Enzyme-linked immunosorbent assay (ELISA)-based quantification and identification of in vitro enzyme-catalyzed glycosphingolipid synthesis and degradation products with carbohydrate sequence-specific monoclonal antibodies.
A new method has been developed to monitor glycosyltransferase and glycosylhydrolase activities. Reaction product identification and quantification are accomplished simultaneously with an enzyme-linked immunosorbent assay (ELISA) using carbohydrate sequence-specific monoclonal antibodies. beta-Galactosyltransferase and alpha-galactosidase reactions were used to illustrate the salient features of the method. These include simple product identification and quantification, no detergent requirement, consumption of small amounts of reagents, and no use of radioisotopes. Furthermore, it is possible to measure substrate disappearance or product formation with this method. Enzyme characteristics such as Km, Vmax, divalent cation requirement, and pH optimum were investigated with this new method.
Stults
C L
CL
Department of Chemistry and Biochemistry, San Francisco State University, California 94132.
Wilbur
B J
BJ
Macher
B A
BA
D000818:Animals; D000911:Antibodies, Monoclonal; D002240:Carbohydrate Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D005700:Galactosyltransferases; D006028:Glycosphingolipids / Q000032:analysis*; D006801:Humans; D000519:alpha-Galactosidase
D000911:Antibodies, Monoclonal; D006028:Glycosphingolipids; D005700:Galactosyltransferases; D000519:alpha-Galactosidase