Liberelle Benoît, Fortier Charles, De Crescenzo Gregory
Department of Chemical Engineering, École Polytechnique de Montréal, succ. Centre-Ville, 6079, Montréal, QC, Canada, H3C 3A7.
Methods Mol Biol. 2014;1172:39-47. doi: 10.1007/978-1-4939-0928-5_4.
In a "sandwich" enzyme-linked immunosorbent assay (ELISA) designed to detect an antigen in a complex protein mixture, the antigen is usually captured via an antibody adsorbed to the wells of a microplate. Plate preparation for standard assay involves a passive adsorption of capture antibodies followed by the incubation of blocking agents. Here, we describe a new strategy that replaces these two time-consuming adsorption steps (up to 15 h) by a unique step corresponding to the covalent grafting of the capture antibody on a carboxymethylated dextran (CMD) layer, a single step completed in 15 min. Taking advantage of the CMD low-fouling properties, blocking agent-free buffer solutions can be used as diluent in the improved approach.
在一种旨在检测复杂蛋白质混合物中抗原的“夹心”酶联免疫吸附测定（ELISA）中，抗原通常通过吸附在微孔板孔中的抗体捕获。标准测定的板制备包括捕获抗体的被动吸附，随后孵育封闭剂。在此，我们描述了一种新策略，该策略通过一个独特的步骤取代了这两个耗时的吸附步骤（长达15小时），该步骤对应于将捕获抗体共价接枝到羧甲基化葡聚糖（CMD）层上，这一步骤在15分钟内即可完成。利用CMD的低污染特性，在改进的方法中可以使用无封闭剂的缓冲溶液作为稀释剂。
