Precise isolation and structural origin of an ultra-specific nanobody against chemical compound

Highly specific antibodies are the key reagents for developing immunoassays with a low false positive rate for environmental monitoring. Here, we provide evidence that nanobodies have the potential to achieve higher specificity than conventional antibodies and explain why from their structural features. Using sulfadimethoxine (SDM) as a model analyte, we constructed an immune phage display library and precisely isolated an ultra -specific nanobody (H1-17) by a crucial homologous antigen counter selection strategy. H1-17 showed no observable cross-reactivity (CR) with other structural analogs of 41 SDM tested, which has never been achieved by conventional antibodies. The structurally original specificity of H1-17 was illuminated and compared with that of one conventional antibody by homology modeling and site-directed mutagenesis validation. It was found that the noncanonical disulfide bond (C50-C104) of H1-17 helped CDR3 form a tailor-made binding pocket and divide it into two parts to accommodate the common structure of sulfonamides and the characteristic methoxyl group of SDM, respectively. Besides, the mutual-checking hydrogen bonds also played important roles in the specific recognition. Lastly, immunoassays with zero false positive rate were developed to screen SDM in water and milk samples, indicating that nanobodies could be reliable reagents for the accurate detection of chemical compounds.

Automated summary

Highly specific antibodies are essential for accurate immunoassays in environmental monitoring. This study demonstrates that nanobodies possess higher specificity than conventional antibodies due to their unique structural features. A specific nanobody (H1-17) was isolated using an immune phage display library and showed no cross-reactivity with other analogs. The structural specificity of H1-17 was compared to a conventional antibody using modeling and mutagenesis validation, revealing the key role of a noncanonical disulfide bond and mutual-checking hydrogen bonds. Immunoassays with zero false positive rate were developed using nanobodies for precise chemical compound detection.