*4.4. Immunoreagent Preparation*

Protein conjugates of the two haptens were obtained by the active ester method. A 50 mM solution of the activated hapten was prepared in DMSO. BSA and OVA solutions were prepared at 15 mg/mL in 50 mM carbonate buffer, pH 9.6. The activated hapten was added to the protein solution at 40-fold molar excess for BSA conjugates. Conjugation reactions to OVA were done with 8- or 11-fold excess for **AL***a* and **AL***b*, respectively. Concerning HRP conjugates, the activated hapten solution (5 mM) was added over a 3 mg/mL enzyme solution in the same carbonate buffer to reach a hapten molar excess of 15. The activated haptens were added slowly to the protein solution (ca. 15–20 μL per hour), and the mixtures were incubated overnight at rt, protected from light, and with gentle stirring. Then, they were centrifuged for 5 min at 6700× *g*, and the conjugates were purified from the supernatant by size-exclusion chromatography using 100 mM phosphate

buffer, pH 7.4, as eluent. Fractions containing BSA or OVA conjugates were pooled and diluted with elution buffer to a final concentration of 1 mg/mL. BSA conjugate solutions were passed through 0.45 μm sterile filters. BSA and OVA conjugate solutions were stored at −20 ◦C. HRP conjugate solutions were 1:1 diluted with PBS containing 1% BSA (*w*/*v*) and 0.02% (*w*/*v*) thimerosal and stored at 4 ◦C. The hapten-to-protein molar ratio of the prepared conjugates was determined by MALDI-TOF-MS and running BSA, OVA, and HRP for reference in the same plate, as previously described [27].

Animal manipulation was performed according to Spanish laws (RD1201/2005 and law 32/2007) and the European Directive 2010/63EU regarding the protection of experimental animals. Polyclonal antibodies to AOH and AME were obtained from the sera of immunized animals. Briefly, two female New Zealand white rabbits—weighing 2 kg at the beginning of the experiment—were immunized by four periodic subcutaneous injections of a 1:1 water-in-oil emulsion containing 300 μg of the BSA-hapten conjugate. The inoculum was prepared with complete Freund's adjuvant for the first injection and with incomplete Freund's adjuvant for subsequent injections. Boosts were applied with 21-day intervals. Animals were exsanguinated by intracardiac puncture 10 days after the last injection, and the blood was left overnight in the refrigerator at 4 ◦C for coagulation. Sera were separated from cells by centrifugation (3000× *g*, 20 min). Finally, immunoglobulins were partially purified by precipitation twice with one volume of cold saturated (3.9 M) ammonium sulphate solution. Antibodies were stored at 4 ◦C as precipitates.

## *4.5. Competitive ELISA Procedures*

Immunoassays were carried out by competitive ELISA using the capture antibodycoated direct format and the conjugate-coated indirect format. After each incubation step, plates were washed three times with a 150 mM NaCl solution containing 0.05% (*v*/*v*) Tween 20. For direct assays, microplates were coated by overnight incubation at 4 ◦C with 100 μL per well of GAR solution (1 μg/mL) in 50 mM carbonate-bicarbonate buffer, pH 9.6. For indirect assays, microwells were coated with 100 μL per well of OVA-hapten conjugate solution in the same coating buffer, and overnight incubation at rt. The competitive reaction was performed by mixing in each well 50 μL of analyte solution in PBS with 50 μL of antibody dilution or enzyme tracer solution in PBS containing 0.05% (*v*/*v*) of Tween-20, and incubating 1 h at rt. For indirect assays, 100 μL per well of GAR-HRP diluted 1/10,000 in PBS containing 0.05% (*v*/*v*) of Tween-20 and 10% (*v*/*v*) of adult bovine serum was added. Signal was obtained using 100 μL per well of TMB as the chromogenic enzyme substrate and incubation at rt during 10 min. Finally, 100 μL of 1 M H2SO4 was added and the absorbance was read at 450 nm using 650 nm as reference wavelength.

Standard mycotoxin solutions were obtained by serially diluting in buffer the most concentrated standard solution, which was prepared from a concentrated stock solution in DMF. Eight-point standard curves were built using those solutions and a blank sample. SigmaPlot software, version 14.0 from Systat Software Inc. (San Jose, CA, USA), was employed to fit the experimental values to a standard four-parameter logistic equation. The half-maximal inhibition concentration (IC50) and the maximum absorbance (Amax) values were considered in order to compare antibody performance. CR was calculated according to Formula (1):

$$\text{CR (\%)}=\text{IC}\_{50} \text{ (AOH)} / \text{IC}\_{50} \text{ (AME)} \times 100\tag{1}$$

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/toxins13120883/s1: General procedures, materials, and equipment; NMR spectra of all of the intermediates and the final activated haptens.

**Author Contributions:** Conceptualization, A.A.-F.; Data curation, A.A.-S., A.A.-F. and J.V.M.; Formal analysis, L.G.A.-M., A.A.-S. and C.A.; Funding acquisition, A.A.-S., A.A.-F. and J.V.M.; Investigation, L.G.A.-M. and C.A.; Methodology, A.A.-S., A.A.-F. and J.V.M.; Supervision, A.A.-S., C.A. and J.V.M.; Validation, L.G.A.-M. and C.A.; Writing—original draft, A.A.-S. and J.V.M.; Writing—review &

editing, A.A.-S., A.A.-F. and J.V.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the SPANISH MINISTERIO DE ECONOMÍA Y COMPET-ITIVIDAD, grant numbers AGL2015-64488 and RTI2018-096121, and cofinanced by EUROPEAN REGIONAL DEVELOPMENT FUNDS. Animal manipulation as well as mass, spectrometric, and proteomic analysis was performed at the SCSIE service of the University of Valencia. The proteomics laboratory is a member of *Proteored*.

**Institutional Review Board Statement:** This study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of GENERALITAT VALENCIANA (protocol code 2019/VSC/PEA/0179) on 20 August 2019.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The technical assistance by Paula Peña-Murgui and José V. Gimeno is greatly appreciated.

**Conflicts of Interest:** The authors declare no conflict of interest.
