ZV-13 antibody purification and quantification

Hybridoma supernatant was harvested as the source of ZV-13 antibody once ~95% cell death was observed. The supernatant was clarified by centrifuging for 10 min at 400 × g at 4°C and then filtered with a 0.22-μm filter prior to antibody purification via protein A affinity chromatography using the Pierce Protein A IgG purification kit and NAb Protein A Plus spin columns (all from Thermo Fisher-Life Technologies). Purified ZV-13 antibody was concentrated and buffer-exchanged into 1× phosphate-buffered saline (PBS; Fisher Scientific) using Amicon Ultra-0.5 100-kDa centrifugal filter units for quantification by enzyme-linked immunosorbent assay (ELISA; Sigma-Aldrich, St. Louis, MO, USA). 96-well Nunc ELISA plates (Fisher Scientific) were coated with goat anti-mouse IgG2c (Jackson ImmunoResearch, West Grove, PA, USA) diluted to 500 ng/mL in 1× PBS and incubated overnight at 4°C. Coated plates were washed with PBS plus Tween 20 (PBST; Fisher Scientific) and blocked for 1 h at room temperature with PBS plus 1% bovine serum albumin (PBSA; Sigma-Aldrich) supplemented with 2% normal goat serum (NGS; Jackson ImmunoResearch). Following blocking, serial dilutions of hybridoma supernatant and purified ZV-13 antibody were added and incubated for 1 h at 37°C. Plates were washed, and biotin-conjugated anti-mouse IgG2c secondary antibody (Jackson ImmunoResearch) diluted 1:5,000 in PBSA was added and incubated for 1 h at room temperature. Plates were then washed and incubated with streptavidin-horseradish peroxidase (HRP) (Thermo Fisher-Life Technologies) diluted 1:20,000 in PBSA for 30 min at room temperature. Plates were washed, developed by adding 3,3′,5,5′-tetramethylbenzinide (TMB; Surmodics, Eden Prairie, MN, USA), and incubated in the dark. The reaction was stopped with the addition of 1 M H2SO4, and absorbance was measured at 450 nm in a microplate reader (BioLegend, San Diego, CA, USA). Serial dilutions of known concentrations of mouse IgG2c (Southern Biotech, Birmingham, AL, USA) were included to generate a standard curve with optical density (OD) values plotted against concentration in GraphPad Prism (version 9, GraphPad, La Jolla, CA, USA). The linear portion of the standard curve was used to derive a least-squares equation to calculate the concentration of purified ZV-13 antibody.