Membrane proteins are important targets for antibody discovery, but they can be challenging to screen. Their structure, conformation, and presentation on the cell surface can be critical for antibody recognition. Antibodies selected against isolated proteins or peptides may therefore not recognize the target in its cell-surface form.
With Xdrop®, membrane proteins can be screened using antigen-expressing cells as targets. This cell-based approach presents the target in a cellular environment, preserving important features of the membrane protein and its extracellular epitopes.
Screening Against Membrane-bound Targets
In the Xdrop workflow, antibody-secreting cells can be co-encapsulated with cells expressing the membrane protein of interest in double-emulsion droplets. Antibodies secreted by individual cells are retained within the droplet, where they can interact with the target displayed on the surface of the co-encapsulated cell.
The resulting antibody–target interaction can be detected by fluorescence, allowing droplets containing target-specific antibody-secreting cells to be identified and sorted.
We demonstrate this approach in the application note Droplet-Based Screening for Membrane Protein Antibody Discovery using HER2 as a model membrane protein.
Plasma cells were co-encapsulated with either HER2-expressing cells or control cells lacking HER2. Antibodies secreted by HER2-specific plasma cells generated a clear fluorescent signal on the HER2-expressing target cells, demonstrating the feasibility of using antigen-expressing cells directly as targets in a droplet-based screening assay.
From Binding to Functional Screening
Cell-based screening can also provide a starting point for assays that assess antibody function in addition to binding.
By incorporating an appropriate cellular readout, the target cell itself or a reporter cell system could be used to identify antibodies that produce a specific biological response. Depending on the target and assay design, this could include blocking receptor–ligand interactions, activating a receptor, or triggering a downstream signaling pathway.
This creates the potential to move from target recognition to functional antibody screening within the same droplet-based platform.
Linking Antibody Function to Sequence
Xdrop can combine cell-based screening with single-cell recovery and V(D)J sequencing, enabling antibody-producing cells identified during screening to be recovered for sequence analysis.
In the HER2 study, approximately 800,000 plasma cells were screened, resulting in enrichment of antibody-producing clonotypes. Of the selected sequences, 67% were subsequently confirmed to bind HER2, with measured affinities ranging from the micro- to nanomolar range.
This workflow connects high-throughput screening, cell recovery, and antibody sequence identification, providing a route from an antibody-producing cell to its corresponding antibody sequence.
Expanding Membrane Protein Antibody Discovery
Using antigen-expressing cells as screening targets provides flexibility in assay design and makes it possible to evaluate antibodies against membrane proteins in a cell-based format.
Combined with appropriate functional readouts, this approach could enable antibody discovery based not only on whether an antibody recognizes its target, but also on what it does.
For membrane protein antibody discovery, Xdrop provides a flexible platform for connecting target recognition, functional screening, cell recovery, and antibody sequence identification.
Read the app note: Droplet-Based Screening for Membrane Protein Antibody Discovery
