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: Cell reports

Article Title: Granulocyte colony stimulating factor promotes scarless tissue regeneration

doi: 10.1016/j.celrep.2024.114742

Figure Lengend Snippet: (A) Parabiosis between WT:WT ( n = 4, red solid line), CXCR2 KO:CXCR2 KO ( n = 3, blue dotted line), and WT:CXCR2 KO mice ( n = 4, black dotted line). Shown is the percentage of ear hole closure. 2-way ANOVA comparing WT:KO pairs to WT:WT pairs. (B) ELISA measuring cytokine expression in injured WT ( n = 4 for day 3, n = 3 for day 7) and CXCR2 KO ( n = 6 for day 3, n = 3 for day 7) plasma. Unpaired two-tailed Student’s t test. (C) WT ( n = 6), CXCR2 KO ( n = 7) and G-CSF depleted CXCR2 KO ( n = 4) plasma was injected daily into the wound bed of WT mice undergoing WIHN for the first 3 days after injury. Shown are representative photographs and quantification of hair follicles. Scale bars, 100 μM. Unpaired two-tailed Student’s t test. (D) G-CSF ( n = 9) or PBS (control, n = 7 ) was injected daily into the wound bed of WT mice undergoing WIHN for the first 3 days after injury. Representative photographs of whole-mount and scanning electron microscopy demonstrating unpigmented hairs in the center of the healed areas. Right: quantification of hair follicles. Scale bars, 100 μM. Unpaired two-tailed Student’s t test. (E) Representative immunofluorescence images of PBS and G-CSF-injected WT wound beds depicting hair follicle structures (Krt14+, Krt6+) Scale bars, 100 μM. (F) Representative photographs and quantification of scar size of G-CSF-treated ( n = 8) or PBS-treated (control, n = 3 ) stented back wounds at day 28 after injury. Scale bars, 1 mm. Unpaired two-tailed Student’s t test. (G) Representative trichrome-stained tissue sections from G-CSF- or PBS-treated stented back wounds. A black line highlights scar size. (H) Quantification of scar diameter for G-CSF ( n = 6) or PBS-treated ( n = 3) mice. Unpaired two-tailed Student’s t test. (I) Wound fibrosis assessed by picrosirius red staining in G-CSF-treated ( n = 14 sections) or PBS-treated ( n = 10 sections). Unpaired two-tailed Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Mean ± SEM are plotted.

Article Snippet: G-CSF , Med Chem Express , HY-P70608.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Clinical Proteomics, Two Tailed Test, Injection, Control, Electron Microscopy, Immunofluorescence, Staining

Journal: Cell reports

Article Title: Granulocyte colony stimulating factor promotes scarless tissue regeneration

doi: 10.1016/j.celrep.2024.114742

Figure Lengend Snippet: (A) Dot plot demonstrating average expression and percentage of immune cells expressing Csf3r . (B) Analysis of key cell-to-cell interactions between immune cells in the ear skin of WT (salmon color) and CXCR2 KO (blue color) mice. Mac, macrophage; T, T cell. (C) Dot plot demonstrating average gene expression of key genes between WT and CXCR2 KO macrophages. (D) GSEA of macrophage populations in WT and CXCR2 KO wounded skin. (E) Representative images and quantification of immunofluorescence of WT and CXCR2 KO wounded skin for CD80 ( n = 4), COX2 ( n = 4), CD163 ( n = 12 for WT and n = 11 for KO), MRC1 ( n = 10 for WT and n = 6 for KO), and ARG1 ( n = 10 for WT and n = 6 for KO). Cell percentages are calculated with total DAPI+ cells as the denominator. Scale bars, 100 μM. Unpaired two-tailed Student’s t test. (F) Representative H&E immunostaining images and quantification of PBS- and G-CSF-injected stented back wounds of WT mice for CD163 ( n = 7 for PBS and n = 5 for G-CSF), MRC1 ( n = 5 for PBS and n = 7 for G-CSF), CD31 ( n = 8 for PBS and n = 7 for G-CSF) and Ki67 ( n = 5 for PBS and n = 4 for G-CSF). Cell percentages are calculated with total DAPI+ cells as the denominator. Scale bars, 100 μM. Unpaired two-tailed Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.01. Mean ± SEM are plotted.

Article Snippet: G-CSF , Med Chem Express , HY-P70608.

Techniques: Expressing, Gene Expression, Immunofluorescence, Two Tailed Test, Immunostaining, Injection

Journal: Cancer Science

Article Title: Novel cancer‐specific epidermal growth factor receptor antibody obtained from the serum of esophageal cancer patients with long‐term survival

doi: 10.1111/cas.15350

Figure Lengend Snippet: Screening flow to identify novel Abs from patients with esophageal cancer who had survived more than 5 years after recurrence. EGFR, epidermal growth factor receptor; SCC, squamous cell carcinoma

Article Snippet: Human EGFR/HER1/ErbB1 protein (His Tag, #10001‐H08H‐20; Sino Biological) and recombinant human EGFR isoform vIII protein CF (#9565‐ER‐050; R&D Systems) were immobilized in a 96‐well immunoplate (MaxiSorp; Nunc).

Techniques:

Journal: Cancer Science

Article Title: Novel cancer‐specific epidermal growth factor receptor antibody obtained from the serum of esophageal cancer patients with long‐term survival

doi: 10.1111/cas.15350

Figure Lengend Snippet: Identification of antigens recognized by KT112 Abs. (A) Results of SDS‐PAGE after immunoprecipitation. Lane 1, OE21_membrane fraction; lane 2, OE21_supernatant fraction; lane 3, OE21_wash fraction; lane 4, OE21_elute fraction; lane 5, MRC5_elute fraction; lane 6, MRC5_wash fraction; and lane 7, MRC5_supernatant fraction. (B) Reactivity of the cell after siRNA treatment by flow cytometry analysis. Thin solid line shows the reactivity of the DNP Ab to cells treated with negative control siRNA, the thick solid line shows the reactivity of the KT112 Ab to cells treated with negative control siRNA, and the dotted line shows the reactivity of KT112 Abs to cells treated with epidermal growth factor receptor (EGFR) siRNA. vIII, variant III; scFv, single‐chain Fv

Article Snippet: Human EGFR/HER1/ErbB1 protein (His Tag, #10001‐H08H‐20; Sino Biological) and recombinant human EGFR isoform vIII protein CF (#9565‐ER‐050; R&D Systems) were immobilized in a 96‐well immunoplate (MaxiSorp; Nunc).

Techniques: SDS Page, Immunoprecipitation, Membrane, Flow Cytometry, Negative Control, Variant Assay

Journal: Cancer Science

Article Title: Novel cancer‐specific epidermal growth factor receptor antibody obtained from the serum of esophageal cancer patients with long‐term survival

doi: 10.1111/cas.15350

Figure Lengend Snippet: Reactivity to recombinant epidermal growth factor receptor (EGFR; left panel) and EGFR variant III (vIII; right panel) was analyzed by ELISA. Black circles, KT112 Ab; black squares, cetuximab; black triangles, AM1; white circles, DNP Ab (5 μg/ml only); white triangles, panitumumab

Article Snippet: Human EGFR/HER1/ErbB1 protein (His Tag, #10001‐H08H‐20; Sino Biological) and recombinant human EGFR isoform vIII protein CF (#9565‐ER‐050; R&D Systems) were immobilized in a 96‐well immunoplate (MaxiSorp; Nunc).

Techniques: Recombinant, Variant Assay, Enzyme-linked Immunosorbent Assay

Journal: Cancer Science

Article Title: Novel cancer‐specific epidermal growth factor receptor antibody obtained from the serum of esophageal cancer patients with long‐term survival

doi: 10.1111/cas.15350

Figure Lengend Snippet: Competitive inhibition of each epidermal growth factor receptor (EGFR) Ab. The reactivity of each EGFR Ab to OE21 cells was analyzed by flow cytometry. The horizontal axis indicates the Ab name, and the vertical axis indicates mean fluorescence intensity. (A) Reactivity of the KT112 Ab in the presence of a competitive Ab is shown. Concentrations of the competitive Abs are 0.3, 1, 3, 10, and 30 μg/ml. 2,4‐Dinitrophenol (DNP) Ab was analyzed only at 30 μg/ml. AM1 Ab was analyzed at 3, 10, and 30 μg/ml. The concentration of the biotinylated KT112 Ab for detection is 0.8 μg/ml. (B) Reactivity of each Ab in the presence of the KT112 Ab. Concentrations of the competitive Abs are 0.3, 1, 3, 10, and 30 μg/ml. Concentrations of the Abs for detection are 0.8 μg/ml for the biotinylated KT112 Ab, 0.1 μg/ml each for biotinylated cetuximab and panitumumab, and 2 μg/ml for the biotinylated AM1 Ab

Article Snippet: Human EGFR/HER1/ErbB1 protein (His Tag, #10001‐H08H‐20; Sino Biological) and recombinant human EGFR isoform vIII protein CF (#9565‐ER‐050; R&D Systems) were immobilized in a 96‐well immunoplate (MaxiSorp; Nunc).

Techniques: Inhibition, Flow Cytometry, Fluorescence, Concentration Assay