Abstract
We developed an HT-2 toxin-specific simple ELISA format with a positive read-out. The assay is based on an anti-immune complex (IC) scFv antibody fragment, which is genetically fused with alkaline phosphatase (AP). The anti-IC antibody specifically recognizes the IC between a primary anti-HT-2 toxin Fab fragment and an HT-2 toxin molecule. In the IC ELISA format, the sample is added together with the scFv-AP antibody to the ELISA plate coated with the primary antibody. After 15 min of incubation and a washing step, the ELISA response is read. A competitive ELISA including only the primary antibody recognizes both HT-2 and T-2 toxins. The anti-IC antibody makes the assay specific for HT-2 toxin, and the IC ELISA is over 10 times more sensitive compared to the competitive assay. Three different naturally contaminated matrices: wheat, barley and oats, were used to evaluate the assay performance with real samples. The corresponding limits of detection were 0.3 ng/mL (13 µg/kg), 0.1 ng/mL (4 µg/kg) and 0.3 ng/mL (16 µg/kg), respectively. The IC ELISA can be used for screening HT-2 toxin specifically and in relevant concentration ranges from all three tested grain matrices.
Highlights
HT-2 toxin (HT-2) and T-2 toxin (T-2) are the most toxic trichothecene mycotoxins [1,2,3]
The production yield was sufficient for noncompetitive enzyme-linked immunosorbent assays (ELISA) assay development
The purity of the scFv-alkaline phosphatase (AP) product was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and no degradation products were observed
Summary
HT-2 toxin (HT-2) and T-2 toxin (T-2) are the most toxic trichothecene mycotoxins [1,2,3]. They occur in grains such as barley, wheat and oats, and are produced by Fusarium species [4]. European Union recommendations for the sum of HT-2 and T-2 in unprocessed barley, oats and wheat have been set to. Most reports of HT-2 and T-2 occurrence have come from northern Europe, France and the UK, and the problem related to these type A trichothecene mycotoxins has previously been associated with a cool climate [4]. In 2014, almost 20% of the samples studied exceeded the EU recommendations (200 μg/kg) for the sum of HT-2 and T-2 in barley [6]
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