IgG, IgM and IgA serum antibody levels to recombinant cervid PrP were determined by a 1:125 dilution of plasma in duplicate, in which 50 μl/well of 1.5 μg/ml cervid recPrP in 50 mM ammonium bicarbonate pH 9.6 was coated at 4°C overnight onto Immulon 2HB 96 well microtiter plates (Thermo Scientific). Bound antibodies were detected by a goat anti-deer IgG, linked to horseradish peroxidase (HRP) (KPL, Gaithersburg, MD) and rabbit anti-goat or anti sheep IgM or IgA linked to HRP (Bethyl Lab. Inc., Montgomery, TX ). The color was developed by 3,3′,5,5′-tetramethylbenzidine (Pierce Biotech. Inc., Rockford, IL) as substrate; the reaction was stopped by 2N sulfuric acid and the plates read at 450nm in an ELISA reader. No commercially available anti-deer IgA or IgM exists; thus, both anti-goat and anti-sheep were used because they gave the best cross-reactivity with cervid Ig. Saliva samples were tested at 1:20 for IgM and IgA using a similar ELISA.
In the feces supernatant extract, IgA levels to recPrP were determined in a 1:30 dilution of the extract in PBST, using the above protocol.
In the feces supernatant extract, IgA levels to recPrP were determined in a 1:30 dilution of the extract in PBST, using the above protocol.