TY  - JOUR
AU  - Filipović, Lidija
AU  - Spasojević, Milica
AU  - Prodanović, Radivoje
AU  - Korać, Aleksandra
AU  - Matijaševic, Suzana
AU  - Brajušković, Goran
AU  - de Marco, Ario
AU  - Popović, Milica M.
PY  - 2022
UR  - https://pubmed.ncbi.nlm.nih.gov/35301156/
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5152
AB  - Correct elucidation of physiological and pathological processes mediated by extracellular vesicles (EV) is highly dependent on the reliability of the method used for their purification. Currently available chemical/physical protocols for sample fractionation are time-consuming, often scarcely reproducible and their yields are low. Immuno-capture based approaches could represent an effective purification alternative to obtain homogeneous EV samples. An easy-to-operate chromatography system was set-up for the purification of intact EVs based on a single domain (VHH) antibodies-copolymer matrix suitable for biological samples as different as conditioned cell culture medium and human plasma. Methacrylate-based copolymer is a porous solid support, the chemical versatility of which enables its efficient functionalization with VHHs. The combined analyses of morphological features and biomarker (CD9, CD63 and CD81) presence indicated that the recovered EVs were exosomes. The lipoprotein markers APO-A1 and APO-B were both negative in tested samples. This is the first report demonstrating the successful application of spherical porous methacrylate-based copolymer coupled with VHHs for the exosome isolation from biological fluids. This inexpensive immunoaffinity method has the potential to be applied for the isolation of EVs belonging to different morphological and physiological classes.
PB  - Elsevier
T2  - New Biotechnology
T1  - Affinity-based isolation of extracellular vesicles by means of single-domain antibodies bound to macroporous methacrylate-based copolymer
VL  - 69
SP  - 36
EP  - 48
DO  - 10.1016/j.nbt.2022.03.001
ER  - 

@article{
author = "Filipović, Lidija and Spasojević, Milica and Prodanović, Radivoje and Korać, Aleksandra and Matijaševic, Suzana and Brajušković, Goran and de Marco, Ario and Popović, Milica M.",
year = "2022",
abstract = "Correct elucidation of physiological and pathological processes mediated by extracellular vesicles (EV) is highly dependent on the reliability of the method used for their purification. Currently available chemical/physical protocols for sample fractionation are time-consuming, often scarcely reproducible and their yields are low. Immuno-capture based approaches could represent an effective purification alternative to obtain homogeneous EV samples. An easy-to-operate chromatography system was set-up for the purification of intact EVs based on a single domain (VHH) antibodies-copolymer matrix suitable for biological samples as different as conditioned cell culture medium and human plasma. Methacrylate-based copolymer is a porous solid support, the chemical versatility of which enables its efficient functionalization with VHHs. The combined analyses of morphological features and biomarker (CD9, CD63 and CD81) presence indicated that the recovered EVs were exosomes. The lipoprotein markers APO-A1 and APO-B were both negative in tested samples. This is the first report demonstrating the successful application of spherical porous methacrylate-based copolymer coupled with VHHs for the exosome isolation from biological fluids. This inexpensive immunoaffinity method has the potential to be applied for the isolation of EVs belonging to different morphological and physiological classes.",
publisher = "Elsevier",
journal = "New Biotechnology",
title = "Affinity-based isolation of extracellular vesicles by means of single-domain antibodies bound to macroporous methacrylate-based copolymer",
volume = "69",
pages = "36-48",
doi = "10.1016/j.nbt.2022.03.001"
}

Filipović, L., Spasojević, M., Prodanović, R., Korać, A., Matijaševic, S., Brajušković, G., de Marco, A.,& Popović, M. M.. (2022). Affinity-based isolation of extracellular vesicles by means of single-domain antibodies bound to macroporous methacrylate-based copolymer. in New Biotechnology
Elsevier., 69, 36-48.
https://doi.org/10.1016/j.nbt.2022.03.001

Filipović L, Spasojević M, Prodanović R, Korać A, Matijaševic S, Brajušković G, de Marco A, Popović MM. Affinity-based isolation of extracellular vesicles by means of single-domain antibodies bound to macroporous methacrylate-based copolymer. in New Biotechnology. 2022;69:36-48.
doi:10.1016/j.nbt.2022.03.001 .

Filipović, Lidija, Spasojević, Milica, Prodanović, Radivoje, Korać, Aleksandra, Matijaševic, Suzana, Brajušković, Goran, de Marco, Ario, Popović, Milica M., "Affinity-based isolation of extracellular vesicles by means of single-domain antibodies bound to macroporous methacrylate-based copolymer" in New Biotechnology, 69 (2022):36-48,
https://doi.org/10.1016/j.nbt.2022.03.001 . .

TY  - DATA
AU  - Filipović, Lidija
AU  - Spasojević, Milica
AU  - Prodanović, Radivoje
AU  - Korać, Aleksandra
AU  - Matijaševic, Suzana
AU  - Brajušković, Goran
AU  - de Marco, Ario
AU  - Popović, Milica M.
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5152
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5153
AB  - Correct elucidation of physiological and pathological processes mediated by extracellular vesicles (EV) is highly dependent on the reliability of the method used for their purification. Currently available chemical/physical protocols for sample fractionation are time-consuming, often scarcely reproducible and their yields are low. Immuno-capture based approaches could represent an effective purification alternative to obtain homogeneous EV samples. An easy-to-operate chromatography system was set-up for the purification of intact EVs based on a single domain (VHH) antibodies-copolymer matrix suitable for biological samples as different as conditioned cell culture medium and human plasma. Methacrylate-based copolymer is a porous solid support, the chemical versatility of which enables its efficient functionalization with VHHs. The combined analyses of morphological features and biomarker (CD9, CD63 and CD81) presence indicated that the recovered EVs were exosomes. The lipoprotein markers APO-A1 and APO-B were both negative in tested samples. This is the first report demonstrating the successful application of spherical porous methacrylate-based copolymer coupled with VHHs for the exosome isolation from biological fluids. This inexpensive immunoaffinity method has the potential to be applied for the isolation of EVs belonging to different morphological and physiological classes.
PB  - Elsevier
T2  - New Biotechnology
T1  - Supplementary information for the article: Filipović, L.; Spasojević, M.; Prodanović, R.; Korać, A.; Matijaševic, S.; Brajušković, G.; de Marco, A.; Popović, M. Affinity-Based Isolation of Extracellular Vesicles by Means of Single-Domain Antibodies Bound to Macroporous Methacrylate-Based Copolymer. New Biotechnology 2022, 69, 36–48. https://doi.org/10.1016/j.nbt.2022.03.001.
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5153
ER  - 

@misc{
author = "Filipović, Lidija and Spasojević, Milica and Prodanović, Radivoje and Korać, Aleksandra and Matijaševic, Suzana and Brajušković, Goran and de Marco, Ario and Popović, Milica M.",
year = "2022",
abstract = "Correct elucidation of physiological and pathological processes mediated by extracellular vesicles (EV) is highly dependent on the reliability of the method used for their purification. Currently available chemical/physical protocols for sample fractionation are time-consuming, often scarcely reproducible and their yields are low. Immuno-capture based approaches could represent an effective purification alternative to obtain homogeneous EV samples. An easy-to-operate chromatography system was set-up for the purification of intact EVs based on a single domain (VHH) antibodies-copolymer matrix suitable for biological samples as different as conditioned cell culture medium and human plasma. Methacrylate-based copolymer is a porous solid support, the chemical versatility of which enables its efficient functionalization with VHHs. The combined analyses of morphological features and biomarker (CD9, CD63 and CD81) presence indicated that the recovered EVs were exosomes. The lipoprotein markers APO-A1 and APO-B were both negative in tested samples. This is the first report demonstrating the successful application of spherical porous methacrylate-based copolymer coupled with VHHs for the exosome isolation from biological fluids. This inexpensive immunoaffinity method has the potential to be applied for the isolation of EVs belonging to different morphological and physiological classes.",
publisher = "Elsevier",
journal = "New Biotechnology",
title = "Supplementary information for the article: Filipović, L.; Spasojević, M.; Prodanović, R.; Korać, A.; Matijaševic, S.; Brajušković, G.; de Marco, A.; Popović, M. Affinity-Based Isolation of Extracellular Vesicles by Means of Single-Domain Antibodies Bound to Macroporous Methacrylate-Based Copolymer. New Biotechnology 2022, 69, 36–48. https://doi.org/10.1016/j.nbt.2022.03.001.",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5153"
}

Filipović, L., Spasojević, M., Prodanović, R., Korać, A., Matijaševic, S., Brajušković, G., de Marco, A.,& Popović, M. M.. (2022). Supplementary information for the article: Filipović, L.; Spasojević, M.; Prodanović, R.; Korać, A.; Matijaševic, S.; Brajušković, G.; de Marco, A.; Popović, M. Affinity-Based Isolation of Extracellular Vesicles by Means of Single-Domain Antibodies Bound to Macroporous Methacrylate-Based Copolymer. New Biotechnology 2022, 69, 36–48. https://doi.org/10.1016/j.nbt.2022.03.001.. in New Biotechnology
Elsevier..
https://hdl.handle.net/21.15107/rcub_cherry_5153

Filipović L, Spasojević M, Prodanović R, Korać A, Matijaševic S, Brajušković G, de Marco A, Popović MM. Supplementary information for the article: Filipović, L.; Spasojević, M.; Prodanović, R.; Korać, A.; Matijaševic, S.; Brajušković, G.; de Marco, A.; Popović, M. Affinity-Based Isolation of Extracellular Vesicles by Means of Single-Domain Antibodies Bound to Macroporous Methacrylate-Based Copolymer. New Biotechnology 2022, 69, 36–48. https://doi.org/10.1016/j.nbt.2022.03.001.. in New Biotechnology. 2022;.
https://hdl.handle.net/21.15107/rcub_cherry_5153 .

Filipović, Lidija, Spasojević, Milica, Prodanović, Radivoje, Korać, Aleksandra, Matijaševic, Suzana, Brajušković, Goran, de Marco, Ario, Popović, Milica M., "Supplementary information for the article: Filipović, L.; Spasojević, M.; Prodanović, R.; Korać, A.; Matijaševic, S.; Brajušković, G.; de Marco, A.; Popović, M. Affinity-Based Isolation of Extracellular Vesicles by Means of Single-Domain Antibodies Bound to Macroporous Methacrylate-Based Copolymer. New Biotechnology 2022, 69, 36–48. https://doi.org/10.1016/j.nbt.2022.03.001." in New Biotechnology (2022),
https://hdl.handle.net/21.15107/rcub_cherry_5153 .

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  - 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 .

TY  - JOUR
AU  - Popović, Milica M.
AU  - Sustar, Vid
AU  - Gricar, Jozica
AU  - Straus, Ines
AU  - Torkar, Gregor
AU  - Kraigher, Hojka
AU  - de Marco, Ario
PY  - 2016
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/3530
AB  - Climate development models predict alterations that will critically influence plant metabolism in southern and central Europe. Although the molecular players involved in the response to climatic stress factors have been well described in crops, little information is available for forest tree species. Consequently, the identification of molecular biomarkers suitable for evaluating the actual impact of different environmental stress conditions on forest plants would be of great importance for monitoring purposes and forest management. In this study, we evaluated a biochemical methodology for the assessment of temperature stress in European beech (Fagus sylvatica L.) and Scots pine (Pinus sylvestris L.) seedlings by analyzing a set of metabolites and enzymes involved in free radical scavenging and cell wall synthesis. The results indicate that the combined analysis of the specific activities and isoform profile of peroxidases, superoxide dismutases, and glutathione peroxidases coupled with the amount variation of phenolic compounds enabled the discrimination between stressed and control seedlings. This approach represents a promising platform for the assessment of temperature stress in forest trees and could also enhance selection and breeding practices, allowing for plants more tolerant and (or) resistant to abiotic stress.
PB  - Canadian Science Publishing, Nrc Research Press, Ottawa
T2  - Canadian Journal of Forest Research = Revue Canadienne de La Recherche Forestiere
T1  - Identification of environmental stress biomarkers in seedlings of European beech (Fagus sylvatica) and Scots pine (Pinus sylvestris)
VL  - 46
IS  - 1
SP  - 58
EP  - 66
DO  - 10.1139/cjfr-2015-0274
ER  - 

@article{
author = "Popović, Milica M. and Sustar, Vid and Gricar, Jozica and Straus, Ines and Torkar, Gregor and Kraigher, Hojka and de Marco, Ario",
year = "2016",
abstract = "Climate development models predict alterations that will critically influence plant metabolism in southern and central Europe. Although the molecular players involved in the response to climatic stress factors have been well described in crops, little information is available for forest tree species. Consequently, the identification of molecular biomarkers suitable for evaluating the actual impact of different environmental stress conditions on forest plants would be of great importance for monitoring purposes and forest management. In this study, we evaluated a biochemical methodology for the assessment of temperature stress in European beech (Fagus sylvatica L.) and Scots pine (Pinus sylvestris L.) seedlings by analyzing a set of metabolites and enzymes involved in free radical scavenging and cell wall synthesis. The results indicate that the combined analysis of the specific activities and isoform profile of peroxidases, superoxide dismutases, and glutathione peroxidases coupled with the amount variation of phenolic compounds enabled the discrimination between stressed and control seedlings. This approach represents a promising platform for the assessment of temperature stress in forest trees and could also enhance selection and breeding practices, allowing for plants more tolerant and (or) resistant to abiotic stress.",
publisher = "Canadian Science Publishing, Nrc Research Press, Ottawa",
journal = "Canadian Journal of Forest Research = Revue Canadienne de La Recherche Forestiere",
title = "Identification of environmental stress biomarkers in seedlings of European beech (Fagus sylvatica) and Scots pine (Pinus sylvestris)",
volume = "46",
number = "1",
pages = "58-66",
doi = "10.1139/cjfr-2015-0274"
}

Popović, M. M., Sustar, V., Gricar, J., Straus, I., Torkar, G., Kraigher, H.,& de Marco, A.. (2016). Identification of environmental stress biomarkers in seedlings of European beech (Fagus sylvatica) and Scots pine (Pinus sylvestris). in Canadian Journal of Forest Research = Revue Canadienne de La Recherche Forestiere
Canadian Science Publishing, Nrc Research Press, Ottawa., 46(1), 58-66.
https://doi.org/10.1139/cjfr-2015-0274

Popović MM, Sustar V, Gricar J, Straus I, Torkar G, Kraigher H, de Marco A. Identification of environmental stress biomarkers in seedlings of European beech (Fagus sylvatica) and Scots pine (Pinus sylvestris). in Canadian Journal of Forest Research = Revue Canadienne de La Recherche Forestiere. 2016;46(1):58-66.
doi:10.1139/cjfr-2015-0274 .

Popović, Milica M., Sustar, Vid, Gricar, Jozica, Straus, Ines, Torkar, Gregor, Kraigher, Hojka, de Marco, Ario, "Identification of environmental stress biomarkers in seedlings of European beech (Fagus sylvatica) and Scots pine (Pinus sylvestris)" in Canadian Journal of Forest Research = Revue Canadienne de La Recherche Forestiere, 46, no. 1 (2016):58-66,
https://doi.org/10.1139/cjfr-2015-0274 . .