A microplate assay for sialidase activity using plant lectin binding to N-acetyllactosamine.
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 microliters) with sialylated oligosaccharides, 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 10,000-fold compared to the colorimetric assay using a synthetic substrate such as 2-O-(p-nitrophenyl)-N-acetyl-alpha-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.
Onodera
S
S
Department of Clinical Chemistry, Showa College of Pharmaceutical Sciences, Tokyo, Japan.
D000606:Amino Sugars / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D006385:Hemagglutination Inhibition Tests; D006863:Hydrogen-Ion Concentration; D009980:Influenza A virus / Q000201:enzymology; D037102:Lectins / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D009439:Neuraminidase / Q000378:metabolism*; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D037121:Plant Lectins; D051381:Rats; D012680:Sensitivity and Specificity; D013291:Streptococcus / Q000201:enzymology; D013379:Substrate Specificity; D014734:Vibrio cholerae / Q000201:enzymology; D000509:alpha-Fetoproteins / Q000737:chemistry
D000606:Amino Sugars; D037102:Lectins; D009844:Oligosaccharides; D037121:Plant Lectins; C017707:Ricinus communis agglutinin-1; D000509:alpha-Fetoproteins; C000458:N-acetyllactosamine; D009439:Neuraminidase