An immune system is present in all species in the animal kingdom and is a

defense against intruding organisms, molecules and malignant cells. The purpose is to protect its host from foreign (i.e. non-self) substances. In a broad sense the immune system in birds is no different from the immune system found inmammals. The immune system can be divided into two parts: the innate (nonadaptive) and the acquired (adaptive) immune system. The innate response compromises many functions and acts as a first line of defense against infections whereas the adaptive immune response is highly specific for a particular antigen. Antibodies belong to the adaptive immune system.

The innate immune system

The skin and mucous membranes act as barriers to prevent invasion of

microorganisms. Cilia, mucus and the cough reflex expel inhaled material from

the respiratory tract. In the gastrointestinal tract the low pH in the stomach and

the normal bacterial flora in the gut also have important protective roles. The

normal bacterial flora acts by competing for essential nutrients or by producing

inhibitory substances against the invading organisms, thereby suppressing the

growth of many potentially pathogenic bacteria. When antibiotics or conditions

like diarrhea disturb the normal bacterial flora the susceptibility for opportunistic microorganisms is increased. Pathogens may also trigger activation of the complement system, which results in the formation of the membrane attack complex, facilitating cell lysis and cell death. There are also several white blood cells involved in the innate immune system. Avian natural killer (NK) cells are large granular lymphocytes that are morphologically similar to mammalian NK cells. The NK cells selectively identify and kill virus-infected and tumor target cells and do not need prior antigenic exposure for target recognition. Unlike cytotoxic T-cells theNK cells are not MHC-restricted. Another important component of avian innate immunity is the monocytesmacrophage system. Macrophages are an important part of the innate immune defense, operating immediately when a microorganism enters the body, thereby limiting the growth of the pathogen. It is also an effector cell during the late phase of the acquired immune response. Macrophages have microbicidal, phagocytic and tumoricidal functions but they also acts as regulatory cells through cytokines and other metabolites. Monocytes are the major phagocytic cell in chicken blood while tissue macrophages are present in almost all organs. Macrophage phagocytic function appears within the first two weeks of chicken embryonic development. The non-specific mechanisms respond rapidly to a foreign invasion but it does not have the ability to respond with increasing strength to repeated challenges from the same organism.

The acquired immune system

Specific immunity depends on the ability to recognize foreign substances,

respond to them and memorize the information in case of repeated exposure. The specific immune system functions through two interacting mechanisms, the humoral and cellular responses. The humoral response involves interaction of B-cells with an antigen and the subsequent proliferation and differentiation into antibody-secreting plasma cells with or without the help of T-helper-cells. Two basic types of lymphocytes are involved in an antigen-specific response. The B-lymphocytes express surface immunoglobulins that are specific to an epitope on the antigen and Tlymphocytes that recognize processed antigens on antigen-presenting cells. The antibody-secreting plasma cells produce soluble antibodies that are identical to the surface immunoglobulin on the original B-cell. The cellular immune response involves interaction of the T-cell receptor and processed antigen. There are two main pathways. The first is reaction of T-cells with antigen and lymphokine secretion that attracts macrophages to the site, which will fagocyte the antigen.The second route is interaction of cytotoxic T-cells with processed antigen presented by MHC class I cells which eventually leads to cell lysis .

IgY in the chicken

Immunoglobulins in the chicken

Three immunoglobulin classes, analogues to the mammalian immunoglobulin

classes have been shown to exist in chicken, IgA, IgM and IgY (IgG). The

presence of antibodies homologous to mammalian IgE and IgD has also been

proposed but has not been proven. The molecular weights, morphology and immunoelectorophoretic mobility of chicken IgA and IgM are similar to mammalian IgA and IgM. IgY is the major low molecular weight serum immunoglobulin in oviparous (egg laying) animals. Chicken IgY is a systemic rather than a secretory antibody but IgY is also found in duodenal contents, tracheal washings and seminal plasma. It is called IgY rather than IgG to distinguish it from its mammalian counterpart. The argument was that the heavy (H) chain of the molecule is larger and antigenically different from the mammalian heavy chain. There is no immunological similarity between chicken IgY and mammalian IgG, and the DNA sequence of chicken

IgY resembles more the sequence of human IgE. There is a lower content of β-sheet structures in IgY that may indicate that the conformation of the IgY domain is more disordered and less stabile compared to that of rabbit IgG domains. The overall structure of IgY is similar to mammalian IgG, with two light (L) and two heavy chains. The molecular mass has been reported to be 167 250 Da, slightly larger than IgG (~160 kDa). Interestingly, the light chain is lighter than its mammalian counterpart. The H chain (Mw 65 105 Da), called upsilon, υ, (capital letter Y) has one variable (V) region and four constant (C) regions. The light chain (Mw 18 660 Da) is composed of one variable and one constant domain. The Cυ3 and Cυ4 of the IgY are most closely related to the Cγ2 and Cγ3 of IgG respectively and the Cυ2 domain is absent in the γ chain. The Cυ2 region was probably condensed to form the hinge region of IgG as studies have shown that IgY is an ancestor to mammalian IgG and IgE and also to IgA. The Fc region of IgY mediates most biological effector functions in the chicken, such as complement fixation and opsonization. IgY is a skin-sensitizing antibody that can mediate anaphylactic reactions, a function that is attributed to IgE in mammals. In many ways IgY combines the functions associated with mammalian IgG and IgE in the chicken.

Immunoglobulin diversity in chickens

Studies of the chicken immune system have contributed substantially to the

understanding of the immune response, including separation of the T- and B-cell lineages. The chicken immune system consists of the bursa of Fabricius, bone marrow, spleen, thymus, the Harderian gland, lymph nodes, circulating

lymphocytes and lymphoid tissue in the alimentary tract. The antibodysynthesizing cells (B-cells) are produced by the bursa of Fabricius. The chicken bone marrow is the source of bursal and thymic stem cells while the spleen is the center for plasma cell proliferation and memory B-cells. Birds without spleen have a lower antibody production. The thymus is a maturation center where stem cells differentiate into T-lymphocytes. The activities of chicken Tlymphocytes are similar to those in mammals. The mechanism of antibody diversity in chicken differs from mammals and is mainly due to somatic hyper conversion. Rearrangement contributes little to the

diversity as both the heavy and the light chain loci consist of only one functional V (variable) gene. There also seems to be a deficiency in the mechanism for selecting higher-affinity somatic mutants. The chicken has solved this deficiency by using three mechanisms that diversify the limited germ-line repertoire: gene hyper conversion. V-J flexible joining and somatic point mutations. Gene hyper conversion starts around day 15-17 of

incubation after the immature B-cell progenitors migrate to the bursa of

Fabricius. During this process blocks of DNA are transferred from pseudo-V

genes to the recombined variable regions of the Ig genes resulting in the

production of mature B-cells competent to form a functional humoral immune

system. The υ heavy chain gene is encoded by three exons separated by only two introns, as there is no intervening DNA sequence between the CH1 and CH 2 alleles. The immunoglobulin heavy-chain constant regions of IgY, IgA and IgM are all located on chromosome E18C15W15. The IgA gene is located upstream the IgY gene in an inverted transcriptional orientation. The distances between the IgA, IgY and IgM genes are about 18 and 15 kilobases, respectively. The size of the whole chicken IGHC locus is approximately 67 kilobases. Furthermore there are 16 alternative diversity (D) segments in the heavy chain locus. However, only V(D)J joining in the chicken can not produce the combinatorial diversity of the large numbers of V and joining (J) segments seen in mammals. Instead, an equivalent degree of diversity is achieved by successive partial conversions of the rearranged V(D) segments by templates in an upstream array of pseudo-V(D) genes. The variable and joining segments of both the heavy and light chain loci undergo V(D)J rearrangement. The entire naive B-cell repertoire of the adult chicken is produced in the Bursa of Fabricius of the young bird.

Transport of IgY from maternal serum to the offspring

The transport of IgY from the hen serum to the offspring is a two-step process.

First IgY is transported from the serum to the egg yolk in analogy to the crossplacental transfer of antibodies in mammals. The second step is the transmission of IgY from the yolk sac to the developing embryo.

The concentration of IgY in the yolk is essentially constant through the oocyte

maturation, and at maturity the yolk will contain about 10-20 mg/ml IgY.

Looking at the egg, IgY is not present in the egg white while IgA and IgM is not present in the yolk. There is about 100-400 mg IgY packed in the egg.

Labeled IgY binds specifically to yolk sac tissue from day 7 up to at least day 18. This binding is saturable, Fc-specific, pH-dependent and reversible.

There is both a high and a low affinity receptor for IgY on the embryo. The low

affinity receptor, (KD 3.4 ×10-7), is present at day eight, whereas the high affinity receptor (KD 3.0×10-8) is detected at day 18. The low affinity receptor has a constant density as the total weight of the yolk sac increases, which implies that the rate change is due to an increase in tissue mass . The receptor binds some heterologous IgY such as pigeon IgY but in a less efficient manner. Molecules such as bovine serum albumin, phosvitin, conalbumin, chicken IgM and chicken Fab fragments does not bind. The IgY receptors on the oocyte bind and move all populations of IgY from the hen serum to the egg. The populations of IgY are transported according to their concentration in the maternal serum. There is no selection nor destruction of IgY during transport and the yolk IgY has the same amount of sialic acid as the serum IgY. The amount of IgY transported is independent of egg size and known to be

proportional to the maternal serum IgY concentration. A delay of three to

four days is found between the appearance of IgY in serum until it is found in the yolk. The concentration of IgY in the yolk is by a factor 1.23 to the serum

concentration. The density of yolk is about 1.1 g/ml. About 50 % of the yolk

is non-aqueous material. The total amount of IgY in the hatched chick has been estimated to be only 2-3 mg, compared to the 100-400 mg present in the yolk. The major part of the IgY probably serves only as nutrition for the developing embryo. In the newly hatched chick the IgY concentration in circulation is about 1-1.5 mg/ml and the circulating half-life of IgY is about 36 h. IgY secreting cells in the offspring are not detectable until six days after hatching.

IgY in vitro

Biochemical properties of IgY

The valency of IgY is two, same as for mammalian antibodies. In place of

the hinge region of mammalian IgG, IgY has a sequence that is more rigid,

giving IgY limited flexibility. This probably is the reason for many of the

different properties of chicken IgY in comparison to mammalian IgG.

The restricted mobility of the hinge region (Cυ2) in IgY heavy chain makes the

antibody more rigid. This affects the capability of the antibody to precipitate or

agglutinate antigens. Only part of chicken antibodies is precipitated at

physiological salt concentrations and approximately 25% of the antibodies

remain in the supernatant at maximum precipitation. The precipitation curve

resembles the curve obtained with horse antibodies with a rapid decline with

antigen excess. The precipitation improves at 1.5 M NaCl. The poor

precipitation properties might be due to steric hindrance of the Fab arms to cross link epitopes of two large antigens. The conditions permitting precipitation might loosen the restricted movement of the Fab arms and give functional independence to the binding sites.

Orally given IgY is generally not immunogenic but IgY injected intravenously is an immunogen and elicits a typical anti-IgY IgM and IgG response in mice.

IgY applied to other endothelial surfaces then the gastrointestinal tract is

probably immunogenic but not yet sufficiently tested. The stability of IgY under acidic conditions and toward pepsin digestion is slightly lower than that of bovine IgG. However, IgY is fairly stable against digestion by internal

proteases such as trypsin and chymotrypsin. There appears to be one

subpopulation of IgY resistant to papain digestion. Immune complexes formed with chicken antibodies are slightly different to those formed with rabbit antibodies. The precipitation curve is steeper and the antigen excess effect on immune complex formation is more pronounced.

Advantages of IgY

As the difference between the antigen and the immunized animal increases, the

immune response usually increases. There is a greater phylogenetic difference

between avian and mammalian species compared to the difference between two

mammalian species. This evolutionary spread means that there is no immunological cross-reactivity between chicken IgY and mammalian IgG. As a result, chicken is a better choice than e.g. rabbits for the production of antibodies against conserved mammalian proteins. Due to this evolutionary difference, chicken antibodies will bind to more epitopes on a mammalian protein than the corresponding mammalian antibody. It has been shown that 3-5 times more chicken antibody than swine antibody will bind to rabbit IgG which will amplify the signal in an immunological assay. Chicken antibodies also recognize other epitopes than mammalian antibodies. This gives access to a different antibody repertoire than the traditional mammalian antibodies.

Cross-reactivity occurs between IgG from different mammalian species. An

increased background binding may result if a secondary anti-mammalian IgG

antibody is used. The secondary antibody may cross-react with IgG that is

present in a histological section or with bovine IgG in the bovine serum albumin solution often used for blocking purposes. Because chicken IgY is so different from mammalian IgG, no cross-reactivity occurs between the two. Therefore, contrary to an anti-rabbit IgG antibody, a secondary anti-chicken IgY antibody will not react with mammalian IgG in the tissue and this may reduce background staining. The use of chicken egg yolk as a source for antibody production represents a reduction in animal use as chickens produce larger amounts of antibodies than laboratory rodents. It also makes it possible to eliminate the collection of blood, which is painful for the animal. The European Centre for the Validation of Alternative Methods (ECVAM) recommends that yolk antibodies should be used instead of mammalian antibodies for animal welfare reasons. IgY can be used in many immunological assays giving a better result than mammalian antibodies used by tradition. Prevalence of human anti-mammalian antibodies varies from 1-80 % in the general population . These human antimammalian antibodies may cause interferences in immunological assays.

IgY avoid complement activation

In clinical laboratories, most analyses are performed on serum samples. A newly obtained serum sample contains an active complement system, but the activity declines during storage and handling. Accordingly, the complement activity may vary between different patients and also between different samples from the same patient. To avoid activation of the complement cascade EDTA is often included in tubes used for blood sampling. EDTA prevents coagulation by sequestering the calcium ions required for clotting. Most of the standards and controls used have been stored and contain an inactive complement system. This difference in activity between the samples and the standards will cause erroneous results. Mammalian antibodies bound to a solid phase and antigen-antibody complexes containing mammalian antibodies will activate the human

complement system. Activated C4 bind to the Fab region of IgG and may

interfere with the antigen binding. Complement components may also

solubilize precipitated immune complexes and prevent soluble immune complexes from precipitating. Such complement activation was shown to

interfere in an immunometric TSH assay and depressed the TSH values by up to 40 % . Because chicken antibodies do not activate the human complement

system, they can be used as to reduce interference by complement activation.

IgY avoid RF and HAMA interaction

Rheumatoid factor (RF) and human anti-mouse IgG antibodies (HAMA) are

probably the most well known causes of false positive or false negative reactions in immunological assays . RF is an auto-antibody that reacts with the Fc part of mammalian IgG. The disease most often associated with RF is rheumatoid arthritis, but RF can be found in serum from patients with many other diseases and also in 3-5% of healthy blood donors. An increasing number of patients are treated in vivo with mouse monoclonal antibodies. This treatment often evokes an antibody response in the patient resulting in production of HAMA. HAMA may also be found in serum from patients who have not been treated with antibodies. However, the increasing use of monoclonal and polyclonal antibodies in vivo will increase the number of patient samples that contain HAMA. RF or HAMA may react with both the capture antibody and the detection antibody in a sandwich assay, thereby mimicking antigen activity. A reaction with the detection antibody, results in formation of an immune complex. This immune complex may influence the activity of the detection antibody. HAMA may also react with the antigen-binding epitopes and inhibit the antigen binding. The problem of RF and HAMA interference will increase as the sensitivity of the assay increases. Interference by anti-IgG antibodies and antibody-binding substances have been demonstrated in approximately 40% of serum samples from healthy individuals in an immunoradiometric assay. RF and HAMA will also give erroneous results in nephelometry and turbidimetry as they change the size of antigen-antibody complex . Chicken IgY does not react with RF or HAMA and can be used to avoid interference due to these factors.

Human Fc receptor interaction

Intact mammalian IgG molecules contain the Fc portion of the antibody. Fc binds to Fc receptors, which are found on many types of blood cells. Human

FcγRI has a high affinity for monomeric mammalian IgG, while FcγRII and

FcγRIII mainly bind mammalian IgG complexes. There is often some aggregated IgG formed during the purification of IgG or during the labeling procedures that will increase the binding to FcγRII and FcγRIII receptors. Interaction with Fc receptors may cause an increased background staining. When working with living cells the interaction with Fc receptors may cause cell activation and changes in the expression of surface proteins. It has been shown that mammalian antibodies used in flow cytometry form immune complexes that cause platelet activation and changes in the expression of the GpIIb-IIIa receptor. No activation was observed when chicken antibodies were used. Immune complexes containing mammalian IgG may also stimulate the production of cytokines. Chicken antibodies do not react with human Fc receptors and their use will avoid these problems.

Bacterial Fc-receptor interaction

Staphylococcal protein A and Streptococcal protein G are Fc-binding bacterial

proteins which are widely used for their ability to bind to IgG. Bacteria of the

Staphylococcus aureus Cowan 1 strain and group C Streptococcus sp. are also

used as immunoadsorbent for mammalian IgG. Staphylococci and Streptococci

are often found in bacterial samples. When present, they may bind detection

antibodies with specificities for other bacteria and cause erroneous results.

Chicken antibodies do not react with protein A or protein G and can be used to

reduce interference problems due to bacterial Fc receptors. There are also other bacteria (e.g. Peptostreptococcus magnus, Streptococcus suis and Actinobacillus actinomycetemcomitans) with Ig-binding capability . The binding of IgG to the Fc receptor probably have a protective function for the bacteria. Bacteria isolated from human specimens will have Fc receptors

with affinity for human immunoglobulin. Due to the immunological similarities

these Fc receptors will often bind other mammalian immunoglobulin but not

avian IgY. On the other hand, bacteria isolated from avian specimens will

probably have Fc receptors for avian immunoglobulin instead of mammalian

IgG.

Antibody production

Chickens can be used for antibody production throughout their entire egg laying period. Animals that are used for antibody production for more than three months should be given booster immunizations every other month to assure that the antibody titer remain high. Chickens can produce high avidity antibodies already after one immunization, compared to sheep whose avidity becomes similar after four immunizations. In response to monthly re-immunizations sheep have been found to produce ten times more specific antibodies than chicken, probably due to the size difference of the animals. Another reason for this might be the differences in antibody half-lives. In sheep the half-life is about 15 days, compared to 36 hours in the chicken. The species may therefore produce immunoglobulins at a comparable rate. The high catabolic rate of the chicken may prevent the accumulation of high titers. However, the avidity in both chickens and sheep after four immunizations was 109 to 1010 l/mol. Freund´s complete adjuvans is quite well tolerated in chickens, as the characteristic local inflammatory response seen in mammals is often not observed. Other types of adjuvant than Freund´s adjuvant can also be used, such as Specol, Hunters TiterMax, and the lipopeptide Pam3-Cys-Ser-(Lys)4 . After immunization with human serum albumin the highest serum IgY titer is found seven to nine days after a single intravenous or intraperitoneal injection. Intramuscular immunization shows a higher antibody level from day 28 after immunization and the specificity is almost more than 10 times higher

compared to sub-cutaneous immunization. Non-reimmunized chickens

investigated more than 200 days later showed a similar high IgY level of specific antibodies. The presence of IgY in the yolk is detected four to seven days after the appearance in the serum. Monoclonal chicken antibodies have also been reported. So far, the number of different chicken monoclonal antibodies is limited, but is certain to increase in the future. This will allow the advantages of monoclonal antibodies to be combined with those of chicken antibodies. There are still not many cell lines well suited for fusion with avian cells. The amount of antigen specific antibodies of the total pool of antibodies in an egg has been reported to be up to 10 % [69], [70]. However, the actual amount of specific antibodies probably varies depending on the individual animal, immunization procedures and the immunogenicity of the antigen itself.

As a laying hen produces approximately 20 eggs per month, over 2 gram IgY per month can be isolated. The IgY concentration in chicken serum is approximately 5-7 mg/ml, therefore 2 gram of egg yolk IgY corresponds approximately to the IgY content of 300 ml of serum or 600 ml of blood. Only larger mammals can produce equal amounts of serum antibodies and compared to rabbits, the chicken antibodies are ten times less expensive.

Purification methods

There are several methods of purification of IgY described. The choice of

method is a matter of yield and purity desired, final use of the IgY as well as

material cost and labor skills. To obtain material from which chicken antibodies can be isolated, either bleeding of the animal or collecting eggs is necessary. However, as the chicken has fragile veins, bleeding is often difficult and results in large haematoma formation. Poor clot retraction can also limit the amount of serum obtained. Sometimes, only 100 μL of serum is obtained from 2 ml of blood. Plasma is therefore more useful than serum.

The best way to obtain antibodies is to purify them from the yolk. Several

methods can be used, even for large-scale purification, of functionally active

chicken antibodies from egg yolk. Over 100 mg of purified IgY can be obtained from a single egg. It is also possible to purify specific antibodies by affinity-chromatography. The antibodies are applied at a neutral pH to a column where the antigen of interest is bound to the matrix. The column is then washed with PBS (0.02 M Na2HPO4, 0.15 M NaCl, pH 7.2) to remove unspecific IgY. Bound antibodies are then eluted with 0.1 M glycine