Preparation and neutralization efficacy of IgY antibodies raised against Deinagkistrodon acutus venom

Journal of venomous animals and toxins

Jia Tang¹  Bo Li¹  Cong Luo¹  Yingfeng Zhang¹  Xiaodong Yu¹  Qiyi He¹  Jinhua Liu¹  Diancheng Chen¹  Wenwen Wang¹  Bin Zhou² 

[1] 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, China;Library, Chongqing Normal University, Chongqing, China

关键词: IgY antibody;    Egg yolk;    Snake venom;    Snakebite;    Deinagkistrodon acutus;    Venom neutralization;   

DOI  :  10.1186/s40409-017-0112-0

学科分类：药理学

来源: BioMed Central

【 摘 要 】

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. IgY from the egg yolk of white leghorn chickens previously immunized intramuscularly with D. acutus venom 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 of D. acutus venom were conducted on mice. For the first time, IgY antibodies against D. acutus venom were raised successfully in egg yolk of chickens injected with D. acutus venom 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 of D. acutus venom. Furthermore, immunodiffusion assay showed that anti-D. acutus venom IgY cross-reacted with the venoms of Trimeresurus albolabris and D. saxatilis Emelianov, but did not react to the venoms of Bungarus multicinctus and Naja atra. In the neutralizing lethal assay, the median effective dose of anti-D. acutus venom IgY was 14.14 mg/kg of mouse body weight under the challenge dose (3 LD50 of D. acutus venom). In neutralizing the hemorrhagic, edema-forming and myotoxic activities of D. acutus venom, IgY showed the characteristic dose-dependent neutralization effects against all these toxic activities of D. acutus venom. Anti-D. acutus venom IgY antibodies with high purity and titer were for the first time raised successfully in egg yolk of chickens immunized with D. acutus venom. They were effective in neutralizing the lethal effects, and the hemorrhagic, edema-forming and myotoxic acitivities of D. acutus venom. IgY could be an effective source to develop a treatment against snake bites in humans or animals in the future.

【 授权许可】

CC BY