On-line coupling of surface plasmon resonance optical sensing to size-exclusion chromatography for affinity assessment of antibody samples

Surface plasmon resonance (SPR) is an optical technique that measures biomolecular interactions. Stand-alone SPR cannot distinguish different binding components present in one sample. Moreover, sample matrix components may show non-specific binding to the sensor surface, leading to detection interferences. This study describes the development of coupled size-exclusion chromatography (SEC) SPR sensing for the separation of sample components prior to their on-line bio-interaction analysis. A heterogeneous polyclonal human serum albumin antibody (anti-HSA) sample, which was characterized by proteomics analysis, was used as test sample. The proposed SEC-SPR coupling was optimized by studying system parameters, such as injection volume, flow rate and sample concentration, using immobilized HSA on the sensor chip. Automated switch valves were used for on-line regeneration of the SPR sensor chip in between injections and for potential chromatographic heart cutting experiments, allowing SPR detection of individual components. The performance of the SEC-SPR system was evaluated by the analysis of papain-digested anti-HSA sampled at different incubation time points. The new on-line SEC-SPR methodology allows specific label-free analysis of real-time interactions of eluting antibody sample constituents towards their antigenic target.

Publication year: 2016
Authors: Dina Lakayan a, b, Rob Haselberg a, Wilfried M.A. Niessen a, c, Govert W. Somsen a, Jeroen Kool a

a Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands
b TI-COAST, Science Park 904, 1098 XH Amsterdam, The Netherlands
c Hyphen MassSpec, Herenweg 95, 2361 EK Warmond, The Netherlands

Published in: Journal of Chromatography A, Vol. 1452, p. 81–88
DOI: 10.1016/j.chroma.2016.05.033


antibody fracments digested anti-HSA by papain human serum albumin antibody (anti-HSA) detection immobilized HSA LC-MP-SPR separation of sample components before interaction size-exclusion chromatography (SEC) on-line with MP-SPR


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