igg1 isotype control  (Bio X Cell)

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

Journal: Science Advances

Article Title: Inflammatory arthritis irAE may represent a unique autoimmune disease primarily driven by T cells but likely not autoantibodies

doi: 10.1126/sciadv.aea4262

Figure Lengend Snippet: ( A ) Expression of CD27 and IgD on CD19 + B cells. Right: Percentage of IgD + CD27 − naïve B cells and IgD − CD27 − DN B cells. ( B ) Expression of CD27 and CD38 on CD19 + B cells. Right: Percentages of CD27 hi CD38 hi ASCs and CD27 + CD38 − memory B cells. ( C ) Expression of CD138 on CD27 hi CD38 hi ASCs. Right: Percentage of CD138 + ASCs and CD138 − ASCs. ( D ) Expression of CD11c and CD21 on CD19 + B cells. Right: Percentage of CD11c + CD21 − CD19 + B cells. [(A) to (D)] HC ( n = 63), irAE ( n = 33), RAC ( n = 46), and ICI ( n = 20). ( E to G ) GSEA was performed on the B cells between irAE and ICI. (E) Significantly enriched pathways in B cells from irAE and ICI. GSEA plots of IFN-α and IFN-γ response (F), and oxidative phosphorylation (G). ( H ) The volcano plots of the citrullinated or noncitrullinated relative IgG or IgM isotype autoantigen levels comparing RA versus HC, irAE versus ICI, irAE versus HC, or irAE versus RA. The autoantigens were labeled when P < 0.01. TNF-α reactivities resulted from the administration of anti–TNF-α therapy for the treatment of RA. ( I ) Immunoglobulin isotype levels in the plasma were measured by multiplex assay. HC ( n = 22), irAE ( n = 34), RAC ( n = 46), and ICI ( n = 26). ( J ) CXCL13 levels in the plasma were measured by enzyme-linked immunosorbent assay (ELISA). HC ( n = 20), irAE ( n = 34) RAC ( n = 47), and ICI ( n = 23). ( K ) B-cell activating factor (BAFF) levels in the plasma were measured by multiplex assay. HC ( n = 21), irAE ( n = 34), RAC ( n = 47), and ICI ( n = 18). Data in graphs represent mean ± SEM. Significance was tested by one-way ANOVA [(A) to (D) and (I) to (K)] and logistic regression (H). [(A) to (D) and (F) to (K)] ICI, ICI control.

Article Snippet: PBMCs from patients with irAE were thawed and rested in a complete medium for 1 hour and then activated with plate-coated anti–human CD3 and anti–human CD28 (10 μg/ml) with IgG1 isotype control (Bio X Cell, catalog no. CP174) or a combination of anti–human IL-6R 50 μg/ml; Bio X Cell, catalog no. SIM0014), anti–human IL-12p40 (50 μg/ml; Bio X Cell, catalog no. SIM0020), and anti–human IFNAR1 (50 μg/ml; Bio X Cell, catalog no. SIM0022) for 3 days.

Techniques: Expressing, Phospho-proteomics, Immunopeptidomics, Labeling, Clinical Proteomics, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Science Advances

Article Title: Inflammatory arthritis irAE may represent a unique autoimmune disease primarily driven by T cells but likely not autoantibodies

doi: 10.1126/sciadv.aea4262

Figure Lengend Snippet: ( A to C ) Human naïve B cells were isolated from PBMCs and cultured under indicated conditions for 7 days; pembrolizumab or isotype control IgG4 (IgG4 Iso) was added on day 2; n = 8. (A) Expression of CD38 and CD27 on B cells. Right: Percentages of CD27 + CD38 − and CD27 + CD38 + B cells. (B) Expression of CD138 on CD27 + CD38 + B cells. Right: Percentage of CD138 + CD27 + CD38 + B cells. (C) Different immunoglobulin isotypes were measured in the culture supernatant of (A) and (B) by multiplex assay. ( D to F ) B cells from Humanized PD-1 (HuPD-1) mice were isolated and cultured with LPS, IL-4, BAFF, or ODN 2006, anti-IgM, IL-21, IL-4 or R848, anti-IgM, anti-CD40, IL-21, and IFN-γ for 3 days; pembrolizumab or isotype control was added on day 1. (D) Expression of IgG2c on activated B cells. FSC-H, Forward scatter height. Right: Percentage of IgG2c + B cells; n = 5. (E) Expression of IgG1 on activated B cells. Right: Percentage of IgG1 + B cells; n = 5. (F) Different immunoglobulin isotypes in the supernatant were measured by multiplex assay; n = 5. Data in graphs represent mean ± SEM. Significance was tested by two-way ANOVA.

Article Snippet: PBMCs from patients with irAE were thawed and rested in a complete medium for 1 hour and then activated with plate-coated anti–human CD3 and anti–human CD28 (10 μg/ml) with IgG1 isotype control (Bio X Cell, catalog no. CP174) or a combination of anti–human IL-6R 50 μg/ml; Bio X Cell, catalog no. SIM0014), anti–human IL-12p40 (50 μg/ml; Bio X Cell, catalog no. SIM0020), and anti–human IFNAR1 (50 μg/ml; Bio X Cell, catalog no. SIM0022) for 3 days.

Techniques: Isolation, Cell Culture, Control, Expressing, Multiplex Assay

Journal: Science Advances

Article Title: Inflammatory arthritis irAE may represent a unique autoimmune disease primarily driven by T cells but likely not autoantibodies

doi: 10.1126/sciadv.aea4262

Figure Lengend Snippet: ( A to G ) PBMCs from the patients with irAE were cultured in the plate coated with anti-CD3 and anti-CD28 (10 μg/ml) in the presence of IgG1 isotype control or anti–human IL-6R (50 μg/ml), anti–human IL-12p40 (50 μg/ml), and anti–human IFNAR1 (50 μg/ml) for 3 days; n = 9. (A) Expression of CD38 and CD127 on CD8 + T cells. Right: Percentage of CD38 + CD127 − CD8 + T cells. (B and C) CD8 + T cells activated for 5 days were restimulated with PMA, ionomycin, and monensin for 5 hours. (B) Expression of granzyme B and IFN-γ. Right: Percentage of granzyme B + IFN-γ + CD8 + T cells. (C) Expression of perforin and IFN-γ. Right: Percentage of perforin + IFN-γ + CD8 + T cells. [(D) and (E)] MFIs of GluCy5 (D), TMRM, and MTDR (E) in CD8 + T cells were presented. [(F) and (G)] CD4 + T cells activated for 5 days were restimulated with PMA, ionomycin, and monensin for 5 hours. (F) Expression of IL-21 and IL-2. Right: Percentage of IL-21 + IL-2 + CD4 + T cells. (G) Expression of CD38 and CXCR5 on CD4 + T cells. Right: Percentage of CD38 + CXCR5 − CD4 + T cells. Data in graphs represent mean ± SEM. Significance was tested paired Student’s t test [(A) to (G)].

Article Snippet: PBMCs from patients with irAE were thawed and rested in a complete medium for 1 hour and then activated with plate-coated anti–human CD3 and anti–human CD28 (10 μg/ml) with IgG1 isotype control (Bio X Cell, catalog no. CP174) or a combination of anti–human IL-6R 50 μg/ml; Bio X Cell, catalog no. SIM0014), anti–human IL-12p40 (50 μg/ml; Bio X Cell, catalog no. SIM0020), and anti–human IFNAR1 (50 μg/ml; Bio X Cell, catalog no. SIM0022) for 3 days.

Techniques: Cell Culture, Control, Expressing