Quantifying Antibody Responses via ELISA and ELISPOT

ELISA and ELISPOT were performed as previously described (25 (link)). For detection of total serum IgM and IgG, serum Ig was captured using goat anti-mouse IgM or IgG(H+L) (1 μg/ml) and detected using polyclonal HRP-conjugated Abs to IgM, IgG1, IgG2a, IgG2b, or IgG3 (SouthernBiotech) at a dilution of 1:3000. For PC ELISA, plates were coated with PC-2–BSA or BSA alone (5 μg/ml), and sera were added at 1:5 starting dilution and serially diluted 2-fold. Titers were calculated by subtracting the BSA signal from the PC BSA signal. NP-binding IgM was captured using plates coated with NP-29–BSA (1 μg/ml) in PBS. For ELISPOT, wells were coated with goat anti-mouse IgM (10 μg/ml). BM and spleen cells were plated in duplicate at 5 × 10⁵ cells per well and 2.5 × 10⁵ cells per well, respectively, and serially diluted 2-fold. Spots were detected with goat anti-mouse IgM-biotin (1:10,000), followed by SA–alkaline phosphatase (1:1,000), and developed with BCIP/NBT Substrate.