Efficient
production
against
MAX
of chicken
a conserved
GASSMANN1,
Department
PIA
of Pharmacology
mammalian
THOMMES,
THOMAS
and Biochemistry,
University
The egg yolk of immunized
chicken is a rich and inexpensive source of specific polyclonal antibodies.
In this
paper
we
show
that
protein,
20-30
tg
of a highly
as exemplified
stein
conserved
by proliferating
cell
response.
Immunoblot
analysis revealed that specific antibodies appeared
20 days after immunization,
reached
130
M.;
mg
TH#{246}MMES,
of
P.;
specific
WEISER,
H#{220}BSCHER,
U. Efficient
production
of chicken
egg yolk antibodies
against a conserved mammalian
protein. FASEBJ
4:
2528-2532;
several
Hemmeler
antibodies
MATERIALS
pro 4ferating
Isolation
EUKARYOTIC
HUBSCHER
Zurich,
Switzerland
(13),
who
showed
that
the
amount
against the poorly
immunogenic
RNA
1990.
Key Words: egg yolk . IgY
chicken antibodies
cell nuclear antigen . conserved protein
which
ULRICH
polymerase
II. By using total amounts
of 30-475
tg
PCNA
and two different
schemes,
we investigated
the
immune
response
of six hens.
Immunoblot
analysis
showed that antibodies
against PCNA
can be detected
16-30 days after the first immunization
even with the
lowest dose of 3 x 10 g antigen injected.
antibodies.-
T.;
and
chicken
a plateau after 30 days, and remained high until at least
day 81. A total amount of 4 g immunoglobulin
was extracted from 62 eggs of one immunized
hen, yielding
approximately
AND
of Z#{252}rich-Irchel,CH-8057
of purified IgY produced
in one month is 18 times higher
than that of IgG produced
in a rabbit. Another
advantage is that conserved
mammalian
proteins
are usually
more immunogenic
in the phylogenetically
distant birds.
Carroll
and Stollar
(5) were successful
in producing
nuclear antigen,are sufficient
to induce an immune
GASSMANN,
WEISER,
protein
sists of two subunits:
a heavy chain of 67,000-70,000
and
a light chain of 22,000-30,000
(11, 12). The quantitative
advantage
of raising IgY has been presented
by Gott-
ABSTRACT
mammalian
egg yolk antibodies
DNA
REPLICATION
enzymatic
15
activities
a multistep
process
are involved.
The
in
key
working
at the replication
fork are DNA polymerase a and t5 (1, 2). The processivity
of DNA
polymerase
can be stimulated
in vitro by addition
of the
proliferatingcellnuclear antigen (PC NA),2 a cell cycleregulated
protein
(3). Structurally,
PCNA is a highly conserved protein in eukaryotic
cells and can be exchanged
functionally
between
yeast and calf (4). Because
birds
enzymes
are known to produce
proteins
of mammals
amount
of calf thymus
response.
The use of chickens
antibodies
against highly conserved
(5, 6), we decided
to titrate the
PCNA required
for an immune
as an alternative
source
AND
METHODS
of antigen
PCNA
from calf thymus was purified
by the following
chromatographic
steps as described
(see ref 14): phosphocellulose
flow through,
DEAE-cellulose,
phenylSepharose, and FPLC
MonoQ.
A partiallypurified
PCNA
fraction
used for immunobiot
analysis
was obtained by chromatography
on DEAE-cellulose,
phenylSepharose,
and phosphocdllulose.
Immunization
of hens
Three
white
brown (Lohmann
Selection
Warren)
and three
(Lohmann
Selection
Leghorn)
laying
hens,
22-24
wk old, were bought
from a local farmer
and
kept isolated on regular
light cycles with free food and
of poly-
cional
antibodies
has been described
by Poison
et al. (7)
and adapted
by many others
(see ref 8). Like mammals,
chickens protect their offspringby transferringmaternal antibodies
from
serum
to egg yolk (9, 10). The
properties
of the 7S Ig species
from avians
are slightly
different
from those of mammalian
IgG, and are called
IgY (11). The
2528
protein
has an
Mr
of 180,000,
which
con-
‘Present
address:
Beckman
Center,
Stanford
University
Medical
Center,
Stanford,
CA 94305-5307,
USA.
2Abbreviations:
PCNA,
proliferating
cell nuclear
antigen;
Ig, immunoglobulin;
PEG, polyethylene
glycol; EDTA, ethylenediamine
tetraacetic acid; SDS, sodium dodecyl sulfate; TBS, Tris-buffered
line; FPLC, fast protein liquid chromatography.
0892-6638/90/0004.2528/$01
sa-
.50. © FASEB
water.
The
antigen
(PCNA)
was diluted
in
potassium
phosphate
buffer
(pH 7.2) containing
NaC1 to a final volume of 750 jLl and emulsified
0.01 M
0.1 M
with an
equal
volume
of complete
Freund’s
adjuvant
(Difco,
Detroit,
Mich.).
The sonified
suspension
(1.5 ml) was
injected
at two sites into the pectoral
muscle.
Further
injections
of the protein,
emulsified
as described
above
with the complete
adjuvant,
were given to the hens 12,
or in some cases, 20 days later (see Table
1). The eggs
were collected
daily, marked
and stored at 4#{176}C
until
use.
Extraction
and purification
of antibodies
purified
from individual
eggs as described (7).
Briefly,the yolk (20 ml) was carefullyseparated from
the white by washing
with deionized
water and collected without
the yolk skin in a graduated
cylinder.
The egg yolk suspension
was filled to 30 ml with buffer
IgY was
A (0.01 M potassium
taining
0.1 M NaC1)
phosphate
and mixed
of 7% (wt/vol)
6000 (Merck,
PEG
buffer
[pH
7.2] conwith 30 ml of a solution
Darmstadt,
W. Ger-
many)
dissolved
in buffer
A. The
precipitate
was
pelleted
by centrifugation
at 14,000
x g for 10
at
4#{176}C.
The
supernatant
was filtered
through
wet baby
napkins,
and solid PEG was added to a final concentration of 12% (wt/vol).
The mixture
was stirred
until all
polyethylene
glycol (PEG)
was dissolved.
mm
The
5
ml
solution
was then centrifuged
at 14,000
pellet containing
IgY was resuspended
mm. The
of buffer
24%
A and
(wt/vol)
precipitation.
ml of buffer
centrifuged
purified
with
solution
an
equal
in buffer
volume
of a
A for a second
Finally,
the pellet was resuspended
in 10
A, dialyzed
against
the same
buffer,
and
as above.
The
supernatant
was
further
on a 10 ml DEAE-cellulose
sorption
8.0) and
phate
mixed
PEG
x g for
in 20
at 0.015
elution
gradient
column
(5) by ad-
M potassium
phosphate
buffer
with
a 0.015-0.2
M potassium
(60 ml).
Part
of the peak
(pH
phos-
fraction
(0.5
ml) was diluted
with 9.5 ml buffer
B (25 mM Tris-HC1
[pH
7.8],
1 mM EDTA, 0.01%
[v/v]
Nonidet
P- 40
[Fluka,
Buchs,
Switzerland],
10% [v/v] glycerol,
and 1
mM
dithiothreitol
liquid
[Fluka]),
chromatography
TABLE
loaded
(FPLC)
onto
MonoQ
1. Physiological data, immunization
a fast protein
column
(Phar-
macia,
Uppsala,
Sweden),
and eluted with a 0-0.5 M
NaCl gradient
(10 ml) in buffer B. IgY appeared
in the
0.24 M NaCl
fraction.
All steps were done at room
temperature
except for MonoQ
FPLC,
which was run
at 4#{176}C.
The buffer
contained
no NaN3
in view
hibiting
effect
on
the
color
reaction
of
of its inthe
im-
munoblots.
To determine
tibodies,
we
the quantity
of specific
anti-PC
conjugated
purified
PCNA
to
activated
cording
Sepharose
4B (0.2 mg protein/mi
to the manufacturer’s
instructions
The column
(0.5 ml) was loaded with
cia).
from the PEG
purifiction
(5). Approximately
3.2%
against
PCNA.
Immunoblot
step
and
eluted
of the antibodies
NA anCNBr-
column)
ac(Pharma10 mg of IgY
as described
was specific
analysis
A partially
purified
PCNA fraction was electrophoresed
through
a 12% (wt/vol) sodium
dodecyl sulfate (SDS)polyacrylamide
gel
membrane
blocked
and
as described
with
5%
transferred
to a nitrocellulose
(see ref 15). The
(wt/vol)
instant
membrane
non-fat
milk
was
powder
and incubated
with the IgY fraction diluted 1:500 in Trisbuffered
saline (TBS,
10 mM Tris-HC1
[pH 7.5], and
0.15 M NaCl).
The
bit against
IgY and
a gift from
Sigma
anti-PC
from
B. Gottstein
(St. Louis,
NA IgG
R. Bravo,
secondary
coupled
(Zurich)
Mo.).
Protein
raised
in a rabphosphatase,
was
or was purchased
A positive
produced
and the anti-rabbit
Sigma.
antibody,
to alkaline
control
from
antibody,
an
in rabbits,
was provided
IgG, F(ab’)2 fragment,
detection
Bio-Rad proteinassay kit using
by
was
was performed
with the
bovine
IgG as a standard.
RESULTS
Immunization
scheme
and isolation
of antibodies
The PCNA that served as antigen was purified
to more
than 95% homogeneity,
as shown in Fig. 1 (lane 2). For
immunization,
different
quantities
of PCNA
were injected either twice or three times into six hens. The immunization
scheme is shown in Table 1. By differential
PEG
90%
precipitation,
purity
(Fig.
the IgY was purified
to better
than
1, lane 3). The antibodies
were fur-
scheme, and immune response of hens#{176}
1
2
3
4
5
6
Color
Brown
White
Brown
White
Brown
White
Weight, g
‘lweight of eggs, g
1790
50.4
1820
51.5
1720
48.2
1540
52.7
1690
57.2
1660
58.1
10
10
20
20
20
10
50
25
200
100
300
175
10
30
20
60
30
75
300
475
20
30
20
25
30
30
16
20
16
25
16
16
Hen (running
Antigen
number)
injected,
g
Day 0
Day 12
Day2O
Total
amount,
cg
Immune
response
First detected
at day
Plateau
level reached
#{176}The
hens were 22-24
ANTIBODIES
PRODUCED
at day
wk old and all laying
IN CHICKEN
before
EGG YOLK
-
-
-
-
immunization.
2529
Mi
2
-R
34
5
_
PCNA-
IiP-i
Figure 1. SDS-polyacrylamide
gel electrophoresis
of antigen and
IgY antibodies.
Lane M) Protein molecular weights: rabbit muscle
phosphorylase
b (97,400),
bovine serum albumin
(66,200),
hen egg
white ovalbumin
(42,700),
bovine carbonic
anhydrase
(31,000),
and
soybean
trypsin inhibitor
(21,500).
Lane 1) Partially purified
PCNA
fraction
(12 g) used as antigen
for immunoblots.
Lane 2) Purified
PCNA
(1.5 gig) used for hen immunization.
Lanes 3-5) IgY fractions (3.5 g) of hen 1(30 days after first injection)
purified
by PEG
precipitation
(Lane 3), DEAE-cellulose
chromatography
(Lane 4),
and MonoQ FPLC
SDS-polyacrylamide
Coomassie
Brilliant
munoglobulin
Large amounts
of antibodies
were obtained
from the egg
yolk by a quick purification
procedure.
All hens showed
an immune
response
independent
of the quantity
of antigen injected.
Hen 1, which was immunized
three times
with 10 tg antigen
at day 0, 12 and 20, showed an immune response after 20 days (Fig. 2A). This suggests that
even two injections of 10 tg antigen at day 0 and 12 might
be enough to induce the production
of specific IgY antibodies. Table 2 compares
the total amount
of antigen
used by different authors for immunization.
In this context, it can be seen that the quantity
of the antigen
can
be reduced 3- to 2600-fold, whereas the immune response
remains
unaffected.
Because we failed to produce antibodies against PCNA
and other highly conserved
mammalian
replication
proteins (e.g., DNA polymerases)
in rabbits (M. Gassmann
and F. Focher, unpublished
results),
we decided
to immunize chickens. The successful exploitation
of the avian
immune
system to produce antibodies
against highly con-
A
10 10
(Lane 5). Electrophoresis
was through a 12%
gel and the proteins
were stained
with
Blue. H, heavy chain; L, light chain of im-
10
vy
Y.
4,
ther purified by chromatography
MonoQ
(Fig. 1, lanes 4 and
Immunoblot
on DEAE-cellulose
5, respectively).
and
-
analysis
Yolk IgY from all six hens at day 0 (preimmune),
12, 16,
20, 25, 30, 40, 49, and 61 after the first immunization
was isolated
up to the PEG step (see Fig. 1, lane 3).
PC NA-specific
antibodies
were detected by reaction with
electrophoretically
separated
PC NA. An example appears
in Fig. 2, which shows the results obtained
with the antibodies
of hens I and 3, both immunized
with a total
of 30 tg PCNA (see Table 1). An immune
response
was
detected
in hen I after 20 days (Fig. 2A) and in hen 3
after 30 days (Fig. 2B). A plateau level was reached
after 30 days. We used a polyclonal
rabbit anti-PC NA IgG
as a positive
control
antibody.
The
time
course
as a positive
control,
the two
positive
(Fig.
3, lanes
1 and
PCNA
preparations
were
2).
DISCUSSION
We have immunized
six laying chickens
to produce
antibodies against a highly conserved
mammalian
protein.
Vol. 4
May
1990
I
B20
‘V
II
‘1
-
___
of the
immunoreaction
of hens 2, 4, 5, and 6 (data of the immunoblots
not shown) is summarized
in Table 1. No
differences
were detected between brown and white hens.
In addition,
immunoblot
analysis showed that IgY fractions purified by chromatography
on DEAE-cellulose
and
MonoQ
were equally
reactive
to partially
purified
or
purified
PCNA (compare
Fig. 3, lanes 3 and 4 or 5 and
6, respectively).
When a rabbit anti-PCNA
IgG was used
2530
H
day
0 12 16 20 25
Figure 2. Time course of anti-PCNA
30 40 49 61
IgY produced
in hens deter-
mined
by immunoblot
analysis.
Electrophoresis
of a partially
purifed
PCNA
was performed
in a 12% SDS-polyacrylamide
gel
and the proteins
were transferred
to a nitrocellulose
membrane
as
described
in Materials
and Methods.
Yolk antibodies
from hen 1
(A) and hen 3(B) were purified by PEG precipitation,
diluted
1:500
in Tris-buffered
saline, and applied
to the nitrocellulose
membrane
for immunoblot
analysis.
The arrows indicate
the time point of immunization
and the quantity
(sg protein)
of antigen
injected.
M:
Protein
molecular
weights
(see legend
of Fig. 1); IgG: rabbit antiPCNA
IgG was used as control
antibody.
The FASEB Journal
GASSMANN ET AL.
5 6
123:
PCNA-.’
-
-
Figure
3. Immunoblot
analysis of purified IgY fractions
and a control rabbit
anti-PCNA
IgG. Electrophoresis
of partially
purified
(lanes 1, 3, 5) and purified
PCNA
(lanes 2, 4, 6) were performed
in a 12% SDS-polyacrylamide
gel and the proteins
were transferred
to a nitrocellulose
membrane
as described
in Materials
and
Methods.
IgY from hen I was further
purified
by DEAE-cellulose
chromatography
(lanes 3 and 4) and MonoQ
FPLC (lanes 5 and 6),
and applied
to the nitrocellulose
membrane
for immunoblot
analysis. A rabbit
anti-PCNA
IgG was used as control
antibody
(lanes
I and 2).
served antigens
that are poorly immunogenic
in mammals has been demonstrated
recently
by other authors
(see Table 2). Yields of IgY were up to 72 mg per egg
at least
until
day
81 (data
not
shown)
when
we stopped
collecting
eggs. For instance,
we were able to isolate
from hen 1 a total of 4 g of IgY, from which 130 mg was
specific against
PCNA
(data not shown).
During
this
period,
the titer of specific antibodies
remained
constantly
high (Fig.
2). This
long-lasting
titer of IgY
without
booster
injections
agrees with the results
of
L#{246}sch
et al. (8). It might be due to mycobacteria
contained
in complete
Freund’s
adjuvant
that lead to development
of granulomes.
Emulsified
antigens
are enclosed in those granulomes
and released
permanently
TABLE
2. Quantity of antigens used by dfferent
authors of immunization
during
a long period
(23-25).
In addition,
the therapeutic
use of egg yolk antibodies
has been proposed
for oral passive
immunization
against
intestinal
diseases of weaning
piglets (23), showing
another
application of the method.
IgY antibodies
do not bind
to protein
A from
Staphylococcus aureus or to streptococcal
protein
G (26).
Nevertheless,
immunoprecipitation
of IgY-antigen
complex with PEG has been described
(12). Because
the
purification
of IgY by affinity
chromatography
with
protein
A is not possible,
other procedures
to extract
avian antibodies
from the egg yolk have been described
(8, 17, 18). The simple PEG precipitation
yields a con-
centrated
IgY preparation,
munoblot
FPLC,
analysis
the IgY are nearly
(M. Gassmann,
eases
of young
1-2 x 1010 cells
3-5 mg
3-5 mg
4 mg
3 mg
6 mg
24 mg’
0.4 mg
amount
PRODUCED
IN CHICKEN
After
unpublished
observations)
animals
(23) or humans
injected’
EGG YOLK
MonoQ
(Fig.
I).
as seen in
(27).
Reference
8
16
16
17
13
18
19
5
0.03 mg
40-80
mg
each 24 mg’
0.09 mgd
0.2 mg
0.1 mg
0.13 mg
0.5 mg
‘Boosters after 45 days are not considered.
‘Immunizations
without the addition
Freund’s adjuvant
was used.
‘Estimated
amount; different antigens were excised
were found after 3 booster injections,
5 months after first immunization.
ANTIBODIES
2).
homogeneous
of laying hens
Brucella abortush
Baaeroides
ruminicola
Immature
protein
of tomato
tubers
Rabbit
IgG
Bovine
RNA polymerase
II
Bovine
proliferating
cell
nuclear
antigen
(PCNA)
Human
serum
albumin6
Human
IgG and 1gM
Human
insulin
receptor
(a subunit)
Human
plasma
kallikrein
Human
parathyroid
hormone
Prostaglandins’1
1,25-dihydroxyvitamin
D’
can be used for im-
rabbits
or other mammals,
and 2) collecting
eggs, in
contrast
to bleeding
animals,
is noninvasive.
In conclusion,
we propose
egg yolk as a convenient
source of polyclonal
antibodies
because:
1) the quantity
of antigen
needed
for an efficient
immune
response
is
much lower than in rabbits
(as an example);
2)
birds
produce
antibodies
against
highly
conserved
mammalian antigens;
3) the use of complete
Freund’s
adjuvant leads to very high and long-lasting
titers of yolk
antibodies,
starting
as early as 16 days after the first immunization
(Table 1); 4) the purification
of antibodies
is simple and quick, and a purity of 90% is easily attained by PEG precipitation;
5)
chickens
are inexpensive to keep and easy to handle; and 6) chicken antibodies are acid- and heat-resistant
(8), and therefore
might
be orally applied
to prevent
or to cure intestinal
dis-
Total
Toxoplasma gondii
Echinococcus granulosus
(Fig.
With regard
to animal
protection
regulations,
the
immunization
of chickens
offers two advantages:
1) the
immunization
with complete
Freund’s
adjuvant
is well
tolerated
and produces
no local inflammatory
reaction
Antigen
(B,4)
Selenomonas ruminantium (D)
which
directly
of complete Freund’s
from a dried gel.
This
adjuvant are not included.
‘Carrier-coupled
hapten.
paper
8
19
12
6
20
21
22
‘Incomplete
1High titers
2531
We are indebted
to B. Gottstein
for advice regarding
immunization of the hens and for the rabbit anti-chicken
antibody.
We also
thank
R. Bravo for providing
the rabbit
anti-PCNA
control
antibody, W. Jeltsch,
E. Ferrari,
and M. Hottiger
for technical
assistance,
and C. C. Kuenzle
for critical comments
and careful reading of the manuscript.
This work has been supported
by the Swiss
National
Science
Foundation
(grant 3.630-0.87)
and by the Krebsliga of the Kanton
Zurich.
A grant-in-aid
was provided
by the
Roche Research
Foundation
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The FASEB Journal
Received for publication January 17, 1990.
Accepted for publication February 23, 1990.
GASSMANN
FT AL.