The discovery of new therapeutic antibodies plays a crucial role in modern medicine, with monoclonal antibodies representing the majority of the 10 best-selling drugs in the world today. These antibodies are used to treat various diseases, including infectious diseases, autoimmune disorders, and cancer. However, current antibody screening methods face limitations. High-throughput antibody screening for binders is dominated by phage display, which commonly screens highly diverse antibody libraries against a single target, limiting its multiplexing capabilities. Functional antibody screening, still predominantly performed using conventional hybridoma technology, remains labor-intensive, costly, and limited in throughput.

This thesis presents proof-of-concept developments of two synergistic approaches leveraging droplet microfluidics to address distinct challenges in current antibody screening methods. Droplet microfluidics enhances the throughput of antibody screening by allowing each droplet to function as an independent microreactor to assess antibody binding and functionality.

The first method presented in this thesis is Phage-Square, developed for highly multiplexed library-against-library screening to identify interactions between two diverse T7 phage libraries. Following incubation and preselection of binding pairs, phages are encapsulated in droplets at low occupancy to promote co-encapsulation of only interacting phages. Their genetic information is fused via emulsion fusion PCR and analyzed using high-throughput sequencing. We provide a first proof-of-concept for this workflow by successfully enriching interacting pairs in a small-scale library-against-library screen (5x4 antibodies against bait proteins). Phage-Square has the potential to accelerate antibody discovery by enabling highly multiplexed screening of large antibody and antigen libraries. To optimize all parameters, we also developed DropQuant, a label-free method for the absolute quantification of infectious phages in droplets, further improving the characterization of phage samples.

The second approach, FuncAb-seq, was developed for functional screening of antibody-secreting cells at the single-cell level. Conceptually, FuncAb-seq, allows for the identification of antibodies that induce a desired effect on the transcriptome of target cells. To achieve this, we designed a microfluidic workflow that enables co-encapsulation and incubation of antibody-secreting cells, target cells, and barcoded beads within droplets. The work also included the development of Link-seq, a method that allows for the simultaneous sequencing of antibody variable regions alongside the transcriptome of target cells using the Drop-seq technology. Together, these methodologies establish a framework for multiplexed functional antibody screening, enabling the evaluation of multiple antibody-target interactions in a single assay without requiring prior knowledge of the targets.

Together, these two platforms pave the way for accelerated screening of antibody specificity and functionality.