Polyclonal Antibodies in Chicken - Beyond Omelets and Sandwiches

Advances made in poultry industry have made chickens an excellent source of daily nutrition. However, few Scientists realize that chickens are an excellent host for raising antibodies as well. If the immunological ability of Chickens is more publicized then more and more researchers will chose Chicken as an antibody production host. This short review will highlight the advantages of chicken-derived antibodies over the more conventional rabbit or goat polyclonal antibodies.

Immune System

The ability to raise antibodies against foreign substances is exclusive to eukaryotes. Most animals and humans encounter hundreds of potentially harmful agents in the form of bacteria, viruses, toxins, allergens, etc on a daily basis. Animals and humans respond to this threat by synthesizing and secreting specialized group of proteins known as immunoglobulins or IgGs. The IgG circulates in the blood and protects against previously encountered and damaging pathogens or molecules derived from them. Theoretically, anything that is not "self" by default is "non-self" and must be attacked and defended against. Therefore higher vertebrates continue to monitor the presence of any foreign substance and react by quickly releasing the neutralizing IgG. By definition, any substance that is capable of invoking ’antibodies or IgGs’ is an antigen. The "Immune System" is one of the most complex and fascinating biological phenomenon. Obviously, any mistakes in the recognition of "self" may lead to ’autoimmune diseases’- the state in which an organism starts attacking its own proteins and cells. Autoimmune arthritis or systemic lupous erythematosus (SLE) are examples of such autoimmune diseases. Many diseases may also compromise the ability of an individual to respond to foreign substances and limit their ability to make antibodies. HIV infection or AIDS suppresses the immune system and the AIDS-infected individuals may succumb due to their inability to handle common illnesses such as common cold or pneumonia.

Antibodies or IgGs have the ability to recognize and bind its specific antigen among thousands and millions of other possible antigens. Antibodies can distinguish subtle changes in the conformation of a molecule or even a single amino acid. No biological interaction is more specific than the ’antigen-antibody interaction". Therefore, antibodies are needed to study location of protein in cells and decipher their mode of action. Many immunological techniques have been developed that make use of specific antibodies (ELISA, Western, Immunohistochemistry, etc.). Advances made so far in biological sciences would not have been made possible without the use of antibodies.

Chickens as Antibody Host and Chicken IgY

How does one go about raising antibodies? All that is needed to make antibodies is to challenge host animal with an antigen, which it would consider considered as foreign. A repeated exposure of antigen (immunization) elicits antibodies in the host animal. These antibodies can be detected in serum by immunological method such as ELISA and Western blotting techniques. Obviously, if the injecting substance is identical or similar to the host, it will be a poor immunogen resulting in low or no antibodies. Many highly conserved proteins such as Insulin and Cytochrome-C are poor antigens in hosts such as rabbits or goats. In these cases it may help to change the host to such as chicken since birds are more distant in evolution than mammals. The fact that many mammalian proteins are less conserved in birds, therefore, chickens may be more desirable as an antibody host may.

Structure and Uses of IgY

IgG’s are produced by mammals. Lower vertebrates (reptiles, amphibian, and birds) produce an equivalent of mammalian IgG known as IgY. IgY simply indicates that the antibody has bee derived from the egg yolk. The IgY has the same general structure as mammalian IgG with 2 heavy chains known as "nu" (~65-70 kDa) and 2 light chains (22-30 kDa). IgY, mol wt ~180 kDa, runs as a broad band because of the presence of substantial amount of carbohydrates (approx. 3%). The nu chains have 4 constant and 1 variable domains. However, IgY lacks the "Fc" domain and can not fix complement or bind to protein-A or G. So Protein A/G can not be directly used to purify IgY or used in immunoprecipitation. Therefore, it will be necessary to modify the IP assay and use another mammalian derived (rabbit/goat etc) anti-IgY and then use Protein A/G for IP’s. Nonetheless, using appropriate secondary antibodies (anti-chicken IgY-Peroxidase or Phosphatase or FITC conjugates), chicken antibodies can be used in most immunological assays.

Since chicken antibodies do not crossreact with mammalian IgGs, bind to Fc receptors, interact with rheumatoid factors or react with HAMA (human anti-murine antibodies), non-specific binding of chicken antibodies is greatly reduced in most immunological assays. In addition, unlike most mammalian secondary antibody-enzyme conjugates, it is not necessary to adsorb the chicken antibody-enzyme conjugate with mammalian protein to reduce background in most immunoassays.

Immunization and harvesting of chicken antibodies (IgY) from Egg Yolk

Chickens are immunized using the same general method as used when utilizing rabbits or goats. Injections are given at multiple sites in the breast muscle. It takes at least 2 weeks or more to initiate immune response. Antibodies are produced and released into serum. Antibody titer increases during subsequent immunization. Although, one may collect chicken serum, it is preferred not to stress the birds since laying hens also concentrate antibodies in the egg yolk. Laying hens produce eggs almost on a daily basis and harvesting eggs is a very noninvasive method of collecting antibodies. On an average, a single egg may contain the equivalent of a standard rabbit bleed, harvesting eggs greatly enhances the amount of antibodies that can be collected from a single chicken. Typically rabbits are bled every 2 weeks yielding ~15-20 ml serum whereas chicken may lay 12-15 eggs during the same 2-week period. Therefore, antibody production in chicken can be 15-20 times of that obtained in rabbit. Antibody concentration in the egg yolk is higher than what is found in chicken serum. This high concentration of antibodies is presumably used to confer passive immunity to newly hatched chicks. It is estimated that a single egg may contain ~100 mg total antibodies of which 1-10 mg (1-10%) may represent the specific antibodies.

Purification of chicken IgY from egg yolk and storage of antibodies

Egg yolk contains large amount of lipids (lipoproteins and phosvitin granules mixed with livetins and low-density lipoproteins) making their direct use in immunoassays problematic. IgY can be isolated from egg yolk by using stepwise ammonium sulfate and polyethylene glycol (PEG) precipitation. Partially purified IgY is quite stable at 4oC for years provided a bacteriostatic agent (Azide or Merthiolate) has been added to prevent microbial growth. The core body temperature of chicken is about 41oC and half-lives of chicken antibodies is in months. Therefore chicken antibodies are also quite stable at room temperature for months. It is, however, advisable to freeze antibodies in suitable aliquots and avoid freeze and thaw. Freeze and thaw will do more harm to antibodies than leaving them at 4oC for weeks and months.

ADI can purify IgY from the egg yolks as an additional service or a kit can be purchased for in-house isolation and purification of antibodies (Chicken IgY purification kit # 60100).

How about secondary antibodies (Anti-chicken IgY-Enzyme or -FITC conjugates).

It is debatable if there is a real difference between anti-chicken antibodies that will distinguish between chicken serum IgG or egg yolk IgY (antibodies derived from egg yolk). Most commonly available anti-chicken IgG antibodies or enzyme conjugates will detect both chicken serum IgG and egg yolk IgY.

Most commonly used anti-chicken secondary antibodies are available from ADI and other manufacturers.

Rabbit Anti-Chicken IgG/Y-HRP conjugate, Cat # 60320;

Rabbit Anti-Chicken IgG/Y-Biotin conjugate, Cat # 60321

Rabbit anti-Chicken IgG/Y-Alk. Phosphatase, Cat # 60322

Rabbit Anti-Chicken IgG/Y-FITC conjugate, Cat # 60323

ELISA kit to detect chicken (serum or IgY) antibodies (the kits contains, coating buffer, blocking buffer, wash buffer, anti-chicken-HRP conjugate, TMB substrate, and stop solution). Sufficient reagents are provided to perform 1000-2000 tests. Cat # 80176.

Further Reading…….

Warr WG et al (1995) Immunology Today 16, 392. (Review)
Larsson A et al (1993) Poultry Sci. 72, 1807 (Review)
Puffinbarger NK (1995) Mol. Pharmacol. 47, 1126
Akita EM et al (1993) J Immunol. Methods 162, 155.
Danielpour, D (1993) J Immunol. Methods 158, 17.
Larson A et al (1993) Hybridoma 12, 143
Lee SC et al (1997) Prep. Biochem. Biotechnol. 27, 227.
Fortgens PH et al (1997) Immunopharmacol. 36, 305.
Gassman M et al (1990) FASEB J 4, 2528

Well folks, think of antibodies when eating eggs.

The egg may be worth its weight in gold and many times over...