Overview

Cellular processes dependent on the presence of precise amounts of specific proteins and their complexes. The stoichiometry for a protein complex is the description of the individual protein subunits. Determining stoichiometry of a given complex, such as the number of antibodies bound to an antigen, helps to understand the overall function of that complex. In drug development, the stoichiometry of a drug–target interaction is often closely linked to the drug’s mechanism of action and is thus crucial for the development of highly effective and safe therapeutics.

MDS provides stoichiometry of challenging interactions

• Affinity determines fibril specificity
• Size provides oligomerization state
• Stoichiometry determines number of antibodies bound giving insights on coverage of fibrils

Figure 1. The NeSt assay using MDS allows the determination of the affinity, size, and binding stoichiometry of anti-amyloid-β antibodies to different oligomeric forms of amyloid-β.

Case Study

Kinetic fingerprints differentiate the mechanisms of action of anti-Aβ antibodies.

Linse et al., Nature Struct. Mol. Biol., 2020, 27, 1125–1133. DOI: 10.1038/s41594-020-0505-6

Linse et al. combine kinetic analyses with MDS binding measurements to address the mechanism of action of four clinical stage anti-Aβ antibodies. They quantify the influence of these antibodies on the aggregation kinetics and on the production of oligomeric aggregates and link these effects to the affinity and stoichiometry of each antibody for monomeric and fibrillar forms of Aβ. Their results reveal that, only one of these four antibodies dramatically reduces the flux of Aβ oligomers which is linked to its inhibitory potential.

“

During our research into protein amyloid formation, we have used many different techniques for assessing protein interactions – all with different limitations and advantages. With diffusional sizing, we were able to confidently generate accurate and complete in-solution data using amyloid proteins to connect stoichiometry and binding affinities with protein self-assembly. The data could provide key insights for novel therapeutic approaches in Alzheimer’s disease.

Professor Sara Linse

Department of Biochemistry and Structural Biology, Lund University

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To study intrinsically-disordered, aggregated proteins, or any biological interaction in their native state in solution we recommend the following: 

Amyloid

Fluidity One-M

Fluidity Intelligence

Results

Affinity, Stoichiometry, Complex Size

Workflow specification and benefits:

• 25 min run time
• K_D range from pM to µM
• Provides stoichiometry
• Amount of antibody or binding partner 1-10µg depending on affinity
• Amount of target 10-25µg
• 12 µl of sample per triplicate
• Use of complex background e.g., serum, lysate, CSF
• Quick & easy to perform.