TY  - JOUR
AU  - Popović, Milica M.
AU  - Mazzega, Elisa
AU  - Toffoletto, Barbara
AU  - de Marco, Ario
PY  - 2018
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/2069
AB  - Background: The thorough understanding of the physiological and pathological processes mediated by extracellular vesicles (EVs) is challenged by purification methods which are cumbersome, not reproducible, or insufficient to yield homogeneous material. Chromatography based on both ion-exchange and immune-capture can represent an effective method to improve EV purification and successive analysis. Methods: Cell culture supernatant was used as a model sample for assessing the capacity of anion-exchange chromatography to separate distinct EV fractions and to isolate nanobodies by direct panning on whole EVs to recover binders specific for the native conformation of EV-surface epitopes and suitable to develop EV immune-capture reagents. Results: Anion-exchange chromatography of cell culture supernatant separated distinct protein-containing fractions and all of them were positive for CD9, a biomarker associated to some EVs. This suggested the existence of several EV fractions but did not help in separating EVs from other contaminants. We further isolated several nanobodies instrumental for implementing immune-affinity protocols. These were able to immobilize EVs from both cell culture supernatant and biological samples, to be used in ELISA, flow-cytometry, and immune-purification. Conclusions: Here we report the first successful isolation of anti-EV nanobodies for the use in immunoaffinity-based EV capture by panning a phage library directly on partially purified EVs. This achievement paves the way for the application of direct EV panning for the discovery of novel antibody-vesicle surface biomarker pairs and represents the preliminary requirement for the development of selective immune-capture that, in combination with anion-exchange chromatography, can simplify the systematic stratification of EV sub-populations and their individual characterization.
PB  - Biomed Central Ltd, London
T2  - Microbial Cell Factories
T1  - Isolation of anti-extra-cellular vesicle single-domain antibodies by direct panning on vesicle-enriched fractions
VL  - 17
DO  - 10.1186/s12934-017-0856-9
ER  - 

@article{
author = "Popović, Milica M. and Mazzega, Elisa and Toffoletto, Barbara and de Marco, Ario",
year = "2018",
abstract = "Background: The thorough understanding of the physiological and pathological processes mediated by extracellular vesicles (EVs) is challenged by purification methods which are cumbersome, not reproducible, or insufficient to yield homogeneous material. Chromatography based on both ion-exchange and immune-capture can represent an effective method to improve EV purification and successive analysis. Methods: Cell culture supernatant was used as a model sample for assessing the capacity of anion-exchange chromatography to separate distinct EV fractions and to isolate nanobodies by direct panning on whole EVs to recover binders specific for the native conformation of EV-surface epitopes and suitable to develop EV immune-capture reagents. Results: Anion-exchange chromatography of cell culture supernatant separated distinct protein-containing fractions and all of them were positive for CD9, a biomarker associated to some EVs. This suggested the existence of several EV fractions but did not help in separating EVs from other contaminants. We further isolated several nanobodies instrumental for implementing immune-affinity protocols. These were able to immobilize EVs from both cell culture supernatant and biological samples, to be used in ELISA, flow-cytometry, and immune-purification. Conclusions: Here we report the first successful isolation of anti-EV nanobodies for the use in immunoaffinity-based EV capture by panning a phage library directly on partially purified EVs. This achievement paves the way for the application of direct EV panning for the discovery of novel antibody-vesicle surface biomarker pairs and represents the preliminary requirement for the development of selective immune-capture that, in combination with anion-exchange chromatography, can simplify the systematic stratification of EV sub-populations and their individual characterization.",
publisher = "Biomed Central Ltd, London",
journal = "Microbial Cell Factories",
title = "Isolation of anti-extra-cellular vesicle single-domain antibodies by direct panning on vesicle-enriched fractions",
volume = "17",
doi = "10.1186/s12934-017-0856-9"
}

Popović, M. M., Mazzega, E., Toffoletto, B.,& de Marco, A.. (2018). Isolation of anti-extra-cellular vesicle single-domain antibodies by direct panning on vesicle-enriched fractions. in Microbial Cell Factories
Biomed Central Ltd, London., 17.
https://doi.org/10.1186/s12934-017-0856-9

Popović MM, Mazzega E, Toffoletto B, de Marco A. Isolation of anti-extra-cellular vesicle single-domain antibodies by direct panning on vesicle-enriched fractions. in Microbial Cell Factories. 2018;17.
doi:10.1186/s12934-017-0856-9 .

Popović, Milica M., Mazzega, Elisa, Toffoletto, Barbara, de Marco, Ario, "Isolation of anti-extra-cellular vesicle single-domain antibodies by direct panning on vesicle-enriched fractions" in Microbial Cell Factories, 17 (2018),
https://doi.org/10.1186/s12934-017-0856-9 . .

TY  - JOUR
AU  - Popović, Milica M.
AU  - de Marco, Ario
PY  - 2018
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/2138
AB  - Extracellular vesicles (EVs) play a pivotal role in cell to cell signalling in both physiological and pathological conditions. Based on their biogenesis, three main classes of EVs are recognized: exosomes, microvesicles and apoptotic bodies. Exosomes are cell-derived vesicles (EVs) present in many body fluids (blood, urine, milk, cerebrospinal fluid) ranging in size from 30 to 150 nm. Due to their involvement in numerous physiological and pathological events, cell derived exosomes in bodily fluids represent a unique source of clinically relevant and non-invasive biomarkers. Since biomolecule content present in exosomes reflects the state of the parent cell, exosome analysis and characterization may provide valuable information about the presence of aberrant processes in the cells from which they originated. Because of the large and heterogeneous scientific community working with exosomes, several purification strategies have been applied so far, which yield EV fractions largely differing for quantity and quality. Most of the present exosome isolation approaches based on ultracentrifugation (UC), ultrafiltration (UF) or precipitation, are inefficient and hard to standardize, thereby creating low reproducibility in sample quality and potentially misleading results because highly sensitivity downstream analytical techniques, such as mass spectrometry, can detect even minute traces of co-isolated contaminants. Furthermore, loss of certain exosomal fractions during purification process or damage of exosomal membrane integrity can also alter final protein and RNA profiles. As a consequence, there is a strong interest in consensus principles for the exosome purification and the search for reliable methods for selective isolation of EV sub-populations. In the present manuscript, we critically overview the most commonly used techniques used for exosome preparation such as ultracentrifugation, size-based isolation methods, precipitation and immunoaffinity (IA) and their respective applicability for purification of exosomes from clinically relevant samples.
PB  - Ame Publ Co, Shatin
T2  - TRANSLATIONAL CANCER RESEARCH
T1  - Canonical and selective approaches in exosome purification and their implications for diagnostic accuracy
VL  - 7
DO  - 10.21037/tcr.2017.08.44
ER  - 

@article{
author = "Popović, Milica M. and de Marco, Ario",
year = "2018",
abstract = "Extracellular vesicles (EVs) play a pivotal role in cell to cell signalling in both physiological and pathological conditions. Based on their biogenesis, three main classes of EVs are recognized: exosomes, microvesicles and apoptotic bodies. Exosomes are cell-derived vesicles (EVs) present in many body fluids (blood, urine, milk, cerebrospinal fluid) ranging in size from 30 to 150 nm. Due to their involvement in numerous physiological and pathological events, cell derived exosomes in bodily fluids represent a unique source of clinically relevant and non-invasive biomarkers. Since biomolecule content present in exosomes reflects the state of the parent cell, exosome analysis and characterization may provide valuable information about the presence of aberrant processes in the cells from which they originated. Because of the large and heterogeneous scientific community working with exosomes, several purification strategies have been applied so far, which yield EV fractions largely differing for quantity and quality. Most of the present exosome isolation approaches based on ultracentrifugation (UC), ultrafiltration (UF) or precipitation, are inefficient and hard to standardize, thereby creating low reproducibility in sample quality and potentially misleading results because highly sensitivity downstream analytical techniques, such as mass spectrometry, can detect even minute traces of co-isolated contaminants. Furthermore, loss of certain exosomal fractions during purification process or damage of exosomal membrane integrity can also alter final protein and RNA profiles. As a consequence, there is a strong interest in consensus principles for the exosome purification and the search for reliable methods for selective isolation of EV sub-populations. In the present manuscript, we critically overview the most commonly used techniques used for exosome preparation such as ultracentrifugation, size-based isolation methods, precipitation and immunoaffinity (IA) and their respective applicability for purification of exosomes from clinically relevant samples.",
publisher = "Ame Publ Co, Shatin",
journal = "TRANSLATIONAL CANCER RESEARCH",
title = "Canonical and selective approaches in exosome purification and their implications for diagnostic accuracy",
volume = "7",
doi = "10.21037/tcr.2017.08.44"
}

Popović, M. M.,& de Marco, A.. (2018). Canonical and selective approaches in exosome purification and their implications for diagnostic accuracy. in TRANSLATIONAL CANCER RESEARCH
Ame Publ Co, Shatin., 7.
https://doi.org/10.21037/tcr.2017.08.44

Popović MM, de Marco A. Canonical and selective approaches in exosome purification and their implications for diagnostic accuracy. in TRANSLATIONAL CANCER RESEARCH. 2018;7.
doi:10.21037/tcr.2017.08.44 .

Popović, Milica M., de Marco, Ario, "Canonical and selective approaches in exosome purification and their implications for diagnostic accuracy" in TRANSLATIONAL CANCER RESEARCH, 7 (2018),
https://doi.org/10.21037/tcr.2017.08.44 . .

TY  - DATA
AU  - Popović, Milica M.
AU  - Mazzega, Elisa
AU  - Toffoletto, Barbara
AU  - de Marco, Ario
PY  - 2018
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/3233
PB  - Biomed Central Ltd, London
T2  - Microbial Cell Factories
T1  - Supplementary material for the article: Popovic, M.; Mazzega, E.; Toffoletto, B.; de Marco, A. Isolation of Anti-Extra-Cellular Vesicle Single-Domain Antibodies by Direct Panning on Vesicle-Enriched Fractions. Microbial cell factories 2018, 17 (1), 6. https://doi.org/10.1186/s12934-017-0856-9
UR  - https://hdl.handle.net/21.15107/rcub_cherry_3233
ER  - 

@misc{
author = "Popović, Milica M. and Mazzega, Elisa and Toffoletto, Barbara and de Marco, Ario",
year = "2018",
publisher = "Biomed Central Ltd, London",
journal = "Microbial Cell Factories",
title = "Supplementary material for the article: Popovic, M.; Mazzega, E.; Toffoletto, B.; de Marco, A. Isolation of Anti-Extra-Cellular Vesicle Single-Domain Antibodies by Direct Panning on Vesicle-Enriched Fractions. Microbial cell factories 2018, 17 (1), 6. https://doi.org/10.1186/s12934-017-0856-9",
url = "https://hdl.handle.net/21.15107/rcub_cherry_3233"
}

Popović, M. M., Mazzega, E., Toffoletto, B.,& de Marco, A.. (2018). Supplementary material for the article: Popovic, M.; Mazzega, E.; Toffoletto, B.; de Marco, A. Isolation of Anti-Extra-Cellular Vesicle Single-Domain Antibodies by Direct Panning on Vesicle-Enriched Fractions. Microbial cell factories 2018, 17 (1), 6. https://doi.org/10.1186/s12934-017-0856-9. in Microbial Cell Factories
Biomed Central Ltd, London..
https://hdl.handle.net/21.15107/rcub_cherry_3233

Popović MM, Mazzega E, Toffoletto B, de Marco A. Supplementary material for the article: Popovic, M.; Mazzega, E.; Toffoletto, B.; de Marco, A. Isolation of Anti-Extra-Cellular Vesicle Single-Domain Antibodies by Direct Panning on Vesicle-Enriched Fractions. Microbial cell factories 2018, 17 (1), 6. https://doi.org/10.1186/s12934-017-0856-9. in Microbial Cell Factories. 2018;.
https://hdl.handle.net/21.15107/rcub_cherry_3233 .

Popović, Milica M., Mazzega, Elisa, Toffoletto, Barbara, de Marco, Ario, "Supplementary material for the article: Popovic, M.; Mazzega, E.; Toffoletto, B.; de Marco, A. Isolation of Anti-Extra-Cellular Vesicle Single-Domain Antibodies by Direct Panning on Vesicle-Enriched Fractions. Microbial cell factories 2018, 17 (1), 6. https://doi.org/10.1186/s12934-017-0856-9" in Microbial Cell Factories (2018),
https://hdl.handle.net/21.15107/rcub_cherry_3233 .