scFv Introduction

2020-12-03 Hits(1106)

A single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids. Single-chain fragment variable (scFv) antibody is an attractive recombinant antibody fragment that can be used in various of medical and biotechnological applications.

 

scFv Structure:

 

A scFv fragment is the smallest unit of immunoglobulin molecule with function in antigen-binding activities. A scFv antibody consists of a variable region of heavy chain (VH) and a variable region of light chain (VL) by a linker that containing 15-30 amino acids. The regions are joined together by a flexible peptide linker that can be easily expressed in functional form in E. coli, allowing protein engineering to improve the properties of scFv such as increase of affinity and alteration of specificity.

In comparison to the full length antibody, these minimized antibodies have several advantages in clinical practices including better tumor penetration, more rapid blood clearance, and lower retention times in non-target tissue and also to reduced immunogenicity. In addition, an ideal antibody at scFv stage is much easier to be transformed to other formats or be humanized.

 

 

 

scFv Construction:

 

A scFv fragment has the characteristics of antigen-binding properties, strong penetrating power, short half-life in vivo, low immunogenicity, and easy to perform genetic engineering operation. Currently, these antibody fragments are commonly generated by phage display technology. According to the source of antibody gene, the antibody library is generally divided into naive antibody library, immune antibody library, synthetic antibody library, semi-synthetic antibody library.

Phage display antibody libraries are widely used in screening high affinity antibodies, it is the method that the most recombinant antibody fragments are generated. Antibody libraries constructed by phage display techniques are usually derived from RT-PCR amplification of peripheral blood mononuclear cells, spleen cells, bone marrow cells and tonsil cells mRNAs to form cDNA fragments. These gene fragments were spliced into phagemid vectors, and then transferred into phages to form covalent structural protein and display in vitro.

Construction Procedure:

1. Plasmid design & synthesis

2. PCR amplification of variable region genes of heavy and light chains using cDNA as a template, scFv gene splicing.

3. Plasmid construction & transformation: after enzyme digestion, scFv and phagemid vector were ligated and transformed into TG1 host bacteria by electric shock to construct antibody libraries.

4. Identification: 20-50 clones were randomly selected, PCR identification, sequencing and analysis of antibody sequences.

 

scFv Application:

 

scFv fragments have good tumor penetration, rapid blood clearance, and low retention times in non-target tissue. Generally, antibody fragments can be utilized for the preparation of immunotoxins, therapeutic gene delivery, and as anticancer intrabodies. Antibody fragments can be fused to a range of toxins such as cytotoxic proteins, radionuclides, or drugs.