Radioimmune assay of sialyltransferase and N-acetylgalactosaminyltransferase activities using specific antibodies on a 96-well filtration plate of a multiscreen assay system.
A new assay method for glycosphingolipid glycosyl-transferase activities was developed using a 96-well filtration plate of a MultiScreen assay system. An acceptor glycosphingolipid and a donor radioactive nucleotide sugar were incubated with an enzyme source in a well of the filtration plate. After incubation, both identification and quantification of the reaction product were carried out simultaneously using a specific antibody for the product which was trapped on a filtration membrane of the plate as a complex with Staphylococcus aureus protein A (IgGSorb). This assay method was used for determining the activity of cytidine 5'-monophosphate-N-acetylneuraminic acid:Lcn4Cer alpha 2----6sialyltransferase and uridine 5'-diphosphate-N-acetyl galactosamine:GM3 N-acetylgalactosaminyltransferase. In addition to the simple and rapid identification and quantification of the product, this method proved to be as reliable and sensitive as the previously published assay procedures. Furthermore, this assay method can be used with a high concentration of detergent which should not be used in the other procedures described previously using enzyme-linked immunosorbent assay methods on a 96-well multiplate even if the enzyme reaction might require a certain percentage of the detergent concentration.
Nakamura
M
M
Division of Hemopoiesis, Jichi Medical School, Tochigi, Japan.
Tsunoda
A
A
Saito
M
M
D000818:Animals; D002241:Carbohydrates / Q000276:immunology*; D003902:Detergents; D005678:G(M2) Ganglioside; D005700:Galactosyltransferases / Q000032:analysis*; D006028:Glycosphingolipids / Q000378:metabolism; D016695:Glycosyltransferases / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis; D009711:Nucleotides / Q000378:metabolism; D011863:Radioimmunoassay / Q000379:methods*; D051381:Rats; D012799:Sialyltransferases / Q000032:analysis*; D012995:Solubility; D013205:Staphylococcal Protein A
D002241:Carbohydrates; D003902:Detergents; D006028:Glycosphingolipids; D008565:Membrane Proteins; D009711:Nucleotides; D013205:Staphylococcal Protein A; D005678:G(M2) Ganglioside; D016695:Glycosyltransferases; D005700:Galactosyltransferases; D012799:Sialyltransferases