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Binding Affinity Measurement of Antibodies from Crude Hybridoma Samples by SPR   

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Original research article

A brief version of this protocol appeared in:
Blood
Jan 2013

Abstract

Surface Plasmon Resonance (SPR) is widely used to generate kinetic and affinity information on specific interactions between biomolecules. This technique is label-free and monitors the binding event in real-time. It is generally used for characterization of monoclonal antibody - antigen interactions. This protocol describes specifically the use of SPR with a Biacore T100 instrument to measure the affinity of crude hybridoma samples to a protein. For that purpose an anti-IgG antibody was firstly covalently immobilized onto a CM5 chip by amide coupling (Canziani et al., 2004; Schraml and Biehl, 2012). Then the antibodies from hybridoma supernatants were captured non-covalently onto the surface via their Fc region providing an optimal analyte-binding orientation. Finally, the resulting complex was stabilized by crosslinking with EDC/NHS to avoid baseline drift during measurement and regeneration (Pope et al., 2009). Then the interaction with the protein was monitored at several concentrations and its affinity towards the immobilized antibodies was determined with the corresponding KD obtained from classical kinetics analysis. This set-up avoids the avidity effects of the bivalent antibodies, allows the use of non-purified analytes with unknown concentrations and the specific capture of the antibodies in a similar stable covalent-orientated manner.

Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Ndao, D. M., Hickman, D. T., López-Deber, M. P., Davranche, A., Pfeifer, A. and Muhs, A. (2014). Binding Affinity Measurement of Antibodies from Crude Hybridoma Samples by SPR. Bio-protocol 4(21): e1276. DOI: 10.21769/BioProtoc.1276.
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