Production of monoclonal antibodies specific for the i and 1,2 flagellar antigens of Salmonella typhimurium and characterization of their respective epitopes

Abstract

Salmonella typhimurium expresses two antigenically distinct flagellins, each containing a different H antigen (i and 1,2), the combination of which is highly specific for this serotype. In this study, overlapping recombinant flagellin fragments were constructed from the fliC (H:i) and fljB (H:1,2) flagellin genes, and the expression products were tested for binding to H antigen-specific monoclonal and polyclonal antibodies. A minimal area, 86 amino acids for H:i and 102 amino acids for H:1,2, located in the central variable domain of each flagellin was required for the binding of serotype-specific antibodies, providing further evidence for the presence of a discontinuous H epitope. Two peptides comprising these areas were shown to be highly suitable for application as antigens in an enzyme-linked immunosorbent assay detecting S. typhimurium-specific antibody.

FIG. 1

Serotyping of Salmonella strains with MAb VB5 and commercial serotyping serum (batch 2) on Western blots. (A) H:i flagellin-containing Salmonella stained with VB5. (B) H:1,2 flagellin-containing Salmonella stained with VB5. (C) H:i flagellin-containing Salmonella stained with commercial H:i-specific serotyping serum. The Salmonella strains used (with the H serotype in parentheses) are as follows: AB, S. aberdeen (i:1,2); BA, S. bandia (i:lw); BE, S. bergen (i:enz15); BO, S. bonariensis (i:enx); EN, S. enteritidis (gm:-); HE, S. heidelberg (r:1,2); JU, S. jukestown (i:enz15); KE, S. kentucky (i:z6); KD, S. kedougou (i:lw); NE, S. newport (eh:1,2); MU, S. muenchen (d:1,2); TA, S. takoradi (i:1,5); TY, S. typhimurium (i:1,2); VI, S. virchow (r:1,2). Molecular size markers in kilodaltons are shown at the right.

FIG. 2

Immunostained cross blots showing the binding of H-specific antibodies to recombinant flagellin expression products. Binding of the H:i (A) and H:1,2 (B) flagellins with H-specific MAbs and commercial serotyping sera. Binding of the MAbs is indicated by the bar (filled, strong; hatched, weak; open, negative). Restriction sites used for the creation of fragments are indicated on the top bar of each panel, which represents the complete flagellin protein. The positions of the primers used for the cloning of the flagellin genes (E1, E2, P1, and P2) are depicted by arrows. Fragments indicated by superscript a were also cloned in pGEX and applied in an ELISA. Both a large fragment of the heterologous flagellin and cro-β-galactosidase (cro-β-gal) expressed from the empty vector pEX13 (indicated by superscript b) were always included as negative control fragments.

FIG. 3

Results of an indirect ELISA with experimental sera from chickens orally infected with S. typhimurium (n = 8), S. enteritidis (n = 5), or S. enterica Pullorum and Gallinarum (n = 7) on H:i-specific fragment pcr6-i (A) or H:1,2-specific fragment pcr2-1,2 (B). OD, optical density.