Discovery of Nanobodies against Tetramethylenedisulfotetramine by Integrating Next-Generation Sequencing and Moderate Random Mutagenesis

Tetramethylenedisulfotetramine (TETS) is a highly neurotoxic rodenticide and a potential terror agent to national security that can cause death within hours. Therefore, antibody-based guidance for timely determination and treatment is urgently demanded. Discovery of antibody against TETS as a hapten molecule remains a highly challenging, since immunized libraries are often recognized chemical linker or carrier epitopes of hapten-protein conjugates, resulting in an exceedingly low frequency of hapten-specific B cells. Here, we proposed a novel integrated strategy combining next-generation sequencing (NGS) and in vitro random mutagenesis, first successfully discovered the TETS-specific nanobodies (Nbs) within 16 days. The workflow began with the antisera evaluation by identification of IgG subclasses, followed by the enrichment of TETS-specific B cells using a fluorescence-activated cell sorter (FACS). Subsequently, these enriched cells were subjected to NGS to discover TETS-specific Nbs. To improve the affinity of Nbs, an error-prone PCR-based random mutagenesis step with an optimized moderate mutation rate was introduced. Through this strategy, we successfully identified the TETS-specific Nb A104, exhibiting a dissociation constant (KD) of 14.9 nM. Sequence analysis revealed distinct amino acid preferences across both the FR and the CDR of Nbs. Notably, inappropriate amino acid substitutions would compromise the affinity of Nbs. Finally, a chemiluminescent ELISA was developed based on the A104, achieving limits of detection (LODs) of 43 μg/L, 64 μg/L, and 101 μg/L for TETS in grain, human plasma, and human urine, respectively. The results demonstrated that this strategy was a feasible, efficient, and robust platform for the discovery of Nbs against haptens.