Immunization and Antibody Quantification

Mice were immunized with 10 μg TNP₆₅-Ficoll, 1 to 5 μg NP₄₀-Ficoll, or the equivalent of 0.125 to 1 μg each PPS within Pneumovax23 (Merck). In one case, a mixture of PPS (type 3, 4, 6B, 8, 9N, 12F, 14, 19F, 23F; ATCC) was used to generate Pneumovax9 due to a Pneumovax23 vaccine shortage. MAR1-5A3 monoclonal Ab mouse IFNAR-1 (BioXcell) or control mouse IgG (Jackson Immunoresearch) was administered i.p. (200 μg on d0 and 100 μg on d2 and 4 of immunization) to block IFNAR1 signaling where indicated. Adjuvant containing 20 μg Salmonella minnesota MPL, 20 μg trehalose-6,6‘-dicorynomycolate (TDCM) in 0.5% squalene/0.05% Tween-80 or 2% squalene/0.2% Tween-80 (Sigma) for intraperitoneal (i.p.) and intramuscular (i.m.) injections, respectively, was mixed with Ag prior to injection. ELISAs were as previously described (6 (link), 10 (link), 11 (link)). TNP- and PPS-specific Ig levels were estimated using a standard curve generated using anti-mouse Ig (H+L) coated wells in conjunction with mouse IgM and IgG isotype standards (Southern Biotechnology Associates). NP-specific Ig concentrations were estimated using NP-specific IgM and IgG standard curves as previously described (11 (link)).