*2.6. FPIA Screening and HPLC Analysis of Blind Samples*

A set of 10 naturally OTA-contaminated rice samples with a contamination levels ranging from 0.98 to 14.6 ng/mL (through HPLC analysis) was analyzed by both an immunoaffinity column clean-up with HPLC and the developed FPIA assay for comparison (Table 2). Linear regression of recovery data showed a good correlation (*r*<sup>2</sup> = 0.9966, Figure 7). An analysis of variance (ANOVA) estimation proved that OTA concentrations results obtained through the FPIA method were a good forecast of the expected values as tested by HPLC (*p* < 0.0001). Data from the two methods showed a good level of correlation and concurred with the spiked quantities, proving the practical applicability of the developed method.

**Table 2.** Quantitative determination of OTA in practical samples with FPIA and HPLC methods.


**Figure 7.** Correlation analysis between HPLC method and the developed FPIA assy.

#### **3. Conclusions**

A sensitive and accurate FPIA analytical method based on a new tracer (OTA-AMF) was developed and optimized for the rapid detection and quantification of OTA in rice and maize samples. The method detected OTA in concentrations ranging from 0.03–0.78 ng/mL and reached an IC50 value of 0.09 ng/mL. Compared to other immunoassays (Table 3), the FPIA method has better sensitivity. Apart from the purification procedure, the whole analytical process was completed within 10 min showing its practical applicability as a rapid screening method. The precision of the method was proven by comparing the results of OTA levels in rice and maize samples obtained by HPLC and results obtained by the FPIA. Furthermore, the proposed technique is inexpensive, user-friendly, and suitable for on-site use. The method is suitable for screening and quantitative determination of OTA in rice and maize at levels lower the EU regulatory limits for OTA in cereal grains and can serve as an alternative to instrument-based methods.


**Table 3.** Comparing the sensitivity with other immuno-assay methods.

<sup>a</sup> The data were not detected or shown in the paper. mAb: monoclonal antibody. Nb: nanobody. FIA: fluoroimmunoassay. ELISA: enzyme-linked immunosorbent assay. TRFICA: time-resolved fluorescent immunochromatographic assay.

#### **4. Materials and Methods**

#### *4.1. Reagents and Chemicals*

Ochratoxin A (OTA), N,N-dimethylformamide (DMF), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), octanoic acid, ammonium sulfate, dimethyl sulfoxide (DMSO), and Freund's incomplete adjuvant liquid (FICA) were bought from Sigma-Aldrich. Modified RPMI medium, HEPES, and penicillin-streptomycin solution were obtained from GE Healthcare-Hyclone. Fetal bovine serum was obtained from Gibco. Cell culture flask (75 cm2) was obtained from CORNING. Dialysis tubing (MWCO 15 KDa) was obtained from Thermo-Scientific. The SDS-PAGE gel preparation kit was obtained from Biosharp Life Sciences. Thin-layer chromatography (TLC) plates were obtained from Darmstadt; the model specifications were silica gel 60, 1 mm, 20 × 20 cm, with fluorescent indicator. Fluorescein thiocarbamoyl ethylenediamine (EDF), fluorescein thiocarbamoyl butane diamine (BDF), fluorescein thiocarbamoyl hexame (HDF), and amino-methyl fluorescein (AMF) were provided by Sergei A. Eremin, a professor of Department of

Chemistry, Lomonosov Moscow State University. All FPIA experiments used sodium borate solution (BB, 0.05 M, pH = 9.0). Phosphate buffer solution (PBS, 0.01 M, pH = 7.4) was used for the mAb dialysis. All of the organic solvents and chemicals were reagent grade or above.

The fluorescence polarizer was a sentinel 200 FP portable unit (Diachemix, Grayslake, 1 L). Measurements of intensity and fluorescence polarization were executed applying the TDx/FLx Analyzer (Abbott, Irving, TX, USA) in an aided Photo Check method. TDx/FLx glass cuvettes were put into the particular turntable up to 10 at a time, then polarization (mP units) and fluorescence intensity (customary units) were measured. The total measurement time of 10 samples was about 7 min.

#### *4.2. Animals and Cells*

Female BALB/c mice(age 8–10 weeks), which were used for the production of antibodies, were purchased from the Institute of Biological Products of Hubei Province (Wuhan, China). OTA hybridoma cell strain 1H2 was developed in our laboratory [37].

#### *4.3. Hybridoma Cell Culture and Antibody Preparation*

OTA hybridoma cell strain 1H2 was taken out of a liquid nitrogen container, melted in a 37 ◦C water bath for 1 min, and washed with modified RPMI medium solution. The cells were resuspended with 1640 complete medium (RPMI medium modified: HEPES: penicillin-streptomycin, *V*:*V*:*V*:*V* = 80:20:1:1), and transferred to a cell culture flask, then incubated in a constant temperature incubator (at 37 ◦C, CO2 5%). Hybridoma cells were collected during the logarithmic phase, and injected into the BALB/c mice that had been treated with FICA. The number of cell injections was maintained at 2 <sup>×</sup> 106. Ascites were harvested with drainage needle about one week later.

The ascites were used to prepare pure OTA antibodies, which were purified by caprylic acid-ammonium sulfate precipitation [38]. Subsequently, the antibodies were freeze-dried in a vacuum, and stored at −20 ◦C for later use. The purity of monoclonal antibody was determined by the SDS-PAGE gel electrophoresis method.
