Tong Qing-He, Tao Tao, Xie Li-Qi, Lu Hao-Jie
Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, P.R. China.
Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, P.R. China.
Biosens Bioelectron. 2016 Jun 15;80:385-391. doi: 10.1016/j.bios.2016.02.006. Epub 2016 Feb 3.
Detection of low-abundance proteins and their post-translational modifications (PTMs) remains a great challenge. A conventional enzyme-linked immunosorbent assay (ELISA) is not sensitive enough to detect low-abundance PTMs and suffers from nonspecific detection. Herein, a rapid, highly sensitive and specific platform integrating ELISA with a proximity ligation assay (PLA), termed ELISA-PLA, was developed. Using ELISA-PLA, the specificity was improved by the simultaneous and proximate recognition of targets through multiple probes, and the sensitivity was significantly improved by rolling circle amplification (RCA). For GFP, the limit of detection (LOD) was decreased by two orders of magnitude compared to that of ELISA. Using site-specific phospho-antibody and pan-specific phospho-antibody, ELISA-PLA was successfully applied to quantify the phosphorylation dynamics of ERK1/2 and the overall tyrosine phosphorylation level of ERK1/2, respectively. ELISA-PLA was also used to quantify the O-GlcNAcylation of AKT, c-Fos, CREB and STAT3, which is faster and more sensitive than the conventional immunoprecipitation and western blotting (IP-WB) method. As a result, the sample consumption of ELISA-PLA was reduced 40-fold compared to IP-WB. Therefore, ELISA-PLA could be a promising platform for the rapid, sensitive and specific detection of proteins and PTMs.
低丰度蛋白质及其翻译后修饰（PTM）的检测仍然是一个巨大的挑战。传统的酶联免疫吸附测定（ELISA）对低丰度PTM的检测不够灵敏，且存在非特异性检测的问题。在此，开发了一种将ELISA与邻近连接测定（PLA）相结合的快速、高灵敏度和特异性的平台，称为ELISA-PLA。使用ELISA-PLA，通过多个探针同时并近距离识别靶标提高了特异性，通过滚环扩增（RCA）显著提高了灵敏度。对于绿色荧光蛋白（GFP），与ELISA相比，检测限（LOD）降低了两个数量级。使用位点特异性磷酸化抗体和泛特异性磷酸化抗体，ELISA-PLA分别成功应用于定量细胞外信号调节激酶1/2（ERK1/2）的磷酸化动力学和ERK1/2的整体酪氨酸磷酸化水平。ELISA-PLA还用于定量蛋白激酶B（AKT）、原癌基因c-Fos、环磷腺苷效应元件结合蛋白（CREB）和信号转导子与转录激活子3（STAT3）的O-连接N-乙酰葡糖胺化（O-GlcNAcylation），这比传统的免疫沉淀和蛋白质印迹（IP-WB）方法更快、更灵敏。结果，与IP-WB相比，ELISA-PLA的样品消耗量减少了40倍。因此，ELISA-PLA可能是一个用于快速、灵敏和特异性检测蛋白质和PTM的有前景的平台。
