Comparison of Three Antihapten VHH Selection Strategies for the Development of Highly Sensitive Immunoassays for Microcystins

Abstract

Owing to their reproducibility, stability, and cost-effective production, the recombinant variable domains of heavy-chain-only antibodies (VHHs) are becoming a salient option as immunoassay reagents. Recently, there have been several reports describing their application to the detection of small molecules (haptens). However, lacking the heavy-light chain interface of conventional antibodies, VHHs are not particularly apt to bind small analytes and failures are not uncommon. Here we describe the construction of a VHH phage display library against the cyanobacterial hepatotoxin microcystin LR and its selection using competitive panning and two novel panning strategies. The outcome of each strategy was evaluated by a large-scale screening using in vivo biotinylated nanobodies. The three methods selected for different nonoverlapping subsets of VHHs, allowing one to optimize the immunodetection of the toxin. The best results were obtained by promoting the isolation of VHHs with the slowest k (off-rate selection). Among these, the biotinylated nanobody A2.3 performed in ELISA with excellent recovery and high sensitivity, IC50 = 0.28 μg/L, with a limit of detection that is well below the most rigorous guidelines for the toxin. While it may be case-specific, these results highlight the importance of exploring different panning strategies to optimize the selection of antihapten nanobodies.

Selective Engagement of FcγRIV by a M2e-Specific Single Domain Antibody Construct Protects Against Influenza A Virus Infection

“…This flexible nature of the M2e peptide could explain why it appears to be difficult to isolate a VHH that recognizes the N-terminal part of M2e. Moreover, due to the absence of the light chain, VHHs typically bind to concave epitopes rather than to convex or protruding peptide termini (59,60). Nevertheless a few VHHs directed against linear peptides have been described (61,62).…”

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

“…Recently, using the cyanotoxin microcystin-LR (995 Da) as a model hapten and a high-throughput screening, we compared this way of selection with two additional strategies. We found that selecting for Nbs with the slowest k off for the immobilized hapten allowed us to attain a detection limit of 50 pg/mL, which was 10-fold better that the one obtained using the Nbs isolated by competitive selection ( 90 ). While the usefulness of this approach still needs to be demonstrated for other haptens, alternative methods for the selection of anti-hapten Nbs are needed, because, conversely to what happens with other antigens, failures in the generation of Nbs that recognize small molecules in solution are common ( 89 ).…”

Current Approaches and Future Perspectives for Nanobodies in Stroke Diagnostic and Therapy

“…Nbs consists of four conserved framework regions and three antigen-binding loops, known as the complementarity determining regions (CDRs). The particularly long CDR3 rends the paratope its convex shape, building protrusions that can reach cryptic epitopes often not accessible to cAbs [27,28,31,32].…”