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

An assay for the neutral glycosphingolipid, globotriaosylceramide (Galalpha1-4Galbeta1-4Glcbeta1-1Cer; GL-3), was developed based on the B subunit of Escherichia coli 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 alpha-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 microg/ml, respectively, whereas normal individuals had 0.9 +/- 0.4 microg/ml. In 5- to 6-month-old mice with alpha-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 microg/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. Copyright 1999 Academic Press.