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REVIEW - Chicken Antibodies- IgY
As early as 1893 Klemprer published his observations that there
must be neutralizing proteins (i.e. antibodies) in the yolk of eggs
laid by immunized hens. On April, 2004 some 250 scientists and industry experts from all over the world
gathered in Banff, Alberta (Egg Nutrition for Health Promotion. The
third international symposium) to discuss eggs as a source of
nutritional, nutraceutical and pharmaceutical products. High on the
agenda was consideration of egg yolk as a source of immune globulins
for diagnostic, prophylactic and therapeutic purposes.
Traditionally mammalian polyclonal and monoclonal antibodies have been
the source of such reagents, so what are the compelling reasons to
develop or advantages of egg yolk antibodies over mammalian
antibodies. To familiarize the reader with a very limited aspect of
avian immunology, suffice to say that while the egg is still in the
ovary, hens transfer their serum immune globulins into the yolk. IgM
and IgA are transferred together with other proteins in the oviduct
into the egg white. IgG (or IgY as suggested in chickens) in the egg
follicle is passed by receptors, hence selectively, in large amounts
into the yolk. The IgY concentration in the yolk is comparable to the
concentration of IgY in the serum; 6-13 mg/ml. Recent genetic research
suggests that the IgY molecule is phylogenetically a progenitor of
mammals' IgG and IgE. Avian sera contain the three principal classes
of immune globulins, namely IgA, IgM and IgY (IgG), however,
structurally there is a big difference between hen IgY and mammals'
IgG as demonstrated in the following figure.
Physico-Chemical Characteristics of IgY
Laying hens are highly cost-effective as producers of antibodies
compared with other mammals traditionally used for such production.
Also chicken antibodies have biochemical advantages over mammalian
antibodies due to the phylogenetical differences between avian and
mammalian species, resulting in increased sensitivity as well as
decreased background in immunological assays. In contrast to mammalian
antibodies, chicken antibodies do not activate the human complement
system nor will they react with rheumatoid factors, human anti-mouse
IgG antibodies, or bacterial and human Fc receptors. Thus chicken
antibodies offer many advantages over mammalian antibodies and are,
even now, beginning to replace conventional sources of custom produced
antibodies.
Research and diagnostic antibodies
Antibodies available to research laboratories generally belong to
one of the three main categories: mammalian monoclonal antibodies,
mammalian polyclonal antibodies, and avian polyclonal antibodies. The
species chosen for antibody production have usually been mammals, most
frequently rabbits. 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. In
the egg, chicken IgY is found mainly in the egg yolk, whereas the
concentration in egg white is very low. Chicken IgA and IgM is found
in the egg white in very low amounts
Chickens and Rabbits
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 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.
Phylogenetic Differences
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.
Avoiding Interferences
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 eh 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 s 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
crossreactivity 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.
Complement
Many immunological assays utilize mammalian capture antibodies
bound to a solid phase. 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. Chicken antibodies do not activate the human
complement system and can thus be used to avoid this interference
problem.
Mammalian Fc-Receptor
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 a Fc-receptor may trigger many
biological responses (phagocytosis, endocytosis, antibody-dependent
cellular cytotoxicity, release of inflammatory mediators, and
enhancement of antigen presentation). Flow cytometry is widely used in
clinical laboratories for the detection of cell surface proteins. The
system utilizes labeled antibodies for the detection of specific cell
markers. When the antibody reacts with the antigen, an immune complex
is formed. 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. It
has been shown that immune complexes containing mammalian antibodies,
but not chicken antibodies, will cause erroneous results when
measuring platelet activation.
Bacterial Fc-Receptor
Antibodies may also be used for the detection of bacterial
infections, but some bacteria have IgG-binding membrane proteins. The
most well known of these proteins are Staphylococcus aureus protein A
and streptococcal protein C. These protein will bind the Fc portion of
IgG from many mammalian species, but they will not bind chicken IgY. A
bacterial specimen usually contains numerous bacteria. If
staphylococci or streptococci are present in the sample, these
bacteria may bind the Fc Portion of the antibody and cause a false
positive reaction.
Stability
Experience with egg yolk antibodies is that they are stable over
time, which contradicts some rumors that chicken antibodies are
labile. IgY antibodies have been stored for over 10 years at 4 degrees
C without any significant loss in antibody activity. Chicken
antibodies have also retained their activity after 6 months room
temperature or 1 month at 37 degrees C. Chicken antibodies are also
useful in immunoprecipitation assays in agar. Chicken antibodies offer
many advantages to mammalian antibodies, and a change from mammalian
to chicken antibodies may in many cases improve an immunological
assay.
From Larsson et al, Poultry Science (1993) 72:1807 with changes
IgY- Prophylaxis Therapy and Diagnosis
Production of IgY in hen's egg yolk is an efficient and economical
method to raise polyclonal antibodies, in that bleeding of hens is not
necessary and purification of IgY is relatively simple.
Chicken IgY has been extensively explored as oral
immunosupplementation for prophylaxis, preventive medicine and therapy
of infectious diseases. Egg yolk antibodies have also great potential
for the isolation/purification of other bioactive compounds or
pathogens and for immunoassays because of their excellent specificity.
To enhance immunity after oral administration of IgY, investigators
prevented infection due to rotavirus in mice and infection of the
human group A rotavirus MO strain (G serotype 3)in 5 day old BALB/c
mice. In a randomized double blind trial researchers evaluated the
therapeutic efficacy of IgY, specific to human rotavirus, in children
with proven rotavirus diarrhea. They observed a modest improvement of
diarrhea in association with IgY therapy in the form of reduction of
stool volume and earlier clearance of rotavirus from stool in children
indicating a potential role of chicken IgY in the management of this
infection. The researchers agreed that better results could be
achieved if the IgY preparation had a higher antibody titer. In other
studies, neonatal calves were protected against bovine coronavirus-induced
diarrhea by passive administration of egg yolk or colostrums antibody
powder. Other data indicate dose dependent effect of specific egg yolk
antibodies on diarrhea of newborn calves. Other workers showed
protection of neonatal pigs from diarrhea by orally administering IgY
raised against enterotoxigenic E. coli. Further, chicken egg
antibodies were used for prophylaxis and therapy of infectious
intestinal diseases due to E. coli strains. Still in another study,
inhibition of diarrhea by immune egg was demonstrated in a castor oil
mouse model. A considerable amount of literature on this subject
matter are available for the interested reader on the appropriate web
sites. Samonella species are notorious for infecting meat, eggs and
other food, yet mice were passively immunized against experimental
salmonellosis by oral administration of hen egg yolk antibodies
specific for 14-kDa fimbriae of salmonella enderitidis. Other studies
have confirmed protective effects of IgY against lipopolysaccharide of
E. Coli and Salmonella typhymurium. Helicobacter pylori, is the most
common cause of gastritis and gastric ulcers and plays a pivotal role
in the development of gastric carcinomas. Successful treatment of
H.pylori most often employs some combination of Metronidazole,
amoxicillin, clarithromycin and either bismuth or a proton pump
inhibitor, however, in a significant number of patients, the bacteria
develop antibiotic resistance and still more patients have aversion
towards antibiotic therapy. An alternative to antibiotic treatment for
control of H. pylori infection is edible egg yolk derived immune
globulins that inhibit proliferation of H.pylori and prevent adherence
of H.pylori to gastric epithelial cells. Thus, anti-adhesion and
mucosal protective agents could represent potential targets for H.
Pylori treatment. Streptococcus mutans is the principal etiologic
agent of dental plaques and dental carries. Passive immunization with
mouth rinse containing S.mutans specific IgY effectively prevented
plaque formation in humans. Similarly an IgY anti S.mutans oral spray
three times a day for three weeks significantly suppressed the number
of bacteria per dental plaque in a group of human volunteers. Glucan
mediated accumulation of cariogenic S.mutans in the dental biofilm is
a critical step in their pathogenesis. Chickens immunized with
S.mutans glucan binding protein B (GbpB). IgY antibody to GbpB were
fed to a rat model for experimental dental caries and subsequently
infected with cariogenic S.mutans Sjr on the first day of the
experiment. The researchers noted significantly lower accumulation of
S.mutans and reduction of dental caries at various times during the
experimental period. A group of Swedish scientists treated Cystic
Fibrosis patients with yolk antibodies to prevent Pseudomonas
aeruginosa infections in their lungs. The patients gargled daily 70 mL
of antibody solution in the evening. The treatment has resulted in
significant reduction in P. aeruginosa positive sputum cultures,
chronic colonization and use of antibiotics.
It is obvious from the above mini review that hens can be the source
of vast number of antibodies to almost any immunogenic stimulant for
preventative and therapeutic purpose.
A substantial amount of information indicates the ease and feasibility
of using IgY antibodies for diagnostic purposes and as powerful
reagents in clinical chemistry and immunology. An extensive discussion
of this subject matter is outside the scope of this writing, however,
suffice to mention that chicken antibodies have biochemical advantages
over mammalian antibodies due to the phylogenetical differences
between avian and mammalian species, resulting in increased
sensitivity as well as decreased background in immunological assays.
Chicken antibodies have been used successfully as secondary antibodies
in radial immunodiffusion, agar gel double immunodiffusion,
immunoelectrophoresis, rocket immunoelectrophoresis two- dimensional
(Crossed) immunoelectrophoresis and ELISAS. Additionally IgY has been
labeled with fluorochromes and enzymes for further immunofluorescent,
immunohistochemical and immunochemical assays. |
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