A lectin-immunofluorometric assay using an immobilized Bandeiraea simplicifolia II lectin for the determination of galactosylation variants of glycoproteins.
The sandwich-type immunometric assay was modified by replacing the solid phase-bound antibody with a lectin for the determination of glycoproteins carrying terminal N-acetylglucosamine residues. Microwells were coated with Bandeiraea simplicifolia II lectin and incubated with glycosylation variants of human serum glycoproteins. The bound glycoproteins were detected by time-resolved fluorometry using europium-labeled antibodies. Agalacto-derivatives of alpha 1-acid glycoprotein and transferrin obtained by neuraminidase and beta-galactosidase treatment bound to the immobilized lectin, whereas the native or desialylated glycoproteins showed no binding. The measuring range of the method for agalacto-alpha 1-acid glycoprotein was 0.01 to 10 micrograms/ml and for agalacto-transferrin 1 to 300 micrograms/ml. The binding of the agalacto-glycoproteins was totally inhibited with 1 to 10 mM N-acetylglucosamine which confirmed the specificity of the method for glycoproteins containing terminal N-acetylglucosamine residues. The results indicate that the novel lectin-immunofluorometric method is sensitive and has a wide measuring range for the determination of glycosylation variants of glycoproteins.
Parkkinen
J
J
Department of Obstetrics and Gynecology, Helsinki University Central Hospital, Finland.
Oksanen
U
U
D000117:Acetylglucosamine / Q000032:analysis*; D005455:Fluorescent Antibody Technique; D005690:Galactose / Q000032:analysis*; D005944:Glucosamine / Q000031:analogs & derivatives*; D006023:Glycoproteins / Q000032:analysis* / Q000097:blood; D006801:Humans; D007074:Immunoglobulin G; D037102:Lectins; D037121:Plant Lectins
D006023:Glycoproteins; C045475:Griffonia simplicifolia lectins; D007074:Immunoglobulin G; D037102:Lectins; D037121:Plant Lectins; D005944:Glucosamine; D000117:Acetylglucosamine; D005690:Galactose