Recognition specificity of monoclonal antibodies which protect mice against infection

The C-Terminal Domain of Salmonella enterica Serovar Typhimurium OmpA Is an Immunodominant Antigen in Mice but Appears To Be Only Partially Exposed on the Bacterial Cell Surface

“…In earlier studies using anti-O4 and antiporin MAbs, 50% inhibition was obtained over a similar range of concentrations of the same polyclonal sera, except for one anti-O4 MAb (42). Thus, the epitope(s) located on the C-terminal domain of OmpA is indeed a major immunodominant antigen, comparable to OmpC porin (recognized by MAb CT21.1 [42]) and O4 antigen (recognized by MAb SH6.11 [41]), in the humoral immune response against whole live or killed cells of Salmonella. Unlike SH6.11, however, CT21.1 and anti-OmpA MAbs (PA 24.1, SH16.2, SH267.15, and SH591.1) did not protect or prolong the survival of mice against as little as 10 50% lethal doses of virulent strain WB600 (40) ( Table 2).…”

“…Serovar Typhimurium strains WB600 (wild-type) and SH5014 (rfa mutant) and E. coli strain HN705 (⌬ompC ompF::Tn5) have been previously described (37,40,42,45); serovar Typhimurium strains SA1627 (rfb [26]) and SL1917 (galE496 ompA202 [44]) were provided by Ken Sanderson and Bruce Stocker, respectively. Clinical isolates of enteric and nonenteric bacteria, as well as the culture media and growth conditions for enteric and nonenteric bacteria, were previously described (41).…”

“…Anti-OmpA MAbs were purified by protein G affinity chromatography and biotinylated with Immunopure NHS-LCBiotin (Pierce) as previously described (41,42).…”

“…Competitive inhibition ELISA. A competitive inhibition ELISA was performed to identify the epitopes that are recognized by both the anti-OmpA MAbs and the polyclonal immune sera (41,42). Antigen-coated 96-well plates were blocked and washed as described above.…”

“…Intact and OM-permeabilized bacteria were stained with anti-OmpA MAbs and fluorescein-conjugated goat anti-mouse Ig (Fisher) and analyzed by cytofluorometry as previously described (41). The OM permeabilization was carried out by an extensive modification (52) of the Ca 2ϩ treatment protocol (5) that was developed for the purpose of introducing proteins into the periplasm of living bacterial cells.…”