Detection of T-2 Toxin in Different Cereals by Flow-Through Enzyme Immunoassay with a Simultaneous Internal Reference

Abstract

In most previously described membrane-based immunoassays a separate negative control assay is always carried out to evaluate the performance of the assay. To overcome this problem, a membrane-based flow-through enzyme immunoassay with an internal control has been developed for the detection of T-2 toxin in cereals (patent pending). An Immunodyne ABC membrane was coated with 2 μL of goat anti-horseradish peroxidase (HRP) (internal control spot) (1:1000) and 2 μL of rabbit anti-mouse (test spot) (undiluted) immunoglobulins, and the free binding sites were blocked. In addition to the antibody-coated Immunodyne ABC membrane, the assay also comprises a plastic snap-fit device, absorbent cotton wool, mouse anti-T-2 monoclonal antibodies (Mab), and T-2−HRP conjugate. The color intensity (Δ ) of the internal control and that of the negative sample showed no difference (P > 0.05), whereas there was a significant difference between the internal control and positive samples (P < 0.05). The minimum detectable limit that could be visually detected with confidence was 50 ng of T-2 per gram of cereal sample. Keywords: Internal control; T-2 toxin; flow-through membrane-based immunoassay; cereals