T-2 Toxin/HT-2 Toxin and Ochratoxin A ELISAs Development and In-House Validation in Food in Accordance with the Commission Regulation (EU) No 519/2014.

Michalina Oplatowska-Stachowiak,Martin Salden,Tim Kleintjens,Willem Haasnoot,Katrina Campbell,Nermin Sajic,Christopher T Elliott

doi:10.3390/toxins9120388

Michalina Oplatowska-Stachowiak, Martin Salden + Show 5 more

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https://doi.org/10.3390/toxins9120388

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Abstract

T-2 toxin/HT-2 toxin (T-2/HT-2) and ochratoxin A (OTA) are mycotoxins that can contaminate a variety of agricultural commodities. To protect consumers’ health, indicative limits for T-2/HT-2 and maximum limits for OTA have been set by the European Commission, requiring food business operators and controlling agencies to conduct routine checks for the presence of these harmful contaminants. Screening methods are increasingly used for monitoring purposes. Due to the demand for new and improved screening tools, two individual detection methods, T-2/HT-2 and OTA enzyme-linked immunosorbent assays (ELISAs), were developed in this study. The T-2/HT-2 ELISA was based on a T-2 monoclonal antibody with an IC50 (50% inhibitory concentration) of 0.28 ng/mL and 125% cross-reactivity with HT-2. As regards the OTA ELISA, a new sensitive monoclonal antibody specific to OTA with an IC50 of 0.13 ng/mL was produced. Both developed ELISA tests were then validated in agricultural commodities in accordance with the new performance criteria guidelines for the validation of screening methods for mycotoxins included in Commission Regulation (EU) No 519/2014. The T-2/HT-2 ELISA was demonstrated to be suitable for the detection of T-2/HT-2 in cereals and baby food at and above the screening target concentration (STC) of 12.5 μg/kg and 7.5 μg/kg, respectively. The OTA ELISA was shown to be applicable for the detection of OTA in cereals, coffee, cocoa and wine at and above the STC of 2 μg/kg, 2.5 μg/kg, 2.5 μg/kg and 0.4 ng/mL, respectively. The accuracy of both ELISAs was further confirmed by analysing proficiency test and reference samples. The developed methods can be used for sensitive and high-throughput screening for the presence of T-2/HT-2 and OTA in agricultural commodities.

Highlights

Mycotoxins are toxic secondary metabolites of fungal origin which have a major effect on agriculture and ecology, as well as human and animal health [1,2]
A new sensitive monoclonal antibody against ochratoxin A (OTA) with an IC50 of 0.13 ng/mL was produced in this study and applied in the development of a direct competitive enzyme-linked immunosorbent assays (ELISAs) for the detection of OTA in cereals, coffee, cocoa and wine
The required sensitivity was achieved using a liquid–liquid extraction approach and diluting the samples to reduce matrix effects. Both assays were validated in accordance with the new guidelines for validation of semi-quantitative screening methods for mycotoxins included in Commission Regulation

Summary

Introduction

Mycotoxins are toxic secondary metabolites of fungal origin which have a major effect on agriculture and ecology, as well as human and animal health [1,2]. T-2 is rapidly metabolised to HT-2 in vivo by animals, plants and fungi and both toxins commonly co-occur in agricultural commodities [7] Due to their high stability T-2 and HT-2 are present in the processed products of the cereal grains. These toxins are harmful to both humans and animals, among which cats and pigs are the most sensitive species. T-2 and HT-2 impair protein and DNA synthesis and interfere with both red and white blood cell formation in bone marrow leading to a number of adverse effects Based on these toxic effects the Panel on Contaminants in the Food Chain (CONTAM Panel) of the European

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