Single-domain antibodies: Rugged recognition elements for tomorrow's biosensors

Camelids and sharks possess a class of unconventional immunoglobulins consisting of heavy-chain homodimers in which antigen binding is mediated through a single variable domain. When expressed recombinantly, these variable domains, referred to as "single-domain antibodies" (sdAb), comprise the smallest known antigen-binding fragments. Both camelid- and shark-derived sdAb have been incorporated into multivalent and/or bivalent constructs. These reagents have the advantage that through avidity, they can have a higher apparent affinity than a monomeric sdAb. Strategies including DNA shuffling, random PCR, and mutational hotspot randomization have been used to generate and select sdAb variants with improved protein expression, stability, and affinity. The ability to design or tailor sdAb to meet an application's particular needs is a strong advantage that favors their future adoption in detection schemes. Furthermore, the biophysical properties of sdAb make them attractive recognition elements in biosensors. However, there are several limitations to the use of sdAb in biosensors. Although sdAb can be rapidly developed from naïve libraries, the highest affinity reagents have been derived from immunized animals. Using animals adds both time and expense to reagent development. Finally, protein engineering offers the potential to create fusions of sdAb with protein domains to impart specific functionalities, such as the construction of monomolecular protein lattices that can be used to pattern surfaces, this cannot currently be accomplished with conventional IgG. Directed evolution enables the selection of sdAb variants with desirable traits such as improved thermodynamic stability and proteolytic stability. These features combined make sdAb ideal reagents for use in the development of tomorrow's rugged biosensors.