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Isolation and characterization of EVs in IVIg and SCIg. (A) EVs were isolated from UHPi (gray), <t>UHPp</t> (green), IVIg (red), and SCIg (blue) by SEC. Fifteen fractions (1 mL) were collected, and protein concentrations were determined by BCA assay. Fractions: 1-4 (void); 5-9 (EV-rich), and 10-15 (protein-rich). (B) Immunoblotting of each fraction with anti-human IgG antibodies. (C-E) NTA profile of pooled, concentrated EV-rich fractions (C), diameter (D), and concentration (particles/mL input volume) (E). (F) Representative TEM image of IVIg EVs showing lipid bilayer. Size bar = 100 nm. P-value: * P ≤ 0.05. BCA, bicinchoninic acid assay; EV, extracellular vesicles; IgG, immunoglobulin; IVIg, intravenous immunoglobulin; NTA, nanoparticle tracking analysis; SCIg, subcutaneous immunoglobulin; SEC, size-exclusion chromatography; TEM, transmission electron microscopy; UHPi, individual <t>unprocessed</t> human plasma; UHPp, pooled unprocessed human plasma; n=3.
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Isolation and characterization of EVs in IVIg and SCIg. (A) EVs were isolated from UHPi (gray), <t>UHPp</t> (green), IVIg (red), and SCIg (blue) by SEC. Fifteen fractions (1 mL) were collected, and protein concentrations were determined by BCA assay. Fractions: 1-4 (void); 5-9 (EV-rich), and 10-15 (protein-rich). (B) Immunoblotting of each fraction with anti-human IgG antibodies. (C-E) NTA profile of pooled, concentrated EV-rich fractions (C), diameter (D), and concentration (particles/mL input volume) (E). (F) Representative TEM image of IVIg EVs showing lipid bilayer. Size bar = 100 nm. P-value: * P ≤ 0.05. BCA, bicinchoninic acid assay; EV, extracellular vesicles; IgG, immunoglobulin; IVIg, intravenous immunoglobulin; NTA, nanoparticle tracking analysis; SCIg, subcutaneous immunoglobulin; SEC, size-exclusion chromatography; TEM, transmission electron microscopy; UHPi, individual <t>unprocessed</t> human plasma; UHPp, pooled unprocessed human plasma; n=3.
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Isolation and characterization of EVs in IVIg and SCIg. (A) EVs were isolated from UHPi (gray), <t>UHPp</t> (green), IVIg (red), and SCIg (blue) by SEC. Fifteen fractions (1 mL) were collected, and protein concentrations were determined by BCA assay. Fractions: 1-4 (void); 5-9 (EV-rich), and 10-15 (protein-rich). (B) Immunoblotting of each fraction with anti-human IgG antibodies. (C-E) NTA profile of pooled, concentrated EV-rich fractions (C), diameter (D), and concentration (particles/mL input volume) (E). (F) Representative TEM image of IVIg EVs showing lipid bilayer. Size bar = 100 nm. P-value: * P ≤ 0.05. BCA, bicinchoninic acid assay; EV, extracellular vesicles; IgG, immunoglobulin; IVIg, intravenous immunoglobulin; NTA, nanoparticle tracking analysis; SCIg, subcutaneous immunoglobulin; SEC, size-exclusion chromatography; TEM, transmission electron microscopy; UHPi, individual <t>unprocessed</t> human plasma; UHPp, pooled unprocessed human plasma; n=3.
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Isolation and characterization of EVs in IVIg and SCIg. (A) EVs were isolated from UHPi (gray), <t>UHPp</t> (green), IVIg (red), and SCIg (blue) by SEC. Fifteen fractions (1 mL) were collected, and protein concentrations were determined by BCA assay. Fractions: 1-4 (void); 5-9 (EV-rich), and 10-15 (protein-rich). (B) Immunoblotting of each fraction with anti-human IgG antibodies. (C-E) NTA profile of pooled, concentrated EV-rich fractions (C), diameter (D), and concentration (particles/mL input volume) (E). (F) Representative TEM image of IVIg EVs showing lipid bilayer. Size bar = 100 nm. P-value: * P ≤ 0.05. BCA, bicinchoninic acid assay; EV, extracellular vesicles; IgG, immunoglobulin; IVIg, intravenous immunoglobulin; NTA, nanoparticle tracking analysis; SCIg, subcutaneous immunoglobulin; SEC, size-exclusion chromatography; TEM, transmission electron microscopy; UHPi, individual <t>unprocessed</t> human plasma; UHPp, pooled unprocessed human plasma; n=3.
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Isolation and characterization of EVs in IVIg and SCIg. (A) EVs were isolated from UHPi (gray), <t>UHPp</t> (green), IVIg (red), and SCIg (blue) by SEC. Fifteen fractions (1 mL) were collected, and protein concentrations were determined by BCA assay. Fractions: 1-4 (void); 5-9 (EV-rich), and 10-15 (protein-rich). (B) Immunoblotting of each fraction with anti-human IgG antibodies. (C-E) NTA profile of pooled, concentrated EV-rich fractions (C), diameter (D), and concentration (particles/mL input volume) (E). (F) Representative TEM image of IVIg EVs showing lipid bilayer. Size bar = 100 nm. P-value: * P ≤ 0.05. BCA, bicinchoninic acid assay; EV, extracellular vesicles; IgG, immunoglobulin; IVIg, intravenous immunoglobulin; NTA, nanoparticle tracking analysis; SCIg, subcutaneous immunoglobulin; SEC, size-exclusion chromatography; TEM, transmission electron microscopy; UHPi, individual <t>unprocessed</t> human plasma; UHPp, pooled unprocessed human plasma; n=3.
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Isolation and characterization of EVs in IVIg and SCIg. (A) EVs were isolated from UHPi (gray), <t>UHPp</t> (green), IVIg (red), and SCIg (blue) by SEC. Fifteen fractions (1 mL) were collected, and protein concentrations were determined by BCA assay. Fractions: 1-4 (void); 5-9 (EV-rich), and 10-15 (protein-rich). (B) Immunoblotting of each fraction with anti-human IgG antibodies. (C-E) NTA profile of pooled, concentrated EV-rich fractions (C), diameter (D), and concentration (particles/mL input volume) (E). (F) Representative TEM image of IVIg EVs showing lipid bilayer. Size bar = 100 nm. P-value: * P ≤ 0.05. BCA, bicinchoninic acid assay; EV, extracellular vesicles; IgG, immunoglobulin; IVIg, intravenous immunoglobulin; NTA, nanoparticle tracking analysis; SCIg, subcutaneous immunoglobulin; SEC, size-exclusion chromatography; TEM, transmission electron microscopy; UHPi, individual <t>unprocessed</t> human plasma; UHPp, pooled unprocessed human plasma; n=3.
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Isolation and characterization of EVs in IVIg and SCIg. (A) EVs were isolated from UHPi (gray), <t>UHPp</t> (green), IVIg (red), and SCIg (blue) by SEC. Fifteen fractions (1 mL) were collected, and protein concentrations were determined by BCA assay. Fractions: 1-4 (void); 5-9 (EV-rich), and 10-15 (protein-rich). (B) Immunoblotting of each fraction with anti-human IgG antibodies. (C-E) NTA profile of pooled, concentrated EV-rich fractions (C), diameter (D), and concentration (particles/mL input volume) (E). (F) Representative TEM image of IVIg EVs showing lipid bilayer. Size bar = 100 nm. P-value: * P ≤ 0.05. BCA, bicinchoninic acid assay; EV, extracellular vesicles; IgG, immunoglobulin; IVIg, intravenous immunoglobulin; NTA, nanoparticle tracking analysis; SCIg, subcutaneous immunoglobulin; SEC, size-exclusion chromatography; TEM, transmission electron microscopy; UHPi, individual <t>unprocessed</t> human plasma; UHPp, pooled unprocessed human plasma; n=3.
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Isolation and characterization of EVs in IVIg and SCIg. (A) EVs were isolated from UHPi (gray), <t>UHPp</t> (green), IVIg (red), and SCIg (blue) by SEC. Fifteen fractions (1 mL) were collected, and protein concentrations were determined by BCA assay. Fractions: 1-4 (void); 5-9 (EV-rich), and 10-15 (protein-rich). (B) Immunoblotting of each fraction with anti-human IgG antibodies. (C-E) NTA profile of pooled, concentrated EV-rich fractions (C), diameter (D), and concentration (particles/mL input volume) (E). (F) Representative TEM image of IVIg EVs showing lipid bilayer. Size bar = 100 nm. P-value: * P ≤ 0.05. BCA, bicinchoninic acid assay; EV, extracellular vesicles; IgG, immunoglobulin; IVIg, intravenous immunoglobulin; NTA, nanoparticle tracking analysis; SCIg, subcutaneous immunoglobulin; SEC, size-exclusion chromatography; TEM, transmission electron microscopy; UHPi, individual <t>unprocessed</t> human plasma; UHPp, pooled unprocessed human plasma; n=3.
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Image Search Results


Isolation and characterization of EVs in IVIg and SCIg. (A) EVs were isolated from UHPi (gray), UHPp (green), IVIg (red), and SCIg (blue) by SEC. Fifteen fractions (1 mL) were collected, and protein concentrations were determined by BCA assay. Fractions: 1-4 (void); 5-9 (EV-rich), and 10-15 (protein-rich). (B) Immunoblotting of each fraction with anti-human IgG antibodies. (C-E) NTA profile of pooled, concentrated EV-rich fractions (C), diameter (D), and concentration (particles/mL input volume) (E). (F) Representative TEM image of IVIg EVs showing lipid bilayer. Size bar = 100 nm. P-value: * P ≤ 0.05. BCA, bicinchoninic acid assay; EV, extracellular vesicles; IgG, immunoglobulin; IVIg, intravenous immunoglobulin; NTA, nanoparticle tracking analysis; SCIg, subcutaneous immunoglobulin; SEC, size-exclusion chromatography; TEM, transmission electron microscopy; UHPi, individual unprocessed human plasma; UHPp, pooled unprocessed human plasma; n=3.

Journal: bioRxiv

Article Title: Translational Opportunity of Engineered IFNγ-eEVs Through Targeted Inhibition of JAK/STAT1 Signaling, Mimicking IVIg Therapy

doi: 10.64898/2026.04.29.721601

Figure Lengend Snippet: Isolation and characterization of EVs in IVIg and SCIg. (A) EVs were isolated from UHPi (gray), UHPp (green), IVIg (red), and SCIg (blue) by SEC. Fifteen fractions (1 mL) were collected, and protein concentrations were determined by BCA assay. Fractions: 1-4 (void); 5-9 (EV-rich), and 10-15 (protein-rich). (B) Immunoblotting of each fraction with anti-human IgG antibodies. (C-E) NTA profile of pooled, concentrated EV-rich fractions (C), diameter (D), and concentration (particles/mL input volume) (E). (F) Representative TEM image of IVIg EVs showing lipid bilayer. Size bar = 100 nm. P-value: * P ≤ 0.05. BCA, bicinchoninic acid assay; EV, extracellular vesicles; IgG, immunoglobulin; IVIg, intravenous immunoglobulin; NTA, nanoparticle tracking analysis; SCIg, subcutaneous immunoglobulin; SEC, size-exclusion chromatography; TEM, transmission electron microscopy; UHPi, individual unprocessed human plasma; UHPp, pooled unprocessed human plasma; n=3.

Article Snippet: Pooled unprocessed human plasma (UHPp) was obtained from Innovative Research (Novi, MI).

Techniques: Isolation, BIA-KA, Western Blot, Concentration Assay, Acid Assay, Size-exclusion Chromatography, Transmission Assay, Electron Microscopy, Clinical Proteomics

Flow cytometry phenotyping of CD63-positive EVs. UHPp, and IVIg EVs isolated using dUC or SEC were labeled with DiD and CD63-PE and analyzed by imaging flow cytometry. (A) Representative images of UHPp and IVIg EVs show morphology, BF, CD63, DiD, and scatter channels. (B) Scatter was used to gate out debris (left, gate R1). Fluorescent dot plots of unlabeled samples show background signal (middle) and labeled samples identified DiD + and CD63 + events (right, gate R2). (C) Summary plots of the frequency of EVs identified by scatter (gate R1, left) or fluorescence (gate R2, right) of UHPp, and IVIg EVs isolated using dUC or SEC. BF, bright field; DiD, 1,1′-dioctadecyl-3,3,3′,3′- tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt; dUC, differential ultracentrifugation; EV, extracellular vesicles; IVIg, intravenous immunoglobulin; PE, phycoerythrin; SEC, size-exclusion chromatography; UHPp, pooled unprocessed human plasma. Note: two different lots of UHPp and IVIg were used for these experiments.

Journal: bioRxiv

Article Title: Translational Opportunity of Engineered IFNγ-eEVs Through Targeted Inhibition of JAK/STAT1 Signaling, Mimicking IVIg Therapy

doi: 10.64898/2026.04.29.721601

Figure Lengend Snippet: Flow cytometry phenotyping of CD63-positive EVs. UHPp, and IVIg EVs isolated using dUC or SEC were labeled with DiD and CD63-PE and analyzed by imaging flow cytometry. (A) Representative images of UHPp and IVIg EVs show morphology, BF, CD63, DiD, and scatter channels. (B) Scatter was used to gate out debris (left, gate R1). Fluorescent dot plots of unlabeled samples show background signal (middle) and labeled samples identified DiD + and CD63 + events (right, gate R2). (C) Summary plots of the frequency of EVs identified by scatter (gate R1, left) or fluorescence (gate R2, right) of UHPp, and IVIg EVs isolated using dUC or SEC. BF, bright field; DiD, 1,1′-dioctadecyl-3,3,3′,3′- tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt; dUC, differential ultracentrifugation; EV, extracellular vesicles; IVIg, intravenous immunoglobulin; PE, phycoerythrin; SEC, size-exclusion chromatography; UHPp, pooled unprocessed human plasma. Note: two different lots of UHPp and IVIg were used for these experiments.

Article Snippet: Pooled unprocessed human plasma (UHPp) was obtained from Innovative Research (Novi, MI).

Techniques: Flow Cytometry, Isolation, Labeling, Imaging, Fluorescence, Size-exclusion Chromatography, Clinical Proteomics