Application of Single-Domain Antibodies ("Nanobodies") to Laboratory Diagnosis

Abstract

Antibodies have proven to be central in the development of diagnostic methods over decades, moving from polyclonal antibodies to the milestone development of monoclonal antibodies. Although monoclonal antibodies play a valuable role in diagnosis, their production is technically demanding and can be expensive. The large size of monoclonal antibodies (150 kDa) makes their re-engineering using recombinant methods a challenge. Single-domain antibodies, such as "nanobodies," are a relatively new class of diagnostic probes that originated serendipitously during the assay of camel serum. The immune system of the camelid family (camels, llamas, and alpacas) has evolved uniquely to produce heavy-chain antibodies that contain a single monomeric variable antibody domain in a smaller functional unit of 12-15 kDa. Interestingly, the same biological phenomenon is observed in sharks. Since a single-domain antibody molecule is smaller than a conventional mammalian antibody, recombinant engineering and protein expression in vitro using bacterial production systems are much simpler. The entire gene encoding such an antibody can be cloned and expressed in vitro. Single-domain antibodies are very stable and heat-resistant, and hence do not require cold storage, especially when incorporated into a diagnostic kit. Their simple genetic structure allows easy re-engineering of the protein to introduce new antigen-binding characteristics or attach labels. Here, we review the applications of single-domain antibodies in laboratory diagnosis and discuss the future potential in this area.

Keywords: Laboratory diagnosis; Monoclonal antibodies; Nanobodies; Single-domain antibodies.

Fig. 1

Comparison of the canonical Ig structure with that of heavy-chain-only antibodies. Abbreviations: V_HH, variable heavy chain-only antibodies; C_H, constant region of heavy chain; V_L, variable region of light chain; V_NAR, variable domain of immunoglobulin new antigen receptor; Ig-NAR, immunoglobulin new antigen receptor; ScF_v, single chain variable fragment.

Fig. 2

Architectures of homodimeric heavy‐chain antibodies (bottom left) with the Ag‐binding single‐domain VHH enlarged on top and classical heterotetrameric antibodies (bottom right) with the Ag‐binding variable fragments (comprising VH and VL domains) enlarged on top. Adapted from Muyldermans (2021, ref 33). (Permission under Creative Commons Attribution license) (https://doi.org/10.1111/febs.15515). Abbreviations: VHH, variable heavy chain-only antibodies; CH, constant region of heavy chain; VL, variable region of light chain; CDR, complementarity-determining region.