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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200047	2/2	Homo sapiens	MCF7	UF DG	Yildizhan, Yagmur	2021	67%
Study summary Full title FO‐SPR biosensor calibrated with recombinant extracellular vesicles enables specific and sensitive detection directly in complex matrices All authors Yagmur Yildizhan, Venkata Suresh Vajrala, Edward Geeurickx, Charles Declerck, Nevena Duskunovic, Delphine De Sutter, Sam Noppen, Filip Delport, Dominique Schols, Johannes V. Swinnen, Sven Eyckerman, An Hendrix, Jeroen Lammertyn, Dragana Spasic Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) have drawn huge attention for diagnosing myriad of diseases, including (show more...)Extracellular vesicles (EVs) have drawn huge attention for diagnosing myriad of diseases, including cancer. However, the EV detection and analyses procedures often lack much desired sample standardization. To address this, we used well‐characterized recombinant EVs (rEVs) for the first time as a biological reference material in developing a fiber optic surface plasmon resonance (FO‐SPR) bioassay. In this context, EV binding on the FO‐SPR probes was achieved only with EV‐specific antibodies (e.g. anti‐CD9 and anti‐CD63) but not with non‐specific anti‐IgG. To increase detection sensitivity, we tested six different combinations of EV‐specific antibodies in a sandwich bioassay. Calibration curves were generated with two most effective combinations (anti‐CD9/Banti‐CD81 and anti‐CD63/Banti‐CD9), resulting in 103 and 104 times higher sensitivity than the EV concentration in human blood plasma from healthy or cancer patients, respectively. Additionally, by using anti‐CD63/Banti‐CD9, we detected rEVs spiked in cell culture medium and HEK293 endogenous EVs in the same matrix without any prior EV purification or enrichment. Lastly, we selectively captured breast cancer cell EVs spiked in blood plasma using anti‐EpCAM antibody on the FO‐SPR surface. The obtained results combined with FO‐SPR real‐time monitoring, fast response time and ease of operation, demonstrate its outstanding potential for EV quantification and analysis. (hide) EV-METRIC 67% (94th 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 Cell culture supernatant Sample origin Rab27B-GFP 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 Ultrafiltration Density gradient Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) 1.086-1.119 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF7 EV-harvesting Medium Serum free medium Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 11 Lowest density fraction 6 Highest density fraction 18 Total gradient volume, incl. sample (mL) 15 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 186700 Duration (min) 116 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200047	1/2	Homo sapiens	HEK293T	UF DG	Yildizhan, Yagmur	2021	57%
Study summary Full title FO‐SPR biosensor calibrated with recombinant extracellular vesicles enables specific and sensitive detection directly in complex matrices All authors Yagmur Yildizhan, Venkata Suresh Vajrala, Edward Geeurickx, Charles Declerck, Nevena Duskunovic, Delphine De Sutter, Sam Noppen, Filip Delport, Dominique Schols, Johannes V. Swinnen, Sven Eyckerman, An Hendrix, Jeroen Lammertyn, Dragana Spasic Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) have drawn huge attention for diagnosing myriad of diseases, including (show more...)Extracellular vesicles (EVs) have drawn huge attention for diagnosing myriad of diseases, including cancer. However, the EV detection and analyses procedures often lack much desired sample standardization. To address this, we used well‐characterized recombinant EVs (rEVs) for the first time as a biological reference material in developing a fiber optic surface plasmon resonance (FO‐SPR) bioassay. In this context, EV binding on the FO‐SPR probes was achieved only with EV‐specific antibodies (e.g. anti‐CD9 and anti‐CD63) but not with non‐specific anti‐IgG. To increase detection sensitivity, we tested six different combinations of EV‐specific antibodies in a sandwich bioassay. Calibration curves were generated with two most effective combinations (anti‐CD9/Banti‐CD81 and anti‐CD63/Banti‐CD9), resulting in 103 and 104 times higher sensitivity than the EV concentration in human blood plasma from healthy or cancer patients, respectively. Additionally, by using anti‐CD63/Banti‐CD9, we detected rEVs spiked in cell culture medium and HEK293 endogenous EVs in the same matrix without any prior EV purification or enrichment. Lastly, we selectively captured breast cancer cell EVs spiked in blood plasma using anti‐EpCAM antibody on the FO‐SPR surface. The obtained results combined with FO‐SPR real‐time monitoring, fast response time and ease of operation, demonstrate its outstanding potential for EV quantification and analysis. (hide) EV-METRIC 57% (92nd 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 Cell culture supernatant Sample origin transfected with gag-EGFP 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 Ultrafiltration Density gradient Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) 1.086-1.119 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293T EV-harvesting Medium Serum free medium Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 108.6 EV concentration Yes Particle yield No NA Particle analysis: flow cytometry Flow cytometer type High resolution flow cytometry Hardware adjustment 200 mW 488 nm laser (Sapphire; Coherent, Santa Clara, CA, USA) and a large-bore nozzle (140 m) were used, sheath pressure was permanently monitored and kept within 4.89 to 5.02 psi, and the sample pressure was set at 4.29 psi, to assure an identical diameter of the core in the jet stream. Forward scattered light was measured with a collection angle of 1525 (reduced wide-angle forward scatter [rw-FSC]). Calibration bead size 0.102 EV concentration Yes EM EM-type Immuno-EM/ Transmission-EM EM protein CD63 Image type Close-up, Wide-field

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EV-TRACK ID	EV200047
species	Homo sapiens
sample type	Cell culture
cell type	MCF7	HEK293T
condition	Rab27B-GFP	transfected with gag-EGFP
separation protocol	Ultrafiltration Density gradient	Ultrafiltration Density gradient
Exp. nr.	2	1
EV-METRIC %	67	57