Chemical evidence for covalent linkages of a semisynthetic glycoconjugate vaccine forHaemophilus influenzae type b disease |
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Authors: | Robert C Seid Jr. Robert A Boykins Dai-Fang Liu Kimberly W Kimbrough Chia-Lung Hsieh Ronald Eby |
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Affiliation: | (1) Praxis Biologics, Inc., 300 East River Road, 14623 Rochester, NY, U.S.A. |
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Abstract: | We have defined the nature of the covalent linkages in aHaemophilus influenzae type b oligosaccharide-CRM197 conjugate vaccine, designated HbOC. The conjugate was acid hydrolyzed to release a novel amino-acid derivative,N-(2-hydroxyethyl)lysine (OHEt-Lys), identifiable with an amino-acid analyzer. This amino-acid derivative was formed by reduction of Schiff bases formed betweenH. influenzae type b oligosaccharides (HbO) and the lysyl -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 gelelectrophoresed HbOC on a Western blot probed with anti-CRM197 and anti-saccharide antisera. |
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Keywords: | chemical analysis saccharide-protein linkages H. influenzae type b glycoconjugate vaccine H. influenzae type b oligosaccharide-CRM197 vaccine semi-synthetic glycoconjugate vaccine |
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