Equine Antibody Library Construction by Phage Display

Background

The horse is an immunologically vulnerable species, prone to infections such as Equine Herpesvirus (EHV-1) and West Nile Virus. Vaccination against such diseases is of low efficacy. There is a clinical need to generate useful therapeutic antibodies. Antibodies isolated from the equine immune system are high-affinity and recognize multiple epitopes within a single antigen. Such targets are less likely to lead to immune escape or mutation than single-epitope-targeting antibodies. The horse is a promising donor species for veterinary antibody generation. The traditional process for generating antibodies, hybridoma technology, is inefficient when adapted to equine cell species, due to difficulties in interspecies fusion of the cells. Antibody library methods, such as recombinant antibody library technologies, overcome this issue, since it is only necessary to be able to select from the target cells in vitro.

Evolution of Antibody Libraries & Equine-Specific Approaches

Traditional hybridoma technology uses mouse cells (derived from myeloma cells and spleen tissue, pioneered in 1975). This approach is difficult to apply to other species. Phage display was developed in 1985 to enable the display of heterologous proteins on the surface of bacteriophages. Approaches such as naïve human antibody libraries (first described in the 1990s with libraries in excess of 10¹⁰ clones) were also introduced, which involved cloning directly from B cells without immunization of a donor. Equine antibody libraries have been constructed using similar technology in the 2020s. This required the use of equine-specific primers and species-specific immunization procedures. There are three approaches to the generation of antibody libraries:

• Naïve Libraries: Generated from PBMCs or spleen tissue from a healthy donor horse. They display a greater diversity than immune libraries, but the affinity of naïve libraries is typically lower, as the in vivo process of affinity maturation has not occurred. Isolated clones will require in vitro affinity maturation.
• Immune Libraries: Use antigen-immunized horses. This benefits from in vivo affinity maturation, which is a greater target-specific enrichment than naïve libraries. Production is dependent on the availability of immunized horses, and the cycles of immunization required.
• Synthetic Libraries/Semi-synthetic Libraries: Designed CDR regions are grafted onto an equine framework. This avoids the animal ethical issues, but requires careful design of the CDRs to ensure function.

Immune libraries (such as the recently published anti-EHV-1 scFv library) are now the most commonly used approach for equine antibody discovery.

Custom Equine Antibody Library Construction

At Creative Biolabs, we specialize in custom equine antibody library construction tailored to meet the unique needs of your research.

Source of Antibody Genes

The construction of an equine antibody library begins with the selection of the source of antibody genes. These can be derived from either immunized animals or naive repertoires. Immunized libraries are generated from animals that have been immunized against a specific antigen, while naive libraries are constructed from unimmunized animals and represent the natural diversity of the antibody repertoire. In the case of single-domain antibody libraries, the source of antibody genes can be either camelized human VH3 in FR2 or a naïve camelid V_HH repertoire. This approach ensures that the library contains a wide range of antibody fragments with high specificity and affinity.

Service Flow

Our process ensures the development of high-quality antibody libraries that are diverse, functional, and ready for screening. The steps for our custom library construction services include:

1. Begin with an initial consultation to fully understand your specific requirements (target antigen, preferred format).
2. If required, we assist in antigen preparation. We ensure you get the properly purified and characterized target antigen.
3. Immunize equines with the target antigen. Multiple immunization protocols can be tailored upon your requirements.
4. B-cell isolation and cDNA synthesis
5. Phage display library construction
6. Using solid-phase or in-solution screening methods for library screening, repeated at least 3-5 cycles.
7. Affibody and affinity maturation if necessary
8. Candidate selection and characterization for best binding specificity, affinity, and stability.

Key Features of Our Services

• Our antibody libraries are huge, typically up to 10¹¹ unique clones, ensuring a broad diversity of potential binders.
• We utilize advanced screening techniques, such as magnetic bead-based selection and in-solution panning, to quickly identify high-affinity antibodies.
• Biopanning strategies can be tailored at Creative Biolabs. We customize the screening process to ensure the highest probability of success in isolating antibodies with the desired properties.

Advantages of Equine Antibody Libraries

• Large serum volume ideal for antibody generation.
• Wide epitope recognition against highly conserved targets.
• Generates high-affinity antibodies.
• Superior specificity

Need horse antibodies built? Creative Biolabs makes the process smooth. Scientists rely on our specialized libraries to find high-quality candidates quickly. Don't let screening slow you down. You can tackle any target (tough pathogens, complex proteins, or elusive small molecules) with our custom equine antibody libraries. They're packed with diverse options, giving you the best launchpad for discovery.

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