Single-Domain Antibodies As Versatile Affinity Reagents for Analytical and Diagnostic Applications

Abstract

With just three CDRs in their variable domains, the antigen-binding site of camelid heavy-chain-only antibodies (HcAbs) has a more limited structural diversity than that of conventional antibodies. Even so, this does not seem to limit their specificity and high affinity as HcAbs against a broad range of structurally diverse antigens have been reported. The recombinant form of their variable domain [nanobody (Nb)] has outstanding properties that make Nbs, not just an alternative option to conventional antibodies, but in many cases, these properties allow them to reach analytical or diagnostic performances that cannot be accomplished with conventional antibodies. These attributes include comprehensive representation of the immune specificity in display libraries, easy adaptation to high-throughput screening, exceptional stability, minimal size, and versatility as affinity building block. Here, we critically reviewed each of these properties and highlight their relevance with regard to recent developments in different fields of immunosensing applications.

Keywords: VHH; haptens; imaging; immunodetection; nanobodies; phage display.

Figure 1

Schematic representation of conventional and heavy-chain-only antibodies (HcAbs). Conventional antibodies, formed by heavy (cyan) and light (blue) chains, are found in all vertebrate, but camelids and cartilaginous fish also have antibodies devoid of light chain, whose antigen-binding site sits exclusively in the heavy chain variable domain. The organization of the heavy chain variable domain of each of these antibodies is shown using three representative structures [PDB IDs: 5WT9 (VH); 5TP3 (VHH); and 2COQ (V-NAR)]. CDR1, CDR2, and CDR3 are depicted in orange, green, and red, respectively, except for V-NAR that lacks CDR2, and the green color is used to denote the HV4 region that sometimes participates in antigen binding. VHHs and V-NARs usually have long CDR3 and non-canonical disulfide bridges (yellow). While the antigen-binding site of conventional antibodies is formed by the combination of the six CDRs of the heavy and light chain, only three and two CDRs are involved in the formation of binding site of camelid and shark HcAb, respectively. Notice that HcAbs do not have the CH1 or an equivalent domain, which forms a strong interaction with the constant domain of the light chain in conventional antibodies.

Figure 2

The typically long CDR3 of VHHs provides flexibility to their epitope recognition. The complex of lysozyme (purple) with two VHHs shows that the long CDR3 may contribute to form a flat paratope (A) or penetrate in concave epitopes (B); based on PDB structures 1OP9 and 1IR8.

Figure 3

Structure and binding characteristics of the VHH domain. The typical organization of the VHH domain is represented using the structure of a VHH to carbazol (PDB 1U0Q). In framework (FR) 2 (dark gray), the amino acids shown in cyan [in this case Phe42, Glu49, Arg50, and Phe52 (IMGT numbering)] represent the hallmark residues that substitute the critical amino acids of VHs that participate in the interaction with the light chain. CDRs 1, 2, and 3 are colored in orange, green, and red, respectively. The loop of FR 3 shown in blue (also known as CDR4) presents significant variability (higher than in VHs of conventional antibodies) and may also interact with the antigen (–43). Frequently, the CDR3 is long and bends over FR 2 shielding its hydrophobic residues and helping to mask Trp118 (cyan–green), which is key for the interaction of VH with the VL chain. The structure of the long CDR3s, much more frequently in camels, may be stabilized by non-canonical disulfide bridge, typically formed between Cys residues of CDR1 and CDR3.

Figure 4

Schematic representation of the construction of heavy-chain-only antibody and conventional antibody libraries. The main steps in the generation of nanobody (Nb) and conventional antibody (scFv) libraries are outlined on the left and right, respectively. The key difference occurs during PCR amplification of the variable gene regions that is done separately for the VH and VL genes. Upon assembly of the VL spacer–VH cassette that encodes the scFv, the VL and VH genes are shuffled, and the original pairing of the light and heavy variable domains occurs only at a very low frequency.

Figure 5

Schematic and surface representation of three VHH structures in complex with haptens (CPK). In the surface representation, CDR1, CDR2, and CDR3 are shown in yellow, orange, and red, respectively. Upon crystallization, two molecules of Reactive Red 6 reacted through the triazine group to form a dimer of the dye.

Figure 6

Sortase-mediated conjugation of nanobodies (Nbs). The procedure is schematized for the Staphylococcus aureus sortase. The Nb is produced with the C-terminal tag (LPXTG) and is incubated with the recombinant sortase that cleaves the Thr–Gly bond forming an acyl intermediate. The nucleophilic attack of the poly-Gly stretch of the partner protein, fluorophore of chelating compound breaks the thioester forming conjugate. The hexa His tag in the Nb and sortase is used to separate the unreacted Nb and the sortase from the final reaction.