A simple and specific assay of glycosyltransferase and glycosidase activities by an enzyme-linked immunosorbent assay method, and its application to assay of galactosyltransferase activity in sera from patients with cancer.
A simple, sensitive, and specific assay method for glycosyltransferase and glycosidase activities has been established by means of an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody, H-11 directed to lactoneotetraosylceramide (nLc4Cer). Enzyme activity was determined by assaying the amount of reaction product, nLc4Cer with the ELISA method. For the assay of galactosyltransferase activity, lactotriaosylceramide (Lc3Cer) immobilized on a 96-well microtiter plate was incubated with bovine milk galactosyltransferase in cacodylate buffer (pH 6.8) containing Triton CF-54, Mn2+, and UDP-galactose. Optimum incubation conditions for the enzyme were determined. Glycosidase activity was also assayed by the ELISA method by using Clostridium perfringens sialidase and neolacto-series gangliosides as substrates, and the substrate specificities towards the gangliosides were examined. By this method, 3-100 pmol of reaction product could be determined. The assay method has several advantages as follows: 1, the method is simple; 2, separation of the reaction product is not required; 3, quantification and identification of the reaction product were done simultaneously; 4, naturally occurring substrates are available (especially for glycosidase); 5, many samples can be assayed in one microplate; 6, sensitivity is very high. The present method was applied for the detection of galactosyltransferase in human sera. Significant elevations of the galactosyltransferase levels were observed in the sera from cancer patients. The formation of nLc4Cer was confirmed by employing the TLC-immunostaining method for bands of Lc3Cer after incubation of the bands with serum and cofactors on an HPTLC plate.
Department of Biochemistry, Faculty of Medicine, Tokyo Medical and Dental University.
T
T
Taki
S
S
Nishiwaki
K
K
Ishii
S
S
Handa
D000911:Antibodies, Monoclonal; D002855:Chromatography, Thin Layer; D003902:Detergents; D004797:Enzyme-Linked Immunosorbent Assay; D005700:Galactosyltransferases / Q000097:blood* / Q000378:metabolism; D005964:Glucosyltransferases / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007120:Immunochemistry; D007700:Kinetics; D009369:Neoplasms / Q000097:blood / Q000201:enzymology*; D009439:Neuraminidase / Q000032:analysis; D013194:Staining and Labeling; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D003902:Detergents; D005700:Galactosyltransferases; D005964:Glucosyltransferases; D006026:Glycoside Hydrolases; D009439:Neuraminidase