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Journal of Immunoassay Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii19

Production and Characterization of Monoclonal Antibodies Reactive with Melatonin a

a

a

Koichi Terazawa , Liying Ji , Hachiro Mikami , b

Takehiro Togashi & Takehiko Takatori

a

a

Departments of Legal Medicine , Hokkaido University School of Medicine , Sapporo, 060, Japan b

Departments of Pediatrics , okkaido University School of Medicine , Sapporo, 060, Japan Published online: 23 Oct 2006.

To cite this article: Koichi Terazawa , Liying Ji , Hachiro Mikami , Takehiro Togashi & Takehiko Takatori (1991) Production and Characterization of Monoclonal Antibodies Reactive with Melatonin, Journal of Immunoassay, 12:3, 413-424, DOI: 10.1080/01971529108055080 To link to this article: http://dx.doi.org/10.1080/01971529108055080

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JOURNAL OF IMMUNOASSAY, 12(3), 413-424 ( 1 9 9 1 )

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PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES REACTIVE WITH MELATONIN

Koichi Terazawa, Liying Ji, Hachiro Mikami, Takehiro Togashi* and Takehiko Takatori Departments of Legal Medicine and *Pediatrics, Hokkaido University School of Medicine. Sapporo 060, Japan. ABSTRACT Monoclonal antibodies(moAbs) reactive with melatonin(MT) were produced using MT. coupled to bovine serum albumin(BSA) with the Mannich reaction, as immunogen and conventional hybridoma techniques. Hybridoma clones secreting the moAbs were selected by an enzyme-linked immunosorbent assay system using MTcarboxymethylchitin and BSA as screening antigens. The moAbs from 6 clones were characterized by a cross1251reactivity test using radioimmunoassay with labelled MT. The moAbs recognized MT but hardly recognized other analogues except for N-acetylserotonin with a crossreactivity of 0.81%. An inhibition curve for MT was obtained in the range of 50 pg to 100 ng and 1.4 ng of MT inhibited the value of the assay by half. There is interference from some unknown source in human serum. (KEY WORDS: Melatonin, Monoclonal antibodies, Radioimmunoassay, Immunoassay.)

INTRODUCTION

Melatonin(MT)

is

a

hormone

secreted

pineal gland into the circulation system. MT

Copyright 0 199 1 by Marcel Dekker, Inc.

the

The level of

in the pineal gland and in the blood is 413

from

circadian-

414

TERAZAWA ET AL.

rhythmic:

high

during

the

night-time but

very

low

during the day (1). Therefore, the determination of the MT

level has been proposed for estimating the time

of

death in the field of forensic practice. Since

the MT level in the pineal gland and

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is very low ( 2 , 3 ) , clonal

antibodies

radioimmunoassays(R1As) using polyhave

investigators ( 4 - 7 ) . MT

blood

been

used

exclusively

by

Arendt ( 8 , 9 ) reviewed assays for

and emphasized the need to improve the

specificity

of RIA.

Since

monoclonal

antibodies (moAbs) may

improve

the specificity achieved with polyclonal antibodies, we describe

the production and characterization of

moAbs

against MT.

MATERIALS AND METHODS Chemicals Melatonin, amine

DL-a-methyltryptamine, Nw-methyltrypt-

and 5-hydroxy-Nw-methyltryptamine

purchased from Aldrich Chemical Co.,

oxalate

Inc.,

were

U.S.A.

5-

Methoxytryptamine was obtained from Fluka A G Chemische Fabrik,

Buchs,

indole,

L-histidine,

aspartic

acid,

Switzerland

and 3-indoleacetic

acid,

tryptophan, choline chloride, L-

L-glutamic acid and glycine from

Pure Chemical Ind., Japan.

Wako

All other compounds listed

415

MONOCLONAL ANTIBODIES REACTIVE WITH MELATONIN

in

1 and 2 were purchased from Sigma

TABLES

Co.,

U.S.A.

Ci/mmol) 2000

activity,

and 2-[1251]iodomelatonin(specific

Ci/mmol) were supplied by Amersham

plc.

85

activity,

International

CM-Chitin 1OC was purchased by Funakoshi PharmaJapan.

ceutical Co.,

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H]Melatonin( specific

[

Chemical

IgM and IgA antisera were obtained from Serotec,

IgG3, Co.,

Anti-mouse IgG1, IgG2a, IgGZb,

Ltd.,

K.

U.

All other reagents were supplied by

Nakarai Chemical Co., Ltd., Japan.

of

Preparation

(i) MT

Antigen Conjugates

An immunogen was prepared by binding MT with [3H] to

bovine

serum albumin(BSA)

with

the

Mannich

reaction as prepared by Grota and Brown(l0). The degree of conjugation was calculated to be about 10 rnol of the

hapten per mol BSA(mo1. wt. 6 8 . 0 0 0 ) on the basis of the radioactivity

measured

in

a

liquid

scintillation

counter (Aloka LSC 903). (ii)

MT-carboxymethylchitin

synthesized three sodium

mg

for moAb screening

solution.

as

follows.

of 4-amino-D,L-phenylalanine and 2.4

bromide

acidified

(MT-CM-chitin)

with

were

dissolved in 1 ml of

0.024 ml of

conc.

was

Twentymg

water

hydrochloric

Then 0.2 ml of water containing 10.8 mg

of and acid

of

41 6

TERAZAWA ET AL.

sodium

nitrite

was added dropwise to the mixture

and

the solution was mixed for 10 min in an ice bath. with

1%

Thirty mg of

MT

containing [3H]MT (250,000 dpm) were dissolved in 4

ml

excess

sodium

ammonium

of

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The

nitrite was then

neutralized

sulfamate aqueous solution.

0.1 M borate buffer (pH 9.0) with 1 ml of dimethyl-

formamide.

The

diazotized

4-amino-D,L-phenylalanine

solution was added to the MT solution.

The mixture was

stirred for 6 h on the ice bath maintaining pH 8.0

and

water

8.5.

Thirty m g of CM-Chitin 1OC in 1 ml

and 3 8 mg of

of

l-ethyl-3-(3-dimethylaminopropyl)-

carbodiimi.de hydrochloride added

between

in 0 . 5

ml

of

water

were

to the diazo-coupled MT derivative solution

stirred

for

between

4.0

imide

2 4 h at room temperature

and 5.0.

maintaining

and pH

A further 19 mg of the carbodi-

were added to the reaction mixture

and

stirred

for 2 4 h followed by dialysis against running water for 48

h.

The

calculated chitin

degree of conjugation with CM-chitin to

(mol.

be about 21 mol of the hapten wt.

per

85,000) on the basis of the

was CM-

radio-

activity.

Production of MoAbs

A

female

immunized

i.p.

six-week-old with

BALB/C

mouse

was

first

0.25 mg of MT-BSA in 0 . 2 5 ml

of

417

MONOCLONAL A N T I B O D I E S R E A C T I V E WITH MELATONIN

emulsion

of saline and Freund's complete

adjuvant(1:l

Thereafter i.p. injections of 0 . 2 5 mg MT-BSA

by vol).

in saline were given monthly over 4 months.

Three days

after the final injection, spleen cells were fused with myeloma cells(5:l by number)

P3U1

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glycol

1500

and

cultured in

using

the

polyethylene

Iscove's

Dulbecco's medium containing hypoxanthine, and

aminopterin

Hybridoma clones secreting

moAbs

selected by an enzyme-linked immunosorbent

assay

thymidine (11).

were

modified

system using MT-CM-chitin and BSA as screening antigens (12) by

except that the MT-CM-chitin was coated on

wells

drying 5 0 0 ng in 0 . 0 5 ml per well at 37 "C for 6 h.

Hybridoma

cells

supernatants

contained in wells in

were

negative

to

BSA,

dilution

method

positive

to

which

MT-CM-chitin

were subcloned twice by a

to ensure their

culture

monoclonal

but

limiting origins.

Isotype analyses were performed by the double-diffusion technique of Ouchterlony(l3). MoAb-secreting hybridomas ( l x 1 0 8 ) were injected i. p. into an adult female BALB/C

mouse

which had been injected i.

p.

Pristane (Aldrich Chemical Company,

with 0 . 5 Inc.,

ml

of

U. S . A.) 3

weeks previously and the ascites produced was used a s a source of antibody.

MoAbs phosphate

and

reagents

buffered

were

diluted

saline (pH 7 . 4 )

in

0.05

containing

M 2.5%

418

TERAZAWA ET A L .

ethanol

0.06% bovine

and

preparation reactivity

of

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of

globulin.

For

an inhibition curve and €or

test,

the following : of MT,

gamma

each assay

0.2 ml of

a

tube (0.5 ml)

cross-

contained

[1251]MT(5000 dpm), 0.2 ml

its analogues or selected compounds and 0.1

the diluted ascites containing

mixture

was

incubated for 2 h

moAbs.

at

room

salting-out method(l4).

ml

This assay temperature.

Antibody-bound [ 1251]MT was separated from the free the

the

by

and its radioactivity was

measured in an Aloka auto well gamma system(ARC-500).

Serum Samples

Individual sera were collected during the from 20 healthy adult women,

pooled and inactivated by

incubation at 56 "C for 30 min to destroy Pooled

serum

(0.1

daytime

ml) was added to

the

complements. RIA

system

instead of unlabelled MT or related compounds.

RESULTS AND DISCUSSION

Six

clones

of

hybridoma

were

selected,

secreting moAbs specific for MT-CM-chitin

all

but not BSA.

The diluted ascites(l:5000) was shown to bind about 50% of

5000

dpm of [ 1251]MT by the

RIA

procedure.

The

MONOCLONAL A N T I B O D I E S R E A C T I V E WITH MELATONIN

419

specificity

the

of each antibody was characterized by

cross-reaction

test,

in

which the moles of

[1251]MT was compared

ment of the binding of moAb to that

of MT,

where the crossreactivity of

chosen as 100%.

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MT

or selected compound which gave 50% displace-

analogue

to

each

MT

was

The six moAbs showed almost the

same

features and representative results are shown in TABLEs 1

and 2.

tested

The immunoglobulin isotype of each moAb was by

the

Ouchterlony

identified

as

establish

whether

recognized

the

from

method

all

IgGl but no experiments were

same

the

moAbs

were

epitope.

were

done

identical

Ascitic fluid derived

medium was used to examine assay

The

(FIGURE 1).

inhibition was

no

in

the

1.4 ng of MT giving 50%

Despite using [ 1251]MT,

more sensitive than

those

performed

previous investigators using polyclonal antibodies [%lMT,

e.

g..

in

sensitivity.

inhibition curve for MT was obtained

range of 50 pg to 100 ng, with

to and

the clone secreting the highest level of moAb

culture

RIA

and

our by and

2 or 25 pg per tube ( 4 ) and 4 0 pg per

tube(5) indicating that the antigen binding affinity of our

moAb

is lower than that of the

polyclonal

anti-

bodies used (15). The MT

anti MT moAb obtained was highly specific

as shown in TABLEs 1 and 2.

to

The only one

slightly

crossreacting analogue was N-acetylserotonin,

which is

420

TERAZAWA ET AL.

O L ,\ 0 b.01 0.1 1 10 Melatonin (ng/tube)

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I

FIGURE 1. Displacement of unlabelled MT in RIA.

'

IIMT bound to moAb

by

TABLE 1 Crossreactivities of Anti-MT MoAb against MT Analogues in RIA Using [I25 IIMT. Compounds

and

its

% Crossreactivity

Melatonin N-Acetylserotonin 6-Hydroxyrnelatonin 5-Methoxytryptophol 5-Hydroxyindole-3-acetic acid 5-Methoxytryptarnine 5-Methyl-3-indoleacetic acid 7-Methyltryptamine DL-a-Methyltryptamine NU-Methyl t ryptarnine 5-rnethyltryptarnine hydrochloride N-Acetyl-L-tryptophan 5-Hydroxytryptophan 5-Methoxytryptophan

100 0.81 0.009

Production and characterization of monoclonal antibodies reactive with melatonin.

Monoclonal antibodies(moAbs) reactive with melatonin(MT) were produced using MT, coupled to bovine serum albumin(BSA) with the Mannich reaction, as im...
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