Journal: Protein engineering, design & selection : PEDS

Article Title: Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules.

doi: 10.1093/protein/gzq118

Figure Lengend Snippet: Fig. 1. (A) Amino acid sequences (one letter code) of the parental Zwt (with randomized positions indicated with closed circles) and phage-display-selected ErbB3-binding Affibody molecules. Representation among sequenced clones is shown after each sequence. Below the sequences of phage-display-selected Affibody molecules is a schematic overview of the design of the affinity maturation Affibody library (ZErbB3LIB) for staphylococcal display selection showing amino acid representation (one letter code) in 12 randomized positions within helices 1 and 2. Note that position 17 (W) is not randomized in the maturation library. Bottom images: immunofluorescence microscopy image of (B) ErbB3-high AU565 cells stained with His6-(Z01814)2-cys. (C) ErbB3-high AU565 cells stained with His6-(Z01820)2-cys. (D) ErbB3-high AU565 cells stained with Anti-ErbB3 antibody (positive control). (E) ErbB3-high AU565 cells stained with (Z01154)2 (negative control). (F) ErbB3-low/ErbB2-high SK-OV-3 cells stained with His6-(Z01814)2-cys. (G) ErbB3-low/ErbB2-high SK-OV-3 cells stained with His6-(Z01820)2-cys.

Article Snippet: An anti-ErbB3 antibody (AF234 Goat-anti-HER3 ab; R&D systems) was included as a positive control in both experiments.

Techniques: Binding Assay, Clone Assay, Sequencing, Selection, Microscopy, Staining, Positive Control, Negative Control

Journal: Protein engineering, design & selection : PEDS

Article Title: Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules.

doi: 10.1093/protein/gzq118

Figure Lengend Snippet: Fig. 2. Density plots showing the results from flow-cytometric sortings of Sc:ZErbB3LIB. FL-4 channel fluorescence intensity corresponding to surface expression level (monitored via HSA binding) on the x-axis and FL-1 channel fluorescence corresponding to ErbB3 binding on the y-axis. The density plots are showing the staphylococcal library before flow-cytometric sorting of rounds 1, 2, 3 and 4, respectively, with regions used for gating outlined in each plot. Note that the concentration of biotinylated ErbB3-Fc is decreased in each sorting round, resulting in decreasing signal intensities on average in subsequent rounds.

Article Snippet: An anti-ErbB3 antibody (AF234 Goat-anti-HER3 ab; R&D systems) was included as a positive control in both experiments.

Techniques: Expressing, Binding Assay, Concentration Assay

Journal: Protein engineering, design & selection : PEDS

Article Title: Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules.

doi: 10.1093/protein/gzq118

Figure Lengend Snippet: Fig. 3. Amino acid sequences of the parental Zwt with randomized positions indicated with closed circles and FACS-isolated potential ErbB3-binding Affibody molecules. Representation among sequenced clones is shown after each sequence.

Article Snippet: An anti-ErbB3 antibody (AF234 Goat-anti-HER3 ab; R&D systems) was included as a positive control in both experiments.

Techniques: Isolation, Binding Assay, Clone Assay, Sequencing

Journal: Protein engineering, design & selection : PEDS

Article Title: Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules.

doi: 10.1093/protein/gzq118

Figure Lengend Snippet: Fig. 4. (A) Histograms from flow-cytometric analysis of ErbB3 binding of staphylococcal cells displaying the four ErbB3-specific Affibody molecules (Z05405, Z05413, Z05416 and Z05417), respectively. Overlay from analysis of cells displaying the first generation binder Z1820 is included in each histogram for comparison. Cells were incubated in 3 nM-labeled ErbB3-Fc prior to analysis. FL-1 channel fluorescence corresponding to ErbB3-Fc-binding on the x-axis and cell counts on the y-axis. (B) On-cell flow-cytometric determination of the apparent equilibrium dissociation constant (KD) for four ErbB3-specific Affibody molecules (Z05405, Z05413, Z05416 and Z05417) from the bacterial surface display selection and one ErbB3-specific Affibody molecule (Z01820) from the phage-display selection. Representative binding isotherms with the normalized MFI on the y-axis and the biotinylated ErbB3-Fc concentration on the x-axis. The MFI is normalized with the MFI for the cell population incubated with the highest concentration of biotinylated ErbB3-Fc in each experiment. Solid line represents the equation from fitting the data to a one-site binding model.

Article Snippet: An anti-ErbB3 antibody (AF234 Goat-anti-HER3 ab; R&D systems) was included as a positive control in both experiments.

Techniques: Binding Assay, Comparison, Incubation, Labeling, Selection, Concentration Assay

Journal: Protein engineering, design & selection : PEDS

Article Title: Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules.

doi: 10.1093/protein/gzq118

Figure Lengend Snippet: Fig. 5 Secondary structure content, thermal stability and SPR kinetic analysis of Z05405, Z05413, Z05416 and Z05417. (A) CD spectrum of the four Affibody molecules, before and after heating to 908C. (B) Variable temperature CD spectra generated by heat treatment from 20 to 908C for the four Affibody molecules. (C) Sensorgrams obtained from surface plasmon resonance kinetic analysis, showing four different concentrations of the four Affibody molecules injected over a surface immobilized with human ErbB3-Fc. Dissociation constants were determined based on kon and koff generated by fitting the data to a one-site binding model. Concentrations of injected Affibody molecules are shown in the figure.

Article Snippet: An anti-ErbB3 antibody (AF234 Goat-anti-HER3 ab; R&D systems) was included as a positive control in both experiments.

Techniques: Circular Dichroism, Generated, SPR Assay, Injection, Binding Assay

Journal: Protein engineering, design & selection : PEDS

Article Title: Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules.

doi: 10.1093/protein/gzq118

Figure Lengend Snippet: Fig. 6 (A) Representative SPR sensorgram from competition analysis of Z05417 with heregulin. Sensorgram showing injections of ErbB3-Fc, either alone or preincubated with 40-fold molar excess of Affibody molecule, over a surface immobilized with human heregulin. (B) Representative SPR sensorgram from competition analysis of Z05417 with heregulin. Sensorgram showing Affibody molecule coinjected in individual duplicates with the ErbB3 ligand heregulin over a surface immobilized with human ErbB3-Fc (i.e. Biacore COINJECT command, resulting in the injection of a second ligand immediately following the first). Each Affibody was injected in five concentrations (10, 5, 1, 0.5 and 0 nM), followed by injection of heregulin (250 nM).

Article Snippet: An anti-ErbB3 antibody (AF234 Goat-anti-HER3 ab; R&D systems) was included as a positive control in both experiments.

Techniques: Injection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CLEC5A Mediates Macrophage Function and COPD Pathologies 1

doi: 10.4049/jimmunol.1500978

Figure Lengend Snippet: Immunofluorescent analysis of CLEC5A in lungs of mice exposed to (A) FA or (B) CS for 6 months. Arrows indicate alveolar macrophage (representative of 4 mice). CLEC5A surface expression was measured on (C) peripheral blood monocytes and (D and E) alveolar macrophage (F4/80hiCD11blo) from FA- and CS- exposed mice. Dashed line represents isotype control, solid line represents CLEC5A (n=5 mice). (F) Average number of CLEC5A+ cells and median fluorescence intensity of CLEC5A on alveolar macrophage. (G) CLEC5A expression on lung macrophage of never smokers (NS) and current smoker (S) (n=4 subjects/group). (H) Average number of CLEC5A+ cells and median fluorescence intensity of CLEC5A on alveolar macrophage.

Article Snippet: CLEC5A was assessed with α-human CLEC5A antibody (clone 283834, R&D Systems) and α-CD45 (eBioscience) using a FACSCalibur and FlowJo software.

Techniques: Expressing, Fluorescence

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CLEC5A Mediates Macrophage Function and COPD Pathologies 1

doi: 10.4049/jimmunol.1500978

Figure Lengend Snippet: BAL macrophage (5×104) were stimulated with (A) control IgG, α-CLEC5A activating antibody (10 ug/ml), LPS (10 ng/ml), or both for 24hrs. Similarly, BAL macrophage (5×104) were stimulated with (B) control IgG, α-CLEC5A activating antibody (10 ug/ml), GM-CSF (20 ng/ml), or both for 24hrs. TNFα was measured by ELISA. Data are mean +/− sem of 3 experiments performed in duplicate. * p<0.05 compared to FA mice.

Article Snippet: CLEC5A was assessed with α-human CLEC5A antibody (clone 283834, R&D Systems) and α-CD45 (eBioscience) using a FACSCalibur and FlowJo software.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CLEC5A Mediates Macrophage Function and COPD Pathologies 1

doi: 10.4049/jimmunol.1500978

Figure Lengend Snippet: (A and B) WT and Clec5a−/− mice were exposed to CS for up to 6 months to evaluate lung remodeling as assessed by MLI (Data are mean +/− SEM, n=7–8). (C) Pulmonary inflammation was assessed by enumerating the total cells in BAL. (D) Cytokine levels in BAL of WT and Clec5a−/− mice (n=5–8). (E) mRNA expression levels in BAL macrophage of WT and Clec5a−/− mice (n=5–6). * p<0.05 compared to FA mice. # p<0.05 compared to wild-type mice.

Article Snippet: CLEC5A was assessed with α-human CLEC5A antibody (clone 283834, R&D Systems) and α-CD45 (eBioscience) using a FACSCalibur and FlowJo software.

Techniques: Expressing