Overview

Neurodegenerative diseases (ND) affect millions of people worldwide and are characterized by the accumulation of neurotoxic aggregates in the brain leading to neuronal death. Immunotherapeutic approaches against NDs focus on antibodies that target the pathogenic aggregated forms of proteins such as α-synuclein or Aß peptide related to Parkinson’s disease and Alzheimer’s disease, respectively. ELISA and other surface-based methods are commonly used to identify antibodies that specifically target these pathogenic aggregates but tethering these sensitive proteins to a surface is often not feasible or induces artificial structures that do not occur in patients. Microfluidic diffusional sizing (MDS) avoids surface immobilization and thus preserves the disease-relevant structures of the aggregates which enables identification of antibodies that bind with high specificity and have the most effective mode of action against disease progression.  

Key advantages of MDS

Direct ELISA

Each species immobilized

• Loss of information through surface-induced conformational bias
• No K_D or stoichiometry
• Assay development required

Competition ELISA

Monomer immobilized

• No K_D or stoichiometry
• Challenging competition experiment
• Assay development required

MDS

Immobilization-free

• K_D – specificity
• Stoichiometry – mechanism of action
• Biological samples (CSF, serum)
• Quick and easy to perform

Fig 1. Unlike ELISA, MDS uniquely measures the most crucial parameters (affinity and stoichiometry) to assess antibody specificity and mechanism of action.

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

Read the paper

Get started

To study intrinsically-disordered or aggregated proteins 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 1-10 µg (affinity dependent)
• Amount of oligomer or fibril 10-25µg
• 12 µL of sample per triplicate
• Use of complex background e.g. serum, CSF
• Quick & easy to perform