A time-resolved immunofluorometric assay of sialyl Lewis x-degrading alpha 2,3-sialidase activity.
We have developed an assay for alpha 2,3-sialidase (EC 3.2.1.18) which employs a biotinylated carbohydrate-polyacrylamide conjugate as substrate for the enzyme. The solution-phase sialidase reactions are followed by a selective capture of biotinylated neoglycoconjugates onto a microtitration plate coated with streptavidin. The amount of the reaction product formed is then rapidly and easily quantified using a product-specific primary antibody and europium chelate-labeled secondary antibody. This method combines the advantages of solution-phase enzymatic reaction and suitability for high-throughput screening typical of solid-phase assays. The assay gives a detectable signal with 0.4% of substrate sites desialylated. We have demonstrated the utility of the assay by measuring alpha 2,3-sialidase activity from crude lysates of cultured rat endothelial cells by using biotinylated sialyl Lewis x glycoconjugate as substrate. Endothelial sialidase(s) showed up to 250-fold higher activity toward soluble compared to immobilized substrate. Product formation detected with an anti-Lewis x antibody was linear in the range of 0.1-4 micrograms/ml of protein in endothelial cell lysate. High sensitivity of the assay was achieved by using solution-phase enzyme reaction and time-resolved fluorometric detection. The same assay format used here is easily adapted to detect activities of several different glycosidases or glycosyl-transferases by using appropriate substrates and antibodies.
Räbinä
J
J
Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Finland.
Pikkarainen
M
M
Miyasaka
M
M
Renkonen
R
R
D000818:Animals; D002457:Cell Extracts; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D006868:Hydrolysis; D009439:Neuraminidase / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D051381:Rats; D000080506:Sialyl Lewis X Antigen; D013379:Substrate Specificity
D002457:Cell Extracts; D009844:Oligosaccharides; D000080506:Sialyl Lewis X Antigen; D009439:Neuraminidase