|  | | | Analytical Biochemistry, 258, pp.362-368, (1998) |
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| A Time-Resolved Immunofluorometric Assay of Sialyl Lewis x-Degrading α2,3-Sialidase Activity. |
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| Rдbinд J.2, Pikkarainen M.1, Miyasaka M.3, Renkonen R.1,* | |
1 Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, P.O. Box 21, Haartmaninkatu 3, FIN-00014 Helsinki, Finland
2 Biocenter 1A, Institute of Biotechnology, University of Helsinki, P.O. Box 56, FIN-00014 Helsinki, Finland
3 Department of Bioregulation, Biomedical Research Center, Osaka University Medical School, 2-2, Yamadaoka, Suita, Osaka 565, Japan
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| We have developed an assay for α2,3-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 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 assay. The assay gives a detectable signal with 0.4% of substrate sites desialylated. We have demonstrated the utility of the assay by measuring α2,3-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 range of 0.1-4 mg/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 is easily adapted to detect activities of several different glycosidases or glycosyltransferases by using appropriate substrates and antibodies. | |
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| *To whom correspondence should be addressed: Fax: +358-9-191 26382. Risto.Renkonen@Helsinki.fi |
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