*Article* **Assessment of the Optimum Linker Tethering Site of Alternariol Haptens for Antibody Generation and Immunoassay Development**

**Luis G. Addante-Moya 1, Antonio Abad-Somovilla 1, Antonio Abad-Fuentes 2, Consuelo Agulló <sup>1</sup> and Josep V. Mercader 2,\***


**Abstract:** Immunochemical methods for mycotoxin analysis require antigens with well-defined structures and antibodies with outstanding binding properties. Immunoreagents for the mycotoxins alternariol and/or alternariol monomethyl ether have typically been obtained with chemically uncharacterized haptens, and antigen conjugates have most likely been prepared with mixtures of functionalized molecules. For the first time, total synthesis was performed, in the present study, to obtain two haptens with opposite linker attachment locations. The functionalized synthetic haptens were purified and deeply characterized by different spectrometric methods, allowing the preparation of bioconjugates with unequivocal structures. Direct and indirect competitive enzyme-linked immunosorbent assays, using homologous and heterologous conjugates, were employed to extensively evaluate the generated immunoreagents. Antibodies with high affinity were raised from conjugates of both haptens, and a structure-activity relationship between the synthetic haptens and the specificity of the generated antibodies could be established. These results pave the way for the development of novel highly sensitive immunoassays selective of one or two of these *Alternaria* mycotoxins.

**Keywords:** alternariol; antibody; ELISA; hapten design; immunoassay; linker site

**Key Contribution:** Two pure regioisomeric alternariol haptens were prepared. Alternariol immunoreagents were generated and characterized.
