antibody phage display library bioinvent n-coder fab lambda library  (BioInvent)

Journal: Nature Communications

Article Title: Targeted inhibition of activated protein C by a non-active-site inhibitory antibody to treat hemophilia

doi: 10.1038/s41467-020-16720-9

Figure Lengend Snippet: a – d Affinity measurement by SPR for type I and type II mAbs: Biacore sensograms showing the association and dissociation curves for antibody binding to hAPC at concentrations of 1.56, 3.125, 6.25, 12.5, 25, 50, 100, and 200 nM ( a , b ) or to hPC at concentrations of 1.56, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400, and 1000 nM ( c , d ). Black traces represent experimental data, and red traces represent the corresponding fits. Type I showed a slower on-rate ( k a , 1.5 × 10 5 M −1 s −1 ) and off-rate ( k d , 1.9 × 10 −3 s −1 ) than type II ( k a , 6.1 × 10 5 M −1 s −1 ; k d , 6.2 × 10 −3 s −1 ). e , f Antibody-binding specificity by ELISA for type I mAb ( e ) and type II mAb ( f ) bound to hAPC (circle) or hPC (square). ELISA results are shown as mean ± SDs from triplicate wells for each antibody concentration, and experiments were repeated >3 times. g Schematic representation of the binding sites on APC protease domain for type I, type II, and R41C17 mAbs. R41C17, an anti-APC Gla-domain antibody, was used as control. h Type II but not type I bound the complex of PPACK-hAPC. PPACK-hAPC or hAPC was coated onto a MaxiSorp plate at 100 ng per well overnight. IgG or Fab at concentrations starting at 20 nM were tested for binding. SPR surface plasmon resonance, ELISA enzyme-linked immunosorbent assay, hAPC human activated protein C, hPC human protein C, RU relative unit, OD490 absorbance at 490 nm, PPACK Phe–Pro–Arg–chloromethylketone, RFU relative fluorescence unit, IgG immunoglobulin G, Fab antigen-binding fragment.

Article Snippet: Type I anti-APC mAb was identified by panning the n-CoDeR ® phage-display library of human antibody Fab fragments (BioInvent International AB).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Control, SPR Assay, Fluorescence