**3. Results and Discussion**

#### *3.1. Selection of Anti-NOD-R Nanobodies from the Phage Display Library*

Considering that MC-LR-KLH previously prepared in our lab as a common immunogen can induce excellent immunization and has been successfully used to generate specific PAbs and MAbs which could recognize NOD-R, in this work, this molecule was chosen as the immunogen for the camel [16]. After six inoculations, the serum titer was up to 1:64,000, and inhibition of NOD-R was higher than 80%. By two-step nested PCR, VHH genes were amplified, and a bacterial library with the capacity of 8.0 × 10<sup>7</sup> cfu/mL was constructed. Then, rescued by helper phage M13K07, the titer of phage-displayed Nbs library was about 1.0 × 10<sup>12</sup> pfu/mL. Furthermore, after four rounds of biopanning, three clones, namely, N4, N56, and N159, were identified as positive ones and exhibited recognition activity for NOD-R (Figure 2). The inhibition rates of these three clones identified by ic-ELISA were all higher than 80%. By sequence alignment analysis, these three clones, composed of 131 amino acid residues, were confirmed to possess the characteristics of Nbs and to have high similarity, except for a few residues in frame regions (FRs) (Figure 3). Therefore, they were considered as the same category of Nbs, and N56 was chosen for further analysis.

**Figure 2.** Positive clones identified by ic-ELISA.

**Figure 3.** Sequence alignments of the three positive clones.

#### *3.2. Preparation and Characterization Analysis of the Nanobody N56*

To better investigate the characteristics of the nanobody N56, it was expressed in 1L of LB medium and purified using the HisPur Ni-NTA resin. The purified N56 were identified by 15% SDS-PAGE (Figure 4a) and Western blotting (Figure 4b). The results showed that the molecular weight of N56 is around 17 kDa, with purity of more than 90%, and the yield was about 4.5 mg/L. It has been observed that Nbs can be highly expressed in *E. coli*, yielding from more than 10 mg to tens of milligrams, thanks to their low molecular weight and good solubility [26]. However, in our work, N56 showed a relatively low yield, not higher than 5 mg/L. We thought it might be related to the ratio of hydrophobic amino acids (22%) in its composition, but after comparison with other Nbs, e.g., Nb-T3-15 (for tetrabromobisphenol A) [27] or Nb-3F9 (for tenuazonic acid) [28], which gave yields up to 30 mg/L and just less then 1 mg/L under the same expression conditions, respectively (Figure S1), this hypothesis was discarded because Nb-T3-15 has a higher hydrophobic amino acids ratio than Nb-3F9. Therefore, maybe the location of the hydrophobic amino acids in the 3D structure of Nbs plays a more important influence on the solubility of nanobodies then their expression level in *E. coli* host cells.

Furthermore, the thermostability and organic solvent tolerance of N56 was evaluated with Mc-MAb as a control. After incubation at different temperatures, from 4 °C to 90 °C, for 10 min, N56 could maintain a binding activity higher than 90%, whereas Mc-MAb lost its activity very quickly. Especially at 25 °C (RT) or 37 °C, two commonly used temperatures during practical detection, N56 exhibited full activity, while Mc-MAb lost nearly 30% of its activity (Figure 5a). Even more, N56 was stable at the high temperature of 90 °C for 1 h, retaining 80% of its activity (Figure 5b). In addition, the tolerance of N56 for the organic solvents methanol (MeOH) and acetonitrile was determined. As shown in Figure 5c,d, with increasing concentration of MeOH or acetonitrile, both N56 and Mc-MAb began to lose their activities; however, N56 as a whole had superior performance than Mc-Mab, in particular, it showed better tolerance to acetonitrile. Generally, the disulfide bonds formed by cysteine

residues present in Nbs are thought to contribute to its excellent stability. According to the sequence of N56, four cysteine residues (cys22, cys37, cys100, cys103) might form two disulfide bonds and maintain the stability of N56 structure in harsh environments, at either high temperature or in the presence of organic solvents (Figure 3). This phenomenon was also observed for other reported Nbs for low-molecular-weight chemical contaminants [29].

**Figure 4.** Identification of N56 by SDS-PAGE and Western blotting. (**a**) SDS-PAGE: lane 1, marker; lane 2, purified N56. (**b**) Western blotting: lane 3, marker; lane 4, purified N56.

**Figure 5.** Thermostability and organic solvent tolerance of N56 and Mc-MAb by ic-ELISA: (**a**) N56 and Mc-MAb (1 mg/mL) were incubated at 25, 37, 50, 65, 80, and 90 °C for 10 min; (**b**) N56 and Mc-MAb (1 mg/mL) were incubated at 90 °C for 10, 20, 30, 40, 50, and 60 min; a series concentrations (10%, 20%, 30%, 40%, 50%, 60%, 70% and 80%) of (**c**) methanol (MeOH) and (**d**) acetonitrile were used to dilute (*v/v*) N56 and Mc-Mab (*n* = 3).

#### *3.3. Establishment of an Ic-ELISA Based on the Nanobody N56*

In order to develop an ic-ELISA for NOD-R with N56 as the recognition element, various reaction conditions, such as concentrations of nanobody and coating antigen, buffers, pH, and ionic strength, were optimized (Figure 6a–c). After the checkboard titration test, the optimal concentrations of N56 and NOD-R-OVA were determined to be 0.125 μg/mL and 2 μg/mL, respectively. The other conditions were further optimized with the highest Amax/IC50 as the selection criterion [28]. Consequently, 10 mM PBS buffer at pH 7.0, at a relative lower ionic strength under a neutral reactive environment, was confirmed as the most suitable buffer for the establishment of ic-ELISA. Finally, an ic-ELISA standard curve based on N56 for NOD-R was established (Figure 7). The IC50 value was 9.94 μg/L, and the linear range of detection was 1.74–56.66 μg/L with a limit of detection (LOD) of 0.67 μg/L, which was lower than that of Mc-MAb for NOD-R (1.25 μg/L). This LOD value can also satisfy the detection requirement of 1 μg/L in drinking water.

**Figure 6.** Optimization of assay conditions for the establishment of ic-ELISA: (**a**) types of buffers (PB, PBS, PBST, Tris-HCl, and HEPES), (**b**) pH value (6.5, 7.0, 7.5, and 8.0), and (**c**) ionic strength (5 mM, 10 mM, 20 mM, and 40 mM PBS). The conditions were evaluated by the Amax (maximal absorbance)/IC50 ratio.

**Figure 7.** Standard curves of ic-ELISA for NOD-R based on purified N56 under the optimized reaction conditions. The standard curves were normalized by expressing the experimental absorbance values (B) as B/B0, where B0 is the absorbance value at zero analyte concentration (*n* = 3).

Besides, the N56-based assay showed relatively significant cross reactivity for MCs. This might result from the common special Adda structural regions ((2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl deca-4,6-dienoic acid). In this work, NOD-R-OVA was used as the coating antigen, and NOD-R at decreasing concentration as the competitive drug to increase the specificity of the eluted phages; yet, the generation of VHHs against the Adda structure in camel by the immunogen MC-LR-KLH was possible, and these antibodies would consequently recognize NOD-R as well as MCs. In addition, among the MCs capable of binding N56, MC-LR, -YR, -RR, -WR exhibited higher cross reactivity than other MCs (Table 2). This is because these four MCs have an arginine residue as NOD-R besides the Adda region. From another perspective, high cross reactivity might be helpful in the detection of multiple residues of cyanobacteria toxins. It was reported that VHHs for MC-LR prepared by Pírez-Schirmer et al. [30] or Xu et al. [31] could recognize other MCs, but there are no data about their cross reactivity with NOD-R. In this study, we, for the first time, obtained N56 that is able to detect NOD-R and MCs, i.e. two groups of cyanobacteria toxins.


**Table 2.** CRs of N56 for MCs analogues (*n* = 3).
