A New Tetradentate β-Diketonate−Europium Chelate That Can Be Covalently Bound to Proteins for Time-Resolved Fluoroimmunoassay
作者:Jingli Yuan、Kazuko Matsumoto、Hiroko Kimura
DOI:10.1021/ac970936z
日期:1998.2.1
A new chlorosulfonylated tetradentate β-diketone, 4,4‘-bis(1‘‘,1‘‘,1‘‘,2‘‘,2‘‘,3‘‘,3‘‘-heptafluoro-4‘‘,6‘‘-hexanedion-6‘‘-yl)chlorosulfo-o-terphenyl (BHHCT), was synthesized as a chelating label for Eu3+. BHHCT can be covalently bound to proteins under mild conditions and forms a strongly fluorescent chelate with Eu3+. Bovine serum albumin (BSA) and streptavidin (SA) were labeled with BHHCH−Eu3+, and the latter was used for time-resolved fluoroimmunoassay of α-fetoprotein (AFP) in human sera. A remarkably high sensitivity was obtained, with a detection limit of 4.1 × 10-3 pg/mL, which corresponds to an improvement of about 4−5 orders of magnitude, compared to those of all conventional immunoassays including fluoroimmunoassay, enzyme immunoassay, and radioimmunoassay. The high sensitivity has been attained both by strong fluorescence of the present label and by the extremely suppressed background level brought about by the direct labeling of proteins with the β-diketone−Eu3+ complex. A general consideration and ideas are given for designing ideal label ligands for strongly fluorescent Eu3+ complexes.
合成了一种新的氯磺酰化四齿β-二酮,4,4’-双(1’,1’,1’,2’,2’,3’,3’-七氟-4’,6’-己二酮-6’-基)氯磺酰化-邻联三苯(BHHCT),作为Eu3+的螯合标记物。乙磺酰化四齿β-二酮可以在温和条件下与蛋白质共价结合,并与Eu3+形成强荧光螯合物。牛血清白蛋白(BSA)和链霉亲和素(SA)被BHHCH-Eu3+标记,后者用于人血清中甲种胎儿蛋白(AFP)的解时间分辨荧光免疫分析。获得了非常高的灵敏度,检测限为4.1 × 10-3pg/mL,与所有传统免疫分析方法(包括荧光免疫分析、酶免疫分析和放射免疫分析)相比,灵敏度提高了约4-5个数量级。这种高灵敏度是通过现有标记物的强荧光和通过β-二酮-Eu3+复合物直接标记蛋白质带来的极低的背景水平实现的。给出了设计强荧光Eu3+复合物理想标记配体的一般考虑和思路。