Antibody Overview | Phage Display Overview | Utility of Selected Antibodies | Glossary

The Phage Display method uses genetically engineered phage, a virus that only infects bacteria, to display and produce human sFv antibody proteins. The phage display process involves the following steps: (1) genetically engineering phage display libraries to "display" human sFv antibodies on the surface of the recombinant phage, (2) selecting antibodies with high affinity and specificity to any given target by screening the antibody phage display libraries, and (3) producing and characterizing the selected antibodies. To learn this process in detail, both an interactive graphic demonstration and a step-by-step text description are provided below.

Construction of human sFv antibody phage display libraries

Human antibody phage display libraries contain antibody genes collected from the blood of either immune or healthy (non-immune) individuals. Through reverse transcription and recombinant PCR, human sFv antibody genes are engineered by amplifying rearranged human antibody V-genes from B-cells of different donors. Once established, sFv genes can be cloned into a phage vector where human sFv antibodies are fused to a phage coat protein encoded by phage gene III, allowing the display of sFv antibodies on the surface of phage particles. One antibody gene is inserted into each phage genome thus each phage expresses a single antibody with specificity to a particular antigen. A pool of phage displaying a collection of diverse antibodies is called an antibody phage display library. The antibody phage display libraries are propagated in bacteria and stored under refrigeration. The large non-immune or naïve human sFv libraries offer a rich source for isolation of antibodies of human origin to virtually any antigen, including self-antigens. The immune libraries created from individuals with autoimmune diseases, tumor transformations or viral infections allow isolation of high affinity disease-specific antibodies at a relatively small scale. The antibody phage display libraries can be viewed as a mimic of the humoral arm of a human immune system in a test tube.

Selection of a human sFv antibody

Selection of antibodies to a target molecule is initiated by first incubating an aliquot of the entire library with its target. The target molecules can be purified proteins that are immobilized onto a solid surface, such as the wells of a plastic 96-well plate or a test tube (Immunotube Selection) or blotted on nitrocellulose membranes. The target molecules can also be biotinylated proteins/peptides in solution (Streptavidin/Avidin Bead Selection), or proteins presented on surface of cells (Pathfinder/StepBack Selection or Subtraction Selection) or liposomes (Paramagnetic Proteoliposomes). Upon incubation, the antigen specific phage antibodies will interact with the target molecule and remain attached. The remaining unbound billions of phages are washed away. The selected antigen specific phage, which both express a unique antibody on its surface and carry the antibody gene in its genome are used to infect bacteria, one single phage per bacterium. Each infected bacterial cell grows into a single colony, a clone of identical cells, upon spreading onto agar plates. Each clone thus produces a single type of monoclonal antibody. Isolated clones are then pooled and further amplified to produce the phage antibodies for the next round of screening (Panning). Selection of those sFv antibodies with the strongest affinity and specificity can be achieved through multiple rounds of panning of antibody-expressing phage particles against antigens of interest by altering selection conditions. Good candidate antibodies will go through additional testing such as ELISA and sequence analysis. The affinity of a chosen antibody can be further analyzed by BiaCore or in appropriate cases, by saturation binding studies to cells as analyzed by FACS. The final selected antibodies can be put into appropriate functional tests.

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