A single-chain variable fragment (scFv) is a small molecule composed of a variable region heavy chain and a variable region of a light chain of an antibody, the two variable regions are linked by a peptide chain. The scFv is a minimal functional structural unit having antibody activity. Single-chain antibodies can be expressed by expression systems or a phage display technology. Because of its small molecular mass, strong penetrability, short half-life and low immunogenicity, it has an important role and broad application prospects in the clinical diagnosis, treatment and prevention of diseases. The article provides an overview of the structure, preparation, and application of single-chain antibodies.

1. The structure of scFv

The intact antibody consists of two heavy chains (H) and two light chains (L), which can be artificially engineered to express only the variable region. The variable region of heavy chain (VH) and the variable region of light chain (VL) of antibody are linked into a recombinant gene by a synthetic linker gene, and the antibody expressed by the recombinant gene is referred to as single chain antibody (scFv). Structurally, the N-terminus of the heavy chain can be linked to the C-terminus of the light chain, or the N-terminus of the light chain can be linked to the C-terminus of the heavy chain. The length of Linker is usually 15~25 amino acids. It is usually composed of glycine (Gly) and serine (Ser), which has a certain elasticity and protease resistance. The function of Linker is to connect VH and VL, and maintain a certain elasticity, so that the functional region of VH and VL can still be paired after folding to form the binding site of the monovalent antigen.

The scFv fragment is monovalent, and a bivalent scFv can be made by concatenating two scFv fragments. Bivalent scFvs generally have two structure, one is to form a single peptide chain containing two heavy chain variable regions and two light chain variable regions, called tandem antibodies (tandem di-scFvs); the other is to shorten the length of Linker from 15 to 3~12, allowing the functional regions of VH and VL from two different molecules to pair with each other to form a dimeric structure called diabodies (if diabodies are composed of variable regions from two different antigens, then a bispecific antibody will be formed). Similarly, a series of tandem antibodies (tandem di-scFvs) with three heavy chain variable regions and three light chain variable regions can be produced, and by further shortening the Linker length, the VH and VL functional regions from three different molecules are paired with each other to form a polymer (triabodies). Compared to the general scFv fragment, the scFv multimeric antigen has an increased binding value and a higher affinity.

2. The preparation of single chain antibody and the screening of high affinity scFv fragments

The scFv can be expressed in multiple expression systems. At present, Escherichia coli expression system and mammalian expression system are commonly used. The scFv fragment can also be produced by phage display technology. The hybridoma cells were obtained from the immune animal experiments, and the cDNA was extracted from the hybridoma cells.

The heavy chain variable region (VH) and light chain variable region (VL) of a full set of antibodies were obtained by reverse transcription PCR (RT-PCR), Overlapping PCR (SOE-PCR) was used to splice and amplify V H and V L fragments to obtain variable fragment gene fragments of single chain variable fragment (scFv). The scFv gene was cloned into a suitable phage vector, The competent Escherichia coli are electroporated and the helper phage are over-stained, and the obtained supernatant is a single-chain antibody library. The specific antigen was taken as the solid phase, after 3 to 5 rounds of "adsorption, elution, amplification" screening, an enriched single-chain antibody that specifically binds to the antigen can be obtained.

3. The purification of scFv

The scFv lacks the Fc fragments contained in intact antibodies, so the common binding site (Protein G) cannot be used to purify antibodies. Typically, 6 histidines are added to the C-terminus of the scFv fragment, and then the scFv can be purified by metal chelating affinity chromatography (IMAC). In addition, protein L can interact with the variable region of the light chain, so protein L can also be used for antibody purification.
Some special ScFv fragments can also be captured and purified by Protein A, but the principles are unknown.

4. The application of scFv

The scFv has the advantages of small molecular mass, strong penetrability and high specificity. It has important applications in targeted therapy, imaging diagnosis, intracellular immunity, and biological detection. In the targeted therapy, drugs and toxins can be linked with scFv to form immunological drugs or immunotoxins. The specific binding of antigens and antibodies can be used to target cells and kill the target cells specifically. In imaging diagnosis, due to the strong penetrating power of scFv, the distribution index in tumor tissues is higher than that of intact antibody molecules.

In radiographic imaging, radionuclides are expelled faster, which is less harmful to the body and can be used for the diagnosis of tumor imaging. In the aspect of intracellular immunity, after the virus infects the organism, the cell expresses an antibody that recognizes a certain virus-encoded protein, thereby preventing the virus from being transmitted between the cells, and the scFv of anti-viral protein can be cloned and expressed in the cell, and can be used in the diagnosis and treatment of viral infectious diseases. In biological monitoring, the scFv has good affinity and is easy to prepare, so it is used to quickly and accurately detect harmful substances in foods and water.

At present, the scFv has become a hot spot in genetic engineering research. With the deepening of the research on scFv, it is believed that in the near future, the scFv will play a greater role in the fields of medicine and food.

Author's Bio: 

Creative Biolabs has established the novel sdAb production platforms that enable the generation of target-specific single domain antibodies within 8 weeks. We have produced a large number of single domain antibodies with high affinity and specificity against a large number of distinct targets, including cytokines, cell surface receptors, tumor cell markers, viral antigens, and enzymes. Of note, single domain antibodies are perfectly stable polypeptides harboring the full antigen-binding capacity of conventional antibodies. This unique structural and functional property render these molecules ideal candidates for new generation of antibody therapeutics.