Overview of Single Domain Antibody (sdAb) Library Screening

Phage Display Screening for sdAb

Phage display involves the co-expression of foreign proteins with phage coat protein on the surface of filamentous phage. As the first well-established screening method for therapeutic antibodies discovery, it combines antibody genotype (sequence) with the phenotype (specificity and affinity). The following are the main screening steps:

• Phage bind presented antigen
• Wash off the unbound phage
• Elute bound phage
• Infect eluted phage to regenerate library
• The next round of screening

Yeast Display Screening

For novel sdAb generation, several in vitro display methods have been developed. A series of sdAbs have been successfully discovered from immune and non-immune libraries via phage display or ribosome display. In fact, target-specific sdAbs can also be obtained via yeast display screening.

Avoiding the lengthy and time-consuming immune process, the yeast display screening method provides the quantitative and multi-parameter analysis offered by flow cytometry. Moreover, binders with different predesignated properties can be obtained.

Semiautomated Panning

Library screening has been an efficient process for antibody discovery but limited by the obtain of pure protein antigens. Consequently, rational designed peptide antigens have been a viable solution. The semiautomated panning is an efficient method to screen a naïve Camelidae library against designed peptide antigens for sdAb generation. The following are the main screening steps:

• Incubation of beads charged with biotinylated peptides with phage library
• Bead removal from binding solution and placement into wash buffer
• Release of beads
• Amplified phage for subsequent rounds of panning

Intracellular Antibody Capture (IAC) Methods

Monoclonal antibodies are important reagents for biological research but restricted in vitro or extracellular molecules on viable cells. IAC technology was developed to obtain intracellular sdAbs while sdAbs are the most effective intracellular antibody fragment.

IAC technology described here is based on the yeast two-hybrid (Y2H) system to obtain intracellular sdAbs against any desired protein antigens.

Bacterial Two-Hybrid Screening

The bacterial two-hybrid system is complementary to yeast two-hybrid system for antibody discovery while the yeast two-hybrid system is difficult to handle large and diverse libraries. The yeast can be replaced by fast-growing bacteria and the novel sdAbs can be used instead of single-chain antibodies (scFvs).

Main steps for bacterial two-hybrid system

• Animal immunization
• sdAb library construction
• Preparation of electrocompetent cells
• Binders selection by bacterial-two-system
• sdAbs expression and purification
• Intracellular functionality of sdAbs retrieved by bacterial-two-system

Mammalian Two-Hybrid System Selection

Protein-protein interactions (PPIs) are promising targets for a variety of diseases. The mammalian two-hybrid system is a powerful tool to identify and characterize protein-protein interactions in transiently transfected mammalian cells. Now the mammalian two-hybrid system has been used for functional sdAbs selection. The main experiment steps contain:

• Plasmid construction
• Transfection into CHO reporter cell lines
• Luciferase reporter assay

Cell-Free Screening by In Vitro Compartmentalization (IVC)

sdAbs present great potentials in immunotherapeutics development and the cell-free screening is a powerful tool to obtain sdAbs with sub-nanomolar affinities.

Main steps of sdAb cell-free screening by IVC

• Library assembly and characterization
• Preparation of sdAb display library in emulsion
• Biopanning
• Assessment of library fitness
• Cloning and screening of library selection output

sdAb Selection Under Application Conditions

The antibody fragments have been widely used in the field of diagnostic and therapeutic. These successful applications are determined not only by the specificity and affinity but also the ability to function in extreme conditions. The characterization of high stability makes it possible to select sdAbs under application conditions.

A variety of sdAbs has been obtained under different application conditions, such as the acidic conditions and proteases, high concentration of detergents, and low pH.