An enzyme-linked lectin assay for alpha1,3-galactosyltransferase.
UDP-Gal:Galbeta1-4GlcNAc alpha1,3-galactosyltransferase (alpha3GalT) is responsible for the synthesis of carbohydrate xenoantigen Galalpha1-3Galbeta1-4GlcNAc. In this work a convenient and sensitive assay system for quantification of alpha3GalT activity by enzyme-linked lectin assay (ELLA) with colorimetric detection is described. Microtiter plate wells whose surface had been coated with the polyacrylamide conjugate of the disaccharide Galbeta1-4GlcNAc (acceptor) are incubated with alpha3GalT in the presence of "cold" UDP-Gal as glycosyl donor. Formation of product by enzymatic extension of the glycan chain is detected by the biotinylated plant lectin Viscum album agglutinin. The standard curve for correct quantification of alpha3GalT activity is completed after running standard assays with no (background) or known quantities of enzyme activity. Product formation detected in this manner is proportional to enzyme activity and the concentrations of the acceptor and the glycosyl-donor UDP-Gal. In accordance with the known specificity of alpha3GalT, no enzymatic conversion of Le(x) into GalalphaLe(x) was observed using this assay. Human alphaGal antibodies were isolated using a disaccharide-exposing affinity adsorbent and their specificity was studied. Relative to the application of these natural immunoglobulins as product-detecting tool, the ELLA proved to be more sensitive.
Khraltsova
L S
LS
Shemyakin Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117871, Russia.
Sablina
M A
MA
Melikhova
T D
TD
Joziasse
D H
DH
Kaltner
H
H
Gabius
H J
HJ
Bovin
N V
NV
D000818:Animals; D000906:Antibodies; D000918:Antibody Specificity; D001681:Biological Assay / Q000379:methods*; D002240:Carbohydrate Sequence; D002417:Cattle; D003124:Colorimetry; D004797:Enzyme-Linked Immunosorbent Assay; D005700:Galactosyltransferases / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D037102:Lectins; D008024:Ligands; D008969:Molecular Sequence Data; D037121:Plant Lectins; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D000519:alpha-Galactosidase / Q000276:immunology
D000906:Antibodies; C045475:Griffonia simplicifolia lectins; D037102:Lectins; D008024:Ligands; D037121:Plant Lectins; D011994:Recombinant Proteins; D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase; D000519:alpha-Galactosidase