Journal: Nature Communications

Article Title: A single-domain antibody targeting factor XII inhibits both thrombosis and inflammation

doi: 10.1038/s41467-024-51745-4

Figure Lengend Snippet: a Schematic representation of the strategy used to identify FXII-specific nanobodies and to design nanobody-Fc fusion after immunizing an alpaca with human FXII. b Characterization of E. coli BL21-expressed Nb and Chinese Hamster Ovary (CHO) cell-expressed Nb-Fc by SDS-PAGE (left panel) and Western blot (right panel). c Binding affinity of Nb or Nb-Fc to human FXII determined by kinetic assays using the Fortebio Octet System. d Dot blot assay to analyze the interaction of native or heat-treated FXII and Nb as indicated. BSA was used as control. e , f FXII epitope mapping. e FXII domains were individually expressed using E. coli BL21, separated by SDS-PAGE under reducing conditions and visualized by Brilliant Coomassie Blue (upper panel), and probed for Nb binding by Western blot (lower panel). f Full-length FXII (FXII_fl) and FXII mutants with indicated deleted domains (FXII_ΔFnII: deletion of FnII; FXII_ΔKringle: deletion of the Kringle domain; FXII_ΔPRR: deletion of the C-terminal part of PRR) were expressed in HEK 293 cells. Binding capacity of a polyclonal anti-FXII antibody as control (upper panel) and Nb (lower panel) to the mutants was evaluated by Western blot under both reducing and non-reducing conditions. g Effects of increasing concentrations of Nb-Fc on ellagic acid (EA, 4 µg/ml)-induced FXII activation using the FXIIa-specific substrate S-2302 (final concentration: 0.8 mM). Isotype control antibody (Iso-Fc) was used as control. h Effects of Nb-Fc on plasma prekallikrein (PK) activation. FXII was pretreated with Nb-Fc, followed by the addition of EA (final concentration: 4 μg/mL) and PK (6.25 μg/mL), then the chromogenic substrate CS-31(02) was used to evaluate the enzymatic activity of activated PK. i Effects of Nb-Fc on activated partial thromboplastin time (aPTT). Citrated human plasma was incubated with increasing concentrations of Nb-Fc (0–31.5 µg/ml) for 5 min prior to aPTT measurements. Experiments in b , d – f were independently performed twice with consistent results. See the Methods for details. Source data are provided as a Source Data file.

Article Snippet: 800 μg of human FXII zymogen (Enzyme Research Laboratories) isolated from human plasma was emulsified with Freund’s complete adjuvant (Millipore Sigma) and used for subcutaneous immunization of an alpaca.

Techniques: SDS Page, Western Blot, Binding Assay, Dot Blot, Control, Activation Assay, Concentration Assay, Clinical Proteomics, Activity Assay, Incubation