Biotinylated anti c tag conjugates

Protein samples were separated by SDS-PAGE and transferred onto PVDF membranes using a Mini-PROTEAN Tetra System (Bio-Rad Laboratories). Membranes were incubated in blocking buffer on a shaker for 30 min and washed three times in TBST. Membranes were probed with biotinylated anti-c-tag conjugates (1:2000; Thermo Fisher) overnight at 4 °C followed by an incubation with IRDye 680RD Streptavidin (1:3000; LI-COR Biosciences) for 2 h at RT, avoiding light. Membranes were imaged using an Odyssey CLx imager (LI-COR Biosciences) and analyzed using Image Studio software.

EpCAM × CD3 BiTE concentrations were quantified by c-tag ELISA developed in house. Briefly, 96-well streptavidin-coated plates (Thermo Fisher) were incubated with biotinylated anti-c-tag conjugates (100 μl/well at 10 μg/ml; Thermo Fisher) diluted in PBS overnight at 4 °C. After blocking the plates with 20% newborn calf serum in PBS, serum samples and protein standards were added to the wells for incubation. Recombinant mouse EpCAM Fc chimera (Bio-Techne) was added to the wells at 5 μg/ml. After extensive washing, HRP Rat anti-Mouse IgG2a antibody (1:7500; Thermo Fisher) was added to the wells. After incubation with TMB substrates, the reaction was stopped, and ODs were determined at 450 nm with wavelength correction at 630 nm using a Synergy H4 Microplate Reader (BioTek). Samples were assayed in duplicate. For in vitro studies, samples were normalized to average values obtained from wells containing cells transfected by control nanoparticles.