Rabbit anti deer hrp

PK and Pefabloc (PK inhibitor) were commercially obtained from (Roche Applied Science, Germany). Primary antibodies used were as follows: anti-PrP mAb 4H11 (47 (link)) anti-PrP mAb 8H4 (Sigma), anti-PrP mAb BAR224 (Cayman Chemical), and anti-β-actin (Sigma). As secondary peroxidase-conjugated antibodies, goat anti-mouse HRP (Jackson ImmunoResearch) and rabbit anti-deer HRP (Kirkegaard & Perry Laboratories) were used.

ELISA was done following the procedure described previously (29 (link)). Briefly, 1 μg of recombinant protein in sodium-carbonate buffer (pH 9.5) was used to coat the wells of high-binding 96-well plates (Greiner Bio-One GmbH-Frickenhausen-Germany) for overnight. Blocking was done using 3% bovine serum albumin (BSA) in PBST for 2 h at 37 °C after washing with PBST. A serial dilution of sera in 3% BSA was prepared, and plates were incubated with sera for 1 h. Following the washing step, either HRP-labeled anti-mouse IgG antibody (Jackson ImmunoResearch, West Grove, PA) or rabbit anti-deer HRP (Kirkegaard & Perry Laboratories) was added as secondary antibody. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) peroxidase (Kirkegaard & Perry Laboratories) was used as substrate for signal detection, and OD was measured at 405 nm using a BioTek Synergy HT reader.