**1. Introduction**

Nodularins (NODs) are harmful cyanobacteria toxins produced by *Nodularia spumigena*, especially when blooms brake out in lakes or rivers [1]. So far, different forms of NOD variants have been found. Among these variants, NOD-R is the most abundant and the only one commercially available [2]. NOD-R is a characteristic cyclic peptide composed of five amino acids (-D-MeAsp–L-Y–Adda–D-Glu–Mdhb), in which the Adda structural region ((2S,3S,8S,9S)-3-amino- 9-methoxy-2,6,8-trimethyl-10-phenyl deca-4,6-dienoic acid) is also found in microcystins (MCs), another group of cyanobacteria toxins composed of heptapeptides. Although NOD-R is similar to MCs in structure (Figure 1), it penetrates more easily into hepatocytes than MCs and exhibits stronger acute toxicity [3]. It has been found that NOD-R can inhibit the catalytic subunit of seine/threonine-specific protein phosphatases (PPs) 1 and 2A (PP1 and PP2A) and lead to severe hepatotoxicity [4–6]. Meanwhile, NOD-R cannot be completely eliminated via routine water treatment processes because of its water solubility and heat stability; therefore, contaminated drinking water and polluted recreational water might increase the risk of human exposure through intake or skin contact. A guideline value specific for NOD-R in drinking water and recreational water has not ye<sup>t</sup> been established by the World Health Organization (WHO); however,

**Citation:** Yang, J.; Si, R.; Wu, G.; Wang, Y.; Fang, R.; Liu, F.; Wang, F.; Lei, H.; Shen, Y.; Zhang, Q.; et al. Preparation of Specific Nanobodies and Their Application in the Rapid Detection of Nodularin-R in Water Samples. *Foods* **2021**, *10*, 2758. https://doi.org/10.3390/foods10112758

Academic Editor: Juliana Moura Luna

Received: 28 September 2021 Accepted: 5 November 2021 Published: 10 November 2021

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many countries commonly take 1.0 μg/L, the maximum residue limit (MRL) for MC-LR, as a reference [7]. Rapid and sensitive detection methods for NOD-R are required to continuously monitor the level of NOD-R in the environment as well as in drinking water or foods [8].

**Figure 1.** Structure of NOD-R.

Recently, several methods for the detection of NOD-R residues have been developed, including high-performance liquid chromatography (HPLC) [9], mass spectrometry (MS) [10], liquid chromatography–mass spectrometry (LC–MS) [11], as well as some rapid detection methods such as immunoassays based on specific antibodies or biosensor assays based on aptamers [12]. Compared with instrumental assay methods, immunoassay technology has the obvious advantages of rapidness, simplicity, and lower cost [13]. For example, only several cents to dollars are commonly spent on each sample when using common ELISA kits, whereas at least tens of dollars are necessary when using UPLC–MS/MS. In addition, antibodies are routinely stable in conformation and common in practical utilization; thus, immunodetection of NOD-R has attracted attention. Both polyclonal antibodies (PAbs) and monoclonal antibodies (MAbs) against NOD-R were obtained, and several detection methods, e.g., indirect competitive enzymatic-linked immunoassay (ic-ELISA), fluorescence polarization immunoassay (FPIA), and optical surface plasmon resonance (SPR) immunosensor detection, were developed, obtaining a limitation of detection (LOD) for NOD-R ranging from 0.051 μg/L to 0.95 μg/L [14,15].

Actually, except for a few examples, most of the antibodies reported above are broadspecificity antibodies originally prepared for MC-LR, but they can recognize also other MCs as well as NOD-R simultaneously. Previously, with MC-LR-KLH as a common immunogen, multi-residue-recognizing PAbs [16] and MAbs [data not shown] were generated in our lab with IC50of 0.29 μg/L and 18 μg/L for NOD-R, respectively.

Obviously, antibodies, as molecular recognition elements, are the core components of immunoassays. In recent years, single-domain antibodies derived from heavy-chain-only antibodies, namely VHH, have been found in camelids, including camels and llamas, as well as VNAR in cartilaginous fishes, including sharks [17]. These antibodies, also called nanobodies (Nbs), have attracted attention for their application in in immunoassays and have shown superiority over traditional PAbs and MAbs because of their small size (−15 kDa) and high solubility and stability [18]. In addition, Nbs can be efficiently generated by the phage display method and can be genetically encoded and expressed in *Escherichia coli* host cells with high yields [19,20]. Until now, several specific Nbs for some chemical contaminants such as parathion [21], aflatoxin B1 [22] and triazophos [23] have been successfully prepared, and related immunoassays have been established.

In this study, an immunized Bactrian camel phage display library was constructed, and then Nbs against NOD-R were selected and prepared. Furthermore, based on optimized reaction conditions, an ic-ELISA for NOD-R in water samples was established. Finally, the performance of the ic-ELISA was measured and validated by UPLC–MS/MS.
