What is a phage or bacteriophage?
Phages are viruses that infect bacterial cells. There are several types of phages that have been used as vehicles for phage display including Ff filamentous phage, Lambda and T7. In phage display, the bacteriophages are used to construct different phage libraries as peptide libraries or antibody library
History of phage display
Phage display technology was first introduced in 1985 by George Smith. After that many advantages have been introduced including the way to construct a phage library like antibody library to select phage display antibodies.
Advantages of phage display
Phage display is a system for large scale study and selection of proteins, peptide and antibodies based on their binding affinity and specificity. One advantage of phage display is the enormous diversity of variant proteins that can be represented in a phage library. Phage display provides a means of rapidly screening large numbers of proteins against potential binding partners.
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Screening of Phage Display Libraries

Library screening

Screening of phage display libraries is usually accomplished by a process which consists of affinity selection steps called panning or biopanning during which phage populations are exposed to targets in order to selectively capture binding phages. In the process of screening libraries involved successive rounds of binding, washing, elution and amplification. Several rounds of this process of the library screening will increasingly enrich the originally very diverse phage population with phages with a propensity to bind to the target in question.

The target molecule of the library screening is immobilized on a solid support by passive or active adsorption. The solid support can be polysterene tubes, MaxiSorp plates, or magnetic beads. The binding steps in the Phage Display library screening consist in presenting the pool of phage particles carrying the displayed peptides, proteins or antibodies to the immobilized target molecules. Knowlegde of the binding properties of the target molecules used in the library screening can facilitate to work out the best binding conditions in order to increase the chance to pick up positive clones. However, not only phages with affinity binding will bind to the target molecules without many phages that bind in a not specific way, in addition phages can bind to the surface of the solid support and other components in the reaction. In order to remove non-binding phages in the screening libraries is necessary several washing steps.

The way of doing the washing in the library screening need to be taken in consideration because it is required a balance between specificity and avidity of selected clones. Some clones may be strong binders with low specificities while others may be weak binders with high specificities. If washing is too stringent in the library screening then highly specific, but weak binders may be lost. If washing is not stringent enough during the screening libraries then populations of selected clones may be dominated by strong binders with low specificity. In practice, this balance is achieved in the Phage Display library screening by adjusting washing times, detergent concentrations and using regimes in which washing stringencies are progressively increased.

Several different treatments can be used to elute bound phages from targets molecules in the library screening such as dramatically lowering or increasing pH, reducing agents, enzymatic cleavage or elution by affinity. However, it is necessary to take in consideration that harsh elution conditions no affect phage integrity. The recovered phage population is commonly amplified before the next round of selection, however, it may be used directly without amplification to reduce background problems caused by non-specific phages that are carried through the panning process. In most cases, the eluted phage are used to re-infect E.coli which be streaked on plate to obtain individual colonies. A large number of single colonies are picked into 96-well plate to rescue the clones as phages. The phage are analyzed to identify positive clones.

Peptide library screening

Peptide library screening of commercially available peptide libraries or in-house made peptide libraries. The peptide library screening can be done with 16-mer or 20-mer  peptide libraries available at the moment.

 

Antibody library screening

Antibody library screening of naive antiboy libraries or immunized libraries

 

 

Vrisko Ltd offers a variety of Custom Services to fit your research and production needs including Screening of phage display libraries Ask for a quote and additional specific details according to your needs in our “contact us” page.