Journal: Journal of Extracellular Vesicles

Article Title: In‐Solution Characterization of Extracellular Vesicles: A New Approach to Evaluating Antibody Binding and Surface Interactions

doi: 10.1002/jev2.70269

Figure Lengend Snippet: Characterization of indicator antibodies by FIDA using different concentrations. A) Taylorgram showing relative fluorescence units (RFU) over time for increasing concentration of anti‐CD63 AF647, 0.88 nM (Rh: 5.24 nm); 1.66 nM (Rh: 5.41 nm); 3.33 nM (Rh: 5.37 nm) and 6.67 nM (Rh: 5.38 nm). B) Taylorgram showing relative fluorescence units (RFU) over time for 0.1–10 nM IgG1‐APC. C) Taylorgram showing relative fluorescence units (RFU) over time for 1–40 nM Cetuximab ALC647.

Article Snippet: EV samples (FLuoEVs, ciMSC‐EVs, Fc‐EVs, and Control EVs) were diluted in assay buffer to the respective concentration based on the particle concentration determined by NTA and/or IFCM followed by incubation with the respective antibodies: mouse anti‐human CD63 AF647 (H5C6, BD Biosciences, Cat. No. 561983, Herlev, Denmark), REA human IgG1 isotype control‐APC (REA293, Miltenyi Biotech, Cat. No. 130‐113‐434, Bergisch Gladbach, Germany) and Cetuximab (ERBITUX, 500 mg/100 mL Cetuximab, Cat. # 06185153) manually labeled with ALC647.

Techniques: Fluorescence, Concentration Assay

Journal: Journal of Extracellular Vesicles

Article Title: In‐Solution Characterization of Extracellular Vesicles: A New Approach to Evaluating Antibody Binding and Surface Interactions

doi: 10.1002/jev2.70269

Figure Lengend Snippet: Binding measurements of increasing concentrations of IgG1‐APC and Cetuximab ALC647 to 1.02 × 10 9 p/mL CD63‐mNG‐Fc EVs. A) Changes of the hydrodynamic radius (Rh) of the complex of CD63‐mNG‐Fc EVs with IgG1‐APC in increasing concentrations of antibody, at 50 mbar ( n = 3). B) Percentage of bound IgG1‐APC by increasing concentrations of antibody to CD63‐mNG‐Fc EVs, at 50 mbar ( n = 3), r 2 = 0.9115. C) Area free/bound IgG1 antibody when incubated with CD63‐mNG‐Fc EVs on increasing concentrations, at 50 mbar ( n = 3), r 2 = 0.9997. D) Changes of the hydrodynamic radius (Rh) of the complex of CD63‐mNG‐Fc EVs with IgG1‐APC in increasing concentrations of antibody, at 100 mbar ( n = 3). E) Percentage of bound IgG1‐APC by increasing concentrations of antibody to CD63‐mNG‐Fc EVs, at 100 mbar ( n = 3), r 2 = 0.8818. F) Area free/bound IgG1 antibody when incubated with CD63‐mNG‐Fc EVs on increasing concentrations, at 100 mbar ( n = 3), r 2 = 0.9996. G) Changes of the hydrodynamic radius (Rh) of the complex of CD63‐mNG‐Fc EVs with Cetuximab in increasing antibody concentrations, at 50 mbar ( n = 3). H) Percentage of bound Cetuximab by increasing concentrations of antibody to CD63‐mNG‐Fc EVs, at 50 mbar ( n = 3), r 2 = 0.9851. I) Area free/bound Cetuximab ALC647 antibody when incubated with CD63‐mNG‐Fc EVs on increasing concentrations, at 50 mbar ( n = 3), r 2 = 0.9950 respectively. J) Changes of the hydrodynamic radius (Rh) of the complex of CD63‐mNG‐Fc EVs with Cetuximab in increasing antibody concentrations, at 100 mbar ( n = 3). K) Percentage of bound Cetuximab antibodies by increasing concentrations of antibody to CD63‐mNG‐Fc EVs, at 100 mbar ( n = 3), r 2 = 0.9537. L) Area free/bound Cetuximab ALC647 antibody when incubated with CD63‐mNG‐Fc EVs on increasing concentrations, at 100 mbar ( n = 3), r 2 = 0.9926, respectively.

Article Snippet: EV samples (FLuoEVs, ciMSC‐EVs, Fc‐EVs, and Control EVs) were diluted in assay buffer to the respective concentration based on the particle concentration determined by NTA and/or IFCM followed by incubation with the respective antibodies: mouse anti‐human CD63 AF647 (H5C6, BD Biosciences, Cat. No. 561983, Herlev, Denmark), REA human IgG1 isotype control‐APC (REA293, Miltenyi Biotech, Cat. No. 130‐113‐434, Bergisch Gladbach, Germany) and Cetuximab (ERBITUX, 500 mg/100 mL Cetuximab, Cat. # 06185153) manually labeled with ALC647.

Techniques: Binding Assay, Incubation