This proposal focuses in the development of immunoanalytical techniques enabling the simple and reliable detection of three of the most relevant mycotoxins in food and feed, that is, zearalenone, alternariol, and patulin. The reasons for the selection of these mycotoxins are diverse. Concerning patulin, it is worth mentioning that, to date, it has not been possible to obtain useful antibodies for the development of rapid methods, most likely because patulin is a very small and low structurally complex molecule. Regarding alternariol, commercial immunoanalytical tests are not available either, although some research groups have made some progress to reach this goal. Nevertheless, the strategies employed so far were essentially based on a single chemical approach, which, in our opinion, is not the most adequate. The same reasoning can be applied to zearalenone, yet in this case the available immunoreagents are sensitive-enough as to allow their implementation in commercial assays. Our long-standing experience in this research field lets us hypothesize that there are alternative, non-explored strategies that should reasonably give rise to antibodies and derived techniques with higher detectability than those developed so far.

As a previous step to the development of the new bioanalytical systems, the generation of the corresponding specific immunoreagents (haptens, bioconjugates, and antibodies) will be necessary. Preserving the physico-chemical properties of the mycotoxin and choosing the correct derivatization site in haptens will be the key factors for the generation of antibodies with the desired properties. Our proposal encompasses the preparation of several regioisomeric derivatives of each toxin with different linker tethering sites, in a way that minimum modifications are introduced and the hapten can be displayed to the immune system in alternative orientations. We are confident that these strategies will culminate with the generation of a wide collection of high-affinity and specific monoclonal antibodies to zearalenone, alternariol, and patulin. Each one of the immunoanalytical techniques included in this proposal, competitive ELISA, immunochromatographic strips, and immunoaffinity columns, will serve for a different analytical demand. Ultimately, the applicability of the new techniques will be validated in a range of products, including fruits, juices, cereals, and feed.

The objective of the project is to develop an immunodiagnostic test that allows quick, simple, in situ and economical analysis of the presence of OTA in wines. The analytical device to be developed is an immunoassay by lateral flow chromatography, a methodology that is also known as immunoreactive strips. These tests, identical in their principle of operation to pregnancy tests, allow to determine the presence of an analyte in a sample without purification in less than 10 minutes, and without the need for special equipment or qualified technical personnel. Consequently, these simple devices would make available to companies in the wine sector a reliable analytical tool to be able to control in the wineries themselves whether a must destined for winemaking, or the wine itself at the end of the elaboration process, complies with the European regulations on food safety on mycotoxins.

Among the biotoxins with the highest incidence and toxicity, two have strongly called our attention, patulin and anatoxin-a, as there are currently no immunoanalytic methods that allow the determination of these biotoxins in a simple and reliable way. The cause of this striking lack is that it has not yet been possible to obtain high-performance antibodies, probably because they are extremely small molecules with very little structural complexity.

Given this situation, we have considered for this project the interest of developing immunoanalytical methods for the selective and sensitive determination of these two important biotoxins in those matrices where their presence could constitute a risk. The strong relationship built by the two participating research groups on the generation of useful immunoreactive substances for the determination of chemical contaminants and residues in food has given us the necessary experience and knowledge to face with sufficient guarantees the ambitious challenge contemplated in this project. The high scientific, social and economic impact that could imply the success of this project is an additional motivating factor.

As a preliminary step to develop bioanalytical systems, it will be necessary to generate the corresponding specific immunoreagents (haptens, conjugates and antibodies). The design and synthesis of functionalized derivatives is a fundamental stage of the project. The generation of suitable bioreceptors depends on the preservation of the physicochemical characteristics of the target biotoxin and on the correct positioning of the spacer arm. Our proposal contemplates the preparation of diverse and stable regioisomeric derivatives of both toxins so that the changes produced by the linker are minimal and the molecule is displayed to the immune system with alternative orientations. The covalent conjugation of the haptens to different proteins will be carried out by coupling methods that were developed in previous projects. We are confident that these strategies will allow us to generate a large collection of high-affinity and specific polyclonal and monoclonal antibodies to each biotoxin. The immunochemical methods included in this project – competitive ELISA, immunochromatographic strips and immunoaffinity columns – are the approximations that we consider most interesting for their particular analytical purposes. The final objective is to validate the applicability of the developed methods for the detection and control of patulin and anatoxin-a in water and food.

Following recent food crises in Europe in recent years, such as bovine spongiform encephalopathy, detection of dioxins in chickens, acrylamide in fried foods, hydrocarbons in sunflower oil, and more recently the 50 deaths in Germany due to the consumption of soybean sprouts contaminated with a verotoxigenic toxin-producing microorganism. Consequently, food safety has become one of the current priorities of European health authorities.

Thus, in recent years there has been a great demand to develop rapid, reliable, sensitive and easy-to-use methods for the analysis of biotic and abiotic contaminants in foods.

This project aims to design a portable and low cost device capable of detecting several analytes of interest in the food safety area. As a model, several allergens (milk and egg) and the two main mycotoxins (aflatoxins and ochratoxin A) have been selected.

It is particularly in this line that the main objective of the present research project has been focused. New rapid and user-friendly specific procedures, based on immunochemical methods, for the analysis of SDH fungicides in horticultural products have been developed. As a previous step in the development of these systems, it has been necessary to generate the corresponding immunoreagents (functionalized haptens, fungicide-protein conjugates and antibodies) specific for each of the three fungicides addressed in the project. By properly combining antibodies and assay conjugates, it has been possible to develop competitive ELISA-based methods capable of determining with high sensitivity (well below established maximum residue limits) and selectivity each of the target compounds in different foods (stone fruits, grapes, juices, and wine).

In addition, we have also carried out the evaluation of new nanomaterials as immunizing vehicles. For this purpose, immunogens have been prepared from multiple wall carbon nanotubes (MWNT) with different surface morphological characteristics and functionalized supeficially to enable the covalent attachment of protein-hapten conjugates or the haptens themselves. These constructs have been shown to be capable of triggering a secondary immune response equivalent in quality and quantity to that obtained with traditional conjugates, even employing doses of hapten up to 300 times lower than usual. In addition, it has been observed that immunization with MWNTs is safer for animals, and that these nanomaterials have some adjuvant character as long as their functionalization is performed with protein conjugates

These compounds are present in approximately 10% of the fruit samples analyzed at European level. In the framework of this project, functionalised derivatives (haptens) have been synthesized, bioconjugates have been prepared, and antibodies (polyclonal and monoclonal) have been generated for all these six compounds. These developments have been opportunely patented, and constitute the first immunoreagents that have been generated for this group of plant protection products. By properly combining antibodies and conjugates, it has been possible to develop rapid methods of analysis based on the ELISA technique, which were capable of determining with high sensitivity and selectivity each of the target compounds in different foodstuffs (fruits, vegetables, juices, and wine) as well as in environmental samples (waters). In addition, multianalyte immunoassays have been developed for the simultaneous analysis of different compounds; either the three fungicides that make up the anilinopyrimidine group (cyprodinil, pyrimethanil and mepanipyrim) using an antibody capable of recognizing the three compounds with similar affinity, or a selection of fungicides commonly found in wine. A validation study was carried out with a large number of international wines.

The analytical performance of these new methods rivals that of conventional instrumental techniques (GC-MS and HPLC-MS) when they are directed towards specific compounds or restricted groups of analytes and a large number of samples. Among the most outstanding advantages of the developed tests are its rapidity, simplicity, high analytical capacity, the possibility of carrying out on-site analysis, and their moderate cost, all that with keeping a very high sensitivity and specificity.

This kit was based on the use of monoclonal antibodies, which were generated by a series of functionalized analogs of CPPU which needed to be synthesized. The actual utility of the kit was tested by determining residues of CPPU in grapes and kiwi fruit and by comparing the results generated with those obtained using a reference technique such as HPLC-DAD.

The ELISA kit for the determination of CPPU that was developed within the framework of this project is the first developed worldwide for the quick, simple and economic quantification of this important plant growth regulator. This kit constitutes a valuable analytical tool for farmers, cooperatives, distribution companies, analytical laboratories and regulatory agencies for the determination of CPPU in fruits.

Exposure of the general population, and especially children, to residues of chemical contaminants through diet due to the application of these compounds in horticultural products is a cause of concern for consumers. Government agencies and producers themselves have an obligation to ensure scrupulous compliance with regulations and the correct application of treatments, so that the residue content of the products that reach the market is the lowest possible and do not exceed the established limits. Therefore, sampling and monitoring programs, combined with prevention measures and effective risk assessment, management and communication, are required in order to increase the confidence of citizens in the safety of the consumed foodstuffs. As the current national and European scientific guidelines state, it would be desirable to have new analytical methods for the determination of residues of this type of products which, in addition to providing results with the necessary accuracy and precision, should be capable of analyzing a large number of samples in a short time, economically and without the need for expensive equipment and/or highly qualified personnel.

It is in this context that this research project has been developed, whose most relevant achievement has been the development of kit immunoassays for the analysis of the most relevant commercial strobilurin fungicides in food samples in a simple, rapid and economic way. These new bioanalytical methods have been shown to have a very high sensitivity and specificity, which has allowed the determination of very low concentrations of these fungicides in juices, cereals, beer, fruit and vegetables. We are confident that in the medium term these analytical tools can arouse the interest of the productive sectors involved and that of the regulatory authorities, becoming useful methodologies that contribute to guarantee the chemical safety of the foods we consume.