ViPS-specific IgM, IgG, IgG1, IgG2b, IgG2c, and IgG3 were measured by coating 96-well microtiter plates (Nunc MaxiSorp; Invitrogen, Carlsbad, CA) with 2 µg/ml Vi PS purified from S. Typhi clinical isolate C652464 (21 (link)) in Dulbecco’s PBS overnight at room temperature. All plates were washed and blocked with 1% BSA in PBS (pH 7.2) (blocking buffer) for 2 h at room temperature. Blood from ViPS- or Typhim Vi– and Typbar TCV–immunized mice was diluted to 1:25 and 1:200, respectively, for IgM and IgG detection. These dilutions were based on a linear range of detection. ViPS-specific mouse IgM, IgG, IgG1, IgG2b, IgG2c, and IgG3 levels were interpreted as nanogram per microliter “equivalents” using normal mouse serum standards (Bethyl Laboratories, Montgomery, TX), mouse isotype-specific capture Abs, and horseradish peroxidase–conjugated anti-mouse IgM, IgG, IgG1, IgG2b, IgG2c, and IgG3 as described previously (22 (link)).
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