Streptavidin sensors

Recombinant human sNRP1 (1.5 nmol/L) spiked in sNRP1‐depleted serum matrix was tested in the novel sNRP1 ELISA with and without GuHCl sample pre‐treatment. The sample was further tested with and without prior addition of a molar surplus of the NRP1 ligands SEMA3A (R&D Systems) or VEGF‐A₁₆₅ (Enzo Life Sciences).
Interferences of SEMA3A or VEGF‐A₁₆₅ with sNRP1 binding of the monoclonal anti‐human NRP1 detection antibody employed in the sandwich ELISA were further tested in bio‐layer interferometry measurements. First, dissociation rates of these two NRP1 ligands were tested. Therefore, biotinylated sNRP1 (4 µg/mL) diluted in PBS was loaded to two streptavidin sensors (ForteBio). Sensors were then either incubated for 10 minutes with 10 µg/mL VEGF‐A₁₆₅ or SEMA3A. Dissociation was performed in PBS for 15 minutes. To further investigate if SEMA3A or VEGF‐A₁₆₅ binding to sNRP1 interferes with antibody binding, four additional sensors were loaded with sNRP1 as described. Two sensors were then either incubated with 10 µg/mL VEGF‐A₁₆₅ or SEMA3A for 10 minutes, while two other sensors were incubated with PBS alone acting as reference. After a one‐minute stabilization phase, association of the monoclonal antibody (2 µg/mL in PBS) was performed for 10 minutes for all four sensors, followed by a 15 minutes dissociation phase.

For Biolayer Interferometry antibody competition assays, we used an Octet RED96 (FortéBio) instrument. All samples were diluted to a final volume of 200 µL in 1X kinetics buffer (FortéBio). For the assay, samples were agitated at 1000 RPM at 28 °C in black 96 well plates (Grenier Bio-One, Monroe, NC). We received GPmperP, a biotinylated, non-prefusion stabilized, uncleaved version of GP that contains a linker between GP1 and GP2 from Dr. Hastie. This protein can adopt both the prefusion and postfusion forms. This protein was immobilized onto streptavidin sensors (FortéBio) and then dipped into serum samples for 900 s for saturation. Next, the saturated probes were washed for 60 s two times, and the binding of potentially competing monoclonal antibodies was assessed. The antibodies chosen were 37.7H (Zalgen Labs) specific for the prefusion LASV GPC, 3.3B (Zalgen Labs) specific for LASV GP1, and 22.5D (Zalgen Labs) specific for LASV GP2. The difference in the level of competitor binding to GPmperP was calculated by subtracting the level of competitor mAb binding observed after preincubation with serum from vaccinated animals from the level of mAb binding observed after incubation with serum from non-vaccinated control animals. Data analysis was completed using version 7.0 Octet software.

The affinity of anti-MERS CoV antibodies to the human FcγRIIIa (CD16a) was determined using the Octet Red96 system (ForteBio). The biotinylated recombinant human CD16a protein (Sino Biological Inc) was equilibrated in kinetics buffer (phosphate-buffered saline, 0.1% bovine serum albumin, 0.02% Tween-20) at a concentration of 5 μg/mL onto the streptavidin sensors (ForteBio). The anti-MERS antibodies were equilibrated with kinetics buffer at two-fold serial dilution concentrations from 31.3 nM to 4000 nM. The sensors were regenerated by dipping in the regeneration buffer (150 mM NaCl, 300 mM sodium citrate, pH 3.5) and wash solution alternately to remove the antibodies but not the CD16a loaded on the sensors. The Octet assay protocol was performed as follows: baseline (kinetic buffer) for 60 s, loading (rhCD16a proteins) for 150 s, baseline 2 (kinetic buffer) for 60 s, association (anti-MERS antibodies) for 60 s, disassociation (kinetic buffer) for 60 s, regeneration (regeneration buffer) for 5 s and wash (kinetic buffer) for 5 s alternately for three times, then the next recycle was started from the association process at a higher concentration of the same sample. The data of the samples were subtracted from the value of the control sensor and were analyzed using the analysis software provided in the Octet Red96 system.