biotinylated egfr antibodies  (R&D Systems)

Journal: ACS sensors

Article Title: Extracellular Vesicle Antibody Microarray for Multiplexed Inner and Outer Protein Analysis.

doi: 10.1021/acssensors.2c01750

Figure Lengend Snippet: Figure 2. EVPio assay optimization for inner protein detection. (A) Illustration of the need for an AR step. (i) The inner proteins of captured EVs are bound by the lipid bilayer membrane and inaccessible to antibodies. (ii) Fixation cross-links inner proteins, while permeabilization allows antibodies to diffuse into EVs, but methylene cross-linking limits the accessibility to epitopes. Permeabilization can be used to allow antibodies to breach the membrane while keeping cytosolic proteins in place, but cross-links can limit the epitope accessibility. (iii) Heat-based AR treatment of EVs induces partial hydrolytic breakage of cross-links, allowing antibody binding of retained and immobilized proteins. (B) AR optimization experiments with different buffers/additives, temperatures and incubation times, and fluorescence detection signal for inner protein HSP90 and outer protein EGFR, with superposed heatmap. The red rectangle indicates the optimal condition that was chosen based on high HSP90 and high EGFR signals. Signal values are above the threshold of NC + 2SD unless grayed. A zero value indicates a negative or zero difference between the signal and its associated no EV control. (C) Fluorescence micrograph of the optimal AR condition: 1 min at 90 °C, in PBS. (D) Bar graph of HSP90 detection signal on spots with CD63 capture antibody and negative control spots with GAM antibody serving as NC. The threshold of NC + 2SD is illustrated as a horizontal line.

Article Snippet: 2022, 7, 3817−3828 3825 Invitrogen), and biotinylated EGFR antibodies (BAF231, R&D Systems) were used at the detection step at concentrations of 5, 5, and 1 μg/mL, respectively, followed by labeling with Alexa Fluor 647- conjugated goat anti-rabbit antibodies (A-21245, Invitrogen) and streptavidin (S21374, Invitrogen).

Techniques: Membrane, Binding Assay, Incubation, Fluorescence, Control, Negative Control

Journal: bioRxiv

Article Title: Chlorotoxin Redirects Chimeric Antigen Receptor T Cells for Specific and Effective Targeting of Glioblastoma

doi: 10.1101/2020.01.24.918888

Figure Lengend Snippet: ( A ) Freshly dispersed viable patient brain tumor (PBT) GBM cells (gated as DAPI-, CD45-, CD31-) were immunostained for expression of IL13Rα2, HER2, EGFR, or binding by Cy5.5-conjugated CLTX. Percentages of stained cells (blue) compared to control staining (grey) are indicated in each histogram. ( B ) Summary of staining results (% positive as in (A)) for 22 primary PBTs ( left ) or 18 PBT tumor spheres (TS) ( right ). ND, not done. ( C ) Representative phenotype of a GBM xenograft established by stereotactic injection of 1 x 10 5 PBT106 TS cells into the right forebrain of an NSG mouse. Tumor-bearing mouse brain was harvested 97 days after cell injection, and paraffin sections were stained with antibodies against IL13Rα2, EGFR, and biotin-conjugated CLTX. Staining was visualized by fluorochrome-conjugated secondary antibody staining and DAPI to identify nuclei. Scale bars: 20μM. ( D ) Freshly-dispersed GBM samples separated into CD44+ and CD44- ( left ) or CD133+ and CD133- ( right ) subpopulations were examined for differences in CLTX-Cy5.5 staining, measured as mean fluorescence intensities (MFI). Only primary PBT samples with >20% of CD44+ or CD133+ fractions were analyzed. ( E ) PBT-TS lines, maintained in neural stem cell medium (TS) or differentiation medium (Dif) for 14 days, were evaluated for CLTX-Cy5.5 and CD133 staining (MFI). ( D, E ) p values are indicated using paired two-way Student’s t-test.

Article Snippet: IF staining was performed on paraffin sections using the procedures of a previous study , with the following antibodies: goat-anti-human IL13Rα2 (R&D systems, polyclonal), mouse-anti-human EGFR (Dako, DAK-H1-WT), and CLTX-biotin.

Techniques: Expressing, Binding Assay, Staining, Injection, Fluorescence

Journal: bioRxiv

Article Title: Chlorotoxin Redirects Chimeric Antigen Receptor T Cells for Specific and Effective Targeting of Glioblastoma

doi: 10.1101/2020.01.24.918888

Figure Lengend Snippet: ( A ) Diagram of a CAR containing a tumor targeting CLTX domain, an IgG4-Fc spacer domain with EQ mutations, a CD4 transmembrane domain, and intracellular signaling domains (CD28 and CD3ζ). ( B ) Immunological synapse formation at 2h after adding CLTX-CAR T cells to GBM cells dissociated from PBT003-4-TS (IL13Rα2-) or PBT106-TS (IL13Rα2+) (E:T=1:1). Top , a representative image of immunological synapse indicated by co-localization of phosphorylated-CD3ζ (p-CD3ζ) and polarized F-actin accumulation at the interface between the CLTX-EQ-28ζ CAR T cell and the tumor cell. Arrowhead: T cell; scale bars: 5 μM. Bottom , Number of immunological-synapses per 5,000 tumor nuclei, when mock-transduced T cells, CLTX-CAR T cells, or IL13Rα2-CAR T cells, were co-cultured with different GBM cells. Five different fields were counted for each group. Mean±SEM is plotted; ***, p<0.001. ( C ) Degranulation of CLTX-CAR T cells (% CD107a) after co-culture with GBM cells from different PBT-TS lines. Mock-transduced T cells, CLTX-CAR T cells or IL13Rα2-CAR T cells were stimulated with either IL13Rα2-negative (PBT003-4, PBT138) or IL13Rα2+ (PBT030-2, PBT106) GBM cells at a 1:1 E:T ratio for 5h, and CD3/CD19t+ gated cells were analyzed for surface CD107a expression as a marker of degranulation. Mean ± S.E.M of % CD107a+ cells in duplicate wells are depicted. ( D ) GBM cells were co-cultured with mock T cells, CLTX-CAR T cells or IL13Rα2-CAR T cells at an effector:target (E:T) ratio of 1:4 for 48 h. CAR T cell activity is shown by the percentage of target cells eliminated. ( C, D ) *, p <0.05; **, p <0.01; ***, p <0.001 compared with mock T cells using one-way ANOVA with Bonferroni’s Multiple Comparison Tests. All PBT numbers indicate PBT-TS lines.

Article Snippet: IF staining was performed on paraffin sections using the procedures of a previous study , with the following antibodies: goat-anti-human IL13Rα2 (R&D systems, polyclonal), mouse-anti-human EGFR (Dako, DAK-H1-WT), and CLTX-biotin.

Techniques: Cell Culture, Co-Culture Assay, Expressing, Marker, Activity Assay, Comparison