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 ALa and ALb, 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.