Getting Around Avidity When Characterizing Nanobody-Based Products
Measuring antibody-antigen binding interactions is common practice in research and development of antibody biologics. But the multivalent nature of antibodies can lead to avidity effects that skew the data. In this exclusive interview, we talk about overcoming these effects in assay design with Michael Metterlein, a scientist at ChromoTek and a protein analytics expert.
This article is posted on our Science Snippets Blog
Affinity vs. avidity
Affinity and avidity are often used interchangeably, but they actually refer to different aspects of molecular interactions.
Affinity is the strength of binding between a single binding site on one molecule and its target on another molecule. It is influenced by hydrogen bonds, electrostatic bonds, Van der Waals forces, and hydrophobic forces. We typically report affinity as a dissociation constant (KD).
Avidity, on the other hand, is the overall strength of binding between multiple binding sites on one molecule and multiple targets on another molecule. It is influenced by factors such as binding affinity, valency, and structural arrangements. Avidity is often reported as an avidity index.
Understanding the difference between these two concepts is important for designing accurate assays to measure binding strength and for developing effective antibody-based drugs and diagnostics. In this interview, ChromoTek (Part of Proteintech Group) scientist Michael Metterlein explains how they tackle avidity effects in studies with nanobody-based products.
Can you describe your work at Proteintech?
“We develop nanobodies and conventional antibodies as new tools for research. My main responsibilities are protein analytics using bio-layer interferometry (BLI), nanoDSF and dynamic light scattering. Further, I am strongly involved in developing immunoassays for antibody validation.”
What is the difference between antibody affinity and avidity?
“Affinity is the strength of a single interaction (1:1 binding), such as the interaction between the epitope on an antigen and the antibody at a single binding site. Avidity, also known as the functional or apparent affinity, describes the cumulative strength of multiple affinities between interacting biomolecules, which arises from two or more interaction sites.
For example, an IgM has 10 binding sites. It is unlikely that all 10 antigens will disengage from the IgM pentamer simultaneously. Therefore, the avidity of IgM can be relatively high, while the binding affinity of a single binding site may be low.”
What is your method of choice for measuring affinity and avidity: ELISA or a label-free real-time approach?
“Analysis of antibody and antigen complexes has traditionally been done using enzyme-linked immunosorbent assays (ELISAs). However, a major shortcoming of this end-point assay is the lack of kinetic, thermodynamic, or stoichiometric information. ELISA cannot accurately describe the affinity or avidity of an antibody. Thus, we use Octet® BLI early on in our antibody discovery process to get important information on affinity and rate constants.”
Can you explain the role of avidity in your work with nanobody-based reagents?
“Most of our nanobody-based products are single-domain antibodies that were derived from Camelid immune libraries and screened for high affinity, which is crucial for immunoassays. These reagents show efficient 1:1 binding in case of monomeric antigens. Nevertheless, in some cases we exploit the avidity effect by using bivalent nanobody formats to increase the apparent affinity.”
How do you recognize interactions that have a bivalent or multivalent component?
“By comparing monovalent and bivalent formats we can observe significant differences in koff. Bivalent proteins usually show much slower koff values than monovalent proteins – but note that this is only true for bivalent proteins used as analytes (free in solution and not when immobilized).”
What are your recommendations for addressing avidity effects in the assay setup?
“The easiest way to prevent avidity effects is to immobilize the bivalent protein sample. Another approach is titrating down a target protein, but this requires a lot of optimizations."
How does the Octet® BLI platform help you to meet your scientific goals?
“We use the BLI platform in early development for off-rate ranking of clones, after ELISA. It accelerates our projects by reducing the number of clones of interest and thus saves us money and time. In addition, we use BLI to characterize final candidates regarding their affinity and kinetics, as well as for epitope binning studies.
Quantification of hybridoma supernatants or binding specificity assays are further important applications of the BLI platform in our workflows.”
To learn more about the Octet® BLI platform for biologics characterization and how to optimize assays to avoid avidity effects check out the hand-picked resources below. For a great overview on designing label-free assays, our new Compendium for Successful BLI and SPR Assays is a great place to start.
