About Us

Nebulabio is a leading biotechnology company specializing in antibody discovery and engineering, providing end-to-end solutions from early-stage development to preclinical research. With an experienced scientific team and state-of-the-art platforms, we offer expertise in phage display, single B cell screening, and antibody humanization, having successfully supported numerous therapeutic candidates into clinical stages.

☞Introduction of Phage Display Technology

Phage display library construction is the foundational step that determines the success of any downstream antibody discovery campaign. The principle centers on creating a vast, diverse collection of bacteriophages, each displaying a unique antibody variant on its surface while carrying the corresponding genetic code inside its viral particle.

 The Core Engineering Process for Different Antibody Formats: 

1.Genetic Source Acquisition:

• Fab & scFv Libraries: We typically source variable region genes (VH and VL) from human B cells (peripheral blood, spleen, bone marrow) for naïve or immune libraries, or design synthetic genes to target specific structural features.

• VHH (Nanobody) Libraries: Genes are obtained from immunized camelids (alpacas or llamas) or designed from synthetic frameworks, capitalizing on their single-domain nature.

2.Vector Assembly & Cloning:

The amplified antibody gene fragments (for Fab, scFv, or VHH) are cloned into a specialized phage display vector. This vector is engineered to fuse the antibody gene with a gene encoding a phage coat protein (e.g., pIII of M13 phage). This ensures that every assembled phage particle physically displays the antibody protein it encodes.

3.Library Transformation & Amplification:

The ligated vector DNA is introduced into competent E. coli cells via highly efficient electroporation. Each bacterium that takes up a single vector gives rise to a clone producing a single type of displayed antibody. The total collection of these bacterial clones constitutes the primary library, with diversities typically ranging from 10⁹ to 10¹¹ unique members.

☞Our Technical Advantages in Library Construction

We don't just build libraries; we engineer high-quality, functional genetic repertoires designed for successful screening outcomes.

■ Format Expertise & Optimization: We possess specialized protocols for constructing robust libraries in Fab, scFv, and VHH formats, understanding the unique expression and folding requirements of each.

■ Maximized Functional Diversity: Our processes are optimized to minimize cloning bias and maximize the percentage of clones that express full-length, well-folded antibody fragments, ensuring your library's diversity is not just in sequence but in functional display.

■ Tailored Library Strategies:

• Naïve Libraries: Sourced from non-immunized donors, offering broad antigen-agnostic diversity.

• Immune Libraries: Built from immunized hosts (animals or humans), providing a head start with pre-enriched, antigen-specific binders of higher affinity.

• Synthetic/Semi-Synthetic Libraries: Designed with controlled diversity in key Complementarity-Determining Regions (CDRs), allowing focus on specific paratope chemistries and enhanced developability profiles.

■ Rigorous QC from Day One: We perform stringent quality control after library construction, including measuring library size (titer), assessing insert rate (>90% typical), and sequencing random clones to analyze framework and CDR diversity, providing you with a Certificate of Analysis for your library.

☞Our Service Workflow: From Concept to Quality Library

Our streamlined, collaborative process ensures your custom library is built to specification, on time, and to the highest standards.Service Flowchart:

 Step 1: Project Consultation & Design 

We begin by understanding your target antigen, project goals (e.g., need for high affinity, specific epitope targeting, developability), and desired antibody format (Fab/scFv/VHH). Together, we determine the optimal library strategy—naïve, immune, or synthetic.

Step 2: Genetic Material Preparation

Based on the chosen strategy:

• Immune/Naïve: We perform RNA extraction from provided tissue (spleen, PBMCs) or source it ethically.

• Synthetic: We design and synthesize gene fragments with optimized codon usage and designed CDR diversity.

Step 3: Library Construction

This core phase involves antibody gene amplification, precise cloning into our high-efficiency phage display vectors, and large-scale electroporation to create the primary, diverse bacterial library.

Step 4: Primary Library Amplification & Phage Rescue

The bacterial library is amplified, and helper phage is added to "rescue" the displayed antibody library, producing the functional phage particle library ready for panning or storage.

Step 5: Comprehensive Quality Control & Delivery

We conduct exhaustive QC: titering for size, checking insert rate via PCR, and performing NGS sequencing on random clones to validate diversity. The final deliverable includes the high-titer phage stock, the corresponding bacterial glycerol stock, and a detailed QC report.

☞Main Process of Phage Display is as Follows:

This robust and well-established laboratory method for antibody discovery consists of two integrated phases, which can be visualized in a typical process schematic with a left and right panel.

 ◆ Phase 1: Library Construction 

In the initial phase, a highly diverse library is built. An extensive repertoire of antibody genes—formatted as scFv, Fab, or VHH fragments—is cloned into a specialized phagemid vector. This genetic material is fused to a gene encoding one of the bacteriophage's coat proteins. The library is then introduced into E. coli host cells via high-efficiency electroporation. During subsequent phage production within the bacteria, each engineered phage particle displays a unique antibody fragment on its surface while encapsulating the corresponding gene inside, creating a direct link between protein function and genetic code.

◆  Phase 2: Affinity Selection & Enrichment 

The constructed library is then subjected to an iterative affinity selection process. The pool of antibody-displaying phage particles is incubated with the immobilized target of interest. Phages displaying well-folded, specific antibody fragments bind to the target, while non-specific or poorly folded variants are removed through controlled washing steps. The specifically bound phages are then recovered (eluted) using various elution strategies. This eluted population, now enriched for target binders, is used to infect fresh E. coli cells, amplifying the selected antibodies for additional rounds of selection to further enrich for the highest-affinity clones. Finally, the output of this selection is characterized in a screening phase to identify and validate individual lead candidates.

Figure 2. Phage display methodology.

☞Why Choose Nebulabio Library Construction Service?

• Proven Success Record: We have built hundreds of successful libraries that have yielded high-affinity leads against diverse target classes, including GPCRs, ion channels, and viral antigens.

• Focus on Functional Output: Our protocols are tuned not just for high DNA diversity, but for maximizing the proportion of clones that display functional, well-folded proteins—increasing your screening hit rate.

• End-to-End Project Support: We are more than a service provider; we are a partner. Our scientists provide expert guidance from design through delivery and are available to support your subsequent screening efforts.

• State-of-the-Art Infrastructure: Our dedicated molecular biology labs, robotic workstations, and Next-Generation Sequencing (NGS) capabilities ensure precision, reproducibility, and deep analytical insights into your library's composition.

• Confidentiality & IP Security: Your project details and the resulting library are handled with the utmost confidentiality. We operate with clear IP agreements, ensuring you retain full rights to all discovered leads.