Multivalent Binding Partners: Binding Affinity and Metacomplex Formation

Standard analytical techniques for measuring macromolecule interactions, for example, SPR and ELISA, entail the immobilization of one binding partner on a surface so as to quantify the binding affinity. In the cases of simple 1:1 association and some 1:n interactions, this immobilization does not normally impact the equilibrium dissociation constants (Kd) that are measured significantly.

However, when both binding partners are multivalent, the immobilization of one ligand can lead to errors regarding estimates of the binding affinity. This occurs due to mass transport limitations,  incorrect assumptions about the stoichiometry of the interaction, and avidity effects at the surface.

Composition-gradient multi-angle light scattering (CG-MALS) has a unique ability to quantify such interactions to yield affinity and stoichiometry of complexes forming in solution. This permits the occurrence of all possible binding stoichiometries and provides simultaneous quantification of self and hetero-interactions, metacomplex formation, and other multivalent interaction.

In this application note, CG-MALS is used to investigate metacomplex formation in the hetero-association of a bivalent antibody (Ab) and a multivalent antigen, streptavidin (SA). A Calypso® connected to an inline UV/Vis concentration detector and DAWN® MALS instrument was used to create automated composition gradients. Any self-association was quantified using single-component concentration gradients, and hetero-association was assessed via a dual-component “crossover” composition gradient.

For details on the methodology, results and conclusions, follow the link to the full application note.

This information has been sourced, reviewed and adapted from materials provided by Waters | Wyatt Technology.

For more information on this source, please visit Waters | Wyatt Technology.

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