*4.1. Materials*

Except where noted otherwise, deionized water (Nanopure II, Thermo Scientific, Waltham, MA, USA) was used in the preparation of all reagents. The primary analytical standard of CTV was produced by Hayashi Pure Chemical Ind., Ltd. (Osaka, Japan). For spiking of rice samples, a standard material containing both CTV and iso-CTV prepared at the USDA-NCAUR (Peoria, IL, USA) was used [21]. Acetonitrile and methanol were HPLC grade and were purchased from Fisher Scientific (Hampton, NH, USA), as was polyvinyl alcohol (PVA). 1-1carbonyldiimidazole (CDI), 4,6-dimethyl-2-oxo-2H-pyran-5-carboxylic acid (IDHA), 4,6-dimethylα-pyrone (DMP), and threose were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were reagen<sup>t</sup> grade or better and purchased from major suppliers.

#### *4.2. Liquid Chromatography-Photodiode Array-Mass Spectrometry (LC-PDA-MS) of CTV Stock Solutions*

Two concentrated stock solutions containing predominantly CTV were prepared and analyzed by liquid chromatography-photodiode array-mass spectrometry (LC-PDA-MS) to determine relative purity. The first of the materials was prepared commercially and was used as the primary analytical standard. The second was prepared from material isolated by the late Robert Stubblefield at USDA-NCAUR (Peoria, IL, USA) and had been stored for many years at −20 ◦C. The latter was used to study the potential cross-reactivity of iso-CTV and was used to spike rice samples. A stock solution of the analytical standard was prepared in acetonitrile and a portion was diluted in methanol to a concentration of approximately 10 μg/mL. The ultraviolet–visible (UV–Vis) absorption spectrum was obtained using an Evolution 201 UV-Visible spectrometer (Thermo Scientific, Waltham, MA, USA). The absorbance at 383 nm was used to determine the concentration of total CTV and iso-CTV based upon the molar extinction coe fficient of 44,000 [20]. A stock solution of the material used for spiking rice was prepared at nominally 0.6 mg/mL in acetone. The concentrations of CTV and iso-CTV in the

spiking material were determined following LC-PDA-MS and were based upon the aforementioned analytical standard.

The LC instrument was a Dionex Ultimate 3000 system (ThermoFisher, Waltham, MA, USA). The column was a Kinetex 1.7 μ XB-C18, 100 Å, 100 × 3.0 mm, with a C18 Security Guard Ultra column (Phenomenex, Torrance, CA, USA) maintained at 30 ◦C. The mobile phase consisted of (A) acetonitrile, and (B) a mixture of 8% acetonitrile in water containing 0.25% acetic acid, with pH adjusted to 4 with ammonium hydroxide. The gradient began as 80% B for 4 min, then 65% B for 9 min. At 13 min the initial condition of 80% B was reinstated and the column was allowed to equilibrate for 7 min before injecting the next sample. For injection, the standards were diluted to 2 μg/mL. Injection volume was 20 μL. Flow rate was 0.7 mL/min, of which approximately 0.4 mL/min was directed to the PDA detector and 0.3 mL/min to the MS. The MS was a model QDa single quadrupole instrument (Waters Corp., Milford, MA, USA) operated in positive ion mode under the following parameters. Single ion monitoring at *m*/*z* = 403.2, cone voltage 6.0 V, probe temperature 425 ◦C, detector gain 1, capillary voltage 1.5 kV, sampling frequency 8 Hz. Under these conditions, CTV eluted at 7.8 min. At 8.5 min a second peak, with the same absorption spectrum as CTV eluted. In previous literature this peak has been termed iso-CTV, a convention also used here. Because no standards for iso-CTV were available the concentration of iso-CTV was calculated relative to that of the CTV analytical standard under the assumption that the ionization efficiencies for the two isomers were the same.

#### *4.3. Preparation of CTV–Protein Conjugates*

Purified CTV was linked to two proteins. One was used as a soluble antigen for immunizing mice and the other was used as an immobilized antigen in indirect ELISAs. The immobilized antigen, CTV-OVA2, was prepared as follows. All reactions were performed under conditions of reduced ambient lighting. CTV (5 mg) was dissolved in 0.25 mL acetone and 50 mg CDI was added and held at ambient temperature for 1 h. Twenty μL of water was added followed by 3.4 mL of OVA solution (50 mg OVA in 0.1 M NaHCO3 buffer, pH 8.6). The mixture was shielded from light and stirred for 28 h at 4 ◦C. The product, CTV-OVA2, was dialyzed extensively against NaHCO3 and phosphate buffers, using a 14 kDa membrane and culminating with 0.1 M PBS. The conjugate was diluted to a concentration of 2 mg/mL, freeze-dried, and stored at −80 ◦C until use. The immunogen, CTV-BSA3, was prepared in a similar fashion. However, instead of OVA the protein was BSA with a short ethylene diamine (EDA) linker attached. The preparation of EDA-BSA has been reported elsewhere [35].

#### *4.4. Immunization of Animals and Isolation of mAb-Producing Clones*

Immunization of mice, splenocyte fusions, cloning operations and antibody production were conducted at Envigo (Madison, WI, USA). Screening of sera and of fusion products were conducted at USDA-NCAUR (Peoria, IL, USA). All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Envigo. Work was performed according to protocols 421-09a – Polyclonal Antibody Production and Hybridoma Development (approved 14 December 2018) and 638-18 Monoclonal Antibody Production (approved 14 December 2018). These protocols were developed in accordance with guidelines established by the U.S. National Institutes of Health- Office of Laboratory Animal Welfare. Ten female Balb/C mice were given a primary immunization of 100 μg CTV-BSA3 using the same procedures as described previously for production of paxilline antibodies [36]. Mouse antisera and hybridoma culture supernatant solutions were tested by competitive indirect ELISA (CI-ELISA). In initial experiments 0.1 mL of CTV-OVA2 (10 μg/mL in 0.05 M sodium phosphate buffer, pH 7.2), was incubated in wells of polystyrene microtiter plates overnight at 4 ◦C. The plates were washed twice with 0.32 mL Tween-PBS (0.02% *v*/*v* Tween-20 in 0.01 M PBS, pH 7.2) and blocked with 0.32 mL of PVA-PBS (1% *w*/*v* PVA in 0.01 M PBS) for 2 h at ambient temperature. Test solutions were prepared by mixing equal volumes of toxin standard solution (or control solution) and antiserum (alternatively, culture supernatant) diluted in BSA-PBS (1% *w*/*v* BSA in 0.01 M PBS) in the wells of a polypropylene microtiter plate (Corning Inc., Corning, NY, USA). The test plate containing immobilized

antigen was washed twice with Tween-PBS and 0.1 mL of test solution was transferred to each well and incubated for 30 min at ambient temperature. The plate was then washed three times and 0.1 mL of a 1:2000 dilution of goa<sup>t</sup> anti-mouse peroxidase conjugate was added. After incubating at ambient temperature for 30 min the plate was washed four times and the substrate o-phenylenediamine (OPD) was added. Preparation of the substrate is described elsewhere [36]. After 5 min 0.1 mL of 1 N hydrochloric acid was added to stop the reaction. Color development was determined by measuring the absorbance at 490 nm using a Synergy Neo microplate reader (Bio-Tek, Winooski, VT, USA).

From the 10 mice two were selected to undergo splenocyte fusion. The animals were sacrificed and the spleens aseptically removed. The splenocytes were chemically fused with NS-1 myeloma cells using polyethylene glycol then plated in HAT selection media. After 10 days, cultures were isolated and screened for anti-CTV activity using the CI-ELISA described earlier in this section. The first fusion yielded only a single product, which did not recognize free CTV. The second fusion also yielded a low number of products (5) of which three recognized free CTV. From these three products two clones were subsequently isolated, expanded, and used to produce larger amounts of antibody for evaluation. The cell lines were designated 2-2.2.2.8 (herein referred to as "2-2") and 2-4.3.5.1.2.1.4 (herein referred to as "2-4"). Ascites fluid from mice administered these cell lines was partially purified by ammonium sulfate precipitation using procedures described previously [37], then dialyzed against 0.1 M PBS and freeze-dried. The protein content was determined using the BCA Protein Assay (Thermo Fisher, Waltham, MA, USA).

#### *4.5. E*ff*ects of Methanol and Acetonitrile on Two CTV mAbs*

The impact of methanol and acetonitrile on the CI-ELISAs with mAbs 2-2 and 2-4 was determined by incorporating the solvents at concentrations from 5% to 30% (*v*/*v*) in the diluent used to prepare the calibration curves. The analytical CTV stock solution (i.e., 93% CTV, 7% iso-CTV) was used for these experiments. CI-ELISAs were performed essentially as in Section 4.4, with a lower concentration of CTV-OVA2 immobilized (4 μg/mL). Preliminary experiments indicated that using mAb 2-2 at a concentration of 12 μg/mL or mAb 2-4 at a concentration of 4.9 μg/mL were su fficient to give color development of approximately 1 absorbance unit with a 5 min substrate incubation. As described in Section 4.4 the antibodies (in 0.1% OVA-PBS) were mixed with the toxin/solvent combinations 1+1 before transferring them to the test plates containing immobilized CTV-OVA2. Note that at 30% methanol, the OVA was very poorly soluble and with 20% acetonitrile it was insoluble. For these reasons the highest solvent concentrations tested were 30% methanol and 15% acetonitrile. Tests were conducted with 4 to 8 replicate microtiter plates. Each CTV concentration level was represented by 4 wells on the plate. To obtain the most accurate comparisons, the two antibodies were compared side-by-side on the same plates. Standards were prepared over the range of 0.2 to 1000 ng/mL. Absorbance of all samples (i.e., "B") were normalized to those of the toxin-free controls (i.e., "Bo") using the equation (B/Bo) × 100%. Calibration curves based upon the transformed data were obtained using a logistic dose-response model and curve fitting software (TableCurve curve 2D, Systat Software Inc., Richmond, CA, USA). Fitted curves were used to calculate the concentrations required to inhibit color development by 50% (IC50s).

#### *4.6. Spiking and Extraction of Rice Samples*

Polished long-grain white rice was kindly supplied by Susan P. McCormick (USDA-ARS-NCAUR). The rice had been milled so the husk, bran, and germ were removed. The rice was ground in a co ffee mill to produce a powder similar in consistency to flour. Ten g of ground rice were spiked with a mixture of CTV and iso-CTV. The spiking solution contained a total of 144.7 μg/mL in methanol, of which 93 μg/mL was CTV and 51.7 μg/mL was iso-CTV. The volumes, amounts, and corresponding levels of CTV and iso-CTV in the rice are summarized in Table 4. Six replicate samples were produced at each spiking level. Samples were kept overnight at ambient temperature to permit the solvent in the spiking solution to evaporate. Samples were extracted with 40 mL of 80% (*v*/*v*) methanol/water by shaking for 2 h on a Burrell model 75 wrist-action shaker (Burrell Corporation, Pittsburgh, PA, USA) for 2 h at ambient temperature. The mixture was filtered through a Whatman 2V filter (Whatman plc, Maidstone, UK). The filtrate was diluted 1+7 (*v*/*v*) with a solution of 1% OVA in 10 mM PBS. The resulting diluted extracts contained 0.03125 g equivalents of rice/mL. Unspiked, control, rice was likewise extracted, diluted, and the diluted extract was used to prepare matrix-matched calibration curves for use in the CI-ELISA experiments.


**Table 4.** Preparation of spiked rice samples.

#### *4.7. CI-ELISA of Rice Samples*

Samples of rice spiked with a mixture of CTV and iso-CTV in the ratio of 1.8:1 were compared to calibration curves prepared using the same spiking mixture. The standards were prepared by diluting the stock solution in a control rice extract consisting of 0.03125 g equivalents of rice/mL (Section 4.6). As this was a 1:8 dilution of an 80% methanol extract of rice, the diluted extract also contained 10% methanol. The concentrations of the standards, represented as the sum of CTV and iso-CTV, ranged from 1.45 ng/mL to 2894 ng/mL. Because the matrix consisted of 0.03125 g equivalents/mL, the calibration curve covered the range from 0.046 to 93 mg/kg rice. To improve quantification, the spiked samples that contained greater than 2 mg/kg were diluted further to keep the resulting signals within the dynamic range of 80% to 20% of the maximal signals (i.e., between the IC20 and IC80). The LOD of the assay in rice matrix was calculated by measuring the standard deviation of the unspiked (toxin-free) controls and calculating the CTV concentration required to observe a signal 3 standard deviations from the mean of the controls.

#### *4.8. E*ff*ects of mAbs and Bovine Serum Albumin (BSA) on CTV Fluorescence*

Preliminary experiments indicated that the mAbs could influence the fluorescence of CTV in aqueous bu ffer. To examine this further, mixtures of mAb or BSA were combined with CTV in aqueous bu ffer (10 mM PBS, pH 7.2). The final concentrations of mAb (or BSA) and CTV were 2.0 μM and 1.25 μM, respectively. The volumes of test solutions were 0.32 mL. Excitation and emission spectra were collected using a Neo microplate reader (BioTek, Winooski, VT, USA). Emission scans were collected using an excitation of 420 nm, with emission monitored over the range of 450 to 700 nm in 1 nm increments. Excitation scans were collected using an emission of 570 nm, with excitation provided over the range of 360 to 500 nm in 1 nm increments. Both excitation and emission data were collected with the top optics of the instrument and the following parameters: gain 150, normal reading speed, optics positioned 4.50 mm above the sample, and temperature 20.5 ◦C. Data from triplicate plates were averaged and imported into TableCurve. Spectra were smoothed using Savitzky–Golay filtering.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6651/11/11/630/s1, Figure S1: E ffect of iso-CTV on the calibration curve in diluted rice matrix using mAb 2-4, Figure S2: E ffect of iso-CTV on the calibration curve in diluted rice matrix using mAb 2-2.

**Author Contributions:** C.M.M. and Y.S.-K. conceived and designed the experiments. C.M.M. conducted the experiments. Y.S.-K., N.K., Y.U., F.K., and C.M.M. contributed reagents/materials/analysis tools, and writing the manuscript.

**Funding:** This work was supported by USDA-Agricultural Research Service project number 5010-42000-0049-00D and by the Health and Labour Sciences Research Grants (Research on Food Safety, H28-shokuhin-ippan-004) from the Ministry of Health, Labour and Welfare of Japan.

**Acknowledgments:** The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. The USDA is an equal opportunity provider and employer.

**Conflicts of Interest:** The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.
