*2.4. Binding Efficency*

To determine the binding efficiency, 500 µg/mL of NPs were incubated with 100 µg/mL of protein (Bet v 1) in the presence of 0.9% NaCl and 10 mM HEPES buffer pH 7.4 (binding buffer) at a volume of 500 µL at 4 ◦C for 17 h on a test tube rotator. The recombinant birch pollen allergen Bet v 1 used in this study was prepared as previously described [28]. To determine the total amount of protein bound to the surface of the NPs, the samples were centrifuged twice for 1 h with 16,000× *g* at 4 ◦C and pellet and supernatant was thoroughly separated. The two-step collection of the supernatant reduces the risk of contaminating the supernatant with the pellet. The bound protein was quantified directly from the pellet using 15% SDS-PAGE by relating it to the concentration of standards using the ImageLab software (version 6.0.1, 2017, Bio-Rad Laboratories, Hercules, CA, USA). In parallel, the unbound protein in the supernatant was quantified using BCA (bicinchoninic acid) assay. From this the percentage of bound protein was estimated indirectly. Hence, testing the amount of proteins left in the supernatant with the BCA assay also allows determine whether the amount of protein in the pellet and supernatant add up to the total amount of Bet v 1 present in the reaction mix during coupling.
