1. health and fitness
2. disease

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MICROCOPY RESOLUTION TEST CHART
BUREAU
OF 6TANQAROR-1963-A
NATIONAL
I'
4.
I
vi
COMMONWEALTH
PATENTS
of
AUSTRALIA
ACT 1952'T
APPLICATrION FOR A STANDARD PATENT
Insert 'ti
YWSA SHOYU KABUSHIKI KAISHA of
Araoi-Cho 2-Chome,
Choshi-Shi,
Chiba-Ken,
Japan.
Insert fu
of dectl
cant(s)A
sir. ,pn
co'mnil
j
MDOAD N?
'Jheeby apply for the grant of a Standard Patent for an~invention entitled:
4
"DIAGNOSTIC AGENT FOR HEART DISEASE
AND USE THERlEOF" t
1(a)or
r(a
eli
by indiv
o
vol
by coir
applicanl
I
which is described in the accompanying -rvaml specification.
complete
Cross or
Details of basic application
Number
58767/1984
Convention Country
Date
Japan
27 March,
2(a) rel,
by inveti
2(b) vol
by0 coml
are not
1984\,
~oSae
a
0
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Dated this
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c
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19
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00
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00CC
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day of
00
CO
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C(
For
insert bi
by date(
The address for service is care of DAVIES
COLLISON, Patent Attorneys, of I1 Little
Collins Street, Melbourne, in ithe State of Victoria- Commonwealth of Australia.
To: THE QOMMISSIONER OF PATENTS
Insert, p1
memer ',th frm of DAVIES&
COLLISON for a)-A1 on behalf of the Applicant).
a
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Signatur
attistatli
Davies
Note:
Collilson, Melbourne and Canberra.
('I
4
4
4
I
la.
COMMONWEALTHJ
OF
AUSTRALIA
PATENTS ACT 1952
DECLARATION IN SUPPORT oF- CONVENTION OR
NON-CONVENTION
APPLICATION FOR A. PATENT
In support of~ the Application made for a..patent for an invention
Insert title of invention,
entitled::DIA; GOSTIC .AGENT,
TH~EREOF"
Insert full riame(s) and address(es)
rL
I
3-e
of declarent(s) being t-he applicant(s) or person(s) authorized to
sigs on behalf of an applicant
compatty.
Croas out whichever of paragraphs
or
does not apply
'*1
the said
YAMVASA SHOYU KABUSHTKI XATSHA.
the applicant
2(a) relates to application made
by inventor(s)
2(b) relates to application mnade
by companyfs) or person(s) who
4
are not inventor(s);
insert full
nm and addres5(es) of inven'Zors.
0.
2. (a)X
4
President of YAMASA SHOYU KABUSHIKI KAISHA
of 10-1, Araoi-Cho 2-Chome, Choshi-Shi,
Chiba-Ken, Japan
-We are
or(b) I am authorized by
Cross out whichever of paragraphs
Va r 2(b) does not apply
.4
Plichio HAMAGUCHI
do solemnly and sincerely declare as follows
1(a) relates to application made
by individual(s)
1(b) relates to application made
by company;
insert name of
applicant company.
.984.
FOR HEART DISEASE AND USE
~~behalf.
for 'the patent to make this declaration o
Yosh -io YAZAKI, of 20-5-805, Koishikawa
4-Cliome, Bunkyo-Ku, Tokyo-To, Japan and
Masahito SUGI, of 2-2, Sakae-Cho 2-Chome,
Choshi-Shi, Chiba-Ken, Japlan
or(b),
a
arethe actual inventor,....of the invention and theIfacts upon which the applicant
are
The applicant is the assignee of the
said actual1 inventors
State manner in which applicant(s)
0 deri~e title from inventor(s)
I 5w5 out paragraphs 3 and 4
*foV dfon-convention
For
convention
S
3.
applications.
applications,
The basic application
as defined by Section 141 of the Act wias made
xrx
InJa~~~pan
e
insert basic country(s) followed
by date(s) and basic al~plicant(s).
nh
h
y
f
AI H
in
arh 18
o n the
by
on
14
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referred to in paragraph 3 of this Declaration was
the first application
made in a Convenition country in respect of the invention the subject
of the application.
IES
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Declared at
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attestation reqt~ired)
Note.
Al
initial' all
Choshi-Shi,
Japan,
this
1 st,
dayof April 1985
(no
6'
alterations.
Michio HAMY6UCH
DAVIES
COLLISON, MELBOU NE and CANBERRA.
Pr
nt
0:
B-
12)
(19)
AUSTRALIAN PATENT ABSTRACT
AU
(11) AUA-40377/85
(54)
DIAGNOSTIC AGENT FOR HEART DISEASE
(71)
YAMASA SHOYU KABUSHIKI
(21)
40377/85
(22) 26.3.85
(31)
59-53767
(32) 27.3.84
(43
3.10.85
(51)4
A61K 49/02
(72)
YOSHIO
GOIN
KAISHA
(24)
(33)
27.3.84
JP
33/577
YAZAKI AND MASAHITO
SUGI
74)
DM
57I
1.
Claim
A diagnostic agent for heart disease 6omprising
a radiolabeled monoclonal antibody having specificity
to human cardiac myosin heavy chain or its
fraghent.
active
A method for the diagnosis of heart disease
which comprises administering a r0adiolabeled monoclonal
antibody having specificity to human cardiac myosin
B%
heavy chain or its active fragment to a subject, and
detectiag the site at which the human cardiac myosin
heavychain is exposed by measuring the radioactivity
orignated from the radiolabeled monoclonal antibody
or its active fragment.
o,
B
2
ND
*57/12
V
34 cell line producing an antibody hav.ihg specificity
23
00;
i-~;i
C 0 MM 0 N V E AL TH
n~li
AUSTRAL'IA
PATENTS ACT 1952
COMPLETE SPECIFICATION
i-i
i:
OF
UE
CE
(Origina
r~
FOR OFFICE USE
Class
-ii
Int. Class
Application Number:
Lodged:
I
ii
tf
a
Complete SpEcification. Lodged:
Accepted:
Published:
(22)/k
Priority:
Related Art:
Name of Applicant:
"'Address
a,4
"e
B
so
cu
YAMASA SHOYU KAB1JSU
KAISHA
of Applicant:
10-1, ARAOI-CHO 2-CHOME,
CHOSHI-SHI,
n
CHIBA-KEN,
JAPAN.
*t.Actual
Inventor(s):
4"
Address for Service:
YOSHIO YAZAKE and
MASAHITO SUGI
DAVIES
COLLISON, Patent Attorneys,
1 Little Collins Street,
.%.gomplete
Melbourne,
3000.
specification for the invention entitled:
0040'DIAGNOSTIC AGENT FOR HEART -DISEASE AND USE THEREOF"
The following statement is a full
description of this invention,
,,.,Including the best method of performing it known to us:
14
EZxample 1
.1
(production of 131 I-labeled anti-human
I
I
'I
I
.1
H
A
w
yi^
y
2laDIAGNOSTIC AGENT FOR HEART
0
DISEASE
AND USE THEREOF
BACKGROUND
o
Field
of the
OF THE INVENTION
Art
The present invention
relates
to a diagnostic,
agent useful for diagnosis of heart disease such as
myocardial infarction and myo.ardial disease by
imaging, and a method for the diagnosis
of heart
disease with use of the diagnostic agent.
In recent years, (in the diagnosis of heart
disease such as myocardial infarction, diagnosis by'
Simaging which involves administering a radiolabeled
Stracers into a body,
detecting y-rays emitted by the
radioisotope to convert the same into an image,
processing the image with a computer to obtain a
two- or three-dimensional image, and diagnosing the
site or size of the myocardial infarction on the
basis of the image thus obtained has made rapid
20
progress.
However, ,tracers used heretofore in
the diciaosis by imaging in cardiac nuclear mediN
-4
i
a
9
a'
S
cine have not always been able to depict specifically the site of myocardial Infarction.
For example, Tl scintigraphy of myocardium
nthalliu-201 (201
using thallium20
Tl) applies the mechanism
at sa*:
wherein the TI behaves in vivo similarly as pd'tasSslum ion and is taken into cells of the heart liver,
1
kidneys.. endocrine organs, tumors and the like where
.in a'
a
I
turnover rate is relatively ofast, whereby normal
a
cardiac muscle is depicted. while the Tl is not
ingested into necrotized or ischem5i?
*4
cardiac muscle
a
at an infarctionsite, which site ('is depicted as a
a
4
a
defect.
Accordingly, the T'1>^does not always depict
a:*Y'
cardiac muscle specifically, and it has also been
difficult to determine by this method whether the
ihfarction occurred recently or in
the past.
Pyrophosphate scintigraphy of myocardium,
on the
01
other hand, utilizes the phenomenon of technetium
1
1
-99m(99 Tc)-labeled pyrophosphate accumulating at an
infraction site, which site is depicted as a positive
scintigram.
This tracer, h6wever, deposits also in
the peripheral region of the infarction site and thus
I.
is liable to overestimate the infarction area.
Prior At
As a method intended to overcome the above
01
described problems accompanying the conventional
00
tracers, a method using a radiolabeled antibody for
cardiac myosin has recently attracted .considerable,
attention.
In this method use is made of an antibody
°obtained by purifying anti-serum prepared by immunizp/ ing animals with purified cardiac myosin or. its active
fragment such as
2 fragment obtained by treating
the antibody with pepsine, both the antibody and its
ij
active fragment beifg radiolabeled and reported to
S accumulate densely at an infarction site.
(cf. U.S.
00
Patent No. 4,036,945)
However, a myosin molecule has a sub-unit
structure comprising two heavy chains 'nd
four
lightschains (two species in the case of human
9
cardiac muscle),
so that the anti-serum obtained
by immunization with the myosin, even if purified
25
by means of affinity chromatography, also contains
4
antibody'molecules specific for light chains.
myoca
ial ifarction,
In
myocardial cell 'embranes
in
a ned1 otized region are destroyed whereby cardiac
4
myosin light chains are released into the blood
#4
44
4
together with polypeptides such as creatine phospho0
0
kinase (CPK) and lactate dehydrogenase.
Thus, the
antibody having specificity to myosin light chain
and its active fragment form an immune complex with
the light chain released into the blood and, are
1
.1
4
4
44
44
4,
4
4,
35
consumed on the route to the infarction site.
,Meanwhile, there is the possibility of the immune
complex inducing an allergic reaction and various
oj.
V
in.
0.-I
o
0
j
for 2 hours at 370C.
r ~ii
i. i
i
I
:i
i
11
I
other biological reactions., A further problem is
that the anti-serum can be prepared only in a limited
quantity and therefore cannot be supplied in a large
quantity for clinical purposes.
SUMMARY OF THE INENTION
'We have carried out considerable research with
a view to developing tracers which will be accumulated
specifically in amyocardial infarction
ing exact diagnosis by iinaging.
site, ensur-
As a result we have
conceived and developed this invention.
More particularly, the present invention provides
a diagnostic agent comprising a radiolabeled monoclonal
antibody having specificity to human cardiac myosin
d
44
heavy chain or its active fragment ard useful for
diagnosis of heart disease such as myocardial infarction, myocardial disease and the like.
0
Theprestent invention, in one aspect "thereof,
further provides, a diagnostic agent for heart disease
com~prising a radiolabeled monoclonal antibody having
specificity to an isozyme of human cardiac-myosin
1eavy chain or its active fragment.
4-
antibody for use in this casF.4,
44-4
4
-4
As the monoclon,al
a monoclonal antibody
having specificity to human cardiac myosin heavy chain
a typeor
25
*ps
type.,is employed 4
The p~resent invention, in another aspect thereof,
5%44
provides a method for the diagnosis of heart disease
with use of the diagnostic. agent.
BRIEF DESCRIPTION OF THE REFEREN-CE
ILLUSTRATIONS
In the illustrations:
4* 4
30
44:,P
'4
FIG.
1 is a photograph wherein FIGS. IA,
1B,
and
1C are planar-images which are respectively an anterior
4c
view, a left anterior oblique view,
*4
444
and a side view of
4
the infarction site in the canine ventricular muscle
obtained in Practice Example by the use of
4
44."
3,5
clonal antibody
1:31
I-mono-
(UHMC-48);
44s
.t0 4*J
-441
4)O
4: 4444
FIG. 2 is a photograph wherein FIGS. 2A, 2B, 2C,
and,2D
are single photon 6missionl\computed tomography
z-i
i
Sepharose CL-4t column equilibrated with 0.1 M Tris-
-4~J
11 -"I
I
of the infarction site in the canine ventricular
muscle obtained in Practice Example by the u-e of
13 1
-monoclonai antibody
(IMC-48);
and.
FIG.
3 is 'a photoraph showing. the radiolabeled
anti-myosin heavy chain monoclonal
accumulating at the myocardial
antibody fragment
infarction
site in
Experimental Example.
DETAILED DESCRIPTION OF THE INVENTION
In myocardial
infarction,
cardiac
yosin light
chains arereleased into the blood while largemoleV
,ules such as
cells.,
cardiac myosinheavy chains stay in dead
By utilizing an antibody having specificity
to cardiac myosin heavy chain as an imagingagent-,
the problem concerning the consumption of an antibody
in
the blpod on the route to the infarction site can
be dissolved,. whereby the
radioisotope can be accumu-
lated specifically at the infarction site, in a high
yield.
Therefore,
it has become possible to obtain a
sharp image of the infarction site 'at a lower dose
0cx
0n
level.
0rws
Further, in comparison with the conventional,,
antibodies derived from anti-serum, this
antibody has
4 ll
the advantageous feature of less undesirable sid~e
effects such as the induction of an allergic reaction
0If
25
and. various other biological reactions
formation of an immune
0:
due to the.
complex with the cardiac myosin
light chain in the blood.
0~
0
Further,
000ri
oo
so
00
concerning cardiac muscles, the exist-
ence of two isozymes, one being a type having a high
ATPase activity and the other being
type having a
low ATPase activity, has been- known, and, generally
speaking,
in humans,
atrial muscles
contain primarily
a type, while ventricular muscles contain substantial-,
ly
35
0
*1
I
.type.
Acq6rdingly, mQnoclonal antibodies specific
for an isozyme of cardiac myosyi-,Uaevy chain ha
the
r
0a 0
0
0aa00n
advantageous
0~i 00
0
0
abovet, of being usable
featuj'in addition
to those
described
for localization o cmyocardial
I
artificallY,induced by ligating the coronary artery.
infarction.
More specifically,
application of mono-
clonal antibodies having specificity to cardiac. myosin
heavy chain a type has facilitated the diagnosis of
atrial myocardial infarction which has heretofore been
extremely difficult.
In contrast, application of mono-
clonal antibodies having specificity to cardiac myosin
heavy chain 8 type makes possible topographic diagnosis
of ventricular myocardial infarction by imaging.
These monocloil antibodies can be supplied
stably in great quantities by known methods as useful
and highly specific antibodies.
The diagnostic agent of the present'invention is
obtained by labeling with radioisotopes monoclonal
antibodies having specificity to human cardiac myosin
heavy chain or human cardiac myosin heavy chain a type
or 8 type (hereinafter, where necessary, referred to
generically as "monoclonal antibodies") or the active
fragments thereof.
These monoclonal antibodies or the
active fragments thereof are not particularly limited
in preparation method and procedure for labeling with
radioisotopes, nor is the diagnostic agent of the
S*
present invention limited in form of preparation,
which can be suitably selected according to the pu pse.
The monoclonal antibody and its
active fragment
ann
25
used in the present invention can be prepared respectively by applying the generally practiced cell fusion
method (cf. G. Kohler, C. Milstein, Eur. J. Immunol.
6 511-519
(19 76
269-270 (1978))
a
30
and M. Shulman et al.,
Nature 276
obtain a hybridoma producing the
antibody and de tiving a monoceonal antibody from the
hybridoma and by subjecting the Jonoclonal antibody
Se
thus derived to hydrolysis to obtain its active fragment.
These procedures will be generally described
as follows.
35
c
Preparation of antibody-producing cells
Preparation of antibody-producing cells is carried
out by immunizing an xenogenic animal such as mouse,
4
rat, rabbit, shepp, horse, bovine, etc,, with a human
G- cardiac myosin heavy cha--'n, human atrial rny2Osin
type),
of monod.iad. mfyosin
humnan ventricular myosin
naosis of
equivalent immunochemically to the human cardiac myosin
S. heavy chain or human, cardiac iyosin a~ type or
tafore been
type
prepared fraom bovineJhorse or hog, and taking antibody-*
producing cells from spleen cells, thymocytes, lymphnode
cells _and/or peripheral blood lymphocytes.
ion of monodiac myosin
ic diagnosis
PreparatioA of myeloma cells
ging.
As myeloma cells,
10
plied
cell lines. originated from
variou~s animals such as mice, rats, rabbits, and
humans, can~be used. The cell l'ine to be used should
as useful
preferably be drug resistant, not Viable in a selective
medium but viable after fusion.
The c!ell line most
vent ion i s
o'clanai
15
Aiac miyosin
'hamn a type
commonlv uised is a 8-azaguanine resitant cell line,
which is defective in .ypozanthine phosphoribosyj,
transferase and cannoz{t be grown in hypoxanthin e't
aminopterine-thyinidine (HAT) medium. The cell line
f erred to
the active
is also -preferably of the "non secretor" type.-
dies or the
ly limited
20
444
eling with
4
~44'<
44
*4
fragment
4
1-7
1580)
.4
4
251-Ag 4
44
44
(19-76))
4444
iL.(NS-lY(European
$p2.10-Ag 14
276,, 269-270
cell fusion
cell line.
Inimunol.
44
4.4
4
4
44
.4
4
4"
(1978))
(SP2)
6 J.
Immunology,
(ATCC CRL3-1581)
4
4
4444
4
44*4
.44
.292-295
4.4
(Nature,
derived from mouse myeloma MOPC-21
Rat myeloma 210 RCY 3 Ag 1-2-3
(Y3 Ag 1-2-3)
4
ure 27G
cing the
P 3 /x63-Ag 8-6-33 (x63.6-5-3) (ATCC CRLImmunology, 123, 1548-1550 (1979))
P 3/NSI-
44.4
(3
ed respec-
(TTCC CRL-1597)
4
4
4
.4
P /x63-Ag 8 U 1 (P U
~(Current Topics in Micr,,biology and Immunology,-81,
44
4
4
se.
(1975))
.4
44
ation,
Typical
exampl~es of'such cell lines are P 3 /x63-Aq 8(Nature 256,
495-437
44
of the
the pu'
type) or a cardiac myosin,
(Nature 277,
o
from the
antibody
4
30
and human myeloma U-266--
AR 1
(Proc. Natl. Acad. Sci>.
77, 1158
(90)
44
4Q
4
.4 .44
and GM 1500
*44*
Some of the cell lines listed above are comme -cially
4
Live f rag-
(1979)),
4
~74
444
4
131-133
4
(Nature, 228, 448
(1980))
are also available.
available.
4
Cell fusion
ascribed
4
4
44
4.4
35
.4
4
4
s is carried
is mouse,
I
4
44
4
4.4
Cell fusion is carried out by mixing 1
to
myeloma cells with antibody 4prfo5 cing' tells at a
mixing ratio of from 1:4 to 1:10 in a medium for
4.4
4
4
.4*4
.44
4
9
culturing animal cells such as Eagle's minimum essen'tial
medium (MEM) and RPMI 1640.
polyethylene glycol
As a fusing aid, a
(PEG) having an average molecular
weight of 1-000 to 6,000, a polyvinyl alcohol, a virus,
or the ike is used.
SeLection of hybridoma in selective medium
Slection of hybridoma from the cells after cell
fusion process is conducted by selective growth in a
selective medium.
For example, the cells are diluted,
appropriately with, for example, RMI 1640 medium
containing 15% fetal calf serum, placed on a microtiter plate tb about,105
selective medium
106 cells/well, and a
HAT medium) is added to each
well, which step is followed b\
of the selective medium.
appropriate exchange
For eample, when an 8-
azaguanine resistant cell line is used as the myeloma
cell and a HAT medium as the selective medium, unfused
myeloma cells will die on about the 10th day after
cultivation, and the antibody producing cells which
are normal cells cannot be grown in vitro for a long
term.
d
Accordingly, the cells growa on the 10th to
14th day are all hybridomas.
Screening for antibody producing hybridomas
A screening foraIybridomas producing anti-cardiac
25
a~a
myosin heavy chai,~agntibody, anti-cardiac myosin heavy
chain a antibody or anti-cardiac myosin heavy chain B
antibody can be carried out according to the Enzyme
Linked Immunosorbent Assay, which will be hereinafter
aa
ane
called "ELISA".
More specifically: a cardiac myosin heavy chain a
type such as bovine atrial myosin or a cardiac myosin
heavy chain 8 type such as human ventricular myosin is
a
L
dissolved previously in a buffer such as phosphate
buffered saline (PBS) or sodium hydrogen carbonate
(pH 8.0) to 10
8 4?
14
4
100
added to a soft plate
Ig/ml; aliquots each of 50 1p are
(96 wells) such as polyvinyl
chloride (PVC) plate for ELISA; and the plate is left
to stand at 4 0C overnight.
Then, the antigen is
discarded, and, after washing with PBS, PBS containing 1
bovine serum albumin (BSA)
is added.
The
mixture is then left to stand at room temperature for
one, hour to block with BSA the sites to which no antigen
is bound.
Aliquots of 50 pl from the supernatant of
each well are added, left to stand at room temperature
for one hour, and washed three times with PBS.
Then,
biotinyl anti-mouse immunoglobulin antiserum (second
antibody) is added, and the mixture is left to stand
at room temperature for one hour.
I
After washing three
times with PBS, avidin D-enzyme complex is added, and
the mixture is left to stand at room temperature for
minutes. After washing four times with PBS, the
optical density is measured with addition, of the substrate for the enzyme.
The well which contains a monoclonal antibody
specific for the antigen can be easily judged adording ,to the procedure as described above, whereby
screening for"hybridoma can be carried out.
S
Cloning
s
There is the possibility that two or more species
«a
r *of
hybridomas are contained in each well, and therefore cloning is conducted according to, for example,
25
the limiting dilution method to obtain a monoclonal
Santibody-producing hybridoma.,
Production of antibody
The most pure monoclonal antibody can be obtained
by culturing the hybridoma producing that monoclonal
S:
30
antibody in a medium for culturing animal cells such
Sas
RPMI 1640 medium containing 10 to 15% fetal calf
2
serum or serum free medium and obtaining the antibody
from the supernatant. For the cell culturing method
and conditions, those conventionally used in animal
cell culturing method may be suitably applied.
On the other hand, as a.method to produce antibodies in a larger amount, it is possible to employ
t
0<U
b
4
*1
a method in which, after a mineral oil such as pristan
Igenl is
(2,6,l0,14-tetramet:hylpe-nt-a-d .ane)
S contain-
intraperitoneally
The
d.
5
iich no antigen
grow
I temperature
in serum and'astitic fluid.
10
stand
Hybridomas will
18 days to produce
(about 1 to 20 mg/xnl)
'When purification is.
required, purification can be carried out after ammo-jz
nium sulfate fractionation by a method such as DEAB
rashing three
cellulose ion exchange column chromatography, affinity
;ad~ded, and
ardiac
column chromatography using Sepharose 4B having.
,rature for
Smyosin bound thereto or the like', or gel filtration
the
PIlS,
o ascitic tumors ;within. 10
antibodies in high concentrations
Then,
-um (second
to
hybridoima is injected intraperitoneally to be proliJ"erated in 2a. large amclint therein.
=ratant of
.t
into syingeneic animals from which the
parental zyeloma of hybridoma has originated, ;the
erature for
PBS.
has been administered
15
of the sub-
column chromatography.
Preparati$/
of active fragments
As active fragments, any of (Fab')2 fragment,
antibody
:.ged adqord-
(I-
(Fab') fragment, Fab~ fragment, and like fragments
that retain the immunullogic4al
hereby
20
4
and there-
with known procedures such as treatments with papa.in,
4
4-44
r example,
4
4
4
4
4
4
These active fragments may be prepared
Yrom purified monoclo nal antiboOies ii accordance
44
more species
04
clonal anEbody used in the present invention'can be
mpl~nyed,.
444
properties of the mono-
"Medicochemical Experi.ment
pepsine an~d trypsin.
4
4
4
4
4
44
25
44
Dnoclonal
Method Series Vol. 4 Immunochemi'Lstry", Kabushiki
Kaisha Nakayati.,a Shoten
"Methods in
4
(August 20, 1972), pp 91-119,
Immunology and Immunochemistry Vol.
1",
pp 422-423, Academic Press, 1967)
be obtained
Lloffoclonal
'ells SUCH
tal
4
N
~44
4
4
4
The monoclonal antibodies and the fragments
4
30
4-
theleothus
prepared can also be labeled, with radio-
4
4
4*
isotopes by various knowf
calf
methods.
Examples of nuclides of radioisotopes ar~e
4
i~e antibody
.ng method
.n ani~mal
netium-99m,
ed.
44
44444
uce anti-
4
o employ
4
4
-4
4
*4
35
galliual-67,
lead-203,
rutl4-enid~m-97,
mercury-197, thallium-201, and bismuth-21.2.
A method
of labeling ,with these radioisotopes can be selected
according to the species thereof.
With respect to
4
radioactive iodine, fo~r instance, the Chioramine T
h as pristan
method,
n administered
'rom which the
the iodine chloride method or the ilactoper-
oxcidase net~od may be employed.
(cf.
"Radioisotope
Drug MetabflismExperilental Method", Maruzen K.K.
.ed,te
(January 30, 1981), pp 95-101, "Methods in Enzymol~ogy
Sbe proli-'
Vol. 70 I=munochemical Tedhniques Part
o0mas will
pp 210-265,
Academic Press, 1980)' With respect to other radio-
to produce
isotopes, a method in which an antibody or its active
to 20 ing/inl)
fragment is covalently bound to a bifunctional chelat-
tion is,
10
after amino-
0
ing agent, and the product thus obtained is labeled
with a radioisotope is applied.
h as DEAE
Examples of typical bifunctional chelating agents
hy, affinity
are l-amino-6 ,17--diydroxy-7 ,l0,28 ,21-tetraoxo-27- (N-
aving cardiac
acetylhydroxyimino) -6 ,ll
iltration
17 ,22-tetraazaheptaeicosane
(desferrioxamine), 8-hydroxyquinoline, ethylenediaminetetraacetic -acid, diethylehetriamninepentaacetic acid (DTPA), diaminocyclohexyltetraacetic acid,
agment,
f3-aminomethaylene-2 ,4-pentanedionebis (thiosemnicarbazone)
and"-the N-alkyl or N-phenyl d~rivatives thereof,
f the mono-
acetylacetone, and-citric &acid.
ion' cani be
U
U
flU
papain,
if U-CU
U
agents are desferrioxamine and dithiosemicarbazone
es for technetium-99m and galliuhi-67 labeling
derivativ,
while DTPA is~ preferred for indium-lll and bismuth-212
Laeling. /e
technetium-99m is-used for labeling, a
U
U
pertechne 'iumate is contacted with a reducing agent
vi th' radio-
stannous chloride, stannous iodide', or stannous
U
U
fluoride)
U
U
to reduce technetium to
a
tri-, tetra- or
pentoxide thereof.
tech-
;pecLt -to
if
UUU
Tment 3
selected
V
Particularly preferred bifunctional chelating
III
A method
U
the acid anhydride m~ethod or the glutaraldehyde method.
U
?p 91-1,19,
-111,
U
U
U
ashiki,
Vol.
U
conventional method such as the carbodiimido method,
U
U
L.EXperiment
U
ing agent may be-carried out in accordance with a
ordance
ith
D
body or its active fragment with a bifunctioirial chelat-
U
repared
Coupling of an anti-
The diagnostic agent of the present invention
35
U
OU
further encdrnPasses as one embodiment thereof a "kit"
4
U
UU
comprising a coupled compound of an antibody-or its
U
U
fragmnent with a bifunctional chelating agent and a
I
I-I
4
t~I~~
radioisotope solution.
This kit may be provided with
a column for chromatography for purifying the nuclide.
The diagnostic agent of the present invention is
administered intravenously into a human body.
This
diagnostic agent is provided in a form suitable for
administration by infjeoion,
examp].
gluco! Eas
and may be prepared,
for
y using a solution of sodium chloride or
a carrier.
The dose,
although varying with
the particular radionuclide used for labeling, is
ordinarily in
the range of from 100 pCi to 30 mci,
preferably from 500 ICi to 3 mCi..
From 1 to 48 hours after administration of the
diagnostic agent of the presen
A C:
invention, the patient's
heart regiod is scanned with a scintilation scanner
or camera to detect radioactivity originated from the
diagnostic agent to Qbtain an image, whereby diagnosis
by imaging will be possible.
Hereinafter, the present invention will be describo
ed in more detail with reference to a Reference Example
illustrating the preparation of'the monoclonal antibodies used in the invention together with Examples,
Practice Example and Experimental Example of the
diagnostic agent of the invention, it being understood
440
that these examples are presented as illustrative only
and not intended t) limit the scope of the invention.
Reference xample
I.
Obtaining hybridoma
Bovine atrial myosin
(1 mg/ml) or human ventri-
cular myosin (1 mg/ml) ws dissolved in a physiological
s
sodium chloride solution and mixed with complete
Freund's adjuvant in a ratio of 1:1 to prepare an
emulsion.
The emulsion was administered intraperito-
neally into a BALB/C mouse
a 4
.4rit
(female,
6
eeks oi,&
several times every two weeks (50 'g/head),
35
30
ad, finally
ig of bovine atrial myosin or human ven-ricular
C
myosin was administered intravenously.
Three days after the final immunization, spleen
cells were taken out of the mouse and washed with MEM.
Mouse,,
."oma
PU,
was washed with MEM and mixed with
3
the spleen cels
in a ratio of 10:1.
After centrifu-
gation, 1 ml of 50% PEG 1000 MEM solution was gradually
added .to a pellet or cake thus obtained to carry ou';
cell fusion.
Further, the MEM solution was gradually
added to obtain a final quantity of 10 ml.
Again,
centrifugation Was conducted, and the pellet was
suspended in RPMI 1640 medium containing 15% fetal
calf serum to 1 x 10
cells/0.1 ml as P3U
and plating
on a 96-well micl\oplate in 0.1 ml/well.
One day latEr, aliquots each of 0.1 ml of HAT
medium were added,
and,
thereafter every 3
rrr
4 days,
CI
half of the mediu" was renewed with fresh HAT medium.
On about the 7th day, growth of hybridoma was recognized in some of the wells.
0.05 ml of the supernatant where hybridoma was
3:
grown were added to a 96-well microplate
previously
a-
coated with bovine atrial myosin (a type) or human
ventricular myosin (8 type).
By using avidin D-
peroxidase '(produced by Vector Co.) as the avidin D4 'enzyme
a"
X
1
conjugate, and hydrogen peroxide, 4-aminoanti-
pyrine and phenol as the substrate and the chromogenic
n~ Ii~
'1
i it
agent, according to the ELISA method as described above,
o
25
the supernatant containing a monoclonal antibody for
cardiac myosin heavy chain which reacts with both
atrial and vedtricular myosins, the supernatant which
8! :i
.tr
r
F
reacts with bovine atrial myosin but does not react
ihuman ventricular myosin
Swi
S
30
h
3
(monoclonal antibody
specificity
e
to cardiac myosin heavy chain a type
i
is contained in this supernatant), and the supernatant
j
which reacts with human ventricular myosin but does
not react with bovine acrial myosin (monoclonal antibody
<o
a:
i;
having specificity to cardiac myosin heavy chain B type
Ss
9
osJ
o
35 .is contained in this supernatant) were selected and the
hybridomas were cloned by limiting dilution.
As a result, hybridomas CMA-25 cell line and CMA-
j/
I
i
ii
"I
N
7/12
34 Qell line producing an antibody havihg specificity
to.adiac myosin heavy chain; C.A-1~9 cell line producing an antibody having specificity to cardiac mnyosin
heavy chain a type; and HMC-14 cell line, HMC-48 cell
2jine and HMC-50
I
cell line pr'oducing an antibody havi-ng
specificity to cardiac myosin heavy chain
type;were
-,jbtained.i
II.
Production of monoclona.
II
antibody
Each of the hybridomas listed above ways cultured
an RPMI 1640 medium containing 15% fetal, calf, serum
2
inia 96-well microplate,
then With scale-up to-25 cm
in
"lask and 75 cm- flask, and. the culture supernatants
were collected.
'~iters of the monoclonal antibodies in these
1S
supDernatants were determined hby the ELISA method.
The
titer is expressed as dilution magnitude of the antibody
sample from the original solution which give's .50% of-the
absorbance, taken as 100%, which is obtained by the
ELISA method for the sample in, which a sufficient amount
of a-atibody exists relative to the coated antiqpn.
4
"was
Further, the subclass of each of the antibodies
determined by means of a MONOABID ETA KIT (supplied
by ZYlvrD Co.)
NI
0
The results obtained are summariz2ed in Tble 1.
Table 1
*Titer with Titer with
respect to respect to
Hyhridoma bovine
hum~an Vent1INricular
cell line atrial
myosi~l
I'R osir l
lP2
C11A 25
CMA- 34
25
CMA-i9
25
HUMC-14
Titer with
respect to
human ventSubclass
ricular myosin
light chain
1>
625
7
1
625
.IgG
K
IaK
-IgG
.625
1
IgG 2 b/<
IgG
-125
I-C4
IgG 1
2 b/K
JIgG
2
/K
14
;Example 1 (production of
131
I-labeled anti-human
ventricular myosin heavy chaini J type
monoclonal. antibody)
'~6
x 10
cells/head of hybridoma HMC-48 cell line
7yere administered into mice which had been previously
administered with
istan -co induce ascitic tumors.
The as'citic fluids
obtained from the mic& 10 to
d.avs after administration were pooled to obtain a
saturated amimonium sulfate fraction.
This fraction
was then su-:bjected to DE52 column chromatography to 2
el~ut'e therefrom a purified monoclonal antibody
bTo
3 mCi of 13 1 were
added,200 pl of the purified
antibody (8.7 mg/ml) thus obtained, 150
T (1 mg/ml),
(HMC-4 8).
4l of chloraxnine
600 p1 of sodium~\metabisulfite (1 mg/ml),
150 jil of potassium iodide
(50 mg/ml)
Id phosphate bufer (pH11 7. 5)
ar~d 150 p1l of a
The mixcture,
after
being, reacted for one minut-e at room temperature, was
to column chromatography by using Sephadex
equilibrated previously with a 0.5% bovine serum
albinnin-phbsphate buffer to, separate free 131i to
4
obtain a, 1I-monoclonal antibody (HMC-48).
Example 2 (Production of ill In- labeled 'anti-humnan
44~'
4.
4
*4*4
*4
44
4
type
!-V~entricular myosin-heavy chain
moioclnalantibody (Fab") 2 fragment)
4
444,4
4
4
25
4 4*4
4
26 mg of thle purified monoclonal antibody
produced ir Examiple
4*44
1were dialyzed against a sodun
acetate-hydr:ochloride
an antibdy solution
buffer.
(HMC-48)
)uffer (pH.
and 2.6 m! of
(10 mg/ml) was formed with the
To this solution was added 0.5 mig of pepsine
(2948 U/mg, supplied by M~illipore
and the mixture
was cause d to react for 18 hour's at 370 C.
*44
*44
4
After reaction, the miixture was dialyzed 'against
p.
1 liter of a borate buffer (pH1
4
*renewed twice duriLng the dialysis.
35
Subsequently, the mixture was stbje~cted to gel
44
44
*4
4a~*
*44
4
the buffer being
-4
*4.4
4*4
filtration column chromatography using Ultrogel AcA 34
(supplied by Bio-Rad Co.) equi'fbrated with 50 mM
C,
phosphate buffer, and fractions having a molecular
weight peak of around 100,000 were collected as
f ragments.
(Fab)
2
The fragment thus obtained wa~s concentrat~d to
5 jng/ml through Amicon B-I5; the concentrate gradually
mixed with a carboxycarbonic ~acd anhydride mixture of
diethylenetriaminepent aacetic aci d (DTPA) by, the
Krejcarek et al. method,:(Biochem. Biophy8. Res. 'Cormun.,
Vol. 77, pp.581-587
(1977)); and the mixture was caused,
to react overnight at 4'C.
The reaction solution was then-dialyzed against 0.1
M acetate buffer (pH 5.0),<,and (Fab') 2 -DTPA fractions
were collected through Sephade;x G-25, which fractions
were dial~yzed against 0.1M glycine-hydrcchloride bu.ffer
The HMC-48,(Fab1
(pH
9 -DTPA
thus obtained was
-mixed with indium chloride 11in in the buffer, and the
mixture was caused to react for 30 miniutes.
As a
result, 1.5 iiCi/mg protein of IMC-48(ab
DTPA-1 1 'In
was obtained.
20
Example 3 '(Production of
iin-labeled anti-human
44
4
4*
4*
44*
cardiac myosin heavy chain monoclonal
44*
antibody (Fab')2 fragment)
.44 4~
.4
4
A monoclonal antibody
4
ly as in Excample 21to obtain a (Fab') 2 fragment.
4
,25
4*44
4
4444
(CMA-34) was treated similar-
To the fragment obtained was then bound DTPA by
1 T.
the-procedure of Example 2 and-reacted with
In
As
a result, CMA-34(Fah').,-DTPA-l~ In having a specific
radio activity of. iL.1 mCi/1wg protein was obtained.
4
.44
4
4
4
30
44
Example 4(Production of
in-labeled anti-human
myosin heavy chain a monoclonal
.atrial
4
antibody Fab fragment)
4
A monoclonal ,antibody (CMA-l9) was purified
similarly as in Example 1 and freeze-dried.
4
44
44
4
444*
*4
4
4*
4
I
44
35
mg of the antibddy thus purified, was added to
2.5 ml or a phosphate b)u fer (iPH
and .to the mixture obtained was adcded
Sigmha
mg of pepsine (supplied by
The resultan ),,mixture was caused to react
complex inducing an allergic
reaction
ad
various
the
for 2 hours at 3700.
Ir
The reaction solution was subjected to affinity
(Fab') 2
column chromatography using a column packed with Protein
A
to
5
lually
Sepharose
(supplied-by Pharmacia Fine Chemicals)
equilibrated previously with a phosphate buffer (PH 7.4")
for adsorption of Fc fragrments and unfragmented antibodies.
ire of
Unadsorbed
fractionswerei collected and concentrated to
5 mg/mi through Amicon B-15.
:oinmun.,
To the Fab fragment thus
obta-Ined was bound DTPA by the procedure of Example 2 to
caused
1
innst
CL-4B
produce CMAk-l9Fab-DTPA7 ill In having a specific radioac:tivity
0.1
Lions
of 1.3 mCi/m3 protein.
In-labeled anti-huan
5(Production OL
ventricularinyosin heavy chain
Lions.
typemono-
clonal antibody Fab fragment)
buffer
15
vwas
To ascitic fluid
line
and the
00
04:
a
Tn.
induced by hybridoma
similarly as in Examnplel
were added an equal
volume of phosphate buffered saline
'a two-fold volume of saturated
000
MC-4E8 cell
(PBS)
(pH 7.0)
and
C'Ximmonium sulfate. 'A
precipitate thus formed was centrifuged off,
and then
the ascitidc fluid was fractionated with 50% saturated
ammonium sulfate.
0
25
nC,
fraction was concentrated and subjected to Ultrogl AcA
0Qb
44(supplied
if ic
by LKB Co.) column chromatography using PBS
to obtain an immunoglobulin fraction having a molecular
d.
weight of 150,00.0 as a purified antibody.
To a solution
o: a
n
40
1
4 04
04I
0
4i 44F
30
purified antibody
chemical Co.,
mM EDTA),
30 m-1M PBS
and
minutes at 37'C.
ed to
35
I
(5 mg/mi) was
of a 0.025% papain solution
00a 40r
4 4,
4f
4.
B
e mix-
(30 m-M PBS plus 5 mM EDTA)
0
of the
01
4
react
0B
4 t
4004a
40#
0400
As
ied by
04
4
The p-ss-through
0L 00
n.
4
4:
subjected to DE52 column chromatography with use of 0.1
M Tris-hydrochloride buffer (pH
Alby
4,4g r
precipitate was dialyz-
ed against 0.1 M J-cis-hydrochloride buffer (pH17.2) and
4
:04pa
0
4
4440
similar-
The resulting
added an equal volume
44:
:O
0Q 04
444~
0" 048;
(supplied by Cooper Bio-
(pH
5 m
cysteine plus 2
ixture was caused to react for
At this stage, 10 mM iodoacetamide
solution Was addedote
mixture to terminate the reac-
4
04
tion.
The reaction
solytionwas
applied to Protein A-
03
0
4*
and-2D, are single photon emission 'poxputed tomography
I
1
Sepharose CL-4t column equilibrated with 0.1 M Trishydrochloride buffer (PH
-ini,ty
:h Protein
The pass-through frac-
tion was concentrated,-''nd was further applied to
,heznicals)
5
(PH 7. 4)
Ultrogel AcA 54
(supplied by LHE Co.) column equili-
brated with PBS
(PH 7.0) to obtain a Fab fragment
having a molecular weight of 50,000.
1 antibod ies.,
T~o the Fab fragment obtained in the manner
described above was bound DT-PA by the procedure of
E;xample 2,.-and the HMC-48Fab-DTPA thus obtained was
:rated to
mnt thus
unple 2 to,
10
radio-
mixed,-," th indium chloride
hydr/)chlride buffer (pH
11
I n in
0.1 M glycine-
The mixture was caused
to re~act for 30 minutes to obtain 1.5 mCi/mg protein'
ian
of 11'In-labeled HMC-48Fab-DTPA.
)e Mono-
1 31
Practice Example (Diagnosis with
15
-4-1 cell
antibody
2 mCi4 of the
lual.
-monoclonal
(ITMC-48)')
1 3 1 I-monoclonal
z ntibody (HMC-48)
obtained in Example 1 was administered intravenously
and
"A
into a dog with myocardial infa.rction artificially
induced by ligating the coronary artery.
idthen
20
:urated
4
after administration,' a planar image and a single,
photon emiss ion computed tomography
(SPECT) were obtained
4,
4
4
4444
4444
-ogeil AcA
by a gamma scintill~ation camera
444,
44
In the planar image, the
4
~444
4
25
1 3 1 I-monoclonal
slightly in the thyroid gland only, but was not found
This antibody was'also found to accumulate
to accumulate in the bones or an~ywherq else as opposed
volume
4 44
4-4
4
4.4
'44
'4
,~4
4
in the computed tomnography, on the other hand, the
of the septum.
amide
Experimental Example
'4
"4
44
'4
.444
A
44
4,44
'4
J
:1
4
44
0
44
4
~'4
4
4,4
monoclonal antibody accumulated in the apex and 'ipart
4
or 30
the reac-
44
444
infarction site wash clearly depicted, and the 131.1-
Bioplus 2
jK\,
to 9MTc-pyrophosphate.,
30
4,
4444
U
4,444
4,
44,4
accumulated at the infarction site in the canine ventri-'
iolecular
4
4*
.444
antibody
cular muscle.
the
044
44
4
supplied-by General Electric Co.).
4
'44
U
(Maxicamera 400 AT,
using PBS
in
4,
4
,e of 0.1
through
f
44
4
4
4,44,
is dialyz7. 2) and
36 hours
35
1 mg of the
mQi
1 1 I n-labeled
HMC-48Fab-DTPA (1.5
obtained in Example 5 was administered int~ra-
venously into a dog with myocardial infarction
0
4,4,
4
4,
'4
18
Sartific
ally induced by ligating the coronary artery,
48 hours Lfter administration,
out of thaf dog.
the heart was excised
From the excised heart, specimens
Ij
each of a size of about 3 x 3 mm were prepared,
and the radioactivity at the infarction site and that
at the non-infarction site were respectively measured.
f
'The
radioactivity was 2879.8 cpn/mg at the infarction site, 563.5 cpm/mg on the periphery thereof, and
7.2 cpm/mg and 67.2 cpm/mg in the normal regions,
about 40-fold radioactivity accumulating at the infarction site in comparison with the nomal regions.
o
z
i
Oo
00 o
A
00
0
0.
I
1
1
4.
a"
11
00
90
0
1,
o.0 S0 9
1
1
*0
A
1 1
11
.0
6
1
1
I
1
1
U
1
1
i
out by immunizing an xenogenic animal such as mouse,
)cardial
r
artery.
excised
,imens,
and thatL
measured.
he inf arc:-eof, and
Tions,
-he infarc-
o
0~
000
0
4
oil
'N'
1
L
_L t= Ua
mixing ratio of fromn
1:4 to 1:10 in a medium for
mouse,
S-I-
V
I
>19
rN
I/
19
r:
THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:1.
diagnostic agentc
agent for heart
heart disese
disease cmprising
comprising
A diagnsic
specificity
having specifiity
antibody
a radiolabeled
antibod ,havng
a
radioabe~d monoclonal
monoonl
its active
or its;
heavy chain or
myosin heavychain:
cardiac mysin
human
to huma
ac~ive
cardiac:
to
fragment.
f ~ragment
2.
2.
A diagnosic
A
diagnostic
:agn
agent
to clim 1, i-n
ccoding
according
to
claim
1,
in
an
recognizes, a~n
antibody recognizes
radiolabeled monoclonal
which
which the
he radiolabeled:
moclo~nal antibodyd
caiac
osin heavy chain.
hai.
isQzyme of humn
isoazyme
human cardiac
myosin
3.
r::'.dianostic
3
claim 1,
agent accoding
accor-dingq to
1J -in
in
aent
to claim
A diagnostic
A
specificity
antibody has speificity
radiolabeled monoclonal
the radiolabeid
which
monocloal atibody
wich te
to human
cardiac mosin
huan Icaiac
myosin heavy cain
chain cO
a type
type.
4,
A diagnostic
inl
diagnostic: agent
agent according
according to
t:oclaim
laim 1,
1, in
ARE
ATTORNEYS FOT
PATENT
E
i:
'I
OLLOWS:
specificity
monoclonal antibody
which the radiolebeled
atibody has
hasS specificit
radioleeled !monclonal
:to
to huma
human cardc
cardiac myosin
chain
myosns heavychain
B
type.
tpe.
the diagnosisof:
:method
A
A method for the
diagnosis of heat
heart isease
disease
a
administeing
administering
a radiolabeled
radiolabeled moocloal~
monoclonal
antibody having
antibdy
seificity to
having specificity
to humda
human cadia
cardiac msin
myosin
which
hich comprises
omp~ies:
00
active fragmnt
Sheav
chain or
heavy chain
orits
its active
fragment ta
to a suject,
subject, and
and
ardiac
mosn
0pO: 1.0detecting
detecting the
te site
iSteat
at wich
which the
the human
human cardiac
myosin
alp
ao1
eavy
chin
isexposed
by
measuring~
the
rdioacivit
h eavy chain is exposed by measuring
the radioactivity
.aroIriinaed
0
thethe radiolabeled
monocihal antibody~
riginated from
from
radiolabeled monoclonal
antibody
o0r
active -fragment.
or :is
its active
fragment.
Dated
this
hi
s
226
day
day
of
f
March
Mac
DAVIES-& COLLISON
DAVIE
COS.LeoLION
0o
c0YAMAS
I"
0
1.
I,
PAENT ATORNEYS OR
KAISHA.
SHOYU KABUSHIKI
YAMASA SHoYU:
KBUSHIKI:KASEA.
1985.
195
4
4
4
44
I ~4
chloride (PVC
plate for ELISA; and the plate is left
ILLOWS:
e comprising
pec if icity
active
recognizes. an
im 1,
in
has specificity
irn L, in
has specificity
4
0
.4
disease
44
*4
04
09
d. monoc lonai
4
4040
-myosin
4
0440
440
ject, and
ac myosin
Lolactivity
antibody
4
4.
00
000
o
0
0
e
4~
*0
00
O
0
S
0
04
04
044*
0@
4
0
0
0
i
jt.
vinyl
bodies in
is lef t
a large~c amount,
it
J9Luuu-e ant-ci-
is possible to emaploy
0
FIG. IA
09
9
090
99
090
00
0*
00
0
o
0004
9
0990
FRONT
0000
B.ACK
T
0
FIG. lB
00
0
000
0
0
0
0
00
0
0
O
00
00
00
0
0
'0
00
0
tote
0
00
9
~1
0
00
*o
BACK
FIG. IC
oz
laoeling with these radioisotopes can be selected
according to the species thereof. With1 "respect to
m
N
8
LL
C?
C,)
LL
(M
0
7--
00
U
8*
8*
0
088
88
88
0
8
0
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8
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0088
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8~ 08
'00
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8
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0
8
8
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8
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8
8
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08
0*00
0*
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88
8
LI
1-4
1)
selected
)ect to
coi~prisiriq a coupled compyound of an antibody- or its
fragjrnent with a bifundtional chelating agent and a
9
9*9
9.
9
9
9*
9*
9
9
*99
9
9
0
9
9
9
9
9
0
9
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999
9
9.
0
9
9
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9
9
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9
9*
999
0
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0
900
9
9
9
0
9
9
9
900
0
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9
9
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0
DoG rEST
'I
I
WALL
M
1-4
2A
F IG. 2 B,,SEPTUM~
0LV
APEXk
FIG. 2
/1
t O r, q
or its
Three days after the final immunization, spleen
and a
j
o
p
0
P
p
p
p
0
mryosin was administered intravenously.
p
0
0
0
0
P
p
DOG TEST
.7
p
7,
7
POSTERIOR
WALL
FIG.
2A
/,SEPTUM
,1
*APE\
G.~C
FG
10
2
1
L±.JZbupurnatant)
were selected and the
hybridonas were cloned by Iimniting dilution.
As a result, hybridoxnas CMA-2$ cell line
and CMA-
WWI.
R-I
W 7i