Monoclonal antibodies against a C-terminal ... - Clinical Chemistry

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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