Horseradish peroxidase

First, the optimal concentration of plant-derived SVP was determined by titration with positive and negative sera for IBDV obtained from vaccinated and healthy chickens from our animal facility. Briefly, 96-well plates (Maxisorp Nunc) were coated with two-fold serial dilutions of SVP in 0.1 M bicarbonate buffer, pH 9.6, overnight at 4 °C. The plate was blocked with 5% skim milk in PBST-ENS (0.05% Tween 20, 5% equine normal serum) and subsequently incubated with anti-IBDV and negative serum diluted 1:100 as primary antibodies and 1:3000 goat anti-chicken IgG antibody coupled to horseradish peroxidase (Bethyl Laboratories Inc cat. # A30-106P). The detection step was performed with H₂O₂/ABTS [2,2′-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt] substrate/chromogen system in citric acid buffer, pH 5. Reading was carried out at 405 nm after 30 min of incubation or once the anti-IBDV serum of each plate (positive control) reached the admission rank of absorbance previously determined (Mean OD value = 0.84–1.07).
The best combination between primary and conjugated antibodies was determined performing a checkerboard titration. This titration consisted of testing 1:50, 1:100, 1:200, 1:300, 1:400 and 1:500 dilutions of the anti-IBDV antibody and 1:3000, 1:4000, 1:5000 and 1:6000 dilutions of the anti-chicken conjugated to peroxidase.

Serum anti-NP antibody levels were measured by ELISA as described below. 96-well flat-bottom plates (Thermo Fisher Scientific) were coated with 10 μg/ml NP₅₄-BSA (Biosearch Technologies), and then the wells were blocked with 5-fold diluted Blocking-One (Nacalai Tesque). Appropriately diluted serum samples were added to the wells and incubated at room temperature for 2 h. The plates were washed and incubated for another 1 h with diluted anti-IgM, -IgG1, -IgG2c and -IgG2b antibodies conjugated with horse radish peroxidase (Bethyl Laboratories), respectively. Bound secondary antibody was assessed by incubation with ABTS substrate (Sigma-Aldrich). Absorbance at 405 nm (A405) in each well was measured.
For the analysis of affinity maturation, appropriately diluted serum samples were assayed using an NP₄-BSA (Biosearch Technologys) -coated 96-well plate and an NP₅₄-BSA-coated plate. The A405(NP₄)/A405(NP₅₄) ratio was calculated as relative affinity.

Antibodies present in the serum were determined by indirect ELISA (iELISA) with the chimeric protein and with anti-pig IgG (H + L) conjugated with horseradish peroxidase (Bethyl, Montgomery, Texas, USA). The plates were coated with 4 µg/mL of the protein (100 µL/well). Then, they were incubated at 4°C overnight. The plates were washed 3 times with 1× Phosphate Buffered Saline with 0.05% Tween-20 (PBS-T). After that, they were blocked with 5% skimmed milk (Svelty-Nestle), allowed to incubate at 37°C for 1 hour and washed 3 times. One hundred microliters of serum diluted 1:2000 from days 0, 10, 21 and 31 post immunization were added. The plates were incubated at 37°C for 1 hour on a rotatory shaker at 200 rpm and washed 3 times. One hundred microliters per well of anti-pig IgG at a 1:200,000 dilution was added and incubated at 37°C for 45 min at 200 rpm, and the washing was repeated. OPD substrate (Sigma Aldrich, Germany) was added and incubated for 25 min, and the optical density (O. D.) was measured at 450 nm. Statistical significance was tested by Tukey’s multivariable comparison test α=0.05.