公开(公告)号：EP2972357A1

公开(公告)日：2016-01-20

申请号：EP14710370.9

申请日：2014-03-11

申请人： Cancer Research Technology Ltd

发明人： LARIJANI, Banafshe ,  PARKER, Peter ,  VEERIAH, Selvaraju

IPC分类号： G01N33/543

摘要： The present invention relates to a method for detecting molecules. The method employs: at least two primary antibodies, wherein the first primary antibody binds to a first site on a molecule and the second primary antibody binds to a second site on a molecule, wherein the second site is different from the first site and wherein the first and second primary antibodies are immunologically distinct; at least two secondary antibodies, wherein the first secondary antibody is labelled with a fluorescence resonance energy transfer (FRET) donor and binds to the first primary antibody; and the second secondary antibody is conjugated or fused to an enzyme and binds the second primary antibody, wherein the first secondary antibody does not bind the second primary antibody and the second secondary antibody does not bind the first primary antibody; a conjugate comprising a FRET acceptor and a substrate specific for the enzyme, wherein when the substrate reacts with the enzyme, an activated conjugate forms, which activated conjugate binds to electron rich moieties on a molecular surface adjacent to the enzyme; wherein the method comprises: contacting a sample with the at least two primary antibodies; contacting the sample with the at least two secondary antibodies; performing a wash step; contacting the sample with the conjugate; and detecting any FRET signal generated by the FRET acceptor.

摘要翻译： 本发明涉及一种检测分子的方法。 该方法采用：至少两种一级抗体，其中第一种一级抗体结合分子上的第一位点，第二种一级抗体与分子上的第二位点结合，其中第二位点不同于第一位点，其中 第一和第二抗体具有免疫学差异; 至少两个二抗，其中所述第一二抗用荧光共振能量转移（FRET）供体标记并结合所述第一个一抗; 并且所述第二次抗体与酶缀合或融合并结合所述第二一抗，其中所述第一二抗不结合所述第二一抗，所述第二二抗不结合所述第一一抗; 包含FRET受体和对酶特异性的底物的缀合物，其中当底物与酶反应时，活化的缀合物形成，其活化的缀合物结合与邻近酶的分子表面上的富含电子部分; 其中所述方法包括：使样品与所述至少两种一抗接触; 使所述样品与所述至少两种第二抗体接触; 执行洗涤步骤; 使样品与缀合物接触; 并检测由FRET接收器产生的任何FRET信号。