MultiScreen ELISPOT plates (MAIPS4510, Millipore) were activated with ethanol (35%), washed four times with PBS, and then coated with 100 μl NP4-BSA (5 μg/ml; N-5050L, Biosearch Technologies), goat anti-mouse IgG1 Ab (5 μg/ml; 1071-01, Southern Biotech), or goat anti-mouse IgM Ab (5 μg/ml; 1121-01, Southern Biotech) in PBS overnight at 4°C. The plates were then washed six times with PBS and blocked with BSA (0.5%) in RPMI/Hepes plus l-glutamine for 1 hour at room temperature. Single-cell suspensions (250,000 or 100,000 cells/ml) from the spleen and bone marrow of immunized mice were cultured in plates at 37°C for 16 hours in FBS-RPMI. The cultures were removed, and the plates were washed six times, incubated with biotin-goat anti-mouse IgG1 Ab (1070-08, Southern Biotech) or biotin-rat anti-mouse IgM mAb (1022-08, Southern Biotech) for 2 hours on a shaker at room temperature, washed six times, incubated with horseradish peroxidase (HRP)–streptavidin (Santa Cruz Biotechnology) for 1 hour on a shaker at room temperature, washed 10 times, and developed using the VECTASTAIN AEC Peroxidase Substrate Kit (SK-4200, Vector Laboratories) following the manufacturer’s protocol. Plates were imaged and quantified using a CTL-ImmunoSpot Analyzer (Cellular Technology). All the Abs and mAbs used in the above experiments are listed in table S1A.

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