Highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation
| Autor: | Padhan, Narendra, Yan, Junhong, Boge, Annegret, Scrivener, Elaine, Birgisson, Helgi, Zieba, Agata, Gullberg, Mats, Kamali-Moghaddam, Masood, Claesson-Welsh, Lena, Landegren, Ulf |
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| Přispěvatelé: | Molecular Biosensing for Med. Diagnostics |
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
Vascular Endothelial Growth Factor A
Medicin och hälsovetenskap Science Colorectal Neoplasms/diagnosis Vascular Endothelial Growth Factor A/pharmacology Sensitivity and Specificity Medical and Health Sciences Article Extracellular Signal-Regulated MAP Kinases/metabolism Signal Transduction/drug effects Human Umbilical Vein Endothelial Cells Humans Electrophoresis Capillary/methods Phosphorylation Extracellular Signal-Regulated MAP Kinases Nucleic Acid Amplification Techniques/methods Human Umbilical Vein Endothelial Cells/metabolism Proteins/analysis Electrophoresis Capillary Proteins Isoelectric Focusing/methods Medicine Nanoparticles Nanoparticles/chemistry Isoelectric Focusing Colorectal Neoplasms Nucleic Acid Amplification Techniques Signal Transduction |
| Zdroj: | Scientific Reports, 7(1):1490. Nature Publishing Group Scientific Reports Scientific Reports, Vol 7, Iss 1, Pp 1-9 (2017) |
| ISSN: | 2045-2322 |
| DOI: | 10.1038/s41598-017-01516-7 |
| Popis: | Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their isoforms with modest sample input. However, insufficient sensitivity prevents detection of proteins present at low concentrations and antibody cross-reactivity results in unspecific detection that cannot be distinguished from bona fide protein isoforms. By using DNA-conjugated antibodies enhanced signals can be obtained via rolling circle amplification (RCA). Both sensitivity and specificity can be greatly improved in assays dependent on target recognition by pairs of antibodies using in situ proximity ligation assays (PLA). Here we applied these DNA-assisted RCA techniques in capillary isoelectric focusing to resolve endogenous signaling transducers and isoforms along vascular endothelial growth factor (VEGF) signaling pathways at concentrations too low to be detected in standard assays. We also demonstrate background rejection and enhanced specificity when protein detection depended on binding by pairs of antibodies using in situ PLA, compared to assays where each antibody preparation was used on its own. |
| Databáze: | OpenAIRE |
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