Semiautomated Panning of Naive Camelidae Library
With the increased requirement of specific binders in biological fields, library screening has been a potential approach. Compared with recombinant target proteins, the designed peptide can be chemically synthesized for library screening. Here, Creative Biolabs describes the novel semiautomated panning of naïve Camelidae library for sdAbs generation against designed peptide antigens.
Background for Semiautomated Panning
Antibodies are naturally designed molecules to characterize and target novel proteins identified from genomic and proteomic initiatives. The in vitro methods for antibodies isolation from naïve and immune libraries also extensively developed. However, the difficulty of obtaining pure protein antigens largely limits library screening purposes.
Based on a combination of bioinformatics, proteomics, glycomics approaches, the rational peptide design is being explored as a viable solution for library screening and antibodies generation. In fact, a list of human putative cell surface and secreted proteins have been created through the bioinformatic approach.
Derived from the heavy-chain-only antibodies produced by camelids, sharks, or the specific developed human VH and VL domain, sdAb is the novel antibody fragment consisting of a single monomeric variable antibody domain. Compared with the conventional antibodies and fragments, sdAbs present great advantages for biological diagnostic and therapeutic. Phage-display technology is a well-established technique for library construction, the target-specific sdAbs can be obtained easily via a series of screening methods.
Fig.1 Schematic representation of the semiautomated panning setup. (Kumaran, et al., 2012)
Semiautomated Panning Procedures
For phage library screening, the traditional solid-phase manual panning is a time-consuming task. Alternatively, the streptavidin magnetic beads-based in-solution screening can be used against multiple targets. In general, the fully automatic panning approaches include multiple automated modules, colony picking via robotic arms, and ELISA screening via robotic systems. With the characterization of large library size, the semiautomated panning approach can be used for sdAb generation against peptide antigens as well as the parental proteins.
The semiautomated panning procedure consists of four main steps: 1). Incubation of beads charged with biotinylated peptides with phage library; 2). Bead removal from binding solution and placement into wash buffer. 3). Release of beads. 4). Amplified phage for subsequent rounds of panning.
Fig.2 Semiautomated panning-based sdAb generation from naïve library.
Features of Semiautomated Panning
- Compared with stable, purified target proteins, the designed peptides can be synthesized easily.
- Biotinylated peptides are suitable for in-solution streptavidin magnetic bead-based panning.
- High efficiency compared with solid-phase manual panning.
- Increased surface area on beads compared with plates.
- Reduced background binding to surfaces.
Reference
- Kumaran, J.; et al. Semiautomated panning of naive camelidae libraries and selection of single-domain antibodies against peptide antigens. Single Domain Antibodies. Humana Press, Totowa, NJ. 2012: 105-124.
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