**1. Introduction**

*Alternaria* sp. fungi, particularly *A. alternata*, are ubiquitous plant pathogens and saprophytes that infect economically relevant crops such as cereals, vegetables, oilseeds, and fruits. Moreover, these microorganisms can contaminate these commodities after harvest even under refrigeration conditions. They are known to produce a wide variety of toxic secondary metabolites [1], and some of them have been identified by the EFSA Panel on Contaminants in the Food Chain (CONTAM) as a potential risk to human and animal health due to their toxicity and occurrence in food and feed. Surprisingly, there are no specific international regulations for any of the *Alternaria* mycotoxins, and the available data on toxicity, occurrence, and dietary exposure are still limited. In 2011, EFSA carried out the first assessment of the risk of these mycotoxins to human and animal health, based on government and published data [2]. More recently, EFSA conducted a survey on the dietary exposure of European consumers to *Alternaria* toxins [3]. This study found that 8% of these mycotoxins are present in food, with infants and other children being the most exposed population group, and fruit and fruit-based products contributing most to dietary

**Citation:** Addante-Moya, L.G.; Abad-Somovilla, A.; Abad-Fuentes, A.; Agulló, C.; Mercader, J.V. Assessment of the Optimum Linker Tethering Site of Alternariol Haptens for Antibody Generation and Immunoassay Development. *Toxins* **2021**, *13*, 883. https://doi.org/ 10.3390/toxins13120883

Received: 9 November 2021 Accepted: 6 December 2021 Published: 10 December 2021

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exposure. It is therefore expected that the European Commission will soon set maximum levels for the most common *Alternaria* mycotoxins in foodstuffs.

Alternariol (AOH) and alternariol monomethyl ether (AME), two of the most important compounds belonging to the group of *Alternaria* mycotoxins, appear to be responsible for the teratogenic effects observed in animals. They have also been shown to inhibit in vitro the catalytic topoisomerase activity, which may be associated with human colon and oesophageal cancer [4]. Frequently, these two mycotoxins are found together in samples because they share most of the biosynthetic pathway [5]. AOH and AME have been detected in a wide variety of products, including lentils, carrots, tomatoes, berries, apples, pears, beer, wines, juices, and various grains and flours [6]. To evaluate the relative hazard level of these toxins to human health, a threshold of toxicological concern (TTC) for AOH and AME in adults of 2.5 ng/kg body weight per day was established as a reference parameter by the CONTAM Panel [2]. With limited data available, a 2016 German survey concluded that the percentage of TTC reached by the average adult daily exposure was 1400% and 280% for AOH and AME, respectively [7].

A variety of analytical techniques have been developed for monitoring *Alternaria* toxins in food, including liquid and gas chromatographic methods coupled to several detection systems, as well as different types of immunochemical assays [8,9]. Molecular affinity techniques nowadays represent alternative strategies for rapid, economical, and/or on-site monitoring of mycotoxins. The first antibodies and immunoassays for AOH were reported in 2011 [10,11]. Since then, a few immunoassays have been described using either polyclonal or monoclonal antibodies specific to AOH [12,13], and only one study has been published using an antibody specific to AME [14]. Additionally, Wang et al. have reported a generic antibody for both mycotoxins [15]. In all these studies, the immunogens used to generate antibodies against AOH were made by attaching the mycotoxin to the carrier protein, either directly by a Mannich-type reaction or by carbodiimide-mediated chemistry after nonselective carboxymethylation of the hydroxyl groups. Neither of these methods can be used to guide the position of attachment of the AOH molecular scaffold to the carrier proteins, so the antibodies were actually generated from an undefined mixture of functionalized haptens. More recently, Yao et al. published an immunoassay for AOH using a carboxymethyl hapten to generate polyclonal antibodies [16]. Disappointingly, no synthetic details and spectrometric data of the prepared hapten were provided in that work.

AOH and AME are the two most representative mycotoxins with a tricyclic benzochromenone chemical backbone. Moreover, these compounds contain hydroxyl, methyl, and other substituents in their chemical structure. It is well known that the orientation of the molecule, i.e., the way the hapten is displayed to the immune system, strongly influences the specificity of the generated antibodies. Thus, the synthesis of haptens with the optimal linker tethering site is critical, although it is hard to predict and challenging to perform. Surprisingly, deeply characterized haptens for AOH or AME with unambiguous chemical structures have not been published so far. The aim of the present study was to prepare, purify, and characterize two rationally-designed synthetic haptens of these *Alternaria* mycotoxins with a functionalized linker located at precise sites of the molecule. The ability of these novel immunoreagents to elicit a potent immune response, ultimately leading to high-affinity antibodies, was investigated. In addition, these bioconjugates also allowed the study of the relationship between the functionalization position in the hapten and the specificity of the resulting antibodies.
