CA2028849A1 - Monoclonal antibodies, assay method, reagent kit, sarching method and drug missiles using them - Google Patents
Monoclonal antibodies, assay method, reagent kit, sarching method and drug missiles using themInfo
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- CA2028849A1 CA2028849A1 CA 2028849 CA2028849A CA2028849A1 CA 2028849 A1 CA2028849 A1 CA 2028849A1 CA 2028849 CA2028849 CA 2028849 CA 2028849 A CA2028849 A CA 2028849A CA 2028849 A1 CA2028849 A1 CA 2028849A1
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- Prior art keywords
- lower alkyl
- monoclonal antibody
- cytidine
- monoclonal antibodies
- recognize
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- Health & Medical Sciences (AREA)
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- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
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- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
ABSTRACT
This invention concerns monoclonal antibodies which can recognize lower alkyl-cytidines; hybridomas which produce said monoclonal antibodies; assay method; reagent kit; immunohistochemical searching method for lower alkyl-cytidines in body fluids and other samples by using said monoclonal antibodies; and drug missiles utilizing said monoclonal antibodies. By this invention, diagnosis and treatment of cancerous diseases are made very easy.
This invention concerns monoclonal antibodies which can recognize lower alkyl-cytidines; hybridomas which produce said monoclonal antibodies; assay method; reagent kit; immunohistochemical searching method for lower alkyl-cytidines in body fluids and other samples by using said monoclonal antibodies; and drug missiles utilizing said monoclonal antibodies. By this invention, diagnosis and treatment of cancerous diseases are made very easy.
Description
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. :~
,: .~ .:: :, , ~ "~
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. ~;;; ^
''' ," ~, J '' "' "~' . ,' ' "~,~';' . r~
`- 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 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 -::
~ ;
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 : ::
.'"' ~' ~
.. . ~'' :
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 `~
:, : ,; . ,. .::
"
: ~ , 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;
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 ~`~
' ~
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 ,; ,"~
',','~` ". ;"",';
! ~ ~ 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.
,.~, :'...:.~, ~`
~`~` 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. ~;;; ^
''' ," ~, J '' "' "~' . ,' ' "~,~';' . r~
`- 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 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 -::
~ ;
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 : ::
.'"' ~' ~
.. . ~'' :
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 `~
:, : ,; . ,. .::
"
: ~ , 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;
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 ~`~
' ~
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 ,; ,"~
',','~` ". ;"",';
! ~ ~ 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.
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 ~ 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.
: : : : ,; :, : ~ -,: ~
: .,: ~.
: ' ;`';'';''"',"`''' 24 ~ ~
Industrial Applicability ~ 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.
: : : : ,; :, : ~ -,: ~
: .,: ~.
: ' ;`';'';''"',"`''' 24 ~ ~
Claims (20)
1. A monoclonal antibody which can recognize a lower alkyl-cytidine.
2. The monoclonal antibody of Claim 1, in which said lower alkyl-cytidine is a 5-lower alkyl-cytidine.
3. The monoclonal antibody of Claim 1, in which said lower alkyl-cytidine is 5-methylcytidine.
4. The monoclonal antibody of Claim 1, in which said lower alkyl-cytidine is contained in a biological sample.
5. The monoclonal antibody of Claim 1, which is produced by a hybridoma obtained by the fusion of spleen cells which have been prepared using a conjugate of a lower alkyl-cytidine and carrier protein-I as an immunogen and myeloma cells.
6. The monoclonal antibody of Claim 5, in which said spleen cells are mouse spleen cells and said myeloma cells are mouse-derived myeloma cells.
7. The monoclonal antibody of Claim 1, in which the class of said antibody is IgG.
8. The monoclonal antibody of Claim 7, in which said IgG
is IgG1.
is IgG1.
9. The monoclonal antibody for use with the imaging diagnosis of Claim 1, said antibody being labeled with one or more substances selected from the group consisting of radioactive substances, paramagnetic substances and fluorescent substances.
10. A hybridoma obtained by the fusion of spleen cells capable of producing antibodies which can recognize a lower alkyl-cytidine, and are prepared by immunizing an animal with a conjugate of a lower alkyl-cytidine and carrier protein-I as an immunogen and myeloma cells.
11. An assay method for lower alkyl-cytidines in samples by an immunological technique, characterized by using a monoclonal antibody which can recognize lower alkyl-cytidines.
12. A reagent kit for immunologically assaying lower alkyl-cytidines in samples, characterized by using a monoclonal antibody which can recognize lower alkyl-cytidine and a conjugate of a lower alkyl-cytidine and carrier protein-II.
13. An immunohistochemical searching method for detecting the presence of a modified nucleoside which is accumulated in tumor tissues as a result of abnormal elevation of metabolism of nucleic acids characterized in that a monoclonal antibody which can recognize said modified nucleoside is bound to a part of tissue by immunoreaction.
14. The searching method of Claim 13, in which said modified nucleoside is a lower alkyl-cytidine.
15. The searching method of Claim 13, in which said modified nucleoside is a pseudouridine.
16. The searching method of Claim 13, in which said modified nucleoside is 1-methyladenosine.
17. A drug missile characterized in that a monoclonal antibody which can recognize a modified nucleoside accumulated in tumor tissues as a result of abnormal elevation of metabolism of nucleic acids is used as a carrier of anticancer or carcinostatic drugs.
18. The drug missile of Claim 17, in which said modified nucleoside is a lower alkyl-cytidine.
19. The drug missile of Claim 17, in which said modified nucleoside is pseudouridine.
20. The drug missile of Claim 17, in which said modified nucleoside is 1-methyladenosine.
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
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