Properties of the avian IgY

The basic chemistry of the chicken IgY has been reviewed by Benedict and Berestecky (Reference BENEDICT and BERESTECKY1987), and comparative immune-chemistry of avian IgYs can be found in a review by Higgins (Reference HIGGINS1996). Schade et al. (Reference SCHADE, BEHN, ERHARD, HLINAK and STAAK2001) has also published a laboratory use manual for working with egg IgY. Furthermore, recent research has been published on the molecular structure and functions of IgY (Warr et al., Reference WARR, MAGOR and HIGGINS1995; Suzuki and Lee, Reference SUZUKI and LEE2004; Taylor et al., Reference TAYLOR, FABIANE, SUTTON and CALVERT2009). No attempt will be made here to provide an exhaustive review of literature on IgY structure and chemistry; however, interested readers are referred to the sources mentioned above.

Physiochemical properties of egg IgY with relevance to commercial application

In general, antibodies have evolved to be more resistant to enzymatic proteolysis and low pH environments than most proteins due to evolutionary demands for passively transferred antibodies to be effective in hostile environments (e.g. gastrointestinal tract). Shimizu et al. (Reference SHIMIZU, HAGASHIMA, SANO, HASHIMOTO, OZEKI, TSUDA and HATTA1992) and Hatta et al. (Reference HATTA, TSUDA, AKACHI, KIM and YAMAMOTO1993a) researched the stability of chicken IgY in a variety of environmental conditions. Heating purified IgY antibodies for 15 minutes showed decreased binding activity at a temperature of 70°C or higher. In studies conducted in our laboratory, egg antibodies added to an animal feed matrix cannot withstand temperatures commonly associated with the steam conditioning (80-95°C) used in the pelleting process (Bobeck and Cook, Reference BOBECK and COOK2005). Bobeck et al. (Reference BOBECK, TROTT, COOK and YANG2006; Reference BOBECK, COOK, GELBACH, YANG and COOK2008) reported that the addition of select carbohydrates to egg antibody preparations was effective at reducing losses in antibody activity at 82-95°C during 10-60 second steam conditioning periods. Hatta et al. (Reference HATTA, TSUDA, AKACHI, KIM and YAMAMOTO1993a) showed that egg antibodies retained most of their anti-rotavirus neutralising activity (85-90%) at a pH 2 for one hour even though the antibody binding, as measured by ELISA, was reduced over 50%. Both neutralizing and ELISA activities decreased when egg antibodies were held at pH 3 or below for more than one hour. Shimizu et al. (Reference SHIMIZU, NAGASHIMA, HASHIMOTO and SUZUKI1994) did show that addition of sugars to IgY preparations in vitro improved stability at reduced pH. It is important to note that purified egg antibodies may not have the same buffering capacity or protective matrix of commercially prepared antibodies contained in the egg matrix. Also, the susceptibility of egg antibodies to the action of low pH in the gastrointestinal tract could be considerably different when administered orally by gavage, with or without food, as compared to their delivery in a component of animal or human food products.

Enzymatic degradation is important when considering the use of egg antibodies for oral use. Egg derived antibodies must partially resist proteolytic degradation in order to provide passive immunity to the gastrointestinal tract of the newly hatched chick; hence, an innate resistance of IgY to degradation would be expected. Several studies have reported the stability of egg antibodies when exposed to gastrointestinal enzymes (Shimizu et al., Reference SHIMIZU, HAGASHIMA, SANO, HASHIMOTO, OZEKI, TSUDA and HATTA1992; Reference SHIMIZU, MIWA, HASHIMOTO and GOTO1993; Hatta et al., Reference HATTA, MABE, KIM, YAMAMOTO, GUTIERREZ and MIYAZAKI1993b; Kovacs-Nolans and Mine, 2005). As with pH (above), in vitro studies on the digestion of immunoglobulins may not always reflect the true antibody level when contained in an egg powder or feed matrix; however, data do suggest that antibodies are somewhat resistant to enzymatic degradation (Shimizu et al., Reference SHIMIZU, MIWA, HASHIMOTO and GOTO1993).

The presence of antibodies in locations of active digestion in vivo provides evidence for a significant level of resistance to degradation. Shawky et al. (Reference SHAWKY, SAIF and McCORMICK1994) found that maternal antibodies to rotavirus in turkey poults could be recovered from the duodenum, jejunum and ileum. Evidence suggested that a significant amount of orally consumed immunoglobulins (as much as 50%) from both avian and bovine sources can be recovered in a biologically active form (Reilly et al., Reference REILLY, DOMING and SANDHU1997; Warny et al., Reference WARNY, FATIMI, BOSTWICK, LAINE, LEBEL, LAMONT, POTHOULAKIS and KELLY1999; Carlander et al., Reference CARLANDER, KOLLBERG, WEJAKER and LARSSON2000; and discussed in Berghman et al., Reference BERGHMAN, ABI-GHANEM, WAGHELA and RICKE2005). Antibodies generated against urease in laying hens (and passed into the gastrointestinal tract) have been shown to suppress ammonia production in hen excreta (Pimentel and Cook, Reference PIMENTEL and COOK1988). These findings suggested that immunoglobulins have value as oral or dietary supplements for the regulation of biologically relevant ligands in the lumen of the intestine.

The bird has evolved to provide the intestinal lumen of the newly hatched chick with maternal antibodies that retain their functional properties in a hostile environment. A purified oral antibody supplement would not have the buffering capacity or the protective matrix of a typical commercial preparation; however, methods of encapsulating purified antibodies are available which improve the retention of biological activity (Shimizu et al., Reference SHIMIZU, MIWA, HASHIMOTO and GOTO1993; Kovacs-Nolan and Mine, Reference KOVACS-NOLAN and MINE2005; Li et al., Reference LI, JIN, McALLISTER, STANDFORD, XU, LU, ZHEN, SUN and XU2007). As with all encapsulation procedures, release of antibody to its strategic location is critical to insure functional effectiveness of antibody. The only way to assure functionality of a product, whether native, purified, or encapsulated is to assess its bio-efficacy in vivo and under the desired conditions.

The commercial targets (ligands or antigens) of egg antibodies used as a dietary supplement or additive to feed/food is primarily limited to ligands associated with the luminal contents of the gastrointestinal tract or luminally oriented cell surface epitopes (e.g. receptors). In mammalian species that have an ‘open gut’ immediately following birth, egg antibodies can be absorbed into circulation; however, absorption of antibodies ceases within 34 hours after birth, at least in the pig (Yokoyama et al., Reference YOKOYAMA, PERALTA, SENDO, IKEMORI and KODAMA1993). Numerous studies investigating the passage of orally fed antibodies to circulation beyond this ‘open gut’ period have failed to provide evidence of effective ‘absorption’ (unpublished findings in our laboratory; Carlander et al., Reference CARLANDER, KOLLBERG, WEJAKER and LARSSON2000). Since humans began eating eggs, the consumption of a wide variety of antibodies in egg products has shown no evidence of ill effects. Thus, the safety and lack of residues of egg antibodies is generally accepted (Kovacs-Nolan and Mine, Reference KOVACS-NOLAN and MINE2004b).

Quantification of egg antibody yield, and storage considerations

The yield of IgY per egg has been reported in ranges as low as 50-100 mg (Schade et al., Reference SCHADE, PFISTER, HALATSCH and HENKLEIN1991; Kovacs-Nolan and Mine, Reference KOVACS-NOLAN and MINE2004a) and as high as 375 mg/egg (Rose et al., Reference ROSE, ORLANS and BUTTRESS1974; Berghman et al., Reference BERGHMAN, ABI-GHANEM, WAGHELA and RICKE2005). Our experiences suggested that the amount of egg IgY is closer to 150 mg/egg in agreement with Tini et al. (Reference TINI, JEWELL, CAMENISCH, CHILOV and GASSMAN2002). The concentration (mg/ml) has been shown to be independent of the rate of lay or egg size (Li et al., Reference LI, NAKANO, SUNWOOD, PAEK, CHAE and SIM1998; Trott et al., Reference TROTT, YANG and COOK2005; Reference TROTT, YANG, UTTERBACK, UTTERBACK, KOELKEBECK and COOK2009a). Hence, the larger the egg yolk, the greater the total gross yield of antibody per egg. The amount of antibodies did not change in subsequent eggs laid after a bird paused in egg production, or laid a double yolked egg. We have recently reported that there were essentially no differences in egg antibody content among different commercial lines, and that the practice of moulting hens before or after the initiation of antibody production, has little effect on the yield of antibody (Trott et al., Reference TROTT, YANG and COOK2005; Reference TROTT, YANG, UTTERBACK, KOELKEBECK and COOK2006; Reference TROTT, YANG, HELLESTAD and COOK2008; Reference TROTT, YANG, UTTERBACK, UTTERBACK, KOELKEBECK and COOK2009a). These data suggested that the hen has a ceiling on the amount of IgY that can be deposited in the egg. We can maximise the rate of this deposition through vaccination procedures and also shorten the days to achieve this maximal production; however, we have not been able to dramatically increase the total IgY level deposited into the egg to any great extent. It has also been observed that the amount of antigen-specific antibodies was approximately 10% of the total IgY, a value which is consistent with the finding of Schade et al. (1991; Tini et al., Reference TINI, JEWELL, CAMENISCH, CHILOV and GASSMAN2002; reviewed by Kovacs-Nolan and Mine, Reference KOVACS-NOLAN and MINE2004a). While these yields of immunoglobulin and antigen-specific antibodies were impressive relative to what has been reported in other species (calculated in Kovacs-Nolan and Mine Reference KOVACS-NOLAN and MINE2004a; Berghman et al., Reference BERGHMAN, ABI-GHANEM, WAGHELA and RICKE2005), a two-fold or more increase in antigen-specific antibodies would dramatically improve the profitability of egg antibodies in select commercial areas.

Another significant advantage of egg antibodies is their ability to be stored for prolonged periods as either shelled eggs or as spray-dried powder. In a natural reproducing situation, birds lays a clutch of eggs over 10 to 14 days, and incubate the eggs for another 21 to 28 days before chicks are hatched. Hence, antibodies should be stable in eggs for 4 to 6 weeks. In our experience, eggs develop mould growth before there is a significant change in the antibody activity in shell eggs. Once the eggs are processed, storage at refrigerated temperatures of 4°C is acceptable; however, microbial growth will limit the length of time of refrigerated storage. Freeze-drying and freeze-thaw (at -80°C, but not at -20°C) conditions can have small, adverse effects on antibody activity. Our own findings agree with those found in the manual published by Schade et al. (Reference SCHADE, BEHN, ERHARD, HLINAK and STAAK2001). Spray dried whole egg is an excellent means of providing extended shelf life of eggs stored at 21°C or lower.

A number of methods of extracting antibodies have been published (Polson et al., Reference POLSON, VON WECHMAR and VAN REGENMORTEL1980; Polson et al., Reference POLSON, COETZER, KRUGER, MALTZAHN and VAN DER MERWE1985; Polson, Reference POLSON1990; Akita, and Nakai, 1992; Chang et al., Reference CHANG, LU, CHEN, TU and HWANG2000; Cook et al., Reference COOK, PAO, FIRCA, ANDERSON and FRYER2001; Schade et al., Reference SCHADE, BEHN, ERHARD, HLINAK and STAAK2001; Chen et al., Reference CHEN, TU, CHEN and CHANG2002; Gee et al., Reference GEE, BATE, THOMAS and RYLATT2003). Each method has unique purposes and intended uses for the final product. The method of purification may affect the storage conditions in which the final product must be held and the usefulness in quantifying the activity of the antibody of interest.

Harnessing eggs for commercial uses

As previously mentioned, there has been little hen breed differences in the ability of most commercial lines to produce antibodies to date. The most important variables governing the efficient production of egg antibodies were egg size and rate of lay. In some situations, it may be more efficient to use young pullets as a source of birds for antibody production, while in other situations, the use of moulted or spent hens may be the most economical route (Trott et al., Reference TROTT, YANG, UTTERBACK, UTTERBACK, KOELKEBECK and COOK2009a). In addition, there are a variety of adjuvants, routes of exposure, and vaccine scheduling protocols that can be used to improve the efficiency of egg antibody production to increase yield (Trott et al., Reference TROTT, YANG, HELLESTAD and COOK2008). Diet may also be a factor that affects antibody production (Cook, Reference COOK1991). No attempt will be made here to describe all the variations that can be used, or explore the most efficient means of making egg antibodies. What is presented below are some simple procedures routinely used in our laboratory for the production of antibodies. Modifications from these procedures will likely improve efficiency for production systems, but following these simple protocols will give a starting point for anyone involved in the production of egg antibodies.

Vaccine preparation and a standard protocol

Since the early 1980s, our laboratory has prepared reagent antibodies, and antibodies which have a potential commercial application. In general, we have followed certain standards for what constitutes an antigen. We have successfully produced antibodies against moulds, bacteria, viruses, protein, small molecules and peptides (Ricke et al., Reference RICKE, SCHAEFER, COOK and KANG1988; Burdsall et al., Reference BURDSALL, BANIK and COOK1990; Pimentel et al., Reference PIMENTEL, COOK and JONSSON1991; Banik et al., Reference BANIK, PAUL, BURDSALL and COOK1993). Peptides and other small molecule hapten antigens (with a molecular weight below 12,000 da) are usually conjugated to a carrier protein. Our preferred carrier protein has been bovine gamma globulin, specifically avoiding bovine serum albumen. Conjugation procedures, when needed, are either standard procedures available through most biological materials companies or done using a glutaraldehyde conjugation. The method used and any subtle changes in the molecular structure of the hapten molecule or peptide can have profound effects on the results achieved. Work has been done with a wide range of hapten/carrier ratios and typically uses 1:1 by weight, but equal success have been seen at 1:10 ratio. The minimal amount of protein in a given vaccine dose (defined as the total amount of vaccine given to a bird at a single time of injection) is 0.1 mg. A vaccine dose of 1 mg per vaccine dose, however, adds a greater level of security for successful results. The aqueous portion of the vaccine dose was diluted to a volume of 0.5 ml in a physiological saline solution (e.g. phosphate buffered saline).

To prepare a single vaccine dose, 0.5 ml of the antigen solution was emulsified with 0.5 ml of Freund's complete adjuvant (first injection) or incomplete adjuvant (second and subsequent injections). The final 1 ml of vaccine was injected intramuscularly into 4 sites (each breast muscle and each leg muscle with 0.25 ml per site). In our standard procedure, the birds received the booster vaccine one week later. We have found that egg antibody titres peak at 21 days after the first inoculation (Trott et al., Reference TROTT, YANG, HELLESTAD and COOK2008). Booster vaccinations (with incomplete adjuvant) can be given every 2 months as warranted.

Over the past 25 years, there have been situations in which the vaccine caused health problems in the birds. In some cases the antigen was an outright toxin to the bird. In these situations, it was much better to prime the bird with denatured toxin or toxoid, and then introduce the toxin slowly, with increasing doses over an extended period of time (Trott et al., Reference TROTT, YANG, GONZALEZ, LARSON, TEPP, JOHNSON and COOK2009b). Occasionally, after immunisation, hens have shown signs of health problems after 14 days, and these adverse events (rarely characterised) have been attributed to auto-antibodies. We have occasionally observed situations in which birds stop laying, become sick and die, develop abnormal organ pathology, and refused to eat beginning on day 21 after the first injection. Hence, it has been important to use a limited number of birds each time a new antigen or method of preparing the vaccine was investigated.

Eggs collected from the hens were usually processed in lots, and changes over time in antibody quality (e.g. avidity and affinity) warrants additional study. Eggs used for laboratory reagents were usually purified according to Polson et al. (Reference POLSON, VON WECHMAR and VAN REGENMORTEL1980) and stored in aliquots so that a sample would not undergo freeze/thaw cycles; however, we have evidence that showed freeze/thaw had no adverse effects on titre for up to twenty-cycles when freezing conditions were -20C. Eggs used in animal experiments were freeze- or sprayed-dried as egg yolks.

Potential targets for antibody synthesis

The targets for making antibodies and the potential uses of them have wide application. A flock, such as the one used at the University of Wisconsin, proved to be a valuable source of birds for the generation of laboratory reagents for scientists across campus. We often found that a single hen or even a single egg supplied enough antibodies to meet the lifetime needs of a laboratory. In a recent Homeland Security funded project for the National Centre for Food Protection and Defence (NCFPD), our laboratory purified 2.2 grams of anti-botulinum neurotoxin from 20 eggs in one afternoon, which had a value of several million dollars when compared to the current pricing for commercial rabbit anti-botulinum neurotoxin (Trott et al., Reference TROTT, YANG, GONZALEZ, LARSON, TEPP, JOHNSON and COOK2009b). Hence, the value of the laying hen can be increased significantly in a very short period of time. Also, there should never be a need to consider antibody cost or availability in the design of detection systems in which antibodies are a key reagent.

The laboratory's primary objective has been the large-scale production system of egg antibody, and not reagent production. In Cook (Reference COOK2004), we estimated that a single human application of egg antibodies that required one egg per person per day, would more than double the U.S. need for laying hens. The opportunities for the entrepreneur to develop specific antibody applications require only imagination. The number of commercial antibody applications will expand with the development of new extraction methods, improved strategies to increase heat-stability, and improved methods to increase the egg yolk antigen-specific antibody levels.

Commercial applications

The use of the laying hen for the production of antibody reagents for laboratory analysis appears to have no limit. A number of companies worldwide have extensive lists of reagents derived from egg antibodies. Most companies that have specialised in producing egg antibodies provide custom synthesis services that produce low-cost antibody to specific proteins or peptides provided by a customer. Most companies currently producing antibodies as reagents commonly included chicken egg antibodies in their product list.

While the production of laboratory reagents using egg antibodies has been an extremely important commercial endeavour, research groups have been intrigued with finding uses for large quantities of low cost antibodies (Cook, Reference COOK2004). The selection of antigens for mass antibody production using laying hens can be categorized into two general areas of use in animal agriculture:

1. 1. Antibodies that target infectious processes in the animal

2. 2. Antibodies that improve animal growth and feed efficiency independent of specific disease organisms.

The latter area has been the primary research focus, specifically ‘Host-Targeted Immunotherapy.’ Most commercial products currently in the marketplace are directed toward pathogenic or disease causing bacteria, viruses and protozoans. Arkion Life Sciences has created a product called Immune 26® that is directed toward 26 enteric pathogens that can affect human gastrointestinal function. Arkion LS manufactured a product for Trouw Nutrition called Protimax®, however this antibody product has been directed toward the F-18 and K-88 strains of E. coli, known to cause scours in pigs and calves, rather than avian applications. Ghen Corp in Japan has produced an antibody cocktail called Globigen® that is specifically designed for enteric organisms in the gastrointestinal tract of chickens (Globigen®22). IgY Biotechnologies has developed a product called AnimalGuard® which is similar to those mentioned, and NeutraGuard® that is being marketed as a functional food or supplement for ‘protection from bacteria, viruses, pollutant or allergens.’ They have also marketed an anti-gluten antibody product for gluten-intolerant people. Good Biotech Corporation is another example of a company producing egg antibodies that target enteric microflora. Their primary product contains duck immunoglobulins that may have some unique properties due to the structure of the duck immunoglobulin molecule (Higgins, Reference HIGGINS1996).

The commercial use of antibodies for the protection against infection is in keeping with the evolutionary basis of passive immunity. There is no question about the efficacy of protection associated with antibodies against enteric pathogens (reviewed by Kovacs-Nolan and Mine, Reference KOVACS-NOLAN and MINE2004a,b). While enteric pathogen-specific antibodies are useful in commercial agriculture and human medicine, their use has one significant limitation. The user of the product must have evidence that the particular pathogen is responsible for decreased health or performance. Since enteric pathogen problems in commercial production settings are unpredictable, the use of commercial products to treat these problems had the same limitation. In the absence of an actual enteric pathogen associated with decreased animal performance, the return on investment associated with use of the product was more difficult to quantify unless the problem was recurring and predictable.

In the mid 1980s, our research group set out to find applications of egg antibodies with a broader value in regards to their use in animal health (Cook, Reference COOK2004). The objective was to produce an antibody-based product that would:

1. 1. Have broad use in animal agriculture and not be limited to a specific disease state

2. 2. Solve a significant problem facing the animal agriculture industry

3. 3. Improve the economical bottom line for all players in animal production

This third requirement caused focus on two critical endpoints: animal growth and feed efficiency. If the antibody product improved growth and/or feed efficiency, then the product would be valued by the animal producer and the major integrator. The safety of the antibody product generated also had to be demonstrated to insure that there were no adverse effects on animal health.

The first product developed was focused on using the passive transfer of antibodies to the enzyme urease (Pimentel and Cook, Reference PIMENTEL and COOK1988; Pimentel et al., Reference PIMENTEL, COOK and JONSSON1991). This concept was largely based on the work of Visek at the University of Illinois (Dang and Visek, Reference DANG and VISEK1960), which showed the ammonia load of the gastrointestinal tract had adverse effects on animal growth. Anitox Corp has been actively working on the development of this antibody product (Pimentel, personal communication).

A more recent focus was on targeting the mechanisms by which antibiotics improved animal growth. More specifically, the antibodies created were designed to prevent the adverse effects of inflammation on animal growth and performance as described by (Cook, Reference COOK2004). Several antibody targets proved to have intended results when fed to animals. Antibodies for gut peptides, cholecystokinin (aCCK, where the small ‘a’ preceding the ligand/antigen targeted signifies ‘antibody’ directed to the ligand/antigen), and neuropeptide Y (aNPY) were found to be effective at improving both growth and feed efficiency in broiler chickens (Cook, Reference COOK2004). Numerous other peptides have been examined and were found to have no benefit in the chick bioassay of growth and feed efficiency. It was hypothesised that these gut antibodies exert their effects by the sequestering of bioactive peptides that regulate appetite. However, data collected from numerous studies suggested that appetite regulation was not the primary mode of action. Basic research awaits further clarification of the mechanism by which these antibodies improve animal performance.

Another group of targets identified in the last seven years was the by-product of other studies being conducted in our laboratory. We identified that antibodies targeting the enzyme phospholipase A₂ (aPLA₂) were an effective means of improving animal performance. In our recent report (Cook, Reference COOK2004), we showed that aPLA₂ improved broiler gain by 5.4% and feed efficiency by 6.2 points in five 3-week chick trials. The company that licensed the aPLA₂ technology (aOvaTechnologies Inc.) provided data from 16 chick trials (the standard bioassay for product efficacy) showing an average improvement of 5.3% in gain and 3.7% (or 5.8 points) in feed efficiency. This same company has shown improvements in the growth of other species, results suggested that the mechanism of action was conserved across species. Data, collected by aOvaTechnologies and participating research groups, suggested that aPLA₂ was additive with antibiotics with regards to improving animal performance. Hence, the aPLA₂ mechanism of action may be more complex than originally thought. One hypothesis we are currently exploring is that the antibodies may be altering the nature of food molecules/peptides and the host response to them.

While this review has focused on the scientific publications and commercial companies involving egg antibody technology, there is another collection of work that could prove useful for readers interested in the commercial aspects of egg antibody. A number of patents have been filed on use of egg antibody technology, and are now listed on government patent office websites. A search of the U.S. Patent and Trademark website www.uspto.gov or Google Patents www.google.com/patents could provide more information on products being developed and companies involved.

Acknowledgments

Mark E. Cook has an ownership interest in aOvaTechnologies Inc., which has licensed from Wisconsin Alumni Research Foundation technology reported in this publication. The contents of this paper were presented at the IV International AMEVEA meeting in Lima Peru, May 15-21, 2007. The authors extend an appreciation for the organizing committee of this meeting and specifically acknowledge Dr Ricardo Valdivia for his interest and thoughts regarding this topic. The authors acknowledge the people in the Cook laboratory that have contributed to the observations reported in this paper: Julio Pimentel, Cheryl Miller, Mingder Yang, Jon Jonssonn, Elizabeth Bobeck, Kevin Rowe, Sharon Worzalla, Monica Vanko, Erica Hellestad, Teresa Casey, Guangming Li, Sean Ryan, Stewart, Covi, J. Gonzales, and Irina Manevska.