BLOG ARTICLE10/21/2022by Mette Soendergaard

Biopanning of Phage Display Libraries

Phage display technology is a laboratory technique that employs genetically modified filamentous bacteriophages to display foreign peptides, antibodies, or other proteins. The phage particles are assembled into phage display libraries, from which researchers can select and identify peptide sequences or antibodies that bind with high affinity and specificity to a target molecule. Phage display technology has been used for many purposes, especially in drug discovery for the development of therapeutic antibodies and peptides.

Biopanning

Biopanning of phage libraries is the most common method of selecting and identifying peptides and antibodies that bind to a target antigen. A traditional biopanning procedure includes the incubation of a phage library with the target of interest followed by the collection of bound phages by elution. Each biopanning protocol contains the following key steps, which are described in more detail below.

Incubation of the phage display library with the target of interest (affinity selection).
Removal of unbound phage by washing.
Collection of bound phage by elution.
Amplification of collected phage.
Repetition of steps 1-4 for typically a total of four rounds.
Identification of collected phage by DNA sequencing.

While phage display biopanning is a relatively simple and efficient technique, it requires careful optimization of several parameters in order to achieve the best results. These include the choice of the type of phage library, phage and target molecule concentration, cell type (tumor cells, bacterial cells, etc.), incubation conditions such as temperature and time, as well as the amplification method of collected phage.

Affinity Selection

The first step of a biopanning process is often referred to as affinity selection since this step allows phage-displayed peptides or antibodies with affinity for the target to bind. Often, the target molecule is an immobilized recombinant protein. However, carbohydrates, lipids, and other biomolecules can also be used. Additionally, whole cells, tissues, and organisms (experimental animals, human patients, etc.) have been used with great success in biopanning protocols to identify binding peptides and antibodies with high affinity and specificity.

Removal of Unbound Phages

Unbound phages are typically removed by extensive washing using a buffer with a low concentration of a detergent. Weakly bound phages, which are typically undesirable in the phage display selection, can be removed by increasing the concentration of detergent or adding additional washing steps.

Elution of Bound Phages

In standard biopanning procedures, bound phages are collected by elution using detergents, changes in pH, or other methods of disrupting the non-covalent interactions between the phage and target moiety. Using such elution methods generally results in the collection of the vast majority of bound phages. However, phage clones with high binding affinity may not be sufficiently eluted by disruption of the non-covalent interactions. Thus, trypsin digestion has become prevalent and ensures equivalent elution of the bound phages.

Amplification of Phages

After each selection round, eluted phages are usually amplified and used in subsequent selections. Amplification of collected phages can be done in different ways and depends on the type of vector used for the phage library. Bacteriophage vectors such as the fUSE5 library are most often amplified by the transduction of E. coli by the phage particle. Phagemid vectors are significantly smaller than bacteriophage vectors and are, therefore, more easily transformed into E. coli cells. However, for the propagation of phage particles, a helper phage is required to ensure the assembly and release of the viral progeny.

Repeating the Selection Rounds

Most biopanning procedures include multiple rounds of affinity selection. Typically, four rounds of biopanning against the target antigen are employed to ensure sufficient selection pressure. Different types of affinity selections may also be used in biopanning to identify phage clones with specific binding characteristics. Such multi-tier biopanning strategies increase the likelihood of selecting peptides and antibodies that have optimal binding properties both in vitro and in vivo.

Identification of Phage Clones

Eluted phages from affinity selections are identified by DNA sequencing of the foreign inserts that encode the phage-displayed peptides or antibodies. It is also advantageous to identify unbound phages that are removed from the selection by washing. This step helps identify target-unrelated peptides (TUP) and antibodies that may otherwise be incorrectly identified as potential leads.

Summary

Biopanning is the most common phage display strategy used to select antibodies and peptides with high affinity and specificity for a target molecule. While biopanning can be straightforward, several considerations are important, such as the choice of peptide library or antibody library, the affinity selection conditions, and the extent of DNA sequencing. Do you need a custom peptide, antibody, or biopanning strategy for your research? Contact us today to discuss your research needs.