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...
