Horseradish peroxidase hrp labeled streptavidin

To determine titers of total or NP-specific IgM, IgG subclasses and IgA, mouse sera were first diluted 1 to 100-fold with PBS (pH 7.4) plus 0.05% (v/v) Tween-20 (PBST). Two-fold serially diluted samples and standards for each Ig isotypes were incubated in 96-well plates coated with pre-adsorbed goat anti–IgM, anti–IgA or anti–IgG Abs (all 1mg/mL) in sodium carbonate/bicarbonate buffer (pH 9.6). After washing with PBST, captured Igs were detected with biotinylated anti–IgM, –IgA or –IgG isotypes Abs, followed by reaction with horseradish peroxidase (HRP)-labeled streptavidin (Sigma-Aldrich), development with o-phenylenediamine, and measurement of absorbance at 492 nm. Ig concentrations were determined using Prism^® (GraphPad Software) or Excel^® (Microsoft) software. To analyze titers of antigen-specific antibodies (high-affinity NP-binding Abs), sera were diluted 1,000-fold in PBST. Two-fold serially diluted samples were incubated in a 96-well plate pre-blocked with BSA and coated with NP₄-BSA (4 NP molecules on one BSA molecule). Captured Igs were detected with biotinylated mAbs. Data are relative values based on end-point dilution factors.

To determine titers of total IgG1, IgG3 or IgA, sera and culture supernatants were first diluted 4 – 100-fold and 10-fold, respectively, with PBS (pH 7.4) plus 0.05% (v/v) Tween-20 (PBST). Two-fold serially diluted samples and standards for each Ig isotypes were incubated in 96-well plates coated with pre-adsorbed goat anti–IgG (to capture IgG1 and IgG3) or anti–IgA Abs (all 1 mg/ml; Table 1). After washing with PBST, captured Igs were detected with biotinylated anti–IgG1, –IgG3 or –IgA (Table 1), followed by reaction with horseradish peroxidase (HRP)-labeled streptavidin (Sigma-Aldrich), development with o-phenylenediamine and measurement of absorbance at 492 nm. Ig concentrations were determined using Prism^® (GraphPad Software) or Excel^® (Microsoft) software.
To analyze titers of high-affinity NP-specific Abs, sera were diluted 1,000-fold in PBST. Two-fold serially diluted samples were incubated in a 96-well plate pre-blocked with BSA and coated with NP₃-BSA (average 3 NP molecules on one BSA molecule). Captured Igs were detected with biotinylated Ab to IgG2b or IgG3. Data are expressed as relative values based on end-point dilution factors.