mark) with. 1 mLIL glutaraldehyde. CF1. X. BALB/c mice were immunized by intraperitonal injections ... electric eel and. Torpedo marenorata have also been used for characteriza- tion of the antibodies, ..... J Neurochem. 1985;. 44:697-704. 12.
Clinical
Chemistry
19-23
(1996)
42:1
Monoclonal antibodies against a C-terminal peptide of human brain acetylcholinesterase distinguish between erythrocyte and brain acetylcholi nesterases NICOLA
BOSCHETTI,’
URS
BRODBECK,’ BENT
Monoclonal tide
antibodies
of
the
10
(mAbs)
C-terminal
acetylchohinesterase
were
to
and
were
distinguish
AChEs. well showed
clear but
190-2
and
binding
not
reacted
with
were
used
fluids,
open
from
normal
nated
analysis,
AChE
neither
AChE neural
ACb.E
serum.
assay
activity
could
tube-defect or
for
and
references
fluid
eel
Both
antibodies in
only
and nor
not
in artificially
blood-contami-
inimunoassay
IEJ%45
nervous
#{149} fetal
(AChE,
Acetylchohinesterase in the
#{149} neural
antibodies
system,
EC
where
tube
defect
3.l.l.7)
is an essential the
by
-20
kDa.
AChE
from
each
subunit forms
ui
Biochemistry
and
Biology,
Molecular
University
Bern,
of
CII-
and
can
of 2 Clinical Biochemistry and ‘Immunology, Statens SerumArtillerivej 5, DK-2300 Copenhagen S. Denmark. Chemical Laboratory II, The H. C. #{248}rsted Institute, University of Copenhagen, DK-2 100 Copenhagen 0, Denmark. Address correspondence to this author. Fax +45 32 611 38 7%. Departments
fluid,
causing
that
Nonstandard
abbreviations:
I)(iCly;
BChE,
EAIA,
Received
AChE,
SDS-PAGE, and
May
acetylcholinesterase;
antigen
butyrylcholinesterase;
acetylcholinesterase; eletrophoresis;
enzyme
ChE, cholinesterase. II, 1995; accepted
immunoassay;
SS-/I)S-AChE, sodium September
Imoc, mAb,
rise
28,
sulfate-polvacrylamide
in
antibodies
of AChE
from
human
in the
erythrocyte
form
Umiless
otherwise
stated,
grade gel
obtained
(Rockford,
MO),
1995.
19
IL), Bio-Rad
this
of the
AChE.
diagnosis
In neural fluid activity
Amnio-
erythrocyte
AChE,
circumvent
brain
form
of AChE
we
produced 10
a sequence
this would
antibodies
C-terminal that
the
compared
pregnancies.
last
tube
into
To
goal,
Such
of neural
results.
of the brain,
141. antibodies
erythrocyte
with
in
of the
erythrocyte
type
to produce
in AChE
for the
amino
is not
present
enzyme.
and Methods all chemicals
either
from
Merck
(Darmstadt,
(Hercules,
splicing
brain
normal
erythro-
but differ
disorders.
AChE
achieve
and
cerebrospinal rise
consisting from
brain
prenatal
contamination
specific
To
a peptide
was and
the
Materials
anti-
salt-soluhle/detergent-soluhle
dodecyl
brain
a sharp
to false-positive
useful.
work
is of
dimer,
the
the
use in the
fluid
result
9-fluorenyl-
monoclonal
present
in amniotic may
than
as of neurological
amniotic
form
membrane
domain
modification,
from
DS
peptide
of different
shorter
are of potential as well
The
catalytic
Because
between
leaks
acids
/3/.
of the
AChE
very
common
acids
distinguish
defects
against
institut,
see
detergent-
The
to the erythrocyte
posttranslational
purpose
enzyme.
moiety.
part
defects,
be
nomenclature
is a disulfide-hinked
is anchored
a large
is 26 amino
with
Switzerland.
methoxycarbonyl;
possess
amitibodies
of acer1’l-
of AChE
a hydrophobic
erythrocytes
of which
C-terminal
tube
is also cells,
forms
of --80%
consists
membrane
difficulty, Bern,
brain
cell
giving Institute
mammalian
to the
centesis
3012
(for
anchored
The
enzymiie
action
of polymorphism
(SS)
AChE
#{149} amniotic
AChE
hematopoietic
molecular
salt-soluble
mRNAs
in fluids
as
The
20%
their
status
it terminates
from (DS)
cyte
in samples
but
degree
However,
such
is unknown.
by a glycophosphatidylinositol
amniotic
pregnancies,
tissues
and
that
#{149} anti-peptide
and
1, 2).
AChE
samples.
INDEXING
KOCH,3
membrane.
nonchohinergic
a large
soluble
bovine
190-1
electric AChE
be found
its function
display
as
postsynaptic
where ability
antibodies
MAbs
at the in
erythrocyte
human from
human
pregnancies
and both
from
CLAUS
JENSEN,2’4
found
(mAbs
their
immunoassay
erythrocytes.
a quantitative
where
from
dot-blot
from
in
for
brain antigen
from
butyrylcholinesterase
choline
brain
clones
tested
enzyme to
to AChE
a pep-
human
positive and
mammalian
a solid-phase
as by Western-
brain
selected
between
In
of
H-Tyr-Ser-Lys-Gln-AspTwo
190-2)
against
acids
(AChE):
Arg-Cys-Ser-Asp-Leu-OH. 190-1
raised
amino
S#{216}REN PETER
N#{216}RGAARD-PEDERSEN2’
Fhuka CA),
used
(Buchs, Germany), or
Milhipore
were
of analytical
Switzerland), Sigma
Pierce
(St. Louis,
(Bedford,
MA).
Boschetti
20
Rabbit
anti-mouse
(Glostrup,
antibodies
were
to the
of
residues
574
to 583
perfornied
Milhipore)
synthetic
peptide,
of bovine
of the
on
human
of
was used with
peptide mass
amino
with
nm).
KA,
/8/. alone.
used
For
the
9-fluesters
the
was
used
by HPLC,
characterized and
amino
resin
teniperature.
The
for conjugation.
The
fast
acid
dried
atom
mmol/L
analysis.
The
of
iodide
per
AChE
activity
under
mouse
imniunoglobulins
buffer
(10
synthesized
protein
peptide
derivative
mark)
with
(Statens
1 mLIL
immunized Al(OH)5
four
times,
2 weeks a booster
antigen.
Fusions [10].
X63-Ag8.653, Culture
and
(10
mL/L
Tween
sodium The
20). (diluted
and
expanded.
were
determined
by ELISA
CA) from
Protein
den)
and
cell culture
according
against the
monoclonal
with
biotinylated Ab-Id
of
them
brain
antibody
were
are
available
bance
San
used
as
Statens
caudate beck
1111,
were
by using
benzamidine
ter
consecutive
6200
of 5000 U/mg
protein
ligand
presence
and
bovine
brain
0.14-8.75
before
AChE.
1.98,
and
were in 10
containing
144 into
with
assay
Triton
washing
For
100
mmol/L sodium Absor-
Devices with
slow
buffer,
X-1001.
Molecular
the
with
times
Reader.
(Sunny-
the
endpoint
assay
buffer
for
assay. performed
controls
as described
were
range
for
prepared the
the concentrations
0.78
plates diluted
iodide and 0.25 a 100 mmol/L
incubated
1 h at buffer
pipetted
of
in
the
were
The
and
five
1 mL/L
was
and
U/L
U/L,
and
AChE
purified
calibrators
in the
four
U/L
for
Control: bovine OS-AChE with mAb 53-4 raised against BChE from human
brain.
Boschetti
Table
1. Results
immunoanalysis with
of dot-blot
the reaction
for
purified
(on
brain
different
eel
acetylcholinesterases
and -2 Denatured
enzyme
last
10
sequence
+
protein
+
+
40%
datal)ase
HSKGIVYRDL
+
+
not
+
+
with
90-99
and
sequence
Human serum BChE
terniinus are,
MAb
53-4
as a control
and,
The
raised
reactions DS-denatured
dot-blot
method. from
both human
and
Toipedo
and
from
also
To
humnan
test
theni
the
when
amniotic
fluid
normal
and
logical
U/L
reacted
used
all
with
mAb
and
the
two
Prenatal
and
with
with
of these
AChE
from
mnAhs,
2).
The
open micural
U/L,
190-1
[up
1:40
0.53-5.45
U/L
sample
is contamiiinated
when
the
this
difficulty,
synthetic brain
study peptide
of the
that
AChE
describes
two
10 C-terminal
distinguish
between
raised
are
mAb
an)in()
acids
the
l)rain
and
against
able
cyte
a
of human
The
AChE
humnami brain
alternative
splicing
erythrocvte
form
However, on,
amid
is 26 amino
mAbs
the
were
acids
emythrocyte
shorter
splicing against
lomig. Because
of
modification,
sequemice
alternative produced
amino
acids
in their
a difference where
is 583
posttranslauonal
than
is found site
brain from
is located
a symithetic
the AChE. position
/4/.
peptide
There-
2. Results
of EAIA
with
pathological
normal,
amniotic
contaminated, Activity,
n
Normal Pathological Contaminated (1:640-1:40,
mAb
350
190-1
mAb
9
190-2
0.53-4.91
0.53-5.45