U.S. patent application number 10/209006 was filed with the patent office on 2003-01-09 for process for selectively isolating igy antibodies from egg yolk of an anseriform bird and igy antibodies obtained thereby.
Invention is credited to Chiou, Victor.
Application Number | 20030009014 10/209006 |
Document ID | / |
Family ID | 24946670 |
Filed Date | 2003-01-09 |
United States Patent
Application |
20030009014 |
Kind Code |
A1 |
Chiou, Victor |
January 9, 2003 |
Process for selectively isolating IgY antibodies from egg yolk of
an anseriform bird and IgY antibodies obtained thereby
Abstract
The present invention mainly relates to a process for isolation
and purification of yolk antibodies from egg yolk of an anseriform
bird 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 the yolk antibodies produced thereby and
various uses of such yolk antibodies.
Inventors: |
Chiou, Victor; (Taichung,
TW) |
Correspondence
Address: |
Y. ROCKY TSAO
Fish & Richardson P.C.
225 Franklin Street
Boston
MA
02110-2804
US
|
Family ID: |
24946670 |
Appl. No.: |
10/209006 |
Filed: |
July 31, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10209006 |
Jul 31, 2002 |
|
|
|
09733210 |
Dec 8, 2000 |
|
|
|
Current U.S.
Class: |
530/389.1 ;
530/417 |
Current CPC
Class: |
Y10S 530/861 20130101;
C07K 2317/23 20130101; C07K 16/065 20130101; C07K 2317/11
20130101 |
Class at
Publication: |
530/389.1 ;
530/417 |
International
Class: |
C07K 016/00; C07K
016/40 |
Claims
What is claimed is:
1. A process for selectively isolating IgY antibodies from egg yolk
of an anseriform bird, comprising: (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-charged absorbent with a buffer to obtain
an aqueous fraction containing the yolk antibodies, wherein the IgY
antibodies comprise antibodies with Fe regions and antibodies that
lack Fe regions.
2. The process of claim 1, wherein the water insoluble non-charged
absorbent is a silicate selected from the group consisting of
synthetic or natural clays, kaolin, talc, and calcium silicate.
3. The process of claim 1, wherein the water insoluble non-charged
absorbent is a silicon compound selected from the group consisting
of fumed silica, amorphous silica, silica dioxide, silica gel,
silicates, diatomaceous earth, and Fuller's earth.
4. The process of claim 1, wherein the water insoluble non-charged
absorbent is comprised of calcium carbonate or barium
carbonate.
5. The process of claim 1, wherein the water insoluble non-charged
absorbent is comprised of calcium sulfate.
6. The process of claim 1, wherein the water insoluble non-charged
absorbent is comprised of calcium phosphate.
7. The process of claim 1, wherein the water insoluble non-charged
absorbent is comprised of activated carbon or carbon fiber.
8. The process of claim 1, wherein the water insoluble non-charged
absorbent is comprised of cellulose powder.
9. The process of claim 1, wherein the water insoluble non-charged
absorbent is comprised of a porous ceramic.
10. The process of claim 1, wherein the water insoluble non-charged
absorbent is comprised of aluminum oxide or titanium oxide.
11. The process of claim 1, wherein the anseriform bird is a duck
or a goose.
12. A method for isolating avian antibodies from egg yolk, the
method comprising: (a) contacting a preparation that comprises
avian antibodies with a water insoluble absorbent that absorbs both
avian antibodies and lipid; and (b) eluting the avian antibodies
from the water insoluble absorbent with an aqueous buffer that
releases the absorbed avian antibodies, but not the lipid, thereby
providing an isolated preparation of avian antibodies.
13. The process of claim 1, wherein the buffer for flowing the
water insoluble non-charged absorbent in step (b) comprises a
chaotropic salt.
14. The process of claim 13, wherein the chaotropic salt comprises
about 3M to about 6M guandine-HCl or about 1 M to about 3 M sodium
thiocyanate.
15. The process of claim 1, further comprising a purification step
by an immunoaffinity chromatography at a pH value ranging from
about 4 to about 7 under an ionic strength of lower than about 50
mM.
16. The process of claim 15, wherein the pH value is ranging from
about 5 to about 6.
17. The process of claim 16, wherein the pH value is ranging from
about 5.6 to about 5.8.
18. The process of claim 1, wherein the aqueous fraction containing
the yolk antibodies in step (b) is a flow-through solution which
has flowed through the water insoluble non-charged absorbent.
19. The process of claim 1, wherein the aqueous fraction containing
the yolk antibodies in step (b) is an eluate eluted from the water
insoluble non-charged absorbent.
20. A process for selectively isolating IgY antibodies from egg
yolk of an anseriform bird comprising performing first salting out
by salting out an aqueous fraction containing yolk antibodies with
(NH.sub.4).sub.2SO.sub.4 of a first concentration ranging from
about 15% (w/v) to about 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 (NH.sub.4).sub.2SO.sub.4 of a second
concentration ranging from about 25% (w/v) to about 40% (w/v) based
on the volume of the aqueous fraction treated in the first salting
out; wherein the IgY antibodies comprise antibodies with Fe regions
and antibodies that lack Fc regions.
21. The process of claim 20, wherein the first concentration is not
more than about 21% (w/v) based on the volume of the aqueous
fraction.
22. The process of claim 20, wherein the second concentration is
not more than about 31% (w/v) based on the volume of the aqueous
fraction treated in the first salting out.
23. The process of claim 20, wherein the anseriform bird is a duck
or a goose.
24. A method for enriching 5.7 S isoform avian antibodies relative
to 7.8 S isoform avian antibodies, the method comprising: (a)
precipitating 7.8 S isoform antibodies using a precipitant salt at
an ionic strength less than the ionic strength of 24%
(NH.sub.4).sub.2SO.sub.4 (w/v) to provide a supernatant that
includes 5.7 S antibodies; and (b) precipitating the 5.7 S isoform
antibodies from the supernatant using a precipitant salt, to
thereby provide an isolated preparation enriched for 5.7 S isoform
antibodies.
25. The process of claim 20, further comprising a purification step
by an immunoaffinity chromatography at a pH value ranging from
about 4 to about 7 under an ionic strength of lower than about 50
mM.
26. The process of claim 25, wherein the pH value is ranging from
about 5 to about 6.
27. The process of claim 26, wherein the pH value is ranging from
about 5.6 to about 5.8.
28. A process for selectively isolating IgY antibodies from egg
yolk of an anseriform bird comprising: (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
(NH.sub.4).sub.2SO.sub.4 of a first concentration ranging from
about 15% (w/v) to about 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
(NH.sub.4).sub.2SO.sub.4 of a second concentration ranging from
about 25% (w/v) to about 40% (w/v) based on the volume of the
aqueous fraction treated in step (c); and wherein the IgY
antibodies comprise antibodies with Fc regions and antibodies lack
of Fe regions.
29. The process of claim 28, wherein the silicate comprises
synthetic or natural clays, kaolin, talc, and calcium silicate.
30. The process of claim 28, wherein the silicon compound comprises
fumed silica, amorphous silica, silica dioxide, silica gel,
silicates, diatomaceous earth, and Fuller's earth.
31. The process of claim 28, wherein the carbonate comprises
calcium carbonate and barium carbonate.
32. The process of claim 28, wherein the sulfate comprises calcium
sulfate.
33. The process of claim 28, wherein the phosphate comprises
calcium phosphate.
34. The process of claim 28, wherein the carbon comprises activated
carbon and carbon fiber.
35. The process of claim 28, wherein the cellulose and synthetic
fiber comprise cellulose powder.
36. The process of claim 28, wherein the ceramics comprises
porosity ceramics.
37. The process of claim 28, wherein the metal oxide comprises
aluminum oxide and titanium oxide.
38. The process of claim 28, wherein the anseriform bird is a duck
or a goose.
39. The method of claim 12, further comprising separating 5.7 S
isoform antibodies in the isolated preparation of avian antibodies
from 7.8 S isoform antibodies.
40. The process of claim 28, wherein the buffer for flowing the
water insoluble non-charged absorbent in step (b) comprises a
chaotropic salt.
41. The process of claim 40, wherein the chaotropic salt comprises
about 3M to about 6M guandine-HCl or about 1 M to about 3 M sodium
thiocyanate.
42. The process of claim 28, wherein the first concentration is not
more than about 21% (w/v) based on the volume of the aqueous
fraction.
43. The process of claim 28, wherein the second concentration is
not more than about 31% (w/v) based on the volume of the aqueous
fraction treated in the first salting out.
44. The process of claim 28, further comprising a purification step
by an immunoaffinity chromatography at a pH value ranging from
about 4 to about 7 under an ionic strength of lower than about 50
mM.
45. The process of claim 44, wherein the pH value is ranging from
about 5 to about 6.
46. The process of claim 45, wherein the pH value is ranging from
about 5.6 to about 5.8.
47. The process of claim 28, wherein the aqueous fraction
containing the yolk antibodies in step (b) is a flow-through
solution which has flowed through the water insoluble non-charged
absorbent.
48. The process of claim 28, wherein the aqueous fraction
containing the yolk antibodies in step (b) is an eluate eluted from
the water insoluble non-charged absorbent.
49. An IgY antibody selectively isolated from egg yolk of an
anseriform bird, the IgY antibody being prepared according a
process comprising: (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
(NH.sub.4).sub.2SO.sub.4 of a first concentration ranging from
about 15% (w/v) to about 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
(NH.sub.4).sub.2SO.sub.4 of a second concentration ranging from
about 25% (w/v) to about 40% (w/v) based on the volume of the
aqueous fraction treated in step (c); and wherein the IgY
antibodies comprise antibodies with Fc regions and antibodies lack
of Fc regions.
50. A preparation comprising 5.7 S isoform avian antibodies,
wherein the 5.7 S are present at a concentration at least 10 fold
greater than any 7.8 S isoform antibodies, if present, the
preparation being prepared by a method comprising: (a) contacting a
preparation that comprises avian antibodies with a water insoluble
absorbent that absorbs both avian antibodies and lipid; (b) eluting
avian antibodies from the water insoluble absorbent with an aqueous
buffer that releases the absorbed antibodies, but not the lipid,
thereby providing an eluate; and (c) separating 5.7S isoform
antibodies from the eluate, the separating being preferential for
5.7 S isoform antibodies relative to 7.8 S isoform antibodies.
51. The IgY antibody of claim 49, wherein the silicon compound
comprises fumed silica, amorphous silica, silica dioxide, silica
gel, silicates, diatomaceous earth, and Fuller's earth.
52. The IgY antibody of claim 49, wherein the carbonate comprises
calcium carbonate and barium carbonate.
53. The IgY antibody of claim 49, wherein the sulfate comprises
calcium sulfate.
54. The IgY antibody of claim 49, wherein the phosphate comprises
calcium phosphate.
55. The IgY antibody of claim 49, wherein the carbon comprises
activated carbon and carbon fiber.
56. The IgY antibody of claim 49, wherein the cellulose and
synthetic fiber comprise cellulose powder.
57. The IgY antibody of claim 49, wherein the ceramics comprises
porosity ceramics.
58. The IgY antibody of claim 49, wherein the metal oxide comprises
aluminum oxide and titanium oxide.
59. The IgY antibody of claim 49, wherein the anseriform bird is a
duck or a goose.
60. The IgY antibody of claim 49, wherein the buffer for flowing
the water insoluble non-charged absorbent in step (b) comprises a
chaotropic salt.
61. The IgY antibody of claim 60, wherein the chaotropic salt
comprises about 3M to about 6M guandine-HCl or about 1 M to about 3
M sodium thiocyanate.
62. The IgY antibody of claim 49, wherein the first concentration
is not more than about 21% (w/v) based on the volume of the aqueous
fraction.
63. A preparation comprising 5.7 S isoform avian antibodies,
wherein the 5.7 S isoform avian antibodies are at least 80% pure
relative to other protein species and are present at a
concentration at least 10 fold greater than any 7.8 S isoform
antibodies, if present.
64. A pharmaceutical composition comprising an IgY antibody
according to claim 49 together with a pharmaceutically acceptable
carrier.
65. A kit for immunoassay, comprising an IgY antibody according to
claim 49 and instructions for use.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part and claims
priority to U.S. application Ser. No. 09/733,210, filed Dec. 8,
2000, the content of which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] 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 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.
[0004] 2. Description of the Related Art
[0005] 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.
[0006] In the art of immunodiagnostics, an intact IgG molecule is
not 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).
[0007] Some of the suggested uses of antibody in immunotherapy
include treating patients with intoxicated bacterial toxins or
snake venoms (see, for example, U.S. Pat. No. 5,340,923 and U.S.
Pat. No. 5,601,823), and protection of neonatal piglets against
fatal enteric colibacillosis (see, for example, H. Brussow et al.,
J. Clin. Microbiol. 25:982, 1987; and C. O. 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(ab').sub.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.
[0008] Recently, the divalent F(ab').sub.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.
[0009] The F(ab').sub.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(ab').sub.2
fragments, which is conventionally made by pepsin digestion of IgG
and subsequent purification via chromatography.
[0010] Ducks and their phylogenetically close relatives and some
reptiles, 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').sub.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(.DELTA.Fc) (equivalent to
5.7 S IgY) to represent both isoforms of IgY (K. E. Magor et al.,
J. Immunol. 149:2627-2633, 1992).
[0011] 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 Fe 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 absence of an
Fc-equivalent region of the IgY(.DELTA.Fc) antibody that
constitutes the quantitatively major component of anseriform bird
antibody response. It is thus believed that the IgY(.DELTA.Fc)
antibody, which appears to be a structural and functional analog of
the F(ab').sub.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.
[0012] 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. Pat. No. 5,340,923; U.S. Pat.
No. 5,585,098; U.S. Pat. No. 5,601,823; and U.S. Pat. No.
5,976,519). Egg yolks derived from a laying hen is inexpensive and
more convenient and safer to handle as compared to the
hyperimmunized mammalian sera. More importantly, yolk antibodies
are able to stand up to the scrutiny under modem 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.
[0013] 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
Feb. 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., Immunol. Invest. 14:323, 1985; see also U.S. Pat. No.
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).
[0014] 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 Fc region) from avian eggs,
rather than on the purification of IgY(.DELTA.Fc) and IgY
antibodies selectively. Moreover, since IgY(.DELTA.Fc) 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(.DELTA.Fc) antibody. IgY(.DELTA.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(.DELTA.Fc) antibody alone was selected isolated from
egg yolk.
[0015] Therefore, there exists a need for a rapid, cost-effective
and high-throughput process that provides easy isolation of the
desired yolk antibody (e.g., IgY(.DELTA.Fc)) from the antibody pool
of anseriform bird egg while maintaining the activity of the
antibody. The substantially purified IgY(.DELTA.Fc) antibody may
acts as a new type of F(ab').sub.2 antibody for various
immunodiagnostic and immunotherapeutic uses.
SUMMARY OF THE INVENTION
[0016] 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(.DELTA.Fc), can be easily obtained with high yield in
an economic manner, and are ready for a wide variety of
immunological uses.
[0017] 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:
[0018] (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
[0019] (b) flowing the water insoluble non-charge absorbent with a
buffer to obtain an aqueous fraction containing the yolk
antibodies.
[0020] In one embodiment, the water insoluble non-charged absorbent
absorbs both the yolk antibodies and the majority of the
water-miscible lipidic substances remaining in the water-miscible
fraction. In another embodiment, the absorbent absorbs the
water-miscible lipid substances, but does not substantially absorb
the yolk antibodies (e.g., absorbs less than 40, 30, 20, or 10% of
the yolk antibodies.
[0021] 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
(NH.sub.4).sub.2SO.sub.4 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 (NH.sub.4).sub.2SO.sub.4 of a second concentration ranging
from about 25% (w/v) to about 40% (w/v), and wherein preferably not
more than about 31% (w/v) based on the volume of the aqueous
fraction treated in the first salting out.
[0022] 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:
[0023] (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;
[0024] (b) flowing the water insoluble non-charged absorbent with a
buffer to obtain an aqueous fraction containing the yolk
antibodies;
[0025] (c) salting out the aqueous fraction containing yolk
antibodies in step (b) with (NH.sub.4).sub.2SO.sub.4 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
[0026] (d) salting out the aqueous fraction containing yolk
antibodies treated in step (c) with (NH.sub.4).sub.2SO.sub.4 of a
second concentration ranging from about 25% (w/v) to about 40%
(w/v), and wherein preferably not more than about 31% (w/v) based
on the volume of the aqueous fraction treated in step (c).
[0027] According to the process of this invention, an abundant
amount of a selected isoform of yolk antibodies, in particular
IgY(.DELTA.Fc) antibody, available for various industrial
applications can be obtained in an economic, efficient and
time-saving manner.
[0028] 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(.DELTA.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 and other applications, or
included in a commercial kit as an active component.
[0029] 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.
[0030] In many implementations, the methods enable the
IgY(.DELTA.Fc) isoform (5.7 S) to be separated from other isoforms,
e.g., the 7.8 S isoforms. In some implementations, the
IgY(.DELTA.Fc) species (5.7 S) is at least 70, 80, 90, or 95% pure
(w/v). It can be enriched at least 10, 20, 30, or 40 fold relative
to the 7.8 S isoform. The highly purified yolk antibodies, in
particular the highly purified IgY(.DELTA.Fc), can be obtained with
high yield in an economic manner, and are ready for a wide variety
of uses.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] 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:
[0032] 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;
[0033] 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;
[0034] 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 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
[0035] 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
[0036] 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(.DELTA.Fc)
isoforms from serum to the egg yolk. In principle, each duck egg
contains about 1 to about 4 mg IgY/ml and about 3 to about 12 mg
IgY(A Fc)/ml 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(.DELTA.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.
[0037] 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 used alone or in combination with the
other, acting as critical steps in the isolation.
[0038] 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 and also trapping water-miscible lipidic
impurities that are normally present in egg yolk.
[0039] 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
may be an important factor during the stage of partial purification
(E. M. Akita and S. Nakai, J. Food Sci. 57:629, 1993), at least in
some implementations. 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.degree.
C. to about 60.degree. 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 precipitant by decanting,
suctioning, or other like methods known in the art.
[0040] 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.
[0041] The adsorption chromatography according to this invention
can be 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.degree. 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.
[0042] Depending upon the capability of the selected absorbent to
capture immunoglobulins, 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.
[0043] 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
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.
[0044] The useful buffer include but are not limited to 0.1 M
glycine-HCl, pH 2.3; 0.1 M glycine-HCl, pH 10.0; 3M to 8 M
guanidine-HCl (e.g., 3 to 6M), pH 3.0; 3.0 M potassium chloride; 5
M potassium iodide; 3.5 M magnesium chloride; 1-3 M
ammonium/sodium/potassium thiocyanate (or e.g., at least 1M) and 6
M urea (or e.g., at least 4, 5, or 6M urea). With respect to the
activity of the recovered antibodies, however, a 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 strength, non-chaotrope-containing,
and weakly acidic buffer.
[0045] 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.
[0046] 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 process that
differentially precipitates or coagulates two or more proteins 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(.DELTA.Fc). Examples of the non-denaturing salts
useful for precipitation of the yolk antibodies include but are not
limited to NaCl, Na.sub.2SO.sub.4, (NH.sub.4).sub.2SO.sub.4, KCl,
CaCl.sub.2, and MgSO.sub.4. Preferably, the non-denaturing salt is
Na.sub.2SO.sub.4 or (NH.sub.4).sub.2SO.sub.4, and
(NH.sub.4).sub.2SO.sub.4 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
(NH.sub.4).sub.2SO.sub.4 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(.DELTA.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(.DELTA.Fc) antibodies from the whole population of yolk
antibodies constituted by both IgY and IgY(.DELTA.Fc).
[0047] 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 immuno-affinity
chromatography.
[0048] 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 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.
[0049] According to the present invention, the immunoaffinity
purification is conducted by use of an "antigen matrix" comprised
of antigen immobilized onto an insoluble support. The type of the
support is not critical to the immunoaffinity purification of the
invention. Any conventional support material suitable for the
covalent attachment of an antigen and inert to the interaction
between the desired antibody and the antigen immobilized thereon is
useful. Usually, the support is made of crosslinked agarose or
crosslinked dextran, such as the CNBr-activated Sepharose 4B
commercially available from Pharmacia.
[0050] The antibodies purified by differential salting-out is
dissolved in a "binding buffer" and applied onto the antigen
matrix, so that the immuno-complexes of the immobilized antigen and
the yolk antibodies are formed. Any buffer system inert to the
antigen-antibody interaction and effective to maintain the desired
binding condition is useful in the present invention. Preferably,
the binding buffer is selected from the group consisting of a
phosphate buffer, an MES (2-[N-morpholino]ethanesul- fonic acid)
buffer and a bis-Tris buffer, among which an MES buffer at a
concentration of 20 mM is the most preferred.
[0051] 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 mM, e.g., less than the ionic strength of a 50
mM NaCl solution. More preferably, the antibodies were allowed to
interact with the 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.
[0052] The IgY(.DELTA.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(.DELTA.Fc) and the mammalian IgG is
not significant. Thus, the invention also provide a new type of
antibody suitable for clinical and research uses.
[0053] The invention also provides a broad variety of clinical and
research uses of the IgY antibody prepared according to the
invention.
[0054] 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.
[0055] The IgY antibody of the present invention is also useful for
detecting an etiological agent of interest, including, for example,
a pathogenic or non-pathogenic organism, such as Escherichia coli,
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.
[0056] The IgY antibody of the present invention is also adapted
for use in biochips and biosensors.
[0057] 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
[0058] 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.degree. 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
[0059] 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.degree. 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
[0060] 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.degree. C. for 60 minutes with gentle stirring.
After completion of the incubation, the absorbents were
precipitated at 4.degree. C. at 20,000 rpm in a Hitachi CR-22F
centrifuge, and the supernatants and pellets were harvested
separately. Ten .mu.l samples taken from each supernatant were
subjected to non-reducing sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE).
[0061] 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.
[0062] 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
[0063] 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 .mu.g of the crude extract of example
2 (lane 1), 10 .mu.l of the flow through harvested in Example 3
(lane 2), 1122.25 .mu.g of the eluate from the fumed silica pellet
(lane 3), and 153 .mu.g and 372.85 .mu.g 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.
1 TABLE 1 IgY (7.8 S) IgY (7.8 S) IgY (.DELTA.Fc) IgY (.DELTA.Fc)
Total 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 1.sup.St precipitation by 30.65 mg
37.82% 11.59 mg 62.18% 19.06 mg 21% (NH.sub.4).sub.2SO.sub.4
2.sup.nd precipitation by 74.57 mg 2.03% 1.51 mg 96.62% 72.05 mg
31% (NH.sub.4).sub.2SO.sub.4
[0064] As illustrated in Table 1, the resulting IgY(.DELTA.Fc)
antibodies are recovered in about 76% yield (72.05 mg/l 19.86
mg.times.100%) in greater than 96% purity. More importantly, this
purification scheme advantageously leads to the essential
separation of the desired IgY(.DELTA.Fc) antibodies from the whole
population of yolk antibodies constituted mainly by both IgY (7.8
S) and IgY(.DELTA.Fc) (5.7 S). In the above example, the ratio of
5.7 S antibody to 7.8 S antibody is at least 40 (i.e., 47.7).
Example 5
Partially Purified IgY(.DELTA.Fc) with Celite Diatomite
[0065] 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 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
.mu.g of the crude extract of Example 2 (lane 1), 1678 .mu.g of the
flow through harvested by Celite diatomite filtration (lane 2),
94.9 .mu.g of the eluate obtained in the first precipitation step
(lane 3), and 169.65 .mu.g and 357.75 .mu.g 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.
2 TABLE 2 Total IgY IgY (.DELTA.Fc) IgY (.DELTA.Fc) protein
percentage IgY 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 1.sup.St precipitation by 21% 18.98 mg 62.60%
11.88 mg 37.40% 7.10 mg (NH.sub.4).sub.2SO.sub.4 2.sup.nd
precipitation by 16% 33.93 mg 6.29% 2.13 mg 77.14% 26.17 mg
Na.sub.2SO.sub.4 2.sup.nd precipitation by 31% 71.55 mg 8.79% 6.29
mg 68.68% 49.14 mg (NH.sub.4).sub.2 SO.sub.4
[0066] As illustrated in Table 2, the resulting IgY(.DELTA.Fc)
antibodies are 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
[0067] 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 mM cold HCl 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.degree. C. overnight. The
antigen matrix was suspended in a solution of 0.5 M ethanolamine in
20 mM Tris-HCl (pH 8.5) in a ratio of 1:1 (v/v) for 2 hours at
4.degree. C. to block remaining protein-reactive sites. The antigen
matrix was then washed with PBS containing 0.02% sodium azide and
stored at 4.degree. C.
[0068] 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-HCl, 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
affinity-purified antibodies are constituted mainly by
IgY(.DELTA.Fc) antibody which is represented by a single band on
the gel.
[0069] All patents and literature references cited in the present
specification are hereby incorporated by reference in their
entirety. In case of conflict, the present description, including
definitions, will prevail.
[0070] 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.
* * * * *