Probing multivalent carbohydrate-lectin interactions by an enzyme-linked lectin assay employing covalently immobilized carbohydrates.
We report here the synthesis of a series of mono- to trivalent N-acetylglucosamine (GlcNAc) derivatives as ligands for the plant lectin wheat germ agglutinin (WGA). Their WGA binding potencies were determined by an established enzyme-linked lectin assay (ELLA) employing microtiter plates with non-covalently immobilized porcine stomach mucin (PSM) as reference ligand and an ELLA with a new GlcNAc derivative covalently immobilized via a thiourea linkage. Comparison of both assays revealed that the type of presentation of GlcNAc residues on the microtiter plates either as part of a glycoprotein or as a covalently immobilized monosaccharide derivative strongly influences the outcome of the assay. Although the apparent dissociation constants K(D)(ELLA) for the interaction of peroxidase-labeled WGA with the microtiter plates are comparable for both surfaces, IC(50) values obtained with the PSM-free ELLA were substantially lower. Even more strikingly, this ELLA displayed a better differentiation between ligands of different valency leading to significantly higher relative inhibitory potencies of multivalent ligands compared to monovalent. Additionally, problems associated with the use of PSM, such as maximum inhibition at considerably less than 100% and poor reproducibility of IC(50) values could be overcome with this type of ELLA.
Maierhofer
Caroline
C
Universität Konstanz, Fachbereich Chemie, Fach M 709, 78457 Konstanz, Germany.
Rohmer
Katja
K
Wittmann
Valentin
V
D000117:Acetylglucosamine / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D002241:Carbohydrates / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D004800:Enzymes, Immobilized / Q000737:chemistry*; D020128:Inhibitory Concentration 50; D037102:Lectins / Q000737:chemistry*; D008024:Ligands; D015336:Molecular Probe Techniques; D015394:Molecular Structure; D014909:Wheat Germ Agglutinins / Q000737:chemistry*
D002241:Carbohydrates; D004800:Enzymes, Immobilized; D037102:Lectins; D008024:Ligands; D014909:Wheat Germ Agglutinins; D000117:Acetylglucosamine