Egg yolk antibodies (IgY) and its relevance in animal and human health-An updated review

aMVSc Scholar, Division of Veterinary Public Health, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly-243 122, Uttar Pradesh, India bAssistant Professor, Department of VPH, CVAS Pookode and PhD Scholar, Division of Veterinary Public Health, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly-243 122, Uttar Pradesh, India cMVSc Scholar, Division of Animal Reproduction, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly-243 122, Uttar Pradesh, India dMVSc Scholar, Division of Animal Nutrition, ICAR-National Dairy Research Institute, Karnal, Haryana


Introduction
IgY-technology (the production and extraction of specific IgY antibodies from egg yolk) is a novel method to produce antibodies for therapy and prophylaxis (Leiva et al., 2020). Protein molecules produced by the body in response to an antigen are called antibodies and are widely used in therapy, diagnosis and research due to their unique ability to bind with the precise targets. In the current scenario major chunk of available antibodies produced are of mammalian origin, especially from small rodents (Michael et al., 2010). One of the major disadvantages in using lab animals for the production of antibodies is the pain and suffering of animals during the procedures such as immunization, blood collection and sacrifice (Narat et al., 2003). Here comes the importance of noninvasive method like the usage of eggs, from which not only produce more amount of antibodies (IgY) with higher specificity compared to lab animals like rodents but also eliminates the ethical issues. (Narat et al., 2003, Schade et al., 1996. In hens, IgY is transferred from blood and accumulates in egg (yolk) and then to their offspring. Hens are capable of producing 100 to 150 mg of IgY antibodies per yolk, in which 1 to 10% are specific IgYs in nature (Michael et al., 2010). In an year, a normal hen produces an average of 300 eggs which yield approximately 18 to 25 g of IgY antibody (Pauly et al., 2011). While coming to the cost of maintenance, a hen is less expensive to maintain than lab animals like rabbits and mice. From the point of animal and human health, IgY antibodies are used in immunotherapy (Rahman et al., 2013), immunodiagnostics (Cai et al., 2012, as a functional food (Horie et al., 2004), in neutralization of bacterial toxins (LeClaire et al., 2002) and venom of animals (Mendoza et al., 2012). Fc portion of IgY immunoglobulin is not capable of activating the complement system in humans (Larsson et al., 1992) by binding with protein G (Akerstrom et al., 1985) and to the rheumatoid factor (Larsson et al., 1991). IgY is more suitable for diagnosis as it displays greater avidity for mammalian conserved proteins and due to phylogenetic distance and structural differences when compared with mammalian antibodies (Gassmann et al., 1993).

Production of IgY immunoglobulin
Single antigens (Nucleic acids, polysaccharides, proteins) and complex type antigen such as bacterial, virus, and parasite are being used to produce specific IgY in birds (Chalghoumi et al., 2009). In hens of seven to eight weeks of age, 10-100 μg (per ml) of antigen is injected in two to three sites (Michael et al., 2010) by intramuscular (I/M) route, usually in the breast muscle and leg muscle is usually avoided as it causes lameness (Schade et al., 1996). Oral administration of antigen can also be performed (Thibodeau et al., 2017). Different antigen concentrations can be combined with adjuvants as the induction of high antibody titer depends on the use of adjuvants and Freund's Complete Adjuvant (FCA) is commonly is used due to its greater potential to induce antibodies in laboratory animals though it can cause severe inflammation at the site of injection (Chalghoumi et al., 2009). The titer of antibodies are influenced by a number of other factors also, like the type of antigen and dose, mode of application, frequency of inoculation, age and development stage of birds (Schade et al., 2005,Chalghoumi et al., 2009 and the higher titer can be maintained by booster inoculations for up to 150 days (Meenatchisundaram et al., 2011).

Inoculation
Minimum of two inoculations should be performed at an interval of four to six weeks before the period of egg laying. After 14 days of last immunization, IgY titer must be assessed. If the antibody titer is found to be decreasing more immunizations should be done during the egg laying period to increase titers (Schade et al., 1996). The number of injections required depends up on the dose and type of the antigen, and the adjuvant used. Highly purified IgY can be obtained by removal of lipid components from egg yolk, followed by precipitation of IgY with ammonium sulfate (NH4)2SO4 (20%) (Araújo et al., 2010) supplemented by sodium chloride (NaCl) and addition or by ultra-filtration technique prior to ion exchange chromatography or gel filtration. Ethanol precipitation method can also be used at lower temperatures for purification of Immunoglobulin (Akita et al., 1992).

Applications of IgY in Therapeutics and prophylactics
The main advantage of IgY polyclonal antibody against infectious diseases is that it minimizes the risk of microbial resistance since it requires multiple jfas.vetinfo.in Sivaprasad et al., 2020;https://doi.org/10.51128/jfas.2020.A015 genes for its synthesis (Rahman et al., 2013). Anti-allergic effect of specific IgY against the cytokines IL-β1 and TNF-α in guinea pigs after inducing allergic rhinitis showed encouraging results (Wei-xu et al., 2016). Advantage of IgY as an antivenom over IgG is that it is easily and sufficiently purified and the absence of other serum proteins that would minimize the side effects due to nonspecific proteins. (Araujo et al., 2010, Sjostrom et al., 1994.

Applications of IgY in diagnosis
Fc portion of IgG react with the protein A of Staphylococcus aureus whereas the Fc portion of IgY does not react with protein A due to structural differences which makes IgY a relevant resource for more specific detection of different S. aureus strains and the toxins (Richman et al., 1982) and it was confirmed by high titer of IgY in ELISA and western blot technique (Walczak et al., 2016). Among the parasitic infections, the ability of IgY to detect the protozoan Toxoplasma gondii was checked by the development of IgY against the surface protein SAG1, which responded to the target antigen in ELISA and Western blot.  Available at http://jfas.vetinfo.in/wp-content/uploads/2020/12/15.pdf

Against SARS-CoV-2
IgY immunoglobulin have excellent pathogenneutralization in the respiratory tract and lungs, and has shown its efficacy to treat and prevent respiratory infections (Abbas et al., 2018). Overall significance for the use of monoclonal chicken egg yolk antibodies (IgY) using phage display method describes their potential passive immunotherapeutic application for the treatment and prevention of SARS CoV-2 infection, an approach that is easy, quick and safe to effectively treat patients. When compared to polyclonal antibodies, chicken monoclonal IgY antibodies proved to be more specific in the identification of a single unique epitope. Chicken scFv IgY antibodies produced against SARS CoV-2 spike protein (S) using phage display technology are expected to be a potential candidate for the development of antibody-based vaccines as an immunotherapy agent model for efficient mass production of high-affinity monoclonal IgY antibodies for the treatment of SARS CoV-2 spike protein (S) with standardized preparation for long-term effective use (Abbas et al., 2018).

As an agent for preservation of food
IgY produced against the Listeria monocytogenes showed an important inhibitory effect on the bacterial growth in the liquid medium and in the fish samples processed between 0°C and 6°C in a dosedependent manner which showed that anti-L. IgY monocytogenesis is a possible antimicrobial agent and can be used in the food industry (Sui et al., 1946).

Protection against bioterrorism agent
IgY produced against the Staphylococcal enterotoxin is capable of saving people exposed to the substance B(SEB) which is a possible biological weapon. The findings with Rhesus monkeys showed the animals received anti-SEB IgY 30 min before or 4 h after lethal exposure to SEB aerosol survived (LeClair et al., 2002).

Conclusion
The benefits of IgY includes, lack of reaction to mammalian Fc receptors, low cost of development and ease of extraction. Compared to IgGs in mammals, they have higher specificity of the target and higher avidity of binding. To obtain IgY from egg yolk, several extraction methods are available and the choice of the appropriate method depends on the purpose, which may require different degrees of purification, as well as the extraction size, cost and available technology. Besides, in view of its proven capacity to neutralize microorganisms, it is a valuable therapeutic resource which can be used in the case of antibiotic resistance and against the development of viral diseases for which there is very less number of anti-viral agents.