mouse tie2 fc chimeric protein  (R&D Systems)

Journal: Scientific Reports

Article Title: An engineered tetra-valent antibody fully activates the Tie2 receptor with comparable potency to its natural ligand angiopoietin-1

doi: 10.1038/s41598-021-93660-4

Figure Lengend Snippet: Characterization of newly identified anti-Tie2 agonistic antibodies. ( a ) Cellular viability as an indicator of agonistic activity of anti-Tie2 antibodies produced by hybridoma clones 2–16 and 2–8 were assessed in human Tie2-expressing Ba/F3 compared to Ang1. Cellular viability (%) was determined using a CellTiter-Glo assay, with basal activity without any ligand (PBS) defined as 0%, and the activity of 17.9 nM (1 µg/mL) Ang1 defined as 100%. Data represent mean ± SD. ( b , c ) Fractionation of the antibody solution using AEX. ( b ) shows SDS PAGE of each fraction from AEX, and ( c ) shows cellular viability as an indicator of the agonistic activity of each fraction in human Tie2-expressing Ba/F3. Solid (left) arrow indicates antibody-rich fractions, dashed (right) arrow indicates the fractions with higher agonistic activity. Cellular viability (%) was determined using a CellTiter-Glo assay, with basal activity without any ligand (PBS) defined as 0%, and the activity in fraction 6 defined as 100%. ( d ) Cellular viability as an indicator of the agonistic activity of purified 2–16 monomer, dimer and HMW human monoclonal antibodies, isolated using SEC (Supplementary Fig. and ), in human Tie2-expressing Ba/F3 compared to Ang1. Cellular viability (%) was determined as described in ( a ).

Article Snippet: Recombinant human, rat and mouse Tie2-Fc chimeric protein (R&D Systems) and recombinant monkey Tie2-Fc chimeric protein (Sino Biological) were each immobilized on a sensor chip CM5 (GE Healthcare) using an Amine Coupling Kit (GE Healthcare) and HBS-EP + buffer (GE Healthcare) in accordance with the manufacturer’s instructions.

Techniques: Activity Assay, Produced, Clone Assay, Expressing, Glo Assay, Fractionation, SDS Page, Purification, Isolation

Journal: Scientific Reports

Article Title: An engineered tetra-valent antibody fully activates the Tie2 receptor with comparable potency to its natural ligand angiopoietin-1

doi: 10.1038/s41598-021-93660-4

Figure Lengend Snippet: Generation and in vitro evaluation of the tetra-valent anti-human Tie2 antibody. ( a ) Structure of the tetra-valent antibody. ( b ) SDS-PAGE of the tetra-valent antibody under non-reduced (left) and reduced (right) conditions. Molecular weights (kDa) are indicated on the left of each gel. ( c ) Binding activity of ASP4021 to human Tie2 and Tie1 by direct ELISA. ASP4021 binding is shown as the absorbance at OD450. Each value is expressed as mean ± SEM. ( d ) Cellular viability as an indicator of agonistic activity of the tetra-valent and bi-valent antibodies in human Tie2-expressing Ba/F3 compared to Ang1. Cellular viability (%) was determined as described Fig. a. ( e ) Anti-apoptotic activity of ASP4021 in human Tie2-expressing Ba/F3 compared to Ang1. Caspase 3/7 activity (%) was calculated using the Caspase-Glo 3/7 assay, with basal activity with IL-3 defined as 0%, and activity without any ligand (PBS) defined as 100%. Data represent mean ± SD. ( f ) Phosphorylation of Tie2 induced by ASP4021 in HUVEC compared to Ang1. HUVEC were treated with ASP4021 or recombinant Ang1 for 30 min. Levels of phosphorylated Tie2 and actin in cell lysates were detected using Western blotting. The assay was performed in duplicate. Full-size versions of Western blots are depicted in Supplementary Fig. .

Article Snippet: Recombinant human, rat and mouse Tie2-Fc chimeric protein (R&D Systems) and recombinant monkey Tie2-Fc chimeric protein (Sino Biological) were each immobilized on a sensor chip CM5 (GE Healthcare) using an Amine Coupling Kit (GE Healthcare) and HBS-EP + buffer (GE Healthcare) in accordance with the manufacturer’s instructions.

Techniques: In Vitro, SDS Page, Binding Assay, Activity Assay, Direct ELISA, Expressing, Caspase-Glo Assay, Recombinant, Western Blot