4.5.4. Immunization and Antibody Production

Rabbit Immunization with *AflR* Recombinant Protein

Ten male New Zealand White rabbits, age 10–16 weeks and weighing 3.5–4.0 kg were used. Physical examinations confirmed lack of abnormalities. Rabbits were housed in stainless steel and polycarbonate cages (Techniplastic, West Chester, PA, USA), at 18–21 ◦C, with 30–70% humidity, and a 12-h: 12-h light: dark cycle (lights on at 0600). Rabbits were fed 250 g of a commercial pelleted rabbit diet (diet 2030, Harlan Laboratories, Madison, WI, USA) twice daily and were allowed free access to municipal water via an automatic watering system (Edstrom Industries, Waterford, WI, USA). After one-week of acclimatization, rabbits were divided into control (4 animals) and treated (6 animals) groups. The latter animals were injected subcutaneously with 500 μL of purified protein (2 mg/mL) following the polyclonal antibody production protocol of Fishback et al. [31] with some modifications (Figure 6). The study was conducted after obtaining approval from the International Animal Care and Use Committees (IACUCs) IACUC # 30-1Y-0521 (date of approval 10 January 2018).

**Figure 6.** Polyclonal antibody production protocol.

Three milliliter of blood was collected from the auricular artery of each rabbit on weeks 1 and 4 to monitor antibody production. On week 6, under deep anesthesia with a mixture of 22–50 mg/kg ketamine and 5–10 mg/kg xylazine, 3 mL of blood was collected by cardiac puncture. Blood was collected into BD Vacutainer serum separation tubes (BD, Franklin Lakes, NJ, USA), and kept upright at room temperature (20 ± 2 ◦C) for serum separation following the manufacturer's instructions. Separated sera were stored at −80 ◦C until further analysis.

#### Serum IgG Purification and Fractionation

Rabbit sera were obtained by centrifugation of immunized rabbit blood at 4000 rpm for 5 min at 4 ◦C. IgG fractions were obtained by loading sera onto an affinity Protein G-Sepharose column. In brief, the IgG1 fraction was eluted with glycine buffer, pH 2.7, and the IgG3 fraction obtained by elution with glycine buffer, pH 3.5. All IgG fractions were immediately neutralized in a neutralization buffer (1 M Tris–HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA) [32].

#### Labelling of Antibodies

Ten mg of alkaline phosphatase (AP) were mixed with purified IgGs (2.5 mg) in 5 mL of 50 mM phosphate buffer, pH 6.8. Mixtures were dialyzed against 2 L of 50 mM phosphate buffer for 24 h at 4 ◦C. One mL of 1.25% glutaraldehyde was added to each mixture and gently stirred for 2 h at room temperature (20 ◦C ± 2). Two hundred fifty μL of 0.2 M glycine solution was added followed by further stirring for 2 h. Mixtures then were dialyzed twice against 2 L of 1.0× PBS containing 1 mM magnesium chloride, followed by centrifugation at 10,000 rpm for 5 min to remove any precipitate [33]. Each conjugate was further purified on a Sephacryl S200 column (5 × 150 mm, GE Health care, Danderyd, Sweden) previously equilibrated with PBS and eluted with the same buffer.

#### 4.5.5. Quality Checks

An indirect enzyme-linked immunosorbent assay (I-ELISA) was used to detect aflatoxin B1 in food samples using polyclonal antibodies. Antibodies were compared using an antiserum produced by Sigma (Berlin, Germany). One gram of contaminated food sample was extracted in 10 mL coating buffer. One hundred microliter of sample extract was added to each well. Plates were then incubated at 37 ◦C for 3 h and blocked with 200 μL of blocking buffer (1× PBS and 0.5% BSA) for 1 h at room temperature (20 ◦C ± 2). One hundred microlite concentration of 1:800 diluted secondary antibody alkaline phosphataseconjugated (anti-rabbit antibody) was added and the mixture was incubated at 37 ◦C for 1 h. All washing steps between incubations used 1× PBS-T buffer. Finally, freshly prepared pNPP substrate was added; plates were incubated at room temperature for 30 min away from direct light, and the absorbance was measured at 405 nm. All experimental steps are summarized in Figure 7.

**Figure 7.** Summary of experimental steps.

#### **5. Statistical Analysis**

Data were statistically analyzed using SPSS software (version 16). One-way analysis of variance was used to assess the significance of differences among means, with a significance threshold of *p* < 0.05. The Pearson correlation coefficient (r) was also calculated (*p* < 0.001) to assess the strength of linear relationships between variables.

**Author Contributions:** Conceptualization, E.H., N.M.A.E.-A., A.M.G.D., M.G.S., A.A.I., A.M.E. and A.N.B.; methodology, E.H., N.M.A.E.-A., A.M.G.D., M.G.S., A.A.I. and A.M.E.; software and static analysis, E.H., N.M.A.E.-A., A.M.G.D., M.G.S., A.A.I., A.M.E. and A.N.B.; formal analysis, E.H., N.M.A.E.-A., A.M.G.D., M.G.S. and A.N.B.; investigation and data curation, E.H., N.M.A.E.-A., A.M.G.D., M.G.S. and A.N.B.; resources, E.H., N.M.A.E.-A., A.M.G.D. and M.G.S.; writing—original draft preparation, E.H., N.M.A.E.-A., A.M.G.D. and M.G.S.; writing—review and editing, E.H., N.M.A.E.-A., A.M.G.D., M.G.S., A.A.I., A.M.E. and A.N.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Animal experiment was conducted upon the approval of International Animal Care and Use Committees (IACUCs) (Permission number: IACUC # 30-1Y-0521, date of approval 10 January 2018).

**Informed Consent Statement:** Not applicable.

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

#### **References**

