Quantitative determination of globotriaosylceramide by immunodetection of glycolipid-bound recombinant verotoxin B subunit

Ken M. Zeidner, Robert J. Desnick, Yiannis A. Ioannou

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Abstract

An assay for the neutral glycosphingolipid, globotriaosylceramide (Galα1-4Galβ1-4Glcβ1-1Cer; GL-3), was developed based on the B subunit of Escherichia colt verotoxin (VTB). The VTB gene was isolated, overexpressed in E. coli, and purified by a single immunoaffinity chromatographic step using a monoclonal anti-VTB IgG-agarose column. Purified recombinant VTB was used to develop an enzyme-linked immunosorbent assay (ELISA) to determine the GL-3 concentrations in plasma and tissue extracts from normal individuals and patients and mice with α-galactosidase A deficiency (human Fabry disease). The mean (±1 SD) plasma GL-3 concentrations in affected male and female heterozygotes with Fabry disease were 12.6 ± 3.7 and 1.1 ± 0.7 μg/ml, respectively, whereas normal individuals had 0.9 ± 0.4 μg/ml. In 5- to 6- month-old mice with α-galactosidase A deficiency, the average GL-3 concentrations in spleen, kidney, liver, heart, and plasma were 2790 ± 400, 1100 ± 93, 378 ± 67, and 196 ± 28 ng/mg wet wt and 5.1 ± 2.0 μg/ml, respectively, whereas tissues from wild-type mice contained very low or undetectable GL-3 levels. This ELISA assay should prove useful for determining the GL-3 levels, as well as for monitoring the effectiveness of therapeutic endeavors in patients with Fabry disease.

Original languageEnglish
Pages (from-to)104-113
Number of pages10
JournalAnalytical Biochemistry
Volume267
Issue number1
DOIs
StatePublished - 1 Feb 1999

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