Kpl abts peroxidase stop solution

Antigen ELISAs were performed as described^{27 (link)}. In brief, high-binding 384-well polystyrene plates (Corning) were coated overnight at 4 °C with 2 µg/ml NPDPNANPNVDPNANP (junction,) (NANP)₅ (repeat), SLGENDDGNNEDNEKLRKPKHKKLKQPADGNPDP (N-CSP), NKNNQGNGQGHNMPNDPNRNVDENANANSAVKNNNNEEPSDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICKMEKCSSVFNVVNSS (C-CSP), 0.7 µg/ml AKFVAAWTLKAAA (PADRE) or 1 µg/ml H. pylori apoferritin in 20 µl. Plates were washed three times with 0.05% Tween 20 in PBS, blocked with 50 µL of 4% BSA in PBS for 1 h at RT, and washed again prior to incubation with 20µL per well of serum samples diluted in 1% BSA in PBS for 90 min at RT. Wells were washed six times and incubated with goat anti-mouse IgG-HRP at 1:1000 (Jackson Immuno Research) in PBS with 1% BSA for 1 h. Wells were washed again and one-step ABTS substrate (RT, 20 µL per well; Roche) and 1× KPL ABTS peroxidase stop solution (RT, 20 µL per well; SeraCare Life Sciences) were used for detection. The concentration of antigen-specific IgG was determined by comparison to an IgG1 standard curve (BD Pharming) of known concentration on each plate.

Antigen ELISAs were performed as described [32 (link)]. In brief, high-binding 384-well polystyrene plates (Corning) were coated overnight at 4°C with 2 μg/mL of NANP₅. Plates were washed three times with 0.05% Tween 20 in PBS, blocked with 50 μL of 4% BSA in PBS for 1 h at room temperature (RT), and washed again. For analysis of serum samples, sera were diluted in 1% BSA in PBS and 20 μL/well incubated for 90 min at RT. For analysis of monoclonal antibodies, 15 μL/well of serially diluted monoclonal antibodies (starting concentration 4.0 μg/mL, 1 in 4 dilution for 8 steps) were incubated for 2 h at RT. Wells were washed six times and incubated with goat anti-mouse IgG-HRP at 1:1000 (Jackson Immuno Research) in PBS with 1% BSA for 1 h. Wells were washed again and one-step ABTS substrate (RT, 20 μL/well; Roche) and 1× KPL ABTS peroxidase stop solution (RT, 20 μL/well; SeraCare Life Sciences) were used for detection. The concentration of antigen-specific IgG in serum was determined using an IgG1 standard curve (BD Pharming) on each plate. Area under the ELISA curve (AUC) was calculated using GraphPad Prism 7.04 (GraphPad).