*4.8. Rabbit Specific Antivenom Production*

The anti-venom serum from the *C. iheringi* centipede was obtained by immunization of rabbits. Two hundred micrograms of the venom and 2.5 mg of aluminum hydroxide (Brenntag Specialties, Inc., South Plainfield, NJ, USA) were added to a final volume of 1 mL of PBS. After this, 250 μL of this mixture was injected intramuscularly. After 1 month, the rabbits received five consecutive boosters of antigen with 15-day intervals. Blood was collected and sera were separated and stored at 20 ◦C until use. Antibodies present in the hyperimmune serum were purified on HiTrapProtein G HP 5 mL column pre-packed with high-performance protein G-Sepharose (GE Healthcare, Little Chalfont, UK) and quantified by BCA (QuantiProBCA Assay Kit, Sigma-Aldrich, St. Louis, MO, USA). To characterize the polyclonal anti-venom, ELISA and immunoblotting assays were performed. The experimental protocols were approved by the Butantan Institute Ethical Committee for Animal Research (certified by CEUAIB n◦ 8172250816).
