Affinity

Applications

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Introduction

Overview

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Case Study

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Workflow specification

Determination of binding affinity is crucial to understanding biomolecular interactions that govern biological processes and for the development of safe and effective therapeutics. Microfluidic Diffusional Sizing is the only method that measures affinity of challenging and disease-relevant targets and avoids immobilization, purification, time-consuming optimization, and large amounts of sample.

Overview

Binding affinity measures the strength of an interaction between a ligand and its binding partner. Since biology is driven by molecular interactions, affinity is essential to understand which molecules interact as part of normal cellular function or in a disease scenario. Affinity also provides a means to evaluate and improve the design and development of therapeutics in fields such as oncology, immunology, and neurodegenerative disease. MDS performs affinity measurements in-solution based on changes in molecular size on disease-relevant molecules such as membrane proteins, amyloid fibrils, or intrinsically disordered proteins. The method comes with purification-free workflows, in-built assay control, easy-to-use consumables in plate format, no-clog design, and low sample usage.

Obtain equivalent measurements in complex backgrounds.

Buffer

Human Serum

Figure 1. Microfluidic Diffusional Sizing demonstrates equivalent affinity of SARS-CoV-2 anti-spike S1 antibody against SARS-CoV-2 RBD in buffer (PBS-T) and > 90% serum matrix.

Case Study

Microfluidic antibody affinity profiling of alloantibody-HLA interactions in human serum.

Schneider et al., Biosensors and Bioelectronics, vol 228, 15 May 2023. DOI: 10.1016/j.bios.2023.115196.

This collaborative paper establishes MDS technology as a proof of principle to provide a more in-depth profiling of the immune response in transplant donor-recipient matching using patient sera samples. Traditionally, evaluation of the humoral immune response involves surface adherence methods giving an antibody titer, a convolution of both affinity and antibody. Implementation of MDS technology provides further insight on the “quality” of the immune response in the form of reliable and reproducible quantification of alloantibody affinity and concentration to Human Leukocyte Antigens, a biomarker used in organ transplantation. This approach allows the quantification of both affinity and alloantibody concentration directly in complex patient samples improving clinician assessment of the reactivity potential of a donor-recipient pair to improve the success of matching and reduce rejection rates.

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We have done a lot of research to understand the affinity of an antibody against primary versus cross-reactive antigens, but measuring antibody affinity in non-purified samples, such as in serum, is very difficult with existing assays. Our desire to overcome these limitations has driven my collaboration with the lab of Tuomas Knowles and this is how we came across microfluidic diffusional sizing and eventually developed antibody affinity profiling.

Vasilis Kosmoliaptsis

Department of Surgery at the University of Cambridge