TY - JOUR
T1 - Nanomolar to sub-picomolar affinity measurements of antibody-antigen interactions and protein multimerizations: fluorescence-assisted high-performance liquid chromatography
AU - Rispens, Theo
AU - Ooijevaar-de Heer, Pleuni
AU - Derksen, Ninotska I. L.
AU - Wolbink, Gertjan
AU - van Schouwenburg, Pauline A.
AU - Kruithof, Simone
AU - Aalberse, Rob C.
PY - 2013
Y1 - 2013
N2 - 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
AB - 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
U2 - https://doi.org/10.1016/j.ab.2013.02.027
DO - https://doi.org/10.1016/j.ab.2013.02.027
M3 - Article
C2 - 23499975
SN - 0003-2697
VL - 437
SP - 118
EP - 122
JO - Analytical biochemistry
JF - Analytical biochemistry
IS - 2
ER -