An antigen attack upon host is followed by Igs or antibodies production in humans, process sustained by plasma cells developing thus the humoral immunity (34). Antibodies engage in the fight against the antigen by an array of mechanisms: neutralization, fixation of pathogen, complement activation or acting like receptors on B cell surface (35). Igs have interspecies differences regarding their structural particularities or classes, thus the term IgY comes from egg yolk where this Ig type is produced for assuring immunity in the hen progeny. IgY represents the functional equivalent of human IgG (29). In the last decade, IgY has gained scientific attention due to its distinctive biological actions largely emerged from its structural particularities (36).
Currently there are several commercial applications where Igs are fully explored in diagnostic and therapy monitoring assays.
Fighting against bacterial infections
Immunization of avian with specific bacterial antigens such as Salmonella sp. would provide a specific IgY against the inductor antigen. Moreover, due its high stability and structural unique features IgY has been applied successfully in diagnostic, prophylactic and therapeutic purposes as well as immunochemical reagents (34).
In addition to chicken models, there are other hands-on and inexpensive models such as the quail model where production of specific IgYs against Salmonella sp. has been recently reported. These quail anti-Salmonella sp. IgYs exhibit a high specificity to their matching immunogens, having the potential to eradicate enterobacterial pathogens. In addition, the oral ingestion of IgYs represent an efficient alternative for annihilation of gastrointestinal pathogens as Salmonella typhimurium and Salmonella enteritidis, bacterial species that raise major concern in health and food industry (37).
IgYs has also been proposed as a strategy for combating infections caused by Pseudomonas aeruginosa known as a common nosocomial pathogen having antibiotic resistance and a frequent infection in acute pneumonia and severely burned patients. A protein called PcrV is a vital part of killing machinery represented by the type III secretion system of P. aeruginosa and therefore PcrV is viewed as a target for neutralizing this infectious agent. Thus, recently recombinant PcrV was used for raising specific IgY. These antibodies displayed a protective effect in both acute pneumonia and burn wound models and moreover IgY anti-PcrV has augmented opsonization capacity and bacterial killing activity of host cells (38). In fact the augmentation of phagocytic killing via IgY was previously explored in an in vitro study targeting P. aeruginosa infection in cystic fibrosis (CF) patients. IgY against P. aeruginosa fulfill their function by opsonizing the pathogen and thus enhancing the neutrophils respiratory burst while further enabling bacterial killing. It was suggested that prophylaxis with anti-P. aeruginosa IgY could lift the innate immunity of CF patients aiding host neutrophils to rapidly clear the bacterial agent (39).
In CF the principal contributor of pulmonary failure is the chronic infection with P. aeruginosa biofilm, which constantly attracts and activates neutrophils sustaining the continuous inflammation. It is suggested that IgY favors bacteria to form aggregates and increase their hydrophobicity enhancing bacterial killing by neutrophils via phagocytosis (40).
Passive immunization with IgY anti-P. aeruginosa could reduce the initial airway settlement with P. aeruginosa in CF patients. Thus in a Balb/c murine P. aeruginosa pneumonia model administration of specific IgY significantly reduced the bacterial load at 24 h after infection along with alleviating the clinical symptoms; in addition an inflammatory cytokine pattern was noted revealing the lung inflammation decrease suggesting that immune-prophylaxis with anti-P. aeruginosa IgY may also function as an adjuvant to antibiotics in lowering primary colonization of lungs (21). In parasitic diseases the possible role of IgY in early diagnosis and therapeutics has been tackled by attempting to obtain polyclonal IgY against parasitic antigens suitable for immunotherapeutic purposes. Although further studies in animal models are indispensable and obtaining a monoclonal IgY anti-parasitic antigens are envisaged, it became obvious that IgY could stand for immunoassay designing also in parasitology area (41).
In immunodiagnostic methods, IgY is an excellent tool in assays involving mammalian sera, due to the discriminative properties of IgY compared to mammalian IgG. IgY has immunological properties which makes it very distinct from mammalian IgG but at the same time very affordable for a plenty of immunological approaches. Foremost, by lacking the hinge region, IgY is less structurally flexible than IgG and retains a different protein content, these structural differences sustaining the differences in immunological behavior (42). IgY has poor cross-reactivity to mammalian IgG, does not activate the complement system similar to IgM/IgG and lacks the reactivity with mammalian Fc receptor (43).
However, there are still incomplete data regarding the three-dimensional structure of Fc-IgY raising the question whether IgY shares a conformational status similar to IgM and IgE whose Fc regions are significantly flexible. However, the evolutionary distance between mammals and birds made possible the feasible generation of IgY against conserved mammal proteins. Thus, the molecular particularities of IgY raised several functional advantages recommending IgY as a versatile tool in biotechnological research, diagnostics and therapeutics (29). One very practical utility is that IgY can be used to generate a specific antibody when an antigen comes in small amounts and additionally is low immunogenic in mammals host (44).
IgY can be generated at low-costs in considerable amounts through an ecologically friedly approach because it is produced in egg yolk so no additional procedures on animals are needed. There are several IgY isolation methods available, mostly based on precipitation from egg extracts using ammonium sulfate or polyethylene glycol although protein impurities still remains in the sample of interest, in addition to time consuming and complexity required (45,46). Chromatographic methods have become very popular in recent years because this methodology generates highly active pure products useful for biomedical applications. A study published in 2020 reports IgY fragment separations by ion exchange column using DEAE-Sepharose leading to Fab and Fc fractions of high purity (88.7 and 90.1%, namely) and intact activity rendering them easily used for medical purposes (47).
Usually, antibodies and/or their active fragments produced in mammals are engaged in diagnostic tests. However, due to animal welfare concerns, technical advantages and the high cost of production, alternatives to the production of antibodies in mammals have been investigated (48).
A constant goal for all current approaches involving IgY applications is to continuously optimize the production and purification of IgY antibodies from egg yolk to achieve high quantities and high active products for research and commercial use.
The most exploited immunoassay format with IgY remains the ELISA platform. In research as well as in clinic, one of the most effective and reliable method for rapid detection remains ELISA which is a versatile, flexible and sensitive method helpful in monitoring various pathologies including infectious diseases. Although IgG is the ʻweapon of choiceʼ in designing such detection formats, in recent years IgY started to replace IgG because this molecule could be easily obtained, through a non-invasive way (basically extracted and purified from egg yolk of chickens immunized with recombinant proteins of interest), in adequate amounts and at lower costs than IgG.
IgY-based ELISA protocols retain all the features related to steps, reagents and applications of classic ELISA formats as IgY molecule can be enzymatically labeled (e.g., with horseradish peroxidase) and further applied in a wide range of immunoassay assessments such as bacterial toxins (49) or tumor antigen detection for cancer diagnosis (50,51).
It is truly noticeable how versatile the use of IgY could be related to area of application, from viruses and bacteria to tumor antigens detection and quantification (42). Thus, diagnosis of cholera is somewhat an intricate attempt. Therefore, a recent study proposed IgY for targeting two proteins contained by the outer membrane of V. cholera (protein W and cytotoxin B). The study proposed two sensitive and specific sandwich ELISA formats, with no cross-reactivity with other bacteria. It was suggested that these IgY based ELISAs could constitute a rapid and specific detection tool for assessing V. cholera in different biological samples (52).
The use of IgY instead of mouse monoclonal IgG has the advantage of limiting the risk of false positive results as well as the reduced costs of the test. For example, an IgY-based sandwich ELISA was developed to quantify the total PSA level in human serum as an alternative to commercially available in vitro diagnosis tests (53).
Taking into account the latest concerns raised by respiratory infections of viral origin IgY might act as a very promising detection agent in this area. Thus, recent studies in swine model suggest that IgY molecule could bind specifically to viral nucleoprotein of influenza virus, which is known for exerting a key role in viral replication and therefore emphasizing the IgY effectiveness for influenza virus diagnosis (48,54).