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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200040	8/8	Homo sapiens	Blood plasma	negative selection immunoaffinity purification qEV	Jung, Stephanie	2020	75%
Study summary Full title Efficient and reproducible depletion of hepatitis B virus from plasma derived extracellular vesicles All authors Stephanie Jung, Karolin Fiona, Kirsten Jacobs, Mikhail Shein, Anne Kathrin Schütz, Fabian Mohr, Herbert Stadler, Daniela Stadler, Aaron Michael Lucko, Sebastian Maximilian Altstetter, Florian Wilsch, Li Deng, Ulrike Protzer Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are emerging fundamental players in viral infections by shuttling viral (show more...)Extracellular vesicles (EVs) are emerging fundamental players in viral infections by shuttling viral components, mediating immune responses and likely the spread of the virus. However, the obstacles involved in purifying EVs and removing contaminating viral particles in a reliable and effective manner bottlenecks the full potential for the development of clinical and diagnostic treatment options targeting EV. Because of the similarities in size, density, membrane composition and mode of biogenesis of EVs and virions there are no standardized approaches for virus‐removal from EV preparations yet. Functional EV studies also require EV samples that are devoid of antibody contaminants. Consequently, the study of EVs in virology needs reliable and effective protocols to purify EVs and remove contaminating antibodies and viral particles. Here, we established a protocol for EV purification from hepatitis B virus (HBV)‐containing plasma by a combination of size‐exclusion chromatography and affinity‐based purification. After purification, EV samples were free of virus‐sized particles, HBV surface antigen, HBV core antigen, antibodies or infectious material. Viral genomic contamination was also decreased following purification. By using appropriate antibodies and size parameters, this protocol could potentially be applied to purification of EVs from other viral samples. In summary, we established a fast, reproducible and robust approach for the removal of HBV from EV preparations. Looking forward to the point of purifying EVs from clinical samples, this method should enable studies shedding light on the underlying mechanisms of EVs in viral infections and their diagnostic and prognostic potential. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin spiked with 3.1*10^8 GE HBV and 767 g anti-HBsAg per ml plasma prior to SEC Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture negative selection immunoaffinity purification Commercial method Protein markers EV: TSG101/ CD63/ Syntenin non-EV: HBsAg/ Calnexin/ Albumin/ anti-HBsAg/ HBcAg Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Other Name other separation method negative selection immunoaffinity purification EV-subtype Distinction between multiple subtypes Size Used subtypes F1-AP (1ml collected after 3ml void volume) Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Syntenin/ CD63/ TSG101 Not detected contaminants Calnexin/ anti-HBsAg/ HBcAg/ Albumin Detected contaminants HBsAg Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) antibody size; < =50.75nm; >50.75nm NTA Report type Not Reported EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1.01E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200040	3/8	Homo sapiens	Blood plasma	qEV	Jung, Stephanie	2020	38%
Study summary Full title Efficient and reproducible depletion of hepatitis B virus from plasma derived extracellular vesicles All authors Stephanie Jung, Karolin Fiona, Kirsten Jacobs, Mikhail Shein, Anne Kathrin Schütz, Fabian Mohr, Herbert Stadler, Daniela Stadler, Aaron Michael Lucko, Sebastian Maximilian Altstetter, Florian Wilsch, Li Deng, Ulrike Protzer Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are emerging fundamental players in viral infections by shuttling viral (show more...)Extracellular vesicles (EVs) are emerging fundamental players in viral infections by shuttling viral components, mediating immune responses and likely the spread of the virus. However, the obstacles involved in purifying EVs and removing contaminating viral particles in a reliable and effective manner bottlenecks the full potential for the development of clinical and diagnostic treatment options targeting EV. Because of the similarities in size, density, membrane composition and mode of biogenesis of EVs and virions there are no standardized approaches for virus‐removal from EV preparations yet. Functional EV studies also require EV samples that are devoid of antibody contaminants. Consequently, the study of EVs in virology needs reliable and effective protocols to purify EVs and remove contaminating antibodies and viral particles. Here, we established a protocol for EV purification from hepatitis B virus (HBV)‐containing plasma by a combination of size‐exclusion chromatography and affinity‐based purification. After purification, EV samples were free of virus‐sized particles, HBV surface antigen, HBV core antigen, antibodies or infectious material. Viral genomic contamination was also decreased following purification. By using appropriate antibodies and size parameters, this protocol could potentially be applied to purification of EVs from other viral samples. In summary, we established a fast, reproducible and robust approach for the removal of HBV from EV preparations. Looking forward to the point of purifying EVs from clinical samples, this method should enable studies shedding light on the underlying mechanisms of EVs in viral infections and their diagnostic and prognostic potential. (hide) EV-METRIC 38% (72nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin spiked with 1.54*10^7 GE HBV and 76.5 g anti-HBsAg per ml input sample after SEC Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture qEV Protein markers EV: None non-EV: HBsAg Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected contaminants HBsAg Detected contaminants HBsAg Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) < =50.75nm; >50.75nm

	EV200040	4/8	Homo sapiens	Blood plasma	negative selection immunuaffinity purification qEV	Jung, Stephanie	2020	38%
Study summary Full title Efficient and reproducible depletion of hepatitis B virus from plasma derived extracellular vesicles All authors Stephanie Jung, Karolin Fiona, Kirsten Jacobs, Mikhail Shein, Anne Kathrin Schütz, Fabian Mohr, Herbert Stadler, Daniela Stadler, Aaron Michael Lucko, Sebastian Maximilian Altstetter, Florian Wilsch, Li Deng, Ulrike Protzer Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are emerging fundamental players in viral infections by shuttling viral (show more...)Extracellular vesicles (EVs) are emerging fundamental players in viral infections by shuttling viral components, mediating immune responses and likely the spread of the virus. However, the obstacles involved in purifying EVs and removing contaminating viral particles in a reliable and effective manner bottlenecks the full potential for the development of clinical and diagnostic treatment options targeting EV. Because of the similarities in size, density, membrane composition and mode of biogenesis of EVs and virions there are no standardized approaches for virus‐removal from EV preparations yet. Functional EV studies also require EV samples that are devoid of antibody contaminants. Consequently, the study of EVs in virology needs reliable and effective protocols to purify EVs and remove contaminating antibodies and viral particles. Here, we established a protocol for EV purification from hepatitis B virus (HBV)‐containing plasma by a combination of size‐exclusion chromatography and affinity‐based purification. After purification, EV samples were free of virus‐sized particles, HBV surface antigen, HBV core antigen, antibodies or infectious material. Viral genomic contamination was also decreased following purification. By using appropriate antibodies and size parameters, this protocol could potentially be applied to purification of EVs from other viral samples. In summary, we established a fast, reproducible and robust approach for the removal of HBV from EV preparations. Looking forward to the point of purifying EVs from clinical samples, this method should enable studies shedding light on the underlying mechanisms of EVs in viral infections and their diagnostic and prognostic potential. (hide) EV-METRIC 38% (72nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin spiked with 1.54*10^7 GE HBV and 76.5 g anti-HBsAg per ml input sample after SEC Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture negative selection immunuaffinity purification Commercial method Protein markers EV: None non-EV: HBsAg Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Other Name other separation method negative selection immunuaffinity purification Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected contaminants HBsAg Detected contaminants HBsAg Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) < =50.75nm; >50.75nm

	EV200040	5/8	Homo sapiens	Blood plasma	qEV	Jung, Stephanie	2020	38%
Study summary Full title Efficient and reproducible depletion of hepatitis B virus from plasma derived extracellular vesicles All authors Stephanie Jung, Karolin Fiona, Kirsten Jacobs, Mikhail Shein, Anne Kathrin Schütz, Fabian Mohr, Herbert Stadler, Daniela Stadler, Aaron Michael Lucko, Sebastian Maximilian Altstetter, Florian Wilsch, Li Deng, Ulrike Protzer Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are emerging fundamental players in viral infections by shuttling viral (show more...)Extracellular vesicles (EVs) are emerging fundamental players in viral infections by shuttling viral components, mediating immune responses and likely the spread of the virus. However, the obstacles involved in purifying EVs and removing contaminating viral particles in a reliable and effective manner bottlenecks the full potential for the development of clinical and diagnostic treatment options targeting EV. Because of the similarities in size, density, membrane composition and mode of biogenesis of EVs and virions there are no standardized approaches for virus‐removal from EV preparations yet. Functional EV studies also require EV samples that are devoid of antibody contaminants. Consequently, the study of EVs in virology needs reliable and effective protocols to purify EVs and remove contaminating antibodies and viral particles. Here, we established a protocol for EV purification from hepatitis B virus (HBV)‐containing plasma by a combination of size‐exclusion chromatography and affinity‐based purification. After purification, EV samples were free of virus‐sized particles, HBV surface antigen, HBV core antigen, antibodies or infectious material. Viral genomic contamination was also decreased following purification. By using appropriate antibodies and size parameters, this protocol could potentially be applied to purification of EVs from other viral samples. In summary, we established a fast, reproducible and robust approach for the removal of HBV from EV preparations. Looking forward to the point of purifying EVs from clinical samples, this method should enable studies shedding light on the underlying mechanisms of EVs in viral infections and their diagnostic and prognostic potential. (hide) EV-METRIC 38% (72nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin spiked with 3.1*10^8 GE HBV and 767 g anti-HBsAg per ml plasma prior to SEC Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture qEV Protein markers EV: None non-EV: HBsAg Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Other Name other separation method qEV EV-subtype Distinction between multiple subtypes Size Used subtypes F2 (0.5 ml fraction collected after 4 ml void volume) Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected contaminants HBsAg Detected contaminants HBsAg Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) antibody size; < =50.75nm; >50.75nm NTA Report type Not Reported EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 5.06E+10

	EV200040	6/8	Homo sapiens	Blood plasma	qEV	Jung, Stephanie	2020	38%
Study summary Full title Efficient and reproducible depletion of hepatitis B virus from plasma derived extracellular vesicles All authors Stephanie Jung, Karolin Fiona, Kirsten Jacobs, Mikhail Shein, Anne Kathrin Schütz, Fabian Mohr, Herbert Stadler, Daniela Stadler, Aaron Michael Lucko, Sebastian Maximilian Altstetter, Florian Wilsch, Li Deng, Ulrike Protzer Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are emerging fundamental players in viral infections by shuttling viral (show more...)Extracellular vesicles (EVs) are emerging fundamental players in viral infections by shuttling viral components, mediating immune responses and likely the spread of the virus. However, the obstacles involved in purifying EVs and removing contaminating viral particles in a reliable and effective manner bottlenecks the full potential for the development of clinical and diagnostic treatment options targeting EV. Because of the similarities in size, density, membrane composition and mode of biogenesis of EVs and virions there are no standardized approaches for virus‐removal from EV preparations yet. Functional EV studies also require EV samples that are devoid of antibody contaminants. Consequently, the study of EVs in virology needs reliable and effective protocols to purify EVs and remove contaminating antibodies and viral particles. Here, we established a protocol for EV purification from hepatitis B virus (HBV)‐containing plasma by a combination of size‐exclusion chromatography and affinity‐based purification. After purification, EV samples were free of virus‐sized particles, HBV surface antigen, HBV core antigen, antibodies or infectious material. Viral genomic contamination was also decreased following purification. By using appropriate antibodies and size parameters, this protocol could potentially be applied to purification of EVs from other viral samples. In summary, we established a fast, reproducible and robust approach for the removal of HBV from EV preparations. Looking forward to the point of purifying EVs from clinical samples, this method should enable studies shedding light on the underlying mechanisms of EVs in viral infections and their diagnostic and prognostic potential. (hide) EV-METRIC 38% (72nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin spiked with 3.1*10^8 GE HBV and 767 g anti-HBsAg per ml plasma prior to SEC Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture qEV Protein markers EV: None non-EV: HBsAg Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Other Name other separation method qEV EV-subtype Distinction between multiple subtypes Size Used subtypes F1 (1ml fraction collected after 3 ml void volume) Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected contaminants HBsAg Detected contaminants HBsAg Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) antibody size; < =50.75nm; >50.75nm NTA Report type Not Reported EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.60E+10

	EV200040	7/8	Homo sapiens	Blood plasma	negative selection immunoaffinity purification qEV	Jung, Stephanie	2020	38%
Study summary Full title Efficient and reproducible depletion of hepatitis B virus from plasma derived extracellular vesicles All authors Stephanie Jung, Karolin Fiona, Kirsten Jacobs, Mikhail Shein, Anne Kathrin Schütz, Fabian Mohr, Herbert Stadler, Daniela Stadler, Aaron Michael Lucko, Sebastian Maximilian Altstetter, Florian Wilsch, Li Deng, Ulrike Protzer Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are emerging fundamental players in viral infections by shuttling viral (show more...)Extracellular vesicles (EVs) are emerging fundamental players in viral infections by shuttling viral components, mediating immune responses and likely the spread of the virus. However, the obstacles involved in purifying EVs and removing contaminating viral particles in a reliable and effective manner bottlenecks the full potential for the development of clinical and diagnostic treatment options targeting EV. Because of the similarities in size, density, membrane composition and mode of biogenesis of EVs and virions there are no standardized approaches for virus‐removal from EV preparations yet. Functional EV studies also require EV samples that are devoid of antibody contaminants. Consequently, the study of EVs in virology needs reliable and effective protocols to purify EVs and remove contaminating antibodies and viral particles. Here, we established a protocol for EV purification from hepatitis B virus (HBV)‐containing plasma by a combination of size‐exclusion chromatography and affinity‐based purification. After purification, EV samples were free of virus‐sized particles, HBV surface antigen, HBV core antigen, antibodies or infectious material. Viral genomic contamination was also decreased following purification. By using appropriate antibodies and size parameters, this protocol could potentially be applied to purification of EVs from other viral samples. In summary, we established a fast, reproducible and robust approach for the removal of HBV from EV preparations. Looking forward to the point of purifying EVs from clinical samples, this method should enable studies shedding light on the underlying mechanisms of EVs in viral infections and their diagnostic and prognostic potential. (hide) EV-METRIC 38% (72nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin spiked with 3.1*10^8 GE HBV and 767 g anti-HBsAg per ml plasma prior to SEC Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture negative selection immunoaffinity purification Commercial method Protein markers EV: None non-EV: HBsAg Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Other Name other separation method negative selection immunoaffinity purification EV-subtype Distinction between multiple subtypes Size Used subtypes F2-AP (0.5 ml collected after 4ml void volume) Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected contaminants HBsAg Detected contaminants HBsAg Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) antibody size; < =50.75nm; >50.75nm NTA Report type Not Reported EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 5.38E+09

	EV200040	1/8	Homo sapiens	Blood plasma	qEV	Jung, Stephanie	2020	17%
Study summary Full title Efficient and reproducible depletion of hepatitis B virus from plasma derived extracellular vesicles All authors Stephanie Jung, Karolin Fiona, Kirsten Jacobs, Mikhail Shein, Anne Kathrin Schütz, Fabian Mohr, Herbert Stadler, Daniela Stadler, Aaron Michael Lucko, Sebastian Maximilian Altstetter, Florian Wilsch, Li Deng, Ulrike Protzer Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are emerging fundamental players in viral infections by shuttling viral (show more...)Extracellular vesicles (EVs) are emerging fundamental players in viral infections by shuttling viral components, mediating immune responses and likely the spread of the virus. However, the obstacles involved in purifying EVs and removing contaminating viral particles in a reliable and effective manner bottlenecks the full potential for the development of clinical and diagnostic treatment options targeting EV. Because of the similarities in size, density, membrane composition and mode of biogenesis of EVs and virions there are no standardized approaches for virus‐removal from EV preparations yet. Functional EV studies also require EV samples that are devoid of antibody contaminants. Consequently, the study of EVs in virology needs reliable and effective protocols to purify EVs and remove contaminating antibodies and viral particles. Here, we established a protocol for EV purification from hepatitis B virus (HBV)‐containing plasma by a combination of size‐exclusion chromatography and affinity‐based purification. After purification, EV samples were free of virus‐sized particles, HBV surface antigen, HBV core antigen, antibodies or infectious material. Viral genomic contamination was also decreased following purification. By using appropriate antibodies and size parameters, this protocol could potentially be applied to purification of EVs from other viral samples. In summary, we established a fast, reproducible and robust approach for the removal of HBV from EV preparations. Looking forward to the point of purifying EVs from clinical samples, this method should enable studies shedding light on the underlying mechanisms of EVs in viral infections and their diagnostic and prognostic potential. (hide) EV-METRIC 17% (46th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin spiked with 3.1*10^8 GE HBV per ml plasma prior to SEC Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture qEV Protein markers EV: None non-EV: HBsAg Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Other Name other separation method qEV EV-subtype Distinction between multiple subtypes Size Used subtypes F1 (1ml fraction collected after 3 ml void volume) Characterization: Protein analysis Protein Concentration Method Not determined Detected contaminants HBsAg Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) antibody size; < =50.75nm; >50.75nm

	EV200040	2/8	Homo sapiens	Blood plasma	qEV	Jung, Stephanie	2020	17%
Study summary Full title Efficient and reproducible depletion of hepatitis B virus from plasma derived extracellular vesicles All authors Stephanie Jung, Karolin Fiona, Kirsten Jacobs, Mikhail Shein, Anne Kathrin Schütz, Fabian Mohr, Herbert Stadler, Daniela Stadler, Aaron Michael Lucko, Sebastian Maximilian Altstetter, Florian Wilsch, Li Deng, Ulrike Protzer Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are emerging fundamental players in viral infections by shuttling viral (show more...)Extracellular vesicles (EVs) are emerging fundamental players in viral infections by shuttling viral components, mediating immune responses and likely the spread of the virus. However, the obstacles involved in purifying EVs and removing contaminating viral particles in a reliable and effective manner bottlenecks the full potential for the development of clinical and diagnostic treatment options targeting EV. Because of the similarities in size, density, membrane composition and mode of biogenesis of EVs and virions there are no standardized approaches for virus‐removal from EV preparations yet. Functional EV studies also require EV samples that are devoid of antibody contaminants. Consequently, the study of EVs in virology needs reliable and effective protocols to purify EVs and remove contaminating antibodies and viral particles. Here, we established a protocol for EV purification from hepatitis B virus (HBV)‐containing plasma by a combination of size‐exclusion chromatography and affinity‐based purification. After purification, EV samples were free of virus‐sized particles, HBV surface antigen, HBV core antigen, antibodies or infectious material. Viral genomic contamination was also decreased following purification. By using appropriate antibodies and size parameters, this protocol could potentially be applied to purification of EVs from other viral samples. In summary, we established a fast, reproducible and robust approach for the removal of HBV from EV preparations. Looking forward to the point of purifying EVs from clinical samples, this method should enable studies shedding light on the underlying mechanisms of EVs in viral infections and their diagnostic and prognostic potential. (hide) EV-METRIC 17% (46th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin spiked with 3.1*10^8 GE HBV per ml plasma prior to SEC Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture qEV Protein markers EV: None non-EV: HBsAg Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Other Name other separation method qEV EV-subtype Distinction between multiple subtypes Size Used subtypes F2 (0.5 ml fraction collected after 4 ml void volume) Characterization: Protein analysis Protein Concentration Method Not determined Detected contaminants HBsAg Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) antibody size; < =50.75nm; >50.75nm

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EV-TRACK ID	EV200040
species	Homo sapiens
sample type	Blood plasma
condition	spiked with 3.1*10^8 GE HBV and 767 g anti-HBsAg per ml plasma prior to SEC	spiked with 1.54*10^7 GE HBV and 76.5 g anti-HBsAg per ml input sample after SEC	spiked with 1.54*10^7 GE HBV and 76.5 g anti-HBsAg per ml input sample after SEC	spiked with 3.1*10^8 GE HBV and 767 g anti-HBsAg per ml plasma prior to SEC	spiked with 3.1*10^8 GE HBV and 767 g anti-HBsAg per ml plasma prior to SEC	spiked with 3.1*10^8 GE HBV and 767 g anti-HBsAg per ml plasma prior to SEC	spiked with 3.1*10^8 GE HBV per ml plasma prior to SEC	spiked with 3.1*10^8 GE HBV per ml plasma prior to SEC
separation protocol	negative selection immunoaffinity purification qEV	qEV	negative selection immunuaffinity purification qEV	qEV	qEV	negative selection immunoaffinity purification qEV	qEV	qEV
EV subtype	F1-AP (1ml collected after 3ml void volume)	NA	NA	F2 (0.5 ml fraction collected after 4 ml void volume)	F1 (1ml fraction collected after 3 ml void volume)	F2-AP (0.5 ml collected after 4ml void volume)	F1 (1ml fraction collected after 3 ml void volume)	F2 (0.5 ml fraction collected after 4 ml void volume)
Exp. nr.	8	3	4	5	6	7	1	2
EV-METRIC %	75	38	38	38	38	38	17	17

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