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The production of avian (egg yolk) antibodies: IgY. The report and recommendations of ECVAM workshop 21
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... Most commonly used animals for the production of immune sera for diagnostic purposes are rabbits and guinea pigs, although horses, goats, and sheep are used to a lesser extent for the same purpose (1). However, chickens are an attractive alternative to mammals as antibody producers because large quantities of antibodies can be produced from the egg yolk making restraint from the blood sampling obsolete techniques to the benefit of the animals used for this purpose (2). ...
Two purification methods of egg yolk immunoglobulin (IgY) based on precipitation using charcoal-PEG and dextran-PEG were developed. These methods were compared with chloroform extraction and polyethylene glycol (PEG) precipitation methods. The results showed that protein contents were high with chloroform method (12.8 mg/ml) followed by dextran-PEG (10.5 mg/ml), charcoal-PEG (8.13 mg/ml) and PEG method (4.4 mg/ml). The purity of resultant IgY was homogeneous with dextran-PEG method followed by charcoal-PEG method, less purity was in PEG method followed by chloroform extraction method.
... IgY Abs are the only Ab isotype present in chicken egg yolk, simplifying processing and purification, and approximately 1500 mg of IgY can be harvested each month from each laying hen (5-25 mg/egg yolk), while 2 to 10% of total Ab are antigen-specific IgY [30]. These properties make IgY production a fast and economic method for polyclonal Ab production [31,32]. It is also advantageous to use avian IgY instead of mammalian IgG based on animal welfare [30]. ...
Bovine Coronavirus (BCoV) is a major pathogen associated with neonatal calf diarrhea. Standard practice dictates that to prevent BCoV diarrhea, dams should be immunized in the last stage of pregnancy to increase BCoV-specific antibody (Ab) titers in serum and colostrum. For the prevention to be effective, calves need to suck maternal colostrum within the first six to twelve hours of life before gut closure to ensure a good level of passive immunity. The high rate of maternal Ab transfer failure resulting from this process posed the need to develop alternative local passive immunity strategies to strengthen the prevention and treatment of BCoV diarrhea. Immunoglobulin Y technology represents a promising tool to address this gap. In this study, 200 laying hens were immunized with BCoV to obtain spray-dried egg powder enriched in specific IgY Abs to BCoV on a large production scale. To ensure batch-to-batch product consistency, a potency assay was statistically validated. With a sample size of 241, the BCoV-specific IgY ELISA showed a sensitivity and specificity of 97.7% and 98.2%, respectively. ELISA IgY Abs to BCoV correlated with virus-neutralizing Ab titers (Pearson correlation, R2 = 0.92, p < 0.001). Most importantly, a pilot efficacy study in newborn calves showed a significant delay and shorter duration of BCoV-associated diarrhea and shedding in IgY-treated colostrum-deprived calves. Calves were treated with milk supplemented with egg powder (final IgY Ab titer to BCoV ELISA = 512; VN = 32) for 14 days as a passive treatment before a challenge with BCoV and were compared to calves fed milk with no supplementation. This is the first study with proof of efficacy of a product based on egg powder manufactured at a scale that successfully prevents BCoV-associated neonatal calf diarrhea.
... A relevant aspect for IgY technology to be considered as a 3R alternative for the production of polyclonal immunoglobulins is the refinement in the procedures to immunize hens (Schade et al. 1996). Thus, the choice of the proper adjuvant is key to the success of any immunization procedure not only to optimize the amount of specific antibodies but also to reduce undesired damage in the animals. ...
The use of egg yolk antibodies—IgY technology—represents an alternative to the production of mammalian immunoglobulins and has several advantages regarding animal welfare and lower costs of production. The use of adjuvants to achieve the hyperimmunization of laying hens plays a key role in the success of the production of high levels of the antibodies. In the present work, two different adjuvant systems (Freund’s adjuvants and MontanideTM ISA 71 VG) were compared to produce IgY anti-Bothrops alternatus. For the first immunization, formalin-inactivated Salmonella was added to MontanideTM ISA 71 VG to emulate Freund’s complete adjuvant which includes a mycobacteria antigen. After eight immunizations, IgY produced by using either adjuvant was able to neutralize the lethal activity of the venom in a mouse model, but differences were found regarding the recognition of components of the venom between the two adjuvants tested. Overall, MontanideTM adjuvant used in this work could be a good alternative choice to produce antibodies capable of neutralizing the lethality of complex antigens. This adjuvant is commercially available and used in the formulation of several poultry vaccines and could be used for the IgY technology instead of traditional immunomodulators such as Freund’s adjuvants.
Key points
• IgY extracts recognized major components of the venom.
• Avidity indexes of the IgY extracts increased after the successive immunizations.
• IgY obtained by two adjuvant systems neutralized the lethal activity of the venom.
... Immunoglobulin Y (IgY) adalah isotop antibodi yang terdapat pada unggas, reptil, dan amfibi yang berfungsi menyediakan pertahanan untuk melawan agen infeksius, sama dengan fungsi IgG pada mamalia (Silveira et al., 2014 (Schade et al., 1996) ...
... IgYs are the main constituent of γ-livetin, and among the most important and most abundant egg yolk proteins [1]. IgY can be isolated from "immune" egg yolk in large quantities in order to avoid animal bleeding, which causes stress on animals, and has been applied in various fields of biotechnology and biomedicine [2][3][4]. A large number of studies have found that IgY has immunological properties, such as not binding to human complement and rheumatoid factors, allowing it to be applied in many areas such as immunological diagnosis and medicine [5]. ...
The preparation of the interaction between polyphenols and protein is of great significance for increasing added value and promoting the application of egg yolk immunoglobulin (IgY). This study systematically investigated the effect of catechin on yolk immunoglobulin structural characteristics and functional properties. The binding conditions, force types, molecular conformation, and residual microenvironment of the interaction between catechin and IgY were analyzed by molecular docking technology, UV-vis absorption and fluorescence spectroscopy studies. The results showed that the main binding forces in the complex were hydrogen bonding and van der Waals forces. After the interaction, fluorescence quenching occurred and the maximum emission wavelength was redshifted. The results showed that the microenvironment around IgY increased polarity, increased hydrophilicity and decreased hydrophobicity, and the structure of the peptide chain changed. The bacteriostatic thermal stability of the compound against Escherichia coli and Staphylococcus aureus was lower than that of catechin IgY. The bacteriostatic acid and base stability were higher than that of catechin and IgY. The antioxidant activity was catechin, complex, and IgY, in descending order. The antioxidant activity of catechin and complex was significantly higher than that of IgY. At the same concentration, the apparent viscosity of the three samples was complex, IgY and catechin, in descending order. G’ was greater than G” indicating that elastic properties dominate in G”. The G’ and G” values of the complex were higher than those of the other groups. Rheological results indicated that the complex may have high physical stability. This study provides theoretical support for broadening the application field of IgY and suggest its properties change in the machining process. It also provides new ideas for the development of functional foods from poultry eggs.
Broiler chickens are the highest source to fulfill animal protein consumption of the Indonesian people. The population of this type of chicken is very high because it does not require a large area in maintenance, has fast growth and good feed efficiency compared to other types of broiler poultry. Broiler farms generally use antibiotics as feed additives to trigger growth, increase body weight and improve the efficiency of food converted into meat. Antibiotics are also used for disease prevention and treatment, especially by commercial chicken farmers. Antibiotics used in livestock production account for about two-thirds of global antibiotic sales and consumption. The unwise use of antibiotics also increases the occurrence of antimicrobial resistance, especially in bacteria. This research goals to analyze the sustainability of using IgY affixes as a substitute for antibiotics in chicken feed. The results of the analysis are grouped based on the value of the sustainability level. The results of Multi Dimensional Scaling (MDS) analysis using the RAPFISH (Rapid Appraisal for Fisheries) analysis tool showed that the level of sustainability of IgY use was 66.49% with a quite sustainable category.
There has been an increasing interest in using Immunoglobulin Y (IgY) antibodies as an alternative to “classical” antimicrobials. Unlike traditional antibiotics, they can be utilized on a continual basis without leading to the development of resistance. The veterinary IgY antibody market is growing because of the demand for minimal antibiotic use in animal production. IgY antibodies are not as strong as antibiotics for treating infections, but they work well as preventative agents and are natural, nontoxic, and easy to produce. They can be administered orally and are well tolerated, even by young animals. Unlike antibiotics, oral IgY supplements support the microbiome that plays a vital role in maintaining overall health, including immune system function. IgY formulations can be delivered as egg yolk powder and do not require extensive purification. Lipids in IgY supplements improve antibody stability in the digestive tract. Given this, using IgY antibodies as an alternative to antimicrobials has garnered interest. In this review, we will examine their antibacterial potential.
The aim of the present study was the development of a sensitive and specific ELISA system for the quantitative and qualitative
assay of chicken Ig Isotypes G, M, and A using monoclonal antibodies. Five hybridoma cell lines were developed that synthesized
specific antibodies against chicken IgG and three lines each producing specific antibodies against IgM or IgA. Using an immunodiffusion
test, the subclasses were determined. Isolation of monoclonal antibodies from ascites was carried out by way of affinity chromatography
with protein G sepharose. The purity of the eluates were determined by both SDS-PAGE and HPLC. A Sandwich ELISA was found
to be the most suitable technique for the assay. Specificity testing was carried out by Western blotting. An epitope analysis
was also carried out. By variation of the single steps concerning incubation times, quantities, and concentrations of the
substances to be applied, the whole procedure was optimized. Assay limits for individual Ig isotypes were determined. The
limits were 20 ng/mL for IgG, 80 ng/mL for IgM, and 160 ng/mL for IgA.
Based on a physiological process, antibodies are concentrated in the yolk of chicken eggs, and can be used as a convenient source of polyclonal antibodies. Hens were immunised with a BSA-cholecystokinin octapeptide conjugate (CCK-8) to obtain antibodies against CCK-8. A simple method is described for extracting the antibodies from egg yolk, which can then successfully be used in immunocytochemical studies. Using these antibodies, CCK-like immunoreactivity was found in brain sections of rats, both by fluorescein isothiocyanate and peroxidase staining. The specificities of these antibodies were different from those of rabbit antibodies against the C-terminal part of CCK-8.
The applicability of egg yolk antibodies in immunological investigations is reviewed and discussed in detail. The properties of avian egg yolk antibodies are compared with those of polyclonal antibodies obtained from mammals (e.g. the rabbit). The more general use of avian antibodies is emphasised as a real alternative, taking into consideration the reduction in animal suffering involved in comparison with the classical immunisation techniques used on rabbits.
Freund's Complete Adjuvant in the Chicken: Efficient Immunostimulation but Severe Local Inflammation
To quantify the enhancing activity of Freund's complete adjuvant (FCA), the humoral immune response of hens to the antigen human serum albumin (HSA) injected intramuscularly combined with FCA was measured. Local tissue reaction was examined on macroscopic and microscopic level. Other hens were injected with HSA in physiological sodium‐chloride solution and served as controls. The immunization of hens without using FCA resulted in a short‐term rise in immunoglobulin G (IgG) antibody titres, while the hens injected with HSA and FCA developed a long‐term response (plateau‐form of antibody titres). Local tissue reaction after injection of HSA in sodium‐chloride solution was a mild and transient form of interstitial myositis. The immunization with HSA combined with FCA regularly resulted in a persisting granulomatous myositis. It is therefore a matter of animal protection to demand restricted use of FCA and to promote adjuvants of comparable efficiency, but better tolerance.
Zusammenfassung
In der vorliegenden Arbeit wurden die humorale Immunantwort von Hühnern gegenüber dem intramuskulär in Kombination mit Freundschem komplettem Adjuvans (FCA) verabreichten Antigen humanes Serumalbumin (HSA) quantifiziert und lokale Gewebereaktionen auf makroskopischer und lichtmikroskopischer Ebene untersucht. Zum Vergleich wurde HSA in physiologischer Kochsalzlösung appliziert. Die Immunisierung von Hühnern ohne FCA führte zu zeitlich sehr begrenzten IgG‐Antikörpertitern. Die Verwendung von FCA bei der Immunisierung mit HSA resultierte in der Ausbildung eines langanhaltenden, hohen Antikörpertiter‐Plateaus. Die lokale Gewebereaktion nach Immunisierung mit HSA in physiologischer Kochsalzlösung war durch eine vorübergehende geringgradige interstitielle Myositis gekennzeichnet. Die Immunisierung unter Verwendung von FCA hatte regelmäßig die Entwicklung einer persistierenden granulomatösen Myositis zur Folge. Uinter dem Aspekt des Tierschutzes ist deshalb zu fordern, auf die Verwendung von FCA soweit als möglich zu verzichten und stattdessen besser verträgliche aber vergleichbar effiziente Adjuvantien zu entwickeln und einzusetzen.
A simple method for the isolation of immunoglobulin (Y) from eggs of immunized hens
Hens were vaccinated with Salmonella typhi-murium and with serum-IgG from a goat. Antibodies (IgY) were isolated from the eggs by ammonium sulphate precipitation. The concentration of egg antibodies was equal or superior to the concentration of serum antibodies of the hen. The longlasting antibody plateau of 9 and 28 weeks, respectively, provides evidence of the high yields of specific IgY which can be collected from a single immunized hen.
Hühner wurden mit Salmonella typhi-murium und Serum IgG einer Ziege immunisiert. Aus den gelegten Eiern wurden Antikörper (IgY) mit Hilfe der Ammoniumsulfat-Präzipitation gewonnen. Die Konzentration der Eigelb-Antikörper entsprach oder übertraf die Konzentration der Serum-Antikörper des Elterntieres. Die langfristige Ausbildung eines Antikörperplateaus von 9 bzw. 28 Wochen gibt einen Hinweis auf die hohen Erträge von spezifischem IgY, die von einem immunisierten Huhn gewonnen werden können.
Platelet function is dependent upon membrane receptors and their interaction with other proteins. Platelet activation appears to cause a structural change of the glycoprotein IIb/IIIa complex that exposes the fibrinogen binding site, which subsequently binds fibrinogen. Fluorescence-activated flow cytometry (FACS) is an efficient method for studying membrane proteins. Flow cytometry gives single-cell data, allowing the detection of only a small proportion of labelled platelets in whole blood without any washing steps. One problem with this method is that the labelled antibodies and the antigen, if present in plasma, form an immune complex, which may cause false positive reactions due to interaction between mammalian IgG and Fcγ receptors on the platelets.
We show that immune complexes with chicken IgG do not activate human platelets. We have developed a method for measuring platelet-bound fibrinogen in whole blood and platelet-rich plasma utilising fluorescein isothiocyanate (FITC)-conjugated chicken antibodies directed towards human fibrinogen. As low as 1% activated platelets could be detected without interference from Fc-interactions.
The egg yolk of immunized chicken is a rich and inexpensive source of specific polyclonal antibodies. In this paper we show that 20-30 micrograms of a highly conserved mammalian protein, as exemplified by proliferating cell nuclear antigen, are sufficient to induce an immune response. Immunoblot analysis revealed that specific antibodies appeared 20 days after immunization, reached a plateau after 30 days, and remained high until at least day 81. A total amount of 4 g immunoglobulin was extracted from 62 eggs of one immunized hen, yielding approximately 130 mg of specific antibodies.
Hens were vaccinated with Salmonella typhi-murium and with serum-IgG from a goat. Antibodies (IgY) were isolated from the eggs by ammonium sulphate precipitation. The concentration of egg antibodies was equal or superior to the concentration of serum antibodies of the hen. The longlasting antibody plateau of 9 and 28 weeks, respectively, provides evidence of the high yields of specific IgY which can be collected from a single immunized hen.