Note: Descriptions are shown in the official language in which they were submitted.
CA 02389897 2002-06-07
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PROCESS FOR SELECTIVELY ISOLATING IGY ANTIBODIES
FROM EGG YOLK OF AN ANSERIFORM BIRD AND IGY
ANTIBODIES OBTAINED THEREBY
BACKGROUND OF THE INVENTION
1.Field of the invention
The present invention relates to a process for rapid isolation and
purification of yolk antibodies, in particular IgY antibody, from anseriform
bird yolk, and the yolk antibodies obtained thereby. More particularly, the
present invention relates to a process for isolation and purification of yolk
o antibodies from anseriform bird yolk by an adsorption chromatographic
procedure using a water insoluble non-charged absorbent to accomplish a
desired separation of yolk antibodies, and by a salting-out procedure that
differentially precipitates the IgY antibodies. The present invention also
relates to uses of the IgY antibodies in quantitative or qualitative
immunoassay or in the preparation of pharmaceutical compositions
directing to an etiological agent of interest.
2.Description of the Related Art
Antibodies are used widely in many biological investigations and
clinical applications. Sera obtained from hyperimmunized mammalians are
the most common source of polyclonal antibodies. Antibodies derived
from such immune sera belong to a group of proteins called
"immunoglobulins," among which the immunoglobulin G (IgG) is the most
abundant. The IgG molecule consists of three domains, namely two Fab
regions and one Fc region. The Fab portion involves mainly in antigen
binding. The Fc portion, though having no ability to bind with an antigen,
directs several biological activity of an antibody, such as complement
fixing and Fc receptor binding.
In the art of immunodiagnostics, an intact IgG molecule is not
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suitable for use in detection systems and immunological assays involving
mammalian sera since the Fc region on an IgG molecule is capable of
binding to Fc receptors, activating the complement system, and reacting
with rheumatoid factor in mammalian sera. Removal of the Fc portion of
an IgG molecule frequently leads to a reduction in the interference (E.
Lamoyi, Methods in Enzymology 121:652-663, 1986).
Some of the suggested uses of antibody in immunotherapy include
treating patients with intoxicated bacterial toxins or snake venoms (see, for
example, U.S. 5,340,923 and U.S. 5,601,823), and protection of neonatal
0 piglets against fatal enteric colibacillosis (see, for example, H.
Brussow et
al., J. Clin. Microbiol. 25:982, 1987; and C. 0. Tacket et al., New Eng. J
Med. 318:1240, 1988). Since the Fc fragment of an antibody molecule is
known to be the most antigenic portion of the immunoglobulin (E. M.
Akita et al., J Immunol Methods. 162:155-164, 1993), cleavage of the
same which results in the formation of an F(a131)2 fragment will reduce
significantly a number of potential allergenic sites on the immunoglobulin
molecule and is thus beneficial to human or an animal administered with
the immunoglobulin.
Recently, the divalent F(ab1)2 antibody fragment has been shown to
be more useful in the immunodiagnostic tests (M. Muratsugu et al., J.
Colloid Interface Sci 147:378, 1991); and J. L. Ortega-Vinuesa et al., J.
Immunol Methods 90:29, 1996) and more suitable for development of the
immunoassays involving mammalian sera than the parent IgG.
The F(ab1)2 antibody fragment, however, has not found widespread
use in clinical immunodiagnostic kits as one might expect. This may be
attributed to the difficulties and cost-ineffectiveness of large scale
production of the F(ab1)2 fragments, which is conventionally made by
pepsin digestion of IgG and subsequent purification via chromatography.
Ducks and their phylogenetically close relatives and some reptiles,
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such as turtles, have three kinds of serum immunoglobulins: a
macromolecular immunoglobulin IgM (800 kDa in duck), and two isoforms
of low molecular weight IgG with sedimentation coefficients of 7.8 S (in
duck, 180 kDa) and 5.7 S (in duck, 130 kDa), respectively. (E. R. Unanue
et al., J Exp. Med. 121:697-714, 1965; H. M. Grey, J. Immunol
98:811-819, 1967); and B. Zimmerman et al., Biochemistry 10:482-448,
1971). Avian IgG is oftentimes called IgY due to their existence in egg
yolk besides in sera. The 5.7 S IgY, constituted with shorter heavy chains,
is structurally and antigenically similar to the F(ab)2 fragment of the 7.8 S
IgY (FIG. 1), and this fact leads to the nomenclature of IgY (equivalent to
7.8 S IgY) and IgY(.Fc) (equivalent to 5.7 S IgY) to represent both
isoforms of IgY (K. E. Magor et al., J. Immunol. 149:2627-2633, 1992).
Studies conducted in the infected or experimentally immunized birds
showed that duck antibodies are deficient in a number of biological effector
functions, including complement fixation and Fc receptors binding, without
sacrificing their binding activity to corresponding antigens (G. W. Litman
et al., Immunochemistry 10:323, 1973; and T. E. Toth et al., Avian Dis.
25:17-28, 1981). This may reasonably result from the apparent lack of
Fc-equivalent region of the IgY(.Fc) antibody that constitutes the
quantitatively major component of anseriform bird antibody response. It is
thus believed that the IgY(.Fc) antibody, which appears to be a structural
and functional analog of the F(ab1)2 fragment, would provide magnificent
advantages in immunological uses, if a promising process for
manufacturing the antibody could be found, and the appropriate physical
requirements for its activity could be identified.
Avian yolk antibodies have been reported to exhibit useful properties
for both research and clinical applications as mammalian antibodies do
(see, for example, U.S. 5,340,923; U.S. 5,585,098; U.S. 5,601,823; and
U.S. 5,976,519). Egg yolks derived from a laying hen is inexpensive and
more convenient and safer to handle as compared to the hyperimmunized
!I !
CA 02389897 2002-06-07
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mammalian sera. More importantly, yolk antibodies are able to stand up to
the scrutiny under modern animal protection regulations (A. Poison et al.,
ImmunoL Commun. 9:475, 1980; and B. Gottstein et al.). These facts
suggest a potential use of egg yolk as a commercial source of antibodies.
However, high contents of lipidic substances, such as fatty acids,
cholesterol and lecithin, in egg yolk make the isolation of yolk antibodies a
cumbersome and laborious task. Many efforts have been made in this
regard. For instance, water soluble precipitants, including agar, pectin
(Japanese Kokai No. 64-38098 published in February 8, 1989), dextran
sulfate (J. C. Jensenius et al., J. Immunol. Methods 46:63, 1981), natural
gums (H. Hatta et aL, J. Food Science 53:425, 1988) and polyethylene
glycol (PEG) (A. Poison et al., lmmunol. Invest. 14:323, 1985; see also
U.S. 4,550,019 issued to A. Poison) were used to precipitate non-aqueous
bio-molecules, mainly lipids and yolk granules, to thereby harvest a water
soluble phase containing abundant yolk antibodies of the entire population.
A. Hassl et al. developed a two-step chromatographic process, comprised
of hydrophobic interaction chromatography and size exclusive
chromatography, for further isolation of yolk antibodies of the entire
population from a PEG-purified fraction (A. Hassl and H Aspock, j.
ImmunoL Methods 110:225, 1988). Akita et al. described an improved
method for isolating IgY, in which yolk antibodies were extracted from
chick eggs by diluting the egg yolks with a large volume of water and
subjecting the resultant supernatant to size exclusive chromatography
and/or ion exchange chromatography (E. M. Akita et aL, J ImmunoL
Methods. 160:207, 1993; and E. M. Akita and S. Nakai, J Food Sci.
57:629, 1993).
However, all these studies and patents focus on the isolation of the
entire population of yolk antibodies (which at least includes IgY present or
absent Fe region) from avian eggs, rather than on the purification of
IgY(.Fc) and IgY antibodies selectively. Moreover, since IgY( Fc)
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antibodies are present only in birds belonging to the Order Anseriformes,
including duck and goose, and since the lipid content in the egg yolk of the
anseriform birds is reported higher than that in the galliform birds, such as
chicken and turkey, the conventional methods described above provide no
suggestion of a successful purification of IgY(.Fc) antibody. IgY(.Fc)
antibody was only purified by co-precipitating with IgY from duck serum
(D. A. Higgins et al., Veterinary Immunology and Immunopathology
41:169-180, 1995) with complexes and expensive procedures, but still no
IgY(.Fc) antibody alone was selected isolated from egg yolk.
Therefore, there exists a need for a rapid, cost-effective and
high-throughput process that provides easy isolation of the desired IgY
antibody from the antibody pool of anseriform bird egg while maintaining
the activity of the IgY antibody. The substantially purified IgY(.Fc)
antibody may acts as a new type of F(ab1)2 antibody for various
immunodiagnostic and immunotherapeutic uses.
SUMMARY OF THE INVENTION
An extensive research has been conducted to fulfill the industrial
requirements for yolk antibodies as indicated above. It is unexpectedly
found that a successful isolation of yolk antibodies from egg yolks of an
anseriform bird can be readily accomplished through an adsorption
chromatographic procedure using a water insoluble non-charged absorbent,
and/or through a simple salting-out procedure that differentiates different
isoforms of the yolk antibodies. According to the process of the present
invention, the highly purified yolk antibodies, in particular the highly
purified IgY(.Fc), can be easily obtained with high yield in an economic
manner, and are ready for a wide variety of immunological uses.
Accordingly, an object of the present invention is to provide a
process for selectively isolating IgY antibodies from egg yolk of an
anseriform bird, which is characterized in:
CA 02389897 2012-11-06
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(a) absorbing yolk antibodies in a water-miscible fraction obtained from
the egg yolk of an anseriform bird with a water insoluble non-charged
absorbent
selected from the group consisting of silicate, silicon compounds, carbonate,
sulfate, phosphate, carbon, cellulose and synthetic fiber, ceramics, and metal
oxide, and wherein the water insoluble non-charged absorbent is at an amount
effective for separating the yolk antibodies from the water-miscible fraction;
and
(b) flowing the water insoluble non-charge absorbent with a buffer to
obtain an aqueous fraction containing the yolk antibodies.
An aspect of the invention is to provide a process for selectively isolating
IgY
antibodies from egg yolk of an anseriform bird, which is characterized in
(a) absorbing yolk antibodies in a water-miscible fraction obtained from
the egg yolk of an anseriform bird with a water insoluble non-charged
absorbent
wherein the water insoluble non-charged absorbent is silicate, silicon
compounds,
carbonate, sulfate, phosphate, carbon, cellulose fiber, synthetic fiber,
ceramics, or
metal oxide, and wherein the water insoluble non-charged absorbent is at an
amount effective for separating the yolk antibodies from the water-miscible
fraction;
and
(b) flowing the water insoluble non-charged absorbent with a buffer to
obtain an aqueous fraction containing the yolk antibodies.
Another aspect of the invention is to provide a process for selectively
isolating IgY antibodies from egg yolk of an anseriform bird, which is
characterized
in performing first salting out by salting out an aqueous fraction containing
yolk
antibodies with (NH4)2SO4 of a first concentration ranging from about 15 %
(w/v) to
about 24 % (w/v), and wherein preferably not more than about 21 % (w/v) based
on
the volume of the aqueous fraction, and then performing second salting out by
salting out the aqueous fraction containing yolk antibodies treated in the
first salting
out with (NH4)2SO4 of a second concentration ranging from about 25 % (w/v) to
CA 02389897 2012-11-06
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about 40 % (w/v), and wherein preferably not more than about 31 c1/0 (w/v)
based on
the volume of the aqueous fraction treated in the first salting out.
Another aspect of the invention is to provide a process for selectively
isolating IgY antibodies from egg yolk of an anseriform bird, which is
characterized
in performing first salting out by salting out an aqueous fraction containing
yolk
antibodies with (NH4)2SO4 of a first concentration ranging from 15 % (w/v) to
24 %
(w/v) based on the volume of the aqueous fraction, and then performing second
salting out by salting out the aqueous fraction containing yolk antibodies
treated in
the first salting out with (NH4)2SO4 of a second concentration ranging from 25
%
(w/v) to 40 % (w/v) based on the volume of the aqueous fraction treated in the
first
salting out.
Still another aspect of the invention is to provide a process for selectively
isolating IgY antibodies from egg yolk of an anseriform bird, which is
characterized
in:
(a) absorbing yolk antibodies in a water-miscible fraction obtained from
the egg yolk of an anseriform bird with a water insoluble non-charged
absorbent
selected from the group consisting of silicate, silicon compounds, carbonate,
sulfate, phosphate, carbon, cellulose and _synthetic fiber, ceramics, and
metal
oxide, and wherein the water insoluble non-charged absorbent is at an amount
effective for separating the yolk antibodies from the water-miscible fraction;
(b) flowing the water insoluble non-charged absorbent with a buffer
to obtain an aqueous fraction containing the yolk antibodies;
(c) salting out the aqueous fraction containing yolk antibodies in step (b)
with (NH4)2SO4 of a first concentration ranging from about 15 % (w/v) to about
24 %
(w/v), and wherein preferably not more than about 21 % (w/v) based on the
volume
of the aqueous fraction; and
(d) salting out the aqueous fraction containing yolk antibodies treated in
step (c) with (NH4)2SO4 of a second concentration ranging from about 25 %
(w/v)
CA 02389897 2012-11-06
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to about 40 % (w/v), and wherein preferably rot more than about 31 % (w/v)
based
on the volume of the aqueous fraction treated in step (c).
Another aspect of the invention provides a process for selectively isolating
IgY antibodies from egg yolk of an anseriform bird, which is characterized in:
(a) absorbing yolk antibodies in a water-miscible fraction obtained from the
egg yolk of an anseriform bird with a water insoluble non-charged absorbent,
wherein the water insoluble non-charged absorbent is silicate, silicon
compounds,
carbonate is sulfate, phosphate, carbon, cellulose fiber, synthetic fiber,
ceramics, or
metal oxide, and wherein the water insoluble non-charged absorbent is at an
amount effective for separating the yolk antibodies from the water-miscible
fraction;
(b) flowing the water insoluble non-charged absorbent with a buffer to obtain
an aqueous fraction containing the yolk antibodies;
(c) salting out the aqueous fraction containing yolk antibodies in step (b)
with
(NH4)2SO4 of a first concentration ranging from 15 % (w/v) to 24 % (w/v),
based on
the volume of the aqueous fraction; and
(d) salting out the aqueous fraction containing yolk antibodies treated in
step
(c) with (NH4)2SO4 of a second concentration ranging from 25 % (w/v) to 40 %
(w/v), based on the volume of the aqueous fraction treated in step (c).
According to the process of this invention, an abundant amount of a selected
isoform of yolk antibodies, in particular IgY(Fc) antibody, available for
various
industrial applications can be obtained in an economic, efficient and time-
saving
manner.
It is still another object of the invention to provide the clinical and
research
uses of the IgY antibody so produced. In addition to the cost-effectiveness
and
ease of preparation, the IgY(Fc) antibody according to the present invention
has
advantages of being inactive to the complement system and rheumatoid factors
in
mammalian sera, and having poor cross-reactivity to mammalian IgG, and is thus
particularly suitable for use in immunological assays involving mammalian sera
with
minimal interference. It would be appreciated by those skilled in the art that
the IgY
antibody can be present in the form of a single reagent for clinical, research
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and other applications, or included in a commercial kit as an active
component.
It is another specific object of the invention to provide a reagent for
immunoassay of an etiological agent of interest, comprising an IgY
antibody obtained by the process according to this invention.
BRIEF DESCRIPTION OF THE DRAWINGS
The above and other objects and features of the present invention
will become apparent with reference to the following description of the
preferred embodiments taken in conjunction with the accompanying
drawings, in which:
FIG. 1 illustrates a SDS-PAGE analysis comparing the antibody
capturing abilities of four absorbents: lane 1, the partially purified
antibody
extract; lane 2, the solution flowing through 2 % fumed silica; lane 3, the
solution flowing through 3 % silica dioxide; lane 4, the solution flowing
through 3 % Celite diatomite; lane 5, the solution flowing through 3 %
Celite diatomite hyflo-Cel; and lane 6, the solution flowing through 5 %
Celite diatomite hyflo-Cel;
FIG. 2 illustrates the electrophoresis results of the purified yolk
antibodies using fumed silica as the absorbent run on an 8 %
SDS-polyacrylamide gel: lane 1, the partially purified antibody extract; lane
2, the solution flowing through 2 % fumed silica; lane 3, the eluate from the
fumed silica pellet; lane 4, the antibody product precipitated with 21 %
(w/v) ammonium sulfate in the first precipitation step; and lane 5, the
antibody product precipitated with 31 % (w/v) ammonium sulfate in the
second precipitation step;
FIG. 3 illustrates the electrophoresis results of the purified yolk
antibodies using Celite diatomite as the absorbent run on an 8 %
SDS-polyacrylamide gel: lane 1, the partially purified antibody extract; lane
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2, Celite diatomite filtrate; lane 3, the antibody product precipitated with
21
% (w/v) ammonium sulfate in the first precipitation step; lane 4, the
antibody product precipitated with 31 % (w/v) ammonium sulfate in the
second precipitation step; and lane 5, the antibody product precipitated with
16 % (w/v) sodium sulfate in the second precipitation step; and
FIG. 4 illustrates the electrophoresis results of the purified yolk
antibodies using fumed silica as the absorbent run on an 8 %
SDS-polyacrylamide gel: M, molecular weight marker; lane 1, the partially
purified antibody extract; lane 2, the solution flowing through 2 % fumed
silica; lane 3, the eluate from the fumed silica pellet; lane 4, the antibody
product precipitated with 21 % (w/v) ammonium sulfate; and lane 5, the
antibody product purified by affinity chromatography.
DETAILED DESCRIPTION OF THE INVENTION
The yolk antibodies are abundant in the bird serum and the eggs laid
by the bird. However, as described above, collection of the antibody from
the egg is usually preferred on account of the cost. The laying hen transfers
both of the IgY and IgY(.Fc) isoforms from serum to the egg yolk. In
principle, each duck egg contains about 1 to about 4 mg IgY/m1 and about 3
to about 12 mg IgY(. Fc)/m1 in the yolk and, therefore, the total quantity of
the antibodies contained in a single egg is estimated to be 15 to 80 mg of
IgY and 45 to 240 mg of IgY(.Fc). The large volume of egg yolk produced
vastly exceeds the volume of the serum that can be safely obtained from the
birds over any given time period. In addition, extraction of yolk antibodies
can be performed on a large scale without costly investment. Preferably,
antibodies in the present invention are obtained from eggs of an anseriform
bird immunized with a specific antigen.
In accordance with the present invention, it provides a process for
efficiently isolating antibodies from egg yolk, in which the so-called
"adsorption chromatography" or "differential salting-out," which may be
I
CA 02389897 2002-06-07
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used alone or in combination with the other, acting as critical steps in the
isolation.
As used herein, the term "adsorption chromatography" is directed to
a type of separation method involving the use of a stationary phase to
selectively take up and concentrate the desired solutes from a mobile phase.
According to one aspect of the present invention, a water insoluble
non-charged absorbent acts as the active constituent in the stationary phase
to separate the yolk antibodies by adsorbing the yolk antibodies in the water
insoluble non-charged absorbent accompanied with traping water-miscible
lipidic impurities normally present in egg yolk.
In a preferred embodiment of the present invention, the yolk is firstly
separated from the egg white, and then washed with distilled water to
remove as much albumen as possible. The
vitelline membrane
encapsulating the yolk is punctured, and the separated yolk fraction is then
diluted with an effective amount of an aqueous buffer or water to form a
suspension of the egg yolk. Preferably, the collected egg yolk is diluted
with an aqueous buffer solution or distilled water ranging from about 2
parts to about 40 parts by volume, more preferably from about 5 parts to
about 30 parts by volume, per 1 part of the egg yolk. Value of pH is
reported to be a critical factor during the stage of partial purification (E.
M.
Akita and S. Nakai, J Food Sci. 57:629, 1993). For the best recovery of
yolk antibodies, pH is preferably set within a range of about 5 to about 7.
Desirably, the temperature in this step is within a range of about 0 C to
about 60 C. The suspension of the egg yolk is gently agitated to form a
homogenous mixture, and then allowed to stand for a period of time
sufficient to form the aqueous and non-aqueous phases. The water
insoluble materials, including non-aqueous bio-molecules such as
lipoproteins, phospholipids, sterols and the like, are then removed from the
aqueous yolk suspension by centrifugation. The
resulting
antibody-containing supernatant may then be separated from the viscous
I.
CA 02389897 2002-06-07
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precipitant by decanting, suctioning, or other like methods known in the art.
In general, the lipid content of the water-miscible fraction thus
obtained is still so high as to be adverse to the subsequent manipulation.
According to the present invention, a stationary phase containing a water
insoluble non-charged absorbent is incubated with the water-miscible
fraction in a sufficient amount to absorb the yolk antibodies and to adsorb
the majority of the water-miscible lipidic substances remaining in the
water-miscible fraction. The suitable absorbents include but are not limited
to silicate, silicon compound, carbonate, sulfate, phosphate, carbon,
cellulose and synthetic fiber, ceramics, and metal oxide, and wherein the
silicate includes synthetic or natural clays, kaolin, talc, and calcium
silicate;
the silicon compound includes fumed silica, amorphous silica, silica
dioxide, silica gel, silicates, diatomaceous earth, and Fuller's earth;
carbonate includes calcium carbonate and barium carbonate; sulfate
includes calcium sulfate; phosphate includes calcium phosphate; carbon
includes activated carbon and carbon fiber, cellulose and synthetic fiber
includes cellulose powder; ceramics includes porosity ceramics; and metal
oxide includes aluminum oxide and titanium oxide. Particularly preferred
absorbents are fumed silica, silica dioxide and diatomaceous earth. The
working ratio of the absorbent to the water-miscible fraction can vary over
a wide range depending upon the properties of the absorbent chosen. When
fumed silica is used in this process, it is preferably added to a
concentration
of equal to or higher than about 0.1 % by weight, and more preferably
ranged between about 0.3 to about 5.0% by weight, based on the volume of
the water-miscible fraction to be treated. When the absorbent is silica
dioxide or diatomaceous earth, the adsorption chromatography according to
this invention is preferably carried out at more than about 1 % by weight,
and more preferably in a range of about 3 to about 20% by weight, of the
absorbent based on the volume of the water-miscible fraction to be treated.
The adsorption chromatography according to this invention can be
CA 02389897 2002-06-07
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effectuated by any conventional ways, such as batch treatment of the
water-miscible fraction with an absorbent or flowing the water-miscible
fraction over a chromatography column packed with the absorbent, as long
as the amount of the yolk antibodies retained on the surfaces of the
absorbent is satisfactory. The reaction time and temperature during the
treatment are not critical to the results, and a reaction temperature of about
4 to about 30 C and a reaction time of about 10 to about 60 minutes are
usually feasible. While the adsorption procedure can be repeated several
times, each with fresh absorbent, if necessary, a single operation is
normally sufficient. By way of this procedure, the lipids and most of the
non-lipid substances can be successfully separated into two immiscible
phases while yolk antibodies can also be absorbed.
Depending upon the capability of the selected absorbent to capture
finmunoglobulins, the yolk antibodies can be recovered from either an
- eluate eluted from the stationary phase or the "flow-through solution"
which, as used herein, is intended to represent the solution passing through
the stationary phase. As shown in the preferred embodiments given in the
text, the yolk antibodies are mainly present in the stationary phase when
fumed silica or silica dioxide is used as the absorbent, whereas
diatomaceous earth leaves more than about 60% of the antibodies in the
flow-through solution.
The choice of a particular method to obtain yolk antibodies can be
determined by the skilled artisan. Typically, the yolk antibodies are
obtained in an aqueous fraction by flowing the water insoluble non-charged
absorbent with a buffer, and wherein the buffer is at a pH of lower than
about 4 or higher than about 8 or containing a chaotropic agent can be
utilized in the present invention to obtain the yolk antibodies from the
stationary phase without substantially dissociating the lipidic substances
from the stationary phase, such that an antibody-containing eluate is
formed. As used herein, the term "eluate" is directed to a solution
;1.11F 1 11
CA 02389897 2002-06-07
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containing the desired substances unbound by the eluent from the stationary
phase. The term "chaotropic agent" or "chaotrope" is directed to a
chemical capable of inducing a conformational change in a protein
molecule, such as an antibody molecule, which is therefore often known as
a protein denaturant. According to the invention, most of the bound
antibodies can be successfully eluted with any neutral buffer containing
moderate concentration (>1 M) of a chaotropic agent. In most instances,
removal of the chaotrope after elution will restore the native protein
structure.
o The
useful buffer include but are not limited to 0.1 M glycine-HC1,
pH 2.3; 0.1 M glycine-HC1, pH 10.0; 3M to 6 M guanidine-HC1, pH 3.0;
3.0 M potassium chloride; 5 M potassium iodide; 3.5 M magnesium
chloride; 1-3 M ammonium/sodium/potassium thiocyanate and 6 M urea.
With respect to the activity of the recovered antibodies, however, a
= 15
moderate-ionic strength, chaotrope-containing, neutral pH buffer, such as 3
M sodium thiocyanate buffered in 20 mM MES buffer (pH 5.8) or 20 mM
Tris(hydroxymethyl)-aminomethane (pH 7.5), is more suitable for
practicing the invention. The active state of the collected antibodies can be
easily restored by, for example, extensive dialysis against a low-ionic
20 strength, non-chaotrope-containing, and weakly acidic buffer.
According to one aspect of the present invention, the aqueous
fraction including the eluate or the flow-through solution, which is enriched
with antibodies, can subsequently be subjected to a procedure of
differential salting-out to separate yolk antibody isoforms.
25 The term
"salting-out" as used herein takes on its common meaning
in the art of protein chemistry and is directed to the addition of a
non-denaturing salt or salts to a mixture or production batch to decrease the
solubility of proteins, which leads to the precipitation or coagulation of the
proteins. By the term "differential salting-out" is meant a salting-out
30 process
that differentially precipitates or coagulates two or more proteins
CA 02389897 2002-06-07
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from a mixture by varying the concentration of the added salt or salts. In
the present invention, the proteins intended to be differentially precipitated
are the isoforms of yolk antibodies, i.e., IgY and IgY(.Fc). Examples of the
non-denaturing salts useful for precipitation of the yolk antibodies include
but are not limited to NaC1, Na2SO4, (NI-14)2SO4, KC1, CaC12, and MgSO4.
Preferably, the non-denaturing salt is Na2SO4 or (NH4)2SO4, and
(=1114)2SO4 is the most preferred. The salt concentration for differentially
precipitating yolk antibody isoforms depends on the type of the salt and can
be determined by a skilled artisan through simple tests. According to a
preferred embodiment of the present invention, in which (NH4)2SO4 is
employed, IgY is firstly salted out at a concentration ranging from about 15
= % (w/v) to about 24 % (w/v), and wherein preferably is equal to or lower
than about 21 % (w/v), of the salt on the basis of the treated volume of the
aqueous fraction, while IgY(.Fc) is precipitated as the concentration of the
salt ranging from about 25 % (w/v) to about 40 % (w/v), and wherein
preferably is about 31 % (w/v) based on the treated volume of the aqueous
fraction. It should be appreciated that the sequence of precipitation of the
two antibody isoforms could be also variable depending on the salt chosen.
The combined use of two or more salts in this procedure, e.g., firstly
precipitating a first isoform with one salt followed by precipitating a second
isoform with another salt, is also feasible. The differential salting-out
procedure according to the present invention dramatically accomplishes a
main object of the present invention, i.e., essential selectively separation
of
the desired IgY comprising IgY and IgY(.Fc) antibodies from the whole
population of yolk antibodies constituted by both IgY and IgY(.Fc).
If obtaining the antibodies with a higher purity is desired, the
precipitated antibodies can be re-dissolved in a suitable buffer system and
subjected to additional purification procedures, such as size exclusive
chromatography, hydrophobic interaction chromatography, ion-exchange
chromatography and inununo-affinity chromatography.
CA 02389897 2010-02-26
As used herein, the term "immunoaffinity purification" or
"immunoaffinity chromatography" is directed to a type of separation
method based on the binding characteristics of antibodies for a specific
antigen. That is, the antibodies that bind to a specific antigen under a
5 particular condition are separated from the unbound antibodies under that
condition. The present invention contemplates the use of immunoaffinity
purification to eliminate irrelevant . proteins, in particular the
non-antigen-binding immunoglobulin.
According to the present invention, the immunoaffinity purification
The antibodies purified by differential salting-out is dissolved in a
"binding buffer" and applied onto the antigen matrix, so that the
Preferably, the immunoaffinity purification is conducted in an
environment of weak acid and low ionic strength, i.e., at pH within a range
of about 4 to about 7 and under an ionic strength of lower than about 50
CA 02389897 2002-06-07
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immobilized antigen at pH within a range of about 5 to about 6 and most
preferably within a range of about 5.6 to about 5.8. The yolk antibodies
can be dissociated from the antigen matrix by a chaotropic salt, or at a pH
of lower than about 4 or higher than about 8. The activity of the collected
antibodies can be restored by, for example, extensive dialysis against a
low-ionic strength, non-chaotrope-containing, weakly acidic buffer.
The IgY(.Fc) purified according the process of the invention neither
activate the complement system nor binds to rheumatoid factor of
mammalian sera. The immunological cross-reactivity between IgY(.Fc)
io and the mammalian IgG is not significant. Thus, the invention also
provide
a new type of antibody suitable for clinical and research uses.
The invention also provides a broad variety of clinical and research
uses of the IgY antibody prepared according to the invention.
For example, the present invention provides a pharmaceutical
composition for treating or prophylaxis an animal (which includes domestic
fowls, livestock and companion animals) or human patient comprising a
therapeutic amount of the IgY antibody of the present invention for
protecting or prophylaxis them from various etiological agents, including
microorganisms, such as bacteria, native, recombinant or peptide-synthetic
viruses, fungi, protozoa, nematodes and the like, and proteinaceous or
non-proteinaceous substances, such as native, recombinant or
peptide-synthetic allergens, toxins, venoms, hormones or any other
immunogen capable of eliciting an immune response. Preferably, the
purified IgY antibody is applied in combination with a pharmaceutically
acceptable carrier such as water, saline and the like. The pharmaceutical
composition can be delivered by ways comprising oral delivery, injection,
external administration, and immunizing treatment.
The IgY antibody of the present invention is also useful for detecting
an etiological agent of interest, including, for example, a pathogenic or
1W I E
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non-pathogenic organism, such as Escherichia coil, Salmonella enterititis,
and other bacterial organisms; a native, recombinant or peptide-synthetic
hormone such as estrogen, progesterone, thyroxin and the like; a major
histocompatibility complex antigen and the like; a native, recombinant or
peptide-synthetic tumor marker such as alpha-fetoprotein, prostate specific
antigen and the like; a disease state marker such as C-reactive protein,
ferritin and the like; an accumulation or a residual of foreign materials such
as drugs of Theophylline and digoxin; in a body sample such as a fluid,
tissue, cell extract and the like, that is derived from the human or animal.
In order to obtain antibodies only specific to the etiological agents, the
etiological agent can be injected into the ducks as antigens for inducing the
production of desired antibodies, and wherein the antigens comprise
naturally purified antigens, recombinant antigens, peptide-synthetic
antigens, and plasmid DNA. Using the IgY antibody obtained according to
this invention, an etiological agent of interest can be quantitatively or
qualitatively detected by any conventional method known in the art, such as
the Ouchterlony methods (MO), the single radial immuno diffusion method
(SRID), the immuno electrophoresis method (IEP), the radioimmuno assay
method (RIA), the enzyme-linked immuno sorbent assay method (ELISA),
the Western blot method (WB), the turbidimetric immunoassay method
(TIA), the particle-enhanced turbidimetric immunoassay method, an
enzymatic immunoassay, a nephelometric immunoassay, a
chemiluminescent immunoassay, an immuno gold assay, or an
immuno-chromatography assay.
The IgY antibody of the present invention is also adapted for use in
biochips and biosensors.
The following Examples are given for the purpose of illustration
only and are not intended to limit the scope of the present invention.
Example 1: Immunization Procedure for Stimulation of Specific Antibody
Production
I 11
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Twelve, 16-week old, domestic ducks (Anas platyrhynchos var.
domestica) were individually housed for antibody and egg production. The
ducks received an initial subcutaneous injection of 1-5 mg/ml of human
C-reactive protein (CRP; purified from human ascites) in phosphate buffer,
pH 7.5 emulsified with an equal volume of complete Freund's adjuvant.
The concentration of the antigen used was generally in the range of 1 to 5
mg/ml. After the initial injection, young hens received three additional
injections of 1-5 mg of antigen every two weeks. One week later, eggs
began to be collected, labeled and stored at 4 C until processed for
extraction and purification of antibody. The booster procedure was
repeated every four weeks during the experiment. Blood was sampled at
the seventh day after each booster injection. Each blood sample was
centrifuged and the resulting serum was collected.
Example 2: Extraction of Antibodies from Duck Yolks
The yolks collected from the eggs laid by the hyperimmunized ducks
of Example 1 were thoroughly washed by a weak jet of distilled water, to
thereby remove albumen. The volume of yolk was measured and then
mixed thoroughly with distilled water in an amount of ten times the
measured amount of yolk. The mixture was then held for at least two hours
under 4 C, and subsequently centrifuged at 10,000 rpm in a Hitachi CR-
22F centrifuge for one hour. A pale supernatant layer and a semi-solid
pliable layer were formed in centrifuge tubes.
Example 3: Treatment with Absorbents
To the crude extract prepared in Example 2 were added with one of
the absorbents: 2 % (w/v) fumed silica (purchased from Sigma), 3 % (w/v)
silica dioxide (sigma), 3 % (w/v) Celite diatomite (purchased from Celite
Corporation), and 3 or 5 % (w/v) Celite diatomite hyflo-Cel (Celite
Corporation). The resultant suspensions were incubated at 4 C for 60
minutes with gentle stirring. After completion of the incubation, the
CA 02389897 2002-06-07
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absorbents were precipitated at 4 C at 20,000 rpm in a Hitachi CR-22F
centrifuge, and the supernatants and pellets were harvested separately. Ten
ill samples taken from each supernatant were subjected to non-reducing
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
As shown in FIG. 1, in terms of the quantity of the antibodies
adsorbed by the absorbents, fumed silica has the best adsorptive activity
and almost no antibody was left in the flow through solution.
Silica dioxide displays a slightly weaker affinity to
immunoglobulins, which perhaps results from its lower porosity (and thus
comprising a less extensive surface area) than fumed silica. On the other
hand, less than 10 % of the yolk antibodies were captured by either type of
the diatomaceous earths.
Example 4: Differential Salting-out of Yolk Antibodies
The fumed silica pellet obtained in Example 3 was treated with 2.5
M sodium thiocyanate (pH 7.5) to elute the antibodies bound thereon. The
resultant eluate was firstly precipitated with ammonium sulfate at a
concentration of about 21 % (w/v) based on the volume of the eluate,
followed by a second precipitation with addition of ammonium sulfate to
about 31 % (w/v). The precipitated antibody products were re-dissolved in
phosphate buffer saline (PBS). Analytical SDS-PAGE was performed on a
8 % non-reducing acrylamide gel, in which 2237 j.ig of the crude extract of
example 2 (lane 1), 10 p,1 of the flow through harvested in Example 3 (lane
2), 1122.25 14 of the eluate from the fumed silica pellet (lane 3), and 153
tig and 372.85 j.ig of the antibody products obtained in the first and second
precipitation steps (lane 4 and lane 5, respectively) were loaded. The result
is shown in FIG. 2. The percentage recovery and purity were determined
by scanning densitometry of the gel and summarized in Table 1.
Table 1
CA 02389897 2002-06-07
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Total IgY IgY
IgY(Fc) IgY(.Fc)
protein percentage yield/egg percentage yield/egg
Crude extract 447.4 mg 4.43% 19.82 mg 26.79% 119.86 mg
Eluate 224.45 mg
8.15 % 18.29 mg 41.65 % 93.48 mg
lst precipitation by 30.65 mg 37.82 % 11.59 mg 62.18 % 19.06 mg
21% (NH4)2SO4
2nd precipitation by 74.57 mg 2.03 % 1.51 mg 96.62 % 72.05 mg
31% (NH4)2SO4
As illustrated in Table 1, the resulting IgY(.Fc) antibodies are
recovered in about 76 % yield (72.05 mg/119.86 mg x 100%) in greater
than 96 % purity. More importantly, this purification scheme
advantageously leads to the essential separation of the desired IgY(.Fc)
antibodies from the whole population of yolk antibodies constituted mainly
by both IgY and IgY(.Fc).
Example 5: Partially Purified IgY(.Fc) with Celite Diatomite
Taking advantage of the ability of diatomaceous earth to attract
lipids and repulse antibodies, the crude extract prepared in Example 2 was
poured onto a filtration column packed with 10 % by weight of Celite
diatomite based on the poured volume of the extract. The solution flowing
through the column was harvested and subjected a first precipitation with
21 % (w/v) ammonium sulfate based on the volume of the flow through
solution. The precipitated antibodies were collected and the supernatant
is was divided into two parts. One part of the supernatant was precipitated
with ammonium sulfate at a concentration of about 31 % (w/v), while the
other part was precipitated with 16% (w/v) sodium sulfate. The
precipitated antibody products were re-dissolved in PBS. Analytical
SDS-PAGE was performed on a 8% non-reducing acrylamide gel, in which
2012.5 p.g of the crude extract of Example 2 (lane 1), 1678 letg of the flow
; r I I
CA 02389897 2002-06-07
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through harvested by Celite diatomite filtration (lane 2), 94.9 ps of the
eluate obtained in the first precipitation step (lane 3), and 169.65 tg and
357.75 1..tg of the antibody products obtained in the second precipitation
step by 31 % ammonium sulfate and 16 % sodium sulfate (lanes 4-5) were
loaded. The result is shown in FIG. 3. The percentage recovery and purity
were determined by scanning densitometry of the gel and summarized in
Table 2.
Table 2
Total IgY IgY IgY(.Fc) IgY(.Fc)
protein percentage yield/egg percentage , yield/egg
Crude extract 405.2 mg 11.60% 46.98 mg 29.01% 117 mg
,
CLT filtrate 335.6
mg 5.08 % 17.05 mg 30.47% 102.25 mg
lstprecipitation by 18.98 mg 62.60% 11.88 mg 37.40% 7.10 mg
21% (NH4)2SO4
28d precipitation 33.93 mg 6.29% 2.13 mg 77.14%
26.17 mg
by 16% Na2SO4
2nd precipitation by 71.55 mg 8.79 % 6.29 mg 68.68% 49.14 mg
31% (NH4)2SO4
As illustrated in Table 2, the resulting IgY(Fc) antibodies are
to recovered
in about 77 % (when sodium sulfate is used in the second
precipitation step) and 69 % purity (when ammonium sulfate is used in the
second precipitation step), respectively, with high yields.
Example 6: Immunoaffinity Purification of Yolk Antibodies
A C-reactive protein (CRP) solution was prepared in 0.1 M
carbonate buffer, pH 8.5 at a concentration of 5 mg/ml. CNBr-activated
Sepharose 4B purchased from Pharmacia was washed initially with 1 tnM
CA 02389897 2010-02-26
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22
cold HC1 in an amount of ten times the matrix volume and allowed to react
with the CRP solution in an amount of two times the matrix volume of at 4
C overnight. The antigen matrix was suspended in a solution of 0.5 M
ethanolamine in 20 mM Tris-HC1 (pH 8.5) in a ratio of 1:1 (v/v) for 2 hours
at 4 C to block remaining protein-reactive sites. The antigen matrix was
then washed with PBS containing 0.02 % sodium azide and stored at 4 C.
The duck antibodies precipitated with 21 % (w/v) ammonium sulfate
in Example 4 and the antigen matrix prepared above were used. One ml of
the antigen matrix was packed into a conventional column and soaked in 20
mM of MES (2-(N-morpholino] ethanesulfonic acid) buffer (pH 5.8). The
antigen matrix was allowed ' to react with 0.25 ml of the antibodies
formulated in the same binding buffer. The antigen matrix was washed
with the binding buffer until the effluent was substantially free of protein.
Bound antibodies were eluted immediately with 6 M guanidine-HCI, and
the optical density thereof was measured at 280 nm after a complete
dialysis. The SDS-PAGE analysis shown in FIG. 4 indicates that the
affmity-purified antibodies are constituted mainly by IgY(.Fc) antibody
which is represented by a single band on the gel.
While embodiments of the present invention have been illustrated
and described, various modifications and improvements can be made by
persons skilled in the art. The embodiments of the present invention are
therefore described in an illustrative but not restrictive sense. It is
intended
that the present invention is not limited to the particular forms as
illustrated,
and that all the modifications not departing from the spirit and scope of the
present invention are within the scope as defined in the appended claims.
