Structurally defined epitopes of Haemophilus ducreyi lipooligosaccharides recognized by monoclonal antibodies

By use of enzyme-linked immunosorbent assay and immunoblotting techniques, the migration patterns and binding epitopes of lipooligosaccharides (LOS) from 10 Haemophilus ducreyi strains were investigated with two monoclonal antibodies (MAbs), MAHD6 and MAHD7, raised against LOS from H. ducreyi ITM 2665. Closely related LOS, with defined structures, from Haemophilus influenzae, Bordetella pertussis, Aeromonas spp., and synthetic glycoproteins were also included in the analyses. The MAbs bound to conserved epitopes of LOS exposed on the surface of H. ducreyi. The MAb MAHD6 reacted with 8 of the 10 LOS from H. ducreyi but with none of the other Haemophilus or Bordetella spp. with structurally defined LOS. It is suggested that MAb MAHD6 binds to a LOS epitope (-DD-Hepp-1-->6-beta-D-Glcp-). This LOS epitope is not present in the hexasaccharide structure of LOS from H. ducreyi ITM 4747 (E. K. H. Schweda, A. C. Sundström, L. M. Eriksson, J. A. Jonasson, and A. A. Lindberg, J. Biol. Chem. 269:12040-12048, 1994). Because MAb MAHD6 reacts with the epitope mentioned above, it also discriminates between the two LOS structures, the hexasaccharide group and the nonasaccharide group, of H. ducreyi strains. MAb MAHD7 recognizes the common conserved inner core region of the LOS because it reacts with all H. ducreyi strains and with LOS with minor components in the inner core epitope structure. Rabbit polyclonal sera raised against the LOS from strains CCUG 4438 and CCUG 7470 were tested with the 10 LOS from the H. ducreyi strains. The antiserum to CCUG 7470 reacted with all H. ducreyi strains as did MAb MAHD7, whereas the antiserum to CCUG 4438 reacted with only its homologous strain and strain ITM 4747. Also, the LOSs of our reference strains CCUG 4438 and CCUG 7470 were structurally analyzed by use of sugar analyses and electrospray ionization-mass spectrometry. The hexasaccharide and nonasaccharide structures obtained from LOS of strains CCUG 4438 and CCUG 7470 were identical to the described LOS structures from H. ducreyi ITM 4747 and ITM 2665, respectively. In conclusion, the MAb MAHD6 recognizes an epitope present in the nonasaccharide LOS group, whereas the MAb MAHD7 recognizes a conserved epitope on LOS of H. ducreyi, which is present in all strains of H. ducreyi tested. Two major groups of oligosaccharides were distinguished by their LOS structures and the reactivity of monoclonal as well as polyclonal antibodies. The majority of H. ducreyi strains possess a nonasaccharide structure of LOS.