Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
A Microplate Assay for Sialidase Activity Using Plant Lectin Binding to N-Acetyllactosamine
Satoshi ONODERA
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1994 Volume 17 Issue 1 Pages 29-33

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Abstract
This paper presents a sensitive assay for sialidase activity based on the specific binding of lecting to N-acetyllactosamine. The substrate used for sialidase assay is fetuin (30-100 ng/50 μl) with sialilated oligo-saccharides, which was then coated on a 96-well microtiterplate. After removing sialic acids from the terminal positions of the glycoconjugate glycans by sialidase, it was subjected to biotin-labeled lectin (Ricinus communis agglutinin 120), which binds specifically to N-acetyllactosamine. This was followed by the addition of a peroxidase conjugated avidin-biotin complex. The amount of bound peroxidase was determined by a colorimetric assay. The sensitivity was enhanced 1000- to 10000-fold compared to the colorimetric assay using a synthetic substrate such as 2-O-(p-nitrophenyl)-N-acetyl-α-D-neuraminic acid (PNPN). In the established method, only very small amounts of substrate and sialidase were required; therefore, it can be applied to the quantitative assay of some sialidases from Vibrio cholerae, streptococcus, the influenza virus and rat liver.
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© The Pharmaceutical Society of Japan
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