Antigen-Specific IgG Antibody ELISA

Sera were tested for IgG antibody response by enzyme-linked immunosorbent assay (ELISA) using standard procedures^{15 (link)}. Briefly, ninety-six-well microtiter plates were coated with recombinant protein PA (BEI, NR-3780), LF (NR-4268), F1 (NR-44223), LcrV (NR-32875), or F1-LcrV monomer (NR-2563) at 1 µg/ml each, or heat-inactivated LVS (HI-LVS, 2 × 10⁷/ml) diluted in carbonate buffer overnight at 4 °C and afterwards processed at ambient temperature. The plates were washed three times with 0.05%Tween20-PBS, blocked in 3% BSA-PBS for 3 h, incubated with each serum sample serially diluted 2-fold twelve times at a starting dilution of 1:20 or 1:50 in 1% BSA-PBS for 90 min, and washed again. Bound antibody was detected by using alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma, St. Louis, MO), subtypes IgG1 (Sigma), IgG2a (Abcam), IgG2b (sigma), or IgG3 (Sigma) diluted in 1% BSA-PBS and incubating for 90 min. Plates were developed with 100 µl of p-nitrophenylphosphate substrate (BioRad), and the A415 was read using a multiscan microplate reader (iMark, BioRad). The results are presented as the mean antibody endpoint titer and SE of the mean (SEM). Antibody endpoint titer is defined as the mean log dilution that yields an OD greater than the mean OD of Sham sera plus three standard deviations at the same serum dilution.