title: Determination of active single chain antibody concentrations in crude periplasmic fractions.
abstract: We have measured active single chain antibody (scFv) concentrations under mass transfer limitation conditions using surface plasmon resonance on the BIAcore. For the creation of a standard curve scFv 4Dwt, derived from monoclonal antibody (mAb) 4D, directed against human chorionic gonadotropin (hCG), was purified by positive affinity chromatography. Determination of the active antibody fraction after purification was performed using anti-FLAG, reactive against a tag sequence C-terminally fused to the scFv. Two independent experiments showed that the activity remaining represented over 75% of the total amount of purified protein. Calibration curves on high density antigen surfaces showed a linear relationship between antibody concentration and binding rates. Periplasmic fractions of six mutant scFvs, also derived from mAb 4D, revealed a clear difference in the amount of soluble active scFv expressed in the periplasm of E. coli compared with the total amount of antibody present, indicating the necessity of measuring active antibody concentrations. This rapid concentration determination method will be particularly useful for accurately comparing affinity constants, using antibody concentrations determined with the BIAcore, of the many different scFv fragments routinely isolated from phage display libraries.
authors.affiliation: Organon Teknika B.V., Boxtel, Netherlands. b.kazemier@organon.akzonobel.nl
authors.forename: B
authors.initials: B
authors.lastname: Kazemier
authors.forename: H
authors.initials: H
authors.lastname: de Haard
authors.forename: P
authors.initials: P
authors.lastname: Boender
authors.forename: B
authors.initials: B
authors.lastname: van Gemen
authors.forename: H
authors.initials: H
authors.lastname: Hoogenboom
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000032:analysis; D015374:Biosensing Techniques; D002138:Calibration; D006063:Chorionic Gonadotropin / Q000276:immunology; D002846:Chromatography, Affinity; D003593:Cytoplasm / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D006063:Chorionic Gonadotropin