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Different Antibody Formats Used for Phage Display
Biointron2025-01-22Read time: 5 mins
Phage display is a widely used technique for selecting and engineering antibodies with high specificity and affinity for target antigens. By fusing antibody fragments to bacteriophage coat proteins, researchers can generate large libraries and screen for desired binding properties. Over the years, different antibody formats have been developed to optimize selection efficiency, stability, and functionality. These formats include single-chain variable fragments (scFv), antigen-binding fragments (Fab), single-domain antibodies (VHH or nanobodies), and others.
DOI: 10.1007/s00284-021-02398-9
VHH Antibodies (Single-Domain or Nanobodies)
VHH antibodies, also known as single-domain antibodies or nanobodies, are derived from camelid heavy-chain-only antibodies. They consist of a single variable domain that retains antigen-binding capability despite their small size (~15 kDa). VHH antibodies have excellent stability, solubility, and tissue penetration, making them ideal for applications where conventional antibodies may be too large or unstable.
In phage display, VHH libraries can be constructed from immunized or naïve camelids, allowing for rapid selection of high-affinity binders. Due to their unique properties, they are increasingly used in imaging, therapeutics, and even intracellular targeting. Their ability to recognize hidden epitopes that traditional antibodies cannot reach makes them particularly valuable for drug discovery.
Single-chain variable fragments (scFv) consist of the variable heavy (VH) and variable light (VL) chains linked by a flexible peptide linker. This format retains the antigen-binding specificity of full-length antibodies while being smaller in size, making it ideal for high-throughput screening and intracellular applications.
One advantage of scFv fragments is their ability to be expressed in bacterial systems without requiring glycosylation. However, their stability can be lower than other formats due to the absence of constant domains. To address this, researchers have engineered scFv variants with improved stability or affinity maturation techniques to enhance binding strength.
Antigen-Binding Fragment (Fab)
Antigen-binding fragments (Fab) are another commonly used format in phage display. They consist of the variable and constant domains of both the heavy and light chains, making them more stable than scFv fragments. Unlike scFv, Fab fragments do not require a linker, which can improve expression and folding.
Fab fragments are widely used in therapeutic and diagnostic applications due to their balanced stability and affinity. They also allow for direct comparison with full-length monoclonal antibodies, making them a preferred format for drug development. However, their larger size compared to scFv can limit penetration in some applications, such as targeting intracellular proteins.
Single-chain Fabs (scFab) are a hybrid format that combines elements of both scFv and Fab fragments. They consist of the VH and VL domains fused to the constant regions, but with a flexible linker connecting the heavy and light chains. This design allows for improved stability compared to scFv while maintaining the flexibility needed for phage display applications.
scFab fragments are particularly useful when dealing with antigens that require higher stability or when engineering bispecific antibodies. Their ability to be expressed in bacterial systems makes them a valuable alternative to traditional Fab fragments.
Variable Heavy Domain (dAb)
The variable heavy domain (dAb) is another minimal antibody format that consists solely of the VH domain. Unlike VHH nanobodies, dAbs are derived from conventional antibodies and retain the ability to bind antigens with high specificity. Their small size (~12–15 kDa) allows for greater tissue penetration and intracellular targeting.
dAbs are used in phage display for selecting binders against complex targets, including membrane proteins and intracellular molecules. Their high solubility and stability make them attractive candidates for therapeutic applications, especially in cases where larger antibody fragments may be less effective.
IgNAR and VNAR Antibodies
Shark-derived immunoglobulins (IgNARs) and their variable domains (VNARs) represent another unique class of antibody fragments. Similar to camelid VHH nanobodies, VNARs consist of a single variable domain, making them exceptionally stable and resistant to harsh conditions.
In phage display, VNARs have been successfully used to develop antibodies against challenging targets, including viral pathogens and toxins. Their rigid structure allows them to bind unique epitopes, including enzyme active sites and recessed pockets that conventional antibodies cannot access.
References:
Anand, T., Virmani, N., Bera, B. C., Vaid, R. K., Medhavi Vashisth, Priyanka Bardajatya, Kumar, A., & Tripathi, B. N. (2021). Phage Display Technique as a Tool for Diagnosis and Antibody Selection for Coronaviruses. Current Microbiology, 78(4), 1124–1134. https://doi.org/10.1007/s00284-021-02398-9