An indirect ECL-based screening assay was developed with a biotinylated-therapeutic antibody as the capture reagent and goat anti-mouse Sulfo-Tag (0.25 µg/mL; Cat#, R32AC-1, MSD, Rockville, MD) detection reagent (Figure ). A TECAN liquid handling system was utilized for in-plate reagent preparations, dilutions and transfers. A reaction mixture was prepared consisting of 25 µl capture reagent and 25 µl detection reagent with or without, 5% (v/v) human serum, +/-soluble antigen and 25 µl of anti-ID antibody at concentrations listed on plate map (Figure ) and incubated on a shaker for 2 h at RT. Streptavidin-coated 96-well MSD plates (Cat# L15SA, MSD, Rockville, MD) were blocked with 1% BSA/PBST on a shaker for 1 h at RT. The reaction mixture was transferred to the pre-blocked MSD plate and incubated on a shaker for 1 h at RT. After 3X wash with PBST, 150 µl of MSD Read Buffer (Cat# R92TC-1, MSD, Rockville, MD) was added to each well and plates were read on a SECTOR ® Imager S 600 plate reader (MSD, Rockville, MD). A total score was calculated based on the listed criteria (Figure ).