*Article* **Preparation of Specific Nanobodies and Their Application in the Rapid Detection of Nodularin-R in Water Samples**

**Jinyi Yang 1,†, Rui Si 1,†, Guangpei Wu 1, Yu Wang 2, Ruyu Fang 1, Fei Liu 1, Feng Wang 1, Hongtao Lei 1, Yudong Shen 1, Qi Zhang 3 and Hong Wang 1,\***


**Abstract:** Nanobodies have several advantages, including grea<sup>t</sup> stability, sensibility, and ease of production; therefore, they have become important tools in immunoassays for chemical contaminants. In this manuscript, nanobodies for the detection of the toxin Nodularin-r (NOD-R), a secondary metabolite of cyanobacteria that could cause a safety risk for drinks and food for its strong hepatotoxicity, were for the first time selected from an immunized Bactrian camel VHH phage display library. Then, a sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for NOD-R, based on the nanobody N56 with grea<sup>t</sup> thermostability and organic solvent tolerance, was established under optimized conditions. The results showed that the limit of detection for NOD-R was 0.67 μg/L, and the average spike recovery rate was between 84.0 and 118.3%. Moreover, the ic-ELISA method was validated with spiked water sample and confirmed by UPLC–MS/MS, which indicated that the ic-ELISA established in this work is a reproducible detection assay for nodularin residues in water samples.

**Keywords:** nodularin; nanobody; detection; ic-ELISA
