Enzyme-Linked Immunosorbent Assay for scFv Binding

The binding of the scFvs to biotinylated rHl and rLr (30 nM) was assessed using a DELFIA‐based assay on black Maxisorp plates (Nunc), as previously described (Laustsen et al., 2018 (link)). Briefly, the scFvs were immobilized with anti‐FLAG M2 antibody (Sigma‐Aldrich #F3165) at 2.5 μg/mL. After immobilization of the scFvs, the biotinylated toxins (rHl and rLr) were added in 3MPBS. Binding was detected using streptavidin conjugated with europium (Perkin Elmer #1244‐360) and DELFIA Enhancement Solution (Perkin Elmer, 4001‐0010). To make the results from different experiments comparable, we included a positive control consisting of an antigen‐scFv pair with known positive binding (TPL 2552_02_B02 and α‐cobratoxin) (Ledsgaard et al., 2022 (link)). The TRF intensity values were then normalized to the TRF signal observed in this control.