Monoclonal antibody:PCDH1 binding ELISA

To determine the capacity of an infection-inhibiting, EC1-specific mAb to bind to sEC1-2 variants, ELISA plates were coated with serial twofold dilutions of WT or mutant sEC1-2 (starting concentration at 400 ng/well) overnight at 4 °C. After briefly washing with PBS, wells were blocked with 5% nonfat dry milk in PBS (1 h at RT) and incubated with anti-EC1 mAb-3305 (1 µg/mL, Donnelly Centre and Department of Molecular Genetics, University of Toronto) for 1 h at RT. After washing with PBS, the bound antibody was detected by incubation with an anti-human IgG HRP pAb (1:10,000 dilution, catalog No. AP112, Sigma-Aldrich) for 1 h at RT. ELISA signal was developed using 1-Step^TM Ultra TMB-ELISA substrate solution (Thermo Scientific) and measured at an absorbance of 450 nm on a Cytation5 cell imaging multi-mode reader (Agilent BioTek Gen5 Microplate Reader and Imager software, V.3.2).