Antibody Advice

When it comes to the production and application of antibody fragments, it pays to be smaller, but more specific, says Shengnan Shao

Antibodies are modular defence systems that identify and neutralise foreign objects such as bacteria and viruses. Each of them could recognise a specific antigen unique to its target as they possess the antigen-binding sites. What’s more, antibodies are now recognised as routine molecules in many therapeutic fields, no longer restricted to oncology and inflammation. To a better specificity, the variable regions are more concerned by scientists.

The antigen-binding fragment (Fab) is a region on an antibody that binds to antigens. It contains one constant and one variable domain of each of the heavy and the light chains. Only the variable regions of the heavy and light chains are fused together to form a single-chain variable fragment (scFv), which is half the size of the Fab fragment, yet the original specificity is retained. Another small antibody fragment, single domain antibody, also known as domain antibody, VHH, VNAR, or sdAb, is a kind of antibody fragment consisting of a single monomeric variable antibody domain and lacking the light chain and CH domain of the heavy chain in conventional Fab region.

Both scFv and VHH nanobodies can be linked to the Fc fragment of the desired species and keep their specificity and binding properties.

All these antibody fragments, Fab, scFv, and VHH, have a smaller size than a whole antibody. It enables their binding to hidden epitopes not accessible to whole antibodies. The smaller size can help to provide lower scattering, thus less contrast in images.

Therapeutic Applications for Antibodies

In the context of therapeutic applications, a small molecular weight also means efficient penetration and fast clearance. Some therapeutic use also needs the fragments due to a better penetration into tissue. It also could lead to the expression of the functional antibody and their fusion in bacteria and also allow their display on filamentous phage. In addition, the combination of small antibody molecules together with the efficient microbial production systems can finally lead to the production of a homogenous protein in sufficient amounts for diagnostic and therapeutic purposes as well as in structural studies.

To date, there are several routes to have an antibody fragment artificially synthesised. Several functional antigen-binding antibody fragments could be engineered by proteolysis of antibodies (papain digestion, pepsin digestions, or other enzymatic approaches), yielding Fab, Fv, or single domains (Fig. 1).

During the past decade, advances in recombinant antibody technology have greatly facilitated the genetic manipulation of antibody fragments. All target genes can now be cloned and expressed successfully as a fragment in bacteria, on mammalian cells, and even insect cells. One advantage of this technology is that it could retain the intact antigen-binding site (paratope) while reducing the size of the antibody molecule.

Shengnan Shao is with Sino Biological

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