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All content in this area was uploaded by Leonardo Guevarra Jr. on May 11, 2017
Content may be subject to copyright.
Immunoglobulin Y and Detection of Hepatitis B Surface Antigen
1
VOL. 46 NO. 1 2012 ACTA MEDICA PHILIPPINA
VOL. 43 N0. 4 2009 ACTA MEDICA PHILIPPINA 13
Heart Failure and Short Stature in a 43 year-old male
transferred to the intensive care unit (ICU) for ventilatory
support and closer monitoring. On bedside cardiac ultrasound,
there was a nding of eccentric left ventricular hypertrophy,
global hypokinesia with depressed overall systolic function
with concomitant spontaneous echo contrast on left ventricular
(LV) cavity suggestive of rheologic stasis, the ejection fraction
was 25%, with moderate mitral regurgitation, moderate
aortic regurgitation with aortic sclerosis, severe tricuspid
regurgitation with mild pulmonary hypertension, pulmonary
regurgitation, and minimal pericardial effusion or pericardial
fat pad. Cardiac enzymes were not consistent with an acute
coronary event (Table 3), however, intravenous (IV) heparin
(overlapping with oral warfarin) was still given to cover for
the presence of a possible LV thrombus as demonstrated
by rheologic stasis on cardiac ultrasound. Medications
were shifted to IV diuretics and inotropes; oral digoxin was
started. IV antibiotics were given for possible pulmonary
infection. The patient later on showed improvement, and was
eventually weaned off from ventilatory support, extubated,
Table 1. Initial Laboratory Results
WBC
RBC
HGB
HCT
MCV
MCH
MCHC
RDW
PLT
RETIC
SEG
LYMPH
MONO
EO
BASO
BLAST
CBC
Reference
Value
5-10
4-6
120-150
0.38-0.48
80-100 FL
27-31 PG
320-360 G/L
11.5-15.5%
200-400
0.005-0.015
50-70%
20-44%
2-9%
0-4%
0-2%
0%
Result
4.5
90
0.27
Inc
48
50
2
0
0
0
RBS
HGBA1C
BUN
CREA
ALB
TAG
HDL
LDL
TOTAL CHOL
AST
ALT
Alk po4
NA
K
CL
CA++
P
MG++
Reference
Value
3.9-6.1
4.27-6.07
2.6-6.4
53-115
34-50
0.34-1.7
0.91-1.56
1.1-3.8
4.2-5.2
15-37
30-65
140-148
3.6-5.2
100-108
2.12-2.52
0.74-1
Result
6.3
6.4
5.0
123
32
0.82
0.67
4.21
5.25
95
91
184
136.9
3.35
86
2.37
2.27
0.83
Blood chem.
Color
Transp
Sp Gravity
pH
Sugar
Protein
RBC
WBC
Cast
Epith cell
Bacteria
Mucus th
Crystals
Am urates
straw
Clear
1.010
8.0
NEG
NEG
0-1
0-2
Rare
Occ’l
Rare
Rare
Urinalysis
pH
pCO2
pO2
HCO3
O2 sat
FiO2
Temp
7.408
49.1
70
31.3
93.6
21%
36.9
ABG
PBS
Slight poikilocytosis,
acanthocytes, ovalocytes,
slight toxic granulation,
slight anisocytosis
Table 2. Thyroid Function Tests
Free T4
TSH
(0.8-2.0)
(0.4-6.0)
0.02 ng/dL
24.75 Uiu/ml
Reference Value Result
Figure 1. Electrocardiogram upon admission
Table 3. Cardiac Enzymes
Qualitative
Troponin I
CK-MB
CK-TOTAL
0-6.0
21-232
POSITIVE
1.14
543
Reference Range (mmoL) Result
Figure 2. Chest radiograph on admission
VOL. 43 N0. 4 2009
12 ACTA MEDICA PHILIPPINA
Heart Failure and Short Stature in a 43 year-old male
Katerina T. Leyritana1, Ma. Czarlota M. Acelajado-Valdenor1, Amado O. Tandoc III2 and Agnes D. Mejia1
1Department of Medicine, College of Medicine and Philippine General Hospital, University of the Philippines Manila
2Department of Pathology, College of Medicine, University of the Philippines Manila
CASE REPORT
Corresponding author: Ma. Czarlota Acelajado-Valdenor, M.D.
Department of Medicine
Philippine General Hospital
Taft Avenue, Manila, 1000 Philippines
Telephone: +632 554-8488
Email: czarlota@yahoo.com
Presentation of the case
This is a case of a 43-year-old male presenting with short
stature and heart failure. The patient was admitted at the
medicine ward of the Philippine General Hospital (PGH)
for dyspnea. This paper will investigate several issues:
differentiating congenital from acquired hypothyroidism,
the relationship between hypothyroidism and the
cardiomyopathies, and the therapeutic options in patients
with cardiomyopathy secondary to hypothyroidism.
The patient had been born full term to a then 31-year-
old Gravida 4 Para 3 (G4P3), the 4th of 9 siblings, with an
apparently unremarkable delivery at home facilitated by
a traditional birth attendant. He was noted to be normal
at birth. The patient was allegedly at par with age both
physically and mentally until eight years old when he was
said to have stopped growing in height. He was brought to
a private doctor, whose diagnosis was undisclosed, and he
was given medications to increase height, which the patient
took for only one month with no improvement. Through the
years, the patient was apparently well, although still of short
stature, with thick lips, coarse facial features and dry skin.
He was notably slow in ambulation. He was said to have
bronchial asthma at age 15 years, and since then he had been
taking salbutamol tablets occasionally for bouts of dyspnea
occurring one to two times annually.
The patient’s symptoms started in 2001 when he was
reported to have sudden loss of consciousness. During this
time, the patient did not have any symptoms of heart failure;
no prior seizures, cyanotic episodes, chest pain, headache, or
blurring of vision. He regained consciousness shortly after and
was brought to a private physician, whose assessment was a
“heart problem”. He was prescribed unrecalled medications
taken for a few months and eventually discontinued when
the syncopal episode did not recur.
In the next four years, the patient would develop
intermittent, progressive exertional dyspnea and bipedal
edema. Later on this would be accompanied by generalized
body weakness, anorexia, and constipation, severe enough to
require regular laxative use. There was also a report of two
more syncopal episodes. He was brought to another doctor
in a private hospital where the assessment was still a “heart
problem”. The patient was again prescribed unrecalled
medications and again was lost to follow-up. This time,
however, symptoms were persistent. He later consulted at
another local hospital, where he was admitted and managed
as a case of anemia and bronchial asthma. He was discharged
slightly improved after four days, only to have recurrent heart
failure symptoms, prompting admission at PGH.
Upon admission the patient was in mild respiratory
distress, with stable vital signs and no note of fever. Pertinent
physical exam ndings included short stature, thick lips, non-
pitting periorbital edema, dry skin, a displaced apical impulse,
crackles on both lung elds, and bilateral non-pitting bipedal
edema. There was also a 3 cm x 3 cm reducible umbilical
hernia. However, there was no pallor, no neck vein distention,
no apparent congenital malformations, no cardiac murmurs
and no clubbing. There was also no note of an anterior neck
mass.
Laboratory workup showed cardiomegaly with pulmonary
congestion, thoracic dextroscoliosis, and atheromatous aorta by
chest radiograph, and left ventricular hypertrophy by 12-lead
electrocardiogram (12-L ECG) (Figures 1 and 2), normocytic
normochromic anemia (Hgb 90 mg/dL), dyslipidemia, and
pre-renal azotemia (serum creatinine 123 mmoL). Electrolytes
on admission showed slight hyponatremia, hypokalemia,
and hypochloremia (serum Na 136, K 3.35, Cl 86). Blood gases
revealed partially compensated metabolic alkalosis with mild
hypoxemia. The patient was noted to be hypothyroid based
on elevated serum thyroid-stimulating hormone (TSH) and
markedly decreased serum free thyroxine (FT4). The exact
values are shown in Tables 1 and 2.
Upon admission to the wards, the patient was managed
as having congestive heart failure from cardiomyopathy
secondary to acquired hypothyroidism. Oral loop diuretics,
angiotensin-converting enzyme (ACE) inhibitors, beta-
blockers, statins, and levothyroxine were started. Electrolyte
correction was instituted. The sections of Endocrinology
and Cardiovascular Diseases were co-managing the patient
together with the General Medicine service.
He soon developed respiratory failure, upon which the
considerations were acute pulmonary congestion, nosocomial
pneumonia, to rule out an acute coronary event. He was later
ORIGINAL ARTICLE
_______________
Corresponding author: Rhodora C. Estacio, MSc
Department of Bi ochemistry and Molecular Biology
College of Medicine
University of the Philippines Manila
547 Pedro Gil St., Ermita, Manila 1000 Philippines
Telephone: +632 5264197
Email address: rhodora .es taci o@yahoo.c om
Production of Immunoglobulin Y (IgY) against Synthetic Peptide Analogs of
the Immunogenic Epitopes of the Hepatitis B Surface Antigen
Leonardo A. Guevarra, Jr,1,2 Milagros B. Leaño,1 Leslie M. Dalmacio,1 Gracia Fe B. Yu,1 Raul V. Destura,3
Bernadette Libranda-Ramirez4 and Rhodora C. Estacio1
1Department of Biochemistry and Molecular Biology, College of Medicine, University of the Philippines Manila
2Department of Biochemistry, Faculty of Pharmacy, University of Santo Tomas
3Institute of Molecular Biology and Biotechnology, National Institutes of Health, University of the Philippines Manila
4IER/TDR/NPR/Lead Discovery and Innovation Research, World Health Organization
Introduction
Immunoglobulin Y (IgY), the avian counterpart of the
mammalian IgG, is the chicken antibody that is exclusively
transferred from the hens to the egg yolks to confer passive
immunity to the embryo.1 Its use in the different fields,
which is referred to as IgY technology, has become popular
because of the several advantages it offers compared to
using mammalian antibody. Among these advantages are:
(1) IgY technology is a cost-effective alternative method of
antibody production; (2) IgY isolation is non-invasive and
more humane; and (3) IgY is non-reactive to rheumatoid
factors (RF) and human anti-mammalian antibody (HAMA),
which makes it a more efficient tool for diagnostics
compared to other mammalian sources.2,3
IgY technology has been used in the field of diagnostics.
Similar to IgG, it has been used for protein detection
assays like enzyme-linked immunoassays (ELISA),
radioimmunoassays (RIA) and immunohistochemical
analysis of biologic compounds and toxins.4 Miyamoto et al
reported the use of IgY in the detection of normal and
abnormal prion proteins, which is essential in the diagnosis
of bovine spongiform encephalopathy in cattle.5 Ruan et al.
showed that IgY can be utilized to determine cell surface
molecules such as major histocompatibility complex
(MHC).6 He et al also reported the potential use of IgY
technology to detect the concentration of serum thymidine
kinase as a diagnostic tool for the recurrence of breast
cancer.7 In the diagnosis of emerging infections, Lee et al
used IgY to detect the presence of SARS Co-V.8 Other
studies that utilized IgY for diagnosis include bovine
leukemia virus,9 Indian cobra venom in forensic samples,10
human thalassemia11 and H. pylori urease B in vitro.12
Immunoglobulin Y and Detection of Hepatitis B Surface Antigen
2VOL. 46 NO. 1 2012
ACTA MEDICA PHILIPPINA
VOL. 43 N0. 4 2009
12 ACTA MEDICA PHILIPPINA
Heart Failure and Short Stature in a 43 year-old male
Katerina T. Leyritana1, Ma. Czarlota M. Acelajado-Valdenor1, Amado O. Tandoc III2 and Agnes D. Mejia1
1Department of Medicine, College of Medicine and Philippine General Hospital, University of the Philippines Manila
2Department of Pathology, College of Medicine, University of the Philippines Manila
CASE REPORT
Corresponding author: Ma. Czarlota Acelajado-Valdenor, M.D.
Department of Medicine
Philippine General Hospital
Taft Avenue, Manila, 1000 Philippines
Telephone: +632 554-8488
Email: czarlota@yahoo.com
Presentation of the case
This is a case of a 43-year-old male presenting with short
stature and heart failure. The patient was admitted at the
medicine ward of the Philippine General Hospital (PGH)
for dyspnea. This paper will investigate several issues:
differentiating congenital from acquired hypothyroidism,
the relationship between hypothyroidism and the
cardiomyopathies, and the therapeutic options in patients
with cardiomyopathy secondary to hypothyroidism.
The patient had been born full term to a then 31-year-
old Gravida 4 Para 3 (G4P3), the 4th of 9 siblings, with an
apparently unremarkable delivery at home facilitated by
a traditional birth attendant. He was noted to be normal
at birth. The patient was allegedly at par with age both
physically and mentally until eight years old when he was
said to have stopped growing in height. He was brought to
a private doctor, whose diagnosis was undisclosed, and he
was given medications to increase height, which the patient
took for only one month with no improvement. Through the
years, the patient was apparently well, although still of short
stature, with thick lips, coarse facial features and dry skin.
He was notably slow in ambulation. He was said to have
bronchial asthma at age 15 years, and since then he had been
taking salbutamol tablets occasionally for bouts of dyspnea
occurring one to two times annually.
The patient’s symptoms started in 2001 when he was
reported to have sudden loss of consciousness. During this
time, the patient did not have any symptoms of heart failure;
no prior seizures, cyanotic episodes, chest pain, headache, or
blurring of vision. He regained consciousness shortly after and
was brought to a private physician, whose assessment was a
“heart problem”. He was prescribed unrecalled medications
taken for a few months and eventually discontinued when
the syncopal episode did not recur.
In the next four years, the patient would develop
intermittent, progressive exertional dyspnea and bipedal
edema. Later on this would be accompanied by generalized
body weakness, anorexia, and constipation, severe enough to
require regular laxative use. There was also a report of two
more syncopal episodes. He was brought to another doctor
in a private hospital where the assessment was still a “heart
problem”. The patient was again prescribed unrecalled
medications and again was lost to follow-up. This time,
however, symptoms were persistent. He later consulted at
another local hospital, where he was admitted and managed
as a case of anemia and bronchial asthma. He was discharged
slightly improved after four days, only to have recurrent heart
failure symptoms, prompting admission at PGH.
Upon admission the patient was in mild respiratory
distress, with stable vital signs and no note of fever. Pertinent
physical exam ndings included short stature, thick lips, non-
pitting periorbital edema, dry skin, a displaced apical impulse,
crackles on both lung elds, and bilateral non-pitting bipedal
edema. There was also a 3 cm x 3 cm reducible umbilical
hernia. However, there was no pallor, no neck vein distention,
no apparent congenital malformations, no cardiac murmurs
and no clubbing. There was also no note of an anterior neck
mass.
Laboratory workup showed cardiomegaly with pulmonary
congestion, thoracic dextroscoliosis, and atheromatous aorta by
chest radiograph, and left ventricular hypertrophy by 12-lead
electrocardiogram (12-L ECG) (Figures 1 and 2), normocytic
normochromic anemia (Hgb 90 mg/dL), dyslipidemia, and
pre-renal azotemia (serum creatinine 123 mmoL). Electrolytes
on admission showed slight hyponatremia, hypokalemia,
and hypochloremia (serum Na 136, K 3.35, Cl 86). Blood gases
revealed partially compensated metabolic alkalosis with mild
hypoxemia. The patient was noted to be hypothyroid based
on elevated serum thyroid-stimulating hormone (TSH) and
markedly decreased serum free thyroxine (FT4). The exact
values are shown in Tables 1 and 2.
Upon admission to the wards, the patient was managed
as having congestive heart failure from cardiomyopathy
secondary to acquired hypothyroidism. Oral loop diuretics,
angiotensin-converting enzyme (ACE) inhibitors, beta-
blockers, statins, and levothyroxine were started. Electrolyte
correction was instituted. The sections of Endocrinology
and Cardiovascular Diseases were co-managing the patient
together with the General Medicine service.
He soon developed respiratory failure, upon which the
considerations were acute pulmonary congestion, nosocomial
pneumonia, to rule out an acute coronary event. He was later
There are also previous studies on the inhibitory effects
of IgY on the activity of infectious agents. In 1999, Carlander
et al produced chicken antibodies against Pseudomonas
aeruginosa, used it in this first oral antibody trial and
reported that no positive cultures were observed on patients
who received oral treatment of the immunoglobulin.13 This
result was consistent with the findings of Nilsson et al on the
therapeutic and prophylactic use of IgY against P.
aeruginosa.14 Sarker et al reported that IgY from egg yolks of
chickens immunized with human rotaviral strain improved
the conditions of children with rotaviral diarrhea.15 Deignan
et al reported that IgY from unfractionated egg yolk reduced
the attachment of Salmonella typhimurium in the intestinal
epithelial cells of murine models in vitro.16 Nomura et al
and Shimamoto et al have investigated and suggested the
curative effect of IgY-specific for the immunodominant
protein of Helicobacter pylori against the disease in animals
and humans respectively.17,18 Sunwoo et al and Cook et al
also reported the growth inhibiting activity of IgY specific
for E. coli 0157:H7.19,20 The group of Sunwoo attribute the
anti-E. coli activity of the IgY to the binding of the avian
immunoglobulin to bacterial surface antigen distorting
therefore the cell surface structure of the pathogen.19 In 2000,
Carlander et al also reported the prophylactic effect of IgY
from rotavirus infection.13 The immunosuppressing effect
of IgY against certain toxins such as snake, scorpion venom21,
22 and botulinum toxin23 has already been reported.
The proven applicability of IgY technology and its
advantages over mammalian IgG offer an alternative in the
detection and prevention of epidemiologically relevant
diseases such as Hepatitis B which have infected
approximately 360 million people as reported by the World
Health Organization.24 Countries like the Philippines which
has intermediate (2-8%) to high (>8%) endemicity of HBV
infection25 must harness its potential and use IgY
technology.
The objective of this study was to produce an IgY
against known immunogenic epitopes of the Hepatitis B
surface antigen using synthetic peptides analogs. The IgY
product of this study can potentially be used in Hepatitis B
diagnostics and therapeutics.
Method
Chicken Immunization and Care
Sixteen to eighteen week-old white leghorn hens
acquired from the National Poultry Research and
Development Center, Bureau of Animal Industry, were
grouped into three namely Negative Control Group, Peptide
1 Immunized Group and Peptide 2 Immunized Group and
were separately immunized with synthetic peptide analogs
comprising of the known immunogenic epitopes of the S
region 39th-147th, herein referred to as Peptide 1, and pre-S1
region (34th-59th), herein referred to as Peptide 2, of the
Hepatitis B Surface antigens (HBsAg) synthesized and
purchased at Genscript Corporation in New Jersey, USA.
The immunogens were prepared by emulsifying the peptide
dissolved in PBS with Freund’s Complete Adjuvant (Sigma)
during the initial immunization and with Freund’s
Incomplete Adjuvant (Sigma) during booster
immunizations. One hundred micrograms of peptide was
injected at four different sites in the pectoral muscle of each
chicken. An initial and two booster immunizations were
administered at two weeks interval.
Chickens were fed with commercially available chicken
laying mash and given purified water ad libitum. Eggs were
collected daily and stored at 4oC until use.
IgY Isolation
The IgY isolation was done as described by Shin et al
with modification.26 The egg yolk was separated from the
egg white, washed with distilled water and poured into a
graduated cylinder for measurement.
The egg yolk was diluted with an equal volume of
distilled water followed by the addition of 0.15% (w/v) of λ-
carageenan (Rico Carrageenan) until a 1:1 diluted egg yolk-
carageenan mixture was achieved. The mixture was
incubated at room temperature for 30 minutes and then
centrifuged at 3,200 x g at 20oC for 30 minutes. The
supernatant was collected and was filtered using Whatman
filter paper No. 1. Sodium sulfate crystals were added
gradually to the water soluble fraction until a total of 19%
(w/v) salt concentration was achieved. The solution was
then centrifuged for 30 minutes at room temperature and the
precipitate was dissolved in 0.01M Phosphate Buffered
Saline pH 7.4 (Sigma) and stored at -20oC prior to use. Total
protein concentration of the IgY isolates was determined by
Bradford method using microtiter plate protocols.
Purity Assessment and Molecular Weight Determination by SDS-
PAGE
Non-denaturing and denaturing Sodium Dodecyl
Sulfate – Polyacrylamide Gel Electrophoresis (SDS-PAGE)
by Laemmli were done to assess purity of isolates and
determine the molecular weight of purified IgY and its
subunits. SDS-PAGE was carried out in the Mini Protean
3Cell (Biorad) using 12% resolving gel and 4% stacking gel
for both the non-denaturing and denaturing SDS-PAGE with
Kaleidoscope Prestained Molecular Weight Markers (Biorad)
and Standard Chicken IgY (Promega) as molecular weight
and purity standards. SDS-PAGE of the samples and
standards was performed at 180 volts for 45 minutes.
Proteins were visualized by staining the gels with
Coomassie Brilliant Blue R-250 Staining Solution (Biorad) for
30 minutes, washing it with distilled water, and destaining it
with Coomassie R-250 Destaining Solution (Biorad).
Immunoglobulin Y and Detection of Hepatitis B Surface Antigen
3
VOL. 46 NO. 1 2012 ACTA MEDICA PHILIPPINA
VOL. 43 N0. 4 2009 ACTA MEDICA PHILIPPINA 13
Heart Failure and Short Stature in a 43 year-old male
transferred to the intensive care unit (ICU) for ventilatory
support and closer monitoring. On bedside cardiac ultrasound,
there was a nding of eccentric left ventricular hypertrophy,
global hypokinesia with depressed overall systolic function
with concomitant spontaneous echo contrast on left ventricular
(LV) cavity suggestive of rheologic stasis, the ejection fraction
was 25%, with moderate mitral regurgitation, moderate
aortic regurgitation with aortic sclerosis, severe tricuspid
regurgitation with mild pulmonary hypertension, pulmonary
regurgitation, and minimal pericardial effusion or pericardial
fat pad. Cardiac enzymes were not consistent with an acute
coronary event (Table 3), however, intravenous (IV) heparin
(overlapping with oral warfarin) was still given to cover for
the presence of a possible LV thrombus as demonstrated
by rheologic stasis on cardiac ultrasound. Medications
were shifted to IV diuretics and inotropes; oral digoxin was
started. IV antibiotics were given for possible pulmonary
infection. The patient later on showed improvement, and was
eventually weaned off from ventilatory support, extubated,
Table 1. Initial Laboratory Results
WBC
RBC
HGB
HCT
MCV
MCH
MCHC
RDW
PLT
RETIC
SEG
LYMPH
MONO
EO
BASO
BLAST
CBC
Reference
Value
5-10
4-6
120-150
0.38-0.48
80-100 FL
27-31 PG
320-360 G/L
11.5-15.5%
200-400
0.005-0.015
50-70%
20-44%
2-9%
0-4%
0-2%
0%
Result
4.5
90
0.27
Inc
48
50
2
0
0
0
RBS
HGBA1C
BUN
CREA
ALB
TAG
HDL
LDL
TOTAL CHOL
AST
ALT
Alk po4
NA
K
CL
CA++
P
MG++
Reference
Value
3.9-6.1
4.27-6.07
2.6-6.4
53-115
34-50
0.34-1.7
0.91-1.56
1.1-3.8
4.2-5.2
15-37
30-65
140-148
3.6-5.2
100-108
2.12-2.52
0.74-1
Result
6.3
6.4
5.0
123
32
0.82
0.67
4.21
5.25
95
91
184
136.9
3.35
86
2.37
2.27
0.83
Blood chem.
Color
Transp
Sp Gravity
pH
Sugar
Protein
RBC
WBC
Cast
Epith cell
Bacteria
Mucus th
Crystals
Am urates
straw
Clear
1.010
8.0
NEG
NEG
0-1
0-2
Rare
Occ’l
Rare
Rare
Urinalysis
pH
pCO2
pO2
HCO3
O2 sat
FiO2
Temp
7.408
49.1
70
31.3
93.6
21%
36.9
ABG
PBS
Slight poikilocytosis,
acanthocytes, ovalocytes,
slight toxic granulation,
slight anisocytosis
Table 2. Thyroid Function Tests
Free T4
TSH
(0.8-2.0)
(0.4-6.0)
0.02 ng/dL
24.75 Uiu/ml
Reference Value Result
Figure 1. Electrocardiogram upon admission
Table 3. Cardiac Enzymes
Qualitative
Troponin I
CK-MB
CK-TOTAL
0-6.0
21-232
POSITIVE
1.14
543
Reference Range (mmoL) Result
Figure 2. Chest radiograph on admission
Table 1. Amino Acid Sequence of Peptides Used for Peptide-Antipeptide Specificity Assay
Peptide Amino Ac id Sequence N-termin al M odific ation C-termin al M od ific ation
Peptide 1 CTKPTDGNC None Amidated
Pepti de 2 FGANSNNPDWDFNIKDHWPAANQVG None Amidated
Peptide 3 ENHSPVNIAHKL None None
Peptide 4 CGTIADKDGC Acetyla ted Amidated
Peptide 5 CGDTKEYGC Acetylated Amidated
Peptide 6 CGDDTGKIGC Acetylated Amidated
Affinity Purification of Peptide Specific IgY
Peptide-specific chicken IgY was purified using the
methods previously described by Camenisch et al. Two and
five tenths (2.5) milligrams of peptide was dissolved in 50ul
of distilled water, vortexed, incubated at room temperature
for 2 hours, and finally brought to 5 ml with coupling buffer
(0.1M NaHCO3 buffer pH 8.3 containing 0.5M NaCl).
Swelling of the Sepharose gel was made according to
manufacturer’s instructions. One gram of freeze-dried
CNBr-activated Sepharose 4B Fast Flow (Sigma) was
dissolved in 5 ml of 1mM HCl. The dissolved Sepharose
was washed with 200 m1 of 1 mM HCl. Immediately, the
swollen Sepharose was combined with the previously
prepared peptide solution and incubated with gentle
shaking at room temperature for 2 hours. The gel was
transferred to Econo-column chromatography column
(Biorad) and washed 15 ml of coupling buffer to remove the
unbound ligands. Excess active groups were blocked for 2
hours with 0.1M Tris-HCl buffer pH 8.0. The gel was
washed 3 times alternately with 10 ml acetate buffer saline
pH 4.0 (0.1M NaCH3COO containing 0.5M NaCl) and 10ml
Tris buffered saline pH 8.0 (0.1M Tris-HCl buffer containing
0.5M NaCl).
Prior to loading into the column, IgY isolates were
pooled at 2 weeks interval and dialyzed against the coupling
buffer for 1 hour. Dialyzed samples were loaded into the
column and fractions were collected at 2ml intervals. The
column with IgY isolates was washed with 50 ml 0.1M PBS
pH 7.4. Peptide-specific IgY was eluted with 8.5 mL 0.2M
glycine pH 2.2 containing 0.15M NaCl at 2ml intervals. To
immediately neutralize the IgY fractions, eluents were
collected in tubes containing 0.8ml of Tris buffered saline pH
8.0.
After elution, the column was immediately regenerated
by washing with 10 ml Tris buffered saline and 10 ml PBS.
The column was cleaned by alternately washing with elution
buffer and Tris buffered saline three times. The column was
stored in PBS containing 0.05% NaN3 at 4oC.27
Antipeptide Activity Determination
Antipeptide activity of purified IgY was done by
Indirect ELISA. The wells of a 96-well microtiter plate
(Corning) were coated with 50 ul of serially diluted synthetic
peptide solutions overnight at 4oC. The wells were then
washed with 0.01M PBS containing 0.05% Tween 20, blocked
with 100 ul 1% ovalbumin, and incubated for 2 hours at
37oC. Excess blocking solution was removed by washing,
and 50 ul of purified IgY was introduced to each well. After
incubation for 1 hour at 37oC and washing of the unbound
IgY, 50 ul of HRP-conjugated anti-chicken IgY was added to
each well. The wells were washed and 100 ul of TMB
substrate solution (1 mg TMB dissolved in 1ml DMSO and
diluted with 9 ml 0.01M phosphate citrate buffer pH 5.0 and
added with 2ul of 30% H2O2) was added to the wells and
incubated for 30 minutes at room temperature. The reaction
was stopped with 50ul 2M H2SO4 and the plate was read at
450 nm in a Model 550 Microplate Reader (Biorad).
Peptide Specificity Assay
Peptide specificity of the affinity-purified IgY was tested
by Indirect ELISA as described above. Six peptides with
sequence shown in Table 1 synthesized by and purchased at
Genscript Corporation (New Jersey) were used for peptide
specificity test.
Statistical Treatment and Analysis
The total protein determination of samples was done in
duplicates. The mean absorbance reading was computed
and used for analysis. A standard curve using gamma
globulin protein standard set (Biorad) ranging from 0.125
mg/ml – 2.0 mg/ml was generated every time total protein
concentration experiment is done and the total protein
concentration of the samples was determined by linear
regression analysis.
The total protein from the crude IgY isolates of eggs
collected weekly was determined. Trends in the protein
concentration per week from each group were observed.
Differences in the mean protein concentration of the three
groups were determined by Analysis of Variance (ANOVA).
For determination of peptide-antipeptide activity and
peptide specificity assay, the mean absorbance readings of
the samples, done at least in duplicate, were computed. Cut-
off values were determined using the formula described by
Frey et al. These were used to discriminate negative and
positive antibody activity. An absorbance reading greater
than the cut off value was interpreted as positive activity
while an absorbance reading less than the cut off value was
interpreted as negative antibody activity. The cut off values
Immunoglobulin Y and Detection of Hepatitis B Surface Antigen
4VOL. 46 NO. 1 2012
ACTA MEDICA PHILIPPINA
VOL. 43 N0. 4 2009
12 ACTA MEDICA PHILIPPINA
Heart Failure and Short Stature in a 43 year-old male
Katerina T. Leyritana1, Ma. Czarlota M. Acelajado-Valdenor1, Amado O. Tandoc III2 and Agnes D. Mejia1
1Department of Medicine, College of Medicine and Philippine General Hospital, University of the Philippines Manila
2Department of Pathology, College of Medicine, University of the Philippines Manila
CASE REPORT
Corresponding author: Ma. Czarlota Acelajado-Valdenor, M.D.
Department of Medicine
Philippine General Hospital
Taft Avenue, Manila, 1000 Philippines
Telephone: +632 554-8488
Email: czarlota@yahoo.com
Presentation of the case
This is a case of a 43-year-old male presenting with short
stature and heart failure. The patient was admitted at the
medicine ward of the Philippine General Hospital (PGH)
for dyspnea. This paper will investigate several issues:
differentiating congenital from acquired hypothyroidism,
the relationship between hypothyroidism and the
cardiomyopathies, and the therapeutic options in patients
with cardiomyopathy secondary to hypothyroidism.
The patient had been born full term to a then 31-year-
old Gravida 4 Para 3 (G4P3), the 4th of 9 siblings, with an
apparently unremarkable delivery at home facilitated by
a traditional birth attendant. He was noted to be normal
at birth. The patient was allegedly at par with age both
physically and mentally until eight years old when he was
said to have stopped growing in height. He was brought to
a private doctor, whose diagnosis was undisclosed, and he
was given medications to increase height, which the patient
took for only one month with no improvement. Through the
years, the patient was apparently well, although still of short
stature, with thick lips, coarse facial features and dry skin.
He was notably slow in ambulation. He was said to have
bronchial asthma at age 15 years, and since then he had been
taking salbutamol tablets occasionally for bouts of dyspnea
occurring one to two times annually.
The patient’s symptoms started in 2001 when he was
reported to have sudden loss of consciousness. During this
time, the patient did not have any symptoms of heart failure;
no prior seizures, cyanotic episodes, chest pain, headache, or
blurring of vision. He regained consciousness shortly after and
was brought to a private physician, whose assessment was a
“heart problem”. He was prescribed unrecalled medications
taken for a few months and eventually discontinued when
the syncopal episode did not recur.
In the next four years, the patient would develop
intermittent, progressive exertional dyspnea and bipedal
edema. Later on this would be accompanied by generalized
body weakness, anorexia, and constipation, severe enough to
require regular laxative use. There was also a report of two
more syncopal episodes. He was brought to another doctor
in a private hospital where the assessment was still a “heart
problem”. The patient was again prescribed unrecalled
medications and again was lost to follow-up. This time,
however, symptoms were persistent. He later consulted at
another local hospital, where he was admitted and managed
as a case of anemia and bronchial asthma. He was discharged
slightly improved after four days, only to have recurrent heart
failure symptoms, prompting admission at PGH.
Upon admission the patient was in mild respiratory
distress, with stable vital signs and no note of fever. Pertinent
physical exam ndings included short stature, thick lips, non-
pitting periorbital edema, dry skin, a displaced apical impulse,
crackles on both lung elds, and bilateral non-pitting bipedal
edema. There was also a 3 cm x 3 cm reducible umbilical
hernia. However, there was no pallor, no neck vein distention,
no apparent congenital malformations, no cardiac murmurs
and no clubbing. There was also no note of an anterior neck
mass.
Laboratory workup showed cardiomegaly with pulmonary
congestion, thoracic dextroscoliosis, and atheromatous aorta by
chest radiograph, and left ventricular hypertrophy by 12-lead
electrocardiogram (12-L ECG) (Figures 1 and 2), normocytic
normochromic anemia (Hgb 90 mg/dL), dyslipidemia, and
pre-renal azotemia (serum creatinine 123 mmoL). Electrolytes
on admission showed slight hyponatremia, hypokalemia,
and hypochloremia (serum Na 136, K 3.35, Cl 86). Blood gases
revealed partially compensated metabolic alkalosis with mild
hypoxemia. The patient was noted to be hypothyroid based
on elevated serum thyroid-stimulating hormone (TSH) and
markedly decreased serum free thyroxine (FT4). The exact
values are shown in Tables 1 and 2.
Upon admission to the wards, the patient was managed
as having congestive heart failure from cardiomyopathy
secondary to acquired hypothyroidism. Oral loop diuretics,
angiotensin-converting enzyme (ACE) inhibitors, beta-
blockers, statins, and levothyroxine were started. Electrolyte
correction was instituted. The sections of Endocrinology
and Cardiovascular Diseases were co-managing the patient
together with the General Medicine service.
He soon developed respiratory failure, upon which the
considerations were acute pulmonary congestion, nosocomial
pneumonia, to rule out an acute coronary event. He was later
were computed from each assay using the method described
by Frey et al.28 The formula used is
Cut off value = X +
SD*f
where X = mean absorbance of the blank,
SD = standard deviation of the absorbance of
the blank, and
f = standard deviation multiplier which is
dependent on confidence interval and
number of replicates
The confidence interval used to compute the cut off value is
99.0%
Results and Discussion
IgY Isolation
Up to 85 milligrams of IgY was isolated in a single egg
yolk using the modified Shin et al IgY isolation technique.
The mean concentrations from the Negative Control Group,
Peptide 1 Immunized Group and Peptide 2 Immunized
Group are 4.61 ± 0.65, 4.93 ± 1.10 and 4.87 ± 0.96 mg/ml egg
yolk, respectively. Comparison of the mean isolated IgY
concentrations from the three groups before immunization
and up to ten weeks after the first immunization revealed
that there is no statistically significant difference among the
three groups.
Akita and Nakai’s comparison of four IgY isolation
techniques namely PEG precipitation by Polson, dextran
sulfate method by Jensenius, Xanthan method by Hatta and
the Water Dilution method by Akita and Nakai revealed that
the water dilution technique was the most efficient in terms
of yield with up to 100 mg/ml of IgY per egg yolk.29 This
was recognized by Shin et al, however, a modification was
done which includes the use of λ-carrageenan to efficiently
remove lipids in the egg yolk which according to Powrie and
Nakai are the most abundant component of the egg yolk.30
Antipeptide Activity Assay of IgY Isolates
To determine antipeptide activity of isolated IgY from
the three groups, absorbance readings of the IgY isolates
were taken and compared against the computed cut off
values for each group. Table 2 presents the absorbance
readings of the anti-Peptide 1 and anti-Peptide 2 activity
assay of IgY isolated from the Negative Control Group,
Peptide 1 Immunized Group and Peptide 2 Immunized
Group. The cut off values for Anti-Peptide 1 activity assay
of IgY isolated from Peptide 1 Immunized group and
Negative Control Group are 0.220 and 0.198, respectively.
For Anti-Peptide 2 activity assay of IgY isolated from
Peptide 2 Immunized Group and Negative Control Group,
the computed cut off values are 0.050 and 0.025, respectively.
Absorbance readings higher than the computed cut off
values were only observed in Anti-Peptide 2 activity assay of
IgY isolated from the Peptide 2 Immunized Group. This
means that IgY isolated from the Peptide 2 Immunized
Group has an anti-Peptide activity against Peptide 2.
However, this was not observed with anti-Peptide 1 activity
assay of IgY isolated from Peptide 1 Immunized Group, as
well as with the anti-Peptide 1 and anti-Peptide 2 activity
assay of IgY isolated from the Negative Control Group.
A concentration dependent increase in the absorbance
readings was also noted in the anti-Peptide 2 activity assay
of IgY isolated from chickens immunized with Peptide 2.
This was not observed with the IgY isolated from chickens
from Negative Control Group (Figure 1). The same was not
detected in the anti-Peptide 1 assay of IgY isolated from
Peptide 1 Immunized Group as well as the IgY isolated from
the Negative Control Group when anti-Peptide 1 activity
assay was done.
Figure 1. Preliminary Anti-peptide Activity Assay of IgY
Isolates from Group P2 and Negative Control Group
against Peptide 2. – IgY from Egg Yolks from Peptide 2
Immunized Chickens, - IgY from Egg Yolks of Negative
Control Group. Peptide 2 concentration-dependent increase
in the absorbance was observed with IgY isolated from egg
yolks of chickens immunized with Peptide 2 – an indication
that the absorbance reading is due to Peptide 2-Antipeptide
2 IgY binding.
Peptides have already been used to elicit immune
response. Use of peptides as immunogen is well described
by the role of Cytotoxic T Lymphocytes (CTL) in presenting
the peptide antigen through the Class I MHC proteins.
However, questions of efficiency of using peptide antigens,
especially synthetic peptide antigens, have been reported
and needs experimental confirmation because of several
factors that must be considered in the immune response
eliciting activity of synthetic peptides, as well as their
clinical applicability. Coligan et al have discussed several
Immunoglobulin Y and Detection of Hepatitis B Surface Antigen
5
VOL. 46 NO. 1 2012 ACTA MEDICA PHILIPPINA
VOL. 43 N0. 4 2009 ACTA MEDICA PHILIPPINA 13
Heart Failure and Short Stature in a 43 year-old male
transferred to the intensive care unit (ICU) for ventilatory
support and closer monitoring. On bedside cardiac ultrasound,
there was a nding of eccentric left ventricular hypertrophy,
global hypokinesia with depressed overall systolic function
with concomitant spontaneous echo contrast on left ventricular
(LV) cavity suggestive of rheologic stasis, the ejection fraction
was 25%, with moderate mitral regurgitation, moderate
aortic regurgitation with aortic sclerosis, severe tricuspid
regurgitation with mild pulmonary hypertension, pulmonary
regurgitation, and minimal pericardial effusion or pericardial
fat pad. Cardiac enzymes were not consistent with an acute
coronary event (Table 3), however, intravenous (IV) heparin
(overlapping with oral warfarin) was still given to cover for
the presence of a possible LV thrombus as demonstrated
by rheologic stasis on cardiac ultrasound. Medications
were shifted to IV diuretics and inotropes; oral digoxin was
started. IV antibiotics were given for possible pulmonary
infection. The patient later on showed improvement, and was
eventually weaned off from ventilatory support, extubated,
Table 1. Initial Laboratory Results
WBC
RBC
HGB
HCT
MCV
MCH
MCHC
RDW
PLT
RETIC
SEG
LYMPH
MONO
EO
BASO
BLAST
CBC
Reference
Value
5-10
4-6
120-150
0.38-0.48
80-100 FL
27-31 PG
320-360 G/L
11.5-15.5%
200-400
0.005-0.015
50-70%
20-44%
2-9%
0-4%
0-2%
0%
Result
4.5
90
0.27
Inc
48
50
2
0
0
0
RBS
HGBA1C
BUN
CREA
ALB
TAG
HDL
LDL
TOTAL CHOL
AST
ALT
Alk po4
NA
K
CL
CA++
P
MG++
Reference
Value
3.9-6.1
4.27-6.07
2.6-6.4
53-115
34-50
0.34-1.7
0.91-1.56
1.1-3.8
4.2-5.2
15-37
30-65
140-148
3.6-5.2
100-108
2.12-2.52
0.74-1
Result
6.3
6.4
5.0
123
32
0.82
0.67
4.21
5.25
95
91
184
136.9
3.35
86
2.37
2.27
0.83
Blood chem.
Color
Transp
Sp Gravity
pH
Sugar
Protein
RBC
WBC
Cast
Epith cell
Bacteria
Mucus th
Crystals
Am urates
straw
Clear
1.010
8.0
NEG
NEG
0-1
0-2
Rare
Occ’l
Rare
Rare
Urinalysis
pH
pCO2
pO2
HCO3
O2 sat
FiO2
Temp
7.408
49.1
70
31.3
93.6
21%
36.9
ABG
PBS
Slight poikilocytosis,
acanthocytes, ovalocytes,
slight toxic granulation,
slight anisocytosis
Table 2. Thyroid Function Tests
Free T4
TSH
(0.8-2.0)
(0.4-6.0)
0.02 ng/dL
24.75 Uiu/ml
Reference Value Result
Figure 1. Electrocardiogram upon admission
Table 3. Cardiac Enzymes
Qualitative
Troponin I
CK-MB
CK-TOTAL
0-6.0
21-232
POSITIVE
1.14
543
Reference Range (mmoL) Result
Figure 2. Chest radiograph on admission
Table 2. Summary of Results for Anti-Peptide 1 and Anti-Peptide 2 Activity of IgY Isolated from Egg Yolks of Chickens from
Negative Control Group, Peptide 1 Immunized Group and Peptide 2 Immunized Group
parameters that must be considered in using synthetic
peptides for the production of antibodies which have
reactivity against the native protein. Among them are (1) the
correct choice of peptide sequence to be mimicked by the
synthetic peptide; (2) length of the synthetic peptide that
may confer immunogenicity to the model animal; and (3)
conformational limitations of synthetic peptide.31
The peptides that were used in this study have already
been identified to be immunogenic in mammalian models.
Of the two peptides used, Peptide 2 is significantly longer,
with 24 amino acid residues, compared to Peptide 1 with
only 9 amino acid residues. This may explain why, in this
study, the IgY isolated from Peptide 2 Immunized Group
had an antipeptide activity against Peptide 2, while no anti-
Peptide 1 activity was observed from IgY isolated from
Peptide 1 Immunized Group. In practice, the usual number
of amino acid residues used as synthetic peptide
immunogen is 10 to 15. However, Coligan et al, also noted
that synthetic peptides ranging from a minimum of 6 amino
acid residues up to 35 amino acid residues have been
successful in eliciting immune response and have produced
anti-peptide antibody.31 The limitation on the number of
amino acid in a synthetic peptide to elicit detectable immune
response might not have been overcome by Peptide 1. Even
conjugating it to a carrier protein, which is the most common
way to resolve the problem of having a shorter peptide as
immunogen, has not been successful in making the peptide
immunogenic in this study.
Amino acid sequence of Peptide 1 and its position in the
tertiary structure may have contributed to non-
immunogenic nature of the synthetic peptide analog of
Peptide 1. The two cysteine residues in the N-terminus and
C-terminus of the synthetic peptide may have a role in its
immunogenicity. Vyas et al reported that reduction of the
disulfide bonds at this position largely destroys the activity
of HBsAg.32 Further, Prince et al inferred that the cysteine
residues are vital for the formation of the loop which is
critical to the antigenic activity of HBsAg.33
Aside from this, there is a possibility that the amino
group of lysine found in Peptide 1, might have also been
conjugated to glutaraldehyde during peptide-BSA
conjugation thus affecting its immunogenicity.
Inter-species variation might also explain the absence of
an immune response of Peptide 1 immunized chickens.
Chickens, the animal which was used in this experiment,
might have a different immune response to the peptide
immunogen. This claim can be supported by studies
concerning variation in the immune response of genetically
different organisms. Though no previous studies have been
cited comparing immune response of chickens and
mammals, Gengozian et al have already reported variation
in the immune responses of two marmoset species both in
vitro and in vivo as early as 1978. Gengozian et al observed
that the marmoset species Saguinus fuscicollis had a
significant immune response to Escherichia coli
lipopolysaccharide and Salmonella typhi flagella compared to
Saguinus oedipus.34 Immunogenetics and immunogenomics
may also explain the heterogeneity of the immune responses
of organisms belonging to different species.35
Affinity Purification of IgY with Anti-peptide Activity
Affinity purification was done to capture the peptide
specific IgY from isolates of peptide 2 immunized chickens.
Total IgY content from the eluates pooled at two weeks
interval were determined by Bradford Total Protein Analysis
Microplate Technique. Figure 2 shows the concentrations of
Anti-peptide Assay Peptide Concentration (ug/ml) Cut-off Values
10.00 5.00 2.50 1.25 0.63 0.31 0.16 0.08 0.04 0.02 0.01
Anti-Peptide 1 Activity
of IgY Isol ated from
Grou p P1
Mean
Absorbance
0.197 0.188 0.195 0.196 0.198 0.199 0.200 0.202 0.208 0.202 0.218
0.220
Interpretati on (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
Anti-Peptide 1 Activity
of IgY Isol ated from
Nega tive Contr ol
Grou p
Mean
Absorbance
0.169 0.165 0.171 0.170 0.171 0.174 0.178 0.180 0.178 0.184 0.184
0.198
Interpretati on (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
Anti-Peptide 2 Activity
of IgY Isol ated from
Grou p P2
Mean
Absorbance 0.251 0.172 0.132 0.109 0.095 0.074 0.063 0.067 0.06 0.051 0.056
0.050
Interpretati on (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+)
Anti-Peptide 2 Activity
of IgY Isol ated from
Negative Contr ol
Grou p
Mean
Absorbance 0.022 0.021 0.022 0.023 0.022 0.021 0.022 0.02 0.022 0.020 0.021
0.025
Interpretati on (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
Immunoglobulin Y and Detection of Hepatitis B Surface Antigen
6VOL. 46 NO. 1 2012
ACTA MEDICA PHILIPPINA
VOL. 43 N0. 4 2009
12 ACTA MEDICA PHILIPPINA
Heart Failure and Short Stature in a 43 year-old male
Katerina T. Leyritana1, Ma. Czarlota M. Acelajado-Valdenor1, Amado O. Tandoc III2 and Agnes D. Mejia1
1Department of Medicine, College of Medicine and Philippine General Hospital, University of the Philippines Manila
2Department of Pathology, College of Medicine, University of the Philippines Manila
CASE REPORT
Corresponding author: Ma. Czarlota Acelajado-Valdenor, M.D.
Department of Medicine
Philippine General Hospital
Taft Avenue, Manila, 1000 Philippines
Telephone: +632 554-8488
Email: czarlota@yahoo.com
Presentation of the case
This is a case of a 43-year-old male presenting with short
stature and heart failure. The patient was admitted at the
medicine ward of the Philippine General Hospital (PGH)
for dyspnea. This paper will investigate several issues:
differentiating congenital from acquired hypothyroidism,
the relationship between hypothyroidism and the
cardiomyopathies, and the therapeutic options in patients
with cardiomyopathy secondary to hypothyroidism.
The patient had been born full term to a then 31-year-
old Gravida 4 Para 3 (G4P3), the 4th of 9 siblings, with an
apparently unremarkable delivery at home facilitated by
a traditional birth attendant. He was noted to be normal
at birth. The patient was allegedly at par with age both
physically and mentally until eight years old when he was
said to have stopped growing in height. He was brought to
a private doctor, whose diagnosis was undisclosed, and he
was given medications to increase height, which the patient
took for only one month with no improvement. Through the
years, the patient was apparently well, although still of short
stature, with thick lips, coarse facial features and dry skin.
He was notably slow in ambulation. He was said to have
bronchial asthma at age 15 years, and since then he had been
taking salbutamol tablets occasionally for bouts of dyspnea
occurring one to two times annually.
The patient’s symptoms started in 2001 when he was
reported to have sudden loss of consciousness. During this
time, the patient did not have any symptoms of heart failure;
no prior seizures, cyanotic episodes, chest pain, headache, or
blurring of vision. He regained consciousness shortly after and
was brought to a private physician, whose assessment was a
“heart problem”. He was prescribed unrecalled medications
taken for a few months and eventually discontinued when
the syncopal episode did not recur.
In the next four years, the patient would develop
intermittent, progressive exertional dyspnea and bipedal
edema. Later on this would be accompanied by generalized
body weakness, anorexia, and constipation, severe enough to
require regular laxative use. There was also a report of two
more syncopal episodes. He was brought to another doctor
in a private hospital where the assessment was still a “heart
problem”. The patient was again prescribed unrecalled
medications and again was lost to follow-up. This time,
however, symptoms were persistent. He later consulted at
another local hospital, where he was admitted and managed
as a case of anemia and bronchial asthma. He was discharged
slightly improved after four days, only to have recurrent heart
failure symptoms, prompting admission at PGH.
Upon admission the patient was in mild respiratory
distress, with stable vital signs and no note of fever. Pertinent
physical exam ndings included short stature, thick lips, non-
pitting periorbital edema, dry skin, a displaced apical impulse,
crackles on both lung elds, and bilateral non-pitting bipedal
edema. There was also a 3 cm x 3 cm reducible umbilical
hernia. However, there was no pallor, no neck vein distention,
no apparent congenital malformations, no cardiac murmurs
and no clubbing. There was also no note of an anterior neck
mass.
Laboratory workup showed cardiomegaly with pulmonary
congestion, thoracic dextroscoliosis, and atheromatous aorta by
chest radiograph, and left ventricular hypertrophy by 12-lead
electrocardiogram (12-L ECG) (Figures 1 and 2), normocytic
normochromic anemia (Hgb 90 mg/dL), dyslipidemia, and
pre-renal azotemia (serum creatinine 123 mmoL). Electrolytes
on admission showed slight hyponatremia, hypokalemia,
and hypochloremia (serum Na 136, K 3.35, Cl 86). Blood gases
revealed partially compensated metabolic alkalosis with mild
hypoxemia. The patient was noted to be hypothyroid based
on elevated serum thyroid-stimulating hormone (TSH) and
markedly decreased serum free thyroxine (FT4). The exact
values are shown in Tables 1 and 2.
Upon admission to the wards, the patient was managed
as having congestive heart failure from cardiomyopathy
secondary to acquired hypothyroidism. Oral loop diuretics,
angiotensin-converting enzyme (ACE) inhibitors, beta-
blockers, statins, and levothyroxine were started. Electrolyte
correction was instituted. The sections of Endocrinology
and Cardiovascular Diseases were co-managing the patient
together with the General Medicine service.
He soon developed respiratory failure, upon which the
considerations were acute pulmonary congestion, nosocomial
pneumonia, to rule out an acute coronary event. He was later
Table 3. Summary of Peptide-Antipeptide Binding Specificity Assay of Affinity Purified IgY from Egg Yolks of Chickens
Immunized with Peptide 2
Peptide Cut-off Value
Peptide 1 Peptide 2 Pept ide 3 Peptide 4 Peptide 5 Pept ide 6
Mean Absorbance Reading 0.181 0.216 0.174 0.170 0.170 0.168 0.185
Anti-peptide bi nding (-) (+) (-) (-) (-) (-)
the putative Peptide 2-specific IgY. As expected, no putative
Peptide 2-specific IgY was observed in the IgY isolated prior
to peptide immunization. A gradual increase in the total
IgY concentration of the pooled samples was observed from
Weeks 1 and 2 up to Weeks 9 and 10. The IgY concentration
peaked at Weeks 5 and 6 at 0.409 mg/ml. However, a
decrease in the total IgY concentration was observed from
Weeks 7 and 8 and Weeks 9 and 10.
Figure 2. Total Protein Concentration of Putative Peptide 2-
specific IgY from Pooled IgY Isolates from Pre-immunization
Up to Week 10 After the First Immunization Dose.
Consistent with previous studies done by Chui et al and
Rahimi et al, IgY with activity against the target antigen was
produced and was detected starting on the first two weeks
after the first immunization. Chiu et al noted the presence of
IgY acting on formalin-inactivated and heat-inactivated
Mycobacterium avium starting Week 2.36 Rahimi et al also
reported that IgY with specific activity on Salmonella species
was observed starting on the first two weeks.37 Rahimi
further reported that titer increased in both the chicken sera
and egg yolk 1 week after administration of Salmonella
enteriditis whole cell antigens.37
In this study, the peak IgY concentration or optimum
density was observed after the second booster at weeks 5
and 6. This is inconsistent with the results of Lee et al, Chui
et al and Rahimi et al, wherein optimum IgY densities were
obtained at weeks 7, 8 and 9 respectively. This might be
attributed to differences in immunization schedules,
laboratory conditions and the composition of feeds given to
the chickens.
SDS-PAGE of Affinity Purified IgY
To assess purity and confirm the presence of the IgY
molecule in affinity purification products, SDS-PAGE was
done to the protein-containing eluates collected after
addition of elution buffer. Denaturing SDS-PAGE revealed
the presence of two distinct bands located at approximately
60 and 28 kilodaltons from Weeks 1 and 2 up to Weeks 9 and
10 (Figure 3). These bands, which have consistent
electrophoretic mobility with the bands observed in IgY
standard, can be inferred as the heavy and light chains of the
IgY. The molecular weights of the whole IgY, as well as the
molecular weights of its heavy chains and light chains, are
consistent with the approximate molecular weights reported
by Leslie and Clem, Akita and Nakai and Schade et al.4,29, 38
Figure 3. Lane 1: Kaleidoscope Molecular Weight Marker,
Broad Range; Lane 2: Pre-immunization; Lane 3: Weeks 1
and 2; Lane 4: Weeks 3 and 4; Lane 5: Weeks 5 and 6; Lane 6:
Weeks 7 and 8; Lane 7: Weeks 9 and 10; and Lane 8:
Standard IgY.
Determination of Peptide Specificity of Affinity Purified IgY
Among the peptides tested, only Peptide 2 gave an
absorbance reading that is higher than the cut off value
which is 0.185 (Table 3)
This means that the IgY that was purified through
affinity chromatography is specific for Peptide 2. This has
Immunoglobulin Y and Detection of Hepatitis B Surface Antigen
7
VOL. 46 NO. 1 2012 ACTA MEDICA PHILIPPINA
VOL. 43 N0. 4 2009 ACTA MEDICA PHILIPPINA 13
Heart Failure and Short Stature in a 43 year-old male
transferred to the intensive care unit (ICU) for ventilatory
support and closer monitoring. On bedside cardiac ultrasound,
there was a nding of eccentric left ventricular hypertrophy,
global hypokinesia with depressed overall systolic function
with concomitant spontaneous echo contrast on left ventricular
(LV) cavity suggestive of rheologic stasis, the ejection fraction
was 25%, with moderate mitral regurgitation, moderate
aortic regurgitation with aortic sclerosis, severe tricuspid
regurgitation with mild pulmonary hypertension, pulmonary
regurgitation, and minimal pericardial effusion or pericardial
fat pad. Cardiac enzymes were not consistent with an acute
coronary event (Table 3), however, intravenous (IV) heparin
(overlapping with oral warfarin) was still given to cover for
the presence of a possible LV thrombus as demonstrated
by rheologic stasis on cardiac ultrasound. Medications
were shifted to IV diuretics and inotropes; oral digoxin was
started. IV antibiotics were given for possible pulmonary
infection. The patient later on showed improvement, and was
eventually weaned off from ventilatory support, extubated,
Table 1. Initial Laboratory Results
WBC
RBC
HGB
HCT
MCV
MCH
MCHC
RDW
PLT
RETIC
SEG
LYMPH
MONO
EO
BASO
BLAST
CBC
Reference
Value
5-10
4-6
120-150
0.38-0.48
80-100 FL
27-31 PG
320-360 G/L
11.5-15.5%
200-400
0.005-0.015
50-70%
20-44%
2-9%
0-4%
0-2%
0%
Result
4.5
90
0.27
Inc
48
50
2
0
0
0
RBS
HGBA1C
BUN
CREA
ALB
TAG
HDL
LDL
TOTAL CHOL
AST
ALT
Alk po4
NA
K
CL
CA++
P
MG++
Reference
Value
3.9-6.1
4.27-6.07
2.6-6.4
53-115
34-50
0.34-1.7
0.91-1.56
1.1-3.8
4.2-5.2
15-37
30-65
140-148
3.6-5.2
100-108
2.12-2.52
0.74-1
Result
6.3
6.4
5.0
123
32
0.82
0.67
4.21
5.25
95
91
184
136.9
3.35
86
2.37
2.27
0.83
Blood chem.
Color
Transp
Sp Gravity
pH
Sugar
Protein
RBC
WBC
Cast
Epith cell
Bacteria
Mucus th
Crystals
Am urates
straw
Clear
1.010
8.0
NEG
NEG
0-1
0-2
Rare
Occ’l
Rare
Rare
Urinalysis
pH
pCO2
pO2
HCO3
O2 sat
FiO2
Temp
7.408
49.1
70
31.3
93.6
21%
36.9
ABG
PBS
Slight poikilocytosis,
acanthocytes, ovalocytes,
slight toxic granulation,
slight anisocytosis
Table 2. Thyroid Function Tests
Free T4
TSH
(0.8-2.0)
(0.4-6.0)
0.02 ng/dL
24.75 Uiu/ml
Reference Value Result
Figure 1. Electrocardiogram upon admission
Table 3. Cardiac Enzymes
Qualitative
Troponin I
CK-MB
CK-TOTAL
0-6.0
21-232
POSITIVE
1.14
543
Reference Range (mmoL) Result
Figure 2. Chest radiograph on admission
been expected since the affinity chromatography procedure
was done to purify the Peptide 2-specific IgY.
Frey et al defined cut off as the upper prediction limit
derived from the negative controls.28 This cut off value is the
absorbance due to background, which is defined as the
reading obtained from the detection system alone exclusive
of any test sera, or in this case, the peptides coated in the
wells. Absorbance readings higher than the cut off value
mean positive peptide-antipeptide binding activity.
The major contributing factor in the specificity of the
affinity purified IgY is the amino acid sequence of the
peptides. Peptide 2, which was used to immunize the
chickens, is intended to generate an antibody which reacts
with it. This is also the reason why the affinity-purified IgY,
which was also purified using Peptide 2 as the capture
molecule in affinity chromatography, was specific to Peptide
2.
Summary and Conclusion
Consistent with the objectives of the study,
Immunoglobulin Y (IgY) with activity against synthetic
peptide analogs of the Hepatitis B Surface Antigen (HBsAg)
was isolated and purified from egg yolks of chickens
immunized with peptide antigens. Based on the indirect
ELISA, only the IgY isolated from Group P2 chickens,
immunized with synthetic peptide analog corresponding to
the 34th – 59th amino acid residue of the pre-S1 region of the
HBsAg, exhibited antipeptide activity. The IgY isolated
from chickens immunized with Peptide 1, the synthetic
peptide analog of the 139th – 147th amino acid residue of the S
region of HBsAg, did not exhibit antipeptide antibody
activity.
The putative Peptide 2 specific IgY was obtained from
affinity chromatography of IgY isolates from Peptide 2
Immunized Group. The affinity-purified IgY, which
demonstrated peak concentration during the 5th and 6th week
after immunization, showed specificity to Peptide 2 when
tested against a battery of synthetic peptides. SDS-PAGE of
the affinity-purified products showed two bands with the
molecular weights of approximately 60 kilodaltons and 28
kilodaltons. The electrophoretic profiles of the affinity-
purified samples are consistent with that of the IgY
standard. Moreover, the molecular weights of the two
bands as shown in the SDS-PAGE are consistent with the
reported molecular weights of the heavy and light chains of
the IgY molecule.
In conclusion, IgY with activity and specificity against
synthetic peptide analog of the immunogenic epitope of the
pre-S1 region was produced using a synthetic polypeptide
whose sequence is similar to the 34th – 59th residue of the pre-
S1 region. However, further investigations must be done to
improve efficiency of the immune induction and IgY
isolation technique to harness optimum amount of specific
IgY in chicken egg yolks.
_________________
Acknowledgement
The authors would like to thank the Office of the Vice President for
Academic Affairs, University of the Philippines System for the
financial support to the IgY Program Project through the Emerging
Fields in Science and Technology Grant.
__________
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Immunoglobulin Y and Detection of Hepatitis B Surface Antigen
8VOL. 46 NO. 1 2012
ACTA MEDICA PHILIPPINA
VOL. 43 N0. 4 2009
12 ACTA MEDICA PHILIPPINA
Heart Failure and Short Stature in a 43 year-old male
Katerina T. Leyritana1, Ma. Czarlota M. Acelajado-Valdenor1, Amado O. Tandoc III2 and Agnes D. Mejia1
1Department of Medicine, College of Medicine and Philippine General Hospital, University of the Philippines Manila
2Department of Pathology, College of Medicine, University of the Philippines Manila
CASE REPORT
Corresponding author: Ma. Czarlota Acelajado-Valdenor, M.D.
Department of Medicine
Philippine General Hospital
Taft Avenue, Manila, 1000 Philippines
Telephone: +632 554-8488
Email: czarlota@yahoo.com
Presentation of the case
This is a case of a 43-year-old male presenting with short
stature and heart failure. The patient was admitted at the
medicine ward of the Philippine General Hospital (PGH)
for dyspnea. This paper will investigate several issues:
differentiating congenital from acquired hypothyroidism,
the relationship between hypothyroidism and the
cardiomyopathies, and the therapeutic options in patients
with cardiomyopathy secondary to hypothyroidism.
The patient had been born full term to a then 31-year-
old Gravida 4 Para 3 (G4P3), the 4th of 9 siblings, with an
apparently unremarkable delivery at home facilitated by
a traditional birth attendant. He was noted to be normal
at birth. The patient was allegedly at par with age both
physically and mentally until eight years old when he was
said to have stopped growing in height. He was brought to
a private doctor, whose diagnosis was undisclosed, and he
was given medications to increase height, which the patient
took for only one month with no improvement. Through the
years, the patient was apparently well, although still of short
stature, with thick lips, coarse facial features and dry skin.
He was notably slow in ambulation. He was said to have
bronchial asthma at age 15 years, and since then he had been
taking salbutamol tablets occasionally for bouts of dyspnea
occurring one to two times annually.
The patient’s symptoms started in 2001 when he was
reported to have sudden loss of consciousness. During this
time, the patient did not have any symptoms of heart failure;
no prior seizures, cyanotic episodes, chest pain, headache, or
blurring of vision. He regained consciousness shortly after and
was brought to a private physician, whose assessment was a
“heart problem”. He was prescribed unrecalled medications
taken for a few months and eventually discontinued when
the syncopal episode did not recur.
In the next four years, the patient would develop
intermittent, progressive exertional dyspnea and bipedal
edema. Later on this would be accompanied by generalized
body weakness, anorexia, and constipation, severe enough to
require regular laxative use. There was also a report of two
more syncopal episodes. He was brought to another doctor
in a private hospital where the assessment was still a “heart
problem”. The patient was again prescribed unrecalled
medications and again was lost to follow-up. This time,
however, symptoms were persistent. He later consulted at
another local hospital, where he was admitted and managed
as a case of anemia and bronchial asthma. He was discharged
slightly improved after four days, only to have recurrent heart
failure symptoms, prompting admission at PGH.
Upon admission the patient was in mild respiratory
distress, with stable vital signs and no note of fever. Pertinent
physical exam ndings included short stature, thick lips, non-
pitting periorbital edema, dry skin, a displaced apical impulse,
crackles on both lung elds, and bilateral non-pitting bipedal
edema. There was also a 3 cm x 3 cm reducible umbilical
hernia. However, there was no pallor, no neck vein distention,
no apparent congenital malformations, no cardiac murmurs
and no clubbing. There was also no note of an anterior neck
mass.
Laboratory workup showed cardiomegaly with pulmonary
congestion, thoracic dextroscoliosis, and atheromatous aorta by
chest radiograph, and left ventricular hypertrophy by 12-lead
electrocardiogram (12-L ECG) (Figures 1 and 2), normocytic
normochromic anemia (Hgb 90 mg/dL), dyslipidemia, and
pre-renal azotemia (serum creatinine 123 mmoL). Electrolytes
on admission showed slight hyponatremia, hypokalemia,
and hypochloremia (serum Na 136, K 3.35, Cl 86). Blood gases
revealed partially compensated metabolic alkalosis with mild
hypoxemia. The patient was noted to be hypothyroid based
on elevated serum thyroid-stimulating hormone (TSH) and
markedly decreased serum free thyroxine (FT4). The exact
values are shown in Tables 1 and 2.
Upon admission to the wards, the patient was managed
as having congestive heart failure from cardiomyopathy
secondary to acquired hypothyroidism. Oral loop diuretics,
angiotensin-converting enzyme (ACE) inhibitors, beta-
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and Cardiovascular Diseases were co-managing the patient
together with the General Medicine service.
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