Single-chain variable fragment
A single-chain variable fragment (scFv) is not actually a fragment of an antibody, but instead is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short peptide linker of ten to about 25 amino acids.
Each variable fragment contains framework regions (FWRs), providing stability to the structure, and complementarity-determining regions (CDRs), which are the regions responsible for antigen recognition.
The linker is usually rich in glycine, which provides flexibility, as well as serine or threonine, that provides solubility. The peptide linker can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa, but changes in the orientation of the VH, peptide linker and VL have been described to affect scFv’s affinity and specificity.
This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. The image to the right shows how this modification usually leaves the specificity unaltered.
These molecules were created to facilitate phage display, where it is highly convenient to express the antigen-binding domain as a single peptide. As an alternative, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma. ScFvs have many uses, e.g., flow cytometry, immunohistochemistry, and as antigen-binding domains of CAR-T cells.
Unlike monoclonal antibodies, which are often produced in mammalian cell cultures, scFvs are more often produced in bacteria cell cultures such as E. coli.