Goat anti mouse igg conjugated to alkaline phosphatase

PMCA substrate homogenates were prepared from mice that had been perfused with PBS containing 5 mM EDTA at the time of death. PrP-null (Prnp^o/o), Tg35 (homozygous for huPrP G127/M129), Tg152 (homozygous for huPrP G127/V129) or Tg183 (homozygous for huPrP V127/M129)) mouse brains^{25 (link)} were homogenised in cold conversion buffer (PBS containing 150 mM NaCl, 1.0% (v/v) Triton X‐100, 4 mM EDTA and 1× Complete protease inhibitor (Roche Applied Science)), using a Duall tissue grinder to give a 10% (w/v) homogenate.
Substrates were seeded with a 1/100 dilution of vCJD (I4618) 10% brain homogenate in PBS. Each reaction mixture was divided in two prior to PMCA with one half stored at −70 °C as a minus PMCA control. PMCA consisted of 96 cycles of 30 s sonication every 30 min in a Misonix S3000 at 75% power output (Misonix, Farmingdale, NY), reactions were carried out with 40 µl substrate in 200-µl thin-walled PCR tubes at 35 °C.
Samples were digested with 50 µg ml⁻¹ proteinase K (PK) for 1 h at 37 °C. The reaction was stopped by the addition of AEBSF in SDS-loading buffer and samples were boiled for 10 min before running on 16% Tris-glycine gels. Western blotting was carried out according to Unit protocol, using 3F4 (Merck Inc, N.J., U.S.A) as the primary antibody and goat anti-mouse IgG conjugated to alkaline phosphatase (Sigma A2179) as the secondary antibody.

Peptide-based ELISA assays were performed as described previously [60 (link)]. Briefly, wells of 4HB NUNC plates for ELISAs were coated with 50 ng of each peptide in 100 µL of coating buffer (Na₂CO₃–NaHCO₃, pH 9.6) overnight at 4 °C. Next, the plates were washed three times using PBS-T washing buffer (PBS with 0.1% Tween^® 20, pH 7.2) and blocked using PBS-T with 2.5% BSA over 2 h at 37 °C. Next, diluted mouse sera (100 µL) in coating buffer was applied and incubated for 2 h at 37 °C. Following several washes with PBS-T, the plates were incubated for 2 h at 37 °C with 100 µL of goat anti-mouse IgG conjugated to alkaline phosphatase (Sigma, St Louis, MO, USA) diluted in coating buffer, washed, and exposed to PNPP substrate (Sigma, St Louis, MO, USA). Absorbance was measured at 405 nm on a FlexStation 3 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). Initially, the optimal serum dilution value of 1:100 was determined by a titration series of vaccinated mouse sera and by constructing the receiver operating characteristic (ROC) curve.