Single domain antibodies (sdAbs) present great advantages compared with conventional antibodies and antibody fragments, such as small size, great solubility, and high stability. In recent years, sdAbs have been served as promising molecules in the field of diagnostic and therapeutic. For sdAb discovery, there are a series of screening methods suitable for different situations and project needs.
Phage Display Screening for sdAbPhage 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:
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Yeast Display ScreeningFor 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. |
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Semiautomated PanningLibrary 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:
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Intracellular Antibody Capture (IAC) MethodsMonoclonal 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. |
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Bacterial Two-Hybrid ScreeningThe 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
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Mammalian Two-Hybrid System SelectionProtein-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:
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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
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sdAb Selection Under Application ConditionsThe 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. |
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