From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
Application
of the
for
Enzyme
the
Linked
Detection
of
By M. Gudino
Many
methods
antibody.
complex
for
enzyme
ies
in
serum.
either
use.
Therefore.
for
The
of
anti-human
lgG,
color
change.
Assay
formaldehyde
fixed
M
is
versus
ANY
LABORATORY
let antibody
are
fixation,’
‘4C labeled
ment
factor
in the
3 release,
detection
bodies.
Fab,89
unfixed
platelets.
nofl
protein
detection
is a modification
all
for
the
of
are
ease
antibody.
assay
and the
with circulating
We
assay
here
the
to
(EL
The
Region,
St.
Submitted
Address
Red
Cross
Boulevard,
(C) 1981
Louis.
April
reprint
Blood
Red
Blood
9, 1980;
requests
Services,
test
Services,
purpuand
was
(LCT)
(PIIFT)
speci-
Also.
(ELISA)
for
4
2 of 2
neonatal
Sensitivity
respectively.
assay
2 of
purpura.
lymphocytotoxicity
commercial
were
Diagnostica,
enzyme
found
to
and
platelet
platelet
be
antibody
METHODS
of
plasma
group
g for
EDTA
for
0
10 mm)
a
of
of the
of patients
wash
step
Missouri-Illinois
was
were
at
without
to
Ind.,
and
less than
0.1%
0.009
discarded
three
times
lOT/cu
were
the erythrocyte
the
fixed,
platelet
sent to our
laboratory
and
at
4#{176}C
for
up to
suspension
contamination
routinely
was
of an
prepared
for testing
Counter
fixed
count
stored
platelet
by a Coulter
1%
the
consisting
were
performed
M
6.8-7.0).
and
Platelets
to be suitable
of
were
each
donors
testing.
found
The
0.0264
pH
platelets
pools
four
for
the platelets
described
Four
from
needed
at 1900 g
and
EDTA,
as before.
mm.
donors
ml of 15%
saline:
M Na2
When
contamination
and
on counts
was
as previously
platelets
(0.07
(phosphate-buffered
used
of
Leukocyte
in EDTA
typed
centrifugation
PR P was centrifuged
twice.
4#{176}C
until
preservative
drawn
repeated
x
1-ILA
differential
was
washed
14 days.
16 healthy,
The
M NaCI,
to 7.5
number
based
conjugated
Elkhart,
after
supernatant
0.14
adjusted
stored
blood
blood).
in PBS-EDTA
platelets
espe-
of fresh
paraformaldehyde’6
equal
from
harvested
at 20#{176}C,
the
resuspended
then
phosphatase
laboratories,
Finland).
(PRP)
was
per 7 ml of whole
7 mm
This
an
(Miles
Preparations
blood
(250
of alkaline
used
Helsinki,
Platelet-rich
advan-
details
AND
sources
lgG
Platelet
Mo.
was
was
less than
Model
1%
ZBI.
Sera
Serum
tion
samples
of
platelet
antibodies
samples
in which
clinical
platelet
antibody
detection
who
antibody.
were
were
information
from
to random
for the determina-
We
and/or
strongly
Sera
refractory
studied.
supported
nine
other
donor
selected
another
10
the
for
presence
of an
polytransfused
platelet
such
technique
transfusions
patients
were
also
A2,
and
referred.
accepted
to
Dr.
August
Maria
Missouri-Illinois
St. Louis.
Mo. 63108.
by Grune
c Stratton.
Inc.
0006-497l/8l/5701_0007$0l.00/0
32
Cross
including
2 of 3 with
98%.
time
in 1 6 of
identification.
Orion
ISA)
ELISA
results
of our investigation
platelet
antibody.
the American
and
the
incubation
obtained
diseases
patients.
immunospecific
superior
antiplatelet
From
various
purpura.
84%
were
thrombocytopenic
all 9 polytransfused
were
and
results
with
idiopathic
post-transfusion
and
Two
time
in which
make
report
patients
anti-human
cially
suitable
for determinations
that are performed
outside
large
centers.22
23 Therefore,
we developed
modification
of the assay
for the serologic
detection
platelet
with
Na2HPO4.2H20,
radiolabel.
of performance
of
concentration
Positive
Conjugate
that
it is less expensive
to perform
and
less
sophisticated
equipment;
a simple
is adequate
for testing.
The stability
of the
and
substrate
MATERIALS
these
very
of the radioimmunoassay
is substituted
reagents
para-
A2’ have been
in other types of
and
1 9 sera
patients
mmunoas-
However,
procedures
are difficult
to set up
consuming.
The enzyme
linked
immuno-specific
and
investigated.
conjugate
radioi
6- 1 7
thrombocytopenia.
tages
are
requires
photometer
of
were
ra,
(ELISA)
V. Miller
immunofluorescence
antibody
use
TESTS
to detect
plateavailable
including
compleserotonin
release,23
platelet
say,’82#{176}and ‘251-staphylococal
reported
useful
for antibody
enzyme
by a
Assay
Antibodies
linked
reaction
the
the
W.
ficity
labeled
techniques
that
include
Fab-antilysis inhibition,’#{176}’4 immunoperox-
mmu
immune
to
as
and
with
by
and 51Cr release.67
These
are useful
of HLA,
drug dependent,
and isoanti-
Recent
complement
IS
platelets
is shown
enzyme
proportional
such
an
antibod-
is indicated
Platelet
dilutions.
too
developed
phosphatase)
product
conditions
or
incubating
of the
reaction
which
concentration.
have
antibody
by assay
The
platelet
of platelet
(alkaline
followed
identify
sensitive
we
detection
attached
enzyme
its substrate.
very
involves
any
an
to
not
the
method
antibody;
addition
described
are
general
serum
with
been
they
immunoassay
with
the
have
but
Immunospecific
27, 1980.
Gudino,
Region,
Positive
the
American
4050
Lindell
control
a polyspecific
made
included
of sera
antibody)
obtained
from
AB,
had never
who
nontransfused
an anti-PV”,
antiserum.
a pool
healthy
from
sera
HLA
A
(negative
five
received
males
an anti
negative
for red cell
healthy
a blood
were
male
HLA
“control”
serum
was
and lymphocytotoxic
donors
transfusion.
of blood
Sera
group
from
50
also tested.
Blood,
Vo). 57, No.
1 (January),
1981
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
ELISA
FOR PLATELET
Performance
The
ofihe
ELISA
platelets.
cells
was determined
The
of
the principles
the
described
tubes,
serum
suspension
was
for
7
mm
at
discarded.
room
Platelets
buffer
by
adjustable
pipetter.
The
were
platelets
1% BSA.
0.2
After
ml
conjugated
was
buffer
incubated
at
were
freshly
prepared
mg/mI,
pH
I
M
was
I 5 mm.
NaOH.
The
300 N)
tests
was resuspended
reaction
for
mm,
45
after
platelet
mixture
incubated
I
room
0. 1 ml
p-nitrophenolate
from
in a spectrophotometer
optical
positive
the
at
by adding
density
control
(OD)
serum
>
control
serum
2 SD above
on the OD
was calculated
from
that
the mean
values
with
of the negative
of 50 healthy
normal
the
controls,
was
tested.
the OD
test
serum.
which
was
donors,
was considered
by
standard
were
lymphocyte
also
screened
immunofluorescence
Borne.’6
To
insure
lymphocytes
used
for
cytotoxicity
indirect
the
inhibited
platelet
cell and
leukocyte
contamination
careful
aspiration
of the
technique
and
and
by
as described
in the
among
platelets
PIIF1’,
(LCT)25
the
test (PIIFT)
uniformity
and/or
for the LCT,
antibodies
results
from
the
same
the
the
NIH
platelet
by Van
dem
three
tests,
individuals
were
ELISA.
Minimal
was
top
3/4
red
best
of the
using
PRP
blood cells. Occasionally,
step was repeated
during
normal
pools
in
reduce
and
the loss of about
by not
fixing
the
the
procedure
time
sera
fixed
unfixed
one
were
plate-
platelets
fixation
50% of the
suspension
about
and by
10 mm)
the second
the platelet
patient
we have used
Paraformaldehyde
by
following
of paraformaldehyde
lets but subsequently
with
equal
success.
resulted
Moreover,
sera
blood
achieved
red
Nonspecific
platelets.
we could
hour.
the development
of the technique,
various
of the assay
were studied
to optimize
the
between
the negative
and positive
reference
Binding
The OD value
obtained
with
platelets
incubated
with normal
serum
represented
nonspecific
conversion
of the substrate.
The presence
of platelets
made
the
greatest
contribution
to nonspecificity;
60%
of the
nonspecific
IgG
that
OD was due
was routinely
in the
33%
attributed
to
incubation
nonspecificity
to normally
bound
platelet
present
despite
three
wash
preparation
Approximately
of the
of
the
adsorption
of
IgG
with
normal
serum.
was due to adsorption
tube,
while
about
to the test
by platelets
platelet
nonspecific
by
suspension.
binding
platelets
About
2%
of serum
was
upon
of the
IgG to
5% represented
adsorption
of
Nonspecific
minimized
conversion
by washing
of
the
tube.
was
platelets
3 times
before
and 2 times
after
incubation
with serum.
Reduced
serum
and conjugate
adsorption
to the test tube was achieved
by albumin
coating
of the
test tubes
prior
testing
and by including
I % BSA in
the wash
If the
binding.
to sediment
centrifugation
the test
sera.
Platelet
antibody
of anticoagulated
whole
blood,
centrifugation
of PRP (250 g for
conjugate
substrate
RESULTS
During
conditions
difference
were not treated
serum
OD
was
reference
serum
when
the same
centrifugation
another
slow
cycles
positive.
Sera
by three
reference
reading.
by substracting
obtained
serum.
For example,
when
platelets
with
NH4CI
the positive
reference
0.614
as compared
to the negative
OD of 0.089.
On the other
hand,
studied
0.2 ml of
phosphate,
was stopped
product,
which
reference
serum.
Removal
from a platelet
preparation
NH4C1
for 5 mm, followed
the OD of the positive
wash procedure.
In this study,
IgG
mixture
button,
(p-nitrophenyl
quantitated
and
containing
The
final
a
in duplicate.
for the test serum
of negative
based
button
in
present.
was added.
to obtain
a negative
performed
resulted
aggregates
l
of ELISA
OD
An OD
nm,
a 200
anti-human
the
reaction
with
to
of
reaction
was
attached
volume
the
and
The
at 405
assay,
were
Evaluation
The
added
serum
PBS-EDTA
platelet
of test
at l900g
centrifugation
tip
with
solution
phosphate,
In each
All
substrate
been
supernatant
times
equal
To
had
volume
negative
red cells
platelets
were
treated
with
NH4CI,
the OD values
were
0.068
and
0.053,
respectively.
It therefore
appeared
that NH4C1
treatment
of platelets
somehow
at 4#{176}C,
0.2 ml
resuspension
temperature
twice.
9.8)
p-nitrophenyl
(Gilford
room
washed
the
no visual
the
an
as the
than
centrifuged
with
phosphatase
for
temperature
wash,
with
used
that
an equal
then
of
three
and
to alkaline
platelets
of
washed
mm)
following
method
the final
of
were
a pipet
suspension
were
overnight
and
with
on
physically
in suspension
75
with
cells
resuspended
This
platelet
in
incubated
pipeting
homogeneous
x
(BSA)
temperature
were
gentle
Perlman2324
antigen
times
(12
Albumin
for 30 mm at 37#{176}C.
The
was based
methods.
tubes
Serum
and
using
platelets
ELISA
polystyrene
Bovine
of platelet
incubation
in other
In disposable
with
whole
shorter
antiplatelet
procedure
the
by treatment
with
washes,
decreased
antiglobulin.
to be the
of
target
to the platelets
conjugated
by Engvall
instead
sera with
bound
assay
we used
Also,
oftest
lgG
considered
The
developed
previously
coated
was
modifications:
antigen.
the
phosphatase
binding
originally
to the
target
and
in the test sera.
following
adsorbed
incubation
washed
alkaline
lgG
activity
involved
were
using
degree
antibody
obtained
with
of contaminating
Assay
procedure
The
33
ANTIBODIES
buffer.
Preparation
platelet
preparation
was
contaminated
with
more
than
1 % red blood
cells,
the OD
difference
between
the positive
and negative
control
sera was less
than
expected.
This
was due to the high OD value
Conjugate
The
Studies
optimal
conjugate
dilution
block
titration
for each new
example
of such a titration
was
determined
by
batch
of conjugate.
One
is illustrated
in Table
I.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
34
GUDINO
Table
1 . R atios
Refere
of Positive
nce Serum
Reference
OD Using
Serum
Two
OD to Negative
Different
Table
2.
Effect
of D ifferent
Substr
ate
Dilution
A
Serum:undil
1:50
Conjugate
Serum
12.09
1 :2
B
Serum:undil
Serum
8.32
18.36
16.71
9.55
5.14
15.65
9.18
1:150
7.07
4.79
12.35
8.75
1:200
6.22
3.50
11.38
6.70
normal
1:250
4.45
3.31
8.89
5.60
different
(different
preparations
lot number)
A was
160
were
U/mI
represent
ratios
over
that
obtained
and
conjugate
and
tested.
dilution.
used for conjugate
density
commercial
activity
B, 270
reading
serum
conjugate
1 : 100 and
1:200,
The
values
reference
serum
at different
serum
dilution
subsequently
respectively.
Platelets
used in this experiment
were not treated
with
paraformaldehyde.
A fixed number
of platelets
(1 .5 x
l0) was tested
with a positive
and a negative
reference serum
in a series
stock conjugate.
The
high
OD
with
and
a low OD
after
1 5 mm
for subsequent
the positive
serum
a negative
of substrate
of the
dilution
(e.g.,
serum
incubation
serum
and the
that yielded
a
1 .0 or greater)
(e.g.,
time
0.2
was
The largest
difference
serum
OD
and the
or less)
selected
OD
was
recorded
at the highest
of conjugate
tested
(Table
1 ), but it
the spectrophotometer.
Also, at low conjuthe negative
reference
serum
OD was
0.2. Consequently,
we selected
a dilution
1 :200 for conjugate
A and B, respectively
1 ) since it permitted
introducing
a dilution
possible
the cost
dilution
of
of this reagent
of the
present
Substrate
investigated.
0.134
1.731
0.119
1 mg/mI
1.023
were
serum.
sensitized
Following
substrate
with
the
1
0.067
:2 dilution
incubation
with
were
tested.
concentrations
of anti-P
1‘
1 : 1 00
diluted
mm.
At
incubation
times
was stable.
However,
the
mm) was chosen
to reduce
of I 5 and
or AB
conjugate.
30 mm,
the
ratio
We also investigated
different
substrate
concentrations,
from
I mg/mi
to 4 mg/ml
(Table
2). Higher
substrate
concentrations
resulted
in overloading
of
spectrophotometer
and therefore
the lowest concentration ( 1 mg/ml)
was chosen
that also lead to reagent
economy.
Substrate
concentration
was not the limiting
factor
for the development
of color, since with increasing incubation
time
and
with
increasing
antibody
concentrations,
greater
OD
values
were
obtained.
Substrate
37#{176}C
gave
incubation
at
similar
results.
room
temperature
and
at
Specificity
To
ELISA
demonstrate
reaction,
the
immunospecificity
we studied
the absence
with platelets
The mean
lacking
(range)
technique.
0 to 60 mm were
were noted
as the
substrate
incubation
time
increased.
However,
the
difference
between
the positive
and negative
reference
serum
OD (expressed
in terms
of a ratio)
decreased
when
the incubation
time
was extended
beyond
30
of
of antigen
the
or
platelet
antibody
in the assay.
As shown
in Fig. 1 , a
much
higher
OD was obtained
when
platelets
were
incubated
with antiplatelet
serum
as compared
to the
the conjugate
we could
per assay.
Moreover,
we
times
from
OD values
serum
shorter
incubation
time ( IS
the overall
procedure
time.
upon
incubation
with
was low when an antibody
Studies
incubation
Increasing
2.243
2mg/mI
Platelets
four
pools
(0-0.04),
of fixed
0.013
normal
serum.
was incubated
the corresponding
antigen.
OD value of 49 normal
sera
platelets
(0-0.05),
were
and
0.005
0.013
with
(0-0.03),
0.006
(0-0.05).
With
one additional
normal
serum,
an OD greater
than the
mean
+2 SD of normal
was obtained
with each
pool
and
was considered
a false
positive
reaction.
This
serum
was also tested
for red cell antibody
and for
platelet
antibody
by LCT and PIIFT
and was found to
be nonreactive.
We also
single
unit rather
than
(not
Substrate
4mg/mI
OD obtained
Also, the OD
a strong
yellow
color
that
could
not be
visually.
Visual
assessment
of the test
felt to be important
since it may facilitate
the application
Control
the measurement
of OD
step. Also, by using the
were even able to visually
interpret
the results
at these
conjugate
dilutions.
Using
diluted
conjugate,
normal
sera imparted
no visually
detectable
color in contrast
to positive
sera. On the other
hand,
at low conjugate
dilution
(e.g.,
1:50),
both
positive
and negative
sera
manifested
differentiated
results
was
Control
between
negative
serum
gate dilution,
greater
than
of 1 : 100 and
highest
reduce
a positive
with
testing.
reference
reference
concentration
overloaded
(Table
without
of dilutions
conjugate
Negative
Concentration
source
for the conjugate
U/mI.
of positive
reference
working
A and B was
a single
conjugate
a negative
The
from
The enzyme
the
for
of optical
with
obtained
Values
Positive
1:2
1:100
Two conjugate
Concentrations
OD
Substrate
Conjugate
MILLER
Conjugate
Preparations
Conjugate
AND
fixed
with
studied
pooled
24 normal
sera from
platelet
preparations
paraformaldehyde)
and
the
mean
OD ± SD was 0.134
±
0.037.
It was apparent
from
these experiments
that normal
control
OD values
were
higher
with unfixed
platelets
compared
to fixed preparation
but correspondingly
higher
OD values
were also
found
with
positive
remained
unaffected.
The reproducibility
sera.
Therefore,
of ELISA
is shown
overall
results
in Table
3.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
ELISA
FOR PLATELET
35
ANTIBODIES
Table
1.4
4.
A Comparative
Platelet
Study
Antibody
of ELISA
with
Detection
1.3
Results
Patient
1. 2
1.1
Idiopathic
thrombo-
cytopenic
purpura
Chronic
1.0
0.9
>
the Standard
Methods
Neonatal
PIIFTt
LCT
ELISA
G.M.
-
-
-
S.H.
-
-
+
MB.
-
+
+
J.W.
-
-
-
Isoantibodies
13
Post-transfusion
purpura
0.7
0.6
Neonatal
purpura
0
0.5
Associated
other
0.4
G.V.
-
+
+
LB.
-
+
+
C.G.
-
-
-
5.5.
+
+
+
CS.
-
-
+
J.F.
-
+
+
H.K.
+
+
+
J.B.
-
+
+
E.J.
+
+
+
K.F.
-
+
+
D.C.
-
-
+
MW.
-
+
+
L.J.
-
+
+
J.B.
+
+
+
JR.
+
with
diseases
Polytransfused
0.3
Acute
leukemia
0.2
0.1
0
Und
2
Reciprocal
4
Dilution
8
ofthe
Lymphoma
(Hodgkins)
Serum
Paroxysmal
Nocturnal
Specificity
of antigen
and antibody
reaction:
low
conversion
was evident
with normal
serum
(‘)
as
to
or polyspecific
anti-HIA
serum
Also. absence
of HLA-A2
antigen
(-U)
resulted
in low
Fig.
1.
substrate
compared
(0-0).
values
as compared
allowed
to react
to its presence
against
anti
(--‘4)
HLA-A2
when
platelets
were
serum.
Hemoglobinuria
Nontransfused
Lymphoma
Total
positive
Total
negative
LCT, lymphocytotexicity
Patient
fPIIFT.
Sera
Nineteen
sera
from
patients
three
techniques.
The
all three
techniques
were
assayed
by
clinical
diagnosis
and results
are listed
in Table
4 and
of LCT
(26%)
Table
3.
and
PuFF
(68%).
Reproducibility
of
the
Positive
Day
Replicate
specific-
Replicate
0.830
0.803
0.816
0.802
1
0.089
0.074
Day 6
0.070
0.085
Day6
Negative
Day
Platelets
HLA-A2
control
from
serum
A 1 : 100
2
control
1
HLA-A2
(positive
conjugate
used.
Platelets
were
under
identical
assay
positive
control)
dilution
stored
and
donors
were
AB serum
1 mg/mI
substrate
at 4#{176}C
and experiment
conditions.
incubated
or normal
(100%),
with
(negative
anti
control).
concentration
repeated
on sixth
and
16
14
6
3
test.
immunofluorescence
technique
was
similar:
PIIFT
(94%)
(Table
test.
was
day
ELISA
(98%),
lIP
were
was positive
LCT
6).
Idiopathic
thrombocytopenic
purpura
(fTP)
tients.
Sera
from
three
patients
with
chronic
and serum
(J.W.)
from the mother
of a patient
MB.)
were positive
Post-transfusion
Vslues
1
ity of each
neonatal
(M.B.)
of ELISA
00
Sara
The
indirect
+
13
all
reactivity
of each platelet
pool by ELISA
is shown
in
Table
5. The sensitivity
of ELISA
was superior
(84%)
to that
platelet
+
5
palIP
with
negative
by LCT;
one serum
with
PIIFT
and two (S.H.
and
with ELISA.
purpura
patients.
Serum
from
both
strong
PIIFT
patients
with
post-transfusion
purpura
gave
positive
results
with all four platelet
pools
by
and ELISA.
Negative
reactions
were seen by
LCT.
with
One sample
gave negative
results
platelets
from
five p1Ai
negative
other
serum
because
was
of an
not tested
with
insufficient
PP’
amount
when
donors.
negative
available
tested
The
platelets
for
test-
ing.
Neonatal
purpura
from
patients
with
patients.
neonatal
Three
purpura
maternal
sera
were
studied.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
GUDINO
36
Table
5.
Results
of ELISA
in
Antibod
Clinical
Disorders
y Against
Assciated
With
Pool
Patient
DISCUSSION
Pool 2
1
Pool
3
the
Pool 4
thrombocytopenic
purpura
G.M.
-
-
-
S.H.
+
+
-
-
MB.
-
-
+
-
J.W.
-
-
-
-
G.V.
+
+
+
+
LB.
+
+
+
+
C.G.
-
-
-
-
5.5.
+
+
-
+
C.S.
-
+
+
-
-
Neonatal
purpura
Associated
ble;22 and one
sent
a hazard
ent
but
diseases
polytransfused
Acute
leukemia
Lymphoma
(Hodgkin’s)
Paroxysmal
J.F.
+
-
+
+
H.K.
+
-
-
+
J.B.
-
+
-
-
E.J.
+
+
-
-
K.F.
+
+
+
+
D.C.
+
+
+
-
MW.
+
-
-
+
L.J.
+
+
+
+
J.B.
+
+
+
+
lymphoma
JR.
Total
negative
One
sample
techniques;
HLA-B5
(5.5.)
this
cross
.ample
(C.S.)
and
the third
gave
serum
reactive
patients
gave
gave
positive
positive
parous
positive
+
+
10
10
6
7
9
9
results
only
nonreactive
Eight
positive
results
reactions
by
of
6.
nine
by all three
as
Bl8.
an anti
Another
Sensitivity
plasma
assessing
polytransfused
and Specificity
Controls
14
IgG
multistrongly
50
and
of the Three
Tee hniques
for
Patients
Platelet
Anti
body
Identification
ELISA
Controls
6
47
Patients
Controls
3
49
-
19
50
--
19
50
68%
84%
Specificity
100%
94%
98%
Lymphocytotoxicity
Platelet
indirect
test.
immunofluorescence
test.
the
in these patients.
It is possible
that
as well the use of an anti C3-conjugate
useful
in such cases.
26%
LCT,
negative
also that
platelet-associated
for the pathogenesis
of
Sensitivity
tPIIFT,
with
or serum
antibody
levels
are less reliable
for
the disease
activity
than
the direct
platelet
determination,
and
be responsible
!
19
fixed platelets,
platelets
gave
negative
reactions
were
seen
by all three
In recent
reports
quantitating
platelet
IgG9’3”8
and
C3,1318 it was
found
that
thrombocytopenia
the direct
assay,
could have been
50
Positive
Total
antigen
PIIFTf
Patients
Negative
appropriate
C3 may
LCT
Test
Result
of an
patient,
techniques.
associated
with ELISA
in all three
a nontransfused
(J.R.)
gave
preit is
to the test tube and the presence
of alkaline
phosphatase normally
found
in platelets27
may also contribute
to the low background
enzyme
activity.
In the cases ofchronic
lIP,
we did not find antibody
in 2 of the 4 sera tested,
however,
one of the patients
(G.l-I.)
was
in clinical
remission
following
steroid
therapy.
The other
patient
(J.W.)
had chronic
ITP for
I 0 yr. was refractory
to cortisone
therapy
and splenectomy, and delivered
two children
neonatal
lIP.
In this
by P11 FT and all of them
ELISA;
only
three
were
by LCT.
Serum
from
woman
with
lymphoma
results
by all methods.
Table
+
12
was
identified
with BW35
and
gave positive
(C.G.)
was
techniques.
Other
disorders.
+
reactions
may
since
control
serum
(Fig.
I ) probably
represents
the normal
platelet
associated
IgG that remains
attached
to platelets after
washing.8#{176}”3”8
Nonspecific
binding
of IgG
13
positive
acid
from
several
donors
to
antibody.
In the pres-
study,
we used paraformaldehyde
in subsequent
study
washed
unfixed
absence
Nontransfused
Totalpositive
assay for
has been
comparable
results
and the assay
time was shortened,
as a consequence,
we no longer
use fixed platelets.
The low enzyme
activity
found
with platelets
in the
nocturnal
hemoglobinuria
reagent,
5-aminosalicylic
to laboratory
personnel
thought
to be carcinogenic.26
We used
platelets
pooled
facilitate
screening
for platelet
with
other
enzyme
labeled
anti-human
IgG
detection
of platelet
antibodies
peroxidase.
This
enzyme
is less expensive
and can
easily be obtained,
but its use of this enzyme
requires
a
multistep
redox reaction;
its substrate,
H2O2, is unsta-
Isoantibodies
Post-transfusion
purpura
A simple,
serologic
developed
in our
laboratory.
We selected
alkaline
phosphatase
as the
marker
enzyme
because
its
substrate
(p-nitrophenyl
phosphate)
is safe and stable.
An alternative
enzyme
that could
have been used
is
Idiopathic
Neonatal
MILLER
Platelets
Results
Chronic
AND
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
ELISA
FOR PLATELET
In one
found
ANTIBODIES
of our
cases
a platelet
terized.
negative
of neonatal
antibody
However,
results
that
and
Bl8
antigen
antigen.
and
In
appears
of
(1:128).
In two
with
patients
demonstrated
while both
of these
antibodies
to
in the
the father
between
the maternal
cytes,
and the titer
ELISA
it is not
with
p1AI
the mother’s
addition
a
( I :32),
since
plate(S.S.),
with
caused
lymphocytes
strong
reaction
the
B5
lacked
was
paternal
was
this
seen
lymphovery high
post-transfusion
identified
is of the IgG type.
nine
polytransfused
patients
donor platelet
transfusions
were
antibody
detected
refractory
to
found to have
by ELISA
and
eight
of these
by PIIFI’.
Since only three of them
antibodies
in their
may have developed
or HLA antibodies
serum,
the remaining
either
platelet
specific
undetected
by LCT.
were
had
also
LCT
six patients
antibodies
ACKNOWLEDGMENT
purpura,
antibody
in high
to react in the LCT.
We are indebted
titer
One
as an anti-PV”.
The
Using
a direct
test,
Cines
et
and
al.’8
characterized.
antiglobulin
found
increased
platelet
associated
lgG and IgG antiplatelet
activity
in the plasma
of a patient
with posttransfusion
purpura.
Our findings
support
their observation
that
circulating
antibody
in post-transfusion
purpura
All
random
leuko-
newborn
because
and newborn
had
platelet
failed
was
we
antiP1At
positive
have
serum
and
the antiserum
sera
other
has not been
indirect
radiolabel
(C.S.),
not yet charac-
with neonatal
purpura
I-ILA-B5
cross
reactive
that
thrombocytopenia
lymphocytes
from
purpura
we have
we know
were
noted
lets. In the other
patient
we identified
an anti
Bw35
37
Center
for
Beverly
Hoover
to Dr. Richard
supplying
anti
for their
immunofluorescence
We thank
review
Aster
PP’
technical
to
assistance
Judy
Blood
Harmon
in the platelet
and
lymphocytotoxicity
Drs. Ram Kakaiya
and Richard
of this
test
of the Milwaukee
antibody,
test,
Kahn
and
indirect
respectively.
for their
helpful
manuscript.
REFERENCES
I . Aster
RH,
Cooper
for the detection
Med63:l6l-172,
2.
and
as
Ri,
a plasma
3.
Shulman
antiplatelet
purpura.
4.
5.
C,
autoantibodies.
Aster
defect
RH,
by
8.
9.
platelets.
Luiken
in idiopathic
thrombocytopenic
platelet-bound
292:230-236,
I 1 . Hedge
bocytopenic
12.
the
antibody
platelet
39:195-207,
I 7. Brand
by
Usefulness
of drug
UM,
for
the
of
anti-
immune
diazepam,
and
associated
21.
Hymes
with
in immune
mune
of serum
N
EngI
J
Gordon-Smith
EC:
Br J Haematol
JG, Neame
Platelet
antibodies
35:1 13-122,
PB: Elevated
Chang
of septicemia.
N EngI
J Med
TW,
Rosse
thrombocytopenia.
Kaden
lymphoproliferative
BR,
W:
Platelet
Blood
Rosse
diseases.
WF:
Blood
bound
and
Med
in throm-
50:1 129-1
Immune
54:545-55
136,
300:760-764,
1, I 979
and
C3
indirect
test.
plateUse of
on platelets.
radioactive
N
Coombs
Vox
Sang
29:253-268,
G:
Studies
on
J Immunol
24.
assay
cyte
in
function
27.
(eds):
and
E,
Clin
1975
the
platelet
Methods
19:1-Il,
(ed):
Proteins.
radioimmunoassay
patients
Med
45
1979
assay, ELISA,
Plenum,
in
of Immo-
1977,
Enzyme-linked
assay
autoim-
Applications
York,
P:
for
with
94:639-648,
Biomedical
New
Quantitative
phase
on
immunosorbent
Perlmann
pp 87-96
immunosorbent
of immunoglobulin
G. Immuno-
1971
Engvall
E,
Perlmann
(ELISA)
III.
Quarititation
P:
anti-immunoglobulin
Enzyme-linked
of specific
in antigen
immunosorbent
antibodies
coated
by enzyme
tubes.
J Immunol
1972
JG, Hare DB, Pedersen
microcytotoxicity
assay
Studies
Swi
8:871-874,
Ray
in
S: A solid
J Lab
Ming
(ELISA).
Techniques.
1977
thrombocytopenia
Optimal
and
thrombocytopenia.
lgG
Schulz
E: Enzyme-linked
109:129-135,
IgG in
(C3)
Immune
grouping.
C,
disorders.
Enzymes
25.
complement
therapy.
1978
C: Platelet
antibody.
Engvall
26.
I 3. Hauch
transfusion
to detect
for PL
Thomas
chemistry
1977
platelet-associated
immunofluoBr J Haematol
I I, 1979
K, Shulman
platelet
23.
of antibody
Correlation
response.
A simple
antibodies.
51:781-788,
Patereau
antiplatelet
assay
Blood 50:3 1 7-325,
1977
Quantitative
determination
of
purpura
of lymphocytotoxicity
radioactiveanti-immunoglobulin
bilized
IgG
Platelet
AD:
Mueller-Eckhardt
22. Engvall
associated
purpura.
JP,
Its utilization
bound
1971
R: Platelet
300:106-I
FW:
of platelet
test
the serum
1978
I 979
14.
Med
labelled
thrombocytopenia
immune
Engli
20.
1977
clinical
Schreiber
antiglobulin
19. Soulier
1969
dependent
lmmunoglobulins
with
I 8. Cines
from
1977
Verheugt
A:
histochemical
antibodies
thrombocytopenic
a combination
tests. Blood
Test.
membrane
AE,
let fluorescence
3 avail-
48:1 199-1210,
diphenylhydantoin,
RS:
of
with
DB,
immunoenzyme
37:265-275,
Borne
1978
A, Leeuwen
selection
An
(autoimmune)
test for the detection
a Coombs
1977
Documentation
2:65-72,
1974
factor
1-233,
donor
of anti-
1975
patients.
Kelton
rescence
problem
granulocyte
Invest
test:
37:316-322,
purpura.
R, Rosse W:
and
Detection
lysis
McMilIan
thrombocytopenic
10. Dixon
33:22
hemoglobinuria:
AV:
Smith
Blood
GA,
Haematol
throm-
JM:
of platelet
Idiopathic
Dem
5-719,
The
with
Bri
release test.
147:71
K:
Sang
J Clin
J 1-lematol
R,
Med
Sorenson
the detection
Von
on the specificity
Biol
A platelet
platelet
induced
Am
McMillan
human
SE:
Pisciotta
thrombocytopenia
sulfisoxazole.
‘4C-serotonin
DY,
for
16.
of
of the platelet
Vox
nocturnal
ttCr
Tate
(ITP).
Idiopathic
problem
Mersch-Baumert
antibodies.
PL,
the
15.
technique
serotonin
1973
The
Studies
I. Evaluation
Enright
ofplatelet
7. Cimo
bodies
B:
Soc Exp
detection.
in Paroxysmal
detection
with
ofthe
GW:
Proc
C,
test for their
6.
Clin
antibodies
platelet
anti-platelet
24:793-802,
Mayser
5, Siskind
Mueller-Eckhardt
ability
test
J Lab
1977
autoantibodies.
platelet
fixation
serum.
use of
in patients
Br J Haematol
33:234-245,
Karpaktin
platelet
The
detecting
II. The applicability
autoantibodies
Sang
NR:
for
factor
Mueller-Eckhardt
Vox
complement
in human
patients
method
a sensitive
bocytopenic
Simplified
antibodies
1964
Hirschman
release
HE:
of platelet
Bethesda,
Bidwell
(ELISA).
DE,
Karpaktin
of platelets,
Hematology.
NIAID
Maryland,
NIH,
Buck
Bull
WHO
AA:
The
Manual
1976,
in Williams
York,
Typing
pp 22-24
immunosorbent
1976
of platelets.
WJ,
Lympho-
of Tissue
enzyme-linked
54:129-139,
5: Composition
New
PD, et al (eds): NIH
technique.
Beutler
McGraw-Hill,
Biochemistry
E, Ersler
1977,
AJ,
pp I 176-I
and
Ct al.
186
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1981 57: 32-37
Application of the enzyme linked immunospecific assay (ELISA) for the
detection of platelet antibodies
M Gudino and WV Miller
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