Liu et al. Journal of Venomous Animals and Toxins including Tropical Diseases (2017) 23:22 DOI 10.1186/s40409-017-0112-0 RESEARCH Open Access Preparation and neutralization efficacy of IgY antibodies raised against Deinagkistrodon acutus venom Jinhua Liu1, Qiyi He1, Wenwen Wang1, Bin Zhou2,BoLi1, Yingfeng Zhang1, Cong Luo1, Diancheng Chen1, Jia Tang1 and Xiaodong Yu1* 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 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. Results: 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.Inthe 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. Conclusion: 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. Keywords: IgY antibody, Egg yolk, Snake venom, Snakebite, Deinagkistrodon acutus, Venom neutralization * Correspondence: [email protected] 1Animal 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 Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. Journal of Venomous Animals and Toxins including Tropical Diseases (2017) 23:22 Page 2 of 9 Background IgY, like mammalian IgG, is a reasonable stable Snake envenomation is an important public health issue protein over time; in the world. There are more than 420 species of ven- the yield of IgY is large and its production may be omous snakes, which are widely distributed throughout readily scalable [17–20]. the globe (including oceans), except for a few islands, frozen environments and high-altitude regions. These In this study, hens were hyperimmunized with low animals provokes 5.4 million bites, about 2.5 million of doses of D. acutus venom to produce specific antibodies. envenomation cases and over 0.125 million of deaths IgY was extracted from egg yolk and further purified by annually. As is well known, the highest burden of affinity chromatography. Subsequently, we assessed and snakebites is found in South Asia, Southeast Asia and examined the purity, the binding specificity, the titer and sub-Saharan Africa [1–4]. In addition, the burden of the neutralization efficiency of IgY. All the results will snakebites is heavy in China, where there are over 50 provide a basis for developing IgY into a clinical agent in species of venomous snakes. One species, the five- the future. paced pit viper (Deinagkistrodon acutus), is responsible for most snakebites in the country. It is a large sized Methods and highly venomous snake, found from the southern Reagent and supplies provinces of China (including Taiwan) to northern Freund’s complete adjuvant (FCA), Freund’sincomplete Vietnam [5, 6]. adjuvant (FIA), rabbit anti-IgY peroxidase conjugate, The intravenous administration of antivenom is the Immobilon®-P (polyvinylidene difluoride, PVDF), a trans- mainstay treatment for envenomated victims. The con- fer membrane with pore size of 0.45 μm, 31,31,51,51-tetra- ventional antivenom is generally produced from the methylbenzidine (TMB), nonfat dry milk and horseradish blood of the large animals, frequently horses or sheep, peroxidase-conjugated rabbit anti-chicken IgY were pur- immunized with the venom of a single snake or mixed chased from Sigma (USA). Polystyrene ELISA plates were venoms obtained from several animals to eliminate the purchased from Corning (USA). Amicon ultra-15 centri- intraspecific diversification [7]. Nevertheless, the serum fugal filter devices were obtained from Millipore (USA). used to treat snakebite victims may cause various side NHS activated Sepharose 4 FF were purchased from GE effects, such as anaphylactic shock, pyrogen reaction and Healthcare (UK). Protein molecular weight markers were serum sickness, which are mainly caused by the nonspe- purchased from Takara (Japan). All the other reagents cific proteins found in commercially available antive- were of analytical grade. noms [8–11]. Moreover, frequent immunization and bleeding during antivenom production may cause great Venom distress to the employed animals. In addition, the pro- Venom was obtained from D. acutus captured at duction of sufficient pure serum antibodies against snake Wulingshan in Chongqing, China. Venom was lyophi- venom may require more investment and time, which lized in a ModulyoD-230 freeze dryer (Thermo Scien- may be conflicting with interests of serum manufac- tific)andstoredat−20 °C until use. turers, especially in developing countries. Thus, it is ne- cessary to find an alternative way of antibody creation, Animal which would be safer, cheaper, short-cycle and non- Seventeen-week-old white leghorn hens weighing invasive. 1.5 kg each, purchased from a local poultry farm, in In recent years, eggs of chicken immunized with anti- good health and laying conditions (laying 5 to 6 eggs gens have been recognized as a good source of IgY anti- per week) were used for the production of IgY against bodies [12–14]. So far, many research results about snake venom. They were kept in individual cages with snake antivenom IgY have demonstrated that IgY in eggs standard food and water. Kunming mice (18–20 g) were of chicken is an excellent alternative to the conventional purchased from experimental animal center of Third antivenom produced in large mammals [15, 16]. The Military Medical University. Mice were kept in plastic main advantages of IgY include: boxes at five per cage, in a room maintained at 20–23 °C on a 12/12-h light/dark cycle with food and water ad collecting eggs instead of bleeding animal meets the libitum. animal welfare requirement; the cost of keeping hens is lower than that of raising Immunization schedule horses or sheep; Based on the LD50 of D. acutus venom for mice (about IgY antibodies do not cross-react with Fc receptors, 2.93 mg/kg, intraperitoneally), the LD50 of D. acutus thereby reducing the cause of false positive results in venom for laying hens was calculated to be 0.72 mg/kg. immunological assays; Each hen was immunized intramuscularly at multiple Liu et al. Journal of Venomous Animals and Toxins including Tropical Diseases (2017) 23:22 Page 3 of 9 sites in the breast region with 0.5 mL saline (containing concentrated and desalted through Amicon Ultra-15 0.29 mg snake venom) emulsified with an equal volume centrifugal filter devices, then stored at 4 °C. of FCA. On the 14th,35th and 56th day after the first immunization, booster doses were administered with Protein concentration 0.5 mL saline (containing 0.58 mg, 1.17 mg and 1.17 mg Protein concentration was measured as described by snake venom, respectively) emulsified with an equal vol- Lowry et al. [22]. ume of FIA. Serum was collected weekly from the first immunization, but after the 10th week serum was col- SDS-PAGE lected every 2 weeks. Eggs began to be collected daily The purity and molecular weight of the IgY were ana- before the first immunization and the collecting eggs lyzed on 12% sodium dodecyl sulfate-polyacrylamide gel sustained for 24 weeks after the first immunization. The electrophoresis (SDS-PAGE), according to the method control group of hens was immunized intramuscularly of Laemmli [23].