title: Preparation and neutralization efficacy of IgY antibodies raised againstDeinagkistrodon acutusvenom.
abstract: BACKGROUND
  The five-paced pit viper (Deinagkistrodon acutus), endemic to China and northern Vietnam, is responsible for most snakebites in the Chinese territory. Antivenom produced from horses is the main treatment for snakebites, but it may cause numerous clinical side effects and have other disadvantages involved in their production such as the welfare of animals. The present study was conducted aiming to develop an alternative antibody (IgY) from the egg yolk of leghorn chickens immunized with snake venom.
  
  
  METHODS
  IgY from the egg yolk of white leghorn chickens previously immunized intramuscularly withD. acutusvenom was extracted by water, precipitated by ammonium sulfate and purified by affinity chromatographic system. IgY was identified by SDS-PAGE, ELISA and Western blot. Finally, IgY neutralization assays to test its efficacy against hemorrhagic, edema-forming and myotoxic activities ofD. acutusvenom were conducted on mice.
  
  
  RESULTS
  For the first time, IgY antibodies againstD. acutusvenom were raised successfully in egg yolk of chickens injected withD. acutusvenom multiple times. By three steps, including caprylic acid extraction, ammonium sulfate precipitation and affinity chromatography, IgY antibodies were isolated and purified from egg yolk, which exhibited a single protein band on SDS-PAGE and two bands (about 65 kDa and 35 kDa, respectively) under reducing conditions, and presented a high titer (1:40,000) tested by ELISA. Immunoblot analysis confirmed that these IgY were polyclonal antibodies since they bound to components ofD. acutusvenom. Furthermore, immunodiffusion assay showed that anti-D. acutusvenom IgY cross-reacted with the venoms ofTrimeresurus albolabrisandD. saxatilisEmelianov, but did not react to the venoms ofBungarus multicinctusandNaja atra. In the neutralizing lethal assay, the median effective dose of anti-D. acutusvenom IgY was 14.14 mg/kg of mouse body weight under the challenge dose (3 LD50ofD. acutusvenom). In neutralizing the hemorrhagic, edema-forming and myotoxic activities ofD. acutusvenom, IgY showed the characteristic dose-dependent neutralization effects against all these toxic activities ofD. acutusvenom.
  
  
  CONCLUSIONS
  Anti-D. acutusvenom IgY antibodies with high purity and titer were for the first time raised successfully in egg yolk of chickens immunized withD. acutusvenom. They were effective in neutralizing the lethal effects, and the hemorrhagic, edema-forming and myotoxic acitivities ofD. acutusvenom. IgY could be an effective source to develop a treatment against snake bites in humans or animals in the future.
authors.affiliation: Animal Toxin Group, Chongqing Key Laboratory of Animal Biology, Chongqing Engineering Research Center of Bioactive Substances, Engineering Research Center of Active Substances and Biotechnology, Ministry of Education, College of Life Science, Chongqing, 401331 China.
authors.forename: Jinhua
authors.initials: J
authors.lastname: Liu
authors.affiliation: Animal Toxin Group, Chongqing Key Laboratory of Animal Biology, Chongqing Engineering Research Center of Bioactive Substances, Engineering Research Center of Active Substances and Biotechnology, Ministry of Education, College of Life Science, Chongqing, 401331 China.
authors.forename: Qiyi
authors.initials: Q
authors.lastname: He
authors.affiliation: Animal Toxin Group, Chongqing Key Laboratory of Animal Biology, Chongqing Engineering Research Center of Bioactive Substances, Engineering Research Center of Active Substances and Biotechnology, Ministry of Education, College of Life Science, Chongqing, 401331 China.
authors.forename: Wenwen
authors.initials: W
authors.lastname: Wang
authors.affiliation: Library, Chongqing Normal University, Chongqing, 401331 China.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhou
authors.affiliation: Animal Toxin Group, Chongqing Key Laboratory of Animal Biology, Chongqing Engineering Research Center of Bioactive Substances, Engineering Research Center of Active Substances and Biotechnology, Ministry of Education, College of Life Science, Chongqing, 401331 China.
authors.forename: Bo
authors.initials: B
authors.lastname: Li
authors.affiliation: Animal Toxin Group, Chongqing Key Laboratory of Animal Biology, Chongqing Engineering Research Center of Bioactive Substances, Engineering Research Center of Active Substances and Biotechnology, Ministry of Education, College of Life Science, Chongqing, 401331 China.
authors.forename: Yingfeng
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Animal Toxin Group, Chongqing Key Laboratory of Animal Biology, Chongqing Engineering Research Center of Bioactive Substances, Engineering Research Center of Active Substances and Biotechnology, Ministry of Education, College of Life Science, Chongqing, 401331 China.
authors.forename: Cong
authors.initials: C
authors.lastname: Luo
authors.affiliation: Animal Toxin Group, Chongqing Key Laboratory of Animal Biology, Chongqing Engineering Research Center of Bioactive Substances, Engineering Research Center of Active Substances and Biotechnology, Ministry of Education, College of Life Science, Chongqing, 401331 China.
authors.forename: Diancheng
authors.initials: D
authors.lastname: Chen
authors.affiliation: Animal Toxin Group, Chongqing Key Laboratory of Animal Biology, Chongqing Engineering Research Center of Bioactive Substances, Engineering Research Center of Active Substances and Biotechnology, Ministry of Education, College of Life Science, Chongqing, 401331 China.
authors.forename: Jia
authors.initials: J
authors.lastname: Tang
authors.affiliation: Animal Toxin Group, Chongqing Key Laboratory of Animal Biology, Chongqing Engineering Research Center of Bioactive Substances, Engineering Research Center of Active Substances and Biotechnology, Ministry of Education, College of Life Science, Chongqing, 401331 China.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Yu
keywords: Deinagkistrodon acutus; Egg yolk; IgY antibody; Snake venom; Snakebite; Venom neutralization