Note: Descriptions are shown in the official language in which they were submitted.
2~88~9
DESC~IPTION
Title of the Invention
MONOCLONAL ANTIBODIES, ASSAY METHOD, REAGENT KIT, SEARCHING
METHOD AND DRUG MISSILES USING THEM
Technical Field
This invention concerns monoclonal antibodies, in
particular monoclonal antibodies against 5-methylcytidine
and other modified nucleosides, which are elevated in the
body fluids of cancer patients.
This invention also concerns hybridomas which produce
such monoclonal antibodies, an immunoassay method, reagent
kit, immunohistochemical searching method and drug missiles
using the monoclonal antibodies.
Background Art
In the cancer diagnosis and postoperational
monitoring, it is very important to assay the cancer-
related substances, or the Ro-called tumor markers, which ; -
. .
are elevated in the patients' serum or urine. The tumor
markers currently assayed include carcinoembryonic antigen
(CEAl, immunosuppressive acidic protein (IAP),
a~fetoprotein (AFP) specific to the liver cancer, and
CA19-9 which is a sugar chain antigen specific to the
- . .: i
pancreatic cancer. Meanwhile, it has long been known that
a variety of modified nucleosides are elevated in the urine ~ ;
of cancer patients. -~
.';
202~$~9 ~:
The present inventors formerly assayed the urines and
sera of cancer patients and tumor-bearing animals by a -
conventional technique of high performance liquid
chromatography (HPLC) in order to determine the usefulness
of modified nucleosides as a tumor marker. As a result, it
was found that 5-methylcytidine was ~bnormally elevated in
cancer patients and tumor-bearing animals.
In this way the usefulness of this compound as a
tumor marker has been proven, but the HPLC analysis ~ ~
requires a complicated sample pretreatment which takes a ~ ;
long time, especlally, when assaying many samples. In
addition, it has been impossible to study the subcellular
localization of 5-methylcytidine and the variation of its
amount on the cellular level by an immunohistochemical
: , . . .
technique.
The present inventors have made extensive studies for
the development of a simplified assay method of
5-methylcytidine and have found that use of a newly
prepared monoclonal antibodies against 6-methylcytidine
makes it possible, without any pretreatment of samples, to
assay many samples at the same time by an modified enzyme
immunoassay technique, thus providing a faster and simpler
assay. This has led us to the completion of this
invention.
. .
. .
2 ~ ~
~" ~
- 20~g49
Disclosure of Invention ~-
This invention concerns monoclonal antibodies which :
can recognize lower alkyl-cytid:ines and hybridomas which
produce said monoclonal antibod:ies.
: This invention also concerns an immunological
technique for estimating lower ~lkyl-cytidines in a sample,
in particular, an assay method of using monoclonal
antibodies which can recognize lower alkyl-cytidines; a
reagent kit for immunologically estimating lower ;
alkyl-cytidines in a sample, in particular, a reagent kit .
: ~:
including monoclonal antibodies which can recognize lower ~- :
alkyl-cytidines and conjugates of lower alkyl-cytidines and : .
:: carrier protein-II; and a method of immunohistochemically
~: searching modified nucleosides which are accumulated in
tumor tissues as a consequence of abnormally elevated -:
metabolism of nucleic acids, in particular, a method ~;
. .
~ wherein monoclonal antibodies which can recognize the
;, ": ~:
modified nucleosides are immunologically bound to a part of
the tissues.
This inventionl furthermore, concerns drug missiles
1 wlith the characteristic feature of using, as a carrier for ;... ;
: anti-cancer or carcinostatic drugs, monoclonal antibodies .`;
: which can recognize the modified nucleosides. :~
,: .~ .:: :,
, ~ "~
3 - ``;;~.
,.~, :'...:.~,
~`
~`~` 2028~9 `
Brief ExPlanation of the Drawln~s
Figure I shows a detection characteristics of
5-methylcytidine detected by MCT-3, one of the monoclonal
antibodies according to this invention. Figure 2 is a
diagram showing the relation between the dilution fold and
absorbance in obtaining the antibody titer of the
monoclonal antibodies according to this invention. Figure ; -`~
3 gives the results of immunological staining of tissues of ;~
esophagus cancer with an anti-1-methyladenosine monoclonal
antibody while Figure 4 shows the results of immunostaining
of the same tissues with anti-pseudouridine monoclonal
antibody. Figure 5 is a photograph showing the result of ~-
immunostaining of a normal esophagus tissue with
anti-l-methyladenosine monoclonal antibody.
Best Mode of the Invention
The monoclonal antibodies according to this invention
are those which can recognize specifically lower
alkyl-cytidines. As the lower alkyl-cytidines may be
mentioned the cytidines in which hydrogen atom(s) is ~;
substituted by stralght or branched chain lower alkyl
groups having 1 - 5 carbon atoms such as methyl, ethyl,
1` ~ ,! ' ~ ' ! j
propyl, isopropyl, butyl, isobutyl, pentyl and isopentyl
groups. Among these lower alkyl groups, methyl is the most
preferred. Favorable positions for the introduction of the
lower alkyl groups are 3-, 5- and 6- positions in the
::;
2 1~ 2 8 g ~ 9
pyrimidine nucleus constituting cytidine, among which
5-position is particularly preferred.
Among the monoclonal antibodies of this invention, th0
most preferable one is the antibody which recognizes 5- . .::
methylcytidine in view of the assay precision of the . ~:~
modified nucleosides as a tumor marker and safety.
As the class for such monoclonal antibodies, IgG is :~
favorable, and IgG1 and IgG2a are particularly preferred. . :. .
The monoclonal antibodies of the class IgG1 show high
reaction specificity in the recognition of 5-methylcytidine
and have high.antibody titers, thus practically ;.
advantageous. ;~
The monoclonal antibodies of this invention can ,~
recogniie lower alkyl-cytidines, and it is desirable that .
they can recognize the alkyl-cytidines present in animals'
urine or in body fluids such as blood and lymph. In
particular, it is praotically preferable that the ~.
antibodies can recognize lower alkyl-cytidines in human
body fluids.
As a favorable example of producing the monoclonal
antibodies which can recogni2e lower alkyl-cytidineslof `
this invention may be given the following method, where a
monoclonal antibody recogni~ing 5-methylcytidine is
prepared~
~. ` ''. ~'`'
:~ 5
~`` 2~2~49 ~
First, an anirnal is immunized with 5-methylcytidine,
and spleen cells are taken out of the animal. In this
procedure, since 5-methylcytidine itself cannot be an
immunogen, it is conjugated with an appropriate carrier
protein-I and used as an immunogen.
5-Methylcytidine to be used in this invention is any
one of those obtainable from the urine of cancer patients
by isolation and purification, obtainable by synthesis or
commercially available authentic preparations. As the
carrier protein-I, such proteins which are used for the -~
purpose of making the immunocompetent cells recognize the
substances actlve as haptens are used, which include ~ ;~
keyhole limpet hemocyanin, bovine serum albumin, human
serum albumin and eggwhite albumin. An examplary method of
conjugating 5-methylcytidine and oarrier protein-I is
oxidizing the sugar moiety of a nucleic acid base with
periodic acld and then making it conjugate with the carrier
protein-I.
Animals to be immunized with the conjugate of
5-methylcytidine and carrier protein-I include mice and ~;
rats, among which mice are practically advisable. -~
Then, spIeen cells of the immunized animal are fused
with myeloma cells to obtain a hybridoma. The cell fusion
~ - .
may be performed by either one of the known methads using
polyethyleneglycol (Marc Shulman, C.D. Wide & G. Kohler, ~;
6 ;~
2028~49
Nature 276, 269 - 270 (1978)), a method of fusion utilizing
Sendai virus tG. Kohler & C. Milstein, Nature 256, 495 ~
497 (~975), a method according to Eur. J. Immunol. 6, 511 - ~ ~ -
519 (1976)), or a method utilizing electric pulses ~J.
Vienken & U. Zimmermann, FEBS Letters 137, 11 - 13 (1982)).
In the screening of hybridomas, it is necessary to remove
the hybridomas that produce antibodies against the carrier
:
protein. For this purpose it is desired to do screening of
the antibody-producing hybridomas, for example, by using,
as an antigen, a substance obtained by conjugating a
protein derived from a species different from that used for
the immunization with 5-methylcytidine.
The spleen cells and myeloma cells used for the
production of the monoclonal antibodies of this invention
via hybridomas are favorably those derived from animals of
the same species. Practically, mouse spleen cells and
myeloma cells derived from a mouse are particularly
preferred.
In the next place, the selected hybridomas or the
transformed cells are cultured to make them form desired
specific monoclonal antibodies. The hybridomas or ~ -
.'~ . ::
trans~ormed cells ~elected by cloning and produce ;~
antibodies which recognize lower alkyl-cytidines may be
preserved frozen. It is also possible to culture them in a
7 `;~
y
. :~ ;;~ ~' .'
`` - 202~9
large scsle by an adequate technique. From the culture
supernatant, monoclonal antibodies that are conjugated
specifically with lower alkyl-cytidines can be obtained.
It is also possible to prepare desired antibodies from
ascites or serum of an animal into which these cells are
transplanted for the formation of tumors. Purification of
the monoclonal antibodies of this invention is performed by ;~
affinity chromatography or other techniques.
This invention also includes hybridomas which are
obtained by the fusion of spleen cells capable of producing
antibodies thQt can recognize lower alkyl-cytidines and
that are prepared by using a conjugate of lower ~;
alkyl-cytidines and carrier protein-I as an immunogen and
myeloma cells. As a method of obtaining such hybridomas,
; the method of preparing hybridomas described above in the
~ production of monoclonal antibodies is favorably employed.
~ ,
~ The inventors of this invention have completed the ~
. .
~ assay method of this invention, where lower
: , . . :- .
alkyl-cytidines, particularly favorably 5-methylcytidine, a
marker of progressive csncer in the patients' urine, serum
and other body fluids can be assayed simply and in high -
precision by using these monoclonal antibodies that can
~ recognize lower alkyl-cytidines.
; Thus the assay method of this invention has a
characteristic ~eature of using monoclonal antibodies that
--` 2Q28~9 ~
' ~ ~
recognize lower alkyl-cytidines among the assay methods of
lower alkyl-cytidines in test samples by the immunological
technique. Favorable test samples (designated simply as
samples hereafter) in this assay method are intact or
processed body fluids of animals, particularly of human ~ -
being, and the use of intact body fluids makes the process --
simpler and more advisable.
In the assay method of this invention, it is -
preferable to use monoclonal antibodies that recognize
~ . .
lower alkyl-cytidines as fixed to a carrier. The fixation
can be performed by a known technique, and as the carrier
such solid ones as balls, beads, gear and microplates made
of polystyrene, polyethylene, polyacrylate, teflon,
polyacetate and other materials may be favorably utilized. `;~
The method and procedures of labeling the monolonal ~ `
antibodies and the method and means of its detection are
not limited at all, and known methods and procedures may be
employed, for example, those using the anti-immunoglobulin
~ , .~ , .
antibody or staphylococcal protein A which are labeled with
a radioactive substance, an enzyme or a flu~rescent
substance. As the labeling substances, horse-radish~ -
peroxidase, ~-D-galactosidase, alkaline phosphatase, and
other enzymes may ordinarily be used in the methods using
an enzyme (enzyme immunoassay, EIA); 125I, 3H and other :
9 " ~' '' :~
',` '''.', ''
-` 2028~49
isotopes in those using a radionctive substance (radio-
immunoassay, RIA); and fluorescein isothiocyanate and other
compounds in those using a fluorescent substance
(fluoroimmunoassay, FIA), but so far as the activity of the
labeling substance is readily measurable, other substances
may also be utilized.
When the labeling substanc,e is an enzyme, a substrate
is used for assaying its activity. Examples of the
substrates for horseradish peroxidase are 2,2'-azinodi-
~3~ethylbenzthiazoline-sulfonic acidJ diammonium salt
(ABTS)-H202, 5-aminosalicylic acid-H202,
O-phenylenediamine-H202 and 4-aminoantipyrine-H202, and
examples of the substrate for ~-D-galactosidase are
fluorescein-di-(~-D-galactopyranoside) and
O-nitrophenyl-~-D-galactopyranoside. For assaying the
enzyme activities, known reagents of solvents, washing
agents and reagents for stopping reaction are used other
than the above reagents. A method utilizing the polarized
light of the antigen-antibody complex may also be used.
For the practice of the immunoassay method of this
inventon, an example of determining 5-methylcytidine is
given:
To a 96-well microplate is added a conjugate of 5~
methylcytidine and bovine serum albumin (BSA) in a quantity
of l ~g/well and the plate is kept at 4 C for
1 0 "
` 2~28~49 :
12-24 hours. Then 100 ~ each of phosphate-buffered
saline (PBS) containing 1% BSA is added to the well to
prevent unspecific adsorption of antibody and other
proteins. A sample (urine, blood or other materials) is
added to the wells in an amount of 50 ~ each. Then, the
equal volume of antibody solution was added to each well.
After through agitation, the plate is left at 4 C for 1
hour. The wells are washed thoroughly with PBS and added
with 100 ~Q each of the 3000-fold diluted solu~ion of
alkaline phosphatese-labeled, goat anti-mouse IgG antibody.
After reaction is run at 4 C for 45 minutes, the wells are
washed with PBS and moisture is removed. 100 ~ each of
the substrate solution (1 M diethanolamine buffer (pH 9.8)
containing p-nitrophenyl phosphate in a concentration of 1 ~-
mg/ml) is added thereto, and the mixture is allowed to
react at 37 C for 30 minutes. By measuring absorbance at
405 nm in each well by an EIA reader, the amount of 5-
methylcytidine present in the sample is determined.
This invention also includes in the scope a reagent
kit with the characteristic feature of using a monoclonal
antibody which can recognize lower alkyl-cytidines and a ;,~
conjugate of lower alkyl-cytidines and carrier protein-II,
.,~
among kits for immunologically assaying lower
alkyl-cytidines in test samples. ~;;; ^
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`- 2028~49
The monoclonal antibodies here mentioned have the
characteristic features as described heretofore. It is
necessary that protein-II which conjugates with lower
alkyl-cytidines be selected from the protein groups similar
to those of the above-mentioned carrier protein-I, and that
the antigenicity of the conjugnte be different from that of
carrier protein~
The reagent kit of this invention may also contain,
other than the monoclonal antibodies and the conjugates, a
labeling agent and a reagent for detection of the labeled
antibodies, if desired.
This invention furthermore includes a method of
immunohistochemically detecting the presence of modified
nucleosides which have been accumulated in tumor tissues as
a result of abnormal elevation of metabolism of nucleic
acids, characterized in that monoclonal antibodies capable
of recogniæing modified nucleosides are bound to a part of
tissues by an immunoreaction.
The tissues in such an immunohistochemical searching
or detecting method mean cells or tissue sections of
animals, in particular of human being which are practically
favorable.
;~ As the monoclonal antibodies to be used in the
searching method of this invention, anti-pseudouridine
monoclonal antibody (Japanese Patent Kokai Publication
12
2~2~9 : ~
No. 299765 (1987), No. 222699 ~1988)), ~;
anti-1-methyladenosine monoclonal antibody (Japanese Patent
Kokai Publication No. 299766 (1987)) and other monoclonal
antibodies against cancer-related, modified nucleosides
may also be mentioned beside the above antibodies against
lower alkyl-cytidines.
In more concrete sense, the searching method of this
invention has the characteristic feature of causing an -~
immunoreaction by allowing the tissues to react with the
monoclonal antibodies capable of recognizing modified
nucleosides, thereby to have the monoclonal antib~dies
bound to the tissues at the site where the nucleosides are -
present. Thereafter, the tissues are treated, for examplei, ~
with a biotinylated antibody that can recognize such ~ ;
monoclonal antibodies and subsequently with enzyme-labeled
avidin for revealing the site of localization of said ~ ;~
modified nucleosides in the tissues by staining or other ~;~
labeling technique so as to make searching the site of
progressive cancers possible.
The immunohistochemical searching method described
below is an example of an embodiment of the searching ; ~;~
method of this invention. In the method of this invention,
cells or tissue sections are fixed by using acetone,
formalin, paraformaldehyde or other chemicals and treated
13
-::
~ ;
2 0 2 8 8 4 9
with PBS containing 1% BSA to prevent nonspecific
adsorption of proteins. Then the monoclonal antibodies are
a~lowed to react and left at room temperature for 30
minutes. After thorough washing with PBS, the biotinylated
anti-mouse IgG antibody is allowed to react and left at
room temperature for 30 minutes. After thorough washing
with PBS, avidin-biotinylated peroxidase complex (ABC) is
allowed to react and left at room temperature for 30
minutes and washed thoroughly with PBS. In the next place,
the substrate solution ~PBS (pH 7.4) containing 0.5 mg/ml
of diamino-benzidine and 0. OlX hydrogen peroxide) is added
for staining. Subcellular localization of desired modified
nucleosides is looked for under an optical microscope.
; In this invention is also included drug missiles with
the characteristic feature of using, as the carrier of
anticancer or carcinostatic drugs, monoclonal antibodies
that can recognize modified nucleosides accumulated as a
result of abnormal elevation of metabolism of nucleic acids
in the tumor tissues.
Such anticancer or carcinostatic drugs may be, for
example, such alkylating agents as chlorambutyl, ACNU, CCNU
and cisplatin; such metabolic antagonists as MTX, 5FU, FUDR
and ARA-C; such vinca alkaloids as vinblastine and
vincristine; and such antibiotics as MMC, Adriamycin and
Daunomycin, and any other agents may be utilized so far as
14
: ::
.'"' ~' ~
.. . ~'' :
2028~4~
their monoclonal antibodies can be used as a carrier, which
should not be construed as limiting this inventian thereto.
Any form of application is possible for using the
monoclonal antibodies as a carrier, including a therapeutic
method utilizing the antibodies as a carrier of
radioisotopes. The radioisotopes may be a-emitters
such AS 212Bi and 211At and ~-emitters such as 131I and
90y, .`'`
By using the drug missiles of this invention, the ;~`~
anticancer and carcinostatic drugs can be brought to the ` i
affected site efficiently, which leads to an enhanced ~`
therapeutic effect and lower adverse side effects. `~
.::
This invention also includes monoclonal antibodies :~`
suitably usable in the imaging diagnosis, which can
recognize lower alkyl-cytidines and which are labeled by ,;
radioactive, paramagnetic or fluorescent substances. ~ ;
The radioactive substances here mentioned are, for ~ ~;
example, 125I, 111In~and 99mTc and an example of the -
paramagnetic substances is gadolinium (Gd). Any substances
may be utilized, so far as it serves as a label for the
monoclonal antibodies of this invention and when the~
labeled antibodies are introduced into animal bodies, ;~
particularly favorably into human bodies, they are brought
: ~ .,, ~ :
to the sites where lower alkyl-cytidines are localized for `~
:, : ,; . ,. .::
"
: ~ ,
2028849
labeling the sites, thus serving for imaging diagnosis with
the aid of radioactive rays and ultrasonic wave.
By using such monoclonal antibodies of this invention
for the imaging diagnosis, the presence of progressive
cancers which are accompanied with the formation of lower
alkyl-cytidines in the body, their localization and their
progression speed can be easily diagnosized.
The antibody titer used in this invention which
indicates the characteristics of the monoclonal antibodies -
is explained taking 5-methylcytidine as an example. A
conjugate of 5-methylcytidine and bovine serum albumin
(BSA) is made to be adsorbed previously to a 96-well
microplate, and to the wells are added serially 2-fold
diluted solutions of the supernatant of a culture medium of
hybridomas which produce the monoclonal antibody of this
invention to allow an immunoreaction to proceed, and then ~ ~
absorbance of each well is estimated. The titer means the ~-
maximum dilution fold that can maintain the maximum
absorbance. A diagrammatic representation of the pattern
of the relation between such a dilution fold and
absorbance, from which the antibody titer is obtained is
~given in Figure 2.
Examples
Below are given examples of the embodiments of this
invention, but they do not limit the scope of this;
16
20%8~g
invention.
Example 1 ~`~
(1) Preparation of Immunogen:
A conjugate of 5-methylcytidine, purchased from ~-~
Sigma, and keyhole limpet hemocyanin, a carrier
protein-I, was prepared by the method of Frlanger and
Bieser (Proc. Natl. Acad. Sci., 52, 68 (1964)).
Namely, 5-methylcytidine was oxidized by periodic ; -
acid, and after decomposing excess periodic acid with l-
ethyleneglycol, it was allowed to react with
hemocyanin under alkaline condition (pH 9 - 9.5).
i``~ Then the reaction mixture was reduced with sodium `-~
borohydride to form a stable compound. The reaction
mixture was dialyzed overnight in PBS (pH 7.4) to ;~
remove unreacted 5-methylcytidine, and after ;~
lyophilization it was stored in a freezer at -20 C.
(2) Preparation of Monoclonal Antibody: ~
(i) The conjugate of 5-methylcytidine and hemocyanin, ~;
which was obtained in (1), was admixed with an ;~
, ~
same amount of the Freund's complete adjuvant to
make an emulsion~ and administered
intraperitoneally to BALB/c mice in a dose of
50 ~g per head. In the second and following
.. . .
administrations, the emulsion made with ~`~
17 -
' ~
j ~ 2028~9
incomplete adjuvant was employed in 50 ~g per
mouse, being given intraperitoneally twice with
a 14 day interval. As a final immunization,
0.2 ml of a 100 ~g/ml solution of the conjugate
of 5-methylcytidine and hemocyanin was
intravenously injected.
(ii) Three days after the final immunization, the
spleen cells isolated from hyperimmunized mouse
and the myeloma cells strain Sp2/0-Agl4, derived ;~
from BALB/c mouse were fused by using
polyethyleneglycol 4000. The cells were placed
in a 96-well microplate in a volume of
: :
100 ~l/well, and after 24 hours, half a volume
of the culture medium was replaced by a HAT
, ~ .- .
medium and this medium replacement was repeated
every third day. After 7 - 10 days, hybridomas
resistant to HAT were found to grow. At this
time the medium was changed to a HT medium and
after 10 days culturing, it was changed to a
hybridoma-growing medium.
(iii) Screening of the antibody-producing cells was
performed by the two-antibody ELISA method by
: ~ :
using a conjugate of 5-methycytidine and
bovine serum albumin (BSA) as an antigen. By ; ;~
this method, abolishing the most worried-about
,; ,"~
18
',','~` ". ;"",';
! ~ ~ 2 0 2 8 8 ~ ~
''~" '
hybridomas which produce the antibodies against
the carrier protein was successfully achieved. ~--;
Then by examining inhibition by 5-methylcytidine,
the hybridomas producing the monoclonal
antibodies specifically reactive with
5-methylcytidine were selected. The hybridomas
here selected were cloned by the limiting
dilution method to establish a hybridoma
clone which produces monoclonal antibody.
Hybridoma MCT-3, one of such clones, have been
deposited as FERM BP-2788 in the Fermentation
Research Institute, Agency of Industrial Science
and Technology.
:: .: -,
(3) Properties of the Monoclonal Antibodies~
The monoclonal antibodies thus prepared were
classified into 3 groups based on their reaction -~
; specificities~
~; (i) The mcnoclonal antibodies that react very
strongly with 5-methylcytidine (MCT-3 and four other
clones), (ii) those showing lower reactivity ~ith ;~
5-methylcytidine than the monoclonal antibodies in (i) but ~ ~
do not react with other nucleosides (MCT-6 and one other ~` ;
clone), ~nd (iii) the monoclonal antibodies that react with
5-methylcytidine, 5-methylcytosine and furthermore
: :
.` '', ,:' '
' ~'';
~ 2~288~9
thymidine and cytidine (MCT-8 and one other clone).
Regarding the monoclonal antibodies showing the highest
reactivity in these groups, namely MCT-3, MCT-6 and MCT-8,
the antibody titers and reaction specificities are shown in
Table l.
Table l ~;
_ ~................ . . '.
i~: MonoclonaI an tibodyMCT-3 MCT-6 ~ICT-8
. ,'
Antibody class IgG1 IgG1 IgG2 A
- . .-
Antibody titer 6~-fold ]6-fold 3~-fold
~ ~ ._ _ ... _ _ __ ..
5-,~1ethy]cytidine + ~ + + ~ + :
Reactinn 5-Methyl cytosi ne _ _ + :~
: : .s~eclficity* Cytidine _ _ ~ N:
. _ Th ym i d i n e ~N
:~: *) +++ : High l~eactivit
+ : Medium reactivity
+ : Lc~ eactivity
No leflctivit
~ ;
:~: 20
',,,' "' ',:
-` ' 2028849
Example 2
To a 96-well microplate which had been coated with
5-methylcytidine-BSA (0.1 ~g/we'll) were added 50 ~ each
of 10 2, 10-3, 10-4, 10-5, 10-6 and 10-7 M solutions of
5-methylcytidine and then 30-fo~d dil,uted solution of
MCT-3 monoclonal antibody to conduct a competitive
reaction. The resurts demonstrated, as shown in Figure 1,
that in proportion to the amount of 5-methylcytidine added,
the binding of MCT-3 antibody and 5-methylcytidine-BSA is
inhibited. When this inhibition curve was used as the
calibration curve, the detection limit of 5-methylcytidine
was 1 pmol, and therefore this assay system has shown to ~-
provide a highly sensitive assay method of minute amounts ;
of 5-methylcytidine. ,
Example 3 ~ -
By replacing the 5-methylcytidine solutions in Example ~ ;
2 with urine of healthy donors and cancer patients, the -~
similar competitive inhibition test was conducted. By
using the calibration curve obtained in Example 2,
5-methylcytidine in the urine samples was assayed. The
results are shown in Table 2.
: I ' !
~. '.; ' ~.
''., ~'
21 '
~` 20288~
Table 2
Assay of 5-Methylcytidine in Healthy Persons and
Cancer Patients by Using MCT-3
_______________________________.__________________________
Casss 5-Methylcytidine
(n mol/~mol creatinine)
_______________________________,__________________________ . -
Healthy person 2.41 + 0.03
Healthy person 3.25 + 0.12 ~ -
Healthy person 1.71 + 0.02 ~
-- ----
`~ Hepatoma 34.92 + 0.89 :~
Gastric cancer35.36 + 1.26
Gastric cancer19.91 + 0.66
:~ Gastric cancer17.38 + 1.06 : -
: Gastric cancer18.75 + 1.39
: Example 4 .
Following the procedures described below,
immunohistological staining of esophageal cancer tissues
` ~ with the use of anti-modified nucleoside monoclonal
antibodies is performed.
1) Esophageal cancer tissue (a preparation of operative .~:;
:isolation) was fixed with formalin and embedded with
paraffin and sections of 3 - 5 ~m size were prepared.
(2) The sections were placed on a slide glass and `~
treated with normal horse serum to prevent nonspecific
~: adsorption. . ~.
(3) Then the sections were treated by allowing to react ~ Y
22 `
:-- :; ::":::
.".'';,; '"'~
,.,. ,',.~'~'.,.
20~88~
with a monoclonal antibody (anti-1-methyladenosine
antibody or anti-pseudouricline antibody, in a ,-~
concentration of 5 ~g/ml) for 1 hour.
(4) The slide glass was thoroughly washed with the buffer
solution and treated with biotinylated anti-mouse IgG
by the reaction at room temperature for 30 minutes.
(5) The slide glass was thoroughly w~ished with the buffer
solution and tre~ted with the horseradish
peroxidase-avidin complex by the reaction at room
temperature for 30 minutes.
(6) After the slide glass WRS thoroughly washed with the
buffer solution, the substrate solu$ion (phosphate
buffer solution containing 0.5 mg/ml of
diaminobenzidine and 0.01% hydrogen peroxide
solution) was added for coloration.
(7) The nucleus was stained with a hematoxylin solution. ;-
The results are shown in Figures 3 - ~. The results -
demonstrated that use of either anti-1-methyladenosine
monoclonal antibody or anti-pseudouridine monoclonal
antibody gave no staining with normal tissues but gave
s~pecific staining with cancerous tissues. In Figures 3 and
4 the parts in dark gray to black colors are the stained
parts (reaction positive sites).
Industrial Applicability
23
~ 2028~4~
The monoclonal antibodies of this invention have
excellent characteristics of recognizing in high
sensitivity lower alkyl-cytidines, in particular
5-methylcytidine. The hybridomas of this inve,ntion have
such useful properties as producing the monoclonal
antibodies having such excellent characteristics. By using
the assay method and the reagent kit of this invention,
lower alkyl-cytidines can be assayed easily without prior
treatment of body fluids and other samples, and thus it is
.... ..
possible to do assaying of many samples simply and promptly
for judging the presence of progressive cancer.
Also by the use of the searching method of this
invention, detection of modified nucleosides which are
accumulated as the result of abnormal elevation of
metabolism of nucleic acids in tumor tissues can be
performed easily. The monoclonal antibodies of this
~ ,:
~ invention for imaging diagnosis make it possible to -~
." ~
diagnose the cancers which are progressive in the body very ~ ~
simply, and the drug missiles provide ~nticancer and other ; ~ -
drugs with higher therapeutic effects by bring them ~`
efficiently to the affected sites.
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24 ~ ~
