Kpl peroxidase substrate

Microtiter plates were coated with H77C gpE1/gpE2 or gpE1/ΔHVR1-gpE2 (amino acids 412 to 656) overnight at 4°C in PBS. For peptide ELISAs, wells coated with an N-terminal biotinylated peptide corresponding to H77C residues 387 to 417 (biotin-CETHVTGGNAGRTTAGLVGLLTPGAKQNIQLINTN; GLBiochem, Shanghai, China) at 2 μg/well were blocked with 4% BSA in PBS for 1 h. Sera from vaccinated mice were diluted in PBST and added to the plates for 1 h (50 μl/well). gpE1/gpE2 and peptide-specific antibodies were detected by using a horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:10,000; Cedarlane Laboratories, Burlington, ON, Canada) and KPL peroxidase substrate (SeraCare Life Sciences, Milford, MA). The absorbance was read at 450 to 570 nm as described above. Absorbance values from three independent experiments are expressed as means ± standard errors of the means (SEM).

Sera were tested using an ELISA assay. In short, Maxisorp 96-well plates (Nunc) were coated over-night at 4°C with 1 µg/mL Polyribosyl Ribitol Phosphate (PRP, NIBSC 12/306). The human anti-Haemophilus influenzae b reference serum (NIBSC 09/222) was used as reference with assigned concentrations of IgG to PRP. After serum incubation for 2 hours at room temperature (RT), plates were incubated for 1 hour at RT with goat anti-human IgG H+L HRP (1:3000, BioRad). Following addition of KPL peroxidase substrate (SeraCare) and 0.5 M sulphuric acid, absorbance was read at 450 nm.

Microtiter plate wells (Corning) were coated with 1 μg GNA-lectin (Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS) overnight at 4°C and then blocked for 1 h with 4% bovine serum albumin (BSA) (Sigma-Aldrich) in PBS containing 0.2% Tween 20 (PBST). After washing of the wells with PBST, WT or ΔHVR1 gpE1/gpE2 antigens (100 ng/well) were added for 1 h. gpE2-specific MAbs (H77.16, AP33, HC33.1, HC33.4, HC84.26, and AR3b) (16 (link), 52 (link), 53 (link), 55 (link), 56 (link)), gpE1/gpE2-specific MAbs (AR4a and AR5a) (54 (link)), or a control MAb (B6) (16 (link)) was added for 1 h (50 μl/well) and detected by an anti-human or anti-mouse horseradish peroxidase-conjugated secondary antibody (1:10,000; Jackson Immuno Research, West Grove, PA, USA) and KPL peroxidase substrate (SeraCare Life Sciences, Milford, MA). The absorbance (450 to 570 nm) was read by using an Enspire plate reader (Perkin-Elmer, Waltham, MA, USA).