Glycoconjugate Journal

, Volume 3, Issue 4, pp 351–362 | Cite as

Immunochemical characterization of antisera reactivities toN-acetyl-d-glucosamine oligosaccharides with theβ(1–4)-glycosidic linkage

  • Samar E Makhlouf
  • Lyman E Davis
  • Deepika Paul
  • Byron Anderson
Article
Received:

Abstract

The β(1–4)-linked oligosaccharides ofN-acetyl-d-glucosamine (GlcNAc) isolated from chitin were used to prepare synthetic immunogens and antigens by reductive amination of (GicNAc)n to bovine serum albumin (BSA). The rabbit antisera produced to the (GlcNAc)n-BSA conjugates were characterized using an enzyme-linked immunosorbent assay (ELISA) system under conditions that, only the antibodies with carbohydrate specificity were reactive with the solid-phase adsorbed (GlcNAc)n-BSA antigens. Inhibition assays using the (GlcNAc)n-BSA, (GlcNAc)n oligosaccharides, and the reduced oligosaccharides showed a relative specificity of the antisera for the chain length of the (GlcNAc)n sequences. For example, the anti-(GlcNAc)5-and anti-(GlcNAc)4-sera were inhibited best by the longer chain (GlcNAc)n ologosaccharides with the antibody combining sites directed mainly to the cyclic GlcNAc residues of the (GlcNAc)n-BSA conjugates. The antibody combining sites were in part directed to the acyclic moiety of the reducing end of the oligosaccharides as shown by the increased inhibitory activities of the reduced (GlcNAc)n oligosaccharides particularly, with the anti-(GlcNAc)2-and anti-(GlcNAc)3-sera. The best hapten inhibitors for the anti-(GlcNAc)2-BSA and anti-(GlcNAc)1-BSA sera were theN-butylamine derivatives of (GlcNAc)2 and (GlcNAc)1, respectively, indicating that the antibodies were also reactive with the secondary amine formed between the reducing end of the oligosaccharides and the ε-amino groups of lysine.

Key words

(GlcNAc)n oligosaccharides antibody specificities antibody combining site 

Abbreviations

ELISA

enzyme-linked immunosorbent assay

BSA

bovine serum albumin

GicNAc

N-acetyl-d-glucosamine

(GlcNAc)n

Oligosaccharides containing GlcNAc in β1–4 linkages

(GlcNAc)2

DGlcNAcβ(1–4)-d-GlcNAc

(GlcNAc)3

(GlcNAc)4 and (GlcNAc)5, the homologous oligosaccharides of (GlcNAc)2

PBS

phosphate buffered saline (0.01 M sodium phosphate, pH73 containing 0.15%M (NaCl)

PBSA

PBS containing 1% BSA and 0.1% Tween-20

ONPG

o-nitrophenyl-β-d-galactopyranoside

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Copyright information

© Glycoconjugate Journal 1986

Authors and Affiliations

  • Samar E Makhlouf
    • 1
  • Lyman E Davis
    • 1
  • Deepika Paul
    • 1
  • Byron Anderson
    • 1
  1. 1.Departments of Molecular Biology, and Otolaryngology and Maxillofacial SurgeryNorthwestern University Medical SchoolChicagoUSA

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