PD-1 Binding Assay Protocol

ELISA was used to evaluate the PD-1 binding activity as previously described^{32 (link)}. For ELISA screening, culture supernatant was used as a sample and detected with goat anti-mouse IgG Fcγ-HRP antibody (Jackson ImmunoResearch, 115-035-164) diluted 1:8,000 in 0.05% Tween-20 PBS buffer (PBST). For the mouse PD-1 binding profile, serial dilutions of purified mouse anti-PD-1 mAbs were used. For the chimeric PD-1 binding profile, serial dilutions of purified chimeric anti-PD-1 mAbs were used and detected with goat anti-human IgG Fcγ-HRP antibody (Jackson ImmunoResearch, 109-005-098) diluted 1:10,000 in PBST. The SIGMAFAST OPD (Sigma-Aldrich, P9187) substrate solution was used, and the reaction was stopped by adding 1 M H₂SO₄. The absorbance was measured at 492 nm by a Cytation 5 cell imaging multi-mode reader (BioTek). Mini-pools providing an A₄₉₂ ELISA signal of more than 1.0 were considered high hPD-1-specific IgG level pools. These mini-pools were further screened for the anti-PD-1 neutralizing antibody (NAb) by flow cytometry.