Antibodies…what incredible molecules! They’re a central arm of biologic therapeutics and they serve as invaluable research tools. There are many commercial sources for antibodies, but what do you do when you need to generate an antibody from scratch? The above infographic walks you through two major strategies for antibody generation: animal immunization and display technology.

Animal Immunization

This traditional approach begins with immunizing a host animal with the antigen of interest. Common host animals include mice (and ‘humanized’ transgenic mice, rats, rabbits, llamas, and goats). Other less common options, but options nonetheless, are chickens, pigs, and cats. Each host species has different nuances to their immune system and the types and features of the antibodies they produce, so your choice in host often comes down to the requirements for the resulting antibody and the specifics of your antigen.

Once the host has been immunized, the process can diverge into three primary tracks depending on your end goal:

• Plasma Isolation involves harvesting plasma and purifying antibodies. This is great when you want to generate polyclonal antibodies (i.e. a mixed population of antibodies, targeting different epitopes on your antigen with varying affinities). This route is often used to generate research tools as this mixed population is not suitable for therapeutic development or applications where purity and specific functionality are required. The entire process from immunization to antibody harvest takes roughly 4–8 weeks.

• Hybridoma Generation is a well-established methodology in which spleens are harvested from the host and B cells are isolated. B cells are the primary antibody producing cells of the immune system, and these spleen-derived lymphocytes are fused with immortal tumor cells to produce hybridomas. This approach makes isolation and functional screening of single clones possible, and enables antibody sequence determination and recombinant production of positive hits for further validation. The hybridoma approach results in generation of monoclonal antibodies and the full process typically requires anywhere from 12–24 weeks.

• B Cell Sorting is a newer method that utilizes single-cell sorting and transcriptomic technologies to isolate and sequence antigen-specific B cells without generating hybridomas. Although this method is relatively more expensive, it allows for more rapid, higher throughput screening and can enable recovery of a more diverse panel of binder sequences. Similar to hybridoma generation, once hits are identified, antibodies can be recombinantly produced and further validated. B cell sorting also leads to generation of monoclonal antibodies, and takes approximately 9–18 weeks.

Display Technology

Display technologies, such as phage or yeast display, utilize antibody libraries derived from naïve or immunized hosts, or from synthetic sources, and these libraries can be comprised of shorter-form antibodies fragments instead of full-length antibodies (i.e. Fab, scFv, VHH). Depending on the desired traits for the final molecule, these libraries can offer a faster path towards binder identification and formatting into multivalent or multi-functional molecules.

The first step in this process involves library generation or selection of a commercial, preassembled library. This library then undergoes iterative panning to enrich for high-affinity binders, followed by hit validation screening (and reformatting, if needed) to yield monoclonal antibodies or the desired molecule format. This process takes roughly 8–12 weeks. Generally, hits coming from a naïve or synthetic library will have a lower affinity to the target antigen compared to those coming from immunization approaches, so it’s common to bring hits through affinity maturation before arriving at the final antibody sequences. This additional step can extend timelines for display generated antibodies by an additional 8+ weeks.

Choosing the Right Strategy

What’s the best strategy for your project? It’s definitely not a one-size-fits-all answer! There are several key factors to consider:

• Project timeline

• Available budget

• Antigen identity and complexity

• Required affinity

• Intended application (i.e. therapeutic, diagnostic, or research use)

Need help navigating an antibody or binder discovery campaign? Reach out to us and schedule a call. We love supporting these projects and we work hard to ensure your project’s success!