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Posted on Thu, Sep 27, 2018

Single Domain Antibody

Until this point we have been thinking about antibodies as one of the five classes, IgG, IgE, IgD, IgA, or IgM. The basic unit of each antibody class is a Y structure, where the base of the Y is known as the Fc region and the arms are the Fab region. The entire IgG antibody is composed of four polypeptide chains (two heavy and two light). The Fc region is composed only of heavy chain, and the variable Fab region is built with heavy chain and light chain. The Fab region is where all of the antigen-binding  occurs because the paratope, or antigen recognition site is located at the tip of each of the two arms of the Y. A single domain antibody paratope is made solely of a single heavy chain.

Free PDF guide:  "Basic Guide to Recombinant Protein Purification" 

Fab fragment vs single domain antibody

A useful derivation from the antibody is called an Fab fragment, and it is often used as a blocking agent in immunohistochemistry because the Fc portion responsible for non-specific interactions is eliminated. An Fab fragment is a piece a full IgG antibody that is made of one light chain and one half of a heavy chain. Fab fragments retain their antigen-binding properties and are about 50 kDa in size—roughly one-third the size of a full IgG antibody. These fragments are derived through  cleavage of an IgG antibody by the enzyme papain. Interestingly, it was through this exact method that the structure of the antibody was elucidated in part by Rodney Porter in 1959 (link to classes of antibodies). Another major asset of Fab fragments is that they are able to penetrate tissues easier due to their smaller size. However, they are still quite large compared to single domain antibodies, which are defined as single heavy chain antibodies that are capable of antigen recognition. Single domain antibodies are naturally produced by the immune systems of sharks and camels, and these special antibodies are typically only 12-15 kDa, or one-tenth the size of a full IgG antibody.

Single domain antibody production for biotechnology

The small size of single domain antibodies turns out to be very useful for the biotech industry. One extremely beneficial property is the thermal and chemical stability due to the absence of the crosslinking disulfide bonds that link the polypeptide chains of full size antibodies together. The small size of the single domain antibodies is useful in applications where the antibody needs to penetrate into tissue or to be rapidly cleared from the system. Also, the single domain antibodies derived from sharks are longer and floppier than regular full-size antibodies, which is useful for reaching antigen epitopes otherwise obscured by steric hindrance.

Single domain antibodies are becoming an attractive option in the development of immune-based biosensors or immunodiagnostics. These sensors rely on the interaction between antibodies and antigens to detect and diagnose disease, infection, or allergic reactions. Sometimes it is more advantageous to use a single domain antibody rather than a full size immunoglobulin. One reason for this could be that the single domain antibody is easier to attach to the sensor surface because of its small size or availability of reactive chemical groups. Another reason could simply be that the small size is more compatible with the geometry of the sensor and could result in a lower limit of detection and greater sensitivity. 

Single domain antibodies can be harvested from camels or sharks who have been intentionally and repeatedly exposed to the target antigen. However, another more humane and in many ways easier method, that is more similar to monoclonal antibody production, involves cloning of the variable domain antibodies from immunized animals and selecting targets from a phage library. Another effective method is the production of recombinant single domain antibodies in E.coli hosts. The recombinant technique allows for the creation of single domain antibodies fused with a fluorescent tag or the creation of linked antibodies with two antigen recognition sites. From there, the door opens to a variety of creative fusion antibodies, especially when a custom antibody can be engineered from a tandem single domain antibody joined to a custom Fc region. Suddenly, anything seems possible.

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