Journal
ANALYTICAL BIOCHEMISTRY
Volume 437, Issue 2, Pages 118-122Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2013.02.027
Keywords
High-performance liquid chromatography; Dissociation constant; Binding assay; High affinity
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Although several techniques exist for the measurement of high-affinity interactions, it is still challenging to determine dissociation constants around or even below 1 pM. 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 0.78 pM could be determined for the anti-adalimumab antibody. (C) 2013 Elsevier Inc. All rights reserved.
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