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Conjugation of LPS-derived oligosaccharides to proteins using oxime chemistry

Joanna Kubler-Kielb1

  • 1Program on Developmental and Molecular Immunity, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA. kielbj@mail.nih.gov

Methods in Molecular Biology (Clifton, N.J.)
|June 16, 2011
PubMed

Conjugates of bacterial polysaccharides covalently bound to a carrier protein are among licensed human vaccines. Immunization of adults and children with these vaccines results in induction of saccharide-specific antibodies composed mainly of the IgG class. Depending on the choice of coupling technique, saccharides can be attached to a protein by either multiple- or single-point attachments. While the first method is suitable for high molecular mass polysaccharides, the second one is beneficial for low-molecular mass compounds such as synthetic carbohydrates or bacterial oligosaccharides obtained by different degradation procedures. This chapter describes a method for coupling low-molecular mass lipopolysaccharide (LPS)-derived oligosaccharides composed of a core or a short O-specific polysaccharide-core fragment (O-SPC) to a carrier protein by a single-point attachment. Conjugation is performed between the carbonyl group of the reducing terminal of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) exposed after acid hydrolyses of LPS and the aminooxy group of a bifunctional linker bound to the protein. This is an efficient reaction that can be carried out quickly and under mild conditions. Conjugates thus prepared using this approach preserve the external nonreducing end of the sugar chain and can induce antibodies to both conjugate components. Consequently, this method is highly suitable for the preparation of LPS-based human vaccines.

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