Immunosorb plates

Immunosorb plates (Nunc) or Immobilon-P plates (Millipore) were coated with 10 µg/ml NP₄-BSA or NP₃₃-BSA (Biosearch Technologies) for ELISA or ELISPOT, respectively, and blocked with PBS containing 2% BSA. For ELISA, sera were incubated for 2 h at room temperature and plates developed with horseradish peroxidase–conjugated anti–mouse IgM or IgG1 antibodies (SouthernBiotech) using 3,3′, 5,5′ tetramethylbenzidine (TMB) substrate (BD). Color development was terminated with 2N H₂SO₄ then read on an EMax microplate reader (Molecular Devices). Purified anti-NP IgM or anti-NP IgG1 used as standards were a gift from G. Kelsoe (Duke University). For ELISPOTs, splenocyte suspensions were incubated for 4–5 h in RPMI/DMEM containing 10% FBS. Plates were developed with biotin-conjugated anti–mouse IgM or IgG1 antibodies (SouthernBiotech), followed by ExtrAvidin-Alkaline Phosphatase (Sigma-Aldrich) using NBT/BCIP substrate (Sigma-Aldrich), and color development was terminated with 1 M NaH₂PO₄. Spots were enumerated on CTL-ImmunoSpot reader (Cellular Technologies).