Overview of PF4-specific rAb generation and PF4 binding properties. (A) Schematic overview of rAb generation and characterization. (B) PF4-binding rAbs were measured by ELISA. PF4 was directly coated onto 96-well ELISA plates, and rAbs were tested in serial dilutions starting from 60 μg/mL. Results are expressed as chemiluminescence (LUM). (C) Surface representations of a model of the paratope of CR22046 and of the structure of PF4 showing the electrostatic potentials. Clustered negatively charged residues in CR22046 and clustered positively charged residues in PF4 are labeled with white characters. For the CR22046 rAb, the numbering is according to Kabat. (D) Real-time binding curves as measured by BLI were obtained by dipping streptavidin sensors with immobilized PF4-biotin into wells containing 5 μg/mL of each antibody (Ab) for 900 seconds, followed by a dissociation of 600 seconds in empty buffer (10× kinetic buffer). (E) BLI-binding responses were obtained by dipping anti-human Fc sensors with immobilized rAbs into wells containing 3.1 μg/mL of each PF4 mutant. The binding of anti-PF4 rAbs to PF4–wild type (WT) was also tested in the presence of 0.25 U/mL heparin (UFH: yellow bars), except for CR22091, which is indicated with an asterisk (∗). Binding responses after 300 seconds were normalized to the binding responses obtained for PF4-WT. H/L, heavy/light; MS, mass spectrometry.