Single-chain variable fragment

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 V_H with the C-terminus of the V_L, or vice versa, but changes in the orientation of the V_H, peptide linker and V_L 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.

Purification

Single-chain variable fragments lack the constant Fc region found in complete antibody molecules, and, thus, the common binding sites (e.g., protein G) cannot be used to purify antibodies. These fragments can often be purified or immobilized using protein L, since protein L interacts with the variable region of kappa light chains. More commonly, scientists incorporate a six histidine tag on the c-terminus of the scFv molecule and purify them using immobilized metal affinity chromatography (IMAC). Some scFv can also be captured by protein A if they contain a human VH3 domain.