Forum :: Measuring avidity

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This forum is intended for questions about kinetics, Surface Plasmon Resonance and the instruments related to these techniques.

TOPIC: Measuring avidity

Measuring avidity 27 Nov 2014 16:01 #1

Dear forum. I am interested in measuring binding avidity and I am unsure if this is possible with SPR.
Basically I have a protein that exists as a monomer, dimer, trimer, tetramer etc. The e,g trimer should not bind to immobilized receptor just as "three monomers" but stronger due to the functional affinity/avidity. I have been told that this "extra" effect is not possible to measure by SPR but I am not sure this is true.

Could anyone elaborate on this?

Best regards

Measuring avidity 02 Dec 2014 20:15 #2

Hi Kristian,
I did copy a part from the SPRpages book for you and added some literature to explore. I hope it will help you.


The intrinsic affinity is the affinity between two sites, which bind to each other in a monovalent interaction. The functional affinity is the total of binding strength due to all binding interactions of two molecules. The term avidity is also used but should be avoided (3). The enhancement factor between intrinsic and functional affinity depends on the intrinsic affinity per binding site, the flexibility and number of the binding sites of the analyte and the number and distances between the binding sites of the ligand.
The first binding depends on the intrinsic affinity of the site. The association of the next site is the favoured because of the high local concentrations of sites. As a rough estimation, the functional affinity is the product of intrinsic affinity and local concentrations (3).
In the BIACORE, the first binding event will produce an increase in response. The subsequent following binding events will not change the signal because the proteins are already within the reach of the sensor and will not cause any mass increase. What will happen is a slowing of the association rate constant. In addition, the multivalent binding will decrease the dissociation rate constant because the dissociation from one of the binding sites will generally not lead to the dissociation of the whole molecule. The possibility of rebinding is high due to high local concentrations.
The stability of the complex is highly dependent on the ligand density and the flow rate (3). To reduce multivalent binding, low ligand concentrations can be used reducing local concentrations. To reduce analyte rebinding, soluble ligand can be injected during dissociation. Rebinding of analyte during the dissociation phase depends also on ligand density and flow rate.
For more information and a model to analyse multivalent binding see Müller (1) and others (2, 4).

1. Muller, K. M., Arndt, K. M. and Pluckthun, A.; Model and simulation of multivalent binding to fixed ligands. Analytical Biochemistry (261): 149-158; 1998.
2. Oda, M. and Azuma, T.; Reevaluation of stoichiometry and affinity/avidity in interactions between anti-hapten antibodies and. Molecular Immunology (37): 1111-1122; 2000.
3. Rheinnecker, M., Hardt, C., Ilag, L. L., et al.; Multivalent antibody fragments with high functional affinity for a tumor-associated carbohydrate antigen. J.Immunol. (157): 2989-2997; 1996.
4. Vauquelin, G. and Charlton, S. J.; Exploring avidity: understanding the potential gains in functional affinity and target residence time of bivalent and heterobivalent ligands. British Journal of Pharmacology; 2013.

Measuring avidity 12 Feb 2015 09:51 #3

Dear Arnoud. Thank you very much for the elaborate respons. I will read the articles asap.
Sorry for the late reply.

Best regards
Moderators: Arnoud