*2.2. Instruments*

A NanoDrop 2000C spectrophotometer (Thermo, Waltham, MA, USA) was used to measure absorbance in the ultraviolet–visible spectrum. Centrifugation of phages and bacteria was performed using SORVALL LYNX 4000 centrifuges (Thermo, Waltham, MA, USA). Biologic LP (Bio-Rad, Hercules, CA, USA) was used to purify VHH antibodies. The absorbance values in ELISA were measured using a Multiskan MK3 microplate reader (Thermo Scientific, Waltham, MA, USA).

#### *2.3. Construction of a Phage-Displayed Nanobody Library*

The procedure for constructing the nanobody library by the phage display technology was similar to that previously published [24]. In practice, a three-year-old Bactrian camel was immunized with 500 μL of MC-LR-KLH(1.0 mg/mL) emulsified with Freund's adjuvant at a ratio of 1:1 (*v/v*) every two weeks for a total of 6 times. Particularly, the first time of immunization was performed with complete adjuvant, and the boost immunizations with incomplete adjuvant. One week after the sixth immunization, 100 mL of fresh peripheral blood was collected, and lymphocytes were isolated, followed by RNA extraction. After the synthesis of cDNA by RT-PCR, the VHH genes were amplified by two-step nested PCR using the primers shown in Table 1. The VH −CH1 −CH2 and VHH −CH2 regions were firstly amplified using the primers CALL001and CALL002. Next, the primers Fr4-*Sfi*I and Fr1-*Sfi*I were used to amplify the VHH genes. The VHH genes and the pComb3XSS vector were digested using the restriction endonuclease *Sfi*I, ligated, and transfected into competent *E. coli* ER2738 cells. All transformants were collected by scraping from LB–agar plates containing ampicillin and tetracycline. After infection with helper phage M13K07, a phage-displayed Nb library was constructed. The capacity and diversity of the constructed library were identified by sequence analysis of 20 randomly selected clones.

**Table 1.** Primers' sequences used to amplify the VHH genes (W = A or T, S = G or C, M=A or C, R = G or A).


#### *2.4. Biopanning and Identification of Nanobody Clones for NOD-R*

Four rounds of biopanning in 96-well microtiter plates were performed, as described in references to select Nb clones for NOD-R [25]. In practice, the antigen NOD-R-OVA was coated at decreasing concentrations (10, 2.5, 0.5, 0.1 μg/mL) in four rounds of biopanning at 4 °C with overnight incubation. Meanwhile, 1 mg/mL of OVA and BSA was coated in the parallel wells under the same condition (4 °C, overnight). After shaking out the excess coating solution, a 1% gelatin solution was used to block the coated wells at 37 °C for 2 h. In each round of biopanning, 100 μL of phage library (about 1 × 1011 pfu/mL) was introduced into OVA-wells and BSA-wells in turn, for 1 h at 37 °C, to pre-absorb the non-specific phages; then, the supernatant was transferred to NOD-R-OVA-coated wells and incubated for another 1 h at 37 °C. After washing with PBST for six times, elution was conducted by incubating with decreasing concentrations of NOD-R (2, 0.5, 0.1 μg/mL) and 10 mg/mL of trypsin under gentle shaking at 37 °C for 1 h in each round. Finally, 10 μL of elution solution was used to test the phage titer, and the remainder was amplified for the next round of biopanning.

After four rounds of biopanning, 20 clones were picked out randomly and induced by 1.0 mmol/L isopropylthio-β-D-galactoside (IPTG) to express Nbs in 96-deep-well plates at 37 °C with shaking at 250 rpm for 16 h. Then, ic-ELISA was used to identify the positive clones. Furthermore, the positive clones were subjected to DNA sequencing.

#### *2.5. Expression and Characteristics of Nanobodies*

The recombinant vector pComb3XSS-vhh with the NOD-R VHH gene was extracted from the positive *E. coli* ER2738 clones and then transfected into competent cells of the host strain *E. coli* BL21(DE3) to express specific Nbs. For scaled-up expression, 10 mL of the overnight culture was added to 1 L of LB medium (with 100 mg/mL ampicillin) and incubated at 37 ◦C with shaking at 250 rpm. When the absorbance of the culture at a wavelength of 600 nm (A600nm) reached 0.6–0.8, 1 mM IPTG was added to induce the expression of the target protein, followed by continuous incubation overnight. Then, the bacteria precipitate was harvested after centrifugation at 12,000 rpm for 15 min. The extraction solution (0.2 M Tris-HCl, pH 8.0, 0.5 mM EDTA, 0.5 M sucrose) was added, and the soluble proteins were isolated via cycles of freezing and thawing and by using the osmotic pressure method. The expressed Nbs were purified using the HisPur Ni-NTA resin, followed by 15% SDS-PAGE and Western blotting identification.

To assess the thermostability of the candidate Nbs, 1 mg/mL of purified Nbs was separately incubated at different temperatures, i.e., 25, 37, 50, 65, 80, 90 ◦C for 10 min, or at 90 ◦C for 10, 20, 30, 40, 50, and 60 min. The organic solvent tolerance of Nbs was evaluated using different concentrations (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 100%) of methanol (MeOH) or acetonitrile. PBS buffer (10 mm PBS, pH = 7.0) was used as the dilution reagen<sup>t</sup> (*v/v*), and Mc-MAb was used as the control.

#### *2.6. Establishment of an Indirect Competitive ELISA Based on the Nanobody N56*

The ic-ELISA for NOD-R was developed under optimized reaction conditions. The concentrations of N56 and coating antigen NOD-R-OVA were optimized by checkboard titration. Different buffers (PB, PBS, PBST, Tris-HCl, HEPES), pH values (6.5, 7.0, 7.5, 8.0), and ionic strengths of PBS (5 mM, 10 mM, 20 mM, 40 mM) were chosen to optimize the conditions based on absorbance values of approximately 1.0 in ELISA at 450 nm wavelength or on the Amax (maximal absorbance)/IC50 ratio. Then, the established ic-ELISA was performed as follows. In practice, 100 μL/well of NOD-R-OVA solution was used to coat a 96-well plate, which was incubated overnight at 4 °C and then blocked with 3% BSA in PBS buffer for 1 h. After five washings with PBST buffer, 50 μL/well of an N56 solution at optimal concentration and 50 μL/well of NOD-R at different concentrations were added into the microwells, which were incubated for 40 min at 37 °C with a slight oscillation. After additional five washings, 100 μL/well of rabbit anti-VHH-HRP polyclonal antibody (1:5000 dilution) was added in the microwells, and incubation was carried out

at 37 °C for 30 min. After 5 more washings, 100 μL/well of TMB peroxidase substrate buffer was added, and the enzyme reaction was stopped after a 10 min incubation by the addition of 50 μL/well of 10% H2SO4 (*v/v*). The absorbance values of the microplates were measured using a microplate reader at 450 nm wavelength. Furthermore, a standard curve was obtained based on the established four-parameter logistic equation utilizing Origin 8.5 (Origin Lab Corp., Northampton, MA, USA). Consequently, the limit of detection (LOD) was defined as the concentration with 10% inhibitory activity and the half-maximal inhibitory concentration (IC50) as the concentration with 50% inhibitory activity. The rate of cross reactivity was calculated according to a previously formula, namely, CR (%) = IC50 (NOD-R, μg/L)/IC50 (other toxins, μg/L) × 100.

#### *2.7. Analysis of Water Samples by Ic-ELISA for NOD-R Detection*

Tap water was from Guangdong Provincial Key Laboratory of Food Quality and safety, College of Food Science, South China Agricultural University (Guangzhou, China), lake water was from NingYin Lake in South China Agricultural University, and river water was from CheBei river. After filtering through a 0.22 μm nitrocellulose membrane, all water samples (5 mL) were spiked with different concentrations of NOD-R standard obtaining concentrations of 0, 0.5, 1, 2 μg/L for assessment of the recovery. Furthermore, the spiked samples were analyzed by the established ic-ELISA. According to the ic-ELISA standard curve, the values of mean recovery, SD, and CV were calculated.

The N56-based ic-ELISA was verified by UPLC–MS/MS operated at the Guangzhou Institute for Food Control, China. The conditions were established according to the literatures [16]: Accucore aQ C18 column (150 mm × 2.1 mm, 2.6 μm, Thermo Scientific, USA) was used for chromatographic separation. The mobile phase A was a MeOH solution containing 0.1% formic acid (*v/v*) and 5 mmol/L ammonium formate, while the mobile phase B was an aqueous solution with the same constituents as the mobile phase A. The gradient elution was 0–0.5 min, 80% B; 0.5–4 min, 80–25% B; 4–7 min, 25–0% B; 7–7.5 min, 0% B; 7.5–8 min, 0–80% B; 8–10 min, 80%B. The flow rate was 0.3 mL/min, and the injection volume of each sample was 5 μL. The temperature of the column oven was set at 40 ◦C. The mass spectra were obtained with the AB TRIPLE QUAD 4500 mass spectrometer under positive ionization mode, with the following parameters' values: curtain gas flow rate, 30 L/min; nebulizer gas flow rate, 50 L/min; auxiliary gas flow rate, 55 L/min; collision gas, 8 L/min; spray voltage, 5.5 kV.
