title: Development of a convenient competitive ELISA for the detection of the free and protein-bound nonhuman galactosyl-α-(1,3)-galactose epitope based on highly specific chicken single-chain antibody variable-region fragments.
abstract: The presence of the nonhuman galactosyl-α-(1,3)-galactose (Gal-α-(1,3)-Gal) carbohydrate epitope on a number of recombinant therapeutic proteins has recently been reported, renewing interest in this immunogenic carbohydrate epitope. It is well-known that this motif is the primary contributing factor in hyperacute rejection of porcine organ xenograft, due to the existence of natural antibodies against this epitope in human serum. Though the number of epitopes on recombinant glycoproteins may be low when compared directly to whole tissue, circulating anti-Gal-α-R immunoglobulins can still induce anaphylaxis. Therefore, there is a need for rapid and convenient methods for detection and monitoring of this epitope in biopharmaceuticals produced in recombinant mammalian systems. To this end, we have generated immune-challenged chicken single-chain antibody variable-region fragment (scFv) libraries targeting the Gal-α-(1,3)-Gal motif and have selected a panel of scFv's that bind the target. We have used one of these antibodies to develop a competitive ELISA for both free and protein-bound Gal-α-(1,3)-Gal and have demonstrated that the ELISA is specific for the target and can be used to determine the loading of the target on glycoproteins. This competitive ELISA will provide a convenient method of detecting and quantifying Gal-α-(1,3)-Gal on therapeutic glycoproteins.
authors.affiliation: Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland.
authors.forename: Stephen
authors.initials: S
authors.lastname: Cunningham
authors.forename: Emily
authors.initials: E
authors.lastname: Starr
authors.forename: Iain
authors.initials: I
authors.lastname: Shaw
authors.forename: John
authors.initials: J
authors.lastname: Glavin
authors.forename: Marian
authors.initials: M
authors.lastname: Kane
authors.forename: Lokesh
authors.initials: L
authors.lastname: Joshi
mesh_terms: D000818:Animals; D002645:Chickens; D004187:Disaccharides / Q000032:analysis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000737:chemistry* / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000276:immunology
chemical_list: D004187:Disaccharides; D006023:Glycoproteins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C055075:galactosyl-(1-3)galactose