Turbo tmb substrate

Wells of a NeutrAvidin-coated 96-well plate or 8-well strips (Thermo Fisher) were coated with 100 µl of biotinylated peptide at 7.5 µg/ml in molecular grade H₂O (GE Healthcare Life Sciences, Logan, UT) for 1 h. Wells were washed five times with TBST. Wells were then blocked with 200 µl of 5% normal goat serum diluted from 10% normal goat serum (Thermo Fisher) in TBS for 1 h. Wells were washed five times with TBST. Antibody was spiked into 2.5% BSA in TBST (2.5% BSA/TBST) or into pooled defibrinated human AB serum (serum; Gemini, Woodland CA) diluted in 2.5% BSA/TBST. The final dilution of serum was 0.4% unless otherwise noted. Wells were incubated with 100 µl of antibody-spiked samples for 1 h and then washed five times with TBST. Bound natalizumab was detected with mouse anti-human IgG4 horse radish peroxidase (anti-IgG4-HRP; Abcam, Cambridge, MA) diluted 1:2000-1:5000 in TBST. Wells were incubated with 100 µl of diluted anti-IgG4-HRP for 30 m and then washed nine times with TBST. Turbo TMB substrate (Thermo Fisher) was added to the wells and allowed to develop for 12–15 m shielded from light. The reaction was stopped with 1 M H₂SO₄ (Thermo Fisher). Optical density (OD) was read at 450 nm on a Multiskan FC plate reader (Thermo Fisher) and data were analyzed with GraphPad Prism v5 or v7 software (Graphpad Software Inc, La Jolla CA).

Binding competence of IT conjugates was confirmed using a GUCY2C extracellular domain (GUCY2C_1-430)-based ELISA [25 (link)]. ITs were incubated in GUCY2C_1-430-coated plates at varying concentrations. The mouse IgG component was detected with HRP-anti-mouse H + L (Jackson ImmunoResearch, #115-035-062). Ricin A chain was detected with rabbit-anti-Ricin antibody (Abcam, ab27169) followed by HRP-anti-rabbit H+L (Jackson ImmunoResearch, #111-035-003). Color was developed with Turbo-TMB substrate (Thermo Scientific, #34022) and quantified at λ480nm.