*2.6. Immune Recognition of FhSOD1 and FhSOD3 in Sera from F. hepatica Experimentally Infected Sheep*

Sera were collected from *F. hepatica* experimentally infected sheep and assayed as previously described by Lopez Corrales et al. (2020). The infections were carried out by Agri-Food and Biosciences Institute (AFBI; Belfast, UK) under license from the Department of Health, Social Services and Public by the *Animal (Scientific Procedures) Act* 1986 (License No. PPL 2771; PPL 2801). FhSOD1 and FhSOD3 total IgG antibodies were analysed by ELISA and Western blot according to standard methods. Briefly, for the ELISA, flat-bottom 96-well microtitre plates (Nunc MaxiSorp, Biolegend, San Diego, CA, USA) were coated with rFhSOD1, rFhSOD3, or recombinant *F. hepatica* cathepsin L1 (rFhCL1) (5 μg/mL in 0.05 M carbonate buffer, pH 9.6) and incubated overnight at 4 ◦C [28]. After incubation in blocking buffer (2% bovine serum albumin in PBS-0.05% Tween-20 (*v*/*v*), PBST, pH 7.4) and washing three times in PBST, sheep sera collected from 10 animals at 0, 3, 7, 11, 15, and 23 weeks post-infection (WPI) was diluted 1:100 in serum dilution buffer (PBS, 0.5% Tween 80, 0.5 M NaCl), added to the antigen-coated wells in triplicate, and allowed to incubate for 1 h at 37 ◦C. After washing five times, 100 μL/well of HRP-conjugated donkey anti-sheep IgG (ThermoFisher Scientific) diluted 1:50,000 in blocking buffer was added, and the plates were incubated for 1 h at 37 ◦C. Following five washes, 100 μL/well of 3,3- ,5,5- -Tetramethylbenzidine (TMB; Sigma-Aldrich) was added, and the plates were incubated at room temperature (RT) for 4.5 min. The reaction was stopped by the addition of 100 μL/well of 1 M sulphuric acid. The optical density was determined at a wavelength of 450 nm (OD450) in a PolarStar Omega spectrophotometer (BMG LabTech, Ortenburg, Germany).

Western blot analysis was performed using standard methods [29]. Briefly, rFhSOD1 and rFhSOD3 (1 μg/lane) was resolved by electrophoresis in 4–20% precast SDS-PAGE gels and electro-transferred onto nitrocellulose membranes prior to incubation in blocking buffer (5% milk in PBST, pH 7.4) for 1 h at RT. rFhCL1 was used as a positive control at 7 WPI. After washing five times with PBST, the membranes were probed with pooled sera from experimentally infected sheep at 0, 7, and 20 WPI diluted 1:1000 in blocking buffer (2.5% milk in PBST, pH 7.4) for 1 h at RT. After washing five times in PBST, the membranes were incubated with the secondary antibody alkaline phosphatase conjugated donkey-antisheep IgG at a 1:10,000 dilution for 1 h at RT. Following a final wash, the immune-reactive bands were visualised using the substrate SigmaFast BCIP/NBT (Sigma-Aldrich).

#### *2.7. Production of Specific Antibodies against rFhSOD1 and rFhSOD3*

Non-homologous sequences at the N-terminal of FhSOD1 (VMSGSSGVQGTVKFVQE-SET) and FhSOD3 (NASYSGQIFVNADGNLLTVR) were identified and protein-specific peptides (pFhSOD1 and pFhSOD3) were synthetically produced coupled with ovalbumin and used to immunise rabbits to generate FhSOD1 and FhSOD3-specific antibodies (Anti-pFhSOD1 and Anti-pFhSOD3) (Eurogentec, Seraing, Belgium). In addition, polyclonal antibodies against the purified recombinant rFhSOD1 and rFhSOD3 proteins (AntirFhSOD1 and Anti-rFhSOD3) were produced in rabbits (Eurogentec). Anti-rFhSOD1 and anti-rFhSOD3 antibodies were adsorbed against recombinant rFhSOD3 and rFhSOD1, respectively, prior to use to ensure specificity. The reactivity of the anti-pFhSOD1 and pFh-SOD3 antibodies and anti-rFhSOD1 and rFhSOD3 polyclonal antibodies was determined by Western blot analysis against 0.05 μg/lane rFhSOD1 and rFhSOD3. Native bovine erythrocyte SOD (BS; 0.05 μg/lane) was used as a negative control. Immuno-detection was conducted as described above, with the following exceptions: membranes were probed with rabbit pre-immune sera, anti-pFhSOD1, anti-pFhSOD3, anti-rFhSOD1 or anti-rFhSOD3 raised in rabbits at a 1:10,000 dilution for 1 h at RT. After washing, the membranes were further probed with the secondary antibody, alkaline phosphatase conjugated goat-antirabbit IgG, at a 1:5000 dilution for 1 h at RT. Following final washes, the immune-reactive bands were visualised using the substrate SigmaFast BCIP/NBT (Sigma-Aldrich).
