length igg antibodies  (New England Biolabs)

Journal: bioRxiv

Article Title: Isolation and Characterization of Antibodies Against VCAM-1 Reveals Putative Role for Ig-like Domains 2 and 3 in Cell-to-Cell Interaction

doi: 10.1101/2024.12.03.626733

Figure Lengend Snippet: (A) Schematics of mVCAM-1 Ig-like domain deletion mutants used in this study. (B, C) Flow cytometry analysis of the binding of the commercial anti-mVCAM-1 antibody (CBL1300) (B, C) , anti-FLAG (B) , and our new full-length IgG2a reformatted antibodies (C) to cells transiently transfected with mVCAM-1-eGFP WT (with or without N-terminal FLAG) or FLAG-tagged Ig-like deletion constructs. Overall VCAM-1 cell surface expression was determined using anti-FLAG antibody or anti-VCAM-1 CBL1300 antibody (B) . CBL1300 required a different secondary antibody control than the FLAG antibody, therefore results with its own secondary antibody-only control is also shown ( B ). (C) Table showing the normalized MFI values for each of the VCAM-1/antibody combinations (also see Supplementary Figure S8 ). The color scheme correlates with MFI value, with lighter colors indicating lower antibody binding and darker colors higher antibody binding. On the right, a schematic representation of the mVCAM1 Ig-like domain structure showing the (tentative) binding location for the different antibodies tested in this study. Displayed is the average normalized MFI-Max-Min (mVCAM-1-eGFP-positive, antibody positive cells normalized to MFI of the mVCAM-1-eGFP-negative, antibody negative cells) from one experiment with 2 replicates. Schematics were created with BioRender.com .

Article Snippet: The scFv sequences obtained were reformatted into full length mouse IgG2a monoclonal antibodies ( Supplementary Table 2 ) using the InFusion HD cloning kit (Takara, 639648), following previously published methodologies [ , ].

Techniques: Flow Cytometry, Binding Assay, Transfection, Construct, Expressing, Control