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.

Nanobodies in the limelight: Multifunctional tools in the fight against viruses

“…Many characteristics of nanobodies render them suitable components in such tests, providing opportunities to utilize them for the detection of other viral antigens in human or animal samples. A comprehensive review on nanobodies for laboratory diagnostic applications can be found here [77].…”

Tripartite split-GFP assay to identify selective intracellular nanobody that suppresses GTPase RHOA subfamily downstream signaling

“…Recombinant antibody technology has so far provided genetically encoded high affinity reagents for common immunological assays used in fundamental and applied research (1)(2)(3). Peculiar applications such as tracing or modulating intracellular proteins in living cells are ascribed to special antibody fragments or alternative scaffolds that rely on single chain binding domains (4).…”

A Nanobody‐on‐Quantum Dot Displacement Assay for Rapid and Sensitive Quantification of the Epidermal Growth Factor Receptor (EGFR)

“…[5][6][7][8][9][10][11] Their much smaller size compared to intact IgG antibodies ( � 150 kDa) combined with simple expression, high stability, high solubility, and many chemical functionalization strategies provide NBs with several advantages for biosensing and bioimaging. [6,9,10] To…”

“…[29] QD FRET acceptor conjugates were prepared depending on the different tags on NB2: i) NB2-H-QD625-CL4: QD625-CL4 were mixed in a 1 : 20 molar ratio with NB2-H, which resulted in efficient metal-affinity mediated self-assembly to the Zn-rich QD surface after � 30 min. [39] Considering the 2.5 nm diameter of NBs, [9] 20 NB2 should take approximately 50 nm 2 of space on the � 265 nm 2 surface of the QD (A = 4πr 2 with r = 4.6 nm), which can be considered as low enough to avoid steric hindrance and allow for efficient self-assembly. ii) NB2-B-QD705-sAv: NB2-B was attached to QD705-sAv via the strong biotin-sAv interaction, [40] also by simple mixing for � 30 min in a 20 : 1 molar ratio.…”

“…[4] Nanobodies (NBs, or variable domains of the heavy chain of heavy-chain-only antibodies-VHH) are genetically engineered small ( � 15 kDa with a cylindrical shape of � 2.5 nm diameter and � 4 nm height) antibodies that have found frequent application in molecular imaging, clinical diagnostics, and disease therapy. [5][6][7][8][9][10][11] Their much smaller size compared to intact IgG antibodies ( � 150 kDa) combined with simple expression, high stability, high solubility, and many chemical functionalization strategies provide NBs with several advantages for biosensing and bioimaging. [6,9,10] To…”