Titration of Known Clonotype Showcases Evercode BCR Sensitivity

Key Takeaways

• Evercode BCR has proven 0.1% clonotype sensitivity. In a controlled spike-in experiment, Evercode BCR detected a known target clonotype present at 0.1% abundance—or 1 in 1,000 cells—delivering measurable, industry-leading BCR sensitivity.
• Whole transcriptome data identifies every single cell. Evercode BCR resolved all expected splenic immune cell lineages, including difficult-to-detect plasmablasts, pairing deep repertoire mining with rich cellular context.
• Faster hit identification. Reliable detection of rare clonotypes paired with Evercode BCR’s high-throughput capabilities enables antibody discovery using fewer animals and screening rounds, accelerating the path to target validation.

The detection of rare clonotypes enables antibody discovery teams to uncover high-affinity antibody candidates the instant they appear, dramatically expanding hit diversity and accelerating screening cycles. Just as important is being confident your assay can capture those one-in-a-thousand cells. In a controlled spike–in titration of enriched B cells carrying a defined clonotype, Evercode BCR delivered industry-leading 0.1% sensitivity–enabling you to confidently find every needle in the haystack.

The HyHEL-10 mouse strain¹ is genetically engineered to express a human anti-HEL (hen egg lysozyme) BCR consisting of a fixed epitope-specific Igkc and rearranged IgM and IgD heavy chains from the immunoglobulin locus in B lymphocytes. Nearly every HyHEL-10 mouse B cell expresses the same, well-defined BCR clonotype (Figure 1), making the strain an ideal reference standard for evaluating clonotype reproducibility and assay sensitivity.

Figure 1. 99.3% of HyHEL-10 B cells express a known and consistent clonotype. To determine purity of HyHEL cells, we used a 100% spike-in condition as a positive control. For both the CDR3 heavy and light chain sequences there was a clear dominant motif. Motif analysis shows that 77.7% of HyHEL-10 cells have the exact matched pair CDR3 sequence whereas only 0.7% of cells have other motifs.

Following Evercode Fixation, we spiked B cell-enriched HyHEL-10 splenocytes (0.1-10%) into B cell-enriched wild type C57/BL6 splenocyte samples and barcoded them using Evercode Mouse BCR (Figure 2).

Figure 2: Schematic of experimental design. B cells from HyHEL-10 and C57BL/6 mice were enriched, fixed, and mixed at defined ratios as outlined in the table.

Across 106,107 cells, unsupervised clustering resolved all major B cell lineages—including plasmablasts, the difficult-to-capture, short-lived mediators of the early antibody response—and validating assay sensitivity and cell-type fidelity (Figure 3).

Figure 3: UMAP of 106,107 B cell–enriched splenocytes from C57BL/6 and HyHEL-10 mouse strains. Unsupervised cluster analysis reveals numerous B cell populations in B cell–enriched splenocytes.

We visualized HyHEL-10 recovery with alluvial plots, which track amino-acid identity at every CDR3 position across clonotypes. The heavy- and light-chain HyHEL sequences—CANWDGDYW and CQQSNSWPYTF, respectively—are highlighted in magenta (Figures 4 and 5). Even at the 0.1 % spike-in, Evercode BCR cleanly separates the HyHEL-10 ribbons from background clones, confirming its rare clonotype sensitivity.

Figure 4: HyHEL-10 light chain CDR3 detection at 10% and 0.1% spike-in reveals sensitive and reproducible clonotype detection. Black arrow shows clonotype.

Figure 5: HyHEL-10 heavy chain CDR3 detection at 10% and 0.1% spike-in reveals sensitive and reproducible clonotype detection. Black arrow shows clonotype.

Evercode BCR pinpoints a clonotype present at just 0.1 %—one cell in a thousand—while still delivering full transcriptome context. That unmatched sensitivity equips discovery teams to tackle the most demanding antibody screens, from early-time-point immunizations to rare lineage hunts, without missing a single high-value clone.

References

1. https://www.jax.org/strain/007783

Sample Names

• HyHel_10 control_100_percent
• HyHel_10 spikein_10_percent
• HyHel_10 spikein_5_percent
• HyHel_10 spikein_1_percent
• HyHel_10 spikein_One_half_percent
• HyHel_10 spikein_One_tenth_percent