1 step ultra tmb elisa hrp substrate solution

EIA and blockade antibody assays were performed at described (Lindesmith et al., 2018a (link)) at 0.25 μg/ml VLP. EIA plates were coated with VLP in PBS for 4 h and blocked over night at 4°C in 5% dry milk in PBS-0.05% Tween-20 before the addition of decreasing two-fold serial dilutions of mAb at 37°C. Bound mAb was detected by anti-human IgG-HRP (GE Healthcare) and color developed with 1-Step Ultra TMB ELISA HRP substrate solution (Thermo-Fisher). Similarly, VLPs were pretreated with decreasing concentrations of mAb for 1 h and added to pig gastric mucin type III (Sigma Aldrich, St. Louis, MO) coated plates for 1 h. Bound VLP was detected by anti-VLP rabbit hyperimmune sera followed by anti-rabbit IgG-HRP (GE Healthcare) and color developed as above. Percent control binding is defined as the binding in the presence of antibody pretreatment compared to the binding in the absence multiplied by 100.

EIAs were performed as described [44 (link)]. EIA plates were coated with VLP (0.25 g/mL) in PBS for 4 h and blocked over night at 4 °C in 5% non-fat dried milk in PBS-0.05% Tween-20 (blotto) before addition of decreasing two-fold serial dilutions of mouse mAb or rabbit pAb. All antibody incubations were conducted at 37 °C in blotto. Bound antibody was detected by either anti-mouse IgG-HRP or anti-rabbit IgG-HRP (GE Healthcare, Chicago, IL, USA), and color developed with 1-Step Ultra TMB ELISA HRP substrate solution (Thermo-Fisher, Waltham, MA, USA). Fifty percent effective concentration (EC₅₀) was determined for antibodies with optical densities (ODs) ≥ 3× background at 2 µg/mL. Data were fit using sigmoidal dose–response analysis of nonlinear data in GraphPad Prism 8.3.0 (GraphPad Software, La Jolla, CA, USA). Monoclonal Abs below the limit of detection were assigned an EC₅₀ of 2× the assay upper limit of detection for statistical comparison.

Antibody blockade of VLP-ligand binding assays were performed as described.³⁹ (link) VLP (0.25 μg/mL) were pretreated with 2-fold serial dilutions of sera for 1 h and then added to pig gastric mucin type III (10 μg/mL in PBS, Sigma Aldrich) coated plates for 1 h. Bound VLP were detected by anti-VLP rabbit hyperimmune sera (Cocalico) followed by anti-rabbit IgG-HRP (Cytiva) and color developed with 1-Step Ultra TMB ELISA HRP substrate solution (Thermo-Fisher). Percent control binding was defined as the ratio of VLP binding in the presence of antibody pretreatment compared to the binding in the absence of pretreatment multiplied by 100. Each serum sample was tested in at least one 10-point dilution series on a plate that included a positive control. If a sample had an R² < 0.85 for the model fit or if the control serum was outside the established range, the sample was repeated.