title: Covalent and oriented immobilization of scFv antibody fragments via an engineered glycan moiety.
abstract: Antibody-derived fragments have enormous potential application in solid-phase assays such as biomarker detection and protein purification. Controlled orientation of the immobilized antibody molecules is a critical requirement for the sensitivity and efficacy of such assays. We present an approach for covalent, correctly oriented attachment of scFv antibody fragments on solid supports. Glycosylated scFvs were expressed in  Escherichia coli  and the C-terminal, binding pocket-distal glycan tag was oxidized for covalent attachment to amine-functionalized beads. The glycosylated scFvs could be immobilized at salt concentrations that precluded nonspecific adsorption of unglycosylated molecules and the covalently attached antibody fragments exhibited 4-fold higher functional activity than ionically adsorbed scFvs. The glyco-tethered scFvs were stable in NaCl concentrations that removed greater than 90% of adsorbed scFvs and they exhibited improved stability of antigen binding over both adsorbed scFvs and soluble, nonimmobilized scFvs in accelerated degradation tests. The simple expression and immobilization approach reported is likely to find broad application in in vitro antibody tests.
authors.affiliation: Medical College, Dalian University, Xuefu Avenue No.10, Dalian Economical and Technological Development Zone, Liaoning 116622, China. huxuejun@dlu.edu.cn
authors.forename: Xuejun
authors.initials: X
authors.lastname: Hu
authors.forename: María J
authors.initials: MJ
authors.lastname: Hortigüela
authors.forename: Sylvain
authors.initials: S
authors.lastname: Robin
authors.forename: Heng
authors.initials: H
authors.lastname: Lin
authors.forename: Yajie
authors.initials: Y
authors.lastname: Li
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Moran
authors.forename: Wenxin
authors.initials: W
authors.lastname: Wang
authors.forename: J Gerard
authors.initials: JG
authors.lastname: Wall
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D016123:Campylobacter jejuni / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000378:metabolism*
chemical_list: D055910:Antibodies, Immobilized; D011134:Polysaccharides; D057127:Single-Chain Antibodies