Journal
ANALYTICAL CHEMISTRY
Volume 89, Issue 12, Pages 6800-6806Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.7b01221
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Funding
- Agencia Nacional de Investigacion e Innovacion, Uruguay [CSIC 149 UdelaR, FMV 104085 ANII]
- PEDECIBA
- ANII
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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) (off-rate selection). Among these, the biotinylated nanobody A2.3 performed in ELISA with excellent recovery and high sensitivity, IC50 = 0.28 mu g/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.
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