Minibody Binding ELISA for PD-L1, PD-1, and CTLA-4

The minibody binding ELISA assay has been described previously [10 (link)]. Briefly, Immulon 2 high binding 96-well plate (VWR, Radnor, PA) was coated with 500 ng/well of recombinant human PD-L1, PD-1, or CTLA-4 (BioVision, Milpilas, CA, USA). After blocking plate with PBS-T containing 3% BSA, serially diluted media containing minibody or media from GFP-transfected cells were added and incubated at 4 °C for 24 h. Serially diluted IgGs (Biolegend, San Diego, CA, USA) were used as positive controls with isotype IgGs serving as negative controls. HRP-labeled anti-human IgG for minibody detection or HRP-labeled anti-mouse IgG (BioRad, Hercules, CA, USA) for anti-human antibody detection were added and incubated at room temperature for 1hr and then developed. Absorbance was measured using Tecan plate reader (TECAN, Männedorf, Switzerland).