Development of an enzyme-linked immunosorbent assay-based method for measuring galactosyltransferase activity using a synthetic glycopolymer acceptor substrate.
A lectin-assisted enzyme-linked immunosorbent assay (ELISA)-based method using a synthetic glycopolymer as an acceptor substrate was developed for measuring beta 1,4-galactosyltransferase (GalT) activity. A polyacrylamide derivative having a beta-linked N-acetylglucosamine (GlcNAc beta) moiety on each monomeric unit was synthesized chemically and immobilized on a polystyrene microtiter plate as an acceptor substrate for GalT. After the plate was incubated with bovine GalT, the enzyme reaction product, beta-linked Gal residue on the polyacrylamide-bound GlcNAc residue, was detected by using Ricinus communis agglutinin 1 (RCA1), rabbit anti-RCA1 antibody, and a peroxidase-labeled anti-rabbit IgG. The lowest GalT concentration detectable by this method was about 0.5 mU/ml, which is comparable to those by the previously reported ELISA-based assays. The unique property of the glycopolymer, PAP(GlcNAc beta), of binding noncovalently but tightly to the polystyrene microtiter plate allowed the use of this acceptor substrate for the GalT activity measurement even in the presence of 1% Triton CF-54 and X-100. Our system was successfully applied to assess GalT activity in milk of various mammals.
Oubihi
M
M
Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, Japan.
Kitajima
K
K
Kobayashi
K
K
Adachi
T
T
Aoki
N
N
Matsuda
T
T
D000117:Acetylglucosamine / Q000737:chemistry*; D000180:Acrylic Resins / Q000737:chemistry*; D000818:Animals; D002162:Camelus; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006041:Goats; D007788:Lactose Synthase / Q000378:metabolism*; D051379:Mice; D008892:Milk / Q000201:enzymology; D011137:Polystyrenes; D012680:Sensitivity and Specificity; D000099166:Polyacrylamides
D000117:Acetylglucosamine; D000180:Acrylic Resins; D007788:Lactose Synthase; D011137:Polystyrenes; D000099166:Polyacrylamides