priority data
  • P2017-021164 (Feb 8, 2017) JP
Title KIT FOR DETECTING OR MEASURING ANTIGEN
Abstract The purpose of the present invention is to develop a kit capable of detecting or measuring an antigen at high sensitivity without requiring an immobilization step or a washing step. The present invention provides a kit for detecting or measuring an antigen, the kit comprising: a conjugate composed of a polypeptide including an antibody light chain variable region and a polypeptide including an antibody heavy chain variable region; and a fragment of protein M marked with a fluorescent pigment, wherein the fragment of protein M is a fragment having the ability to bond with the conjugate.
Outline of related art and contending technology BACKGROUND ART
Current, are becoming increasingly important in clinical diagnosis and immune measuring method is a measurement technique has been. Is adopted to avoid individual immunoassay, the sensitivity, as well as improve specificity, rapid measurement, and also simplification has been an important factor. Immunoassays in the current mainstream, a sandwich method for detecting protein biomarkers, the conflict can be detected by a low molecular weight used as a measurement principle of the method. However both of which, after the reaction and washing several times in the main measuring the enzymatic activity using a label enzyme immunoassay method in many cases, few hours of labor and the measurement is a problem that it takes. In contrast, the measurement sample by mixing a reaction reagent, homogeneous immunoassay detection have been developed.
The present inventors have, in recent years, in such a homogeneous immunoassay can be used as a measuring device with high sensitivity quickly binds to the antigen antibody Quenchbody shines successful construction of a (Q-body), such as paper and patent application publication (WO2011/061944,WO2013/065314,R. Abe et al., J. Am. Chem. Soc., 2011, 133 (43), 17386-17394) been done. Q-body is, in the vicinity of the antigen binding site of a particular 1 location 2 of the location, via a short linker such as a fluorescent dye TAMRA-labeled modified antibody and fluorescent, dye (mainly tryptophan) amino acids of an antibody interacting with, the quenched state (extinction), the antigen is added to the light-off is released. However, conventionally in the production of the antibody gene cloning is essential to construct was a problem that it takes time and cost.
In order to solve this problem, recently, the present inventors, paying attention to the fact that PAxPG (J. Dong et al., J. Biosci. Bioeng., 2015, 120, 504-509) antibody-bound protein, the protein with a fluorescent dye and the linker is coupled via a fluorescent probe, a mixture of an antibody, the same Q-body, is released by the addition of the antigen in the production of the complex quencher was successful (non-patent document 1).
Scope of claims (In Japanese)請求の範囲 [請求項1]
 抗体軽鎖可変領域を含むポリペプチドと抗体重鎖可変領域を含むポリペプチドからなる複合体と、蛍光色素で標識されたプロテインMの断片とを含む抗原検出又は測定用キットであって、前記プロテインMの断片は、前記複合体と結合能を有する断片である、抗原検出又は測定用キット。

[請求項2]
 プロテインMの断片が、プロテインMのC末端側ドメインの全部又は一部を含む断片である、請求項1に記載の抗原検出又は測定用キット。

[請求項3]
 プロテインMの断片が、プロテインMにおけるN末端側の50~100アミノ酸残基及びC末端側の50~100アミノ酸残基を欠損させた断片である、請求項1に記載の抗原検出又は測定用キット。

[請求項4]
 抗体軽鎖可変領域を含むポリペプチドと抗体重鎖可変領域を含むポリペプチドからなる複合体が、全長抗体である、請求項1乃至3のいずれか一項に記載の抗原検出又は測定用キット。

[請求項5]
 蛍光色素が、プロテインMの断片のC末端側に標識されている、請求項1乃至4のいずれか一項に記載の抗原検出又は測定用キット。

[請求項6]
 プロテインMの断片が、2種類の蛍光色素で標識されている、請求項1乃至4のいずれか一項に記載の抗原検出又は測定用キット。

[請求項7]
 2種類の蛍光色素が、ローダミン系蛍光色素、及びGFP又はその変異体である、請求項6に記載の抗原検出又は測定用キット。

[請求項8]
 試料中の抗原を検出又は測定する方法であって、以下の工程(1)~(3)を順次行うことを特徴とする抗原の検出又は測定方法、
(1)試料を、蛍光色素で標識されたプロテインMの断片の存在下で、抗体軽鎖可変領域を含むポリペプチドと抗体重鎖可変領域を含むポリペプチドからなる複合体と接触させる工程であって、前記プロテインMの断片は、前記複合体と結合能を有する断片である工程、
(2)前記蛍光色素の蛍光強度を測定する工程、
(3)前記蛍光強度から試料中の抗原の存在を判定、又は前記蛍光強度から試料中の抗原量を算出する工程。

[請求項9]
 プロテインMの断片が、プロテインMのC末端側ドメインの全部又は一部を含む断片である、請求項8に記載の抗原の検出又は測定方法。

[請求項10]
 プロテインMの断片が、プロテインMにおけるN末端側の50~100アミノ酸残基及びC末端側の50~100アミノ酸残基を欠損させた断片である、請求項8に記載の抗原の検出又は測定方法。

[請求項11]
 抗体軽鎖可変領域を含むポリペプチドと抗体重鎖可変領域を含むポリペプチドからなる複合体が、全長抗体である、請求項8乃至10のいずれか一項に記載の抗原の検出又は測定方法。

[請求項12]
 蛍光色素が、プロテインMの断片のC末端側に標識されている、請求項8乃至11のいずれか一項に記載の抗原の検出又は測定方法。

[請求項13]
 プロテインMの断片が、2種類の蛍光色素で標識されている、請求項8乃至11のいずれか一項に記載の抗原の検出又は測定方法。

[請求項14]
 2種類の蛍光色素が、ローダミン系蛍光色素、及びGFP又はその変異体である、請求項13に記載の抗原の検出又は測定方法。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • TOKYO INSTITUTE OF TECHNOLOGY
  • Inventor
  • UEDA, Hiroshi
  • OHMURO, Yuki
  • MIYAKE, Chihiro
  • TSUKAHARA, Tomoya
IPC(International Patent Classification)
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