Elsevier

Analytical Biochemistry

Volume 265, Issue 2, 15 December 1998, Pages 282-289
Analytical Biochemistry

Regular Article
Carbohydrate-Based Probes for Detection of Cellular Lectins

https://doi.org/10.1006/abio.1998.2859Get rights and content

Abstract

Carbohydrate (spacered saccharide residue, Glyc) probes with various tags were synthesized as analytical tools for study of cellular lectins, i.e., Glyc–polyacrylamide–3H, Glyc–PAA–biotin, Glyc–PAA–fluorescein (flu), and Glyc–PAA–digoxigenin, where PAA is a soluble polyacrylamide carrier of ≈30 kDa. Binding of all types of probes, where Glyc is the sialyl Lewis X (SiaLeX) tetrasaccharide or a blank saccharide, was assessed using Chinese hamster ovary (CHO) cells either transfected with the E-selectin cDNA or mock-transfected. High binding of SiaLeX–PAA–3H to E-selectin-transfected cells and absence of binding to control cells (both native and permeabilized) allowed the conclusion that the polyacrylamide carrier and the spacer arm do not contribute significantly to the binding. The biotinylated probe showed a high level of nonspecific binding in cell enzyme-linked assays. A similarly built digoxigenin-labeled probe was significantly better. In flow cytometry assays, the fluorescein probe demonstrated a specific binding to E-selectin-transfected cells of a similar level to that given by an anti-E-selectin antibody. In addition, it could be inhibited by the anti-E-selectin antibody, further demonstrating specificity. Tumors were obtained from nude mice by injection of CHO E-selectin or mock-transfected cells. The fluorescent SiaLeX–PAA–flu probe could bind to tumor sections from E-selectin-positive CHO cells, but not from control CHO cells. These probes can thus be used to reveal specifically complex carbohydrate-binding sites on cells either in culture or on tissue sections.

References (17)

  • D. Storm et al.

    J. Immunol. Methods

    (1996)
  • A. Adam et al.

    J. Pharm. Biomed. Anal.

    (1996)
  • A. Krzeslak et al.

    Mech. Ageing Dev.

    (1996)
  • T.M. Carlos et al.

    Blood

    (1994)
  • G. Weitz-Schmidt et al.

    Anal. Biochem.

    (1996)
  • S.M. Game et al.

    Anal. Biochem.

    (1998)
  • Q. Zhou et al.

    Cell Surface Carbohydrates and Cell Development

    (1992)
  • H.-J. Gabius

    Eur. J. Biochem.

    (1997)
There are more references available in the full text version of this article.

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Fukuda, M.

1

To whom correspondence should be addressed. Fax: 33-02 40 08 40 82. E-mail:[email protected].

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