Detection of human Parvovirus B19 DNA by using the polymerase chain reaction

Department of Pediatrics, Children's Medical Center, Medical College of Virginia, Richmond 23298.
Journal of Clinical Microbiology (Impact Factor: 3.99). 02/1990; 28(1):65-9.
Source: PubMed
ABSTRACT
The polymerase chain reaction (PCR) was investigated for detecting human parvovirus B19 (B19) DNA in sera. Three pairs of oligonucleotides were evaluated as primers. The best oligonucleotide pair spanned 699 nucleotides, including the region common to VP1 and VP2. After PCR amplification of B19 DNA in serum, a 699-nucleotide DNA fragment was detected on agarose gels. This DNA fragment was B19 DNA, because after Southern transfer it hybridized to a 19-nucleotide internal probe and contained a single PstI cleavage site. Dot blot hybridization with a radiolabeled cloned portion of the B19 genome as a probe was compared with PCR. PCR was 10(4) times more sensitive than dot blot hybridization and, with an internal radiolabeled probe, 10(7) times more sensitive than dot blot hybridization. Of 29 serum specimens from 18 patients with proven B19 infections, 24 were PCR positive. None of 20 serum samples from uninfected controls were positive. Of 22 serum samples positive for immunoglobulin M to B19, PCR detected B19 DNA in 17. Seven serum samples lacking immunoglobulin M were PCR positive. PCR detected B19 DNA in urine, amniotic fluid, pleural fluid, ascites, and leukocyte extracts. PCR is a rapid and simple method for diagnosing infections with human parvovirus B19 but must be combined with serologic tests for immunoglobulin M to B19, especially when testing only a single serum sample.

Full-text

Available from: William C Koch, Nov 12, 2014
JOURNAL
OF
CLINICAL
MICROBIOLOGY,
Jan.
1990,
p.
65-69
0095-1137/90/010065-05$02.00/0
Copyright
©
1990,
American
Society
for
Microbiology
Detection
of
Human
Parvovirus
B19
DNA
by
Using
the
Polymerase
Chain
Reaction
WILLIAM
C.
KOCH*
AND
STUART
P.
ADLER
Division
of
Infectious
Disease,
Department
of
pediatrics,
Children's
Medical
Center,
Medical
College
of
Virginia,
Richmond,
Virginia
23298
Received
16
June
1989/Accepted
28
September
1989
The
polymerase
chain
reaction
(PCR)
was
investigated
for
detecting
human
parvovirus
B19
(B19)
DNA
in
sera.
Three
pairs
of
oligonucleotides
were
evaluated
as
primers.
The
best
oligonucleotide
pair
spanned
699
nucleotides,
including
the
region
common
to
VP1
and
VP2.
After
PCR
amplification
of
B19
DNA
in
serum,
a
699-nucleotide
DNA
fragment
was
detected
on
agarose
gels.
This
DNA
fragment
was
B19
DNA,
because
after
Southern
transfer
it
hybridized
to
a
19-nucleotide
internal
probe
and
contained
a
single
PstI
cleavage
site.
Dot
blot
hybridization
with
a
radiolabeled
cloned
portion
of
the
B19
genome
as
a
probe
was
compared
with
PCR.
PCR
was
104
times
more
sensitive
than
dot
blot
hybridization
and,
with
an
internal
radiolabeled
probe,
107
times
more
sensitive
than
dot
blot
hybridization.
Of
29
serum
specimens
from
18
patients
with
proven
B19
infections,
24
were
PCR
positive.
None
of
20
serum
samples
from
uninfected
controls
were
positive.
Of
22
serum
samples
positive
for
immunoglobulin
M
to
B19,
PCR
detected
B19
DNA
in
17.
Seven
serum
samples
lacking
immunoglobulin
M
were
PCR
positive.
PCR
detected
B19
DNA
in
urine,
amniotic
fluid,
pleural
fluid,
ascites,
and
leukocyte
extracts.
PCR
is
a
rapid
and
simple
method
for
diagnosing
infections
with
human
parvovirus
B19
but
must
be
combined
with
serologic
tests
for
immunoglobulin
M
to
B19,
especially
when
testing
only
a
single
serum
sample.
Human
parvovirus
B19
(B19)
causes
several
syndromes,
including
erythema
infectiosum,
chronic
arthritis
in
adults,
aplastic
crisis
in
patients
with
hemolytic
anemias,
fetal
death,
and
chronic
anemia and
neutropenia
in
immunocom-
promised
patients
(2,
3,
7,
10,
15, 17,
20, 22,
25).
The
virus
replicates
only
in
erythroid
precursor
cells
derived
from
bone
marrow
(17,
19).
In
vitro
culture
systems
producing
human
parvovirus
B19
have
not
been
developed.
Conse-
quently,
diagnostic
tests
for
this
infection
are
not
widely
available.
The
few
laboratories
that
do
these
tests
must
rely
on
antigen
obtained
from
the
serum
of
infected
individuals
(4).
In
addition,
we
and
others
have
observed
chronic
B19
infection
of
immunocompromised
patients
whose
serum
lacked
immunoglobulin
G
(IgG)
and
IgM
to
the
virus
(15,
16;
W.
C.
Koch
and
S.
P.
Adler,
J.
Pediatr.,
in
press).
Diagnosis
was
made
by
detecting
B19
DNA
in
their
sera
by
dot
blot
analysis.
To
circumvent
these
problems
in
diagnosis
and
because
the
polymerase
chain
reaction
(PCR)
is
sensitive
and
specific
for
detecting
several
other
viruses,
we
evaluated
the
PCR
for
the
detection
of
human
parvovirus
B19
in
serum
and
other
body
fluids
(1,
8,
13, 18,
23).
MATERIALS
AND
METHODS
Selection
of
sequences
for
primers
and
probes.
B19
has
a
single-stranded
DNA
approximately
5,400
nucleotides
long
(12).
The
B19
genome
encodes
two
capsid
proteins,
VP1
(84
kilodaltons)
and
VP2
(58
kilodaltons)
and
a
nonstructural
protein
(77
kilodaltons)
(11).
VP1
and
VP2
share
common
carboxy-terminal
amino
acids.
VP2
composes
96%
of
the
viral
capsid.
We
evaluated
three
sets
of
primers
and
probes:
one
set
located
within
VP1,
one
set
within
the
nonstructural
gene,
and
one
set
within
VP2
(Table
1).
Primers
and
probes
were
synthesized
on
a
DNA
synthesizer
(model
380A;
Applied
Biosystems,
Foster
City,
Calif.)
and
purified
by
high-pressure
liquid
chromatography
before
use.
*
Corresponding
author.
Amplification.
Amplification
of
target
sequences
occurred
in
a
500-,ul
polypropylene
microfuge
tube
with
a
total
reac-
tion
volume
of
100
,ul.
The
reaction
mixture
contained
200
,uM
each
dATP,
dCTP,
dGTP,
and
dTTP;
oligonucleotide
primers
(each
at
1
FjM);
50
mM
KCl;
10
mM
Tris
(pH
8.3);
2.0
mM
MgCl2;
0.01%
gelatin;
and
1
to
3
,ul
of
sample.
After
the
mixture
was
heated
to
94°C
for
3
min,
2.0
U
of
Taq
polymerase
(Perkin-Elmer
Cetus,
Norwalk,
Conn.)
was
added.
The
mixtures
were
overlaid
with
100
,ul
of
mineral
oil,
and
then
thermal
cycling
was
carried
out
in
a
programmable
heat
block
(Perkin-Elmer).
Each
cycle
consisted
of
2
min
at
94°C,
2
min
at
37°C,
and
3
min
at
72°C.
An
additional
7
min
was
added
at
the
end
of
the
cycling
to
complete
extension
of
the
primers.
Thirty-five
cycles
were
performed
in
6
h.
Detection
of
amplified
B19
sequences
in
reaction
products.
After
cycling,
10
,uI
of
each
amplified
mixture
was
electro-
phoresed
on
a
4%
agarose
minigel
(3%
NuSieve,
1%
SeaKem;
FMC
Corp.,
Rockland,
Maine).
After
gels
were
stained
with
ethidium
bromide,
they
were
viewed
under
UV
light
and
photographed.
Southern
transfer
and
hybridization
with
a
B19-specific
probe
were
performed
by
electrophoret-
ically
transferring
DNA
fragments
from
the
agarose
gels
to
nylon
membranes
(Nytran;
Schleicher
&
Schuell
Co.,
Keene,
N.H.)
in
TAE
buffer
(10
mM
Tris
hydrochloride,
5
mM
sodium
acetate,
0.5
mM
EDTA
[pH
7.8]).
Before
transfer,
the
gels
were
soaked
in
0.2
N
NaOH-0.5
M
NaCI
for
30
min,
followed
by
two
washes
of
10
min
each
in
500
ml
of
4x
TAE
buffer
and
a
final
10-min
wash
in
1
x
TAE
buffer.
After
the
membrane
was
presoaked
in
1
x
TAE
buffer
for
5
min,
the
membrane
and
gel
were
assembled
as
a
sandwich
between
filter
paper
and
placed
in
the
middle
slot
of
a
Trans-Blot
tank
(Bio-Rad
Laboratories,
Richmond,
Calif.),
and
2.5
to
3.0
liters
of
lx
TAE
buffer
was
added.
After
electrophoretic
transfer
overnight
at
250
mA,
the
mem-
branes
were
baked
at
80°C.
The
membranes
were
prehybrid-
ized
at
42°C
for
at
least
3
h
in
hybridization
solution,
which
contained
the
following:
6x
SSPE
(20x
SSPE
is
3.6
M
NaCl,
65
Vol.
28,
No.
1
Page 1
66
KOCH
AND
ADLER
TABLE
1.
Location
in
the
B19
genome
and
sequences
of
oligonucleotide
primer
pairs
and
probes
Primer
or
Sequence
(5'-3')
Product
length
Nucletide
Los.aGene
probe
(base
pairs)
~~~~~~~~~~~~~Nucleotide
nos.
Gene
K-1
primer
ATAAATCCATATACTCATT
699
2936-2954
VP1
K-2
primer
CTAAAGTATCCTGACCTTG
3617-3635
K-5
probe
CTAACTCTGTAACTTGTAC
3222-3240
K-6
primer
AAACTATGGTAAACTGGTT
442
1085-1103
NS1
K-7
primer
TGCTACATCATTAAATGGA
1509-1527
K-11
probe
TTACTTTGTCAAAACTATG
1321-1339
K-8
primer
AGCTACAGATGCAAAACAA
308
4702-4720
VP2
K-9
primer
TAACCACAACAAATGTTTA
4992-5010
K-12
probe
CTATAAGACAGCCTAACAC
4873-4891
200
mM
NaH2PO4
[pH
7.4],
and
20
mM
EDTA
[pH
7.4]),
1%
sodium
dodecyl
sulfate
(SDS),
10x
Denhardt
solution
(0.2%
bovine
serum
albumin,
0.2%
polyvinylpyrrolidone,
and
0.2%
Ficoil),
20
,ug
of
tRNA
per
ml,
and
50
,ug
of
sheared
salmon
sperm
DNA
per
ml.
Then
0.2
,ug
of
each
probe
was
end
labeled
to
a
specific
activity
of
at
least
2
x
108
with
gamma-labeled
[32P]ATP
by
using
T4
kinase
(Bethesda
Re-
search
Laboratories,
Gaithersburg,
Md.).
The
labeled
probe
was
purified
on
a
Nensorb
column
(Nensorb
20;
Du
Pont
Co.,
Wilmington,
Del.).
Labeled
probe
(5
x
107
cpm)
was
added
to
the
hybridization
solution
(6x
SSPE,
1%
SDS)
and
incubated
overnight
at
5°C
less
than
the
melting
temperature
of
each
probe.
After
hybridization
the
membranes
were
washed
in
6x
SSPE-1%
SDS
three
times
at
room
tempera-
ture
and
once
at
hybridization
temperature.
After
washing,
the
membranes
were
exposed
to
Cronex
X-ray
film
(Du
Pont)
for
3
to
18
h
at
-70°C
with
an
intensifying
screen.
Dot
blot
hybridization.
Dot
blot
hybridization
was
per-
formed
as
described
by
Clewley;
plasmid
pSP321,
which
contains
the
middle
one-half
(2.7
kilobases)
of
the
B19
genome
cloned
into
the
PstI
site
in
the
ampicillin
resistance
gene
of
pBR322
(9),
was
used
as
a
probe.
Briefly,
10
pul
of
sample
was
added
to
200
pul
of
2x
SSC
(0.3
M
NaCI,
0.03
M
sodium
citrate
[pH
7.0]),
followed
by
the
addition
of
200
puI
of
1
M
NaCl-0.1
M
NaOH.
After
10
min
at
room
tempera-
ture,
1.8
ml
of
2x
SSC
was
added
and
then
filtered
over
a
nitrocellulose
membrane
(Schleicher
&
Schuell)
that
had
been
prewet
with
20x
SSC
for
30
min.
The
membrane
was
washed
briefly
in
2x
SSC
and
baked
at
80°C
for
2
h
in
a
vacuum
oven.
The
membrane
was
prehybridized
for
4
h
at
65°C
in
10
ml
of
hybridization
solution
(6x
SSC,
0.5%
SDS,
1
x
Denhardt
solution,
100
,ug
of
sheared
salmon
sperm
DNA
per
ml).
The
membrane
was
transferred
to
fresh
hybridiza-
tion
solution
with
106
cpm
of
[32P]dCT-labeled
probe
(2
x
106
cpm/,ug).
The
filter
was
hybridized
overnight
at
65°C
with
mixing.
After
hybridization,
the
filter
was
washed
at
65°C
for
2
to
4
h
with
serial
changes
of
lx
SSC-0.1%
SDS,
allowed
to
dry
at
room
temperature
for
10
to
20
min,
and
exposed
to
X-ray
film.
Autoradiograms
were
developed
after
3,
18,
and
72
h
and
7
days
at
-70°C
with
an
intensifying
screen.
pBS321
was
labeled
with
[32P]dCT
by
nick
translation
(21).
Unincorporated
[32P]dCT
was
removed,
and
the
plas-
mid
was
concentrated
with
an
Centricon
30
filter
(W.
R.
Grace
and
Co.,
Danvers,
Mass).
Before
use,
the
labeled
plasmid
was
added
to
0.5
ml
of
sheared
salmon
sperm
DNA
(10
mg/ml),
placed
in
a
boiling
water
bath
for
3
min,
and
then
placed
on
ice
for
5
min.
Serologic
assays.
IgG
and
IgM
to
B19
in
human
sera
were
detected
as
previously
described
(4,
14).
Specimens.
Serum
samples
for
PCR
were
obtained
from
18
patients
at
Medical
College
of
Virginia
Hospital
with
hema-
tologic
problems
compatible
with
B19
infection.
Six
of
the
18
patients
had
at
least
onç
specimen
positive
for
B19
DNA
by
dot
blot
hybridization,
including
three
patients
with
sickle
cell
anemia
and
aplastic
crisis,
two
patients
with
acute
lymphoblastic
anemia,
and
one
patient
with
systemic
lupus
erythematosus
and
autoimmune
hemolytic
anemia.
The
other
12
patients
had
at
least
one
serum
sample
containing
IgM
to
B19;
8
of
these
12
had
sickle
cell
disease,
3
had
hereditary
spherocytosis,
and
1
adult
had
erthyma
infectio-
sum
with
arthritis.
Additional
B19-positive
sera
were
provided
by
B.
J.
Cohen
(Wi;
London),
A.
M.
Courouce
(REM,
DES,
and
LEC;
Paris),
and
N.
Young
(Minor;
Bethesda,
Md.).
Other
specimens
included
ascites
and
pericardial
fluid
from
a
hydropic
fetus
and
urine
and
peripheral
blood
mono-
nuclear
cells
from
a
patient
with
aplastic
crisis.
Peripheral
blood
mononuclear
cells
were
obtained
from
heparinized
blood
samples
by
using
Sepracell-MN
(Sepratech
Corp.,
Oklahoma
City,
Okla.);
5
,ul
of
the
cell
suspension
was
used
in
the
PCR
assay
without
prior
DNA
extraction.
For
amplification
of
sera
and
other
body
fluids,
1
to
3
,ul
was
added
directly
to
the
PCR
reaction
mixture.
Control
serum
samples
were
obtained
at
random
from
20
obstetric
patients
when
blood
was
drawn
for
other
purposes.
Ail
serum
was
stored
at
-70°C.
RESULTS
Each
of
the
three
primers
worked
effectively
for
the
detection
of
B19
DNA
(Tables
1
and
4).
Primers
Ki
and
K2
TABLE
2.
PCR
results
of
B19
antigen-positive
sera
tested
with
three
different
sets
of
primer
pairs
Results
with
primer
pair:
Serum
Yr
Location
Ki,
K2
K6,
K7
K8,
K9
Wi
1973
United
Kingdom
+
+ +
REM
1978
France
+
-
+
DES
1978
France
+
+
+
LEC
1979
France
+
+
+
Minor
1984
Ohio
+
+
+
KM
1987
Richmond
+
+ +
CS
1988
Richmond
+
+
+
KT
1988
Richmond
+
+
+
KA
1989
Richmond
+
+
J.
CLIN.
MICROBIOL.
Page 2
PCR
DETECTION
OF
B19
DNA
67
di
lution
4..30
300
Se
rum
Plasmid
ONA
10000
0.6rig
70Pg
FIG.
1.
Ethidium
bromide-stained
agarose
gel
of
PCR
amplified
reaction
products
of
two
different
sera
(lanes
1
and
2
and
lanes
3
and
4).
The
left
lane
(unlabeled)
contains
base
pair
(bp)
marker
frag-
ments,
with
the
sizes
of
two
fragments
indicated.
Undigested
(-)
reaction
products
for
both
serum
samples
contained
a
699-base-pair
fragment
when
Ki
and
K2
were
used
as
primers
(lanes
1
and
3)
and,
for
one
of
the
two
serum
samples,
a
442-base-pair
fragment
when
K6
and
K7
were
used
as
primers
(lane
5).
For
both
serum
samples
after
PstI
digestion
(+),
two
fragments
of
the
predicted
length
(194
and
505
base
pairs)
appeared
when
Ki
and
K2
were
the
primers
(lanes
2
and
4),
but
the
K6-K7-primed
fragment
lacked
a
PstI
restriction
site
(lane
6).
were
selected
for
complete
characterization
of
the
PCR
reaction.
To
find
optimal
reaction
conditions,
three
anneal-
ing
temperatures
(37,
40,
and
42°C)
and
three
different
numbers
of
total
cycles
(25,
30,
and
35)
were
investigated.
The
combination
of
35
cycles
and
37°C
produced
the
best
level
of
sensitivity
and
specificity.
The
expected
amplified
product
of
699
base
pairs
was
observed
after
amplification
of
a
dot-blot-positive
serum
sample
(Fig.
1).
PstI
digestion
of
the
699-base-pair
fragment
generated
by
primers
Ki
and
K2
in
two
serum
samples
yielded
a
194-base-pair
fragment
and
a
505-base-pair
fragment,
as
predicted
from
the
nucleotide
sequence
(Fig.
1).
PCR
performed
with
primers
K6
and
K7
yielded
a
DNA
fragment
of
the
predicted
size,
442
base
pairs,
that
lacked
a
PST1
site
(Fig.
1).
None
of
the
primer
sets
produced
B19-specific
DNA
fragments
when
either
cytomegalovirus
DNA,
simian
virus
40
DNA,
or
human
cellular
DNA
(from
MRC-5
fibroblasts
and
from
lympho-
cytes)
was
used.
To
determine
the
sensitivity
of
PCR,
PCR
was
compared
with
dot
blot
hybridization.
The
serum
from
a
patient
with
aplastic
crisis
due
to
human
parvovirus
B19
was
serially
diluted.
The
dot
blot
assay
detected
approximately
0.2
ng
of
B19
DNA
in
10
,uI
of
the
diluted
serum
with
an
exposure
of
the
nitrocellulose
filter
for
7
days
at
-70°C
(Fig.
2).
In
contrast,
when
1-pul
samples
of
the
same
dilutions
of
this
serum
were
amplified
by
using
PCR
and
the
agarose
gels
were
stained
with
ethidium
bromide,
B19
DNA
was
detected
in
the
1:107
dilution
(Fig.
3A).
When
the
agarose
gel
was
probed
with
the
internal
probe
K5,
both
single-
and
double-
stranded
fragments
were
detected
in
a
1:1010
dilution
of
this
serum
with
an
exposure
at
-70°C
for
3
h
(Fig.
3B).
There-
fore,
with
ethidium
bromide
staining
the
PCR
detected
approximately
0.02
pg
of
B19
DNA,
and
with
a
radiolabeled
internal
probe
the
PCR
detected
0.02
fg
of
B19
DNA.
Dot
blot
hybridization
and
PCR
were
compared
by
using
15
serum
samples
from
six
patients
with
B19
infections.
All
450ng
FIG.
2.
Autoradiogram
after
dot
blot
hybridization
of
plasmid
pSB321
and
serum
containing
B19
virus.
Serum
and
plasmid
were
diluted
in
2x
SSC,
and
10
,ul
of
each
dilution
was
denatured
and
filtered
over
nitrocellulose.
For
the
serum
the
fold
dilution
is
indicated
above
three
of
the
dots,
and
for
the
plasmid
the
quantity
of
DNA
on
each
dot
is
indicated
below
three
of
the
dots.
32P-labeled
pSP321
was
the
probe,
and
the
nitrocellulose
sheet
was
exposed
to
X-ray
film
for
1
week
at
-70°C.
six
patients
had
at
least
one
serum
sample
positive
for
B19
DNA
by
dot
blot
hybridization.
Of
11
dot-blot-positive
serum
samples,
all
were
also
positive
by
PCR.
Of
four
serum
samples
negative
by
dot
blot
hybridization,
three
were
positive
by
PCR.
One
serum
sample
was
negative
by
both
tests.
In
addition
we
assayed
20
serum
samples
from
20
pregnant
women
selected
randomly.
All
20
serum
samples
lacked
IgM
to
B19,
and
8
contained
IgG
to
B19.
All
20
serum
samples
were
negative
by
dot
blot
hybridization
and
also
negative
by
PCR.
Twenty-nine
serum
samples
from
18
patients
with
proven
B19
infections
were
assayed
for
IgM
and
IgG
to
B19
and
by
PCR
(Table
3).
Of
22
serum
samples
that
were
IgM
positive,
17
were
PCR
positive.
All
five
serum
samples
(from
five
B
Log
dil
ution
-11
-10
-9
-8
-
7
-6
FIG.
3.
PCR
amplification
of
serially
diluted
patient
serum.
Samples
(1
,uI)
of
the
same
dilutions
of
the
serum
used
for
dot
blot
hybridization
(Fig.
2)
were
amplified
with
primers
Ki
and
K2.
After
amplification
and
agarose
gel
electrophoresis,
the
gel
was
stained
with
ethidium
bromide
(A).
The
DNA
fragments
of
this
gel
were
transferred
to
a
nylon
membrane
and
probed
with
32P-labeled
internal
probe
K5
(B).
Ethidium
bromide
stained
only
the
double-
stranded
fragment
(B)
in
serum
diluted
i0-'.
The
radiolabeled
probe
detected
single-
and
double-stranded
fragments
in
serum
diluted
10-10
after
3
h
of
exposure
to
X-ray
film
at
-70°C.
VOL.
28,
1990
Page 3
68
KOCH
AND
ADLER
TABLE
3.
Association
of
IgM
positivity
and
PCR
in
sera
of
patients
infected
with
B19
No.
of
serum
samples
by
PCR
result
by:
IgM
status
Positive
Negative
Ethidium
bromide
staining
Positive
13
7
Negative
9
O
Radiolabeled
probe
Positive
17
7
Negative
5
°
patients)
that
contained
IgM
but
were
PCR
negative
were
obtained
during
the
convalescent
phase
of
illness
(.3
days
after
the
onset
of
symptoms)
and
contained
IgG
to
B19.
Seven
serum
samples
from
five
patients
lacked
IgM
to
B19,
and
all
were
PCR
positive.
One
of
these
five
patients
was
immunocompromised
and
had
an
impaired
ability
to
make
IgG
and
IgM
to
B19
(Koch
and
Adler,
in
press).
The
other
four
serum
samples
that
lacked
IgM
but
were
PCR
positive
were
from
four
patients
with
sickle
cell
disease
and
aplastic
crisis.
Each
of
these
four
serum
samples
was
obtained
during
acute
infection
(<4
days
after
the
onset
of
symptoms)
and
lacked
IgG
to
B19.
Of
24
serum
samples
positive
by
PCR,
20
were
positive
when
only
ethidium
bromide
was
used
to
detect
B19
DNA
after
amplification
(Table
3).
Four
serum
samples
were
positive
only
after
a
radiolabeled
probe
was
used
for
detec-
tion.
These
four
serum
samples
(from
four
patients)
were
obtained
during
the
convalescent
phase,
and
each
contained
IgG