At DuneX Biosciences, our mission is to provide clients with highly precise, data-driven, and scalable antibody discovery solutions. Among the technologies we leverage, FACS-based yeast display stands out as one of the most powerful tools for isolating high-value antibody candidates—especially when the goal is to recover extremely rare, high-affinity binders hidden in massive libraries.

While panning-based methods (e.g., phage display) have long been a staple in discovery workflows, they lack the quantitative resolution required for modern therapeutic engineering. Yeast display, when combined with analytical FACS, allows us to enrich true signal over noise with single-cell granularity, enabling recovery of a single high-performance variant in a background of one million non-binders.

This article outlines the DuneX approach to FACS-based yeast display, why it consistently delivers ultra-rare binder identification, and how it integrates into our broader antibody engineering pipeline.

Why DuneX Uses FACS-Integrated Yeast Display

Yeast display is not new—but how it’s executed varies drastically between labs and CROs. At DuneX, we implement a rigorously optimized and QC-driven platform with:

• Single-cell quantitative selection

• Dual- or tri-color FACS gating strategies

• High-throughput sorting (≥10⁷ cells per round)

• Precise antigen titration down to picomolar levels

• Standardized induction and display QC

• NGS-integrated enrichment analysis

These elements allow us to deliver exceptional sensitivity and reproducibility for clients seeking best-in-class antibody leads.

The Scientific Basis Behind 1-in-a-Million Recovery

True Single-Cell Resolution

Each engineered yeast cell displays a single antibody variant.
FACS interrogates tens of thousands of cells per second, measuring:

• Binding signals

• Expression (via epitope tags)

• Specificity via multi-color labeling

• Affinity shifts under titrated antigen conditions

This enables clean separation of ultra-rare binders from vast noise populations.

Tight FACS Gating Gives Full Control

DuneX applies multi-parameter, multi-fluor gating:

• High display expression

• High antigen binding

• Off-target depletion

• Competitive binding selection

This level of control is simply not possible with wash-based panning systems.

Tunable Antigen Concentration for Affinity Discrimination

Clients often need antibodies with specific affinity properties, not just “any binder.”
By adjusting antigen concentration from nanomolar to picomolar, we selectively enrich variants with:

• Faster on-rates

• Slower off-rates

• Higher thermodynamic stability

Yeast display is unmatched for controlled affinity maturation [Boder & Wittrup, 1997].

What “1-in-a-Million” Means Operationally at DuneX

For a rare clone at 10⁻⁶ frequency:

• Sorting 10 million cells yields ~10 occurrences

• Modern sorters (Sony SH800, BD Aria) complete this within minutes

• With optimized gating, those 10 events are resolvable from millions of noise cells

This is the foundation for DuneX’s high-confidence enrichment.

DuneX’s End-to-End FACS Enrichment Workflow

We follow a multi-round selection strategy that steadily amplifies true binders while eliminating background.

Library QC & Induction

Every project begins with rigorous QC:

• Transformation efficiency

• Display percentage

• Expression homogeneity

• Reference controls for antigen binding

We routinely achieve >50–70% display-positive cells for high-quality starting populations.

High-Sensitivity Antigen Labeling

DuneX uses best-in-class fluorophores (AF647, PE, APC) and biotin/streptavidin multistep conjugation for:

• Increased brightness

• Reduced photobleaching

• Superior signal-to-noise ratios

Labeling reproducibility is critical for rare-variant detection.

Multi-Gate FACS Strategy

Gate 1 — Live, Single Cells

Removes doublets and dead cells, ensuring accurate fluorescence attribution.

Gate 2 — Display Expression

We use tag-based detection to isolate clones with reliable display levels.

Gate 3 — Antigen Binding (Primary Selection)

This fast, precise gate isolates the top 0.01–0.1% performers—often where the rare high-value clones reside.

With this combination, we routinely enrich ultra-rare variants in a single round.

Round-Based Enrichment at DuneX

Round 1: Broad Capture

Antigen concentration: high
Goal: retrieve all potential binders from noise
A rare 1-in-10⁶ clone rises to ~1-in-10⁴.

Round 2: Selective Enrichment

Antigen concentration: moderate
Goal: enrich medium- and high-affinity variants
Signal separation significantly increases.

Round 3: High-Stringency Selection

Antigen concentration: low
Goal: refine top performers
Rare binders may now represent 5–20% of the pool.

Round 4: Ultra-Fine Discrimination

Antigen concentration: sub-nM
Goal: isolate highest-affinity clones
This is where FACS resolves even subtle affinity differences.

Core Determinants of Successful Ultra-Rare Recovery

Signal Resolution

High fluorescence resolution enables clear separation between:

• True binders

• Weak binders

• Background/noise

A bright, stable antigen label is essential.

Expression Uniformity

DuneX ensures consistent induction so binders don’t masquerade as low-signal clones due to poor display.

High-Throughput Sorting

We sort 10–30 million cells per round, enhancing recovery probability and lowering stochastic error.

Kinetic & Competitive Selection Options

To refine affinity or specificity:

• On-cell KD titrations

• Off-rate–based kinetic selections

• Competitive binding with unlabeled antigen

• Negative selection against homologs

This level of control is crucial for therapeutic antibody programs.

Advanced Multi-Parameter Sorting at DuneX

Dual-Color Sorting (Expression + Binding)

This ensures accurate normalization and protects against false positives.

Specificity Engineering with Negative Gates

We can deplete binders to:

• Homologous proteins

• Conserved domains

• Highly similar isoforms

This is essential for clinical specificity engineering.

Off-Rate Sorting (Kinetic Gates)

A powerful method for selecting antibodies that retain antigen binding after extended washout [Yang et al., 2019].

Proven Scientific Basis: Key Studies Supporting Ultra-Rare Binder Recovery

The scientific literature validates FACS-based yeast display as the gold standard for ultra-rare binder identification:

• Boder & Wittrup, 1997 — Foundational demonstration of quantitative affinity discrimination

• Feldhaus et al., 2003 — Direct recovery of rare human scFvs using high-resolution FACS

• Chao et al., 2006 — Comprehensive human antibody engineering workflow on yeast

• McMahon et al., 2018 — Rapid discovery of novel scaffolds via yeast display

• Yang et al., 2019 — Fine epitope mapping & off-rate selection

• Stevens et al., 2017 — NGS-coupled yeast display for deep mutational scanning

These studies form the backbone of the DuneX platform.

Why Biotech Teams Choose DuneX for FACS-Based Yeast Display

We deliver higher sensitivity than panning-based CROs

FACS enables true single-cell precision rather than wash-dependent enrichment.

We support full antibody engineering cycles

From initial library screening to affinity maturation and developability checks.

We implement rigorous QC across all steps

Ensuring reproducibility and transparency for clients.

We integrate sequencing-based analytics

Including lineage tracking, mutational analysis, and enrichment dynamics.

We operate with therapeutic engineering in mind

Every step is optimized for downstream CHO/HEK expression, not just binder identification.

Conclusion

At DuneX Biosciences, we invest in platforms that deliver clarity, depth, and actionable results.
FACS-based yeast display is one of the most powerful and precise discovery technologies available today—and our optimized workflows allow reliable recovery of 1-in-a-million antibody candidates with confidence.

Whether your goal is novel target discovery, affinity maturation, specificity tuning, or engineering of complex therapeutic modalities, the DuneX FACS-based yeast display system provides the precision, control, and data resolution required for modern antibody programs.

We don’t just find binders—we find the right binders.

References

1. Boder, E. T., & Wittrup, K. D. (1997). Yeast surface display for screening combinatorial polypeptide libraries. Nature Biotechnology, 15(6), 553–557.

2. Feldhaus, M. J. et al. (2003). Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nature Biotechnology, 21(2), 163–170.

3. Chao, G. et al. (2006). Isolating and engineering human antibodies using yeast surface display. Nature Protocols, 1(2), 755–768.

4. McMahon, C. et al. (2018). Yeast surface display platform for rapid discovery of novel binding proteins. Nature Chemical Biology, 14(5).

5. Yang, Z. et al. (2019). Yeast display enables fine epitope mapping and affinity tuning of therapeutic antibodies. mAbs, 11(5).

6. Stevens, A. J. et al. (2017). Yeast display + NGS for deep mutational scanning. PNAS, 114(19).

7. Wang, X. et al. (2020). Advances in display technologies for antibody discovery. Frontiers in Immunology, 11.

8. Koide, S., & Koide, A. (2007). Monobody engineering using yeast display. FEBS Journal, 274(19).