Data analysis software v8.2

To determine the affinities of the recombinant nanobodies, BLI was done using a ForteBio Octet RED96 biolayer interferometer (Pall ForteBio). Streptavidin-coated BLI biosensor tips (ForteBio) were soaked in the assay running buffer [PBS with 0.05% Tween-20 and 1% recombinant human albumin (Sigma)] for 10 minutes. Biotinylated EIIIB was then immobilized on streptavidin-coated BLI biosensor tips by immersion in a 2 μg/mL solution. The association and dissociation were analyzed for different concentrations of analyte ranging from 0.1 to 350 nM. Association and dissociation rate constants were determined using the ForteBio data analysis software (V8.2) using the 1:1 binding model and a global fit analysis with double referencing.

BLI binding experiments were performed using an Octet RED96 instrument. Fabs and purified recombinant Pfs48/45-D3 or Pfs48/45-D3 expression supernatants were diluted in kinetics buffer (PBS, pH 7.4, 0.01% (w/v) BSA, and 0.002% (v/v) Tween-20) as previously described.¹¹ (link) Binding kinetics parameters were determined using Ni-NTA biosensors loaded with 10 μg/ml of purified His-tagged Pfs48/45-D3 or expression supernatant followed by a 30 s baseline and an association phase in serially diluted Fab from 500 nM to 15 nM. Biosensors were then dipped into wells containing kinetics buffer for a dissociation step. Epitope binning experiments were done by loading 10 μg/ml of His-tagged Pfs48/45-D3 followed by a 30 s baseline, a 10 min association phase in RUPA-117 or TB31F Fab, and another association phase in the second Fab, as previously described.³⁷ (link) Data were analyzed using FortéBio’s Data Analysis software V8.2.

Bio-layer interferometry (BLI) was done using a ForteBio Octet RED96 bio-layer interferometer (Pall ForteBio) to determine the affinities of the hTNC-specific nanobodies to full-length human TNC protein. Streptavidin-coated BLI biosensor tips (Forte Bio) were soaked in the assay running buffer consisting of PBS supplemented with 0.05% Tween-20 and 1% recombinant human albumin (Sigma) for 20 minutes. To capture the nanobodies the biosensor tips were immersed in 100 nmol/L solution of biotinylated nanobodies NJT3, NJT4, or NJT6. The nanobody-loaded tips were then dipped into wells containing different concentrations of purified hTNC protein (Sigma-Aldrich). The data were reference-subtracted and the association and dissociation rate constants were analyzed using the ForteBio data analysis software (V8.2) using a 1:1 binding model and global fit analysis.