Iscove s modified dulbecco s minimal medium

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).

Monoclonal antibodies were produced and purified by previously described methods [8 (link), 21 (link)]. Briefly, clonal hybridoma lines were grown in complete hybridoma medium (Iscove’s modified Dulbecco’s Minimal medium [Sigma Aldrich] containing NaHCO₃ [36 mM] and 1x Glutamax [Invitrogen, Calsbad, CA], supplemented with 10% heat-inactivated fetal calf serum [FCS] [Invitrogen]). The purification of monoclonal antibody was conducted as described [8 (link)]. Briefly, 250–450 mL of antibody-containing media (hybridoma cells grown for 4–5 days) was passed through a Protein G column (GE Healthcare). Antibody was eluted with 0.1 M glycine (pH 2.7), resulting in 3–11 mg of purified Stx2e antibody. Protein concentration was determined using the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). Biotinylation of antibodies was conducted using the Lightning-Link Biotin Conjugation Kit (Innova Biosciences, Cambridge, UK). Antibodies were isotyped by ELISA using Stx2e (E167Q) toxoid and horseradish peroxidase (HRP)-conjugated isotype-specific antibodies (Southern Biotech, Birmingham, AL).