Antigen-specific Antibody ELISA Protocol

Maxisorb Plates (Nunc) were coated with 0.05 μg/well CTH522 or polyclonal goat anti-mouse IgG (Southern-Biotech), diluted 1:1000, overnight at 4 °C. Individual mouse sera were analyzed in duplicate. After blocking, serum was added in PBS with 2% BSA, starting with a 30-fold dilution for antigen-specific IgG or IgG subclasses. For analysis of total IgG2c secreted by in vitro stimulated murine B cells, the supernatant was added in 10-fold dilutions, starting from undiluted sample. HRP-conjugated secondary antibodies, goat anti-mouse IgG (Zymed), IgG2c (Invitrogen), or IgG1 (Southern Biotech), were diluted in PBS with 1% BSA. After 1 h of incubation, antigen-specific Abs were detected using TMB substrate as described by the manufacturer (Kem-En-Tec Diagnostics). ELISA data were plotted as the sum of absorbances as described previously^{60 (link)}, as a simple method to visualize antibody responses.