Journal of Immunological Methods 296 (2005) 199 – 209

www.elsevier.com/locate/jim

Research paper

Microencapsulation for the gastric passage and controlled

intestinal release of immunoglobulin Y
Jennifer Kovacs-Nolan, Yoshinori Mine*
Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1
Received 11 August 2004; received in revised form 12 November 2004; accepted 16 November 2004
Available online 16 December 2004

Abstract

Avian immunoglobulin (Ig) Y is a promising alternative for the treatment and prevention of enteric infections, and has
shown to be effective against a number of gastrointestinal pathogens, including Escherichia coli, Salmonella, and rotavirus.
However, its application is limited by its sensitivity to human gastrointestinal conditions. Here, we report on the enteric coating
of IgY-containing granules with a pH-sensitive methacrylic acid copolymer. A 35% (w/w) polymer coating was found to protect
IgY from gastric inactivation in vitro, maintaining greater than 95% activity after 6 h in simulated gastric fluid. The IgY was
slowly released from the microencapsulated granules upon exposure to simulated intestinal fluid, and retained 80% activity after
8 h exposure to pancreatic enzymes. In vivo, using pigs as a model of human digestion, the encapsulated IgY retained
significantly more activity than non-encapsulated IgY, indicating that microencapsulation with a methacrylic acid copolymer
may be an effective method of protecting IgY from gastrointestinal inactivation, enabling its use for oral passive
immunotherapy.
D 2004 Elsevier B.V. All rights reserved.

Keywords: Immunoglobulin Y; Gastro-resistance; Methacrylic acid copolymer; Human rotavirus; VP8; Passive immunotherapy

1. Introduction

Passive immunization may be one of the most

Abbreviations: BSA, bovine serum albumin; ELISA, enzyme-
linked immunosorbent assay; GI, gastrointestinal; HRP, horseradish
peroxidase; HRV, human rotavirus; Ig, immunoglobulin; MWCO,
molecular weight cut-off; PBS, phosphate-buffered saline; PEG,
polyethylene glycol; SGF, simulated gastric fluid; SIF, simulated
intestinal fluid; TBS, Tris-buffered saline; TBST, Tris-buffered
saline containing Tween-20.
* Corresponding author. Tel.: +1 519 824 4120x52901; fax: +1
519 824 6631.
E-mail address: ymine@uoguelph.ca (Y. Mine).
0022-1759/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.jim.2004.11.017
valuable applications of antibodies (Sim et al., 2000).
Oral immunotherapy, using pre-formed pathogen-
specific antibodies, has been examined as a method
of establishing passive immunity against enteric
pathogens (Carlander et al., 2000), and has the
advantage of reduced cost, ease of administration,
and the potential to treat localized conditions in the
gastrointestinal tract (Reilly et al., 1997). However, in
order to be effective, the antibodies must survive the
200 J. Kovacs-Nolan, Y. Mine / Journal of Immunological Methods 296 (2005) 199–209
gastrointestinal environment and reach their target
areas with their biological properties intact (Bogstedt
et al., 1997).
Avian immunoglobulin (Ig) Y, the functional
equivalent of IgG found in mammals, is ideal for
passive immunotherapy applications, as it may be
readily obtained in large quantities from egg yolk, and
presents a more cost-effective, convenient, and
hygienic alternative to mammalian antibodies (Car-
lander et al., 2000). IgY has been shown to be
effective against a number of enteric pathogens,
including Escherichia coli, Salmonella spp., and
rotavirus, by preventing infection and inhibiting
growth in vitro, and preventing or reducing symptoms
in animal models (Kovacs-Nolan and Mine, 2004).
However, to maximize the potential of IgY for
immunotherapeutic use in humans would require
protection of the IgY from acidic and enzymatic
degradation in the stomach, in order to reach the small
intestine, the site of infection, intact.
While the survival of IgG has been reported in
individuals fed antibodies of human or bovine origin,
IgY is a much less stable molecule, owing to its higher
molecular weight (180 kDa), lower amount of h-sheet
structure, and reduced flexibility (Warr et al., 1995;
Shimizu et al., 1992), and therefore may be more
susceptible to degradation in the gastrointestinal tract.
Although the stability of both immunoglobulins
has been found to be similar when subjected to
alkaline conditions, IgY is denatured much more
readily than IgG in acidic conditions (Shimizu et al.,
1992, 1993a). IgY has been found to be relatively
resistant to trypsin and chymotrypsin digestion, but
sensitive to pepsin digestion. However, it is more
susceptible to pepsin, trypsin, and chymotrypsin
digestion than IgG (Shimizu et al., 1993b). Hatta et
al. (1993) found that digestion of IgY with pepsin at
pH 2 resulted in complete hydrolysis of the antibody,
with a corresponding loss in activity, but activity
remained even after 8 h incubation with trypsin or
chymotrypsin. Therefore, an effective method of
protecting the IgY from pepsin degradation is
necessary for the successful application of IgY to
treat and prevent enteric infections.
The microencapsulation of IgY using liposomes
(Shimizu et al., 1993b) and multiple emulsions
(Shimizu and Nakane, 1995) have been previously
described; however, these have shown limited effec-
tiveness, in some cases destroying antibody activity by
the encapsulation procedure itself. The macroencap-
sulation of IgY, using enteric-coated gelatin capsules,
has also been examined, and was found to significantly
improve antibody stability (Akita and Nakai, 2000).
The anionic methacrylic acid–ethyl acrylate
copolymer, EudragitR L100-55, is a commonly used
pharmaceutical coating. It is insoluble in acid medium,
but will begin to dissolve above pH 5.5. Coating with
this enteric polymer results in products which are
resistant to gastric conditions, and will dissolve readily
in the neutral to slightly alkaline environment of the
small intestine (Lehmann et al., 1999).
Previously, we reported on the production of IgY
against the recombinant VP8 subunit of the human
rotavirus (HRV) (Kovacs-Nolan et al., 2001). HRV is
the major etiologic agent of acute infantile gastro-
enteritis, infecting up to 90% of children under the age
of three (Kapikian and Chanock, 1996), and is
characteristically localized to the epithelial cells of
the gastrointestinal tract (Kapikian and Chanock,
1996; Clark et al., 1999). Currently no effective
vaccine exists. Passive immunization, to prevent and
treat rotavirus gastroenteritis, would require the
delivery of virus-neutralizing antibodies to the small
intestine, the site of viral replication.
Here, we describe the microencapsulation of anti-
VP8 IgY using an anionic methacrylic acid–ethyl
acrylate copolymer (EudragitR L100-55), and the
characterization and evaluation of the protective
efficacy of the enteric polymer coating under gastro-
intestinal conditions.
2. Materials and methods
2.1. IgY production and separation
Polyclonal antibodies (IgY) against recombinant
HRV subunit protein, VP8, were produced in chick-
ens, as previously described (Kovacs-Nolan et al.,
2001). Crude IgY was separated from the yolks of
immunized hens using a modification of the method
of Akita and Nakai (1992). Briefly, egg yolks were
separated from the egg white, diluted 1:4 with
distilled water (which had been acidified with 0.1 M
HCl to give a final pH of 5–5.2 after dilution), and
kept at
20 8C for 48 h. The frozen yolk solutions
 J. Kovacs-Nolan, Y. Mine / Journal of Immunological Methods 296 (2005) 199–209
were thawed at 4 8C, and centrifuged at 12 000g, for
1 h at 10 8C, to precipitate lipoproteins (Jensenius et
al., 1981). The resulting supernatant was then filtered
and lyophilized.
2.2. Preparation of IgY granules
IgY granules were prepared by Snow Milk, Tokyo,
Japan. The crude IgY was first blended with corn
starch (National Starch, Tokyo, Japan), at a ratio of 1
part IgY to 4 parts starch. Granules ranging in size
from 2 to 3 mm in diameter were formed using a
Yokomizo Granular (Model FR160 60, Tokyo,
Japan), at a temperature of 32 8C, humidity of 78%,
and rotation time of 5 min.
2.3. Coating of the IgY granules with EudragitR
L100-55
The IgY granules were first sealed with a 5% (w/
w) coating of PEG 6000 (USP, Fluka Chemie GmbH,
Buchs, Switzerland), applied by immersing the
granules in a 10% (w/v) aqueous solution of PEG
6000, followed by air drying at 37 8C.
A 15% (w/v) aqueous polymer dispersion was
prepared according to the manufacturer’s instructions.
In short, EudragitR L100-55 (Rfhm GmbH, Darm-
stadt, Germany) powder was added slowly to the
appropriate volume of water, at room temperature,
with stirring, and 1 M NaOH was added to the polymer
solution to partially neutralize 5–15% of the carboxyl
groups. The PEG-coated granules were coated with the
polymer solution by repeated immersion in the 15%
polymer dispersion, followed by air drying at 37 8C, to
give final polymer coatings of 5%, 10%, 15%, 20%,
25%, 30%, 35%, and 40% (w/w).
2.4. In vitro stability of IgY to simulated gastric
conditions
The stability of the encapsulated IgY to gastric
conditions was evaluated using simulated gastric
fluid (SGF). SGF was prepared as described in the
United States Pharmacopoeia (United States Pharma-
copeial Convention Council of Experts, 2004a), and
consisted of 3.2 mg/mL pepsin (Sigma Chemical, St.
Louis, MO) in 0.03 M NaCl, at pH 1.2. The SGF
was added to the IgY to give an enzyme to substrate
201
ratio of 1:250 (Eggum and Boisen, 1991), and
incubated, with shaking, at 37 8C. At intervals of
0, 0.5, 1, 2, 3, 4 and 6 h, samples were neutralized
with 0.1 M sodium carbonate buffer, pH 9.6, and the
antibody activity was assessed by ELISA. Activities
were expressed as a percent relative to an untreated
control sample.
2.5. In vitro stability of IgY to simulated intestinal
conditions
Simulated intestinal fluid (SIF) was prepared as
described in the United States Pharmacopoeia (United
States Pharmacopeial Convention Council of Experts,
2004b), consisting of 10 mg/mL pancreatin (USP,
Sigma Chemical) in 0.05 M KH2PO4, at pH 6.8, and
was used to simulate small intestinal conditions. The
encapsulated IgY was first incubated in SGF for 2 h.
The granules were then filtered and suspended in 0.05
M KH2PO4, pH 6.8, and SIF was added to give an
enzyme to substrate ratio of 1:50 (Mouecoucou et al.,
2004). Samples were incubated, with shaking, at 37
8C. At intervals of 0, 0.5, 1, 2, 3, 4, 6, and 8 h,
samples were removed and placed on ice to stop the
reaction. Non-encapsulated IgY was incubated in SIF
only. The antibody activity was assessed by ELISA.
Activities were expressed as a percent relative to an
untreated control sample.
2.6. In vivo gastrointestinal stability of IgY
Six 28-day-old weaned Yorkshire pigs (weighing
6–7 kg) were obtained from Arkell Research Station,
Swine Unit (University of Guelph, Guelph, Ontario),
and were kept in accordance with the Canadian
Council on Animal Care Guide to the Care and Use
of Experimental Animals. Water and food were
provided ad libitum throughout the experiment, in
order to maintain normal digestive functions.
Pigs were divided into two groups of three pigs
each. Control pigs were each given 250 mg of non-
encapsulated IgY, suspended in 30 mL of 0.05 M
citrate buffer, pH 3.7, administered by oral gavage,
using a 10-mL syringe attached to a length of tubing.
Test pigs were given 2 g of encapsulated IgY granules
(equivalent to 250 mg of IgY), flushed down the
tubing with 30 mL of 0.05 M citrate buffer, pH 3.7.
After 3 h, the pigs were euthanized, and necropsy was
202 J. Kovacs-Nolan, Y. Mine / Journal of Immunological Methods 296 (2005) 199–209
immediately carried out to collect tissue samples from
the small intestine (duodenum, jejunum, and ileum)
and large intestine, as well as stomach contents.
Tissue samples were added to 1–2 volumes of PBS,
containing Completek protease inhibitor cocktail
tablets (Roche Diagnostics GmbH, Mannheim, Ger-
many), and kept on ice. Stomach contents were added
to 3–6 volumes of protease inhibitor-containing PBS,
to neutralize stomach acid.
Samples were homogenized using a PolytronR PT
2000 homogenizer (Kinematica AG, Littau-Luzern,
Switzerland) at 15 000 rpm, and centrifuged at
5500g for 10 min at 4 8C. Supernatants were passed
through a 1.0-Am glass fiber filter to remove
particulate matter, and were then concentrated to 1/5
of their original volume by ultrafiltration using a 50-
kDa MWCO cellulose membrane (Millipore, Bedford,
MA). IgY activity determination and IgY quantifica-
tion were done by ELISA, using standard curves of
anti-VP8 IgY and pure IgY, respectively.
2.7. In vitro IgY release
The release of IgY from the encapsulated granules
was studied by incubating 100 mg of granules in 1 mL
SGF, at 37 8C, with shaking. After 2 h, the granules
were filtered and transferred to 1 mL of 0.05 M
KH2PO4, pH 6.8, and incubated at 37 8C, with
shaking. At 0, 0.5, 1, 2, 3, 4, 6, and 8 h, 50-AL
aliquots were removed and assayed for protein
concentration using the DC Assay (BioRad, Hercules,
CA). The amount of protein released was expressed as
a percent relative to the total protein in the sample.
2.8. IgY ELISA
To assess IgY activity, 96-well microwell plates
(Corning Costar, Cambridge, MA) were coated with
0.25 Ag/well of purified recombinant HRV subunit
protein, VP8, in 0.1 M sodium carbonate buffer, pH
9.6, and incubated at 4 8C overnight. The plate was
washed with Tris-buffered saline (TBS) containing
0.05% (v/v) Tween-20 (TBST), and blocked with 150
AL/well of 2% (w/v) BSA (Fisher Scientific, Fair
Lawn, NJ) in 0.1 M sodium carbonate buffer, at 37 8C
for 2 h, with shaking. The plate was then washed with
TBST, and incubated at 37 8C, for 1 h, with 100 AL/
well of the IgY solution to be tested. The plate was
washed again, and 100 AL/well of horseradish pero-
xidase (HRP)-conjugated anti-chicken IgG (Sigma
Chemical), diluted 5000 in TBS containing 1% (w/
v) BSA, was added and incubated for 1 h, at 37 8C.
The plate was washed and developed using 100 AL/
well of 3,3V,5,5V-tetramethylbenzidine substrate sol-
ution (TMB) (Sigma Chemical). The reaction was
stopped by the addition of 1 M H2SO4 (100 AL/well),
and the absorbance at 450 nm was read using a BioRad
model 550 plate reader.
For IgY quantification, plates were coated with 0.01
Ag/well of rabbit anti-chicken IgY (Jackson Immu-
noResearch Laboratories, West Grove, PA) in 0.1 M
sodium carbonate buffer, pH 8.6. A standard curve was
prepared using pure chicken IgY (Jackson ImmunoR-
esearch Laboratories), at concentrations from 0 to 32
ng IgY/mL in TBS containing 1% (w/v) BSA.
Detection was carried out as described above.
2.9. SDS–PAGE
Sodium dodecyl sulfate polyacrylamide gel elec-
trophoresis (SDS–PAGE) was performed according to
the method of Laemmli (1970). Samples were
prepared under non-reducing conditions, and resolved
using 10% acrylamide.
2.10. Statistical analysis
Statistical analyses were performed using SPSS
7.5.2 for Windows, and statistical significance was
determined using a paired-samples t-test.
3. Results
Initial coating experiments were carried out to
optimize EudragitR L100-55 concentration and coat-
ing conditions for the anti-VP8 IgY granules. A first
coat of PEG 6000, to seal the granules before polymer
application, was found to be necessary to improve the
resistance of the coated granules to gastric conditions.
Polymer coatings of 5–40% (w/w) were examined for
their ability to confer gastric stability to the IgY, and
the results can be seen in Fig. 1. Initial samples were
screened for IgY activity after 1 h in simulated gastric
fluid (SGF). Antibody activity was defined as the
ability of the anti-VP8 IgY to bind to recombinant
 J. Kovacs-Nolan, Y. Mine / Journal of Immunological Methods 296 (2005) 199–209
100
90
80
Relative activity (%)
70
60
50
40
30
20
10
0
PEG
Fig. 1. Optimization of methacrylic acid copolymer coating concentration. The ability of the different coating concentrations to protect anti-VP8
IgY against gastric inactivation was assessed by incubating samples for 1 h in SGF. Antibody activity was measured by ELISA, and expressed as
% activity relative to an untreated sample. Data are presented as meansFstandard errors (n=2).
VP8, and was measured by an antibody capture
ELISA. The relative activities were expressed as a
percentage of an untreated, positive control, sample
(100% activity). A decrease in absorbance, as a result
of decreased antibody binding, was attributed to a loss
of antibody activity.
A minimum 35% (w/w) polymer coating was
required for stability to gastric conditions, resulting
in greater than 95% retention of IgY activity after 1 h
in SGF. This polymer concentration was chosen for all
of the subsequent IgY microencapsulation experi-
ments. The microencapsulated IgY was tested in
parallel with non-encapsulated IgY to determine
efficacy of the polymer coating under each of the
conditions being tested.
Gastro-resistance, or stability of the IgY under
simulated gastric conditions, was assessed by incubat-
ing the IgY in SGF for 6 h. At each time interval, the
samples were neutralized to inhibit pepsin activity,
and IgY activity remaining was determined by ELISA
(Fig. 2A). After 30 min of SGF exposure, the non-
encapsulated IgY demonstrated a significant decrease
(86%) in IgY activity, which rapidly decreased to zero
following continued incubation. The microencapsu-
lated IgY maintained greater than 95% activity after 6
h in SGF. The corresponding intact IgY was visualized
by SDS–PAGE, and can be seen as a band around 180
kDa (Fig. 2B). As this was a crude IgY preparation,
additional smaller proteins present in the water soluble
fraction, including other livetins, can also be seen.
203
5% 10% 15% 20% 25% 30% 35% 40%
% L100-55 (w/w)
The stability of the IgY to small intestinal conditions
was examined using simulated intestinal fluid (SIF).
IgY samples were first incubated in SGF, and then SIF,
and monitored for antibody activity over a period of 8
h. Non-encapsulated IgY was incubated only in SIF,
with no prior treatment in SGF. The percent of IgY
activity remaining after treatment was determined by
ELISA (Fig. 3A). IgY activity in both non-encapsu-
lated and encapsulated samples was not significantly
affected ( pb0.005) by incubation in SIF, retaining
around 90% and 80% antibody activity, respectively,
after 8 h. The microencapsulated IgY did not differ
significantly ( pb0.005) from the non-encapsulated IgY
in its susceptibility to protease digestion by trypsin and
chymotrypsin, as both samples retained similar levels
of activity after 8 h of SIF exposure. The intact IgY was
visualized by SDS–PAGE (Fig. 3B), and can be seen as
a band around 180 kDa.
The in vivo stability of the IgY was examined by
feeding microencapsulated and non-encapsulated IgY
to weaned pigs, and observing the progress of the IgY
through the gastrointestinal tract. Three hours after
feeding of the IgY, samples were taken from several
sites along the gastrointestinal tract. The pH of the
samples was immediately measured following tissue
collection. The pH of the small and large intestine
samples ranged from 6 to 7; the stomach pH was
around 2, and sufficient PBS was added to adjust to
neutral pH. The IgY was extracted from the tissue
samples, and IgY activity was determined by an
204 J. Kovacs-Nolan, Y. Mine / Journal of Immunological Methods 296 (2005) 199–209

A
100

Relative activity (%)

80

60

40

20

0
0 1 2 3 4 5 6
Time (hours)
B
kDa
205
IgY
116
97
84
66
55
45
36

mwm IgY 0h 1h 2h 3h 4h 6h
std

Fig. 2. In vitro stability of microencapsulated (4) and non-encapsulated (5) anti-VP8 IgY to simulated gastric conditions. (A) IgY activity
remaining over 6 h incubation in SGF was measured by ELISA, and expressed as % activity relative to an untreated sample. Data are presented
as meansFstandard errors (n=2). (B) Intact IgY was visualized by SDS–PAGE.

antibody capture ELISA. Because the IgY activity
observed in the samples from the gastrointestinal tract
could not be expressed as a percentage relative to the
initial dose administered, the samples were instead
compared to a standard anti-VP8 IgY curve, of known
dilutions, and reported as relative amounts of active
antibody, as determined from the curve. Throughout
the gastrointestinal tract, significantly more activity
( pb0.05) was retained by the microencapsulated IgY
than the non-encapsulated IgY, which retained very
little activity (Fig. 4). The highest IgY activity
appeared to be in the stomach, and, to a lesser extent,
in the jejunum and ileum.
The in vivo gastrointestinal distribution, or transit,
of IgY in the gastrointestinal tract was visualized by
quantifying the IgY in each of the regions of the
gastrointestinal tract. The results were expressed as ng
IgY per gram of tissue sample (Fig. 4), and indicated
that a large amount of the IgY was still present in the
stomach, although a small portion appeared to have
traveled distally to the jejunum and ileum.
In the case of the microencapsulated sample, there
appeared to be a positive correlation (r=0.996)
between the IgY activity and the amount of IgY in
the different regions of the gastrointestinal tract. In
contrast, the amount of non-encapsulated IgY in the
gastrointestinal tract did not correlate as well with IgY
activity (r=0.615), showing high quantities of IgY yet
low IgY activity, suggesting that although the anti-
bodies were present, they had lost their activity.
The sustained release characteristics of the encap-
sulated IgY granules were examined by incubating the
encapsulated IgY in SGF and subsequently in SIF, and
measuring the percent of protein released over time
 J. Kovacs-Nolan, Y. Mine / Journal of Immunological Methods 296 (2005) 199–209 205

A
100

Relative activity (%)

80

60

40

20

0
0 0.5 1 2 3 4 5 6 8
Time (hours)
B
kDa
205
IgY

116
97
84
66

55
45
36

mwm IgY 0h 1h 2h 4h 6h 8h
std

Fig. 3. In vitro stability of microencapsulated ( ) and non-encapsulated (5) anti-VP8 IgY to simulated intestinal conditions. (A) IgY activity
remaining over 8 h incubation in SIF was measured by ELISA, and expressed as % activity relative to an untreated sample. Data are presented
as meansFstandard errors (n=2). (B) Intact IgY was visualized by SDS–PAGE.

(Fig. 5). After transfer to SIF, the granules began to
disintegrate, releasing over 80% of protein within 2 h of
incubation in SIF. The granules continued to disinte-
grate, reaching 100% release after 4 h incubation.
4. Discussion
Ingested proteins are typically denatured by the
acidic pH of the stomach, and degraded by the
proteolytic enzymes present in the stomach and small
intestine (Reilly et al., 1997), thereby rendering
difficult the use of orally administered antibodies for
the passive immunotherapy of enteric infections.
Studies of the passage of IgG, derived from bovine
colostrum, through the gastrointestinal tract of
humans have demonstrated only 4–19% survival of
antibody activity (Hilpert et al., 1987; Roos et al.,
1995; Pacyna et al., 2001). Bogstedt et al. (1997)
reported the detection of only a minute amount of
antibodies, both IgG and IgY, following digestion.
And Kelly et al. (1997), who investigated the survival
of bovine antibodies against Clostridium difficile
toxin, observed that loss of antibody activity follow-
206 J. Kovacs-Nolan, Y. Mine / Journal of Immunological Methods 296 (2005) 199–209

4.50 45

4.00 40

IgY concentration (ng/g)

3.50 35
Relative activity (x10-4)

3.00 30

2.50 25

2.50 20

1.50 15

1.00 10

0.50 5

0.00 0
stomach duodenum jejunum ileum lg intestine
Region of GI tract
Fig. 4. In vivo stability of microencapsulated ( ) and non-encapsulated (5) anti-VP8 IgY. Anti-VP8 IgY activity in each region of the
gastrointestinal tract was assessed by ELISA, and expressed as the relative amount of IgY retaining activity. The amount of microencapsulated
(.) and non-encapsulated (E) IgY was determined by IgY quantification ELISA. Data are presented as meansFstandard errors (n=3).

ing passage through the human gastrointestinal tract stomach, and enable it to be released in the small
corresponded to a loss of toxin-neutralizing activity. intestine, the site of viral replication.
In order to passively prevent or treat enteric Pepsin, which is optimally active in acidic con-
infections, such as that caused by human rotavirus, ditions ranging from pH 1 to 3.5, is the predominant
the IgY must resist degradation in the stomach, and digestive enzyme in the stomach (Castro, 1981; Reilly
reach the small intestine with its activity intact. An et al., 1997). Transit time through the stomach is
effective method of IgY microencapsulation would highly variable, ranging from 0.5 to 4.5 h (Davis,
protect the IgY from the digestive conditions of the 1989a; Madsen, 1994) with an average time of 1 to

SGF SIF

100
90
Percent release (%)

80
70
60
50
40
30
20
10
0
0 1 2 3 4 5 6 7 8
Time (hours)
Fig. 5. Dissolution of encapsulated anti-VP8 IgY granules. Granules were first incubated in SGF, and then transferred to SIF, and the amount of
protein released over time was measured. Data are presented as meansFstandard errors (n=2).
 J. Kovacs-Nolan, Y. Mine / Journal of Immunological Methods 296 (2005) 199–209
1.5 h (Fallingborg et al., 1990; Madsen, 1994). The
activity of pepsin is terminated when the stomach
contents are mixed with alkaline pancreatic juice in
the small intestine. The principal enzymes present in
the small intestine are trypsin, chymotrypsin, elastase,
and carboxypeptidase (Castro, 1981). Small intestinal
transit times typically range from 2 to 6 h, but it may
be as short as 1 h (Davis, 1989b).
A simulated gastric solution, mimicking conditions
in the stomach, was used to assess the stability of IgY
encapsulated with a pH-sensitive methacrylic acid
copolymer (EudragitR L100-55) in vitro. Similar to
previous reports (Hatta et al., 1993), the non-
encapsulated IgY was extremely sensitive to gastric
conditions, and was rapidly inactivated. We have
demonstrated, however, that a 35% (w/w) enteric
polymer coating resulted in the retention of greater
than 95% of IgY activity after 6 h incubation in SGF,
which is longer than the typical observed gastric
transit time of humans.
Once the microencapsulated IgY was exposed to
the simulated intestinal conditions (pHN5.5), the
polymer coating gradually disintegrated, releasing
the IgY from the granules. The sustained release of
the IgY from the microencapsulated granules would
ensure that, in vivo, the antibodies would be
distributed slowly throughout the small intestine
during transit, available to bind and neutralize the
target organism. Here, over 50% was released after the
first hour, and 100% release from the encapsulated
granules occurred between 3 and 4 h. The dissolution
profile of the granules, however, may be further
modified, or delayed, by additional polymer coatings,
possessing different pH-release characteristics tailored
to the increasingly alkaline pH of the small intestine.
Furthermore, it should be noted that the disintegration
times of polymer films are often somewhat longer in
vivo than in vitro (Lehmann et al., 1999).
To more accurately simulate gastrointestinal con-
ditions, the encapsulated IgY was first exposed to
SGF, before incubation with pancreatic enzymes in
the SIF. The non-encapsulated IgY was, however,
exposed only to the SIF, as it would have been
degraded completely in the SGF, making it impossible
to assess the effects of the pancreatic enzymes. As the
polymer coating disintegrated in the SIF, the IgY was
brought into direct contact with the pancreatic
enzymes. As expected, the simulated intestinal con-
207
ditions had little effect on IgY activity, indicating that
the IgY remained unaffected by the digestive enzymes
of the small intestine. As well, the activity of the
encapsulated and non-encapsulated IgY did not differ
significantly, suggesting that the structure of the IgY
was not altered by encapsulation, or by the prior
exposure to SGF.
Several studies have been carried out to examine
the survival of immunoglobulins of human or bovine
origin through the gastrointestinal tract (Bogstedt et
al., 1997); however, less is known about the gastro-
intestinal fate of IgY. In order to assess the stability of
the microencapsulated IgY in vitro, under physiolog-
ical conditions, an animal model was used.
The anatomy, physiology, and biochemistry of the
gastrointestinal tract differ considerably among mam-
malian species. A number of physiological factors can
modify dissolution rates and solubility, and transit
times can be significantly different between species
due to different dimensions and propulsive activities
of the gastrointestinal tract. It is imperative, then, to
select the correct animal model to study gastro-
intestinal function in the human (Kararli, 1995). Pigs
are considered the best models for the study of
digestion in humans, due to similar morphology and
physiology of the gastrointestinal systems (Miller and
Ullrey, 1987; Rowan et al., 1994), and so were
utilized here to study the gastric stability of IgY.
The microencapsulated IgY retained significantly
more activity than did the non-encapsulated IgY after
3 h of digestion, indicating that the polymer is
capable of protecting IgY from degradation under
physiological digestive conditions. The decrease in
activity of the non-encapsulated IgY following in
vivo digestion is in agreement with the results of
Yokoyama et al. (1993), who found that antibody
activity decreased several fold after passage through
the stomach of pigs.
A greater amount of non-encapsulated IgY
appeared to be present in the distal portion of the
gastrointestinal tract, which may be due to the
sustained release characteristics of the microencapsu-
lated sample. However, despite the larger quantities of
non-encapsulated IgY detected in the intestines, and
in contrast to the observations for the microencapsu-
lated antibodies, its activity was disproportionately
lower, indicating that it had lost activity upon passage
through the stomach.
208 J. Kovacs-Nolan, Y. Mine / Journal of Immunological Methods 296 (2005) 199–209
The results demonstrate that the microencapsula-
tion of IgY with an a pH-sensitive methacrylic acid
copolymer (EudragitR L100-55) can successfully
protect the antibodies from gastric inactivation, and
may provide an effective means of controlling HRV
infection, as well as having significant implications
for passive immunization techniques in general, for
the widespread prevention of enteric pathogens.
Acknowledgements
This work was supported by the Natural Sciences
and Engineering Research Council of Canada
(NSERC), the Ontario Egg Producers Marketing
Board, Agriculture and Agri-Food Canada, and the
Ontario Ministry of Agriculture and Food (OMAF).
We would like to thank Dr. Y. Takada, Snow Brand
Milk, Tokyo, Japan, for his technical support with the
IgY granulation, and the staff at the Central Animal
Facility, Animal Care Services, University of Guelph,
for their assistance with the in vivo experiments.
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