Rapid immunochemical tests for qualitative and quantitative determination of T-2 and HT-2 toxins

Abstract

Rapid immunotests were developed for qualitative and quantitative detection of T-2 and HT-2 toxins in cereal samples. Specific antibodies were immobilized either onto polyethylene filters or sepharose gel inside the transparent column. The tests combined the basic functions of purification, concentration and immunodetection of the target analytes through the enzymatic reaction of horseradish peroxidase (HRP). To make the test suitable for detection of both T-2 and HT-2 toxins, anti-T-2 toxin antibody was combined with HT-2 toxin-HRP conjugate. Matrix interference was eliminated by appropriate dilution of sample extracts with polyethylenglycol solution followed by filtration through a solid sorbent in a separate clean-up column. For the qualitative assay (visual detection) the cut-off level was 2 ng mL À1 of T-2 and HT-2 toxins, while the quantitative assay enabled the detection of 0.55 ng g À1 T-2 toxin and 1.7 ng g À1 HT-2 toxin in spiked wheat samples. Analysis of naturally contaminated wheat samples resulted in a good agreement between the developed immunotest and confirmatory LC-MS/ MS. The proposed immunotest is fast, easy-to-use and suitable for rapid screening of T-2 and HT-2 toxins in wheat.

High Specific Monoclonal Antibody Production and Development of an ELISA Method for Monitoring T-2 Toxin in Rice

“…Besides, published immunoassays for T-2 toxin mainly focused on the sum of T-2 toxin and HT-2 toxin. This is mainly due to the similar structure between T-2 toxin and HT-2 toxin, and the broad specificity mAb used in those methods could not distinguish one from another. − In this research, a high specific antibody was produced, which could recognize T-2 toxin specifically. The developed ELISA using this mAb could detect T-2 toxin in rice specifically, rather than the sum of T-2 toxin and HT-2 toxin.…”

Quantum Dot Loaded Liposomes As Fluorescent Labels for Immunoassay

“…The frit-based immunoassay procedure was described in our previous publications. , Anti-ZEN antibody (200 μL, dilution 1/2 000 for ZEN-QD, 1/10 000 for ZEN-LSQD, and 1/30 000 for ZEN-LIQD) was loaded onto the frit and the column was incubated during 6 min. Excess of unbound antibody was removed by washing with PBST, and the column was ready for application.…”

Molecular super-gluing: a straightforward tool for antibody labelling and its application to mycotoxin biosensing

“…5). This behaviour can be explained considering that although the primary Fab recognises both HT-2 and T-2 toxins, the secondary antibody, anti-IC HT2 (10) scFv, binds selectively to the bio-Fab-HT-2 complex. Thus, the use of this approach allows a significant increase in the selectivity of the method in comparison to traditional competitive immunoassays [9,32].…”

“…Moreover, conjugations are time-consuming, and the reaction may suffer from high batch-to-batch variations, which will affect the reproducibility of the bioassay. In addition, labelling may modify the toxin structure and its affinity for the antibody compared to the free toxin [9][10][11][12]. The implementation of non-competitive immunoassays based on a sandwich format avoids the use of toxin-conjugates for analyte detection and usually provides higher selectivity and, in many cases, sensitivity than competitive assays [13].…”