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IgY, an immunoglobulin produced by chickens and other avian species are detectable in egg yolks as well as their blood. These avian antibody, IgY bears some resemblance to mammalian IgG, but also displays some unique structural and functional characteristics that are sufficiently different and antigenically distinct from IgG. Chicken IgY is the functional equivalent to mammalian IgG. It is found in the serum of chickens and is passed from the mother chicken to the embryo via the egg yolk, imparting a high concentration of chicken IgY to developing embryo. Avian sera contain the three principal classes of immune globulins, namely IgA, IgM and IgY (IgG), however, structurally there is a big difference between them. The major differences observed are:
Chicken antibodies have several distinct biochemical advantages over mammalian antibodies due to the phylogenetical differences between avian and mammalian species. In contrast to mammalian antibodies, Chicken antibodies do not react with mammalian IgG nor IgM1, neither with human anti-mouse IgG antibodies (HAMA)2 ,3, bacterial , human Fc receptors, nor binding to the rheumatoid factor (RF), which is an anti-immunoglobulin autoantibody found in many different diseases4. IgG molecules often give false positive results by interaction with RF in immunoassays 5. The lack of cross-reactivity between IgY and IgG can be utilized in many ways to reduce unwanted reactions in assays, in which anti-IgG antibodies are used. IgY does not activate mammalian complement factors6 , which also helps to reduce the assay interference by complement factors in mammalian serum sample, resulting in increased sensitivity as well as decreased background in immunological assays.
A laying hen produces approximately five to six eggs per week with a yolk volume of approximately 15 ml per egg, the antibody concentration of which is comparable to that of serum. Therefore, in one week a hen produces egg antibodies equivalent to 75 to 90 ml of serum or 150 to 180 ml of whole blood. This could be compared to an immunized rabbit, which yields approximately 20 ml whole blood per week. Only large mammals such as cows or horses can produce more antibodies than a laying hen. The blood collection procedure is time consuming and stressful for the animal. Furthermore, the cost of feeding and handling is considerably lower for a hen than for a rabbit. Crude egg yolk may be used as an antibody source, but the lipids in the yolk may interfere with the antibody activity. Therefore, avian antibodies are usually purified from the yolk prior to use.
In general terms, the antibody response will improve as the phylogenetic distance between the immunized animal and the species providing the antigen increases. Because of the evolutionary distance between birds and mammals, a chicken is often a better choice for antibody production than a mammal when the antigen is of human or other mammalian origin. This is of particular relevance for highly conserved proteins, such as hormones, which demonstrate few differences in amino acids between closely related species. For instance, there is only one amino acid that differs between human and porcine insulin, but seven amino acids differ between human and chicken insulin. Chickens will recognize more sites on the insulin molecule as foreign, and more antibodies will bind to the molecule, which will result in an amplification of the signal in an immunoassay. If a second antibody of mammalian origin is used, the phylogenetic difference will result in further amplification, as more secondary antibody is bound to chicken IgG than to rabbit IgG. Antibody diversity is achieved differently in chickens than in mammals. The chicken light chain locus consists of a single J-C unit to which the same V gene becomes rearranged in nearly all bursal cells. The chicken light chain repertoire thus appears to be of an extremely somatic type. This, combined with the observation that the chicken will frequently recognize more sites on a mammalian protein as foreign, will give access to a different antibody repertoire. It may therefore be possible to produce antibodies in chicken that are difficult or impossible to produce in a mammal.
A frequently used approach for the detection of antigens is to create a so-called sandwich assay (immobilized capture antibody, antigen, and labeled detection antibody). The antibodies in such assays are usually derived from mammals, and the samples to be tested are often serum or plasma. If anti-mammalian IgG antibodies are present in the samples, they may simulate the behavior of the antigen by linking the detection antibody to the capture antibody, thus causing false positive reactions. Such false positive reactions occur in sandwich assays whether or not the assay utilizes mammalian polyclonal or monoclonal antibodies. The most well-known of these anti-mammalian IgG antibodies is rheumatoid factor, which is an IgM antibody reacting with the Fc fragment of mammalian IgG. The disease usually associated with rheumatoid factor is rheumatoid arthritis, but it can also be found in serum from patients with other diseases and also in sera from healthy individuals. As the sensitivity of the assay increases, so will the interference by anti-IgG antibodies. Another cause for the presence of anti IgG antibodies is the in vivo use of heterophilic antibodies. Mammalian antibodies have been used in vivo for the treatment of patients for more than 100 years, but during the last decade there has been an increasing use of such antibodies. Mouse monoclonal antibodies are now widely used in vivo for diagnosis and treatment of patients, and will probably be used even more in the future. When such antibodies are given to the patient, the patient normally responds by producing human anti-mouse Ig antibodies (HAMA). The HAMA will react with mouse antibodies but also with structurally related proteins such as IgG of other mammals. Thus, the presence of HAMA might give false positive reactions with all types of sandwich assays based on mammalian antibodies. Chicken IgG have no immunological cross reactivity with mammalian IgG and can thus be used to avoid interference due to rheumatoid factor HAMA. The cross reactivity between different mammalian IgG may also cause problems in histochemical staining. If a mammalian IgG is used as primary antibody in histochemistry, the secondary anti IgG antibody may also react with IgG in the mammalian tissue section, which will result in an increased background staining. This can be avoided if chicken IgG is used as the primary antibody, due to the lack of cross-reactivity between chicken and mammalian IgG.
Many immunological assays utilize mammalian capture antibodies bound to a solid surface. When a serum sample is added to the immobilized antibodies, the complement system in the samples is activated and the complement components are bound to the antibodies. This binding may block the antigen binding sites, and it has been shown that complement activation may interfere with antigen binding to the capture antibody and significantly reduce the number of positively reacting samples. The complement system is inactivated during storage. The standards used in immunological assay have usually been stored for some time and will thus have an inactive complement system, whereas the complement activity in the patient samples vary. This will cause an analytical error. IgY was shown to have good stability after being applied to latex microspheres, due to its more hydrophobic surface compared to mammalian antibodies which prevent activation of human complement system 1. Immobilized IgY antibodies were also shown to improve the detection of serum antigens with surface plasmon resonance 2.
Receptors for the Fc domain of IgG provide an important link between specific humoral responses and the cellular branch of the immune system. The binding of IgG to an Fc-receptor may trigger many biological responses (phagocytosis, endocytosis, antibody-dependent cellular cytotoxicity, release of inflammatory mediators, and enhancement of antigen presentation). Detection methods such as flow cytometry often cause erroneous results when mammalian antibody reacts with antigen, an immune complex forms. Immune complexes containing mammalian antibodies may interact with Fc or complement receptors on the cell, which can cause cell activation and changes in the expression of surface proteins. Chicken IgY has less cross-reactivity toward mammalian protein because it does not bind to mammalian Fc-Receptor, but also bacterial Fc receptors such as staphylococcal protein A or streptococcal protein G1,2 , indicating the immunological difference of the Fc region from that of IgG.
Chicken antibodies offer many advantages to mammalian antibodies, and a change from mammalian to chicken antibodies may in many cases improve an immunological assay. IgY is stable at pH 4-9 and temperature up to 650C in aqueous condition, which is different from the stability of IgG at pH 3-10 and temperature up to 700 oC1,2 . However, the resistance of IgY to the more extreme pH ranges increases if high salt conditions or stabilizing reagents such as sorbitol are present 3. IgY was reported to be stable at 40 oC for an extended period 4. Chicken antibodies are also useful in immunoprecipitation assays in agar.
Avian antibodies have been recognized for several decades and offer many advantages to mammalian antibodies. The only avian species from which antibodies are highly defined and easily accessible is the chicken. The major serum antibody in chicken is IgY, but antibody is also actively transported to the egg in a manner similar to the placental transfer of IgG in mammals. The protection against pathogens that the immuno-incompetent newly hatched chick has is through transmission of antibodies from the mother via the egg. IgY has been successfully used in conventional immunoassays such as ELISAs, Western blots, immunohistochemistry, immunoprecipitation, immunocytochemistry, cell and tissue staining, and cell sort etc.1,2,3 The quality of assay using IgY antibodies is either comparable to, or better than, using conventional IgG antibodies. It also been use in application such as xenotransplantation, diagnostics and antibiotic-alternative therapy. 4,5,6
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