BINDING-SPECIFICITY FOR 4 MONOCLONAL-ANTIBODIES RECOGNIZING TERMINAL GAL-ALPHA-1-]4GAL RESIDUES IN HAEMOPHILUS-INFLUENZAE LIPOPOLYSACCHARIDE

Four murine monoclonal antibodies (MAbs) reactive with the outer-core region of the lipopolysaccharide (LPS) from Haemophilus influenzae wer e generated after immunization with azide-killed H. influenzae RM.7004 AH1-2 and their epitope specificities studied. The monoclonal antibod ies: MAHI 6 (IgM), MAHI 5 (IgG2a), MAHI 8 (IgG3), and MAHI 11(IgG2b) b ound to synthetic glycoconjugates or glycolipids with terminal galabio syl (Gal alpha 1 --> 4Gal beta 1-) or globotriaosyl (Gal alpha 1 --> 4 Gal beta 1 1 --> 4GLc) residues as evaluated in enzyme immunoassays (E IA). Glycoconjugates or glycolipids with internally placed galabiose e lements were not active, indicating selectively of the MAbs for recogn ition of the epitope. Nine LPSs from H. influenzae inhibited the bindi ng of the four MAbs. The presence of the galabiosyl disaccharide eleme nt in these nine LPSs was evidenced by the binding of I-125-labeled Sh iga toxin isolated from the bacterium Shigella dysenteriae type 1, rep orted to have as receptor the Gal alpha 1 --> 4Gal beta disaccharide ( Lindberg et al., J Biol Chem, 1987, 262: 1779-85). Structural studies of these H. influenzae LPSs were also in accord with the presence of t he galabiosyl disaccharide, in addition H-1-NMR spectroscopy showed th e presence of O-acetyl groups in the RM.7004 AH1-2 LPS. However, diffe rential binding specificities of the MAbs to modified RM.7004 AH1-2 LP Ss were observed. MAHI 6 and MAHI 11 bound equally well to LPS, polysa ccharides obtained after mild acidic treatment, and dephosphorylated L PS samples as shown in inhibition EIA. In contrast, both dephosphoryla ted LPS samples and polysaccharides were poorer inhibitors of the bind ing of MAHI 5 and MAHI 8 to native RM.7004 AH1-2 LPS. Neither the de-O -acylated nor the de-O,N-acylated LPSs were effective inhibitors of an y of the four MAbs. These results suggest that the MAbs recognition in volves Gal alpha 1 --> 4Gal and O-acetyl and other saccharide residue( s) from the oligosaccharide moiety of the LPS. The epitopes are also e xpressed and accessible to recognition in clinical isolates coming fro m different sources of Neisseria spp., Haemophilus spp., and Moraxella catarrhalis, but not in Bordetella spp., Aeromonas spp. or Enterobact eriaceae as evaluated by whole-bacteria EIA and colony-dot-immunoblott ing. (C) 1995 Academic Press Limited