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.
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.