This article was downloaded by: [University of Kiel] On: 24 October 2014, At: 10:29 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
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
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages,
and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [University of Kiel] at 10:30 24 October 2014
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
JOURNAL OF IMMUNOASSAY, 12(3), 413-424 ( 1 9 9 1 )
Downloaded by [University of Kiel] at 10:30 24 October 2014
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
Downloaded by [University of Kiel] at 10:30 24 October 2014
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.,
Downloaded by [University of Kiel] at 10:30 24 October 2014
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
Downloaded by [University of Kiel] at 10:30 24 October 2014
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
Downloaded by [University of Kiel] at 10:30 24 October 2014
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
Downloaded by [University of Kiel] at 10:30 24 October 2014
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%.
Downloaded by [University of Kiel] at 10:30 24 October 2014
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)
Downloaded by [University of Kiel] at 10:30 24 October 2014
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