Sabine I Brett1,2, Fabrice Lucien1,2,3, Charles Guo4, Karla C Williams1,2, Yohan Kim1,2,3, Paul N Durfee5, C J Brinker5,6, Joseph I Chin1,2, Jun Yang4, Hon S Leong1,2,3. 1. Translational Prostate Cancer Research Laboratory, Lawson Health Research Institute, London, Ontario. 2. Department of Surgery, Schulich School of Medicine and Dentistry, Western University, London, Ontario. 3. Department of Urology, Mayo Clinic, Rochester, Minnesota. 4. Department of Mechanical and Materials Engineering, Western University, London, Ontario. 5. Department of Chemical and Biological Engineering, University of New Mexico, Albuquerque, New Mexico. 6. Sandia National Laboratories, Albuquerque, New Mexico.
Abstract
BACKGROUND: The ability to isolate extracellular vesicles (EVs) such as exosomes or microparticles is an important method that is currently not standardized. While commercially available kits offer purification of EVs from biofluids, such purified EV samples will also contain non-EV entities such as soluble protein and nucleic acids that could confound subsequent experimentation. Ideally, only EVs would be isolated and no soluble protein would be present in the final EV preparation. METHODS: We compared commercially available EV isolation kits with immunoaffinity purification techniques and evaluated our final EV preparations using atomic force microscopy (AFM) and nanoscale flow cytometry (NFC). AFM is the only modality capable of detecting distinguishing soluble protein from EVs which is important for downstream proteomics approaches. NFC is the only technique capable of quantitating the proportion of target EVs to non-target EVs in the final EV preparation. RESULTS: To determine enrichment of prostate derived EVs relative to non-target MPs, anti-PSMA (Prostate Specific Membrane Antigen) antibodies were used in NFC. Antibody-based immunoaffinity purification generated the highest quality of prostate derived EV preparations due to the lack of protein and RNA present in the samples. All kits produced poor purity EV preparations that failed to deplete the sample of plasma protein. CONCLUSIONS: While attractive due to their ease of use, EV purification kits do not provide substantial improvements in isolation of EVs from biofluids such as plasma. Immunoaffinity approaches are more efficient and economical and will also eliminate a significant portion of plasma proteins which is necessary for downstream approaches.
BACKGROUND: The ability to isolate extracellular vesicles (EVs) such as exosomes or microparticles is an important method that is currently not standardized. While commercially available kits offer purification of EVs from biofluids, such purified EV samples will also contain non-EV entities such as soluble protein and nucleic acids that could confound subsequent experimentation. Ideally, only EVs would be isolated and no soluble protein would be present in the final EV preparation. METHODS: We compared commercially available EV isolation kits with immunoaffinity purification techniques and evaluated our final EV preparations using atomic force microscopy (AFM) and nanoscale flow cytometry (NFC). AFM is the only modality capable of detecting distinguishing soluble protein from EVs which is important for downstream proteomics approaches. NFC is the only technique capable of quantitating the proportion of target EVs to non-target EVs in the final EV preparation. RESULTS: To determine enrichment of prostate derived EVs relative to non-target MPs, anti-PSMA (Prostate Specific Membrane Antigen) antibodies were used in NFC. Antibody-based immunoaffinity purification generated the highest quality of prostate derived EV preparations due to the lack of protein and RNA present in the samples. All kits produced poor purity EV preparations that failed to deplete the sample of plasma protein. CONCLUSIONS: While attractive due to their ease of use, EV purification kits do not provide substantial improvements in isolation of EVs from biofluids such as plasma. Immunoaffinity approaches are more efficient and economical and will also eliminate a significant portion of plasma proteins which is necessary for downstream approaches.
Authors: Judith M Silverman; Darren Christy; Chih Cheih Shyu; Kyung-Mee Moon; Sarah Fernando; Zoe Gidden; Catherine M Cowan; Yuxin Ban; R Greg Stacey; Leslie I Grad; Luke McAlary; Ian R Mackenzie; Leonard J Foster; Neil R Cashman Journal: J Biol Chem Date: 2019-01-11 Impact factor: 5.157
Authors: Jaena Park; Rebecca L Kamerer; Marina Marjanovic; Janet E Sorrells; Sixian You; Ronit Barkalifa; Kimberly A Selting; Stephen A Boppart Journal: Am J Cancer Res Date: 2022-05-15 Impact factor: 5.942