Affinity vs Avidity — what’s the difference?

Nadia Shakir


May 14, 2024

Affinity and avidity are an example of those pesky terms that seem interchangeable but in reality, are quite different. In this blog post we will be exploring the differences of affinity vs. avidity, and hopefully dispelling that vagueness you feel next time you come across them in a paper.

avidity vs affinity - a figurative comparison

What is affinity?

Interactions are widespread throughout biology and affinity is the crucial parameter that describes them. The binding affinity indicates the strength of interaction at a single site between two species. These species could both be proteins, or one or both species could be a small molecule, a lipid particle, a sugar, or a nucleic acid.

A classic example of a protein-protein interaction is that between an antibody and its antigen. Interactions are site specific – there are particular regions on each of the binders that interact with each other specifically. In the antibody-antigen example, the epitope of the antigen binds to the variable domain of the antibody.

Importantly, binding interactions are non-covalent as no interatomic bonds are formed. The interaction instead relies upon weaker bonds such as hydrophobic interactions, hydrogen bonds and electrostatic forces. Because interactions are non-covalent, they are also reversible; binding interactions are thus an example of an equilibrium reaction.

Selecting first a simple example of two generic species that interact at one site only, we can write the chemical reaction for their interaction:

A + B ⇌ AB

This equation tells us that A and B combine to form the complex AB, and that the complex can dissociate to release free A and B. In the case of a tight binding interaction the equilibrium would lie to the right, favoring the AB complex, whereas for a weak interaction the complex would be less prevalent.

The strength of the binding interaction can be numerically described by examining the relative concentrations of the free and bound species according to the following equation:




  • [A] and [B] are the concentrations of A and B respectively
  • [AB] is the concentration of AB complex

The dissociation constant, KD tells us directly how strong an interaction is, with a low value describing a tight binding interaction and a high value for weak interactions. Some examples of interactions and their affinities are shown below in Table 1.


Table 1. Literature reported affinities of selected interactions. Note that in many cases the binders are not full-length due to experimental constraints.

Range Binder A Binder B Affinity Ref. 
millimolar Nck-2 PINCH-1 3 mM [1] 
hemagglutinin α-methylsialic acid 2.8 mM [2] 
micromolar PD-1 PD-L1 8.2 uM [3] 
p53 (92-360)  BCL-xL 4.1 uM [4] 
nanomolar CCR5 gp120 4 nM [5] 
KRAS RAF1 (52-188) 152 nM [6] 
picomolar adalimumab soluble TNFα 127 pM [7] 
insulin insulin receptor 42 pM [8] 
femtomolar streptavidin d-biotin 40 fM [9] 


What is avidity?

Many binders are multivalent, meaning they possess more than one binding site. While affinity is the strength of a single bond or interaction, avidity is the total strength of all non-covalent interactions between two species that interact multivalently.

The pre-requisite for avid binding is simultaneous interaction of two species at more than one independent site (Figure 1). Avidity always provides stronger binding than the affinity of either of the independent binding sites. The reason for this is that if one site unbinds the species are not free to diffuse apart as they are constrained by the remaining binding site(s), and so there is a higher chance of the unbound site rebinding than if the interaction involved just a single site.

avid binding explanation

Figure 1. Different pathways of dissociation for a monovalent interaction (top row) and a divalent interaction (bottom row). For the divalent interaction, the competition between the second unbinding event and rebinding reduces the likelihood of overall dissociation and so this interaction will be tighter than the monovalent interaction due to the avidity of the interaction.

A real-world analogue of avid binding is velcro, where a single hook and loop provides negligible strength but when acting in large numbers (simultaneous interaction at multiple independent sites) the overall binding strength can be very strong.

Avidity is determined by three parameters:

  • The affinity of each individual binding site
  • The number of sites that can be bind simultaneously
  • The orientation of the sites

Thus, an avid binding system will bind more tightly if the binding affinity of each interacting site is tighter, if there are more sites able to bind at the same time, and if the sites are spaced and oriented to facilitate binding with no need for structural flex or rearrangement.

Avid binding can provide important functionality in biology be ensuring selectivity for multivalent or clustered interactions. For example, activation of the immune complement cascade relies on binding of multiple antibodies to a target, with the cluster of antibodies then recognized by the C1q protein. This selectivity is ensured by C1q having a low affinity for a single IgG (KD ~ 100 µM) but when multiple, ideally six, IgG molecules mediate binding the binding is much tighter (KD ~ 10 nM) [10]. Viruses also can exploit avidity to enter cells presenting a high concentration of a low affinity receptor. For example, human adenoviruses can infect CD46-expressing cells despite the affinity for this receptor being low (KD ~ 100 µM) by binding avidly to multiple receptors to give sufficient overall binding strength for prolonged attachment and cell entry [11].


Affinity vs. Avidity: a conclusion

To summarize, binding affinity is the strength of an interaction between two species, whereas avidity is the total strength of all non-covalent interactions between two species that interact multivalently.

Care must be taken, particularly when working with surface immobilization technologies, to ensure that experiments are measuring the desired parameter. Microfluidic Diffusional Sizing (MDS) is a powerful solution-phase method for affinity determination that does not suffer the risk of unintentionally measuring avidity instead of affinity. Furthermore, as MDS measures the size of complexes formed, it is ideally suited to detection of network formation in solution.

One way to circumvent this experimental challenge is to work in solution where avidity is only relevant for binding partners that are both multivalent and thus employ avid binding in their natural context. Microfluidic Diffusional Sizing (MDS) is a powerful solution-phase method for affinity determination that does not suffer the risk of unintentionally measuring avidity instead of affinity. Furthermore, as MDS measures the size of complexes formed, it is ideally suited to detection of network formation in solution.

If you are interested in measuring avidity and affinity with MDS, learn more about the technology below.



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  2. Sauter, N.K.; Bednarski, M.D.; Wurzburg, B.A.; Hanson, J.E.; Whitesides, G.M.; Skehel, J.J.; Wiley, D.C. Hemagglutinins from Two Influenza Virus Variants Bind to Sialic Acid Derivatives with Millimolar Dissociation Constants: A 500-MHz Proton Nuclear Magnetic Resonance Study. Biochemistry 2989, 28, 8388–8396.
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