Fluorescence Polarization Immunoassay for the Determination of T-2 and HT-2 Toxins and Their Glucosides in Wheat

Abstract

T-2 and HT-2 toxins and their main modified forms (T-2 glucoside and HT-2 glucoside) may co-occur in cereals and cereal-based products. A fluorescence polarization immunoassay (FPIA) was developed for the simultaneous determination of T-2 toxin, HT-2 toxin and relevant glucosides, expressed as sum. The developed FPIA, using a HT-2-specific antibody, showed high sensitivity (IC₅₀ = 2.0 ng/mL) and high cross-reactivity (100% for T-2 toxin and 80% for T-2 and HT-2 glucosides). The FPIA has been used to develop two rapid and easy-to-use methods using two different extraction protocols, based on the use of organic (methanol/water, 90:10, v/v) and non-organic (water) solvents, for the determination of these toxins in wheat. The two proposed methods showed analytical performances in terms of sensitivity (LOD 10 µg/kg) recovery (92-97%) and precision (relative standard deviations ≤13%), fulfilling the criteria for acceptability of an analytical method for the quantitative determination of T-2 and HT-2 toxins established by the European Union. Furthermore, the methods were then validated in accordance with the harmonized guidelines for the validation of screening methods included in the Regulation (EU) No. 519/2014. The satisfactory analytical performances, in terms of intermediate precision (≤25%), cut-off level (80 and 96 µg/kg for the two methods) and rate of false positives (<0.1%) confirmed the applicability of the proposed methods as screening method for assessing the content of these toxins in wheat at the EU indicative levels reported for T-2 and HT-2 toxins.

Keywords: HT-2 glucoside; HT-2 toxin; T-2 glucoside; T-2 toxin; fluorescence polarization immunoassay; screening method; validation study; wheat.

Figure 1

Chemical structures of T-2 and HT-2 toxins and their main modified forms T-2 and HT-2 glucosides.

Figure 2

Normalized calibration curve of the selected FPIA obtained with mixed standard solutions of T-2, HT-2, T2-glucoside and HT2-glucoside (expressed as sum, ratio 1:1:0.5:0.5) in PBS-A solution ([Anti-HT2] = 8 µg/mL; [HT2-FL_1a] obtained after dilution 1:3000 (v/v) of the stock solution, see Table 1). The FP linearity range vs. log [T-2 + HT-2 + T-2G + HT-2G] is reported in the insert. Values of the x-axes are the toxin concentrations in the final test solution.

Figure 3

Contents of T-2, HT-2, T-2G and HT-2G (expressed as sum) of 20 artificially contaminated wheat samples at STC (100 µg/kg) and of 20 blank wheat samples analyzed under repeatability conditions on 5 different days: (a) FPIA using protocol A; (b) FPIA using protocol B.