Phage biopanning

Key Words Epitope conformational sequential blotting phage biopanning. 

Between successive rounds of phage biopanning, the output phage sample (and/ or the acid wash sample, if desired) must be amplified. This procedure may be performed in parallel with the phage titering as detailed in Subheading 3.3 or may be performed independently. 

The selectivity determination is performed in a manner similar to the phage biopanning protocols for adherent and nonadherent cells detailed in Subheadings 3.1 and 3.2, with some important modifications. 

Figure 16.4 Graph depicting the percentage of lysine residues among peptides that bind to the indicated monoclonal antibodies. The peptides were isolated after affinity selection (biopanning) from a phage-displayed combinatorial peptide library. The peptides are grouped as to whether they are susceptible to formalin fixation, resulting in a loss of immunoreactivity.

The procedure for epitope mapping by the phage display method involves biopanning, amplification, assay for positive colonies, DNA isolation, and sequencing. Some steps of the procedure must be performed at the same time. Therefore, we describe the protocols in Subheadings 3.1. and 3.2. on a day-by-day manner. All steps of biopanning and phage amplification should be carried out under sterile conditions, when possible. 

Assuming that the amount of free target and the fraction of nonspecifically bound phage remain the same each round, the enrichment over r rounds of biopanning is (h(Ka))r. 

FIGURE 19.1 Biopanning on plates. The target, immobilized on a Petri dish, is incubated with the library in solution. Phages displaying the active sequence are eluted, amplified, and submitted to subsequent biopanning cycles. 

Alternatively, biopanning on plates is based on the strong biotin-streptavidin interaction to isolate target-binding phages [6]. Figure 19.4 shows a schematic representation of the procedure. 

FIGURE 19.4 Biopanning selection through biotin-streptavidin interaction. The library is incubated with the biotinylated target in solution phages displaying the active binding peptide are captured on a streptavidin-coated Petri dish, eluted, and amplified. 

FIGURE 19.6 Biopanning on a column. The target is covalently immobilized on a chromatographic support. The phage library is then applied to the column and, after an extensive washing step, bound phages are recovered by elution, amplified, and submitted to a successive selection cycle. 

FIGURE 19.7 Competitive biopanning on a column. The library in incubated with a competitor in solution, then applied to the column. Competitor-bound phages are not available to interact with the immobilized target, allowing the isolation of specific binding phages. 

FIGURE 19.8 Biopanning procedure on cells. The binding of the phage library, followed by elution and amplification steps, allows the selection of ligands directed against specific cell markers. 

FIGURE 19.9 Biopanning in vivo. The phage library is injected intravenously into mice, allowing the identification of phage-peptide motifs that have been retained in specific organs. 

Fig. 5.1. The principle of phage display is based on in vitro selection of peptides or proteins expressed at the surface of filamentous phages. In a biopanning experiment, specific binders can be captured from a library and their genes can be amplified by infection.

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