hepha2  (R&D Systems Hematology)

Journal: PLoS ONE

Article Title: Engineering of monobody conjugates for human EphA2-specific optical imaging

doi: 10.1371/journal.pone.0180786

Figure Lengend Snippet: (A) Western blot analysis of hEphA2. Lysates of PC3 and HeLa cells (8 × 10 5 ) were separated by SDS-PAGE and transferred to nylon membranes. Membranes were stained with rabbit anti-hEphA2 or β-actin antibodies. (B) Flow cytometric analysis of cells. Cells (5 × 10 5 ) were stained with mouse anti-hEphA2 and Alexa 488-conjugated anti-mouse IgG antibodies (anti-hEphA2), and fluorescence was measured by flow cytometry. Unstained cells or cells stained with Alexa 488-conjugated anti-mouse IgG antibody (2 nd Ab) alone were used as controls.

Article Snippet: Cells (1 × 10 4 ) were cultured on cover glasses on 6-well plates for 24 h. To detect bound E1-Rluc8, cells were incubated with 25.5 nM E1-Rluc8 and rabbit anti-hEphA2 antibody (1:10000 dilution, Santa Cruz Biotechnology), in 3 mL of culture medium containing 3% FBS at 37°C for 2 h. After the plates were washed with 1% BSA in PBS-T, they were incubated in FITC-conjugated anti-6×His (1:1000 dilution) and Alexa 555-conjugated anti-rabbit IgG antibodies (1:1000 dilution) for 2 h in the dark at room temperature (RT).

Techniques: Western Blot, SDS Page, Staining, Fluorescence, Flow Cytometry

Journal: PLoS ONE

Article Title: Engineering of monobody conjugates for human EphA2-specific optical imaging

doi: 10.1371/journal.pone.0180786

Figure Lengend Snippet: (A) Flow cytometric analysis of cells treated with E1-Rluc8 and E1-EGFP. PC3 and HeLa cells (5 × 10 5 ) detached from culture plates were treated with E1-Rluc8 and E1-EGFP. The cells treated with E1-Rluc8 were stained with FITC-conjugated mouse anti-6×His antibody. Unstained cells or cells stained with FITC-conjugated secondary antibody (2 nd Ab) alone were used as controls. (B) Fluorescence microscopy images of the cells. PC3 and HeLa cells were stained with mouse anti-hEphA2 antibody and E1-Rluc8, and then detected with Alexa 555-conjugated anti-mouse IgG (red) and FITC-conjugated anti-6×His antibodies (green). Cell nuclei were stained with DAPI (blue). Scale bar, 10 μm. (C) Fluorescence microscopy images of xenograft tumor tissues. PC3 tumor tissues from transplanted nude mice were stained with the antibody combinations used in (B). Scale bar, 10 μm.

Article Snippet: Cells (1 × 10 4 ) were cultured on cover glasses on 6-well plates for 24 h. To detect bound E1-Rluc8, cells were incubated with 25.5 nM E1-Rluc8 and rabbit anti-hEphA2 antibody (1:10000 dilution, Santa Cruz Biotechnology), in 3 mL of culture medium containing 3% FBS at 37°C for 2 h. After the plates were washed with 1% BSA in PBS-T, they were incubated in FITC-conjugated anti-6×His (1:1000 dilution) and Alexa 555-conjugated anti-rabbit IgG antibodies (1:1000 dilution) for 2 h in the dark at room temperature (RT).

Techniques: Staining, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Engineering of monobody conjugates for human EphA2-specific optical imaging

doi: 10.1371/journal.pone.0180786

Figure Lengend Snippet: (A) Measurement of luminescence in mice injected with different concentrations of E1-Rluc8. PC3 and HeLa cells were transplanted into 6-week-old Balb/c nude mice via subcutaneous injection (n = 3). After tumor formation, the indicated amounts of E1-Rluc8 and coelenterazine were intravenously injected via the tail vein, and luminescence images were acquired at the indicated times with the NightOWL in vivo imaging system (left). The luminescence intensities measured in tumor areas at 6 h were graphed (right). (B) Luminescence maintenance in mice injected with E1-Rluc8 for 24 h. E1-Rluc8 (60 μg) and coelenterazine were intravenously injected via the tail vein into PC3 tumor mice (n = 5), and luminescence images were obtained at the indicated times (left). The luminescence intensities in tumor tissues were measured with the NightOWL in vivo imaging system and graphed (right). (C) Detection of the remaining E1-Rluc8 in PC3 tumor tissues. Tumor tissues were collected at the indicated times from mice injected with E1-Rluc8 and stained with FITC-conjugated anti-6×His antibodies (green). hEphA2 was stained with mouse anti-hEphA2 antibody and Alexa 555-conjugated anti-mouse IgG (red). Cell nuclei were stained with DAPI (blue). Scale bar, 10 μm.

Article Snippet: Cells (1 × 10 4 ) were cultured on cover glasses on 6-well plates for 24 h. To detect bound E1-Rluc8, cells were incubated with 25.5 nM E1-Rluc8 and rabbit anti-hEphA2 antibody (1:10000 dilution, Santa Cruz Biotechnology), in 3 mL of culture medium containing 3% FBS at 37°C for 2 h. After the plates were washed with 1% BSA in PBS-T, they were incubated in FITC-conjugated anti-6×His (1:1000 dilution) and Alexa 555-conjugated anti-rabbit IgG antibodies (1:1000 dilution) for 2 h in the dark at room temperature (RT).

Techniques: Injection, In Vivo Imaging, Staining

Journal: PLoS ONE

Article Title: High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting

doi: 10.1371/journal.pone.0135278

Figure Lengend Snippet: Phage supernatants of the seven clones with different loop DE sequences were tested by ELISA on plates coated with mEphA2-Fc, hEphA2, DR6-Fc and BSA to asses specificity of binding and crossreactivity.

Article Snippet: Chinese Hamster Ovary K1 cells (CHOK1), (ATCC, Manassas, Virginia, USA) were transfected with transfection-ready hEphA2 cDNA (OriGene Technologies, Rockville, Maryland, USA) according to the manufacturer’s instructions.

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

Journal: PLoS ONE

Article Title: High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting

doi: 10.1371/journal.pone.0135278

Figure Lengend Snippet: Phage supernatants of the seven clones with different loop DE sequences were tested by cell-based ELISA on HEK293 cells expressing doxycycline-inducible hEphA2. All the Abdurin variants specifically recognize the hEphA2 receptor in its native conformation on cells. Assays were done in the absence or presence of doxycycline with concentrations of the phage ranging from 25 ng/ml to 100 ng/ml.

Article Snippet: Chinese Hamster Ovary K1 cells (CHOK1), (ATCC, Manassas, Virginia, USA) were transfected with transfection-ready hEphA2 cDNA (OriGene Technologies, Rockville, Maryland, USA) according to the manufacturer’s instructions.

Techniques: Clone Assay, In-Cell ELISA, Expressing

Journal: PLoS ONE

Article Title: High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting

doi: 10.1371/journal.pone.0135278

Figure Lengend Snippet: Affinity matured binders to EphA2, selected by CIS display, were tested in end-point titration ELISA assays in 96-well plates coated with hEphA2-Fc (a), mEphA2-Fc (b) or strepavidin (c). The purified matured Abdurin clones D2, G7, B6 and B11 were tested alongside the purified non-matured parental EphA2 binding clones selected by phage display, E10par and H3par, as detected by anti-FLAG M2-HRP conjugated antibody. Binding of Abdurin binders and the truncated wild-type human CH2 (shWTCH2) to CHO cells transfected with human EphA2 (d) or to non-transfected cells (e), was assessed by FACS analysis.

Article Snippet: Chinese Hamster Ovary K1 cells (CHOK1), (ATCC, Manassas, Virginia, USA) were transfected with transfection-ready hEphA2 cDNA (OriGene Technologies, Rockville, Maryland, USA) according to the manufacturer’s instructions.

Techniques: Titration, Enzyme-linked Immunosorbent Assay, Purification, Clone Assay, Binding Assay, Transfection

Journal: PLoS ONE

Article Title: High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting

doi: 10.1371/journal.pone.0135278

Figure Lengend Snippet: K D measured using Biacore 3000.

Article Snippet: Chinese Hamster Ovary K1 cells (CHOK1), (ATCC, Manassas, Virginia, USA) were transfected with transfection-ready hEphA2 cDNA (OriGene Technologies, Rockville, Maryland, USA) according to the manufacturer’s instructions.

Techniques:

Journal: PLoS ONE

Article Title: High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting

doi: 10.1371/journal.pone.0135278

Figure Lengend Snippet: Affinity measurement of binding to hEphA2 transfected cells.

Article Snippet: Chinese Hamster Ovary K1 cells (CHOK1), (ATCC, Manassas, Virginia, USA) were transfected with transfection-ready hEphA2 cDNA (OriGene Technologies, Rockville, Maryland, USA) according to the manufacturer’s instructions.

Techniques: Binding Assay, Transfection

Journal: PLoS ONE

Article Title: High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting

doi: 10.1371/journal.pone.0135278

Figure Lengend Snippet: A) HEK293-hEphA2 cells were seeded in 96 well plates and either, not treated (top panel), or treated (bottom panel) with 100 nM doxycycline for 24 hours at 37°C to induce over-expression of the transfected EphA2 receptor. Cells were incubated with 1μg/well B11 for 30 minutes at 4°C after which time cells were fixed in 2% PFA. B11 binding was monitored by anti-FLAG antibody and anti-mouse AF488 secondary antibody. B) PC-3 cells seeded in 96 well plates were incubated with 1μg/well B11 and 10 μg/well of the anti-EphA2 monoclonal antibody (mAb) (molar ratio 1:1; bottom panel) or an unrelated mAb (top panel) for 30 minutes at 4°C followed by 1 hour incubation at 37°C to allow internalization. Cells were then fixed in 2% PFA and B11 binding monitored by anti-FLAG-AF488 antibody. Nuclei were stained with DAPI. Images were acquired at IN Cell 2000.

Article Snippet: Chinese Hamster Ovary K1 cells (CHOK1), (ATCC, Manassas, Virginia, USA) were transfected with transfection-ready hEphA2 cDNA (OriGene Technologies, Rockville, Maryland, USA) according to the manufacturer’s instructions.

Techniques: Over Expression, Transfection, Incubation, Binding Assay, Staining