title: ELISA-PLA: A novel hybrid platform for the rapid, highly sensitive and specific quantification of proteins and post-translational modifications.
abstract: 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.
authors: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, P.R. China.
authors: Qing-He
authors: QH
authors: Tong
authors: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, P.R. China.
authors: Tao
authors: T
authors: Tao
authors: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, P.R. China. Electronic address: xieliqi@fudan.edu.cn.
authors: Li-Qi
authors: LQ
authors: Xie
authors: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, P.R. China; Department of Chemistry, Fudan University, Shanghai 200433, P.R. China. Electronic address: luhaojie@fudan.edu.cn.
authors: Hao-Jie
authors: HJ
authors: Lu
mesh_terms: D000215:Acylation; D000818:Animals; D015374:Biosensing Techniques / Q000191:economics / Q000379:methods; D017362:Cyclic AMP Response Element-Binding Protein / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000191:economics / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry; D048052:Mitogen-Activated Protein Kinase 3 / Q000737:chemistry; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry*; D051057:Proto-Oncogene Proteins c-akt / Q000737:chemistry; D016760:Proto-Oncogene Proteins c-fos / Q000737:chemistry; D050796:STAT3 Transcription Factor / Q000737:chemistry
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D011506:Proteins; D016760:Proto-Oncogene Proteins c-fos; D050796:STAT3 Transcription Factor; D051057:Proto-Oncogene Proteins c-akt; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3
keywords: ELISA; O-GlcNAcylation; Phosphorylation; Proximity ligation assay