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

An assay for the neutral glycosphingolipid, globotriaosylceramide (Gal alph a 1-4Gal beta 1-4Glc beta 1-1Cer; GL-3), was developed based on the B subun it of Escherichia coli verotoxin (VTB), The VTB gene was isolated, overexpr essed in E. coli, and purified by a single immunoaffinity chromatographic s tep using a monoclonal anti-VTB IgG-agarose column. Purified recombinant VT B was used to develop an enzyme-linked immunosorbent assay (ELISA) to deter mine the GL-3 concentrations in plasma and tissue extracts from normal indi viduals and patients and mice with alpha-galactosidase A deficiency (human Fabry disease). The mean (+/-1 SD) plasma GL-3 concentrations in affected m ale and female heterozygotes with Fabry disease were 12.6 +/- 3.7 and 1.1 /- 0.7 mu g/ml, respectively, whereas normal individuals had 0.9 +/- 0.4 mu g/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 we re 2790 +/- 400, 1100 +/- 93, 378 +/- 67, and 196 +/- 28 ng/mg wet wt and 5 .1 +/- 2.0 mu g/ml, respectively, whereas tissues from wild-type mice conta ined very low or undetectable GL-3 levels. This ELISA assay should prove us eful for determining the GL-3 levels, as well as for monitoring the effecti veness of therapeutic endeavors in patients with Fabry disease. (C) 1999 Ac ademic Press.