Journal: Chemistry of Materials

Article Title: Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET

doi: 10.1021/acs.chemmater.6b03198

Figure Lengend Snippet: Figure 2. Representative normalized PL intensity decay curves (measured at 659 ± 10 nm) of AB−QD and Tb−AB conjugates mixed with increasing concentrations of EGFR (0 nM, black; 0.9 nM, red; 2.25 nM, blue; 4.5 nM, orange; and 9 nM, green) for the Tb-Cet/Mat-QD650 (A), Tb-Cet/MatFab-QD650 (B), and Tb-EgA1/EgB4-QD650 (C) FRET pairs. Yellow arrows indicate QD FRET sensitization with increasing EGFR concentration. The magenta curves present the mathematical sum of only AB-QD650 and only the Tb−AB conjugate. Although the optical bandpass filters (cf. Figure 1B) were selected to minimize Tb crosstalk into the QD detection channels, there was still significant Tb PL detectable. For intensity normalization, all curves within one graph where multiplied by the value that led to unity intensity for the magenta curve at 0.5 ms. PL decay curves (Tb donors and QD acceptors) of all immunoassays can be found in the Supporting Information (Supporting Figures S1−S8).

Article Snippet: Recombinant human EGFR Fc chimera (#344-ER-050) and HER2 Fc chimera (#1129-ER-050) dimers were purchased from R&D system.

Techniques: Concentration Assay

Journal: Chemistry of Materials

Article Title: Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET

doi: 10.1021/acs.chemmater.6b03198

Figure Lengend Snippet: Figure 3. Homogeneous FRET immunoassay calibration curves against EGFR using different Tb−AB and AB−QD conjugates (blue, Tb-Cet/MatFab-QD605; green, Tb-Cet/MatFab-QD650; cyan, Tb- Cet/Mat-QD605; black, Tb-Mat/Cet-QD650; red, Tb-Cet/Mat- QD650; orange, Tb-EgA1/EgB4-QD650; brown, Tb-EgA1/EgB4- QD605; magenta, Tb-Cet/MatFab-QD650 measured in serum samples). [EGFR] corresponds to the variable EGFR dimer concentrations (recombinant human EGFR Fc chimera) in the 50 μL EGFR samples (in buffer or serum), whereas the overall measuring volume of 150 μL also contained 100 μL of a constant assay solution (50 μL of Tb−AB conjugate with 9 nM AB and 50 μL of AB−QD conjugate with 1.5 nM of QD650 or 3 nM of QD605 for all samples). Individual curves for the Tb and QD signals (that lead to the calculation of FR) and for the determination of LODs can be found in the Supporting Information (Supporting Figures S11−S18).

Article Snippet: Recombinant human EGFR Fc chimera (#344-ER-050) and HER2 Fc chimera (#1129-ER-050) dimers were purchased from R&D system.

Techniques: Recombinant

Journal: Chemistry of Materials

Article Title: Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET

doi: 10.1021/acs.chemmater.6b03198

Figure Lengend Snippet: Figure 5. Specificity of the different AB pairs for EGFR (red) or HER2 (blue). Tb-Cet/Mat-QD650 (A) and Tb-Tras/Pert-QD650 (B) showed a concentration-dependent FRET-ratio increase only for their respective receptors. Tb-Pert/Mat-QD650 (C) and Tb-Tras/Mat-QD650 (D) showed a concentration-dependent FRET-ratio increase for HER2, which indicates a cross-reactivity of the EGFR-specific Mat to HER2. Tb-Cet/Pert-QD650 did not lead to any concentration dependent FRET-ratio increase (Supporting Figure S27).

Article Snippet: Recombinant human EGFR Fc chimera (#344-ER-050) and HER2 Fc chimera (#1129-ER-050) dimers were purchased from R&D system.

Techniques: Concentration Assay

Journal: Chemistry of Materials

Article Title: Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET

doi: 10.1021/acs.chemmater.6b03198

Figure Lengend Snippet: Figure 6. Duplexed immunoassay (all samples containing Tb-Cet, Tb-Tras, Mat-QD605, and Pert-QD650) calibration curves of EGFR (FRET-ratio of QD605 and Tb PL intensities, A) and HER2 (FRET-ratio of QD650 and Tb PL intensities, B) for increasing (0 nM, black; 0.3 nM, green; 0.6 nM, magenta; 1.2 nM, red; 1.8 nM, cyan; 2.4 nM, brown; 3 nM, blue) HER2 (A) and EGFR (B) concentrations.

Article Snippet: Recombinant human EGFR Fc chimera (#344-ER-050) and HER2 Fc chimera (#1129-ER-050) dimers were purchased from R&D system.

Techniques:

Journal: Chemistry of Materials

Article Title: Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET

doi: 10.1021/acs.chemmater.6b03198

Figure Lengend Snippet: Figure 7. Duplexed FRET immunoassay for the quantification of EGFR (blue squares) and HER2 (red dots) within 19 different samples. Dotted lines represent the known concentrations, and data points represent the measured concentrations (±10% error bars).

Article Snippet: Recombinant human EGFR Fc chimera (#344-ER-050) and HER2 Fc chimera (#1129-ER-050) dimers were purchased from R&D system.

Techniques:

Journal: Biomedicines

Article Title: A Rational Designed Novel Bispecific Antibody for the Treatment of GBM

doi: 10.3390/biomedicines9060640

Figure Lengend Snippet: ( a ) EGFRvIII antigen expression level on mutant GBM cell line U87MG.ΔEGFR and wild-type GBM cell line U87MG. ( b ) A binding activity comparison of EGFRvIII-BsAb and CD3 mAb with Jurkat cells (CD3-positive) (upper), as well as a binding activity comparison of the EGFRvIII-BsAb and the EGFRvIII mAb with U87MG.ΔEGFR cells (EGFRvIII-positive) (lower). ( c ) Photographs of the redirection of T cells to cancer cells by 0.01 ng/mL EGFRvIII-BsAb or EGFRvIII mAb. ( d ) FACS analysis of the redirection of CD3+ Jurkat cells to cancer cells by EGFRvIII-BsAb. Jurkat (CD3+) cells labeled by PKH26 (PE-A), as well as U87MG.ΔEGFR cells labeled by CFSE (FITC-A).

Article Snippet: The EGFRvIII antigen (AVI10494; R&D System, Minneapolis, MN, USA) and extracellular domain of human CD3D/CD3E heterodimer (CT038-H2508H; Sino Biological, Beijing, China) were immobilized to a CM5 chip (29149603; GE Healthcare, Chicago, IL, USA) surface using standard protocols with 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) amine.

Techniques: Expressing, Mutagenesis, Binding Assay, Activity Assay, Comparison, Labeling

Journal: PLoS ONE

Article Title: A comparative analysis of EGFR-targeting antibodies for gold nanoparticle CT imaging of lung cancer

doi: 10.1371/journal.pone.0206950

Figure Lengend Snippet: (A) shows the ELISA signal (reflecting EGFR binding) normalized by nanoparticle concentration. Both C225 and VHH-122 have high binding, while PEG has almost none. Differences in binding between all three nanoparticle types are statistically significant. (B) shows the ELISA signal normalized by antibody concentration. Here, C225 has much higher signal than VHH-122, consistent with its higher binding affinity and lower number of antibodies per nanoparticle.

Article Snippet: Antibody-conjugated AuNPs were blocked with 10% donkey serum in PBS, then incubated with a soluble recombinant human EGFR extracellular domain-Fc chimera (Sino Biological) at 10 nM in blocking buffer for 2 hours.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay

Journal: PLoS ONE

Article Title: A comparative analysis of EGFR-targeting antibodies for gold nanoparticle CT imaging of lung cancer

doi: 10.1371/journal.pone.0206950

Figure Lengend Snippet: AuNPs appear as bright yellow-orange dots in the dark field images. PEG-AuNPs (A) showed little to no cell binding, VHH-AuNPs (B) showed a moderate level of cell binding, while C225-AuNPs (C) had a high number of nanoparticles bound to the cells in culture. This demonstrates effective EGFR-targeting, particularly for the C225-AuNPs. Scale bars are 30 μ m.

Article Snippet: Antibody-conjugated AuNPs were blocked with 10% donkey serum in PBS, then incubated with a soluble recombinant human EGFR extracellular domain-Fc chimera (Sino Biological) at 10 nM in blocking buffer for 2 hours.

Techniques: Binding Assay

Journal: Molecular Cancer

Article Title: Lung endothelial PEAR1 induces tumor cell dormancy

doi: 10.1186/s12943-025-02488-3

Figure Lengend Snippet: PEAR1 sequesters CTSD and LOXL2 to promote tumor cell dormancy. A , MDA-MB-231 cells were treated with increasing concentrations of the extracellular domain of recombinant human PEAR1 containing a His-tag and immobilized on Ni-NTA beads. Cells incubated with Ni-NTA beads only served as a control group. Cell count was determined by flow cytometry analysis ( n = 3 independent experiments). B , MDA-MB-231 cells were treated daily for 4 days with PEAR1 immobilized on Ni-NTA beads, and cells were analyzed by immunoblotting using anti-GAPDH and anti-p27 antibodies. Shown is a representative immunoblot and the statistical analysis ( n = 3 independently performed experiments). C , D , MDA-MB-231 cells expressing mVenus-p27K − were transfected with control siRNA or siRNA directed against SVEP1 or LOXL2 and were incubated in the absence or presence of PEAR1 (100 nM). After 36 h, the cell count (C) and the percentage of p27-positive cells (D) was determined ( n = 3 independent experiments). E , Procedure to search for interaction partners of PEAR1 within the conditioned medium of tumor cells using a pull-down assay with PEAR1 immobilized on Ni-NTA beads. Control Ni-NTA beads and the recombinant extracellular part of human epidermal growth factor receptor immobilized on Ni-NTA beads served as controls. F , The plot shows the proteins that were specifically co-precipitated with PEAR1 compared to control samples. Proteins only found with PEAR1 are indicated by green dots, and proteins found with both PEAR1 and EGFR are represented by grey dots. G , Recombinant CTSD, SEMA6D or LOXL2 were incubated alone or together with purified His-tagged PEAR1. PEAR1 was precipitated with Ni-NTA beads, and precipitates were analyzed by immunoblotting. Shown are representative of 3 independently performed experiments. H , I , MDA-MB-231 cells co-expressing mCherry-luciferase and mVenus-p27K − were co-cultured with HUVECs for the indicated time periods, and cells were treated daily without or with CTSD (100 nM), LOXL2 (100 nM) or SEMA6D (1000 nM). The cell count (H) was assessed using flow cytometry. At the end of the experiment, the percentage of p27-positive cells (l) was determined by flow cytometry ( n = 3 independent experiments). J , K , Co-cultures of HUVECs and MDA-MD-231 cells were incubated for the indicated time periods with 100 nM PEAR1 alone or in combination with 100 nM CTSD or LOXL2. Assessment of cell count (J) was conducted utilizing flow cytometry, and, after 4 days, the percentage of p27-positive cells (K) was analyzed by flow cytometry ( n = 3 independent experiments). L , M , MDA-MB-231 cells co-expressing mCherry and mVenus-p27K − were transfected with control siRNA or siRNA directed against CTSD, LOXL2 or both, and were cultured in the absence or presence of the recombinant extracellular part of human PEAR1 (rhPEAR1; 100 nM). After 96 h, cells were counted (L), and p27 expression was determined (M) ( n = 3 independently performed experiments). Shown are mean values ± S.E.M.; * , p ≤ 0.05; ** , p ≤ 0.01; *** , p ≤ 0.001; n.s., non-significant (2-way ANOVA and Bonferroni’s post-hoc test (A-D, H, J, L, M); multiple two-tailed t-test, Bayesian moderated, Benjamin, Krieger, and Yekutieli-corrected, false discovery rate = 1% (F); one-way ANOVA with Tukey’s multiple comparison test (I, K))

Article Snippet: Human recombinant PEAR1 extracellular part (catalog #4527-PR-050), human Sema6D extracellular part (catalog #2095-S6), human cathepsin D (catalog #1014-AS), the extracellular part of the human EGF receptor (EGFR) (catalog # AVI10493 ), mouse recombinant Sema6D extracellular part (catalog #7067-S6), human recombinant LOXL2 (catalog #2639-AO-010), and mouse recombinant LOXL2 (catalog #9259-AO) were purchased from R&D systems.

Techniques: Recombinant, Incubation, Control, Cell Counting, Flow Cytometry, Western Blot, Expressing, Transfection, Pull Down Assay, Purification, Luciferase, Cell Culture, Two Tailed Test, Comparison