A sialylation-sensitive cell-based in vitro bioassay for erythropoietin; incorporation of the galactose-binding Erythrina crista-galli lectin.
The in vivo biological activity of erythropoietin (Epo) is dependent on its being adequately sialylated. Current in vitro bioassays for Epo do not correlate with the in vivo bioassays as the former do not take into account the role the liver plays in clearing desialylated glycoproteins from the circulation. Here we describe a sialylation-sensitive cell-based Epo bioassay. In the first instance, Epo activity in vitro was measured using proliferation of AS-E2 cells, and in vivo using the polycythaemic mouse bioassay. Activity in vivo was progressively abolished by controlled desialylation, whereas activity in vitro was essentially unaffected. Incorporation of an incubation step with a solid-phase galactose-binding lectin (Erythrina crista-galli), effectively mimicking passage through the liver in vivo, renders the in vitro bioassay sensitive to desialylation, such that Epo desialylated almost to completion had <10% of the activity of untreated Epo. These studies offer proof of principle, that rational manipulation of in vitro bioassays can allow prediction of activity in vivo without the use of live animals.
Liefooghe
Emily C
EC
National Institute for Biological Standards and Control (NIBSC), Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK.
Tiplady
Richard
R
Gerson
Peter
P
Lloyd
Pauline
P
Heath
Alan
A
Bristow
Adrian F
AF
D000818:Animals; D001681:Biological Assay; D015153:Blotting, Western; D004921:Erythropoietin / Q000032:analysis* / Q000737:chemistry; D005690:Galactose / Q000378:metabolism*; D066298:In Vitro Techniques; D007525:Isoelectric Focusing; D051379:Mice; D019158:N-Acetylneuraminic Acid / Q000737:chemistry*; D037121:Plant Lectins / Q000737:chemistry* / Q000378:metabolism
D037121:Plant Lectins; C045629:erythrina lectin; D004921:Erythropoietin; D019158:N-Acetylneuraminic Acid; D005690:Galactose