In vivo pharmacokinetic study

Mouse experiments were performed at Stanford University in compliance with ethical regulations approved by the Administrative Panel on Laboratory Animal Care (APLAC) under protocol 31511. Mice were housed in the veterinary service center facility at Stanford where they were kept at a constant temperature and humidity, exposed to 12 h cycles of alternating light and dark, and were continuously provided water and standard rodent food. Female BALB/c (6–8 weeks old, Jackson Laboratory) were injected intraperitoneally with 5 mg/kg Ctx or Ctx conjugates. Blood was collected at 6, 24, 48, 72h via tail-bleed and plasma was separated. 2 μl of plasma was loaded onto SDS-PAGE (4–12% Bis-Tris gel) and transferred onto a nitrocellulose membrane for detecting human antibody presence (800CW goat anti-human, LI-COR). Organs were harvested at 72 hours, lysed with RIPA buffer supplemented with protease inhibitor and 0.1% Benzonase. Protein concentration was determined using BCA and 50 μg of total protein were loaded onto SDS-PAGE (4–12% Bis-Tris gel) and transferred onto a nitrocellulose membrane for detecting human antibody presence (800CW goat anti-human, LI-COR).