Biolayer interferometry kinetic binding assay

Antibody binding kinetics were measured using biolayer interferometry (BLI) on an Octet HTX instrument (FortéBio) using streptavidin-capture biosensors (fortéBio). PfCSP mAb solutions were plated in black tilted-bottom 384-well microplates (fortéBio); assays were performed with agitation at 30°C. mAb serial concentrations used are as follow: 1.25, 0.625, 0.3125, and 0.15625 μg/mL. Loading of biotinylated peptide 22 was performed for 300 sec, followed by dipping of biosensors into buffer (PBS + 1% BSA) for 60 sec to assess baseline assay drift. Association with whole IgG (serially diluted from 16.67 to 1.04 μM) was done for 300 sec, followed by a dissociation step in buffer for 600 sec. Background subtraction of nonspecific binding was performed through measurement of association in buffer alone. Data analysis and curve fitting were performed using Octet software, version 7.0. Experimental data were fitted with the binding equations describing a 1:1 analyte-ligand interaction. Global analyses of the complete data sets, assuming binding was reversible (full dissociation), were carried out using nonlinear least-squares fitting allowing a single set of binding parameters to be obtained simultaneously for all concentrations of a given mAb dilution series.