Abstract
SummaryMonoclonal antibodies are a main player in modern drug discovery. Many antibody screening formats exist, each with specific advantages and limitations. Nonetheless, it remains challenging to screen antibodies for the binding of cell-surface receptors (the most important class of all drug targets) or for the binding to target cells rather than purified proteins. Here, we present a high-throughput droplet microfluidics approach employing dual-color normalized fluorescence readout to detect antibody binding. This enables us to obtain quantitative data on target cell recognition, using as little as 33 fg of IgG per assay. Starting with an excess of hybridoma cells releasing unspecific antibodies, individual clones secreting specific binders (of target cells co-encapsulated into droplets) could be enriched 220-fold after sorting 80,000 clones in a single experiment. This opens the way for therapeutic antibody discovery, especially since the single-cell approach is in principle also applicable to primary human plasma cells.
Highlights
Monoclonal antibodies are the biggest class of biopharmaceuticals used clinically against cancer, autoimmune diseases, inflammatory diseases, and several other clinical conditions (Nelson et al, 2010; Scott et al, 2012; Weiner, 2015)
Outline of the Antibody Binding Assay in Droplets Our antibody binding assay is based on the co-encapsulation of an antibody-secreting cell and a target cell into the same microfluidic droplet
We chose OKT9 hybridoma cells releasing antibodies binding to the transferrin receptors on leukemic K562 cells (Sutherland et al, 1981)
Summary
Monoclonal antibodies are the biggest class of biopharmaceuticals used clinically against cancer, autoimmune diseases, inflammatory diseases, and several other clinical conditions (Nelson et al, 2010; Scott et al, 2012; Weiner, 2015). More than 50% of the currently marketed therapeutic antibodies are targeted against cell-surface receptors (Reichert, 2012, 2016, 2017). Despite the major clinical importance of such therapeutics, there are several challenges in isolating antibodies against cell-surface proteins: a major requirement is the availability of a conformationally stable, native, and pure receptor molecule as a target antigen. Since the surface-expressed proteins are embedded in the lipid bilayer, their soluble forms are not always conformationally stable (Hutchings et al, 2010) Many of these surface molecules are expressed at a low level, and there are difficulties in obtaining their purified forms in abundant amounts for screening purposes (Midgett and Madden, 2007). It is imperative to use whole-cell antigen target for antibody screening
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