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You searched for: EV230715 (EV-TRACK ID)

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Results list Study Tree
Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV230715 1/17 Escherichia coli JC8031 (d)(U)C
DG
Filtration 		Valentine JL 2016 33%

Study summary

Full title
Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies.
All authors
Valentine JL, Chen L, Perregaux EC, Weyant KB, Rosenthal JA, Heiss C, Azadi P, Fisher AC, Putnam D, Moe GR, Merritt JH, DeLisa MP
Journal
Cell Chem Biol
Abstract
The development of antibodies against specific glycan epitopes poses a significant challenge due to (show more...)The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens. (hide)
EV-METRIC
33% (74th 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
Control condition
Focus vesicles
outer membrane vesicles
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
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Function
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
JC8031
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28 Ti
Pelleting: speed (g)
141000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
8
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Top-down
Speed (g)
180000
Duration (min)
180
Fraction volume (mL)
0.5
Fraction processing
None
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Other 1
dot blot
Not detected EV-associated proteins
T antigen
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
20-60
EM
EM-type
Transmission-EM
Image type
Wide-field
EV230715 2/17 Escherichia coli JC8031 (d)(U)C
DG
Filtration 		Valentine JL 2016 33%

Study summary

Full title
Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies.
All authors
Valentine JL, Chen L, Perregaux EC, Weyant KB, Rosenthal JA, Heiss C, Azadi P, Fisher AC, Putnam D, Moe GR, Merritt JH, DeLisa MP
Journal
Cell Chem Biol
Abstract
The development of antibodies against specific glycan epitopes poses a significant challenge due to (show more...)The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens. (hide)
EV-METRIC
33% (74th 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
pTF mutation
Focus vesicles
outer membrane vesicles
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
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Function
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
JC8031
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28 Ti
Pelleting: speed (g)
141000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
8
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Top-down
Speed (g)
180000
Duration (min)
180
Fraction volume (mL)
0.5
Fraction processing
None
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
OmpA, T antigen
Other 1
Western blot, dot blot
Detected EV-associated proteins
T antigen
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
20-60
EM
EM-type
Transmission-EM
Image type
Wide-field
EV230715 3/17 Escherichia coli JC8032 (d)(U)C
DG
Filtration 		Valentine JL 2016 33%

Study summary

Full title
Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies.
All authors
Valentine JL, Chen L, Perregaux EC, Weyant KB, Rosenthal JA, Heiss C, Azadi P, Fisher AC, Putnam D, Moe GR, Merritt JH, DeLisa MP
Journal
Cell Chem Biol
Abstract
The development of antibodies against specific glycan epitopes poses a significant challenge due to (show more...)The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens. (hide)
EV-METRIC
33% (74th 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
pTF mutation
Focus vesicles
outer membrane vesicles
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
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Function
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
JC8032
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28 Ti
Pelleting: speed (g)
141000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
8
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Top-down
Speed (g)
180000
Duration (min)
180
Fraction volume (mL)
0.5
Fraction processing
None
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Other 1
dot blot
Not detected EV-associated proteins
T antigen
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV230715 4/17 Escherichia coli JC8033 (d)(U)C
DG
Filtration 		Valentine JL 2016 33%

Study summary

Full title
Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies.
All authors
Valentine JL, Chen L, Perregaux EC, Weyant KB, Rosenthal JA, Heiss C, Azadi P, Fisher AC, Putnam D, Moe GR, Merritt JH, DeLisa MP
Journal
Cell Chem Biol
Abstract
The development of antibodies against specific glycan epitopes poses a significant challenge due to (show more...)The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens. (hide)
EV-METRIC
33% (74th 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
Control condition
Focus vesicles
outer membrane vesicles
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
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: T antigen
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Function
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
JC8033
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28 Ti
Pelleting: speed (g)
141000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
8
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Top-down
Speed (g)
180000
Duration (min)
180
Fraction volume (mL)
0.5
Fraction processing
None
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
20-60
EM
EM-type
Transmission-EM
Image type
Wide-field
EV230715 5/17 Escherichia coli JC8033 (d)(U)C
DG
Filtration 		Valentine JL 2016 33%

Study summary

Full title
Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies.
All authors
Valentine JL, Chen L, Perregaux EC, Weyant KB, Rosenthal JA, Heiss C, Azadi P, Fisher AC, Putnam D, Moe GR, Merritt JH, DeLisa MP
Journal
Cell Chem Biol
Abstract
The development of antibodies against specific glycan epitopes poses a significant challenge due to (show more...)The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens. (hide)
EV-METRIC
33% (74th 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
pNeuD
Focus vesicles
outer membrane vesicles
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
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: T antigen
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Function
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
JC8033
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28 Ti
Pelleting: speed (g)
141000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
8
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Top-down
Speed (g)
180000
Duration (min)
180
Fraction volume (mL)
0.5
Fraction processing
None
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV230715 6/17 Escherichia coli JC8033 (d)(U)C
DG
Filtration 		Valentine JL 2016 33%

Study summary

Full title
Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies.
All authors
Valentine JL, Chen L, Perregaux EC, Weyant KB, Rosenthal JA, Heiss C, Azadi P, Fisher AC, Putnam D, Moe GR, Merritt JH, DeLisa MP
Journal
Cell Chem Biol
Abstract
The development of antibodies against specific glycan epitopes poses a significant challenge due to (show more...)The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens. (hide)
EV-METRIC
33% (74th 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
pPSA
Focus vesicles
outer membrane vesicles
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
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Function
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
JC8033
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28 Ti
Pelleting: speed (g)
141000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
8
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Top-down
Speed (g)
180000
Duration (min)
180
Fraction volume (mL)
0.5
Fraction processing
None
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Other 1
dot blot
Not detected EV-associated proteins
PSA
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV230715 7/17 Escherichia coli JC8033 (d)(U)C
DG
Filtration 		Valentine JL 2016 33%

Study summary

Full title
Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies.
All authors
Valentine JL, Chen L, Perregaux EC, Weyant KB, Rosenthal JA, Heiss C, Azadi P, Fisher AC, Putnam D, Moe GR, Merritt JH, DeLisa MP
Journal
Cell Chem Biol
Abstract
The development of antibodies against specific glycan epitopes poses a significant challenge due to (show more...)The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens. (hide)
EV-METRIC
33% (74th 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
pPSA pNeuD
Focus vesicles
outer membrane vesicles
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
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: T antigen
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Function
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
JC8033
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28 Ti
Pelleting: speed (g)
141000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
8
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Top-down
Speed (g)
180000
Duration (min)
180
Fraction volume (mL)
0.5
Fraction processing
None
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
OmpA, PSA
Other 1
Western blot, dot blot
Detected EV-associated proteins
PSA
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
20-60
EM
EM-type
Transmission-EM
Image type
Wide-field
EV230715 8/17 Escherichia coli JC8033 (d)(U)C
DG
Filtration 		Valentine JL 2016 33%

Study summary

Full title
Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies.
All authors
Valentine JL, Chen L, Perregaux EC, Weyant KB, Rosenthal JA, Heiss C, Azadi P, Fisher AC, Putnam D, Moe GR, Merritt JH, DeLisa MP
Journal
Cell Chem Biol
Abstract
The development of antibodies against specific glycan epitopes poses a significant challenge due to (show more...)The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens. (hide)
EV-METRIC
33% (74th 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
pPdelC pNeuD
Focus vesicles
outer membrane vesicles
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
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: T antigen
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Function
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
JC8033
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28 Ti
Pelleting: speed (g)
141000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
8
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Top-down
Speed (g)
180000
Duration (min)
180
Fraction volume (mL)
0.5
Fraction processing
None
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Other 1
dot blot
Not detected EV-associated proteins
PSA
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV230715 9/17 Escherichia coli JC8033 (d)(U)C
DG
Filtration 		Valentine JL 2016 33%

Study summary

Full title
Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies.
All authors
Valentine JL, Chen L, Perregaux EC, Weyant KB, Rosenthal JA, Heiss C, Azadi P, Fisher AC, Putnam D, Moe GR, Merritt JH, DeLisa MP
Journal
Cell Chem Biol
Abstract
The development of antibodies against specific glycan epitopes poses a significant challenge due to (show more...)The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens. (hide)
EV-METRIC
33% (74th 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
pPdelL pNeuD
Focus vesicles
outer membrane vesicles
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
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Function
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
JC8033
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28 Ti
Pelleting: speed (g)
141000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
8
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Top-down
Speed (g)
180000
Duration (min)
180
Fraction volume (mL)
0.5
Fraction processing
None
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Other 1
dot blot
Not detected EV-associated proteins
PSA
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV230715 10/17 Escherichia coli JC8033 (d)(U)C
DG
Filtration 		Valentine JL 2016 33%

Study summary

Full title
Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies.
All authors
Valentine JL, Chen L, Perregaux EC, Weyant KB, Rosenthal JA, Heiss C, Azadi P, Fisher AC, Putnam D, Moe GR, Merritt JH, DeLisa MP
Journal
Cell Chem Biol
Abstract
The development of antibodies against specific glycan epitopes poses a significant challenge due to (show more...)The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens. (hide)
EV-METRIC
33% (74th 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
pPdelCL pNeuD
Focus vesicles
outer membrane vesicles
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
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: T antigen
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Function
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
JC8033
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28 Ti
Pelleting: speed (g)
141000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
8
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Top-down
Speed (g)
180000
Duration (min)
180
Fraction volume (mL)
0.5
Fraction processing
None
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Other 1
dot blot
Not detected EV-associated proteins
PSA
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV230715 11/17 Escherichia coli JC8034 (d)(U)C
DG
Filtration 		Valentine JL 2016 33%

Study summary

Full title
Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies.
All authors
Valentine JL, Chen L, Perregaux EC, Weyant KB, Rosenthal JA, Heiss C, Azadi P, Fisher AC, Putnam D, Moe GR, Merritt JH, DeLisa MP
Journal
Cell Chem Biol
Abstract
The development of antibodies against specific glycan epitopes poses a significant challenge due to (show more...)The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens. (hide)
EV-METRIC
33% (74th 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
pPSA pNeuD
Focus vesicles
outer membrane vesicles
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
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: T antigen
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Function
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
JC8034
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28 Ti
Pelleting: speed (g)
141000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
8
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Top-down
Speed (g)
180000
Duration (min)
180
Fraction volume (mL)
0.5
Fraction processing
None
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Other 1
dot blot
Detected EV-associated proteins
PSA
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV230715 12/17 Escherichia coli JC8035 (d)(U)C
DG
Filtration 		Valentine JL 2016 33%

Study summary

Full title
Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies.
All authors
Valentine JL, Chen L, Perregaux EC, Weyant KB, Rosenthal JA, Heiss C, Azadi P, Fisher AC, Putnam D, Moe GR, Merritt JH, DeLisa MP
Journal
Cell Chem Biol
Abstract
The development of antibodies against specific glycan epitopes poses a significant challenge due to (show more...)The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens. (hide)
EV-METRIC
33% (74th 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
pPSA pNeuD
Focus vesicles
outer membrane vesicles
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
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Function
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
JC8035
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28 Ti
Pelleting: speed (g)
141000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
8
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Top-down
Speed (g)
180000
Duration (min)
180
Fraction volume (mL)
0.5
Fraction processing
None
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Other 1
dot blot
Detected EV-associated proteins
PSA
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV230715 13/17 Escherichia coli EV36 (d)(U)C
DG
Filtration 		Valentine JL 2016 33%

Study summary

Full title
Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies.
All authors
Valentine JL, Chen L, Perregaux EC, Weyant KB, Rosenthal JA, Heiss C, Azadi P, Fisher AC, Putnam D, Moe GR, Merritt JH, DeLisa MP
Journal
Cell Chem Biol
Abstract
The development of antibodies against specific glycan epitopes poses a significant challenge due to (show more...)The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens. (hide)
EV-METRIC
33% (74th 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
Control condition
Focus vesicles
outer membrane vesicles
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
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Function
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
EV36
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28 Ti
Pelleting: speed (g)
141000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
8
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Top-down
Speed (g)
180000
Duration (min)
180
Fraction volume (mL)
0.5
Fraction processing
None
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Other 1
dot blot
Detected EV-associated proteins
PSA
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV230715 14/17 Escherichia coli MG1655-ves (d)(U)C
DG
Filtration 		Valentine JL 2016 33%

Study summary

Full title
Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies.
All authors
Valentine JL, Chen L, Perregaux EC, Weyant KB, Rosenthal JA, Heiss C, Azadi P, Fisher AC, Putnam D, Moe GR, Merritt JH, DeLisa MP
Journal
Cell Chem Biol
Abstract
The development of antibodies against specific glycan epitopes poses a significant challenge due to (show more...)The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens. (hide)
EV-METRIC
33% (74th 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
Control condition
Focus vesicles
outer membrane vesicles
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
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Function
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
MG1655-ves
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28 Ti
Pelleting: speed (g)
141000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
8
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Top-down
Speed (g)
180000
Duration (min)
180
Fraction volume (mL)
0.5
Fraction processing
None
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Other 1
dot blot
Not detected EV-associated proteins
PSA
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV230715 15/17 Escherichia coli MG1655-ves (d)(U)C
DG
Filtration 		Valentine JL 2016 33%

Study summary

Full title
Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies.
All authors
Valentine JL, Chen L, Perregaux EC, Weyant KB, Rosenthal JA, Heiss C, Azadi P, Fisher AC, Putnam D, Moe GR, Merritt JH, DeLisa MP
Journal
Cell Chem Biol
Abstract
The development of antibodies against specific glycan epitopes poses a significant challenge due to (show more...)The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens. (hide)
EV-METRIC
33% (74th 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
pPSA pNeuD
Focus vesicles
outer membrane vesicles
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
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Function
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
MG1655-ves
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28 Ti
Pelleting: speed (g)
141000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
8
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Top-down
Speed (g)
180000
Duration (min)
180
Fraction volume (mL)
0.5
Fraction processing
None
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Other 1
dot blot
Detected EV-associated proteins
PSA
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV230715 16/17 Escherichia coli ClearColi (d)(U)C
DG
Filtration 		Valentine JL 2016 33%

Study summary

Full title
Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies.
All authors
Valentine JL, Chen L, Perregaux EC, Weyant KB, Rosenthal JA, Heiss C, Azadi P, Fisher AC, Putnam D, Moe GR, Merritt JH, DeLisa MP
Journal
Cell Chem Biol
Abstract
The development of antibodies against specific glycan epitopes poses a significant challenge due to (show more...)The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens. (hide)
EV-METRIC
33% (74th 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
Control condition
Focus vesicles
outer membrane vesicles
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
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: T antigen
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Function
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
ClearColi
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28 Ti
Pelleting: speed (g)
141000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
8
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Top-down
Speed (g)
180000
Duration (min)
180
Fraction volume (mL)
0.5
Fraction processing
None
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Other 1
dot blot
Not detected EV-associated proteins
PSA
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV230715 17/17 Escherichia coli ClearColi (d)(U)C
DG
Filtration 		Valentine JL 2016 33%

Study summary

Full title
Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies.
All authors
Valentine JL, Chen L, Perregaux EC, Weyant KB, Rosenthal JA, Heiss C, Azadi P, Fisher AC, Putnam D, Moe GR, Merritt JH, DeLisa MP
Journal
Cell Chem Biol
Abstract
The development of antibodies against specific glycan epitopes poses a significant challenge due to (show more...)The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens. (hide)
EV-METRIC
33% (74th 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
pPSA pNeuD
Focus vesicles
outer membrane vesicles
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
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Function
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
ClearColi
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28 Ti
Pelleting: speed (g)
141000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
8
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Top-down
Speed (g)
180000
Duration (min)
180
Fraction volume (mL)
0.5
Fraction processing
None
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Other 1
dot blot
Detected EV-associated proteins
PSA
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
1 - 17 of 17
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV230715
species
Escherichia
coli
sample type
Cell
culture
cell type
JC8031
JC8031
JC8032
JC8033
JC8033
JC8033
JC8033
JC8033
JC8033
JC8033
JC8034
JC8035
EV36
MG1655-ves
MG1655-ves
ClearColi
ClearColi
condition
Control
condition
pTF
mutation
pTF
mutation
Control
condition
pNeuD
pPSA
pPSA
pNeuD
pPdelC
pNeuD
pPdelL
pNeuD
pPdelCL
pNeuD
pPSA
pNeuD
pPSA
pNeuD
Control
condition
Control
condition
pPSA
pNeuD
Control
condition
pPSA
pNeuD
separation protocol
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
Exp. nr.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
EV-METRIC %
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33