Abstract
The analysis of extracellular vesicles (EVs) typically requires tedious and time-consuming isolation process from bio-fluids. We developed a nanoparticle-based time resolved fluorescence immunoassay (NP-TRFIA) that uses biotinylated antibodies against the proteins of tetraspanin family and tumor-associated antigens for capturing EVs from urine samples and cell culture supernatants without the need for isolation. The captured-EVs were detected either with Eu3+-chelate or Eu3+-doped nanoparticle-based labels conjugated either to antibodies against the tetraspanins or lectins targeting the glycan moieties on EVs surface. The NP-TRFIA demonstrated specific capturing and detection of EVs by antibodies and lectins. Lectin-nanoparticle based assays showed 2–10 fold higher signal-to-background ratio compared with lectin-chelate assays. The nanoparticle assay concept allowed surface glycosylation profiling of the urine derived-EVs with lectins. It was also applied to establish an assay showing differential expression of tumor-associated proteins on more aggressive (higher ITGA3 on DU145- and PC3-EVs) compared to less aggressive (higher EpCAM on LNCaP-EVs) PCa- cell lines derived-EVs. This NP-TRFIA can be used as a simple tool for analysis and characterization of EVs in urine and cell culture supernatants. Such approach could be useful in identification of disease-specific markers on the surface of patient-derived urinary EVs.
Highlights
Extracellular vesicles (EVs) are 40 to 1000 nm sized membranous particles which are secreted by most of the cells and found in the bodily fluids like urine, plasma and saliva[1]
The S/B ratios obtained from lectin-NP tracers were systematically 2–10 folds higher compared to lectin-Eu tracers (Fig. 1a–c), even though the S/B ratios obtained with NP-tracers varied considerably depending on the lectin used
We reported a lanthanide chelate-doped nanoparticle aided TRFIA (NP-TRFIA) approach to detect the presence of specific proteins and glycans on the surface of extracellular vesicles (EVs) from minimally preprocessed urine samples and cell culture supernatants
Summary
Extracellular vesicles (EVs) are 40 to 1000 nm sized membranous particles which are secreted by most of the cells and found in the bodily fluids like urine, plasma and saliva[1]. The surface of the EVs is enriched with integral membrane proteins of tetraspanin family, such as CD9, CD63 and CD818 and with mannose- and sialic acid-containing glycoproteins[9]. Lectin microarrays have been used in various studies for the surface glycosylation profiling of the EVs of urine[13,15]. Such approach requires purified EVs from large sample volumes to work with. Achieve this, we explored the use of various reporter molecules targeting EVs, which include antibodies specific to tetraspanin family proteins and tumor-associated membrane antigens as well as lectins recognizing the glycan moieties. The assays were tested for surface glycosylation profiling of urinary EVs and for the characterization of EVs derived from prostate cancer cell line medium
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
