IgY Technology Research Articles

Environmental and occupational respiratory diseases – 1034. A novel ELISA method for the detection of dust mite allergens using IgY technology

Background One of the main strategies to reduce the expression of allergic symptoms in atopic patients sensitized to house dust mites is the use of detection system for monitoring and reduce environmental allergens in indoor spaces. In this context the IgY technology represent a valuable tool. The specific aims of this study was to standardize a in house method of indirect ELISA assay for detection of mite allergens using mite IgY antibodies.

Chicken Egg Yolk Antibody (IgY) Controls Solobacterium moorei Under In Vitro and In Vivo Conditions

Solobacterium moorei is a causative agent in diseases such as oral halitosis, bacteremia, and necrobacillosis-associated thrombophlebitis. The objective of this study was to determine the effectiveness of chicken egg yolk antibody (IgY) in controlling S. moorei. Intact S. moorei cells were used as an immunogen to immunize four White Leghorn laying hens. IgY, extracted from egg yolks obtained from these immunized hens, was purified using water dilution, two-step salt precipitation, and ultrafiltration. The purity of the IgY obtained was approximately 87.3 %. The antibody titer of the IgY was determined by enzyme-linked immunosorbent assay. The antibody titer peaked at 10,000 following the third immunization. In order to evaluate the inhibitory effects of the specific IgY, the growth of S. moorei in liquid media was measured every 12 h using a microplate reader at 600 nm. Biofilm formation of S. moorei was quantified by staining with crystal violet. The specific binding ability of IgY was further confirmed by the use of immunofluorescence and immunoelectron microscopy. Growth and biofilm formation of S. moorei were significantly (P<0.05) inhibited by 20 and 40 mg/ml specific IgY compared with the control. The specific IgY also decreased the bacterial level in the oral cavity of mice after infection with S. moorei. This study demonstrates that the growth and biofilm formation of S. moorei can be effectively inhibited by specific IgY. As a result, IgY technology may have application in the control of diseases caused by S. moorei.

Brucella spp. Lumazine synthase as a novel immunomodulator to produce egg yolk antibodies

Lumazine synthase from Brucella spp. (BLS) is a highly immunogenic decameric protein. It has been previously described as a carrier of peptides or proteins to increase their immunogenicity in different animal species, but its activity has never been evaluated in chickens. In this work, the use of BLS to improve the antibody response against bovine rotavirus (BRV) VP8d protein in laying hens was assessed. VP8d is the inner domain of the VP8 spike protein which preserves the sialic acid binding activity and the neutralizing epitopes present in the viral protein. Hens were immunized three times with 2 μg of VP8d alone or fused to BLS. Hens inoculated with BLSVP8d developed higher antibody titers (evaluated by ELISA and viral neutralization test) than hens immunized either with VP8d alone or the mixture of VP8d and BLS. Furthermore, IgY antibodies against BLSVP8d were able to fully protect mice against challenge with virulent BRV in a dose-depent-manner. Overall, these results demonstrate that BLS is a potent immonumodulator that enhances the antibody response in hens, thus increasing the concentration of specific IgY in the egg yolk, one of the main issues to be adressed in order to improve the use of the IgY technology.

Antibodies anti-Shiga toxin 2 B subunit from chicken egg yolk: Isolation, purification and neutralization efficacy

Shiga toxins (Stx1 and Stx2) are the main virulence factors of enterohemorrhagic Escherichia coli (EHEC), a foodborne pathogen associated with diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. The aim of this study was to evaluate the antibodies against Stx2 obtained from egg yolks of laying hens immunized with a recombinant Stx2B subunit. A high specific response in serum was observed 25 days after the first immunization and IgY antibodies were extracted from day 47th and purified from egg yolk. A concentration of 0.84 mg of total IgY/ml of egg yolk was obtained, of which 8% were antigen specific. The ability of anti-Stx2B IgY to recognize Stx2B and Stx2 either in solid-phase or in solution were evaluated and compared with anti-Stx2B rabbit antibodies by Western blotting and ELISA. The protective efficacy of IgY against Stx2 was determined by in vitro and in vivo experiments. The results show that IgY was able to recognize Stx2B and Stx2 in denatured conditions, attached to a solid-phase and free in solution. The anti-Stx2B IgY could effectively block the biological activity of Stx2 on Vero cells and protect mice from Stx2 challenge. The data suggest that immunization of hens with Stx2B could be a strategy to obtain at low cost a relatively high concentration of anti-Stx2 egg yolk IgY, able to neutralize Stx2 lethal activity. IgY technology could be an useful tool for research, diagnosis and therapy of EHEC infection.

Neutralization effects of egg yolk immunoglobulin (IgY) and Fab' fragment against lipopolysaccharide (LPS) in burned mice model

The aim of this study is to evaluate the neutralization effects of egg yolk immunoglobulin (IgY) and Fab’ fragment against lipopolysaccharide (LPS), and identify possible approaches to prevent and treat LPS related injuries. Mice with third-degree burns covering 30% of the total body surface and exposed to LPS were orally administered with the IgY or Fab’ fragment of egg yolk immunoglobulin at 6, 12, 24 and 48 h after burning. The mortality rates and circulating levels of LPS, tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) were determined. The mortality rate of mice in the IgY-treated groups and Fab’treated groups were significantly lower when compared with the control groups (without treatment). The levels of LPS, TNF-α and IL-6 in the IgY-treated group and Fab’-treated group were lower than those in the control group, and Fab’-treated group posses much lower levels of LPS, TNF-α and IL-6 than IgYtreated group. The IgY and Fab’ fragment of egg yolk immunoglobulin raised against LPS may be used to inhibit and treat tissue injury caused by LPS. IgY technology has significant future opportunities for the prevention and treatment of LPS related infectious gastrointestinal diseases in humans and animals.

Egg yolk IgY: Protection against rotavirus induced diarrhea and modulatory effect on the systemic and mucosal antibody responses in newborn calves

Bovine rotavirus (BRV) is an important cause of diarrhea in newborn calves. Local passive immunity is the most efficient protective strategy to control the disease. IgY technology (the use of chicken egg yolk immunoglobulins) is an economic and practical alternative to prevent BRV diarrhea in dairy calves. The aim of this study was to evaluate the protection and immunomodulation induced by the oral administration of egg yolk enriched in BRV specific IgY to experimentally BRV infected calves. All calves in groups Gp 1, 2 and 3 received control colostrum (CC; BRV virus neutralization Ab titer – VN = 65,536; ELISA BRV IgG 1 = 16,384) prior to gut closure. After gut closure, calves received milk supplemented with 6% BRV-immune egg yolk [(Gp 1) VN = 2048; ELISA IgY Ab titer = 4096] or non-immune control egg yolk [(Gp 2) VN < 4; ELISA IgY Ab titer < 4] twice a day, for 14 days. Calves receiving CC only or colostrum deprived calves (CD) fed antibody (Ab) free milk served as controls (Gp 3 and 4, respectively). Calves were inoculated with 10 5.85 focus forming units (FFU) of virulent BRV IND at 2 days of age. Control calves (Gp 3 and 4) and calves fed control IgY (Gp 2) were infected and developed severe diarrhea. Around 80% calves in Gp 1 (IgY 4096) were infected, but they showed 80% (4/5) protection against BRV diarrhea. Bovine RV-specific IgY Ab were detected in the feces of calves in Gp 1, indicating that avian antibodies (Abs) remained intact after passage through the gastrointestinal tract. At post infection day 21, the duodenum was the major site of BRV specific antibody secreting cells (ASC) in all experimental groups. Mucosal ASC responses of all isotypes were significantly higher in the IgY treated groups, independently of the specificity of the treatment, indicating that egg yolk components modulated the immune response against BRV infection at the mucosal level. These results indicate that supplementing newborn calves’ diets for the first 14 days of life with egg yolk enriched in BRV-specific IgY represents a promising strategy to prevent BRV diarrhea. Moreover a strong active ASC immune response is induced in the intestinal mucosa following BRV infection after the administration of egg yolk, regardless the specificity of the treatment.

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

Hens can be immunized by means of i.m. vaccination (Musculus pectoralis, left and right, injection volume 0.5-1.0 ml) or by means of Gene-Gun plasmid-immunization. Dependent on the immunogenicity of the antigen, high antibody-titres (up to 1:100,000 - 1:1,000,000) can be achieved after only one or 3 - 4 boost immunizations. Normally, a hen lays eggs continuously for about 72 weeks, thereafter the laying capacity decreases. This protocol describes the extraction of total IgY from egg yolk by means of a precipitation procedure (PEG. Polson et al. 1980). The method involves two important steps. The first one is the removal of lipids and the second is the precipitation of total IgY from the supernatant of step one. After dialysis against a buffer (normally PBS) the IgY-extract can be stored at -20°C for more than a year. The purity of the extract is around 80 %, the total IgY per egg varies from 40-80 mg, dependent on the age of the laying hen. The total IgY content increases with the age of the hen from around 40 mg/egg up to 80 mg/egg (concerning PEG precipitation). The laying capacity of a hen per year is around 325 eggs. That means a total potential harvest of 20 g total IgY/year based on a mean IgY content of 60 mg total IgY/egg (see Table 1).

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

Hens can be immunized by means of i.m. vaccination (Musculus pectoralis, left and right, injection volume 0.5-1.0 ml) or by means of Gene-Gun plasmid-immunization. Dependent on the immunogenicity of the antigen, high antibody-titres (up to 1:100,000 - 1:1,000,000) can be achieved after only one or 3 - 4 boost immunizations. Normally, a hen lays eggs continuously for about 72 weeks, thereafter the laying capacity decreases. This protocol describes the extraction of total IgY from egg yolk by means of a precipitation procedure (PEG. Polson et al. 1980). The method involves two important steps. The first one is the removal of lipids and the second is the precipitation of total IgY from the supernatant of step one. After dialysis against a buffer (normally PBS) the IgY-extract can be stored at -20°C for more than a year. The purity of the extract is around 80 %, the total IgY per egg varies from 40-80 mg, dependent on the age of the laying hen. The total IgY content increases with the age of the hen from around 40 mg/egg up to 80 mg/egg (concerning PEG precipitation). The laying capacity of a hen per year is around 325 eggs. That means a total potential harvest of 20 g total IgY/year based on a mean IgY content of 60 mg total IgY/egg (see Table 1).

Chicken IgY: utilizing the evolutionary advantage

Laying hens are highly cost-effective producers of polyclonal antibodies in comparison with mammals as the antibodies can be purified from egg yolk. The antibodies are called IgY to differentiate them from mammalian IgG antibodies. Avian antibodies represent both a reduction and refinement in animal use. The cost for producing IgY is lower than for mammalian antibodies as chicken housing is cheap and the isolation/separation process is economical, high yielding, uncomplicated and fast.IgY antibodies do not cross-react with Fc receptors, mammalian immunoglobulins such as RF and HAMA or activate the complement system and thereby reducing the cause of false positive results in immunological assays. These properties have also made them attractive for peroral immunotherapy for a variety of gastrointestinal diseases, such as human rotaviruses, enterotoxigenic Escherichia coli and Salmonella spp. In the future the IgY technology offers great opportunities to be more widely used in immunotherapy, diagnostics and research.

Passive Immunization Through Avian Egg Antibodies

Oral administration of specific antibodies is an attractive approach to establish passive immunization against various pathogens in humans and animals. Egg yolk contains over 100 mg of IgY per egg and is considered to be a more hygienic and economical source of antibodies, than antibodies from mammalian sera. Recently, considerable research has focussed on the use of IgY as an alternative to mammalian antibodies for several applications, most notably for oral passive immunization against various bacteria and viruses. Oral administration of IgY has proven to be successful for the treatment of a variety of gastrointestinal (GI) pathogens such as bovine and human rotaviruses, bovine coronavirus, Yersinia ruckeri, enterotoxigenic Escherichia coli, Salmonella spp., Edwardsiella, Staphylococcus, and Pseudomonas. Recent advances in hybridoma technology, for the production of monoclonal IgY, also present many benefits for its universal application. IgY technology has significant future opportunities for the design of strategies for the prevention and treatment of infectious gastrointestinal diseases in humans and animals.

Chicken egg yolk antibodies as therapeutics in enteric infectious disease: a review.

Passive immunization by oral administration of specific antibodies has been an attractive approach against gastrointestinal (GI) pathogens in both humans and animals. Recently, laying chickens have attracted considerable attention as an alternative source of antibodies for the prevention and treatment of infectious GI diseases. After immunization, the specific antibodies (called IgY) are transported to the egg yolk, from which the IgY then can be separated without sacrificing chickens. A chicken usually lays about 280 eggs in a year, and egg yolk contains 100-150 mg of IgY per yolk, suggesting that more than 40 g of IgY per year can be obtained from each chicken through eggs. IgY is also an alternative to antibiotics for treatment of enteric antibiotic-resistant pathogens. Oral administration of IgY has proved successful for treatment of a variety of GI infections, such as bovine and human rotaviruses, bovine coronavirus, Yersinia ruckeri, enterotoxigenic Escherichia coli, Salmonella spp., Edwardsiella tarda, Staphylococcus, and Pseudomonas. The IgY technology offers great future opportunities for designing prophylactic strategies against infectious GI diseases in humans and animals. However, there is still controversy regarding the stability of IgY through the GI tract. Finding an effective way to protect the antibodies from degradation in the GI tract would open the door for significant advances in IgY technology and nutraceutical applications.

Comparison between ‘IgY technology’ from chickens and ‘IgG technology’ from mice for production of tailor-made antibodies

Antibodies collected from chickens immunized towards a β-ketoalcohol, key intermediate in the synthesis of chrysanthemic acid, which have been shown to possess similar behavior to those elaborated from mice, offer the enormous advantages of no bleeding and access to large quantities of antibodies by easy extraction of the yolk of the eggs.