Chicken Egg Yolk Antibodies or Immunoglobulin Y (IgY) production Technology and its applications in healthcare Management
Eggs are laid by animals of different species, including birds. Avian eggs consist of an egg shell, egg yolk, and egg white, which constitute the embryo from which a chick develops. From day zero, the avian egg contains a powerful immune system comprising antibodies against each foreign agent to which her mother (laying hen) has been in contact with. Egg yolk possesses a high titer of immunoglobulin Y (IgY), which is equivalent to immunoglobulin G (IgG) in mammalian species, and egg white contains immunoglobulin M (IgM) and immunoglobulin A (IgA).
Immunoglobulins are glycoproteins called antibodies, which are secreted by plasma cells in response to antigen exposures and are considered a product that majorly effects humoral immunity. The property of antibodies is recognized small specific structures on other molecules which let them an indispensable tool in laboratory in various applications such as research, diagnostic and therapy. Antibodies presently available for these purposes are mostly mammalian monoclonal or polyclonal antibodies. The production of these antibodies requires normally the use of laboratory animals. Nowadays, most classical chosen mammals for polyclonal and monoclonal antibodies are rabbits and mice, respectively. The procedure involves two steps, each of which causes distress to the animals: the immunization itself and repeated bleeding or sacrifying for spleen removal, which is a prerequisite for antibodies preparation. The commercially available immunoglobulins play critical roles in diagnostic assays, therapy, and purification of specific target compounds.1 Hens egg yolk immunoglobulins or IgY have been studied intensively due to their importance. Serum IgG of the hen is transferred from the mother hen to the offspring egg yolk to acquire immunity. Antibodies are transferred from hen to chick via the latent stage of the egg, and play an important role in immunological function for the relatively immunoincompetent chick to resist various infectious diseases. Immunoglobulin is called IgY because it is present in the egg yolk and due to the differences in protein nature compared to that of the mammalian immunoglobulins.1 The greater molecular mass of IgY is due to an increased number of heavy-chain constant domains and carbohydrate chains. In addition, the hinge region of IgY is much less flexible compared to that of mammalian IgG. Recently, it has also been suggested that IgY is a more hydrophobic molecule than IgG.2 Therefore, IgY has been applied successfully in scientific, diagnostic, prophylactic, therapeutic purposes, immunochemical reagents, and in food formulation or supplements due to the stability of IgY under food processing conditions.3 In the immunodiagnostic technologies, IgY is an excellent antibody for using in immunological assays involving mammalian sera, due to discriminative properties of IgY compared to mammalian IgG, as IgY does not react with the rheumatoid factor and human anti mouse IgG antibodies do not activate the complement system and do not bind to Fc receptor.4 Also, they have poor cross reactivity to mammalian IgG due to immunological differences. IgY is usually low-cost and can be generated through convenient production processes that make it an attractive antibody for research and diagnosis. The advantages can be concluded as; IgY is produced in egg yolk; so there is no need to bleed animals, considerable amounts of antibodies can be obtained in a fairly low cost, usually rapid production process, IgY can be stored in eggs at 4 °C for at least one year, it is achievable to produce a specific antibody to small amounts of antigen that is poorly immunogenic in mammals.
IgY technology more popular and to convince the scientific community of its significant advantages. The significant potential of avian antibodies for further use in immunodiagnostics and identification of disease markers, immunotherapy and the treatment and prevention of disease is expected. Since lot of benefits of IgY technology and its universal application in both research and medicine, it is expected that IgY will play an increasing role in research.
Powdered whole eggs or yolks have been used as an inexpensive alternative for the IgY treatment of enteric diseases in veterinary medicine Kumaran et al. The most famous example of a successful therapeutic/prophylactic use of IgY is the treatment of calves and piglets with specific Abs against Escherichia coli, rotaviruses and corona virus. Studies using both animal models and trials in field herds have been carried out. These studies confirmed that treatment of diarrhea in calves and piglets with specific egg yolk Abs has achieved significant prophylactic and therapeutic benefits. Another effect of IgY binding to bacterial surface antigens is a marked impairment of bacterial attachment to the intestinal mucosa Lee et al. Thus, therapeutic IgY administration could reduce the clinical use of antibiotics, and so could lower the risk that bacteria will develop antibiotic resistance.
IgY-technology (the production and extraction of specific IgY antibodies from egg yolk) is an innovative method to produce antibodies for therapy and prophylaxis. Also,it represents an alternative to the conventional mammalian antibody production due to ethical, productive and economic advantages.
Antimicrobial resistance is a growing global challenge and IgY-technology can be used to address this issue by obtaining alternative products to antibiotics. For instance, in Canada, patients that have failed to respond to antibiotic treatments for C. difficile infection were treated with oral IgY powder for 10 days and significantly improved their clinical symptoms.
In the egg yolk several different types of antibodies can be found and monitored. The main antibody classes present in the egg yolk are:
1. IgY (Immunoglobulin Y):
– IgY is the predominant antibody isotype found in the egg yolk.
– It is the functional equivalent of mammalian IgG and is the main antibody transferred from the hen to the developing embryo and chick.
– Monitoring the levels of IgY in the egg yolk provides insight into the overall maternal antibody status and the potential passive immunity that will be conferred to the offspring.
2. IgM:
– IgM is the first antibody class produced in response to an infection or vaccination.
– Detecting the presence and levels of IgM in the egg yolk can indicate a recent exposure to a pathogen or the effectiveness of a vaccination program.
– Monitoring IgM levels can help identify early-stage immune responses in the breeder flock.
3. IgA:
– IgA is the predominant antibody class found in mucosal surfaces, such as the respiratory and digestive tracts.
– The presence of IgA in the egg yolk suggests that the hen has been exposed to pathogens that can infect the mucosal tissues.
– Monitoring IgA levels can provide insights into the hen’s local immune responses and the potential protection that may be conferred to the chicks against mucosal infections.
4. IgG:
– Although IgG is the major antibody class in mammals, it is present in relatively lower concentrations in the egg yolk compared to IgY.
– Nevertheless, monitoring IgG levels in the egg yolk can provide additional information about the maternal antibody profile and the potential protection againQst certain diseases.
Chicken egg yolk antibodies (IgY) are the functional equivalent to IgG found in mammalian serum, placenta and colostrum. In avians, the IgY antibodies are selectively transferred by certain specific receptors from the hens serum to the egg yolk . One chicken egg yolk contains the actual amount of IgY equivalent to that as 30ml of blood/serum . Due to this high quantity of consistent chicken egg yolk antibodies (IgY) and their high epitope specificity and affinity has led to the broad use of IgY antibodies in scientific and medicine therapeutic fileds. It is also found that IgY antibodies lack cross reactivity with human rheumatoid factors and they do not activate mammalian complement system, which is a major advantage for antigen-specific treatments of bacterial, fungal, viral and protozoan infections and diseases in human beings . The production and amount of IgY produced from Chciken egg yolk can be affected by certain different factors, such as the breed, age, antigen used, adjuvants selected for immunization, site of immunization, maintainence, climatic conditions, chronobiological impacts and so on. It is found that the concentrations of chicken egg yolk antibodies (IgY) ranges from 2.8-7mg/ml to upto 10mg/ ml from non-specific and specific groups of chickens that are non immunized and immunized . There are numerous studies have been conducted with antigen-specific IgY antibodies for topical and oral applications in human against various diseases . Over the last 20 years, Chicken egg yolk antibodies (IgY) have received a major importance as an safe and economical source of antigen speicific antibodies.
Chicken Antibodies (IgY)
Immunoglobulin’s are classified into three different types namely IgA, IgM and IgY in Chickens that are the analogues to mammalian antibody classes. Interms of chicken antibodies, often IgG and IgY are interchanged because they are funtionally equivalent to each other and also the antibodies in hens serum is IgG and once when it is maternally transferred from hens serum to egg yolk it is called as IgY, which is an egg yolk antibody in high concentrations . IgY antibodies exhibits distinct structural and functional characteristics which differentiates IgY from IgG, mammalian antibody. IgY is classified as a primitive antibody or may be a precursor to mammalian antibody and is found to have different molecular weights than other mammalian antibodies respectively. Although IgY and IgG are structurally similar, their heavy chains holds some modifications with heavier and varies antigenically that makes it to be different from IgG heavy cahins. IgY antibodies molecular weight is found to be 180kDa whereas, IgG vries from 180 – 150kDa. Chicken egg yolk antibodies does not bind to Fc receptors on cell surfaces having different immunological responses whereas, IgG binds to Fc receptors of specific mammalian cells.
IgY production:
Immunization of the Hens: Specific IgY development and production can be achieved by immunizing laying hens with the target antigen. However, the resulting immune response of the immunized hens cannot be very predictable. Mainly five factors influence this response: the antigen (dose and molecular weight), the type of adjuvant used, the route of application, the immunization frequency, and the interval between immunizations (Schadeet al., 1996).
Antigen:
The immune response is triggered by contact of the organism with antigen, which is a structure that is recognized by the immune system as foreign (“nonself”).The dose of antigen influences significantly the immune response and the antibody titre that is evoked. Too much or too little antigen may induce suppression, sensitization, tolerance or other unwanted immune modulation found that the injection of antigen concentrations ranging between 10 μgand1 mg elicited good antibodies responses, and this was also reported by other researchers (Mahn, 1998).
Adjuvant:
The induction of high and sustainable egg yolk antibody titre reclaims the use of adjuvant. There are more than 100 known adjutants, which differ in their chemical characteristics, their efficacy in stimulating the immune system, and their secondary side effects. Freund’s complete adjuvant (FCA) remains the most effective adjuvant for antibodies production in laboratory animals. In mammals, the use of this adjuvant leads systematically to severe inflammation at the injection site. In birds, the use of FCA does not seem to result in the same severe lesions as in mammals. The results of Gassmannet al., (1990) suggest that chickens show higher resistance to tissue damaging potency of FCA than rabbits. Svendsenet al., (1996) also support this finding.
Route of application:
The most common route for antigen injection in hens for IgY production is the intramuscular route. Injection is usually performed in the breast muscle. Chicken can also be injected subcutaneously in the neck. With very young animals, it may be preferable to inject intramuscularly into the breast muscle, because subcutaneous injection is more difficult to perform and can therefore cause more distress (Schadeet al., 1996).
Immunization Frequency and Interval between Immunizations:
The total number of immunizations required depends on the type and dose of the antigen as well as the adjuvant employed. At least two immunizations have to be given. Yolk antibody titres should be checked 14 days after the last immunization. The success of an immunization protocol depends also on the interval between the first and second and subsequent immunizations. Often reported interval is two to four weeks (Tiniet al., 2002).
Immunoglobulins:
The discovery and use of antibiotics and vaccination in animal agriculture have evolved from the management of small poultry flocks in the era prior to 1890s (Wehman, 1892) to the large consolidated units of today. The antibodies present in egg yolk have been termed IgY (Hattaet al., 1990). Thus, it is possible to obtain pathogen specific IgY antibody from eggs laid by hens immunized against antigen (Shimizu et al., 1988). Since poultry farming is carried out on a large scale globally, eggs may be a suitable source of antibody for passive immunization, which requires large amounts of antibodies. Over the past few years, we have successfully used the chicken egg yolk system to produce polyclonal antibodies to enamel proteins and other calcified tissue matrix proteins (Nanciet al., 1996). Furthermore, the amount of antibodies produced from an egg is equivalent to that from 200 to 300 ml of mammalian blood, and the costs for animal care per unit production of antibodies are much lower in chicken than in mammals. However, the practical use of IgY in research and diagnostics is limited due to complex and time-consuming purification steps associated with the further purification of IgY (Akita and Nakai, 1992).
Isolation and Purification Methods for IgY: Several methods were described in the 1950s for purifying IgY based on the strategy of separation of proteins (levitins) from lipoproteins (lipovitellins) and the rest of the yolk lipids using extraction with organic solvents with rather low yields of antibody. However, purification methods based on organic solvents like chloroform remain in use. Other methods are based on affinity chromatography or on dilution of the yolk followed by a freezing-thawing process after which the process consists of ion exchange chromatography and salt precipitations often combining a number of salts like for e.g. polyethylene glycol (PEG), dextran sulfate, dextran blue, sodium sulfate, ammonium sulfate, caprylic acid and sodium citrate. Hatta (1990) reported that the IgY remaining in this supernatant was isolated by DEAE-Sephacel column followed by salting-out with sodium sulfate resulting in almost pure IgY (98%) and the yield was 70 100 mg per egg. Water dilution method found to be superior in terms of ease of use and large scale production of IgY. This is simple rapid and efficient means of purifying IgY with high activity.
Antibody Stability: IgY is fairly heat stable and most antibody activity remain after 15 min at 70°C. Incubation of IgY at pH above 4 is well tolerated, but at pH 2 and 37°C the activity is rapidly decreased. The rapid activity loss is probably due to conformational changes, as the polypeptide is not broken down as observed by SDS-PAGE. The immunological activity of IgY is not affected by pasteurization at 60°C for 3.5 min. Addition of high concentrations of sucrose stabilizes IgY regarding heat denaturation, acid environment as well as high pressure. IgY fractions have been stored in 0.9% NaCl, 0.02% NaN3 at +4°C for over 10 years without any significant loss of antibody titer. An egg can be stored in +4°C, with just a small loss of IgY activity for at least six months (Carlander, 2002).
Why Choosing IgY?
Egg laying Chickens like White Leghorns, as a bioreactor for large scale antibody production and becomes a cost effective source. Huge amount of acquiring antibodies and maintaining the Chickens becomes more easier and efficient than maintaining large number of mammals for antibody production. Chicken eggs are considered to be a daily source of large amount of nutrients and immunoglobulin’s that can be efficiently collected and processed in a humane way which also proves to be very practical and inexpensive source of specific and non-specific immunoglobulin’s in larger quantities effectively. Chicken egg yolk antibodies (IgY) have wider applications in diseases prevention, control and treatments where they are systematically used to treat victims of Snake venom, CPV, Rabies, Respiratory syncytial virus infections and so on . It is also reported used in many other applications such as passive protection against Avian Infuenza and Rotavirus infections, potential use in preventing and treating upper respiratory tract infections and gastrointestinal infections which led to a breakthrough in passive therapy approach with Chicken egg yolk antibodies (IgY). Other studies showed the efficacy of IgY antibodies against various infections caused by coronavirus , Escherichia coli , Salmonella , Canine Parvo-virus, Helicobacter pylori in gastrointestinal tracts and Pseudomonas aeruginosa infections in cystic fibrosis pateints , therapeutic effects in preventing dental caries and also in preventing acne as an topical application. Additionally IgY antibodies have also shown its potential to be as an useful tool in cancer research and its applications.
It has a distinguished wide range of applications for several substances found in eggs, mainly in case of food, pharmaceutical and biotech industrial applications to control antibiotic resistant microbes and also to prevent them from emerging into new strains. To have a better understanding about the importance of IgY research, Chicken egg yolk antibody (IgY) has all the properties of anti-infective, especially against various infections caused by bacteria, viruses and antibiotic resistant fungi. Since, IGY being an alternative to antibiotics it can be effectively used in human and animal treatment of diseases which may play a vital role in preventing the serious global threat, the emergence of new multidrug resistant bacterias. The IgY technology is an effective, easier, environmental friendly, non-hazardous, large source and economical alternative to more pharmaceutical industries and food industries. Summary of these studies proves that the potential use of IgY antibodies in therapeutics and neutraceutical applications are wider and huge into many promising substantial benefits to be commercialized by industries and in clinical applications in both humans and animals effectively.
IgY Antibodies for Therapeutic use in Healthcare Sectors for Humans—
IgY research studies has shown that the antigen-specific IgY antibodies raised against specific pathogens are effective in treating viral, bacterial and fungal diseases both in in vitro and in vivo analysis which could be immediately applicable in a straight way to humans and it does not have any toxic or harmful side effects .
IgY in Digestive Care
Rotavirus Infection: Abundant research studies with IgY antibodies therapeutic applications in humans with specific prophylaxis and therapy has been conducted globally. It has been shown that the specific antibodies against Salmonella antigens were able to inhibit their adhesion to epithelial cell linings . Sarker demonstrated the action of Hyperimmune bovine colostrum (HBC) and Human rotavirus infection in children were controlled by the use of IgY in passive therapy . A randomized placebo controlled clinical trial has been conducted with specific chicken egg yolk antibodies (IgY) as an adjunct to protective therapy for rotavirus-associated diarrhea in infected children and observed for the signifcant reduction of diarrhea and the safe, effective use of specific IgY for managind acute diarrhea in the pediatric patients .
Helicobacter pylori Infection: Studies with anti-Helicobacter pylori IgY antibodies has proven significant therapeutic protection in both humans as well as in animals . Specific IgY antibodies raised against particular protein of this pathogen were found to be more effective as an prophylactic agent than as an whole cell lysate and also studies shows that there were some immunodominant protein are present in Helicobacter pylori . Horie et al. study results showed that there is a suppressive effect of infection in 42 human volunteers with H.pylori positive after regular consumption of the functional yoghurt drink fortified with anti- urease IgY antibodies and also by obtaining a significant reduction in their urea breath test effectively .
IgY in Oral Care
Streptococcus mutans: Dental caries is a major problem in humans and maintaining a good oral health plays a vital role in human life. Anti-Streptococcus mutans IgY was used as an effective local protection agaisnt dental plaque and dental caries and found to achieve good results as a passive protection approach to prevent and control caries . Zhou et al. tested anti-S.mutans IgY spray in adult volunteers to identify its protective effect and found that there was a significant decrease in S.mutans colonies after three weeks of IgY application . Active immunization against Streptococcus mutans glucan binding protein B (GBP-B) was found to induce good protection against dental caries in experimental conditions . Carlander et al. studied the residual activity and the results suggest that the anti-S.mutans IgY antibodies may bind to the pathogen and prevent it from adhering to the oral cavity .
IgY in Respiratory Care
Pseudomonas aeruginosa: Carlander et al. investigated the advantages of IgY antibodies as a prophylactic tool against Pseudomonas aeruginosa infection in Cystic fibrosis patients which is most commonly found in USA and Caucasian populations in Europe . In another study 38 human volunteer patients with cystic fibrosis were made to gargle with anti-P.aeruginosa IgY anibody solution for two minutes and found that prolonged use resulted in no adverse effects and none of the IgY treated patients were chronically clonized with P. aeruginosa respectively .
By analyzing the levels and patterns of these different antibody types in the egg yolk, broiler breeders can gain valuable insights into the immune status of their flock, the effectiveness of their vaccination programs, and the potential passive immunity that will be conferred to the chicks. The production of polyclonal antibodies through the chicken immunization proved to be an excellent alternative, producing the antibodies in large amount and quality from the simple methods of production without the need for invasive. IgY should be used as an alternative to mammalian antibodies, and it is better to immunize chickens before they begin to produce eggs, since the stress induced by handling them could have an adverse effect on egg production. The benefits of IgY technology and its universal application in research and medicine is expected to expand at a large-scale. It is expected that IgY will play an increasing role in research, diagnosis, and immunotherapy in the future.
Future Prospects: Today, there is no doubt that chicken Abs can be produced and used, with minor modifications, in similar ways to the use of mammalian Abs. It is to be expected that studies on the therapeutic or prophylactic use of IgY Abs will be intensified in future. In particular, because of the increasing resistance of microorganisms to antibiotics, research on all aspects related to the development of specific IgY against pathogenic microorganisms will have to be intensified. IgYs can be used both in veterinary medicine and in human medicine. IgY technology is a fast developing field and we have tried to cover most of its aspects in this review. We are convincedthat, once accepted and widely used, the technology will offer alternatives and solutions toscience, to medicine and to society as a whole.
Compiled ,Edited & Shared by-Team LITD
Reference source-To be shared on request
