From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOO
VOL.
D
XL, NO.
The Journal
1
Characterization
Human
Isadore
M. Pike,
of
Antibody
William
was
mixed
with
J. Yount,
an
excess
Elliot
for yA,
yM,
7D, yE, yGI,
of
yG2, and
yG3
heavy
chains
and
kappa
light
chains
had no effect
on the inhibitor.
On preparative
zone
electrophoresis,
C
IRCULATING
ANTICOAGULANTS
factor
V (accelerin),
factor
VIII
[plasma
thromboplastin
component
scribed.”6
Most
Antibodies
to factor
chain
types,
of
and
Other
From
the
Depart
November
Supported
in part
Isadore
M.
Professor
Chapel
of
Hill,
Medicine,
M.D.:
Medicine,
of
Puritz,
North
of
Vol. 40, No. 1 (July),
have
both
kappa
to
contain
Pathology,
as
in
found
antibodies.
lambda
of
light
to factor
light
a mixture
University
and
immunoglobulin
antibodies
and
N.
of
North
N.
chains.4
light
chain
Carolina
and
of
North
Fellow
of
Pathology,
February
HE-05652,
William
C.
of
Research
School
accepted
1972;
Department
Hill,
University
M.D.:
Carolina
Medicine
7,
HE-06350,
Fellow,
Chapel
Bacteriology,
M.
Hill,
R. Roberts
as to heavy
restricted
however,
February
Postdoctoral
and
characterized
be
AI-10327-O1,
Medicine,
Professor
Chapel
Grants
M.D.:
C. Elliot
revised
1971;
of
to
shown
and
Harold
School
C.
USPHS
Medicine
been
and
characterized
found
to contain
Medicine
N.
16,
by
School
N.
of
Hill,
University
Roberts,
Blood,
Pike,
Carolina
has
inents
Chapel
Submitted
North
of
V antibody
Medicine,
been
and
been
further
investigators,
to be heterogeneous
One
factor
types.3
of
have
IX
M. Puritz,
have
been
A
that specifkally
inhibit
fibrinogen,
[antihemophilic
factor,
(AHF)],
factor
IX
(PTC)1,
and
factor
XIII have
been
de-
inhibitors
have
some
composition.5’7’9
VIII
these
VIII
G4,
inhibitor
activity
was
localized
in a
sharp
band
in the anodal
portion
of the
‘/G peak,
an electrophoretic
distribution
paralleling
that
of yG4.
Precise
localization
of the inhibitor
activity
on
calibrated
gel
filtration
columns
revealed
a relatively
narrow
zone of fractions
containing
the inhibitor,
wherein
the proteins
have an estimated
molecular
weight
of 145,000.
The
relative
homogeneity
of the antibody
may
reflect
specificity
for a uniform,
discrete
portion
of the human
factor
IX molecule.
specific
antiserum
and
then
assayed
for residual
inhibitor
activity.
Inhibitor
activity
was
completely
removed
by
antisera
specific
for yG4 heavy
chains
and lambda
light
chains.
Antisera
spe-
cific
a Monoclonal
to Factor
A circulating
inhibitor
specific
for factor IX in a patient
with
hemophilia
B
was characterized
with
antisera
to human
immunoglobulins.
Inhibitor-rich
plasma
1972
JULY
linmunochemical
By
of Hematology
and
Pathology,
J. Yount,
Carolina
in
Medicine,
University
University
M.D.:
School
Medicine,
Department
Hill,
of
of
Associate
of
Dermatology,
Chapel
1972.
9,
AM-05298-09.
North
N.
C.
Carolina
of
Harold
R.
School
C.
1972
1
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2PIKEETAL.
Antibodies
In
the
to
present
factor
IX
study,
hemophilia
B was
have
not
a factor
found
IX
been
previously
inhibitor
that
to be predominantly,
characterized
developed
if not
in
in
detail.
a patient
exclusively,
with
a 7G4
lambda
antibody.
MATERIALS
AND
METHODS
Patient
Citrated
plasma
inhibitor
of
studies
and
(8
factor
Purified
of
Antisera
or
and
for
rabbits,
goats,
tolerance
mg
fragments,
‘yGi
II,
of
modified
determination16
was
not
fractions
globulin
Ammonium
sulfate
detectable
experiments,
in
fractions
were
globulin)
and
heated
at
56#{176}Cfor
was
The
inhibitor
the PTT
inhibitor
was
after
mixed
1-mm
was
normal
Table
mg
BaSO4/ml
the
material
clotting
NaCl
immuno-
monkey
(PTT)
sera,
in
no
The
parts
of
fraction).
the
globulin
antiserum
After
and
sulfate
neutralization
glycol.
centrifuged,
but
sulfate.
until
used
five
the
described
time
dialyzed
globulin
obtain
been
ammonium
polyethylene
was
to
digestion
radial
and
were
plus
The
or
single,
saturated
and
oxalate
factor
has
rabbit
BaCl2,
continuous
supernatant
activity.
taken
plasma
were
made
with
with
equal
parts
of
at
shown
as
by
1. Neutralization
Mixture
1: Neutralization
Inhibitor
plasma
+
Assay supematant
insure that mixture
Mixture
2: Assay for
Supernatant
from
Residual
factor IX
(100
mm,
by
order
normal
with
sodium
incubation
been
plasma
M
adjuvant.
as
thromboplastin
aggre-
immunization
in
partial
50%
with
M
destroy
out
‘1G3
im-
Inhibitor
previously
titer
of
to
IX
of
determined
mm
20
45
0.15
concentration
0.1
BaSO4
for
in
sera
of
of
and
proteins
specificity
with
precipitation
part
with
by
Because
Freund’s
myeloma
carried
The
precipitation
after
(one
dilution
had
by
footpad
Gm
was
redissolved
‘1G2
by
whole
or
adversely
by
purified
complete
of
whole
technique
of
selected
subclass,
the
followed
of
and
isolated
fragments.13
subclass,
proteins,
‘yG, or
‘yG
affected
prepared
temperature
dilutions
Inhibitor
be
instances
of Factor
Each
to
absorbed
room
volume
Jones
kappa
immunization
with
digestion
chains,
described.10
by
baboons
‘1G1
milligrams
previously.10
dialysate
some
at
Serial
found
oxalated
mixing
Titration
described
the
equal
heavy
previously
prepared
or
tolerance,
diagnosis
and
subclasses,11’12
were
the
immunoglobulin
as
fractions,
in
for
Five
normal
described.2
established
as
heavy-chain
enzymatic
Bence
type,
Quantitation
diffusion,15
7.2.17
isolated
light-chain
C
an
isolated,
prepared
monkeys,
induce
an
specific
B. Clinical
been
with
were
markers
rhesus
isolated
a
hemophilia
previously
patients
“yE classes,
to
with
have
were
genetic
rabbits.14
i.v.
with
class,
from
or
containing
severe
of Antisera
specific
in
protein
Fraction
previously.13
was
used
adsorbed
heavy-chain
‘yD,
chains
given
IX
fragments
Cm
citrate),
with
Proteins
antisera
was
sodium
P.W.B.
factor
prepared
monkeys,
were
of
was
by
Preparation
‘yM,
preparing
proteins
0.2
and
polypeptide
in
unmologic
antisera
for
and
cynomolgus
difficulty
with
‘yA,
types,
immunoglobulins,
gate-free
inhibitor
digestion
yG,
3.2%
patient
macroglobulinemia.
enzymatic
specific
light-chain
part
from
were
myeloma
lambda
the
the
immunoglobulins
chains,
1
obtained
Isolation
multiple
light
blood:
was
specificity
Immunoglobulin
of
parts
IX,
the
at
be
30
sec.
constant
dilution
of Factor
imidazole
citrated
37#{176}C.Neutralization
to
highest
least
citrated
normal
from
of
IX Inhibitor
inhibitor
With
of
1-
saline
plasma,
to
plasma
Specific
Factor
120-mm
that
and
IX
buffer,
pH
the
PTT
with
this
incubation.2
would
prolong
Antisera
phase
specific
antiserum
-#{247} preciptate.
Ratio, 1:19.
for residual
antigen
and antibody
to
was In zone of antibody
excess.
residual
inhibitor
mixture
I + normal
plasma
-+
assay residual
factor
IX. Ratio
Is directly
proportional
to amount
of inhibitor
neutralized.
1:1.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
MONOCLONAL
Factor
ANTIBODY
The
specific
assay
time
as
1,
of
as
an
3
one
Controls
for
IX
inhibitor
plasma
using
to
and
the
double
diffusion
experiments
reported,
the
the
1),
supernatant
in
the
amount
antiserum
the
partial
thrombo-
for
IX
of
for
at
antibody
excess.
using
all
mixture
2
incubating
factor
IX
mixture
plasma
In
In
by
in
inhibitor
as
immunization
diffusion.10
inhibitor
IX. Residual
neutralized
Ratios
antiserum.
and
radial
antibody
factor
used.
were
incubated
by centrifugation
antigen
of
plasma
experiments,
before
mixtures
removed
residual
zone
dilution
rabbit
animals
single
residual
serial
normal
was
concentrated
The
was
whole
most
highly
from
instances,
in
For
1)19
antiserum
or
obtained
residual
factor
of inhibitor
included
and
specific
the
(either
(Table
antiserum.
precipitate
in
for
inhibitor
inhibitor
some
were
determining
to the
experiments
parts
assayed
and,
assayed
kappa-specific
19
sera
was
neutralization
on
Antisera
the
antisera
to the
4#{176}C.The
mixtures
supernatant
normal
plasma
and
is directly
proportional
based
Specific
purified
included
Ouchterlony
(Table
test
neutralize
specific
partially
experiment
g for 30 mm,
one-stage
With
to
using
and processed
in a manner
identical
1 hr at 37#{176}C
and then
overnight
at
30,000
the
made
by
possible
each
was
Inhibitor
was
inhibitor)
part
1 :3 were
IX
previously.18
attempt
purified
ratio
factor
of Factor
mixture
or partially
low
for
described
Neutralization
a
IX
IX Assay
plastin
In
TO FACTOR
in
in
with
mixture
Additional
1.
globulins
2
from
serum.
RESULTS
Heavy-chain
Class
Initial
inhibitor
If
myeloma
of
the
protein,
was
due
to the
obtained
from
animals
The
antibody
was
subclasses
(Table
3).
subclass,
as previously
trols,’#{176}’2#{176}
are shown.
of the
0.45
total
mg/mi
normal
on the
yG.
or
After
the
yG
antithe
the
anti-
antisera
on
further
The
(Table
2).
for the
excess,
yA,
no
was
with
were
the
determined
in
yG4 subclass
the
yG.
a large
usually
total
yGi,
Class
Factor
<1
<1
‘yE
<1
‘yG
123,104
purified
‘yG)
and
of Factor
(#{176}Io
control)
with
specific
standard
for yGi,
yG2,
antisera
specific
‘yA
‘yM
antisera
<1
<1
fractions
inhibitor.
for
deviation
mg/ml,
yG3
were
and
for
adult
4.2
±
and
also
yG3
yG4
the
for
of normal
for only
7.81
yG
neutrali-
globulin
on the
yG was
yG2,
purified
that
no effect
number
accounts
specific
completely
with
indicating
control
and
‘yD
‘yG (adsorbed
adsorbed
lost,
using
Residual
Anti.
was
had
percentage
2. Immunoglobulin
antisera
inhibitor
In addition,
characterized
total
different
the
was
to immunization
P.W.B.,
a yG
two
first
inhibitor
normal
of the
globulin
with
antisera
specific
in the zone of antibody
however,
proportions.
Antisera
specific
factor
IX inhibitor.
However,
Table
inhibitor
mixed
yG specificity.
prior
The
IX Inhibitor
mixing
class,
yG
effect
In patient
5.8%
that
plasma
was
of immunoglobulins
neutralized.
chains
neutralized.
of Factor
indicated
IX inhibitor
yM classes
was
heavy
zation
Subclass
experiments
When
factor
-yD, yE, and
for
and
the
con2.6%
7G4
present
had no
neutralized
IX Inhibitor
IX
Residual
7G
each
Inhibitor
Present
Present
Present
Undetectable
Present
Present
in
effect
81
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4
PIKE
ET AL.
k
YG2
Fig. 1. Ouchterlony
analysis
of ‘yG4specific
antiserum
before
and
after
neutralization
of factor
IX inhibitor.
Well
A contains
antiserum
to 7G4
Hchains
prepared
in a rhesus
monkey.
There
is no
precipitin
line
between
purified
yGl,
yG2,
and yG3
myeloma
proteins,
nor do these
proteins
inhibit
the
lines
with
purified
yG4
myeloma
proteins
or P.W.B.
inhibitor
plasma.
Supernatant
after
neutralization
of inhibitor
plasma
(S) retains
yG4
specificity,
indicating
that
mixture
was
in
zone
of antibody
excess.
In addition,
no yG4
antigen
is detected
in (S) by
antiserum
(A).
There
is
complete
fusion
of precipitin
lines
with
inhibitor
plasma
and
purified
yG4
myeloma
proteins.
1G2,X
Anti
A
S
TG4
Supernatant
with
and
of
90%
after
only
proteins,
antiserum
of
by
not
before
Type
prior
two
experiments.
heavy
chains.
adsorption
with
those
and
after
of Factor
Neutralization
inhibitor
molecules
4). Two
antisera
IX
specificity
The
The
the
other
factor
with
IX
antibody,
neutralization
antiserum
was
with
subclasses.
mixing
The
inhibitor
com-
purified
yG4
specificity
plasma
of
is
the
shown
in
look
Inhibitor
specific
for
kappa
antisera
to light
chains
exclusively
of lambda
determinants
for
small
dilutions
of
amounts
of
inhibitor
of normal
tical
curves
at any
were
rabbit
human
were
kappa
plasma
preimmunization
level
obtained,
among
made
in both
At
was
Table
each
determined
indicating
A second
lambda
Several
that
approach,
3. ‘yG Subclass
Residual
also
of Factor
Factor
(#{176}/o
control)
Anti-
‘yGI
‘yG2
‘yG3
‘yG4
(adsorbed
with
purified
‘yG4)
<I
<1
<1
81,90
<I
on
for lambda
(Table
4).
Bence
approaches
inhibitor
inhibitor.
protein,
employed
the
to
molecules.
the
effect
As
can
2).
that
the
(Table
chains
comWhen
lambda
Serial
antikappa
dilution,
antiserum
using
the
Jones
were
rabbit
(Fig.
kappa
indicated
light
chains
no effect
specific
activity
chains
globulin.
plasma
dilution.
had
different
antisera
factor
IX inhibitor
was
adsorbed
with
purified
to neutralize
inhibitor
was lost.
capacity
‘yG4
of
of
experiments
with
were
composed
On the other
hand,
three
pletely
neutralized
all the
effect
in
yG4
1.
Light-chain
and
ppt
activity
composed
blocked
myeloma
yG4
Fig.
A
plasma
inhibitor
was
therefore,
pletely
of
inhibitor
whole
had
antiserum
on
be
factor
seen,
no
IX
iden-
detectable
inhibitor
plasma,
IX Inhibitor
IX
Normal
Residual
Inhibitor
Present
Present
Present
Undetectable
Present
Total
#{176}/,
of
‘yG ± SD
66±8
23±8
7.3 ± 3.8
4.2 ±
2.6
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
MONOCLONAL
Fig.
2.
ANTIBODY
Lack
of
TO FACTOR
kappa
IX
molecules
5
in
so
factor
IX inhibitor
from patient
P.W.B.
Serial
dilutions
of
P.W.B.
inhibitor
plasma
in kappa-specific
rabbit
globulin and preimmunization
normal
rabbit
globulin.
All dilutions
were
in zone of
antibody
excess.
Per cent IX inhibited
at each
with
bit
dilution
kappa
was
virtually
antiserum
40
identical
and
normal
rab-
globulin.
C
320
1160
FINAL
was
the
search
lambda
for
inhibitor
plasma
amounts
of
centrated
with
an
to
Partial
and
plasma
was
the
migrates,
Sephadex
total
volume,
tions
Vt,
elution
radial
cular
by
The
peak
portion
lambda
inhibition
volume
of
of 145,000.
lambda
of the
of inhibitor
to
The
antiserum,
Table
column
fractions
isolated
4. Light-Chain
x(Higher
x(Adsorbed
with
purified
IX)
A
The
and
described
above.
Type
of Factor
of
4).
the
The
exclusion
marker,
A narrow
zone
to peak
gave
of 7G
an
was
Factor
<1
<1
and
(frac-
II OD
280
as determined
estimated
mole-
completely
neu-
IX Inhibitor
control)
7G4
with
peak
a calibrated
blue
inhibitor
103
Bence Jones)
(Fig.
to that
102, 99
ratio Inhibitor:
half
a dextran
<1,
K
:A-anticompletely
electrophoresis.
corresponding
(‘I.
X
antiserum.
inhibitor
anodal
buffer.
Residual
Anti-
lambda
was
that
IX Antibody
8.0
identical
partially
as
globulin
three
fractions
passed
over
pH
barbital
PTT
con-
achieved
plasma
indicated
antiserum
the
with
was
part
small
30-fold
was
of
zone
The
and
NaC1
with
1),
in three
fractions
(Fig. 3). These
of the yG peak,
where
yG4
localized
Tris-HC1
determined
the
ratio
preparative
simultaneously
immunodiffusion
weight
tralized
was
showed
64-68)
The
by
found
by
a 1:2
of Factor
in a sharp
anodal
in
With
one
preimmunization
neutralized
1:32),
with
of
1 (Table
detected.
whereas
excess.
inhibitor.
fractionated
determined
mixture
20
neutralization
dilution
neutralization
Characterization
column
was
Vo.
in
be
by radial
immunodiffusion.
were
pooled,
concentrated,
G-200
volume,
1:3,
was
were
usual
40
Inhibitor
titer in unneutralized
be detected
at 1:300.
This
(titer
found
extreme
and
peak
determined
inhibitor
activity
of
inhibitor
antibody
purified
not
after
the
inhibitor
inhibitor
Further
was
IX inhibitor
localized
to
normally
by
ratio
had no effect.
inhibitor
could
sufficient
for
the partially
Inhibitor
Factor
were
might
antiserum
the
inhibitor
Using
antiserum
complete
purified
Isolation
anti-A
inhibitor
of
partially
residual
30-fold.
antiserum,
30-fold
traces
of
serum
was
neutralized
of
parts
kappa-type
98-99%
Using
19
inhibitor:
at least
titers
concentrated
lambda
concentrated
1:64,
and
mm.
low
antiserum
80
OF INHIBITOR
DILUTION
IX
Residual
Inhibitor
Present
Undetectable
Undetectable
Present
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
6
PIKE ET AL.
240-
230220
210
-.
TOTAL PROTEIN (mg/mU
761mg/mI)
7 64 (mg/mI)
INHIBITOR
FUNCTION
(PTT I
-
-
20090
30
Fig.
80
Sharp
ITO
76 781 mg/mI
PLASMA
764
045 mg/mI
PLASMA
I60
l
‘5#{176}E
140
I-
z
30
20
Ui
I-
plasP.W.B.
of inhibitor
by
radial
immunodiffusion. There was no overt
evidence
of a mono-
0
110
peak
zone
of
activity
was
found
in
anodal
portion
of
yG
peak
and was
restricted
to anodal
portion
of yG4
subgroup
as determined
20’-
(1,
.
3. Preparative
electrophoresis
ma from
patient
100.
00-
clonal
90
band
using
either
total protein, yG, or yG4
quantitation,
indicating
80
70
that
monoclonal
antibody
represented
a minor
population
of total
immunoglobulin.
60
0
2
4
6
lOT
8
12
14
IS
16
FRACTION
20
22
24
28
26
30 32
NUMBER
DISCUSSION
The
studies
show
that
the
factor
body
consisting
predominantly,
lambda
light
chains.
It is of note
preparative
zone
electrophoresis,
of whole
plasma,
with
particular
no indication
of a monoclonal
antibodies
to
factor
VIII.8’9
IX inhibitor
emphasis
band.
Thus,
Five
of
240
(Fig.
200
on
with
dextran
blue,
total
volume
(Vt)
with barbital
and
determined
buffer.
Peak
Ill did not contain
inhibitor and was not further
identified.
the
six
antibodies
to
Factor
VIII
anti-
charac-
-
V0
210-
i
MW. lEST
45,000
-
75
-
70
-
.65
90
-
60
ISO
-
55
170’
-50
PEAK
1761
§
-
.45
Oj
ISO-
ii:
-40
40
-
110
00
025
OO0
-30
,,
120
6 050
35
a.
130
volume
determined
is an
on 7G4 and lambda
antisera,
gave
this factor
IX antibody
resembles
220-
calibrated
Exclusion
(Vo) was
P.W.B.
230-
Sephadex
G-200 column
equilibrated
with
TrisHCI 0.1 M, NaCI 0.5 M,
pH 8.0. Sharp
inhibitor
peak
corresponded
in
elution volume to peak II
and coincided
with ‘yG
peak.
patient
if not exclusively,
of yG4 heavy
chains
and
that no 7G4 monoclonal
band
was found
on
and
careful
immunoelectrophoretic
studies
Fig. 4. Gel filtration
of
pooled and concentrated
zone
electrophoresis
fractions
17, 18, and 19
3)
from
-
-
I
90-
::
I’..
)“:1_i’
2030
405060
IS
........t........
....
0
25
-20
7080
FRACTION
-0
-
05
-00
90
NUMBER
00
HO
120130l40150
I
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
MONOCLONAL
terized
In the
ANTIBODY
as to yG
experiments
neutralization.
TO FACTOR
subclass
have been
reported
here,
Thus,
produced
not
complete
tively
inhibitor
amounts
It is of interest
and
with no affinity
the most
likely
that
the
antisera
glomerular
basement
the
IX
IX
presence
of
iv. with
inhibitor
complex
disease,
small
amount
of
administered,
antibody
and the
yG2 binds
for antibodies
circulating
particularly
it
complexes.
presently
may
nephritis.
antibody
also
To
reported
reflect
or
the
to
lack
among
the
VIII,
antibodies.22
periodic
titer,2,
sickness
This
may
the
small
fourth
factor
The
in
relationship
VIII and
no
relafor
proof
residual
predominate
that
yG4
it does
between
this
IX is uncertain.
administration
the
nor
of
patient
evidence
relate
with
for
to the
amount
of complement
date,
antibodies
to
antibody
to factor
had
of small
activity.
represents
to factor
subgroups
and
antibody
serum
yG3
and are
specificity
presence
inhibitor
should
poorly.
The
to factors
antibody
rise in
neither
and
90%
antiserum
no experimental
presence
of
the
retain
IX. This
antibodies
other
circulating
but
subclass
antinuclear
subsequent
manifested
mune
is
and
to Clq,23’24
although
and the propensity
factor
factor
yG
VIII
and
factor
the yG4 subclass:
the
that
unlike
not bind
property
yG
yG2,
Although
for
biologically
is known
Despite
to be
to yGi,
chains.
and
81
However,
membrane,2’
subclass
heavy
are highly
adsorbed
antisera
utilized
had
for Fab.
explanation
minor
of yG4
produced
neutralized.
experiments
complexes
most
both
factors
activity
for
primarily
antiserum
antisera
two yG4
in the
yG4
neutralization
of soluble
inhibitor-anti-yG4
antibodies
to
known
biologic
IX,
composed
the yG4
was
neutralization
yG4 Fc determinants
is available,
we feel
7
all inhibitor
The
yG4 subclass-specific
weakly
precipitating.
The
effect.
IX
relatively
of
&xation
the
im-
factor
by
IX
antigen
clotting
factors
other
than
VIII
IX have
not been
characterized
as to subclass.
All
nantly
experiments
lambda
antibodies
have
affinity
most
antibodies
the
light
factor
zation
antiserum.
inhibitor
inhibitor
The
chain
that
Some
the factor
investigators
IX
type5’1’9
When
light-chain
were
sought,
factor
VIII
V antibodies
and was
however,
contain
could
be
several
detected;
higher
experiments;
serial
The availability
in patient
P.W.B.
molecules
content
properties
found
genic
specificity.
nature
and
confirmatory
populations
dilutions
lambda
of
the
were
of inhibitor
antisera
estimate
light
factor
light-chain
type,
in myeloma
Two genetic
proteins,
markers
found
that
types.4
One
be characterized
To explore
this
utilized:
studies
titer
the
of lower
light-chain
isolated
factor
of antibody
of inhibitor
of higher
permitted
contained
monoclonal
of
approaches
predomifactor
VIII
investigators
a mixture
concentrations
and
minor
recently
reported
could
found
to be heterogeneous
IX antibody,
so that
antibody
contained
have
found
that
other
using
whole
plasma
rather
than
of antisera,
so that minor
populations
chains
inhibitor,
to
factor
content
experiments
centration
indicated
chains.
a single
antibodies
of the two
light-chain
with
light
partially
be
plasma
plus
that
neutralization
IX antibody;
containing
with
could
as to
point
used
conkappa
isolated
in neutrali-
in kappa-specific
high
titer
factor
at least
98%
of
IX
his
chains.
IX
suggests
antibody,
both
further
i.e., allelic
exclusion
for yG4 have been
regarding
study
regarding
heavyother
and individual
antidescribed
recently,25
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PIKE ETAL.
8
but
typing
P.W.B.
etic
was
markers
Cm (a+g+f+b+n
of the yGi
and
the
4a
4b
will
requires
and
require
separation
markers
sence
of
All normal
indicating
or
can
persons
that
02
contain
one
of
the
clonal,
variable
regions
chains
or Bence
these
prototype
Antisera
to
the
antigenic
property
same
or very
specificity,
regions,
e.g.,
yG monoclonal
have
light
sequence
homologies
chains.30’-33
appears
to have
The
the
Unfortunately
agglutinins.29
factors
found
same
IX
specificity
high-titer
for
antibody
the
combining
the
in
IX as that
was
of cross-
combining
the
by
normal
factor
IX
of
these
clonal
nature
of the
antigenic
limited
site
structural
CRM
factor
with
and
tetanus
response
in
which
Isolated
human
teichoic
acid
stricted
in light-chain
yG4
been
on the
between
complex
factor
such
produces
in
carbohydrate
to be
when
restricted
further
factor
relate
to
its
and
IX and
as
R.T.J.2
may
factor
group
minor
polymers,
to the
fractionated
The
mononature
reflect
very
IX found
antibody
proportions.35
dextrans,
yG2
studies
as well.
shared
substances,
a heterogeneous
normal
thus,
specificity
with
hemoinactive
discrete
the
blood
study
and
IX.
the
IX molecule
usually
occurs
to the
shown
factor
antigens,
subgroup
content
inactive
heavy
patient
two patients.34
Family
members
are CRM
antigenic
determinants
may
normal
toxoids,
antibodies
have
biologically
IX antibodies
recognized
difference
in CRM
patients.
Immunization
diphtheria,
and
of
available
could
neither
be characterized
in the present
study,
nor its individual
compared
with
that
of P.W.B.
antibody.
Ten
per cent
of males
philia
B possess
cross-reactive
material
(CRM)
that
is biologically
but combines
with antibody
from either
of these
of CRM
patients
have
shown
that
all affected
These
antibodies
appear
to have
specificity
for
human
purpura
present
from
not
site
purified
regions
reported
R.T.J.
Antibodies
phenomenon
in
of
reflecting
site.28
hypervariable
factor
from
to one
populations
The kappa
chains
of
in hypergammaglobulinemic
antibody
should
conform
probably
the
to
mono-
characterized,
similarities
in
factor
proto-
restricted
antibody
exhibit
studies
of five
If truly
specificity,
sequence
molecules,
are either
sequence
are
should
homogeneous
specificity
to
on
when
to the
amino
190.2627
(arg)
chains
chains
region
related
a single,
subgroups.
antibody,
antigenic
related
related
human
cold
rheumatoid
exhibited
and
closely
probably
to
composed
lambda
glutaralde-
at position
based
region
and
of sequences
relates
are
genfor
isolated
antibody,
presence
or ab-
chains
chains,
variable
individual
With
regarding
proteins,
isolated
the
donor
exclusion
with
(lys)
and Oz (-)
myeloma
lambda
variable
proteins
of
uniqueness
of the
or
The
allelic
progress
antigen
monoclonal
and
sequences.
myeloma
(+)
but
of
in
of lambda
Jones
of the
or O(-),
Oz
Oz
individual
chains
are
of lambda
sequences
lambda
O(+)
exhibit
and
prototype
the
be either
both
allotypic,
not
subclasses.
Demonstration
studies
portion
The
sequences,2#{176}
locus.
completed.
light
(-).
yG
IX is an immunoadsorbent.
the lambda
light
chains
constant
is
other
and therefore
was heterozygous
for
Thus,
he may well be heterozygous
loci.
yG4
from
such
be
in the
of myeloma
type
of
)
+
the
factor
yG4
yG3
antibody;
antigen
Oz
substitution
(+)
of
purified
hyde-insolubilized
further
characterization
acid
of
subgroup
levan,
and
as to oligosaccharide
and
re-
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MONOCLONAL
ANTIBODY
specificity.35
of allelic
In two
exclusion
viously
been
clonal
heavy-
nature
and
to
of the
light-chain
variable
IX
heterozygous
so characteristic
shown
of antibody
will
allelic
exclusion,
to the
TO FACTOR
9
donors,
of
exhibit
the antibodies
exhibited
the property
myeloma
proteins.
Several
had
pre-
individual
antigenic
presently
reported
homogeneity
in
permit
binding
many
affinity,
specificity.28
factor
IX
neutralization
The
antibody
is
experiments.
additional
studies
exploring
and antigenic
and structural
mono-
indicated
by
Isolation
the questions
of
studies
relating
region.
ACKNOWLEDGMENT
It is a pleasure
Ronald
to acknowledge
the
technical
assistance
of Mr.
Randall
Fuller
and
Mr.
Neal.
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1972 40: 1-10
Immunochemical Characterization of a Monoclonal γG4, λ Human Antibody
to Factor IX
Isadore M. Pike, William J. Yount, Elliot M. Puritz and Harold R. Roberts
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