Production Of Egg Yolk Antibodies Research Articles

Production of egg yolk antibody (IgY) against a chimeric protein containing IpaD, StxB, and TolC antigens from Shigella: An investigation of its prophylactic effects against Shiga toxin (Stx) and Shigella dysenteriae in vitro and in vivo

Shigella is a major problem in developing countries. Immunoglobulin Y (IgY) can be used for prophylaxis and neutralize bacteria. The aim of this study was to produce IgY against the chimeric protein containing IpaD, StxB, and TolC antigens from Shigella, investigate its prophylactic and neutralizing effects against Stx and Shigella dysenteriae. The nucleotide sequence corresponding to the chimeric protein was cloned into pET28a plasmid and expressed in E. coli BL21 (DE3). Protein expression was confirmed by SDS-PAGE and the recombinant protein was purified by Ni-NTA affinity chromatography. The 150 μg of chimeric protein was mixed with Freund's adjutant and injected into laying hens (Leghorn). IgY was purified using PEG6000 precipitation. Antibody titer in the serum and egg yolk was evaluated by ELISA. IgY challenge against 1,10 and 50 LD50 of Stx and S. dysenteriae was investigated. A 60.6 kDa recombinant protein was confirmed by SDS-PAGE. ELISA showed that the antibody titer was significantly increased. MTT assay [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] showed that at 16 μmol/L, IgY protected HeLa cells against Stx. Treatment of mice with 1000 and 1500 μg IgY leads to complete survival of the mice against 1LD50 toxin and 4000 μg of IgY led to complete survival against 1LD50, also 70% and 30% survival against 10 and 50 LD50S. dysenteriae. This study showed that IgY produced against Stx and Shigella virulence factors could cause high protective effects against bacteria and toxins.

Production of egg yolk antibody against A.fumigatus and its therapeutic potential for treating A.fumigatus keratitis

ObjectiveTo prepare specific IgY against A. fumigatus and verify its specificity and antifungal effect on A. fumigatus keratitis. MethodLay hens were immunized with the suspension of inactivated A. fumigatus hyphae which mixed with Freund's complete adjuvant or incomplete Freund's adjuvant. The IgY protein specific for A. fumigatus was extracted by ammonium sulfate salting-out method at the fifth to eighth week after immunization. Bradford method and indirect ELISA were used to determine the concentration and titer of IgY. To verify the inhibitory effect of specific IgY on fungal growth, 1 × 105 CFU/mL A. fumigatus hyphae suspension and specific IgY of different concentrations were mixed and cultured for 24 h, 48 h and 72 h to measure the absorbance. Using specific IgY to treat A. fumigatus keratitis in mice, we observed the cornea under a slit lamp at 24 h, 72 h, and 120 h after treatment. Clinical score was used to assess the disease severity of fungal keratitis in mice cornea. The indirect ELISA method was used to determine the titer of specific IgY stored at room temperature and 4 °C for 1, 2, 3, 4, 5, 6, 7, 8, and 9 months. ResultsThe protein concentrations of specific IgY at the fifth, sixth, seventh and eighth weeks after immunization were 5.46 mg/mL, 5.79 mg/mL, 26.98 mg/mL, 28.71 mg/mL. The titer of the specific IgY of A. fumigatus can reach 1:10000, and the antifungal effect of the specific IgY is dose dependent within a certain range. Specific IgY treatment alleviated the severity of fungal keratitis of mice and reduced the clinical score. Moreover, there were no significant change in the titer of specific IgY after storage at room temperature for 2 months and storage at 4 °C for 6 months. ConclusionThe specific IgY can be successfully prepared by ammonium sulfate salting-out method. And it has excellent stability and significant antifungal effect on A. fumigatus keratitis.

Production of egg yolk antibody (IgY) against shiga-like toxin (stx) and evaluation of its prophylaxis potency in mice

ObjectiveEnterohemorrhagic Escherichia coli (E. coli O157: H7) is an enteric pathogen, transmitted through contaminated water and food. Pathogenic factors include bacterial adhesion, invasion of intestinal epithelial and epithelium cells. The pathogenicity of EHEC is due to the production of Shiga-like toxin (Stx). This toxin binds to the ribosome and inhibits the synthesis of proteins. EHEC causes hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). The EHEC treatment with antibiotics leads to resistance. The best way to solve this problem is to use specific antibodies and prophylaxis. Egg yolk antibody (IgY) is a suitable method for prophylaxis. Hence, the aim of this study was to investigate the production of IgY against Stx toxin and its prophylaxis. ResultThe produced antibodies were confirmed by SDS-PAGE and ELISA. IgY was obtained at a concentration of about 5 mg/ml (30 mg of each egg) and a purity of more than 90%. Toxin and antibody challenge was performed in mice. The obtained IgY was able to neutralize the effect of Stx at 2 mg/mice. ConclusionThis challenge showed that an antibody produced with an acceptable percentage was able to neutralize the effect of Stx.

Production of chicken hyperimmune egg for immunological purposes in animals

The present study was aimed to produce hyperimmune chicken egg against rabbit hemorrhagic disease virus (RHDV) as well as studying immunogenic characters of the extracted IgY using antigen-antibody reaction tests (agar gel immunodiffusion and ELISA). The study was carried out on ten white Hy-Line layers which received initial immunization of 300µl of RHDV vaccine subcutaneously followed by three booster doses at time intervals of 2, 5 and 8 weeks after initial immunization. Throughout the experiment (14 weeks), regular blood sampling was made every week with daily collection of egg till further purification and detection of the purity of the isolated IgY by SDS-PAGE. Also, determination of the specificity and titers of purified IgY in hens' sera and their purified egg yolk samples was carried out using agar gel immunodiffusion test and indirect ELISA. Results showed presence of considerable titers of isolated IgY against RHDV. Therefore, the current study recommends immunization of hens with RHDV for production of egg yolk antibodies against such pathogen.

Shrimp protected from WSSV disease by treatment with egg yolk antibodies (IgY) against a truncated fusion protein derived from WSSV

White spot syndrome virus (WSSV) is a virulent pathogen known to affect penaeid shrimp. It has a bacilliform morphology and a tail-like appendage at one end. The virus particle consists of at least four major proteins. VP28 and VP19 reside in the envelope, while VP26 and VP24 are present in the nucleocapsid. Truncated VP28 (TrVP28) gene was fused to a truncated VP19 (TrVP19) to form TrVP28:19. This together with TrVP28, TrVP24, and TrVP26 were expressed in Escherichia coli. The panel of resulting recombinant proteins was immunoblotted with serum from rabbit anti-glutathione S-transferase (GST), rabbit anti-WSSV, and normal rabbit serum. TrVP28:19 immunoreacted strongly with rabbit anti-WSSV. When TrVP28:19 was used as an antigen, dramatically increased titres against it were obtained when compared to other antigens for the production of egg yolk antibodies (IgY). The fusion protein was used as an immunogen with chickens. Shrimp used in WSSV neutralization assays was P. chinensis. Shrimp treated with 0.01 mg/10 μl of IgY exhibited a survival rate of 50% at day 15 post-challenge with WSSV while those treated with 0.1 and 0.5 mg/μl exhibited survival rates of 85% and 83%, respectively. The results showed that WSSV can be neutralized by the IgY against TrVP28:19 and that it has potential for immunotherapeutic application to prevent WSSV infection in P. chinensis.

Production of an egg yolk antibody specific to microbial uricase and its inhibitory effects on uricase activity

Ammonia gas produced from poultry manure can be a potential source of environmental pollution. Microbial uricase in poultry manure is an important target enzyme to reduce ammonia production because ammonia is mainly generated from the microbial decomposition of uric acid in the manure. Thus, the inhibition of microbial uricase is critical in preventing NH3 volatilization. A potential method of inhibiting uricase activity is the use of antibodies specific to microbial uricase. Therefore, this study was designed to evaluate 1) the production of the uricase-specific egg yolk antibody (IgY) from immunized hens and 2) the effect of the uricase-specific IgY on the activity of uricase. A total of 12 Single Comb White Leghorn hens were injected intramuscularly with uricase from Arthrobacter globiformis. The hens were immunized a second and third time at 1 and 2 wk, respectively, after the initial injection. The production of uricase-specific IgY was first detected at 2 wk after the initial immunization, and levels increased more than threefold at 4 wk. The method including water extraction, ammonium sulfate precipitation, and ethanol precipitation showed the most acceptable IgY purity and over 97% uricase-specific IgY recovery. Finally, the effect of the uricase-specific IgY on uricase activity was determined by a uricase assay. The slope ratio showed that the immune-IgY from the uricase-immunized hens and nonimmune IgY from nonimmunized control hens reduced uricase degradation by 58 and 43%, respectively, compared to the uricase treatment. The regression slope indicated that the immune IgY and nonimmune IgY treatments had a significant inhibitory effects on uricase activity compared to the uricase.

Effective methods for the production of immunoglobulin Y using immunogens of Bordetella bronchiseptica, Pasteurella multocida and Actinobacillus pleuropneumoniae

Swine respiratory diseases induce severe economic losses in the swine industry worldwide. Several methods have been developed and applied to control these diseases. However, there are still problems of disease control in the swine industry. Recently, egg yolk antibodies have been found to offer several advantages for disease control in animals and humans. In a previous study (24), antibodies to several causative pathogens of swine respiratory diseases were developed. However, several problems remained, especially in terms of reduced laying rates. Therefore, experimental vaccines were reformulated with various bacterial antigens of the swine respiratory diseases. After immunizing hens with the antigens, antibody profiles and other effects including laying rates were investigated and compared to those of the previous study. Profiles of antibody titers were very similar with those of the previous study. However, side effects, such as depression, weakness, reduction of laying rates and mortality, were dramatically lowered and laying rates were increased in hens injected with certain experimental vaccines. In particular, laying rates of hens injected with vaccines against atrophic rhinitis were increased up to 84% by injecting a vaccine composed of only the DNTs of B. bronchiseptica and P. multocida D:4. Efficacies of the vaccines against swine pneumonic pasteurellosis and pleuropneumonia were very similar with those of the previous study. These results suggest that new vaccines could be effective in the production of egg yolk antibodies against the causative agents of swine respiratory diseases.

鶏卵抗体の大量生産および産業利用技術の開発

鶏卵抗体の大量生産および産業利用技術の開発