Ovalbumin (ova)

Total egg extract and proteolytic products were mixed with an equal volume of 2x sample buffer (62 mM Tris, 50 mM DTT, 0.2% SDS, 10% glycerol, and 0.01% bromophenol blue). Proteins were resolved by SDS-PAGE using a 12% gel [35 (link)] and visualised by staining with 0.2% Coomassie Brilliant Blue R250 (w/v) dissolved in ethanol, acetic acid, and water (45:10:45, v/v/v) (Bio-Rad Laboratories, Brazil), and the molecular masses were estimated using the following protein standards: phosphorylase B (101.4 kDa), bovine serum albumin (87.5 kDa), ovalbumin (52.7 kDa), carbonic anhydrase (35.8 kDa), soybean trypsin inhibitor (22.8 kDa), and lysozyme (18.8 kDa) (Bio-Rad Laboratories, USA).
Total ovary extracts were resolved using 8% SDS-PAGE [35 (link)] by the same process described above, and molecular masses were estimated using the following protein standards: myosin (202.8 kDa), β-galactosidase (115.5 kDa), bovine serum albumin (98.2 kDa), ovalbumin (51.4 kDa) (Bio-Rad Laboratories, USA).

SDS–PAGE (Laemmli, 1970 (link)) was used to determine the subunit molecular mass of the enzymes. Myosin (200 kDa), β-galactosidase (116.3 kDa), phosphorylase B (97.4 kDa), serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa), and aprotinin (6.5 kDa) were used as molecular mass standards (Bio-Rad, Tokyo). The native molecular mass of PH0782 was determined using gel filtration chromatography with a HiLoad 26/60 Superdex 200 column (GE Healthcare, Tokyo). Thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), and Myoglobin (17 kDa) were used as molecular mass standards (Bio-Rad).

Antibodies against OVA were detected using ELISA plates (Maxisorp; Nunc) coated for 1 h at room temperature with 100 μL/well of 20 μg/mL OVA (Sigma-Aldrich, Saint Louis, MO, USA) in 0.1 M carbonate buffer (pH 9.6). Serial dilutions of sera from inoculated sheep were added to the plates and incubated for 1 h at room temperature, after blocking with blocking buffer (PBS with 0,05% (vol/vol) Tween-20 and 4% (wt/vol) skimmed milk) for 1 h at room temperature and washing five times with 1% (vol/vol) Tween-20 in PBS. The presence of OVA-specific IgG or IgM was detected using a secondary donkey anti-sheep IgG conjugated to HRP (1/2,000; Serotec, Corston Bath, UK) or rabbit anti-sheep IgM also conjugated to HRP (1/20,000; Bethyl, Montgomery, Texas, USA). After washing 10 times with 1% (vol/vol) Tween-20 in PBS, signal was developed using TMB Liquid Substrate System (Sigma-Aldrich, Saint Louis, MO, USA) and the reaction was stopped with 3 M sulfuric acid before reading. OD was determined at 450 nm on a FLUOstar Omega (BMG Labtech, Ortenberg, Germany) ELISA plate reader. All IgG/IgM measurements were made in triplicate, and assays were only considered valid when SDs were below 10% of the average. IgG/IgM binding to OVA was considered positive only when the OD obtained was at least twice the OD obtained with the preimmune serum from the same sheep.