title: A flow cytometric assay enabling specific detection of the human lysosomal enzyme, beta-glucocerebrosidase.
abstract: We have developed a flow cytometric assay specific for human lysosomal beta-glucocerebrosidase (hGC) which is the enzyme deficient in Gaucher disease, a lysosomal storage disorder. The assay is based on the primate-specific monoclonal antibody 8E4 and thus allows detection of endogenous hGC and primate GC protein at a single cell level. We demonstrate that detection of endogenous hGC is possible in rhesus and human cells. Since antibody 8E4 does not bind to rodent GC, hGC detection in murine cell lines and primary cells upon transduction with a retrovirus carrying the hGC cDNA is possible. Comparison of this assay to a flow cytometric method which detects enzymatic GC activity shows that the 8E4-based assay is significantly more sensitive. We also show that multiparameter analyses in combination with hGC detection are feasible. This enables hGC detection in different lineages of complex cell populations. The increased sensitivity in combination with the specificity for hGC makes the 8E4-based flow cytometric assay ideally suited to monitor hGC expression. This assay is therefore of significant value to monitor the success of therapeutic strategies for Gaucher disease such as enzyme supplementation therapy, allogeneic bone marrow transplantation, and gene therapy.
authors.affiliation: Gene Therapy Section of the Department of Molecular Cell Biology, Leiden University Medical Centre, The Netherlands.
authors.forename: M J
authors.initials: MJ
authors.lastname: Havenga
authors.forename: A B
authors.initials: AB
authors.lastname: Werner
authors.forename: D
authors.initials: D
authors.lastname: Valerio
authors.forename: H H
authors.initials: HH
authors.lastname: van Es
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002452:Cell Count; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods*; D005776:Gaucher Disease / Q000235:genetics; D005962:Glucosylceramidase / Q000032:analysis*; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D008253:Macaca mulatta; D051379:Mice; D012680:Sensitivity and Specificity; D014161:Transduction, Genetic / Q000235:genetics
chemical_list: D000911:Antibodies, Monoclonal; D005962:Glucosylceramidase