Monoclonal Antibody Purification and Characterization

After clonal hybridoma lines were isolated, cells were grown in complete hybridoma medium (Iscove's modified Dulbecco's Minimal medium [Sigma-Aldrich] containing NaHCO₃ [36 mM] and 1x Glutamax [Invitrogen, Carlsbad, CA], supplemented with 10% heat-inactivated fetal calf serum [FCS] [Invitrogen]). The purification of monoclonal antibody was conducted as described [28] (link). Briefly, 400 mL of antibody-containing media (hybridoma cells grown for 2–3 days) was passed through a Protein G column (GE Healthcare). Antibody was eluted with 0.1 M glycine (pH 2.7), resulting in 5–8 mg of purified Stx1 antibody. Protein concentration was determined using the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). Biotinylation of antibodies was performed using the Lightning-Link Biotin Conjugation Kit (Innova Biosciences, Cambridge, UK). Antibody isotyping was conducted by ELISA using Stx1 (E167Q) toxoid and horseradish peroxidase (HRP) -conjugated isotype-specific antibodies (Southern Biotech, Birmingham, AL).