Recent Advances in Antibody Discovery Using Ultrahigh-Throughput Droplet Microfluidics: Challenges and Future Perspectives

Abstract

Droplet microfluidics has emerged as a transformative technology that can substantially increase the throughput of antibody "hit" discovery. This review provides a comprehensive overview of the recent advances in this dynamic field, focusing primarily on the technological and methodological innovations that have enhanced the antibody discovery process. This investigation starts with the fundamental principles of droplet microfluidics, emphasizing its unique capabilities for precisely controlling and manipulating picoliter-volume droplets. This discussion extends to various assay types employed in droplet microfluidics, including binding assays, functional assays, Förster Resonance Energy Transfer (FRET) assays, internalization assays, and neutralization assays, each offering distinct advantages for antibody screening and characterization. A critical examination of methods to improve droplet encapsulation is presented, besides addressing challenges such as reducing the leakage of small molecules from droplets and explaining what a "hit" droplet looks like. Furthermore, we assess design considerations essential for implementing high-throughput fluorescence-activated droplet sorting (FADS) workstations and emphasize the need for automation. This review also delves into the evolving commercial landscape, identifying key market players and emerging industry trends. This review paper aims to catalyze further research and innovation, ultimately advancing the field towards more efficient and robust solutions for antibody identification and development.

Keywords: FADS; FRET assay; antibody discovery; binding assay; droplet microfluidics; internalization assay; neutralization assay; sorting workstation; “hit” droplet.

Figure 1

Overview of the various on-chip microfluidic operations or capabilities that can be used at single-cell resolution for antibody discovery. DLD = Deterministic lateral displacement.

Figure 2

Schematic of a bead-based binding assay format where Droplet 1 contains Antibody (Ab)-secreting cells (ASCs) secreting non-specific antibodies and Droplet 2 contains ASCs which secret antigen(Ag)-specific antibodies. Only Droplet 2 will be sorted due to its signal intensity being above the threshold due to the localization of the fluorophore-conjugated secondary antibodies (Ab) on the beads.

Figure 3

Schematic of functional assays where (a) sorting is performed to isolate enzyme-secreting antibodies. Only Droplet 1 would be sorted due to its lower signal intensity below the threshold due to the lower concentration of the fluorogenic product of angiotensin-converting enzyme (ACE). (b) Effector cells were encapsulated and incubated with target cells to activate them for secreting IFN-γ molecules. Only the effector cells coated with fluorescently labeled IFN-γ detection antibodies and those above the signal threshold were sorted. A commercial Fluorescence-Activated Cell Sorting (FACS) system performed the sorting.

Figure 4

Schematic of functional assays where (a) sorting is performed to isolate bispecific T cell engager (BiTE) antibodies. Only Droplet 2 would be sorted due to the presence of the GFP signal of the “activated” reporter cell, (b) sorting is performed to isolate CD40 agonist antibodies. There are six possible signal intensity-based voltage traces. Only Droplet 6 would be sorted due to the overlap of the GFP signal, localization of cross-linking detection antibodies, reporter cell’s code, and the droplet code.

Figure 5

Schematic of (a) FRET assay: target-specific antibodies bring the FRET donor and acceptor nearby, giving rise to two different kinds of FRET signals—Droplet 2 (membrane-bound) and Droplet 3 (high secretion, non-membrane specific); (b) internalization assay: two scenarios with membrane-bound antibodies in Droplet 1 and internalized antibodies in Droplet 2. Secreted antibodies are labeled with green fluorescent secondary antibodies, and target or reporter cells are labeled with a Deep Red Cell tracker. Compared to antibodies on the surface of the cells, internalized antibodies resulted in a higher green fluorescence peak above the sorting threshold.

Figure 6

Schematic of (a) neutralization assay: Only Droplet 1 has the viral particles bound to the neutralizing antibodies. The unbound antibodies present in Droplet 2 were removed by ultrafiltration. Finally, the enzymes labeled on the neutralizing antibody would convert the fluorogenic substrate into its product in Droplet 1, which would increase the signal intensity above the threshold and would be sorted. (b) Infection vs. neutralization assay: two scenarios using droplets encapsulating viral particles, host cells, and ASCs. Unlike Droplet 2, the Infected host cells in Droplet 1 expressed GFP (green fluorescent protein), indicating viral infection. In Droplet 2, neutralizing antibodies bind to the viral particles, mitigating their pathogenic properties. Consequently, the host cells do not become infected or express GFP, and these droplets would be sorted.

Figure 7

Block diagram of the sorting workstation, comprising four commonly used laser excitation wavelengths (405 nm, 473 nm, 561 nm, 638 nm) and four corresponding PMTs for collecting some of the most popular fluorophores. The sorting chip has been shown with its emulsion and spacer oil inlets, and the waste and collection outlets. Whenever “hit” droplets are detected by any of the PMTs with its signal intensity above a user-defined threshold, a field programmable gate array (FPGA) triggers the sorting pulse via the high voltage amplifier. Droplets, electrodes, channels not drawn to scale.