Solid-phase Assay for Protein Detection

Solid-phase assays were performed as described previously (Michele et al., 2002 (link); Goddeeris et al., 2013 (link)). Briefly, WGA eluates were diluted 1:50 in TBS and coated on polystyrene ELISA microplates (Costar 3590) overnight at 4°C. Plates were washed in LBB and blocked for 2 hr in 3% BSA/LBB at RT. The wells were washed with 1% BSA/LBB and incubated for 1 hr with L9393 (1:5000 dilution) in 3% BSA/LBB followed by incubation with Horseradish Peroxidase (HRP)-conjugated anti-rabbit IgG (Invitrogen, 1:5000 dilution) in 3% BSA/LBB for 30 min. Plates were developed with o-phenylenediamine dihydrochloride and H₂O₂, and reactions were stopped with 2 N H₂SO₄. Absorbance per well was read at 490 nm by a microplate reader.