About Us

Nebulabio is a world-leading Contract Research Organization (CRO) specializing in advanced single-domain antibody discovery and engineering. Our Premade Phage VHH Library platform is the culmination of over a decade of dedicated research and development in camelid immunology, synthetic biology, and high-throughput screening technologies. We have built one of the industry's most extensive and deeply characterized collections of ready-to-screen VHH (Nanobody) libraries, serving pharmaceutical giants, innovative biotech startups, and academic pioneers worldwide. By providing immediate access to billions of pre-constructed, quality-validated VHH clones, we empower our partners to bypass the lengthy timeline of custom library generation and launch discovery campaigns against high-value targets within days. Our expertise ensures that clients not only access superior genetic diversity but also leverage optimized selection protocols specifically designed for the unique advantages of the VHH format.

☞Introduction to the Phage VHH Library Platform

The Phage VHH Library platform is a powerful fusion of a revolutionary biological discovery—single-domain antibodies—and a robust in vitro selection technology.

VHH/Nanobodies are the variable antigen-binding domains of heavy-chain-only antibodies found naturally in camelids (llamas, alpacas). These compact, ~15 kDa proteins are characterized by exceptional stability (resistance to temperature, pH, and chemical denaturants), high solubility, and the ability to bind unique and cryptic epitopes (e.g., enzyme active sites, G-protein-coupled receptor cavities) due to their elongated CDR3 loops.

In our phage display system, the genes encoding these VHHs are cloned and fused to a gene of the M13 bacteriophage coat protein (pIII). Each resulting phage particle displays a unique VHH on its surface while packaging the gene that encodes it inside the viral capsid. This direct phenotype-to-genotype linkage enables biopanning: an iterative selection process where a library of billions of VHH-displaying phages is incubated with a target, non-binders are washed away, and specific binders are eluted and amplified. Over 3-4 rounds, the population becomes highly enriched for high-affinity, target-specific VHHs.

Our Premade libraries take this powerful technology to the next level. These are not custom-built for a single project but are large-scale, pre-constructed, and exhaustively quality-controlled libraries made available for immediate screening. This eliminates the 3-6 months typically required for animal immunization programs, lymphocyte harvesting, and primary library construction, offering an unparalleled fast-track to lead discovery.

☞Classification of VHH Libraries

Our portfolio of premade VHH libraries is strategically categorized to meet the specific needs of diverse discovery projects. Clients can select the optimal starting repertoire based on their target and desired lead profile.

 1. By Immunization Status: 

• Immune VHH Libraries: Generated from camelids hyper-immunized with specific antigens or antigen classes (e.g., human cell-surface receptors, viral proteins, tumor lysates). These libraries are pre-enriched for high-affinity (nanomolar to picomolar) binders against the immunogen, offering the fastest and most direct route to potent leads.

• Naïve VHH Libraries: Constructed from the natural, non-immunized B-cell repertoires of healthy camelids. These libraries offer universal, unbiased diversity against any antigen, making them the ideal choice for novel target discovery, multi-target projects, or when immunization is not feasible.

• Synthetic VHH Libraries: Built de novo using rational design. Diversity is introduced into the CDRs of one or more optimized, stable VHH frameworks. These libraries can be designed to be fully humanized, minimize immunogenicity risk, and incorporate tailored chemical diversity for specific applications.

 2. By Target Focus & Design: 

• Universal Naïve Libraries: Our largest libraries (>10^10 clones), designed for maximum generic diversity.

• Target-Focused Immune Libraries: Specialized libraries built from camelids immunized with common therapeutic target classes (e.g., GPCR peptides, kinase domains, cytokine families).

• Stability-Optimized Synthetic Libraries: Libraries designed on super-stable frameworks known for high expression yields and resilience, yielding leads with outstanding developability.

☞Phage VHH Library Construction Process

The superior quality of our premade libraries stems from a rigorous, multi-stage construction pipeline, ensuring maximum functional diversity and performance.

 Phase 1: Repertoire Acquisition 

• Immune Libraries: We manage ethical camelid immunization programs with optimized antigen formulations and schedules to elicit a broad, high-titer immune response. Lymphocytes are harvested from blood or spleen.

• Naïve Libraries: PBMCs are sourced from a diverse panel of healthy, non-immunized camelids.

• Synthetic Libraries: Gene fragments are designed in silico and synthesized with controlled CDR diversity.

 Phase 2: Library Build – Molecular Cloning 

• RNA/DNA Extraction: Total RNA is isolated from lymphocytes for immune/naïve libraries.

• VHH Gene Amplification: cDNA is synthesized, and VHH genes are specifically amplified using primers that target the unique splicing site and structure of camelid heavy-chain-only antibody mRNA.

• Cloning into Phagemid Vector: The purified VHH PCR products are cloned into a high-efficiency phagemid vector (e.g., pHEN series). The vector fuses the VHH gene to gene III of M13 phage and contains all necessary elements for expression and antibiotic selection.

• High-Efficiency Transformation: The ligated vector DNA is introduced into electrocompetent E. coli via electroporation. This critical step is optimized to achieve the highest possible transformation efficiency, resulting in a primary bacterial library of immense diversity (>10^10 independent clones).

 Phase 3: Master Stock Creation & Quality Control

• The transformed bacteria are expanded and preserved as a master bacterial glycerol stock.

• A representative aliquot is infected with helper phage to "rescue" the VHH-displaying phage particles, creating the working library stock.

• Comprehensive QC Suite:

1. Library Titer: Quantification of the total number of unique clones.

2. Insert Rate & Sequence Analysis: PCR and Sanger sequencing of random clones to confirm correct VHH insertion and framework integrity.

3. Next-Generation Sequencing (NGS): Deep sequencing of the library pool is performed to provide a detailed diversity report, including CDR3 length distribution, amino acid composition, and clonal evenness.

4. Functional Validation: Successful test pannings against control antigens confirm the library's "pan-ability" and ability to yield specific, high-affinity binders.

☞Our Service Workflow

Our client-centric service provides a seamless, transparent, and efficient pathway from target to validated VHH leads.

 Step 1: Project Consultation & Library Selection 

We discuss your target biology, desired affinity range, and application (therapeutic, diagnostic, imaging). Based on this, we recommend the optimal premade VHH library and outline a tailored screening strategy.

 Step 2: Antigen Submission & Protocol Design 

You provide the purified target antigen. Our screening scientists design a customized panning protocol, selecting the best method for antigen presentation (immobilized, biotinylated, or on cells) and defining the washing stringency.

 Step 3: Immediate Screening Launch 

Using the pre-qualified, ready-to-ship premade phage library, we initiate the biopanning campaign without delay. We perform 3-4 rounds of selection, monitoring enrichment after each round via polyclonal phage ELISA and providing progress reports.

 Step 4: Hit Isolation & Sequence Analysis 

Individual clones from the final enriched pool are screened using monoclonal phage ELISA. All positive clones are sequenced. Our bioinformatics team analyzes the sequences, clusters them into families based on CDR3 homology, and identifies a diverse set of unique VHH leads.

 Step 5: Soluble Expression & Lead Validation 

The genes of selected unique VHH leads are subcloned for soluble expression in E. coli or yeast. Purified VHHs undergo a robust validation cascade:

• Binding Confirmation: ELISA or flow cytometry.

• Affinity Measurement: Kinetic analysis using Biolayer Interferometry (Octet) or Surface Plasmon Resonance (SPR).

• Specificity Assessment: Profiling against related proteins to ensure target selectivity.

• Stability Check: Thermal shift assay to determine melting temperature (Tm).

 Step 6: Final Delivery & Knowledge Transfer 

You receive a comprehensive final project report, including all screening data, sequence files, bioinformatics analysis, and validation results. Physical deliverables include expression plasmids for all lead VHHs and purified protein samples. We conclude with a project review meeting to discuss the results and potential next steps.

 Our Integrated Service Flowchart: 

Partner with Nebulabio for a reliable, expert-led journey from antigen to purified, functional VHH antibody. Our integrated service provides the unique advantages of Nanobodies with the assurance of professional production standards.