High-throughput Antigen ELISA Protocol

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.