Immunogenic Bacterial Factors Elicit IgA Responses

Ninety-six-well plates were coated with 2.0 μg/ml of lipopolysaccharide (LPS, Salmonella enterica serovar Typhimurium, Sigma), salmochelin receptor (IroN), aerobactin receptor (IutA), or 0.25 μg/ml unlabeled chicken IgA (i.e., total IgA; H+L, Thermo Fisher Scientific) overnight at 4°C. LPS is common in gram-negative bacteria, and IroN and IutA are virulence factors involved in iron acquisition. Recombinant IroN and IutA proteins were purified from culture of E. coli BL21 containing the pET-101/D-TOPO vectors (Invitrogen) carrying iroN or iutA genes as previously described (Mellata et al., 2016 (link)). SISs were diluted 1:1 in SEA blocking buffer (Thermo Fisher Scientific), serially diluted 1:2, and incubated for 1 h at room temperature. Goat-anti-chicken-IgA-AP (H+L, Thermo Fisher Scientific) was added, followed by PNPP substrate (Thermo Fisher Scientific), and absorbance was measured at 405 nm. To measure antibody titer, the reciprocal of the highest dilution values doubling the control value (i.e., CON birds) were considered positive. ELISAs were done in duplicate per individual bird and independently replicated twice.