To move from immunization to nanobody discovery, we construct high-diversity phage libraries from isolated PBMCs. Each library undergoes stringent validation to ensure quality and performance throughout downstream screening.

Library Construction Highlights

Our platform ensures high-quality phage libraries through rigorous optimization and validation.

1. Primer Optimization – Proprietary primers are designed to comprehensively cover all HcAb germline sequences.

2. Zero-Background Cloning – Background-free cloning technology enables 100% VHH insertion efficiency.

3. High Library Capacity – We guarantee a library size exceeding 1×10⁹ CFUs.

4. Clonal Uniqueness – Sanger sequencing of 48 random clones confirms 100% clonal uniqueness, with no sequence duplication.

5. Sequencing Depth Analysis – ORF correctness exceeds 90% based on sequencing of the sampled clones.

Phage Display Library Construction Workflow

Case Study: Improved Primer Design for Broader VHH Coverage

Standard VHH primers from literature often fail to capture the full diversity of camelid germline sequences. To overcome this limitation, we developed optimized primers that deliver higher PCR amplification efficiency and broader clone diversity.

Figure 1. Agarose gel showing PCR products generated using published VHH primers (top) vs. RayBio’s optimized primers (bottom). Our primers produced more abundant and diverse bands, indicating better amplification and broader germline coverage.

Phage Display Screening Options

Depending on the type and format of your antigen, we offer two proven methods to screen phage libraries:

● Solid-Phase Screening
Antigens are coated directly onto a solid surface (e.g., plate wells). This method is widely used for its simplicity and compatibility with various antigens.

● Liquid-Phase Screening
Biotinylated antigens or antigen-expressing cells are incubated with phage in solution. Phage-antigen complexes are then captured with streptavidin-coated beads. This method better preserves antigen conformation and allows tighter control over concentration—ideal for delicate or complex targets.

Figure 2. Workflow diagram of phage display screening. Shown here are iterative rounds of target binding, washing, elution, and phage amplification to enrich for high-affinity binders.

View More

Yeast Surface Display (YSD) is a widely used method for screening and selecting high-affinity antibody fragments and other binding proteins. The technique works by inserting the gene of interest into a yeast expression vector, allowing the encoded protein to be displayed on the yeast cell surface. This system enables rapid identification of candidates with desired binding properties, while preserving protein folding and functionality in a eukaryotic environment.

Library Construction Highlights:

Our platform ensures high-quality yeast libraries through rigorous optimization and validation:

● Primer Optimization – Proprietary primers provide broad coverage of HcAb germline sequences

● Zero-Background Cloning – 100% VHH insertion efficiency with background-free cloning

● Large Library Size – Guaranteed capacity exceeding 1×10⁸ CFUs

● Clonal Uniqueness – 100% uniqueness confirmed by Sanger sequencing of 48 random clones

● High ORF Accuracy – >90% correctness rate in open reading frames from validated clones

Yeast Surface Display Library Construction Workflow

Yeast Display Screening Workflow

Following library construction, VHH candidates are screened using dual-selection:

● MACS (magnetic-activated cell sorting) for initial enrichment

● FACS (fluorescence-activated cell sorting) to isolate high-affinity binders

● Binders are validated via sequencing and recombinant expression

Figure 2. Yeast Surface Display Screening Workflow
Overview of the yeast display screening process, from library generation and enrichment to FACS-based selection and hit analysis.

View More