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: 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

Journal: British journal of cancer

Article Title: Quantification of HER1, HER2 and HER3 by time-resolved Förster resonance energy transfer in FFPE triple-negative breast cancer samples.

doi: 10.1038/s41416-019-0670-8

Figure Lengend Snippet: Fig. 1 Calibration curve plots showing the limit of detection (LOD), lower limit of quantification (LLOQ), linear dynamic range, and upper limit of quantitation (ULOQ) of the HER1, HER2 and HER3 TR-FRET assays. For each assay, the LOD was calculated as the average signal of 20 replicate-negative samples plus three standard deviations. The lower limit of quantification (LLOQ) and the upper limit of quantification (ULOQ) were the lowest and highest standard point that can be measured with <15% coefficient of variance (CV).

Article Snippet: Recombinant HER1, HER2 and HER3 were purchased from Origene technologies (Catalog No.: TP710011; TP710032 and TP710089, respectively).

Techniques: Quantitation Assay

Journal: British journal of cancer

Article Title: Quantification of HER1, HER2 and HER3 by time-resolved Förster resonance energy transfer in FFPE triple-negative breast cancer samples.

doi: 10.1038/s41416-019-0670-8

Figure Lengend Snippet: Fig. 2 Quantitative measurement of HER1, HER2 and HER3 expression by TR-FRET in 51 TNBC samples.

Article Snippet: Recombinant HER1, HER2 and HER3 were purchased from Origene technologies (Catalog No.: TP710011; TP710032 and TP710089, respectively).

Techniques: Expressing