Production and evaluation of egg derived hot start antibodies

BACKGROUND: Hot start can greatly improve specificity, sensitivity and yield of PCR. Non-specific amplification can occur in PCR when reaction mixture is prepared at room temperature, because Taq DNA polymerase is active and the primers can hybridize non-specifically. Hot start Taq DNA polymerases remain inactive at room temperature and are activated after heating at 95°C preventing non-specific amplification. Monoclonal antibodies against Taq DNA polymerase is the first line of reagents used for turn on regular Taq DNA polymerase into Hot start one. The goal of this research was to produce and evaluate Hot Start antibodies derived from chicken eggs. RESULTS: We performed affinity purification of yolk immunoglobulin (IgY) and obtained polyclonal Hot Start antibodies. The yield of specific antibodies was 0.36 mg per egg or 0.2% of total yolk antibodies. The protocol for real time measurement and Hot start IgY activity assessment was developed. We found that Hot start IgY can reversibly block Taq DNA polymerase activity at 50°C and have no negative impact neither on the Taq DNA polymerase activity after denaturation nor on the reverse transcriptase. We estimated that 1.0 µg of Hot start IgY effectively blocks 5 U activity of Taq DNA polymerase. CONCLUSIONS: Egg derived Hot Start polyclonal antibodies are the cheapest source of Hot start antibodies, from one immune egg we can isolate 0.36 mg IgY, this quantity is enough for producing 1800 U activity of Hot start Taq DNA Polymerase.

Chicken antibodies against venom proteins of Trimeresurus stejnegeri in Taiwan

The venom of bamboo vipers (Trimeresurus stejnegeri - TS), commonly found in Taiwan, contains deadly hemotoxins that cause severe envenomation. Equine-derived antivenom is a specific treatment against snakebites, but its production costs are high and there are some inevitable side effects. The aim of the present work is to help in the development of an affordable and more endurable therapeutic strategy for snakebites. Methods: T. stejnegeri venom proteins were inactivated by glutaraldehyde in order to immunize hens for polyclonal immunoglobulin (IgY) antibodies production. After IgY binding assays, two antibody libraries were constructed expressing single-chain variable fragment (scFv) antibodies joined by the short or long linker for use in phage display antibody technology. Four rounds of biopanning were carried out. The selected scFv antibodies were then further tested for their binding activities and neutralization assays to TS proteins. Results: Purified IgY from egg yolk showed the specific binding ability to TS proteins. The dimensions of these two libraries contain 2.4 × 107 and 6.8 × 107 antibody clones, respectively. An increase in the titers of eluted phage indicated anti-TS clones remarkably enriched after 2nd panning. The analysis based on the nucleotide sequences of selected scFv clones indicated that seven groups of short linkers and four groups of long linkers were identified. The recombinant scFvs showed significant reactivity to TS venom proteins and a cross-reaction to Trimeresurus mucrosquamatus venom proteins. In in vivo studies, the data demonstrated that anti-TS IgY provided 100% protective effects while combined scFvs augmented partial survival time of mice injected with a lethal amount of TS proteins. Conclusion: Chickens were excellent hosts for the production of neutralization antibodies at low cost. Phage display technology is available for generation of monoclonal antibodies against snake venom proteins. These antibodies could be applied in the development of diagnostic kits or as an alternative for snakebite envenomation treatment in the near future.(AU)

Gallus gallus domesticus: immune system and its potential for generationof immunobiologics / Gallus gallus domesticus: sistema imunológico e seu potencial para geração de imunobiológicos

ABSTRACT: Gallus gallus domesticus' immune system is a promising tool for generation of antibody-based immunobiologics. Immunoglobulin y (IgY) is extracted from egg yolk and has equivalent functions to mammal's igg antibody. Avian immune system can be stimulated to produce a high-quality antibody repertoire. In this review, we present an overview of avian immune system emphasizing igy and its applications as an immunobiologic.

RESUMO: O sistema imunológico deGallus gallus domesticus é uma ferramenta promissora para a geração de imunobiológico a partir de anticorpos. A imunoglobulina Y (IgY) é extraída da gema do ovo e apresenta funções equivalentes ao anticorpo IgG dos mamíferos. O sistema imune aviário pode ser estimulado para produzir um repertório de anticorpos de alta qualidade. Nesta revisão apresentamos aspectos gerais do sistema imune aviário enfatizando o IgY e suas aplicações como um imunobiológico.

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

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. 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. 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.

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

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. 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. 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.(AU)

The experimental study on IgY against tumour necrosis factor alpha and interleukin-1beta allergic bronchial asthma of guinea pigs / 中国免疫学杂志

Objective:To investigate the mechanism of immunoglobulin Y antibodies(IgY) against tumour necrosis factor alpha(TNF-α) and Interleukin-1beta(IL-1β) in treating allergic bronchial asthma through nebulization inhalation.Methods:The allergic bronchial asthma model was established with Hartley guinea pig by ovalbumin nebulizating inhalation.The animals were randomly divided into 4 groups: normal control group(group A)，allergic bronchial asthma group(group B),0.1% anti-TNF-α and IL-1β IgY treating group(group C),1.0% anti-TNF-α and IL-1β IgY treating group (group D).The animals were killed after treatment being accomplished for 2 h,4 h,8 h,24 h and the lungs were made pathological,which were then stained by hematoxylin-eosin(H.E.).The bronchoalveolar lavage fluid (BALF) was collected and the deposited cells were stained by Wright's.Results:①The histological appearance of lung: In group B the histological structure of alveolar ducts and alveolar walls was damaged,the alveolar space was full of transudate and lots of alveolar epithelial cells and leucocytes.The pulmonary interstitial edema,inflammatory cells infiltration,distorted or dilated capillaries and reducing capillary numbers of effective blood stream were observed in alveolar walls.In group C and group D the damage degree of alveolar ducts and alveolar wall was slighter than that in group B and there were few inflammatory cells in alveolar space.In bronchial lumen and pulmonary alveoli the sticky mucus plug was obviously less in group C and group D than in group B.Moreover,inflammatory cell infiltration was seldom observed aroud bronchia,and restoration of bronchial tunica mucosa epithelium was obviously observed in group C and group D.②The cytology appearance of BALF: In group C and group D the numbers of eosinophils,neutrophils,lymphocytes were significantly fewer （2 h,4 h,8 h,P＜0.05）,however,the number of macrophage was significantly more（2 h,4 h,8 h,P＜0.05）than in group B.Conclusion:The anti-TNF-α and IL-1β IgY can obviously alleviate pathological extent of inflammatory reaction in allergic bronchial asthma of guinea pigs by nebulization inhalation therapy.The therapeutic effect of anti-TNF-α and IL-1β IgY between 0.1% and 1.0% concentration is not obvious difference for pathology changes.