title: Site-directed immobilization of a genetically engineered anti-methotrexate antibody via an enzymatically introduced biotin label significantly increases the binding capacity of immunoaffinity columns.
abstract: In this study, the effect of random vs. site-directed immobilization techniques on the performance of antibody-based HPLC columns was investigated using a single-domain camelid antibody (VHH) directed against methotrexate (MTX) as a model system. First, the high flow-through support material POROS-OH was activated with disuccinimidyl carbonate (DSC), and the VHH was bound in a random manner via amines located on the protein's surface. The resulting column was characterized by Frontal Affinity Chromatography (FAC). Then, two site-directed techniques were explored to increase column efficiency by immobilizing the antibody via its C-terminus, i.e., away from the antigen-binding site. In one approach, a tetra-lysine tail was added, and the antibody was immobilized onto DSC-activated POROS. In the second site-directed approach, the VHH was modified with the AviTag peptide, and a biotin-residue was enzymatically incorporated at the C-terminus using the biotin ligase BirA. The biotinylated antibody was subsequently immobilized onto NeutrAvidin-derivatized POROS. A comparison of the FAC analyses, which for all three columns showed excellent linearity (R(2)>0.999), revealed that both site-directed approaches yield better results than the random immobilization; the by far highest efficiency, however, was determined for the immunoaffinity column based on AviTag-biotinylated antibody. As proof of concept, all three columns were evaluated for quantification of MTX dissolved in phosphate buffered saline (PBS). Validation using UV-detection showed excellent linearity in the range of 0.04-12μM (R(2)>0.993). The lower limit of detection (LOD) and lower limit of quantification (LLOQ) were found to be independent of the immobilization strategy and were 40nM and 132nM, respectively. The intra- and inter-day precision was below 11.6%, and accuracy was between 90.7% and 112%. To the best of our knowledge, this is the first report of the AviTag-system in chromatography, and the first application of immunoaffinity chromatography for the analysis of MTX.
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115-2862, USA.
authors.forename: Kaitlynn R
authors.initials: KR
authors.lastname: Davenport
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115-2862, USA.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Smith
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115-2862, USA.
authors.forename: Heike
authors.initials: H
authors.lastname: Hofstetter
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115-2862, USA.
authors.forename: James R
authors.initials: JR
authors.lastname: Horn
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115-2862, USA. Electronic address: ohofst@niu.edu.
authors.forename: Oliver
authors.initials: O
authors.lastname: Hofstetter
mesh_terms: D000818:Animals; D055910:Antibodies, Immobilized / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001710:Biotin / Q000737:chemistry* / Q000378:metabolism; D002162:Camelus; D002846:Chromatography, Affinity / Q000379:methods*; D057230:Limit of Detection; D016014:Linear Models; D008727:Methotrexate / Q000276:immunology* / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D055910:Antibodies, Immobilized; D001710:Biotin; D008727:Methotrexate
keywords: AviTag; Camelid antibody; Immunoaffinity chromatography; Methotrexate; Oriented antibody immobilization