Zebrafish Antibody Library Construction by Phage Display

Zebrafish (Danio rerio) are becoming a powerful tool for antibody discovery. These transparent, striped little fish bridge the gap between basic laboratory research with animals like mice and human health. Zebrafish and humans have about 85% of their genes in common. And that rises to nearly 99% when looking at genes involved in human disease. Because the core cellular pathways that underpin life work in almost exactly the same way in zebrafish, we use them to find antibodies that will work in humans. At Creative Biolabs we combine our antibody development experience with state-of-the-art phage display technology, applying it to zebrafish to find higher quality antibodies more quickly and cost effectively, even against challenging targets like cell surface proteins or complicated-shaped molecules.

Why Use Zebrafish?

Zebrafish antibodies are a powerful tool for many reasons:

• Their immune systems are similar to our own: When a zebrafish's immune system detects a foreign molecule, it responds much like humans do.
• They develop quickly, and you can see through them: Zebrafish embryos are clear. That means that scientists can directly observe where antibodies are getting to and how they work in a living animal.
• They're very responsive to antigens: When zebrafish are exposed to foreign proteins, their immune cells churn out a rich diversity of antibodies.
• They make a unique mix of antibodies: In addition to IgM, zebrafish also make two other antibody isotypes: IgD and IgZ (also called IgT). Zebrafish IgD arises via non-canonical splicing, fusing the third IgD exon to the IgM constant region, bypassing a mutation-prone exon. IgZ functions in mucosal immunity.
• Zebrafish antibodies often bind human disease targets well: In some cases, they find unique binding sites on targets that can be missed by other systems completely, making them useful for drug target study or disease marker detection.

Why We Use Phage Display with Zebrafish & Our Step-by-Step Service

• Speed: Linking each antibody to its DNA allows us to rapidly identify the best candidates.
• Scale: We build libraries that contain billions of antibody variants. This allows us to cover more of the target space.
• Hybridoma-free: This method works out of the box because zebrafish aren't amenable to traditional hybridoma technology.
• Format-flexible: We can produce any format needed, from scFv or Fab all the way up to smaller sdAb variants.
• Super binders: We can take the antibodies we start with and further improve them using directed evolution strategies.

Immunization & Library Preparation

• We first identify ideal epitopes on your target protein.
• Over the course of 4-6 weeks, zebrafish are dosed with low quantities (~10µg) of your antigen of interest.
• We isolate high-quality RNA from the fish's immune tissues.

Library Construction & Screening

Our zebrafish antibody discovery service includes:

• cDNA Synthesis
• VH/VL Amplification
• scFv/Fab Ligation
• Phagemid Transformation
• 3-Round Panning
• ELISA Screening

Fig.1 Library construction & screening workflow at Creative Biolabs.

Quality control checks are performed at each step to ensure proper diversity and function are maintained. We can also reformat selected antibodies into full IgG format if desired.

Screening Key Antibodies

• We measure binding strength (affinity)
• Determine if they are competing for the same epitope
• Confirm function in living zebrafish with human tumor xenografts

Library Options

You can choose the best library to start with:

• Immune Library: Generated using your specific antigen
• Naïve Library: Sourced from healthy zebrafish tissues
• Synthetic Library: Rationally generated using CDR design
• Semi-Synthetic: Natural scaffold with synthetically designed CDRs

Phage Display Options

• M13 (pIII and pVIII fusion)
• T7 (rapid cycle, ideal for cDNA libraries)
• T4 (dual display on HOC/SOC proteins)

Biopanning Methods

We choose the best method to expose the antibodies to the target:

• Solid-phase: Solid, works best for purified proteins
• In-solution: Keeps fragile proteins in their natural form
• Cell-based: Intact live cells to test membrane proteins
• In vivo: Assays targeting in living zebrafish tissues

We repeat the binding steps 3-4 times to isolate antibodies with strong binding.

What You Get?

• Ready to use phage library (titered & quality checked)
• DNA sequences of 50-200 clones
• Key monoclonal antibodies
• Option to make full IgG or Fc-fused antibody
• Detailed quality control report

Why Creative Biolabs?

• In-depth experience with zebrafish and immune system biology
• Tailored methods for small source tissues like zebrafish spleen
• Build antibody libraries containing >1 billion unique clones
• Use proven methods to further improve binding affinity
• Assistance with humanization and antibody production

Whether you're working on zebrafish biology or using them as a surrogate model to discover antibodies for human disease, we can help you find better antibodies more quickly and cost effectively. Contact Creative Biolabs today to get started on your project.

Please kindly note that our services can only be used to support research purposes (Not for clinical use).