title: Nanomolar to sub-picomolar affinity measurements of antibody-antigen interactions and protein multimerizations: fluorescence-assisted high-performance liquid chromatography.
abstract: Although several techniques exist for the measurement of high-affinity interactions, it is still challenging to determine dissociation constants around or even below 1pM. During the analysis of several human-derived monoclonal antibodies to adalimumab, we found a clone with a very high affinity that could not be measured using conventional surface plasmon resonance assays. We developed a straightforward and robust method to measure affinities in the nanomolar to sub-picomolar range. The assay is based on separation of bound and free fluorescently labeled antigen using size exclusion chromatography and quantification by in-line fluorescence detection. We describe optimal conditions and procedures that result in a very sensitive assay that can be used to reliably determine ultra-high affinities. Using the method described in this article, a dissociation constant of 0.78pM could be determined for the anti-adalimumab antibody.
authors.affiliation: Sanquin Research, 1066 CX Amsterdam, The Netherlands. t.rispens@sanquin.nl
authors.forename: Theo
authors.initials: T
authors.lastname: Rispens
authors.forename: Pleuni
authors.initials: P
authors.lastname: Ooijevaar-de Heer
authors.forename: Ninotska I L
authors.initials: NI
authors.lastname: Derksen
authors.forename: Gertjan
authors.initials: G
authors.lastname: Wolbink
authors.forename: Pauline A
authors.initials: PA
authors.lastname: van Schouwenburg
authors.forename: Simone
authors.initials: S
authors.lastname: Kruithof
authors.forename: Rob C
authors.initials: RC
authors.lastname: Aalberse
mesh_terms: D000068879:Adalimumab; D000906:Antibodies / Q000737:chemistry / Q000502:physiology*; D061067:Antibodies, Monoclonal, Humanized / Q000276:immunology* / Q000627:therapeutic use; D000915:Antibody Affinity / Q000502:physiology*; D018501:Antirheumatic Agents / Q000627:therapeutic use; D001172:Arthritis, Rheumatoid / Q000188:drug therapy; D001402:B-Lymphocytes / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005453:Fluorescence; D006801:Humans; D015850:Interleukin-6 / Q000276:immunology; D012680:Sensitivity and Specificity; D013745:Tetanus Toxoid / Q000276:immunology
chemical_list: D000906:Antibodies; D061067:Antibodies, Monoclonal, Humanized; D018501:Antirheumatic Agents; D015850:Interleukin-6; D013745:Tetanus Toxoid; D000068879:Adalimumab