title: High throughput ranking of recombinant avian scFv antibody fragments from crude lysates using the Biacore A100.
abstract: Advances in molecular evolution strategies have made it possible to identify antibodies with exquisite specificities and also to fine-tune their biophysical properties for practically any specified application. Depending on the desired function, antibody/antigen interactions can be long-lived or short-lived and, therefore, particular attention is needed when seeking to identify antibodies with specific reaction-rate and affinity properties. Surface plasmon resonance (SPR) biosensors routinely generate sensitive and reliable kinetic data from antibody/antigen interactions for both therapeutic and diagnostic applications. However, many kinetic-based screening assays require rigorous sample preparation and purification prior to analysis. To ameliorate this problem, we developed a rapid and reliable assay for characterising recombinant scFv antibody fragments, directly from crude bacterial lysates. Ninety-six scFv antibodies derived from chickens immunised with C-reactive protein (CRP) were selected by phage display and evaluated using the Biacore A100 protein interaction array system. Antibodies were captured from crude bacterial extracts on the sensor chip surface and ranked based on the percentage of the complex left (% left) after dissociation in buffer. Kinetic rate constants (k(a) and k(d)) and affinity (K(D)) data were obtained for six clones that bound monomeric CRP across a broad affinity range (2.54 x 10(-8) to 3.53 x 10(-10) M). Using this assay format the A100 biosensor yielded high quality kinetic data, permitting the screening of nearly 400 antibody clones per day.
authors.affiliation: Biomedical Diagnostics Institute, Dublin City University, Dublin 9, Ireland.
authors.forename: Paul
authors.initials: P
authors.lastname: Leonard
authors.forename: Pär
authors.initials: P
authors.lastname: Säfsten
authors.forename: Stephen
authors.initials: S
authors.lastname: Hearty
authors.forename: Barry
authors.initials: B
authors.lastname: McDonnell
authors.forename: William
authors.initials: W
authors.lastname: Finlay
authors.forename: Richard
authors.initials: R
authors.lastname: O'Kennedy
mesh_terms: D000818:Animals; D002097:C-Reactive Protein / Q000276:immunology; D002645:Chickens / Q000276:immunology*; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000302:isolation & purification*; D007700:Kinetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification*; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D002097:C-Reactive Protein