We used synthetic mono-to hexasaccharides that mimic the fragments of the O-antigen of Ogawa and Inaba O-polysaccharides (2-4), together with certain analogs of their monosaccharides to evaluate specificity. The binding of three immunoglobulins G (two specific for Ogawa and one specific for Ogawa/ Inaba) and of two immunoglobulins A (one specific for Ogawa and one specific for Inaba/Ogawa) were characterized by ligand-induced fluorescence titration or ELISA inhibition. The cDNA sequences of these antibodies are also presented in this report. MATERIALS AND METHODSMonoclonal Antibodies-Murine ascites fluids of A-20-6 and S-20-4 both contain vibriocidal IgG 1 specific for Ogawa-LPS. I-24-2, in contrast, contains IgG 3 specific for both serotypes Ogawa and Inaba-LPS, and it has low vibriocidal activity (5) (clone S-20-4 comes from the same hybridoma cells as clone S-20-3 described in this reference). Murine ascites fluid 2D6 and ZAC-3 contain IgA specific for Ogawa-LPS and IgA specific for Inaba/Ogawa-LPS, respectively. The latter two hybridomas were gifts from Drs. Marian Neutra, Harvard Medical School, and Dr. Richard Weltzin, Oravax, Cambridge, MA (1, 6) and were grown in BALB/c mice. IgGs were purified using ImmunoPure® (G) IgG purification kits (Pierce). Briefly, ascites fluid (2 ml, clarified by centrifugation) was mixed with ImmunoPure® (G) binding buffer (2 ml) and applied to a protein G column. After washing the column with 5 ϫ 2-ml aliquots of the ImmunoPure® (G) binding buffer, the bound IgG was eluted with 6 ml of ImmunoPure® (G) elution buffer, dialyzed against PBS, pH 7.4 (2000 ml) for three changes at 0°C, frozen, and labeled. The purified A-20-6, S-20-4, and I-24-2 showed a single arc of precipitation versus goat anti-mouse IgG 1 and IgG 3 , respectively (heavy chainspecific), and goat anti-whole mouse serum (Sigma) by immunoelectrophoresis. IgAs were purified from ascites fluid by 40% ammonium sulfate precipitation and anion-exchange DEAE-Sephadex A-25 chromatography (7). Monomeric IgA was obtained by mild reduction with 5 mM 1,4-dithiothreitol (Sigma) and alkylation with 11 mM 2-iodoacetamide (Sigma), followed by re-adsorption of the sample on DEAESephadex A-25 and elution with PBS, pH 7.4. The purity of IgAs was also verified by immuno-electrophoresis against anti-mouse IgA and serum and SDS-polyacrylamide gel electrophoresis.LPS and Synthetic Oligosaccharides-V. cholerae O:1 LPSs were obtained from acetone-treated cells of strain 569B, classical biotype, serotype Inaba, lot VC1219; strain 3083, classical biotype, serotype Ogawa; and V. cholerae O:139 Bengal, strain 4450. Salmonella paratyphi A LPS was a field isolate in Nepal, strain NTP-6. All LPSs were purified as described (8) and at 2 mg/ml showed negative tests (Coomassie Blue) for protein. Severely base-degraded V. cholerae O:139 LPS (9) was a gift from Dr. Andrew D. Cox, National Research Council, Ottawa, Canada. De-O-acylated Ogawa-LPS (10) was oxidized in aqueous 0.8% periodate solution for 3 days in the dark, dialyzed, and freezedried a...

Molecular mechanics and dynamics calculations were carried out on the disaccharides α‐L‐Rhap‐(1 → 2)‐α‐L‐Rhap‐(1 → OMe) (1) and α‐L‐Rhap‐(1 → 3)‐α‐L‐Rhap‐(1 OMe) (2), and the trisaccharide α‐L‐Rhap‐(1 → 2)‐α‐L‐Rhap‐(1 → 3)‐α‐L‐Rhap‐(1 → OMe) (3). The semiflexible conformational behavior of these molecules was characterized by the occupation of a combination of different glycosidic linkage and side‐chain conformational positions whose relative occupations were sensitive to dielectric screening. Molecular dynamics simulations of the trisaccharide 3 showed little difference between the linkage conformations in the trisaccharide and the component disaccharides 1 and 2. Experimental optical rotation data of 1 and 2 were obtained as a function of temperature in varying solvents. The molecular models were combined with the semiempirical theory of Stevens and Sathyanarayana to yield calculated optical rotations. Interpretation of the data of both 1 and 2 implied that a combination of conformations, both in glycosidic and side‐chain positions, could explain the experimental data. Solvents effects were important in influencing the conformational mix and averaged optical rotation. Three‐bond heteronuclear coupling constants 3JC, H were obtained for the glycosidic linkages of 1 and 2 in D2O and DMSO. Analysis of the coupling constants with a Karplus curve showed that small reductions in the glycosidic torsion angles of the conformations of the models used here of ca. 10°–15° in ϕ and 5°–10° in ψ were required to give better agreement with experiment; a combination of conformations for both 1 and 2 was consistent with the data. There was a negligible influence on the coupling constants of 1 on changing the solvent from D2O to DMSO. © 1997 John Wiley & Sons, Inc.

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