Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 95/07290 PCT/US94/09148
METHOD FOR PURIFYING EGG YOLK IMMUNOGLOBULINS
BACKGROUND OF THE INVENTION
Immunoglobulins play an important role in
diagnostic and therapeutic applications. Sera obtained
from hyperimmunized mammals has been extensively used to
provide immunoglobulins for use in such applications.
In certain cases, avian-derived immunoglobulins provide
significant advantages over their mammalian
counterparts. For example, avian-derived
immunoglobulins may provide a higher level of
specificity and a reduced amount of undesirable side
effects as compared to immunoglobulins derived from
mammalian serum.
A useful source of avian IgG immunoglobulins is
the yolk of avian eggs. Not only does egg yolk contain
high levels of IgG immunoglobulins but it is less labor
intensive to collect immunoglobuli.n-containing eggs from
birds than serum from mammals. However, it is necessary
to separate the immunoglobulins from other egg yolk
constituents such as lipids and lipoproteins to
effectively use avian IgG immunoglobulins in assays and
therapeutics.
The IgG immunoglobulins in bird egg yolks are
hydrophilic and interspersed with non-aqueous components
of the yolk. Present methods for separating IgG
immunoglobulins from lipids, lipoproteins and other non-
aqueous components in egg yolks use multiple treatments
with a separating agent. Such methods are time
consuming, and often require the use of specialized
equipment. For example, i.n ane technique, the aqueous
IgG (IgY) immunoglobul~ns of the egg yolk are separated
from the non-aqueous lipids and other components by
multiple precipitation extractions using polyethylene
glycol (PEG). Polson et al., Inmuno. Communications,
9:495-514 (1980). The remaining PEG is removed by
precipitation of the IgG immunoglobulin fraction with
ammonium sulfate or ethanol at subzero temperatures.
In another method, the lipids and lipoproteins
of the egg yolk are precipitated using multiple
WO 95/07290 PCT/US94/09148
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2
extractions with dextran sulfate and calcium chloride.
Jensenius et al., J. of Immuno. Methods,.46:63-68
(1981). The IgG immunoglobulins are then precipitated
with sodium sulfate, and resolubilized to provide a 70-
80~ yield of total IgG immunoglobulin concentration in
the egg yolk. Another method involves multiple
extraction steps using organic solvents at -20°C. Bade
et al., J. of Immuno. Methods, 72:421-426 (1984). Yet
another method utilizes pH adjustment of the egg yolk to
separate out the yolk proteins. Jensenius et al., J. of
Immuno. Methods, 46:63-68 (1981). The yield of IgG
immunoglobulins by this method is only about 50-70~ of
the total IgG in the egg yolk.
Other methods use separation techniques such as
hydrophobic interaction chromatography and gel
filtration chromatography. Hassl et al., J. of Immuno.
Methods, 110:225-228 (1988). While such methods purport
to provide a relatively pure IgG product, purification
of the IgG fraction requires expensive separation
equipment, and the overall yield of IgG is relatively
low compared to other separation procedures.
Accordingly, an object of the invention is to
provide a method for separating and purifying a high
percentage of the total IgG immunoglobulin from the yolk
of an egg without the need for multiple extraction
steps, or expensive separation equipment.
SUM~SARY OF THE INVENTION
The present invention is directed to a method
for separating IgG immunoglobulins from the yolk of an
egg, a composition containing the egg yolk-derived IgG
immunoglobulins, and methods of using the IgG
immunoglobulin composition in diagnostic assays and
therapeutic applications. The method of preparing an
IgG immunoglobulin fraction according to the invention,
includes the use of a protein non-denaturing, nonionic
detergent in a single application to phase separate an
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3
aqueaus suspension or clarified supernatant of the egg
yolk into an aqueous phase containing a major portion of
the IgG immunoglobulins of the yolk and a non-aqueous
detergent phase containing a major portion of the
lipids, lipoproteins and other like constituents. The
phase separating conditions used in the method are
substantially non-denaturing to the IgG immunoglobulins.
The method of the invention provides a high yield of the
total IgG immunoglobulins from the egg yolk starting
material of about 75-99~, preferably about 85-95~.
In the method of the invention, an egg yolk is
combined with an effective amount of an aqueous medium,
preferably a buffer solution, to provide an aqueous
suspension of the yolk. Preferably, the yolk suspension
is clarified, for example, by centrifugation, and the
supernatant collected. An effective amount of a
nonionic detergent, with octoxynol-8 (TRITON~ X-114)
preferred, is added to the yolk suspension or
supernatant to form a homogeneous mixture, and the
mixture is allowed to separate into an aqueous phase and
a non-aqueous detergent phase. The separated aqueous
phase contains a major proportion of the IgG
immunoglobulins of the egg yolk, while the detergent
phase contains a major amount of the lipids,
lipoproteins and other hydrophobic substances of the
yolk. The aqueous phase may be removed from the non-
aqueous detergent phase, for example, by suctioning,
decanting, and the like, and further remaining detergent
in the aqueous phase may be removed, for example, by a
membrane dialysis technique using a physiologically-
acceptable dialyzing buffer. The IgG immunoglobulins in
the separated aqueous phase may then be used immediately
in a diagnostic assay or therapeutic procedure, or may
be stored for future use, by known procedures in the art
for example, lyophilization or a cryopreservation
technique, and the like.
The nonionic detergent used in the method of
WO 95!07290 ' ' ~ ~ ' " PCT/US94/09148
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the invention is substantially protein non-denaturing
and capable of providing a two-phase separation of the
egg yolk suspension or clarified supernatant when
combined therewith. Preferably, the nonionic detergent
is capable of causing such phase separation at a
temperature which is substantially non-denaturing to the
IgG immunoglobulins in the mixture. According to the
invention, it is preferred that the temperature at which
the detergent causes phase separation of the mixture is
from about -25°C to about 60°C, preferably about -10°C
to about 55°C, preferably about 0-50°C.
The invention also provides a composition
containing the substantially pure IgG immunoglobulin
fraction, isolated and purified according to the
invention. Preferably, the IgG immunoglobulin fraction
is combined with an aqueous, pharmaceutically-acceptable
carrier such as water, saline, and the like, and other
additives and adjuvants as desired. A composition
useful according to the present invention includes an
amount of the purified IgG immunoglobulin fraction
effective to provide passive immunity in the bird or
mammal against an etiological agent of interest.
The invention also provides a method for
immunizing a bird or mammal with the IgG immunoglobulin
fraction prepared according to the invention, to provide
passive immunity protection against a bacterial or viral
pathogen, or other etiological agents. The method
includes administering to the animal, an effective
amount of the IgG immunoglobulin fraction to provide
passive immunity against the etiological agent. The
antibodies may be administered orally (liquid or bolus),
parenterally, (intravenous, intramuscularly,
subcutaneously, respiratory aerosolization,
metabolizable implant, and other known parenteral
routes), or by egg inoculation.
Another therapeutic application in which the
IgG immunoglobulin fraction prepared according to the
c
WO 95/07290 PCT/US94/09148
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invention may be used includes tumor immuno-diagnosis
and/or immuno-toxin delivery. For example, antibodies
may be made against specific antigens on target cells in
the body, such as epitopes on tumor cells. The antibody
5 may be linked to a drug, an isotope, or other labeling
system providing for recognition and/or destruction of
the targeted cell bearing the antigen to which the
specific antibody will bind.
The invention further provides an in vitro
method of detecting an etiological agent of interest
through the use of the purified IgG immunoglobulin
fraction. For example, the yolk-derived IgG
immunoglobulin fraction may be combined with a labeling
agent such as a radioactive isotope, an enzyme, a
dyestuff, a fluorescent group, and the like, or any
combination thereof, and used in a diagnostic assay, as
for example, an immunofluorescent assay, a
radioimmunoassay, and the like, or in an immunoassay
such as an immunodiffusion assay, an immunoagglutination
assay, and the like.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term "cloud point" of a
detergent means the point of separation of an aqueous
mixture containing the detergent into an aqueous phase
and a non-aqueous phase, resulting, at least in part,
from the reaction of the detergent with hydrophobic
components in the mixture. By the term "substantially
pure," it is meant that the IgG immunoglobulins of the
egg yolk have been extracted and isolated from their
natural association with other substances and elements
of the egg yolk to provide a fraction containing
essentially IgG, heavy and light chains of IgG and other
proteins less than about 50,000 molecular weight.
In the method of the invention, one or more egg
yolks which have been separated from the albumen
fraction of the egg, and suspended in an aqueous medium
WO 95/07290 PCT/US94/09148
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6
are treated with a single,~.application of a phase-
separating nonionic detergent to separate and purify the
IgG immunoglobulins from the egg yolk.
Using known techniques, an egg-laying female .'
bird such as a turkey, chicken, duck, goose, pigeon,
pheasant, quail, and other like birds, or an egg-laying '
reptile such as a snake, turtle, and the like, may be
immunized with an antigen to stimulate production of an
IgG immunoglobulin of interest. The antigen may be a
pathogenic gram negative or gram positive bacteria, or
subunit thereof (e. g., outer membrane protein), a toxin,
an allergen, a hormone, or any other material capable of.
stimulating immunoglobulin production.
Examples of gram negative bacteria which may
serve as antigens for immunizing the animal include
Escherichia coli, Salmonella spp., Bordetella avium,
Pasteurella spp., Hemophilus spp., Pseudomonas spp.,
Neisseriaceae spp., Shigella spp., Vibrio spp., and the
like. Examples of gram positive organisms which may be
antigens include Staphylococcus spp., Streptococcus
spp., Erysipelothrix spp., Clostridium spp., and the
like. Other organisms which may serve as antigens
include, for example spirochetes such as Borrelia spp.,
and the like; fungi such as Aspergillus spp., Fusarium
spp., Trichophyton spp., Candida spp., and the like;
protozoa including Treponema spp., Toxoplasm spp.,
Giardia spp., Cryptococcus spp., Coccidia spp., and the
like; nematodes including Ascaris spp., Trichinella
spp., and the like; Platyhelminthes such as flukes,
tapeworms, and the like; and RNA and DNA viruses.
Toxins which may serve as antigens include
venoms from reptilia such as snakes, lizards, and the ,
like; and venoms from arachnida such as spiders,
scorpions, and the like. Examples of other toxins which ,
serve as suitable antigens include bacterial endotoxins
and/or exotoxins such as those produced by Escherichia
coli; enterotoxins including heat labile enterotoxin
wo 9s/o~290
PCT/US94/09148
7
(LT) and heat stable enterotoxin (ST); and verotoxin
(VT). Also included are diphtheria and tetanus toxin,
and other toxins including algal and fungal toxins, and
the like.
Allergens may also serve as antigens for
producing antibodies according to the method of the
invention. Examples of allergens useful in the present
method include pollens, mold spores, dust, and the like.
Hormones are also useful as antigens for
antibody production in the method of the invention.
These include testosterone, prolactin, estrogen
progesterone, follicle stimulating hormone (FSH),
luteinizing hormone (LH), prostaglandins and the like.
Also, major histocompatibility complex antigens may
serve to stimulate IgG immunoglobulins such as Class I,
II and III antigens. In addition, B and T cell markers,
lymphokines such as interferon, interleukins, tumor
necrosis factor (TNF), prostaglandins, and the like, may
also be used as antigens.
Such immunization will stimulate the female
animal to produce eggs containing a high level of the
IgG immunoglobulin of interest. The IgG immunoglobulins
in the resulting eggs may then be separated and purified
according to the method of the invention to provide a
purified IgG immunoglobulin fraction containing high
levels of the immunoglobulin of interest. It is
understood, according to the invention, that IgG
immunoglobulins may be isolated from egg yolks derived
from an animal which has not been hyperimmunized to
produce a high level of IgG immunoglobulins.
According to the invention, the source of egg
yolk may be derived from a single species or a
combination of different species. The albumen fraction,
located external to the yolk within the shell of an egg,
generally contains a major amount of IgM and IgA
immunoglobulins, and a minor amount of IgG
immunoglobulins, and the egg yolk contains a major
WO 95/07290 PCT/US94/09148
8
amount of IgG immunoglobulins. The yolk portion may be
separated from the albumen of the egg by known
techniques including, for example, breaking the egg in
half and separating the albumen from the yolk by passing .'
the yolk from one half shell to the other half shell, by
means of an egg yolk separating device which has a '
recessed surface portion to receive the egg such that
the albumen drops off the side edges and the yolk
remains in the recessed portion (Egg Separator), such as
that commercially available from EKCO Housewares,
Franklin Park, IL, by means of an automatic egg breaking
and separating machine such as that commercially
available from Sanova Engineering, Salt Lake City, UT,
and other like separation methods.
It is preferred that any albumen remaining on
the yolk is removed, for example, by gently applying a
stream of water onto the yolk, by gently rolling the
yolk on a paper towel, and the like. If desired, the
vitelline membrane encasing the yolk may be allowed to
separate out at centrifugation or can be removed
manually. ,
The separated yolk fraction is then combined
with an aqueous medium such as water, an aqueous buffer,
and the like, to form a suspension of the egg yolk.
Preferably, the aqueous medium is a physiologically-
acceptable buffer solution formulated to maintain the
egg yolk suspension at about pH 5.5-9.5, preferably
about pH 6.0-9.0, preferably about pH 6.5-8.5. Buffers
suitable for use according to the invention include, for
example, phosphate-buffered saline (PBS), Tris
(hydroxymethyl) aminomethane (iris), tris-buffered
saline (TBS), N-2-hydroxyethylpiperazine-N'-2-
ethanesulfonic acid (HEPES), 3-(N-morpholino)
propanesulfonic acid (MOPS), and the like. Preferably,
the egg yolk fraction is combined with the aqueous
medium in a ratio of egg yolk:aqueous media of about 1:1
to about 1:40, preferably about 1:10 to about 1:30 v/v,
WO 95107290 '~' PCT/US94/09148
9
preferably about 1:15 to about 1:25, preferably about
1s20.
It is preferred that insoluble materials such
as lipoproteins, phospholipids, sterols, and the like,
are removed from the aqueous yolk suspension. For
example, the yolk suspension may be centrifuged at about
20,000-30,000 X g. The clarified supernatant which
contains the IgG immunoglobulins may then be separated
from the precipitant or sediment, for example, by
decanting, suctioning, and other like methods.
Optionally, the supernatant may be re-centrifuged one or
more times to further clarify the supernatant fraction
as desired.
The clarified yolk supernatant is then treated
with a nonionic detergent to cause a phase separation of
the suspension or supernatant into an aqueous phase and
non-aqueous detergent phase. The nonionic detergent is
substantially non-denaturing to the IgG immunoglobulin
proteins of the suspension or supernatant. The
detergent is also capable of forming a mixture with the
aqueous yolk suspension or clarified supernatant, and
then separating the mixture into two phases, an aqueous
phase and a non-aqueous detergent phase. Preferably,
the nonionic detergent is capable of combining with a
major portion of the lipids, lipoproteins and other
hydrophobic constituents of the earQ vnlk +~ fnrm
non-aqueous detergent phase. It is further understood
that multiple detergents sequentially mixed with the egg
yolk suspension'or clarified supernatant, may be
utilized to enhance IgG immunoglobulin extraction.
Preferably, the nonionic detergent is capable
of causing phase separation of the egg yolk suspension --
or clarified supernatant under conditions such as
temperature, pH and salt concentration which are
substantially protein non-denaturing, particularly non-
denaturing to the IgG immunoglobulins. As used herein,
the term "protein non-denaturing~~ means that the protein
WO 95/07290 ~ f ~~ '~~ 9 ~ PCT/US94/09148
substantially maintains its structure and/or functional
characteristics.
Preferably, the nonionic detergent is
capable of causing separation of a detergent mixture
5 containing the yolk suspension or clarified supernatant
at a temperature of about -25°C to about 60°C,
preferably about 10-55°C, preferably 0-50°C, a pH of
about 5.5-9.5, preferably about pH 6.0-9.0, preferably
about pH 6.5-8.5.
10 Nonionic detergents useful according to the
invention include, for example, octoxynol-8 (TRITON~
X-114), and other like polyoxyethylene ethers available
commercially in the TRiTON~ X series (Union Carbide) or
IgePal CA series (GAF). A highly preferred nonionic
detergent according to the invention, is TRITON~ X-114
or TRITON~ X-100, added to the yolk suspension or
supernatant as an about 0.5~-13~ solution, preferably an
about 2-12~ solution.
Preferably, the nonionic detergent is combined
with a compatible, physiologically-acceptable aqueous
medium such as water, a buffer solution, and the like.
Suitable buffers for combining with the detergent
include, for example, Tris(hydroxymethyl)aminomethane
(Tris), tris-buffered saline (TBS), phosphate-buffered
saline (PBS), N-2-hydroxyethylpiperazine-N'-2-
ethanesulfonic acid (HEPES), 3-(N-
morpholino)propanesulfonic acid (MOPS), and the like.
Preferably, the detergent is combined with the aqueous
medium to provide a mixture containing about 100 to
500,000 ppm detergent. For example, for Triton~ X-114,
a preferred mixture contains the detergent at a
concentration of about 1000 to 100,000 ppm, preferably
about 5000 to 80,000 ppm, preferably about 10,000 to
70,000 ppm.
The aqueous yolk suspension or, preferably, the
clarified supernatant, is combined with an effective
amount of the nonionic detergent to cause phase
WO 95/07290 PCT/US94/09148
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separation of the resulting mixture within about 5
minutes to about 2 hours, preferably about 15-60
minutes, preferably about 30-45 minutes, with a major
portion of the lipids, lipoproteins and other
hydrophobic constituents of the egg yolk combining with
the detergent in a non-aqueous phase separate from the
aqueous phase containing a major portion of the IgG
immunoglobulins of the yolk. The amount of detergent
combined with the yolk suspension or supernatant will
vary according to the concentration of the detergent,
the concentration of the yolk in the suspension or
supernatant, and the salt concentration to provide
substantially complete separation of the IgG
immunoglobulins into the aqueous phase, and apart from
other non-aqueous substances of the yolk. Suitable
concentrations of detergent are about 1-12~ w/v. In
a preferred method according to the invention, Triton
X-100 or Triton X-114 is added to the clarified egg
yolk supernatant until the desired w/v percentage
concentration of detergent is obtained. For example, to
cause phase separation of an about 250-300 ml clarified
yolk supernatant, about 6-12~k w/v of octoxynol-8
(TRITON X-114) may be added to the supernatant.
It is preferred that the detergent mixture is
gently agitated to provide a homogenous mixture of the
detergent and the yolk suspension or supernatant,
preferably for about 10 minutes to about 3 hours,
preferably about 15 minutes to about 2 hours, preferably
about 30-60 minutes. It is also preferred that the
mixing speed used is effective to thoroughly mix the
detergent with the suspension or supernatant, yet avoid
denaturing the IgG immunoglobulins. Preferably, the
mixing speed is about 50-150 rpm, preferably about 60-
100 rpm, preferably about 70-85 rpm. It is also
preferred that the temperature of the detergent mixture
is maintained at about -25°C to about 60°C, preferably
about -10-55°C, preferably about 0-50°C, and the pH is
WO 95/07290 PCT/US94/09148
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maintained at about pH 5.5-9.5, preferably about pH 6.0-
9.0, preferably about pH 6.5-8.5.
The detergent mixture is then allowed to
separate into a relatively clear aqueous phase and a '
non-aqueous, relatively viscous, detergent phase. The
mixture is maintained at a temperature of about -25°C to
about 60°C, preferably about -10°C to about 55°C,
preferably about 10-50°C, during the phase separation
step. According to the invention, it is preferred that
about 90-95~ of the detergent phase falls out of the
mixture within about 30-60 minutes at a temperature of
about 30-50°C. To enhance separation of the two phases,
the mixture may be centrifuged at a low speed,
preferably about 500-1,000 rpm for about 10-30 minutes.
The phase separation by the nonionic detergent
may also be enhanced and/or activated, for example, by
raising or lowering the temperature, pH and/or salt
concentration of the detergent mixture. For example,
the temperature of the detergent mixture may be raised
up to about 40-60°C, preferably up to about 45-50°C, or
lowered down to about 0°C to about -25°C, preferably
down to about -4°C to about 5°C. The pH of the
detergent mixture may be adjusted to effect phase
separation by adding an effective amount of acid and/or
base at about 0.01 to 0.1 normality. In addition, the
salt concentration of the mixture may be altered, higher
or lower, to enhance phase separation by adding an
effective amount of potassium chloride or sodium
chloride, as desired.
After the mixture is separated into the two ,
phases, the aqueous layer is removed from the non-
aqueous detergent layer by known techniques, for
example, by decanting, suction pipetting, and the like.
According to the invention, the aqueous layer contains a
major portion of the total IgG immunoglobulins of the
egg yolk starting material, IgG extraction from the
aqueous phase is about 51-99~, preferably about 70-97~,
WO 95/07290 PCT/US94/09148
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preferably about 80-95~. The detergent layer contains a
major portion of the total lipids and lipoproteins of
the egg yolk starting material, or about 51-99~,
preferably about 70-97~, preferably about 80-95~.
Residual nonionic detergent remaining in the
aqueous phase may optionally be removed by known
methods, for example, by membrane dialysis, gel
filtration, ion exchange chromatography, affinity
chromatography, and other like methods for separating
molecules of different molecular weight and/or polarity.
For example, the aqueous phase may be treated by gel
filtration using a gel made of dextran and acrylamide
such as SEPHACRYL S-300~, available commercially from
Pharmacia, Piscataway, New Jersey.
Preferably, residual nonionic detergent is
removed from the aqueous phase by membrane dialysis.
For example, the aqueous phase may be diluted with a
physiologically-compatible buffer such as phosphate
buffered saline (PBS), and placed into a dialysis
membrane in the form of a tube or bag, and submerged in
a compatible physiological dialysis buffer, as for
example, a physiological saline solution (0.850 , a 5~
glucose in physiological saline solution (0.85 0 ,
Tris(hydroxymethyl)aminomethane (Tris), tris-buffered
saline (TBS), phosphate-buffered saline (PBS),
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
(HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS),
and the like. Detergent molecules in the aqueous phase
within the dialysis tube will then pass through the
dialysis membrane into the dialysant outside the tube.
Preferably, the pore size of the dialysis membrane is
effective to retain IgG immunoglobulins having a
molecular weight of about 150,000-180,000 daltons within
the bag, and allow passage of detergent molecules having
a molecular weight of about 1-145,000 daltons,
preferably about 1,000-125,000 daltons, preferably about
5,000-100,000, out of the bag into the dialysis solvent.
WO 95/07290 PCT/US94/09148
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14
Dialysis membranes suitable for use in the present
method include, for example, SPECTRA POR~ MWCO 3500,
SPECTRA~POR #7~ MWCO 50,000, and the like, available
commercially from Spectrum Medical Industries Inc., Los
Angeles, California.
The dialysis treatment of the aqueous phase, or
other like treatment to remove the residual detergent,
results in a substantially pure aqueous IgG
immunoglobulin fraction comprising a major amount of the
IgG immunoglobulins of the yolk starting material, or
about 51-99~, preferably about 60-95~, preferably about
70-90~.
A composition according to the invention
contains the resulting substantially pure IgG
immunoglobulin fraction in combination with a
physiologically-acceptable carrier, and other additives
and adjuvants as desired. Suitable carriers include,
for example, water, saline, phosphate buffered saline
(PBS), Tris(hydroxymethyl) aminomethane (Tris), tris-
buffered saline (TBS), N-2-hydroxyethylpiperazine-N'-2-
ethanesulfonic acid (HEPES), 3-(N-morpholino)
propanesulfonic acid (MOPS), and the like. Additive
agents which may be included in the composition include,
for example, an effective amount of a preserving agent
such as bacteriostats, fungistats, and the like. In
addition, to prevent precipitation of the globulin, an
effective amount of glucose (5~), and the like may be
added.
The purified IgG immunoglobulin fraction may be
used immediately or stored for future use. For example,
the IgG immunoglobulin fraction may be cryopreserved by
combining the fraction with a compatible isotonic
solution, and stored at a temperature about -45°C to
about 4°C, preferably about -30°C to about -10°C. To
prevent precipitation of the IgG immunoglobulins upon
thawing of a cryopreserved solution, a compatible
protein precipitation-preventing agent may be included
R'U 95/07290 PCT/US94/09148
in the composition, as for example, an aqueous solution
of about 5~ glucose w/v in physiological saline (.85~),
and the like.
The invention also provides a method for
5 immunizing domestic fowl, livestock, horses, companion
animals and humans with the purified IgG immunoglobulin
fraction prepared according to the invention to provide
passive immunity protection against various etiological
agents, including, for example, bacteria, viruses,
10 fungi, protozoa, nematodes, platyhelminthes, and the
like. In addition, the method of the invention provides
for passive immunization against allergens, toxins,
venoms, hormones, or any other immunogen capable of
eliciting an immune response. The method includes
15 administering to the animal, an effective therapeutic
amount of the substantially pure IgG immunoglobulin
fraction to provide passive immunity against the
etiolagical agent. Preferably, the animal is
administered the purified IgG immunoglobulin fraction
combined with a pharmaceutically-or physiologically-
acceptable carrier such as water, saline, and the like.
The composition may be administered to the
animal orally or parenterally, by suppository, by
injection, by aerosolization or by other suitable,means
and techniques known in the art. For example, the
composition may be formulated with conventional
pharmaceutically- or physiologically-acceptable
parenteral vehicles for administration by injection.
These vehicles comprise substances that are essentially
nontoxic and nontherapeutic such as water, saline,
dextrose solution, Hank's Solution, and the like. It is
to be understood that immunoglobulin formulations may
also include small amounts of diluents such as buffers
and preservatives to maintain isotonicity, physiological
pH and stability. The composition may be administered
to an animal on a periodic or continuous basis.
Preferably, the purified IgG immunoglobulin
WO 95/07290 ~ PCT/US94109148
16
fraction is administered to the animal orally,
parenterally, (intravenously, intramuscularly,
subcutaneously, respiratory aerosolization), by
metabolizable implant, and the like. In such
application, the IgG immunoglobulin fraction is a
constituent in a suitable liquid media such as water and
the like, or a feed in an appropriate dry format, or a
tablet or other oral form understood by those skilled in
the art. As described hereinabove, the oral composition
can include suitable compatible diluents. The specific
IgG immunoglobulin of interest is contained in the
purified immunoglobulin fraction provided to a patient.
In such form, the immunoglobulin concentration provided
to the animal is about 0.25-20 grams per day. For
example, about 0.5-1 grams of immunoglobulin could be
given to a animal at 3 to 4 times per day. The doses of
the immunoglobulin formulation to be administered will
depend upon the type of animal, size, and the like.
In a preferred method according to the
invention, a therapeutically effective amount of the
purified immunoglobulin fraction of IgG can be given
passively to the developing turkey or chicken embryo
during the incubation period. The immunoglobulins can
be administered in ova into the albumin which is
swallowed by the developing chick, or into the yolk sac
where it is absorbed, to provide systemic and intestinal
passive protection. The method of in ova injection by
modular injection system is described, for example, in
U.S. Patent No. 5,136,979 the disclosure of which is
incorporated by reference herein.
The IgG immunoglobulin fraction of the present
invention may also be used for in vitro detection of an
etiological agent, for example, a pathogenic organism
such as Escherichia coli, Salmonella enterititis,
Bordetella avium, Pasteurella ruultoc.ida, and other
bacterial organisms; a hormone such as estrogen,
progesterone and the like; a major histocompatibility
WO 95/07290 pCT/US94/09148
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complex antigen, and the like; in a body sample such as
a fluid, tissue, cell extract, and the like, that is
obtained from the human or animal for testing. A method
for in vitro detection of an etiological agent in a body
sample would include combining the body sample with an
effective amount of the purified IgG immunoglobulin
fraction prepared according to the method~of the
invention, with the IgG immunoglobulin in the fraction
being labeled. The label may be any labeling group that
may be suitably bound to the IgG immunoglobulin or
second immunoglobulin or antibody, and which will allow
for the reaction with the antigen of interest in the
sample. The label may be, for example, a radioactive
group such as 1241, 14C, 3I~, and the like; an enzymatic
label such as horseradish peroxidase, a catalase, a
glucose oxidase, and the like; a fluorescent label such
as rhodamine, fluorescein, and the like; a fluorescent
label such as rhodamine, fluorescein and the like; an
immuno-electron microscopy conjugate such as a gold
conjugate and the like; and an immuno diagnostic label
for protein blotting, and the like, using alkaline
phosphates, biotin, and the like; and other like
labeling agents. The body sample is combined with the
labeled IgG immunoglobulin for a time effective to allow
binding of the labeled IgG immunoglobulin with the body
sample evidencing the presence of the etiological agent.
The presence of the labelled IgG immunoglobulin bound to
the body sample is then detected, for example, by
techniques known in the art, including color changes,
autoradiography, positron emission tomography, nuclear
magnetic resonance imaging, a gamma counter, a
scintillation counter, and the like. The detection
method may also include quantifying, by known
techniques, the amount of labeled antibody that is bound
to the sample.
An example of an in vitro method of detecting
an etiological agent in a body sample is by immobilizing
ltd'=~'A~ :.
WO 95107290 PCT/US94109148
18
the IgG immunoglobulins of the purified fraction on a
solid phase support, as for example, immunomagnetic ,'
beads, a resin test plate such as polyvinylchloride,
polystyrene and the like, or a nitrocellulose carrier '
and the like, in an amount effective to bind with a
fluid, tissue, or other body sample that evidences the
presence of the antigen of interest, such as that
associated with Borrelia burgdorferi (Lymes Disease),
Mycobacterium tubercu.Zosis (TB), Histoplasmosis spp., or
any other etiological agent expressing antigens capable
of eliciting an immune response. An example of a useful
composition for detecting an antigenic etiological agent
in a sample in vitro is one that includes about 1 to 100
ug of the IgG immunoglobulin per 2 x 108 immunomagnetic
I5 beads.
In another example of detecting an etiological
agent in a body sample in vitro, the purified IgG
immunoglobulin fraction.may be combined with the sample,
and after a suitable reaction time, the IgG
immunoglobulin that is bound to the sample is detected
by a suitable method known in the art. In one example
of such an assay, the purified IgG immunoglobulins of
the purified fraction may be labeled and immobilized
onto a solid phase carrier according to techniques known
in the art, and then reacted with the antigen of
interest in the sample, or fragment thereof that
includes the epitopal-binding sites) for the IgG
immunoglobulin, to form a complex between the labeled
IgG immunoglobulin and the antigen. The carrier with
the bound IgG immunoglobulin/antigen complex would then
be washed to remove unbound materials, and the labeled
IgG immunoglobulin in the complex detected by
conventional methods known in the art. As another
example, the sample may be immobilized onto a solid
phase carrier according to conventional methods in the
art, and reacted with the labeled IgG immunoglobulin.
The carrier with the bound complex would then be washed
WO 95/07290 ~ ~ i 3 ~ 7
PCT/US94/09148
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to remove unbound material and the labeled IgG
immunoglobulin in the complex would be detected.
EXAMPhE 1
IgG Immunoglobulin Purification Using
6% Triton X-114
Twenty eggs were collected from twenty female
chickens, and the egg yolks were separated from the
albumen fraction of the egg by cracking the egg into an
EKCO~ Egg Separator (EKCO Housewares, Franklin Park,
IL). This application holds the yolk stationary while
allowing for the separation of albumen. The yolk, while
still in the applicator is rinsed in a stream of
distilled water to remove any excess albumen that may
cling to the yolk. The washed egg yolks (100 grams; 4
egg yolks) were then combined together in a beaker,
combined with an equal amount (100 ml) of tris-buffered
saline (TBS; i.e., Tris{hydroxymethyl) aminomethane)
(pH 7.4), and mixed thoroughly using a magnetic stirrer
at 100-200 rpm for 10 minutes to form a suspension of
the egg yolks in the buffer.
The egg yolk/buffer suspension (20 grams) was
diluted 1:10 in tris-buffered saline TBS buffer (pH
7.4), mixed thoroughly using a magnetic stirrer at 200
rpm far 5 minutes, and centrifuged at 22095 X g for
25 minutes in a Beckman J2-21M centrifuge (Beckman
Instruments, Palo Alto, California), to remove insoluble
material. The supernatant was collected by suctioning
the supernatant, and the insoluble material was
discarded. The supernatant was re-centrifuged at 22095
X g for 25 minutes to further clarify the supernatant.
The clarified supernatant (270 ml) was combined with
180m1 of 6% Triton X-114~ in tris-buffered saline (TBS)
(pH 7.4). The buffer mixture was held at 4°C in a 500
ml screw capped erlenmeyer flask while continuous
stirring at 200 rpm, for 30 minutes.
The mixture was allowed to warm to 37°C by
WO 95107290 PCT/US94/09148
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>.
maintaining the mixture at a stationary condition. As
the mixture warmed, phase separation occurred. At the '
same time, the water phase (containing the IgG
immunoglobulins) formed a layer over the TRITON~
detergent layer. The water phase was relatively clear
while the non-aqueous TRITON~ X-114 detergent phase was
viscous and appeared yellowish in color. The upper
aqueous layer was collected by suction into a reservoir
vessel.
The aqueous phase was then clarified by
centrifuging at 15344 X g for 10 minutes at 25°C. After
the centrifugation, glucose, at 5~ w/v concentration was
added to the aqueous solution (17.5 grams) was added to
350 ml aqueous phase supernatant to prevent
precipitation of the immunoglobulins from solution. The
supernatant was then dialyzed against a physiological
saline solution (0.85 0 containing 5~ glucose (Sigma
Chemical Co., St. Louis, Missouri) for 48 hours at 4°C,
to remove residual contaminating TRITON~ detergent in
the aqueous IgG immunoglobulin fraction.
The process yielded an aqueous solution (350
ml) containing about 85~ of the total IgG
immunoglobulins of the egg yolk starting material.
E~VTIIflT L. H
Immunization Procedure for Stimulation
of Specific Antibody Production
Eighty, 12-week old, shaver white chickens were
equally divided among ten groups. Chickens were
vaccinated with the following antigens: outer membrane
proteins of Escherichia coli, Bordetella avium, r
Staphylococcus areus; pili antigens of Escherich.ia coli
(K88, K99, 987P and F41); New Castle Disease Virus (NDV)
and hemorrhagic enteritis. The birds were given three
additional boosters at fourteen day intervals.
One week after the last booster injection, eggs
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were collected from all groups of birds over eighteen
days. On the eighteenth day all groups were again
vaccinated with the appropriate antigen and eggs were
again collected. This procedure was repeated for the
duration of the experiment.
Yolks were harvested and processed as described
in Example 1. The IgG was collected from each group and
stored at -95°C. The affinity of the IgG for each group
was determined using a standardized Enzyme Linked
Immunoadsorbent Assay (ELISA) for each group of
antigens.
The invention has been described with reference
to various specific and preferred embodiments and
techniques. These examples are not meant to limit the
scope of the invention that has been set forth in the
foregoing description. It should be understood that
many variations and modifications may be made while
remaining within the spirit and scope of the invention.
The disclosures of the references cited in the
disclosure are incorporated by reference herein.
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