IgG/IgA Titration in STm Vaccine Mice

Titration of serum IgG from STm vaccine-immunized mice, monoclonal antibodies (mAbs) (anti-S. Typhimurium O4 IgG [SC5223, Santa Cruz Biotechnology, CA]) and anti-S. Typhimurium O5 IgA (Sal4), or polyclonal anti-SE COPS sera (described previously [18 (link)]) was accomplished using an enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well, medium-binding, microtiter plates (Greiner Bio-One, NC) were coated with either COPS antigens (1925wzzB-COPS, dOAc-1925wzzB-COPS, or SE COPS) or COPS conjugates (STm-COPS^KDO:CRM₁₉₇, or dOAc-STm-COPS^KDO:CRM₁₉₇) at a concentration of 5 μg polysaccharide/mL and incubated overnight at 4°C. Plates were washed with PBST and blocked with PBS + 10% Omniblok non-fat, dry milk for 2 h at 37°C. Serum samples and monoclonal antibodies were serially diluted in PBST + 10% Omniblok, transferred to blocked ELISA plates, and incubated for 1 h at 37°C. Plates were washed, and incubated for 1 h at 37°C with horseradish peroxidase (HRP)-labeled anti-mouse IgG (for O4 and mouse serum; 1:1,000) (KPL, MD) or HRP-labeled anti-mouse IgA (for O5; 1:500) (Southern Biotech, AL). After washing, substrate (3,3’,5,5’-tetramethylbenzidine, KPL, MD) was added, and the plates were incubated on a rocker at ambient temperature for 15 min in darkness. The reaction was stopped with the addition of 1 M H₃PO₄, and the absorbance at 450 nm was recorded using an Ascent microplate reader (Thermo Scientific, MA). Endpoint titers, represented as ELISA units (EU) per mL, were defined and calculated as previously described [18 (link)]. Analyses were conducted with positive sera as plate controls with acceptance criteria of < 15% variance between plates. Proportional levels of epitope-specific antibody in sera were calculated by subtracting the serum IgG titers for various COPS antigens as follows: O1,12,core antibody levels were defined as the SE COPS titer; O4 antibody levels were calculated as (dOAc-1925wzzB-COPS titer)–(SE COPS titer); O-acetyl specific antibody levels were calculated by (1925wzzB-COPS titer)–(dOAc-1925wzzB-COPS titer). Any negative values were assigned a titer of 0. The relative levels of each antibody population were calculated as a percentage of the sum of all three calculated titers.