Biolayer interferometry

The binding affinity of AfEno1 and factor H, C4BP, or plasminogen were determined by biolayer interferometry (Forte Bio, Menlo Park, CA) as previously described (19 (link), 34 (link)). For each concentration, Ni-NTA biosensors were hydrated in DPBS (0.001% gelatin) for 10 min; then recombinant AfEno1 was bound to biosensors for 120 s. After washing the sensor (30 s) with DPBS (0.001% gelatin), factor H, C4BP, or plasminogen at various concentrations were added as analytes. For each concentration, the association of the complexes was followed for 250 s. After washing the sensor, the dissociation of the complexes was followed for another 250 s. All interactions were performed at RT. As a non-binding control, heat inactivated regulators were used. The data were obtained after subtracting the buffer blank using a 1:1 model of interaction. Graphs were plotted in Graph Pad Prism 5.

Binding of Factor H to immobilized Aspf2 was analyzed in real time by biolayer interferometry (Forte Bio, Menlo Park, CA, USA). The Ni(II)-NTA biosensor was loaded with recombinant Aspf2. Before measurement, biosensor tips were briefly washed (30 s) to remove free Aspf2, and Factor H at concentrations from 190, 375, 750, 1,500, and 3,000 nM was added as an analyte. For each concentration, the association of Factor H to Aspf2 was followed and after removal of the analyte the dissociation of the complexes was followed for 150 s. Denatured Factor H (95°C for 10 min) was used as a non-binding control.
Binding of plasminogen to Aspf2 was determined by adding plasminogen at 687.5, 1370, 2,750, and 5,500 nM as an analyte. For each concentration, the association and dissociation of plasminogen to Aspf2 was followed for 200 s. The K_d values were determined by subtracting the values from buffer control, using the BLITZ software. The association and dissociation curves were plotted using Graphpad5.

Recombinant purified human PD-1 protein, residues Leu25-Thr168, with a C-terminal 6-His tag was purchased from R&D Systems (catalog# 8986-PD, lot# DDJM0417041), and its molecular weight confirmed by HPLC-MS and shown to be 99% pure by SEC-HPLC. It was used as an analyte in the affinity determinations and binning experiments. Monomeric human PD-1 Fc fusion protein was expressed as a murine IgG1 fusion by pairing FLAG-murine IgG1 Fc with His-tagged human PD-1 murine IgG1 Fc. The fusion protein was purified by a two-step process (anti-FLAG; Ni-NTA), was sequence confirmed by HPLC-MS and shown to be 96% pure by SEC-HPLC. It was used as a bead-coating reagent in the KinExA experiments. Recombinant purified human PD-L1 Fc chimera protein was purchased from R&D Systems (catalog# 156-B7). It was used as a competitor in the binning assays. His-tagged (9-His) N-terminal truncated PD-1, residues Pro35-Glu167, was expressed in HEK293 suspension cells, purified in a single step (Ni-NTA) to a monomeric purity of 42% (SEC-HPLC) and shown to bind (Bio-layer Interferometry, ForteBio) to pembrolizumab (100 nM) but not to nivolumab (100 nM). It was used as an analyte for affinity determinations.

The binding affinities and kinetic constants of geraniin for the spike protein RBD and hACE2 receptor were evaluated using a BLItz system based on biolayer interferometry (Pall FortéBio, Fremont, CA, USA). After the pre-equilibration of the streptavidin BLI sensor (Pall FortéBio) in PBS buffer for 10 min, biotinylated spike protein S1 RBD and hACE2 receptor were fully loaded onto the sensors by soaking the sensor in 4 μL of 50 μg/mL spike protein RBD and hACE2 receptor solution. The geraniin solutions for kinetic analysis were prepared by diluting the stock solution in PBS with 1% DMSO to achieve 0, 50, 100, and 200 nM.
The protein-immobilized sensor was first equilibrated in PBS buffer containing 1% DMSO for 20 s. Then, association in 4 μL of geraniin solution for 20 s was measured. Next, the dissociation in PBS containing 1% DMSO for 20 s was evaluated. The kinetic constants were calculated using BLItz Pro by fitting the association and dissociation data to a 1:1 model. The equilibrium dissociation constant, K_D, was calculated as dissociation constant (k_d)/association constant (k_a).

Purified monoclonal antibodies were buffer exchanged into PBS using equilibrated Zeba Spin columns. The protein concentration was determined and the antibodies biotinylated by incubating for 30 min at room temperature with EZ-Link NHS-PEG4-Biotinylation reagent (Thermo Scientific) at a 10:1 biotin-to-protein ratio. Free biotin was removed by repeating the buffer exchange step in a second Zeba Spin column equilibrated with PBS. Affinity of interactions between biotinylated antibodies and purified soluble RBD proteins were measured by Biolayer Interferometry (BLItz, ForteBio). Streptavidin biosensors were rehydrated in PBS containing 0.1% BSA for 1 h at room temperature. Biotinylated antibody was loaded onto the sensors “on-line” using an advanced kinetics protocol, and global fits were obtained for the binding kinetics by running associations and dissociations of RBD proteins at a suitable range of molar concentrations (2-fold serial dilution ranging from 800 nM to 50 nM). The global dissociation constant (K_D) for each 1:1 binding interaction was determined using the Blitz Pro v1.2.1.3 (Forte Bio) software. For competition assays, RBD at 400 nM was pre-incubated with competitor (ACE2 Fc or unconjugated antibody) at 1 µM for 10 min at room temperature.

A reference curve was obtained by defining relocation values for anti-IgG (Alexa Fluor 647) and RBD with a panel of 7 anti-RBD mAbs with a 1 log K_D range, determined using bio-layer interferometry (ForteBio). Droplet populations were generated with different concentrations of each mAb or buffer and the DropMap bioassay. A curve was defined by plotting the relocation from the anti-IgG (Alexa Fluor 647) against relocation of RBD, and the slope of the resulting line was calculated and termed “DropMap slope.” The reference curve was defined by the linear relationship between K_D for RBD as defined by bio-layer interferometry and the DropMap slope of each mAb.