Cp004

Streptavidin-coated 96-well plates (R&D Systems, CP004) were coated with 50 ng of biotinylated HLA-A*02:01 pHLA monomers in 50 μl of blocking buffer (PBS with 0.5% BSA, 2 mM EDTA, and 0.1% sodium azide) or 25 ng of recombinant human CD3_ε/δ (Acro Biosystems, CDD-H82W6) at 4°C overnight. Plates were washed with 1X TBST (TBS + 0.05% Tween-20) using a BioTek 405 TS plate washer. Serial dilutions of scDb or IgG were incubated on plates for 1 hour at room temperature (RT) and washed. For scDbs, the plate was then incubated with 1 μg/ml recombinant protein L (Thermo Fisher Scientific, 77679) for 1 hour at RT, washed, followed by incubation with antiprotein L HRP (1:10000, Abcam, ab63506) for 1 hour at RT. For IgG, the plate was incubated with anti-human IgG HRP (1:1000, Thermo Fisher Scientific, 62-8420) for 1 hour at RT. Plates were washed, 50 μl of 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (BioLegend, 421101) was added to each well, and the reaction was quenched with 50 μl 1N sulfuric acid (Thermo Fisher Scientific, SA212-1). Absorbance at 450 nm was measured with a Synergy H1 Multi-Mode Reader (BioTek).