From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
Immunodepletion
of Human
By F. Ofosu,
Affinity
chromatography
of
human
detectable
factor
VIIIC,
VIIIR:Ag.
hemophilic
plasma
when
used
K. Cassidy,
cryosupernatants
and
as substrate
M.A.
on
VIIIR:WF
activities.
in factor
Plasma
VIII
Blajchman,
anti-human
plasma
tor
VIII,
strated
immobilizing
plasma
tigations
factor
and
antibodies
factor
factor
supports
VIII
those
by immunoabsorption.
of l-lolmberg
and
raised
VIII
VIII
for
against
each
the
removal
Ljung3
heparin
from
are capable
of removing
activity
from
plasma
of
entity
all
when
nogen
of
aspects
of
the
anti-
human
Montreal,
Quebec;
and
strated
that
on the
example,
For
rabbit
and
effect
concentrations
of other
Furlan
et al.2 demon-
human
anti-factor-VILI
immo-
bilized
on Sepharose
CL-2B
removed
both factor
VIII
and
fibrinogen
from
plasma.
In this
report,
we
compare
the relative
effectiveness
of CL-Sepharose
4B, AH-Sepharose
4B, CH-Sepharose
4B, and Affi10 as immobilizing
of plasma
factor
supports
VIII and
for the immunodeplethe suitability
of the
I 0 were
the
the
Blood
Canadian
Products
McMaster
Cross
Laboratory
University.
Supported
Red
of
by Grant
Transfusion
The
Department
Ontario.
Canada.
PR646Cfrom
the
Hamilton,
in part
Blood
Service
and
of Pathology.
Ministry
of Health.
Address
Blood
October
reprint
Transfusion
23.
1979;
requests
Service,
Ontario,
Canada
LSN 1H8.
5:) 1 980 by Grune
& Stratton.
0006-4971/80/5604-0004$01.00/0
604
accepted
to Dr. Fred
299
Inc.
Main
May
Ofosu,
Street
21. 1980.
Canadian
East,
Cross
Hamilton,
pertussis
Toronto,
Quebec.
Rabbit
from
Behring
vaccine
from
Ontario;
was
macroglobulin,
Inc.,
from
Springfield,
Difco
Labora-
Connaught
Laboratogel electropho-
plasma
plasma
0.001
was
M Na2HPO4,
also
contained
following
frozen
and
EDTA,
the
7000
g. Ten
of
0.15
cryoprecipitate
cryoprecipitate
10 mm
0.1
The
1 hr. thawed
adsorbed
plasma
was
packed
M
containing
in the
was
eluates,
60%
located
electrophoresis
2-macroglobulin.
volume
on
VIII
4B
Blood,
on
I liter
the
of
of 80 mI/hr.
7.35,
NaN3.
with
congenital
concentrated
elution
was
Factor
buffer
and
determined
containing
by
sodium
dode-
rocket
immunoelectrophoreanti-lgM,
and anti-alpha-
eluted
and
AI(OH)3
at 20#{176}C
to
pH
0.02%
VIII
gels
by quantitative
antifibrinogen,
Factor
Sepharose
rate
assay
against
on 4% polyacrylamide
cyl sulfate
(SDS)’4
and
sisll.Is
with monospecific
v/v
from
was
factor
the
of CL-Sepharose
at a flow
and
7.35,
to
diethylbarbiturate,
at 4#{176}C,
dialyzed
the
10%
supernatant
as
of
pH
at 37#{176}C.The
on 2 liters
a one-stage
plasma
PEG
incubated
g for 20 mm
EACA,
was
added
with
column
by
was
at 18,000
sodium
0.5%
NaCI,
and
resulting
cm
buffer,
EACA,
filtration
0.03
-60#{176}C. Purity
centrifugation
absorbed
centrifuged
factor-VllI-deflcient
at
4#{176}Cat
dissolved
x 100
the
at
by
thus
0. 1 5 M
heparin,
bath,
of plasma
was
mg/mI;
water
1 liter
buffer
and
aliquots,
0.5%
The
plasma
20,000)
M diethylbarbiturate
to gel
citrate,
in 200-ml
from
in a 5 cm
wt
0.01
plasma,
and
proteins.
M sodium
platelet-poor
at 4#{176}C
in a circulating
NaC1
subjected
eluting
SBTI,
recovered
at 20#{176}C
and
remove
mol
platelet-poor
so anticoagulated,
0.012
(average
0.5%;
mM.
When
acid,
The
EACA,
to 20 ml of 0.03
containing
CPD.
cryoprecipi-
fresh
M glucose.
PEG
cryoprecipitate
of human
from
M citric
0.018
1% w/v
at -60#{176}Cfor
and
The
and
inhibitors;
2 U/mI;
with
0.002
Affi-
Mississauga,
VIII
filtration
obtained
anticoagulated
contained
Laboratories,
Factor
by gel
to
and
BioRad
purified
Phar-
Diagnostics,
for polyacrylamide
from
unfla-
antiserum
alpha-2
obtained
was
Mo.;
from
Laboratories,
Reagants
Cryoprecipitate
human
stored
Red
VIII
tate.6’’2
against
Submitted
Ltd.,
CH-Sepharose
was
Fisher
plasma
and
to human
ofPurified
Factor
VIII
Ontario
from
Wyeth
Meloy
adjuvant
purchased
Preparation
4B
From
cya-
20,000)
l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide,
Gel-
for
antisera
Ontario.
diethylbarbiturate,
City,
obtained
from
Mich.;
Toronto,
resis,
and
Freund’s
Detroit,
ries,
was
Mo.;
ethylene-
Kansas
Montreal,
1gM
Complete
tories,
from
inhibi-
Louis,
Ontario;
(PEG
Biomedical,
AH-Sepharose,
prothrombin
the
of immunodepletion
clotting
factors.
for
trypsin
St.
Factor-VIlI-deficient
King
Chemicals,
fibrinogen,
Va.
sodium
glycol
[Al(OH)3]
4B,
soybean
Chemicals,
Brantford,
Ontario.
George
Fine
and
Sigma
polyethylene
Toronto,
from
macia
(EACA)
from
acid-(EDTA);
amphogel
In addition,
on the
plasmas
METHODS
Laboratories,
and
CL-Sepharose
Ontario.
is presented
Harris
bromide,
factor-VIII
antisera
are insolubilized
on Sepharose
or
acrylamide.
Although
Bouma
et al.’ stated
that recycled
immunodepleted
plasma
could
be used
as a
reagent
for the determination
of factor
VIII activity,
no further
information
about
such use was presented.
no information
of
congenital
substrate
assays.
AND
acid
obtained
diaminetetraacetic
vored
Their
invesshow that
functional
plasmas
as
VIII coagulant
caproic
were
obtained
by several
of factors
VII, and XII.’5
Bouma
et al.’ have
demonthat
both Sepharose
4B and acrylamide
are
suitable
Geltion
amino
(SBTI)
Scientific,
been used
depleted
devoid
severe
assays.
Epsilon
assays.
have
plasmas
a plasma
from
Materials
cially
depleting
human
plasma
of factor
VIII
by
immunoabsorption
with rabbit
anti-human
factor
VIII
insolubilized
on Sepharose
4B for use in factor
VIII
antibodies
to derive
yielded
indistinguishable
MATERIALS
cryoprecipitates,
and factor
VIII
concentrates.
Such
plasma
is sometimes
difficult
to obtain
in large quantities. We have therefore
developed
a method
for artifi-
Insolubilized
investigators
VIll-Sepharose
was
immunodepleted
one-stage
factor
LASMA
FROM
INDIVIDUALS
with
severe
hereditary
deficiency
of factor
VIII are required
to assay
the levels of coagulant
factor
VIII in plasma,
VIII
and J. Hirsh
factor
This
coagulant
P
Factor
as
a sharp
concentration
Vol.
56,
peak
to
No.
in
0.4-1
4 (October),
the
void
mg/mI
1980
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
IMMUNODEPLETION
OF FACTOR
VIII
reacted
only
with
immunoelectrophoresis.
rabbit
Preparation
ofAnti-Factor-
Immobilization
VIII
605
anti-human
factor
VIII
on
quantitative
Table
1 . Coagulation
Cryosupern
atant
Factor
Plasma
Assays
of Cryosupernatant
Immunodepleted
of Factor
Factor-VlIl-CL-Sepharose
New
and
Zealand
were
white
male
by
intradermal
immunized
human
factor
rabbit
was
vaccine
with
weekly
and
with
the
antiserum
was
isolated
IgG/g
AH-Sepharose
and
the
wet
gels
the
manufacturers.
coupling
Affi-Gel-
factor
This
at
VIII
the
It was
then
lgG
was
to be subjected
CPD.’7
Fifty
donors
were
20 normal
to remove
aliquots
at
of 10-15
coupling
methods
methods
a
ratio
aliquot
and
10-1
and
debris,
pooled,
as the
to clotting
factor
IX,
X,
were
collected
The
The
levels
brand
cofactor18
were
also
tants,
50-mi
aliquots
stored
After
its
o_
use
4B
with
OD,nm
in
was
CPD,
of the
at
10-15
CPD,
mm.
The
pH
7.4,
gel was
containing
The
plasma
of factor
CL-Sepharose
Only
this
of
The
due
were
10
column
tLower
*Not
using
Wille-
the
as follows.
to immuno-
7.4,
containing
less
than
pH
3.5,
then
anti-factor-VIIl-CLThe
0.15
0. 1
in a 250
washed
M
NaCI.
gel
was
the
,
under
column
was
the
suspended
ml Buchner
suction
devel-
When
in
funnel
with
1-2
for
liters
of
immunodepletion
process
from
were
obtained
from
values
limit
of detection
Factor
VI!!
depleted
the
regeneration,
could
be
loss
of
4B resulted
x
in addition
VII,
IX, and
of depleted
and factor
the plasma.
cryosuperna-
for substrate
use.
similarly
processed,
depleted
of factor
was
not
associated
When
50
only the
VIII. The
with
cryosupernatant
the
are
immunodepletion
9 to
1 2 batches
clotting
factors
(Table
after
immunodepletion
dilution
immunodepletion
of
the
reused
the
anti-factor-V
up to 6
in the
to factor
X were
VIII
of
cryoprecipitates,
VIIIC
levels
normal
plasma
mined.
II, VII,
VIII.
The levels
reduced
to less
VI!!
contained
and
II,
of
than
as
Assays
plasmas
plasmas
or fresh-frozen
mixtures
of
(factor
VIIIC
immunodepleted
substrates
patient
plasmas.
hemophilic
plasmas
as well as mixtures
and immunodepleted
plasma
plasma
or
as substrate.
less
Vhf-Deficient
Plasma
<
for the
plasmas,
The
and
factor
pooled
of pooled
normal
were also deter-
In Fig. I are shown
the results
obtained
VIIIC
levels were determined
with either
philic
plasmas
IX,
of factors
than
40%
When
plasma
was
on anti-factor-VIll
4B or Affi-Gel-lO,
were compared
as
VIIIC
in various
in
to AH-Sepha-
of factors
ofSevere
Factorand Immunodepleted
a
capacity
coupled
loss
plasma
Factor
III-Sepharose
times,
before
factor-VIll-binding
plasma
in each experiment.
to immunodepletion
to either
CH-Sepharose
Comparison
Hemophilic
I ). The
repre-
of the plasma
procedure.
25%
the
the factor-VIII-depleted
10% factor
XI.
of factor
quantitation
cryosuper-
starting
ml
assay.
of the
Two severe
hemophilic
I %) and 6 cryosupernatant
VIIIC,
factor
VIIIR:Ag,
the first 25-30
ml of
fraction
resulting
mean
Substratesfor
0. 15 M NaCI.
tant plasma
was suitable
ml of whole
plasma
was
first 10-I 5 ml was totally
to the
dilution
mg/100
determined.
normal
subjected
coupled
to cryosuperna-
subjected
relative
25%
sents
an approximately
that occurred
during
rose
starting
von
obtained
became apparent.
The use of anti-factor-VIII
II, V, VII,
and
180.0
8.5
plasma.
significant
subjected
the
The
After
columns
50-mI
of 8 ml/hr
antigen
also
I 5 to
previously
to
values
ml
significant
loss of other
lower
values
obtained
Two-
relative
low
<6t
mg/100
to an approximate
immunodepleted
in
in 50-mI
of factors
225
procedure.
of
g for
then
<6t
12.2
anticoagu-
each
54
±
NaCl.
In addition
4B completely
25-30-ml
42.0
Fibrinogen
of 50 ml of pooled
cryosupernatant
a 10-mi column
of anti-factor-VIII-
natant
of factor
VIIIR:WF
from
VIIIC
7.8
lgG/g
and
was
levels
VIII
plasma
ofPlasma
passage
through
8.8
8.2
RESULTS
Immunodepletion
±
±
by
from
M
rate
plasma
the
was
I 50 ml of I M glycine-HCI,
78.0
77.8
lgG/g
frozen
4B
a flow
immunodepletion,
pH
90.0
100.0
employed
from
0.15
fibrinogen
regenerated
eluate
8.8
IX
II
depletion.
Sepharose
±
was
specified
on a I 0-mI
containing
determined.
of the
78.2
±
to
I 7,000
obtained
resulting
and
cryosupernatant.
at
and
to determine
XII,
100.0
±
coupled
was
CL-Sepharose
7.4,
eluant.
XI,
8.3
x
61.4
of
plasmas
chromatography
pH
assays
8.4
±
ND
VIII
centrifuged
was
to
CPD,
fractions
CPD/NaCl
VIII,
pooled
coupled
with
±
70.1
V
coupled
5 mg
to immunodepletion
to affinity
anti-factor-VIlI
equilibrated
milliliter
Factor
-70#{176}C. Cryosupernatant
subjected
71.0
81.0
The
IgG
mg
were
of
to 75 ml of platelet-poor
cellular
95.0
Xl
73.8
at a ratio
also
at a ratio
ofPlasma
plasma
with
(U/lOOmI)
XII
ND
95
by absorp-
and
Immunodepleted
Plasma
(U/lOOmI
± 1 SD)
VlllR:WF
VII + X
I 0.
The
of
and
immunized
of Cuatrecasas”
manufacturers’
Affi-Gel-lO
the
of Al(OH)3,
carbodiimide
Immunodepletion
lated
were
of cryosupernatant.”
method
Sepharose.
The
to
pertussis
injection
from
on DEAE-cellulose.
the
of
initial
obtained
to human
CH-Sepharose
using
lgG
was
10% v/v
by the
wet
ml
Each
Rabbits
the
precipitate
with
4B
mg
mm
VIII
by chromatography
to CL-Sepharose
10-15
4 wk after
purified
VIIIR:Ag
ethanol
absorbed
0.5
Fact
weight)
of
adjuvant.
immunization.
monospecific
3%-8%
g
mo.
factor
rendered
with
initial
VIII
for 4-6
anti-human
rabbits
the
40 .tg of factor
thereafter
Rabbit
of
40
Freund’s
intradermally
time
2 kg
of
in complete
injected
the
(approximately
injections
emulsified
also
at
boosted
tion
VIII
rabbits
4B
Cryosupernatant
Prior to Immunodepletion
Coagulation
and
VIII on Anti-
when
severe
factor
hemo-
immunodepleted
cryosupernatant
There
is an excellent
correlation
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
OFOSU
606
ET AL.
tor in the immunodepleted
plasmas.
Plasma
immunodepleted
of factor
VIII did not support
the aggregation
of fixed human
platelets
in the presence
of ristocetin.
Factor-VIlI-depleted
plasma
was
also
used
as a
diluent
of pooled
normal
plasma
instead
of buffer
in
I
deriving
standard
curves
for
cofactor.’8
The
predicted
were achieved
in all cases,
able
VIII
anti-factor-VIII
column.
When
used
as a diluent
17 separate
U-
levels
for pooled
±
of
ristocetin
cofactor
levels
that no detect-
was eluted
from
factor-VIII-depleted
contain
20% VIIIR:WF,
was found
to be 17.4
4
the
ristocetin
indicating
normal
the
plasma
anti-factorplasma
was
adjusted
to
the mean
factor
VIIIR:WF
2.3 (1 standard
deviation)
determinations
performed
in
over
a 10-wk
coupled
to CL-
period.
0.5
1.0
1.5
DISCUSSION
FACTOR
VIIIC (UNITS/mi)
WITH
HEMOPHILIC
PLASMA
Fig.
1.
A comparison
assays
when
either
plasma
or severe
(r=0.94)
pleted
of the
hemophilic
between
plasma
the values
cryosupernatant
was
using
the
factor
the substrate
and
plasmas
level
in
VIIIC
cryosupernatant
obtained
as
were
no statistically
values obtained
with
at the 0.05
obtained
plasmas
factor-VIII-deficient
There
between
results
factor-VIII-immunodepleted
Rabbit
plasma.
with
immunode-
severe
congenital
plasma.
significant
the two types
differences
of plasmas
test.’9
Similar
results
were obtained
when
whole
plasma
completely
immunodepleted
offactor
VIII was used as
substrate
plasma.
However,
the use of whole
plasma
required
a lower flow rate (3 ml/hr)
and gave substantially
reduced
volumes
of immunodepleted
plasma.
When
whole
plasma
was subjected
to immunodepletion
was
at a flow
completely
processed
depleted
rate of 3 ml/hr,
only 10-I
depleted
of factor
VIII.
at 8 ml/hr
of factor
VIII.
S ml of plasma
Whole
plasma
yielded
plasma
only
This plasma
was thus
partially
unsuita-
ble for one-stage
factor
VIII assays.
of the partially
depleted
plasma
on
Sepharose
was required
to achieve
of factor
VIII.
This
recycling
of
plasma
then
resulted
in a further
A second
passage
anti-factor-VIlIcomplete
removal
partially
depleted
dilution
of the
plasma.
Cryosupernatant
plasma
immunodepleted
factor-VIII
immobilized
on AH-Sepharose
Sepharose
substrate
4B,
plasma
loss of additional
pletion.
and
Affi-Gel-lO
for factor
VIII
clotting
were
assays
factors
during
Willebrand
The
employed
method
Factor
of
in determining
on anti4B, CHunsuitable
as
because
of the
immunode-
groups
rose
on this
4B
the
and
levels
Zucker’t
of ristocetin
and
anti-factor-VIII,
bly resulted
type
was
from
was
cofac-
When
were
used
This is
amino
CH-Sephato
immobilize
the loss of factor
XI activity
probathe presence
of residual
carboxylate
groups.
Anti-factor-VIlI
pleted
VIIIC
factor
VIII.
of residual
of Sepharose.
Affi-Gel-lO
was
not detected
in the immunode-
plasmas,
as no detectable
lowering
or VIIIR:WF
values
was evident
of the factor
in compara-
tive assays.
Immobilization
ofanti-factor-VIlI
on CLSepharose
4B thus permits
the removal
of factor
VIII
in its various
functional
forms without
the introduction
of antibodies
in the plasma,
as is the case
when
antibody
is simply
added
to the plasma
to derive
substrate
plasma
for factor
VIII assays.2#{176}In addition
to its use as a substrate
for factor
VIIIC
assays
and as
a diluent
for ristocetin
cofactor
assays,
the plasma
immunodepleted
on Sepharose
4B has also been used
as an immunoabsorbent
in the preparation
of monospecific
antisera
to human
factor
VIIl.
Solid-phase
immunoabsorption
has been
used
to
derive
factors
VII and XII deficient
plasmas
for use as
substrate
plasmas
respectively.4’5
This
for factors
procedure
derive
factor-VIII-deficient
VIII coupled
to CL-Sepharose
Assays
McFarlane
VIII
clotting
factors
in addition
to the
most
likely
due to the presence
Sepharose
von
factor
4 B, A H-Sepharose,
C H-Sepharose
and
Of the four types
of immobilized
antiused, only anti-factor-VIII
coupled
to CL4B resulted
in the specific
immunodeple-
tion of plasma
factor
VIII.
The use of AH-Sepharose
4B resulted
in the removal
of the vitamin-K-dependent
substrate
Wilcoxon
anti-human
Sepharose
Affi-Gel-lO.
factor-VIII
Sepharose
2B,
however,
VII and
XII
has also been
plasma
using
2B.2 The
resulted
in the
fibrinogen
in addition
to factor
VIII.
tors”3
have
used
anti-factor-VIII-Sepharose
immunodeplete
plasma
of factor
VIII.
Other
assays,
used to
anti-factoruse of CLremoval
of
investiga4B to
However,
these
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
IMMUNODEPLETION
OF
investigators
FACTOR
VIII
explored
against
specific
determi
ne
607
the
use
functional
of
entities
structure-function
antisera
of
raised
factor
VIII
relationships.
to
While
thus
yielded
larger
volumes
substrate
plasma
for one-stage
Thus,
the
process
of
plasma
factor
of immunodepletion
suitable
as
VIII
assays.
may
prove
to be
these
investigators
suggest
that
antibody
coupled
to
Sepharose
4B could
provide
factor-VIII-immunodepleted
plasma
for reagent
use, no data are presented.
The use of cryosupernatant
plasma
where
the initial
levels of factor
VIII was reduced
before
immunodepletion made
it possible
to increase
the flow rate to 8
of general
applicability
for the production
of various
deficient
plasmas
where
the plasma
constituent
in
question
occurs
in the microgram
per milliliter
range.
These
results
show
that
the immobilization
of anti-
ml/hr
from the 3 ml/hr
required
with whole
plasma.
The
use
of cryosupernatant
plasma
also
enabled
immunodepletion
of factor
VIII
in a single
filtration
step,
thus
eliminating
the
need
for recycling,
as
reagent
suggested
tants
as
von
by
the
Bouma
starting
et al.’ The use of cryosupernaplasmas
for immunodepletion
factor-VIll
step for the
antibodies
preparation
on CL-Sepharose
of factor-VIII-free
4B is a useful
plasma
for
use.
ACKNOWLEDGMENT
The
help
of J. Russett
Willebrand’s
factor
and
M. Johnston
and
coagulation
in the
performance
factor
assays
of the
is gratefully
acknowledged.
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1980 56: 604-607
Immunodepletion of human plasma factor VIII
F Ofosu, K Cassidy, MA Blajchman and J Hirsh
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