title: Optimization of the enzyme-linked lectin assay for enhanced glycoprotein and glycoconjugate analysis.
abstract: Lectins are proteins capable of recognizing and binding to specific oligosaccharide structures found on glycoproteins and other biomolecules. As such, they have utility for glycoanalytical applications. One common difficulty encountered in the application of these proteins, particularly in multiwell plate assay formats known as enzyme-linked lectin assays (ELLAs), is finding appropriate blocking solutions to prevent nonspecific binding with plate surfaces. Many commonly used blocking agents contain carbohydrates and generate significant background signals in ELLAs, limiting the utility of the assays. In this study, we examined the suitability of a range of blocking reagents, including protein-based, synthetic, and commercially available carbohydrate-free blocking reagents, for ELLA applications. Each blocking reagent was assessed against a panel of 19 commercially available biotinylated lectins exhibiting diverse structures and carbohydrate specificities. We identified the synthetic polymer polyvinyl alcohol (PVA) as the best global blocking agent for performing ELLAs. We ultimately present an ELLA methodology facilitating broad spectrum lectin analysis of glycoconjugates and extending the utility of ELLAs.
authors.affiliation: Irish Separations Science Cluster, National Centre for Sensor Research, Dublin City University, Glasnevin, Dublin 9, Ireland.
authors.forename: Roisin
authors.initials: R
authors.lastname: Thompson
authors.forename: Aileen
authors.initials: A
authors.lastname: Creavin
authors.forename: Michael
authors.initials: M
authors.lastname: O'Connell
authors.forename: Brendan
authors.initials: B
authors.lastname: O'Connor
authors.forename: Paul
authors.initials: P
authors.lastname: Clarke
mesh_terms: D000818:Animals; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006001:Glycoconjugates / Q000032:analysis*; D006023:Glycoproteins / Q000032:analysis*; D006031:Glycosylation; D006801:Humans; D037102:Lectins / Q000737:chemistry*; D011142:Polyvinyl Alcohol / Q000737:chemistry / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013499:Surface Properties
chemical_list: D004800:Enzymes, Immobilized; D006001:Glycoconjugates; D006023:Glycoproteins; D037102:Lectins; D012710:Serum Albumin, Bovine; D011142:Polyvinyl Alcohol