title: Chemical evidence for covalent linkages of a semi-synthetic glycoconjugate vaccine for Haemophilus influenzae type B disease.
abstract: We have defined the nature of the covalent linkages in a Haemophilus influenzae type b oligosaccharide-CRM197 conjugate vaccine, designated HbOC. The conjugate was acid hydrolyzed to release a novel amino-acid derivative, N epsilon-(2-hydroxyethyl)lysine (OHEt-Lys), identifiable with an amino-acid analyzer. This amino-acid derivative was formed by reduction of Schiff bases formed between H. influenzae type b oligosaccharides (HbO) and the lysyl epsilon-amino groups of CRM197 (a non-toxic, cross-reactive variant of diphtheria toxin), followed by acid hydrolysis of HbOC. Quantification of OHEt-Lys per CRM197 molecule allowed the determination of a covalency ratio, a useful parameter for evaluating the stoichiometry and consistency of HbOC preparations. Covalent association between HbO and CRM197 was also demonstrated by the coincidence of immunoreactivity of gel-electrophoresed HbOC on a Western blot probed with anti-CRM197 and anti-saccharide antisera.
authors.affiliation: Praxis Biologics, Inc., Rochester, NY 14623.
authors.forename: R C
authors.initials: RC
authors.lastname: Seid
authors.forename: R A
authors.initials: RA
authors.lastname: Boykins
authors.forename: D F
authors.initials: DF
authors.lastname: Liu
authors.forename: K W
authors.initials: KW
authors.lastname: Kimbrough
authors.forename: C L
authors.initials: CL
authors.lastname: Hsieh
authors.forename: R
authors.initials: R
authors.lastname: Eby
mesh_terms: D000596:Amino Acids / Q000032:analysis; D001428:Bacterial Vaccines / Q000737:chemistry*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D006001:Glycoconjugates / Q000737:chemistry*; D006192:Haemophilus Infections / Q000276:immunology*; D006193:Haemophilus influenzae / Q000276:immunology*; D006801:Humans; D009844:Oligosaccharides / Q000737:chemistry; D014614:Vaccines, Synthetic / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D001428:Bacterial Vaccines; D006001:Glycoconjugates; D009844:Oligosaccharides; D014614:Vaccines, Synthetic