Normal mouse serum standards

ViPS-specific IgM and IgG were measured by coating 96-well microtiter plates (Nunc MaxiSorp™; Invitrogen, Carlsbad, CA) with 2 µg/ml of ViPS purified from S. Typhi clinical isolate C6524^{64 (link)} in DPBS overnight at room temperature. All plates were washed and blocked with 2% Bovine serum albumin (BSA) in PBS pH 7.2 (blocking buffer) for 2 h at room temperature. Blood from immunized mice was diluted to 1:25 for IgG detection and 1:50 for IgM detection in blocking buffer; ViPS-specific mouse IgM and IgG levels were interpreted as ng/μl “equivalents” using normal mouse serum standards (Bethyl Laboratories, Montgomery, TX), as described previously^{35 (link)}.

Unconjugated serotype polysaccharides MenA (13/262), MenC (08/214), MenW (16/152), or MenY (16/206) were purchased from the National Institute for Biological Standards and Control (Hertfordshire, UK). Serotype polysaccharide-specific IgM and IgG were measured by coating 96-well microtiter plates (Nunc MaxiSorp; Invitrogen, Carlsbad, CA) with 1 µg/ml MenA, MenC, MenW, or MenY in PBS, pH 7.2, 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 immunized infant and adult mice was diluted to 1:25 and 1:50, respectively, and serogroup-specific IgM and IgG levels were interpreted as ng/µl “equivalents” using normal mouse serum standards (Bethyl Laboratories, Montgomery, TX), mouse isotype–specific capture Abs, and HRP-conjugated anti-mouse IgM, IgG, IgG1, IgG2b, IgG2c, and IgG3 as described previously (33 (link), 34 (link)).

ViPS-specific IgM, IgG, IgG1, IgG2b, IgG2a, IgG2c, and IgG3 were measured by coating 96-well microtiter plates (Nunc MaxiSorp™; Invitrogen, Carlsbad, CA) with 2 µg/ml of ViPS purified from S. Typhi clinical isolate C652464 (22 (link)) in DPBS overnight at room temperature. All plates were washed and blocked with 1% bovine serum albumin 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, IgG2a, IgG2c, and IgG3 levels were interpreted as ng/μl “equivalents” using normal mouse serum standards (Bethyl Laboratories, Montgomery, TX), mouse isotype-specific capture antibodies, and HRPO-conjugated anti-mouse IgM, IgG, IgG1, IgG2b, IgG2a, IgG2c, and IgG3 as described previously (12 (link), 21 (link)).