Parque Lecocq, Intendencia de Montevideo , Montevideo, Uruguay , 12600.
Anal Chem. 2017 Jun 20;89(12):6800-6806. doi: 10.1021/acs.analchem.7b01221. Epub 2017 Jun 2.
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.
由于重链抗体（VHH）的重现性、稳定性和成本效益生产，它们作为免疫分析试剂正成为一个突出的选择。最近，有几篇报道描述了它们在小分子（半抗原）检测中的应用。然而，由于缺乏常规抗体的重轻链界面，VHH 不太适合结合小分子分析物，失败并不罕见。在这里，我们描述了针对蓝藻肝毒素微囊藻 LR 的 VHH 噬菌体展示文库的构建及其使用竞争性淘选和两种新的淘选策略的选择。每种策略的结果都通过体内生物素化纳米抗体的大规模筛选进行了评估。这三种方法选择了不同的不重叠 VHH 子集，允许优化毒素的免疫检测。通过促进最慢 k（离解速率选择）的 VHH 分离，获得了最佳结果。其中，生物素化纳米抗体 A2.3 在 ELISA 中表现出色，回收率高，灵敏度高，IC50=0.28μg/L，检测限远低于毒素的最严格准则。虽然这可能是具体情况，但这些结果强调了探索不同淘选策略以优化抗半抗原纳米抗体选择的重要性。
