title: Improved detection of domoic acid using covalently immobilised antibody fragments.
abstract: Antibody molecules, and antibody fragments in particular, have enormous potential in the development of biosensors for marine monitoring. Conventional immobilisation approaches used in immunoassays typically yield unstable and mostly incorrectly oriented antibodies, however, resulting in reduced detection sensitivities for already low concentration analytes. The 2H12 anti-domoic acid scFv antibody fragment was engineered with cysteine-containing linkers of two different lengths, distal to the antigen binding pocket, for covalent and correctly oriented immobilisation of the scFvs on functionalised solid supports. The Escherichia coli-produced, cysteine-engineered scFvs dimerised in solution and demonstrated similar efficiencies of covalent immobilisation on maleimide-activated plates and minimal non-covalent attachment. The covalently attached scFvs exhibited negligible leaching from the support under acidic conditions that removed almost 50% of the adsorbed wildtype fragment, and IC50s for domoic acid of 270 and 297 ng/mL compared with 1126 and 1482 ng/mL, respectively, for their non-covalently adsorbed counterparts. The expression and immobilisation approach will facilitate the development of stable, reusable biosensors with increased stability and detection sensitivity for marine neurotoxins.
authors.affiliation: Microbiology, NUI Galway, University Road, Galway, Ireland.
authors.forename: María J
authors.initials: MJ
authors.lastname: Hortigüela
authors.forename: J Gerard
authors.initials: JG
authors.lastname: Wall
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods*; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004784:Environmental Monitoring / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D007118:Immunoassay / Q000379:methods*; D007128:Immunoglobulin Fragments / Q000737:chemistry; D020128:Inhibitory Concentration 50; D007608:Kainic Acid / Q000031:analogs & derivatives* / Q000032:analysis; D008387:Marine Toxins / Q000032:analysis*; D009498:Neurotoxins / Q000032:analysis
chemical_list: D055910:Antibodies, Immobilized; D007128:Immunoglobulin Fragments; D008387:Marine Toxins; D009498:Neurotoxins; D003545:Cysteine; C012301:domoic acid; D007608:Kainic Acid