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Monoclonal antibodies are the most widely used reagents for specific detection and quantification of proteins. However, their production is long, time-consuming and requires animal immunization. In addition, some proteins are not immunogenic or reveal toxic to the animals and cannot lead to efficient antibody selection. Finally, monoclonal antibodies are large molecules of about 150 kDa and it sometimes limits their use in assays with several reagents competing for close epitopes recognition. Because of these limitations, the use of smaller antibody-derived molecules has emerged.
Single-chain variable-fragment (scFv) antibodies have been commonly used as alternatives. ScFv consist of only the light chain and heavy chain variable regions of immunoglobulins connected by a peptide linker. Their average molecular weight is about 27 kDa. ScFv contain the antigen-binding site and are asspecific and affine as intact antibodies. In addition, they can be easily expressed in yeast or in E. coli with a high production yield.
More recently, single domain antibodies were isolated from camelid animals; the so-called VHH. A VHH antibody corresponds to the variable region of a heavy chain of a camelid antibody and has a very small size of around 15 kDa. This type of affinity molecules are easy to express in E. coli, to link to a fluorescent marker or to conjugate with an enzyme to be used in various assays.
The advantage of these antibody-derived molecules is their small size which enables their binding to hidden epitopes not accessible to whole antibodies. In the context of therapeutic applications, a small molecular weight also means an efficient penetration and fast clearance. A VHH antibody has bigger probability to adopt identical intra and extracellular folding. Therefore, it is a qualified candidate for intracellular probing (Intrabody).
Being a single-domain molecule, a VHH antibody is expressed in cell without the need for a supramolecular assembly in contrast to a full immunoglobulin made of 4 chains, 2 light chains and 2 heavy chains. A VHH antibody is more stable and robust than a whole antibody
Both scFv and VHH can be linked to the Fc fragment of the desired species and keep their specificity and binding properties (Minibody).
* The name "Nanobody" belongs to ablynx