Hrp conjugated goat anti human iga igg igm

ELISA plates were coated with 50 µL of 1 µg/mL rL-HDAg in coating buffer (13 mM Na₂CO₃, 88 mM NaHCO₃, pH 9.2) and blocked with 200 µL/well blocking buffer (1% w/v casein sodium salt, 0.05% v/v Tween^® 20, PBS). Patient sera were serially diluted 1:8 in dilution buffer (0.1% w/v casein sodium salt, 0.05% v/v Tween^® 20, PBS), starting at a 1:100 dilution. A total of 50 µL/well was incubated for 1 h at 37 °C. All washing steps were performed with 200 µL/well washing buffer (0.05% Tween^® 20 v/v, PBS). Secondary binding was performed with 1:10,000 HRP-conjugated goat anti-human IgA/IgG/IgM (Jackson Immunoresearch, West Grove, PA, USA) in dilution buffer for 1 h at 37 °C. Signals were obtained by addition of 100 µL/well TMB substrate (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min and reactions were stopped with 100 µL/well 1 M H₃PO₄. The OD450 was quantified using the EnVision HTS multilabel reader (Perkin Elmer, Waltham, MA, USA).