JPH1175839A - Monoclonal antibody, cell strain and measurement of n1,n12-diacetylspermine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for measuring N<1> ,N<2> -diacetylspermine to be expected as a marker. SOLUTION: N<1> ,N<12> -Diacetylspermine in a specimen is measured by utilizing an immune reaction of N<1> ,N<12> -diacetylspermine which is made into a solid phase or labeled or N<1> -dacetylspermine which is made into a solid phase or labeled and anti-NN<1> ,N<12> -diacetylspermine monoclonal antibody. In the operation, an anti-NN<1> ,N<12> -diacetylspermine monoclonal antibody having the inhibitory activity of the immune reaction of N<1> ,N<12> -diacetylspermine under measuring conditions of >=20 times as much as the immune activity of the immune reaction by N<1> -acetylspermine is used.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an anti-N¹, N¹²-Zia
Cetyl spermine monoclonal antibody, anti-N¹, N ¹²−
Acetylspermine monoclonal antibody producing cell line and
And N using the antibody¹, N¹²-Measurement of diacetylspermine
It is about a statute.

[0002]

2. Description of the Related Art Polyamines are putrescine (Pu).
t), cadaverine (Cad), spermidine (Sp
It is a general term for d), spermine (Spm) and their derivatives, and is a physiologically active substance widely distributed in a living body. Considerable amounts are synthesized in proliferating cells, including cancer cells, and are localized in these cells and act as factors that promote cell proliferation (Annu. Rev. B).
iochem. 53 p749-790 (198
4)).

In 1971, Russell et al. Found that polyamines increased in urine of cancer patients (Canc).
er Res. 31 p1555 (1971)), urinary polyamines are currently used as tumor markers. However, there have been some limitations to the significance of measuring "total polyamines" so far (Pro).
g. Drug Res. 39 p9-33 (199
2)).

[0004] Polyamines exist as free forms as they are, as well as as acetylated or bound forms. The like Abdel-Monem, polyamines excreted in the urine mainly N ¹ mono acetyl form - acetyl spermidine (N ¹ -Ac-Spd), acetyl putrescine ^{(Ac-Put), N 8} - acetyl spermidine ( N ⁸ -Ac-Spd), and it has been reported that an increase in these components or a change in the component ratio is a better tumor marker (J. Pharm. Sci. 6).
7 p 1671-1673 (1978), Cancer
Res. 42p2097-2098 (1982))
However, there are objections to this (J. Biochem. 118).
p1211-1215 (1995)).

[0005] Recently, Hitoshi Hiramatsu, N ¹ of diacetyl of polyamines in urine, N ⁸ - diacetyl spermidine (N ^1, N ^{8
-2Ac-Spd), N 1,} N 12 - found that Diacetylspermine ^{(N 1, N 12 -2Ac-} Spm) are present (J.Biochem.117 p107-112 (1
995)), which are expected to be new tumor markers because they significantly increased in urinary tumors and surpassed the fluctuations of conventional polyamines and monoacetylated polyamines (J. Cancer Res. Cli).
n. Oncol. 121 p317-319 (199
5)).

Conventionally, N ¹ , N ⁸ -2 of these diacetyl forms
Ac-Spd, N ¹ , N ¹² -2 Ac-Spm were measured by capillary gas chromatography (Clin. Ch.
em. 32 p1930-1937 (1986)) and liquid chromatography (J. Biochem. 117).
pp. 107-112 (1995)).

[0007]

SUMMARY OF THE INVENTION
Chill N¹, N¹²Capillary for measuring -2Ac-Spm
Lee gas chromatography and liquid chromatography
Method requires complicated sample pre-processing operations and equipment maintenance.
It requires considerable skill, such as the need for technical skills.
In addition, the measurement is time-consuming due to the complexity,
As a measurement method, it is not very acceptable.
Therefore, we aim to develop a simple measurement method that can be used clinically.
Then, an immunoassay using an antibody was examined. Fujiwara et al.
Immunized the albumin complex of Spm with N¹, N¹²-2
A monoclonal antibody reactive with Ac-Spm was prepared.
Is the monoacetyl form of N¹-Ac-Spd and N¹−Aceti
Ruspermine (N¹-Ac-Spm).
As a result, the most abundant of these three components in urine
Many, N ¹-Can only measure Ac-Spd
(J. Biochem. 118 p1211-1)
215 (1995)). Therefore, N¹, N¹²-2Ac-S
An immunoassay that can measure pm has not been completed yet.
No.

[0008]

Means for Solving the Problems The present inventors have described the above-mentioned.
As a result of intensive research to solve such problems, N ¹
Immunization with an albumin complex of Ac-Spm, substantially
Urine N¹, N¹²-2 Items that can be used for measuring Ac-Spm
Create a clonal antibody and check the assay using that antibody.
This has led to the present invention.

That is, the present invention relates to (1) immobilization or
Intellectualization N¹, N¹²-Diacetylspermine (N¹, N¹²-2A
c-Spm) or immobilized or labeled N¹-Acetyl
Spermine (N¹-Ac-Spm) and anti-N¹, N¹²-Zia
Utilizes immune response of cetylspermine monoclonal antibody
N in the sample¹, N¹²-Measurement system for diacetylspermine
, N ¹, N¹²-By diacetylspermine
The inhibitory activity of the immune response is N¹-Acetyl spermidine
(N¹-Ac-Spd)
20 times or more, preferably 30 times or more, more preferably 4 times or more.
It is possible to select measurement conditions that are 0 times or more.
N¹, N¹²A diacetylspermine monoclonal antibody,
(2) Immobilized or labeled N¹, N¹²-Diacetylspec
Lumin or immobilized or labeled N¹-Acetyl spelling
50% inhibition of immune response with Min¹, N¹²−Diase
The concentration of tilspermine is 20 μM or less, preferably 15 μM.
μM or less, more preferably 1 μM or less
Monochrome as described in (1) above, which can be selected
Null antibody, (3) above, wherein the sample is a urine sample or
(2) the monoclonal antibody according to the above, (4) the monoclonal antibody
0520, 4914 or 8624, (5) above
The monochromator according to (1), (2), (3) or (4)
Cell line producing antibody, (6) immobilized or labeled
N¹, N¹²-Diacetylspermine or immobilized or labeled
Intellectualization N¹-Acetyl spermine and anti-N¹, N¹²-Diaceti
Utilizing the immune response of Ruspermine monoclonal antibody
N in the sample¹, N¹²-When measuring diacetylspermine
And N under the measurement conditions¹, N¹²-Diacetyl spelling
The activity of inhibiting the immune response by¹-Acetyl spec
More than 20 times the activity of inhibiting the immune response by lumidine.
Preferably it is 30 times or more, more preferably 40 times or more.
Anti-N¹, N¹²-Diacetylspermine monoclonal antibody
N characterized by using¹, N¹²-Diacetyl spelling
Method for measuring min, (7) anti-N¹, N¹²-Diacetyl sperm
Monoclonal antibody is immobilized under measurement conditions
Or labeled N¹, N¹²-Diacetyl spermine or solid phase
Or labeled N¹-Immune reaction with acetylspermine
Inhibits 50% of N¹, N¹²-Concentration of diacetylspermine
Degree is 20 μM or less, preferably 15 μM or less, more preferably
Or less than 1 μM anti-N¹, N¹²-Diacetyl spelling
The measurement according to (6) above, which is a min monoclonal antibody.
Law, (8) Anti-N¹, N¹²-Diacetyl spermine monochrome
Monoclonal antibody 0520, 4914 or
Is 8624, (9) Sample
Is the urine sample, the measurement according to (6), (7) or (8) above.
Related to the law.

[0010]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
Immobilized N ^1, N ¹² -2Ac-Spm or immobilized N ¹ -Ac
The ^{-Spm, N 1, N 12 -2Ac} -Spm or N ¹
-Ac-Spm bound to the surface of an insoluble substance through a spacer. The bonding position of the spacer may be at its end in the case of N ¹ , N ¹² -2Ac-Spm or at any position between both ends, but in the case of N ¹ -Ac-Spm, It is desirable to attach to the terminal amino group. Many methods are known for the type and introduction method of the spacer, and any of these methods may be used. For ^{example, N 1, N 12 -2Ac-} Spm
Is introduced into any position of ( ¹⁾ , when the spacer is derived from the acetyl group of N ¹ , N ¹² -2Ac-Spm, the N ¹ -Ac-Spm N
^The acyl group introduced into the ¹² -amino group serves as a spacer), and binds to a protein or a synthetic polymer via this reactive group to form a complex of the formed N ¹ , N ¹² -2Ac-Spm with the protein or the synthetic polymer. A method of adsorbing a body on a solid support that serves as a site for an immune reaction, and a method in which N ¹ , N ¹² -2Ac-Spm itself, or N ¹ There is a method of reacting the terminal amino group of -Ac-Spm, but the method is not limited thereto. N ^1, N ¹² -2Ac-Spm or N ¹ -Ac-S
What kind of spacer should be introduced at which position in pm may be appropriately selected depending on the properties of the antibody to be used.

As a spacer, for example, when glutaraldehyde (GA) is used, a formylbutyryl chain is used.
When N- (4-maleimidobutyloxy) succinimide (GMBS) is used, the maleimidobutyryl chain is
When succinic anhydride is used, a carboxypropionyl chain and the like can be mentioned, and any known one can be used. Examples of the protein and the synthetic polymer include albumin and polylysine, but are not limited thereto. Examples of the solid phase carrier include, but are not limited to, a microtiter plate having 96 holes or the like, polystyrene beads, various latex particles, a nitrocellulose membrane, and the like.

As the labeled N ¹ , N ¹² -2Ac-Spm or the labeled N ¹ -Ac-Spm, N ¹ , N ¹² -2Ac-Spm or N ¹ -Ac-
Spm itself can be labeled, N ¹ , N ¹² -2Ac-Sp
m or N ¹ -Ac-Spm, a compound containing a radioactive element, a compound capable of holding an element having delayed fluorescence such as europium, a compound capable of holding an element serving as a catalyst for electrochemical luminescence such as ruthenium, a fluorescent substance, etc. And N ¹ , N ¹² -2Ac-Spm or N ¹ -Ac-Sp
m is labeled with an enzyme via a spacer as described above, or N ¹ , N ¹² -2Ac-Spm or N ¹ -Ac-Spm
Are labeled with biotin, reacted with an avidin enzyme-labeled substance after an immunoreaction, and indirectly detected as a biotin-avidin complex.

The monoclonal antibody of the present invention may be any cloned immunoglobulin antibody, regardless of the animal species from which the antibody is derived, the type and subtype of the immunoglobulin, and the method of producing the antibody. Also included are those obtained by fragmenting an antibody to leave an immunoreactive site, modified products of these fragments, modified products of the antibody itself, and chimeric antibodies in which two types of antibodies are bound. The monoclonal antibody of the present invention can be produced by a tissue culture method of an antibody-producing strain, a production method using genetic engineering using DNA predicted from the amino acid sequence of the antibody, or the like, using genetic engineering.

The antibody-producing strain producing the monoclonal antibody of the present invention can be prepared by a method according to a known method, that is, mouse,
It can be obtained by immunizing an animal such as a rat with an immunogen, fusing B cells of the immunized animal with myeloma cells, and selecting a cell line that produces the monoclonal antibody of the present invention from the obtained hybridomas. it can.

The immunogens include haptens (N ¹ , N ¹² -2Ac)
-Spm or N ¹ -Ac-Spm) and a carrier substance (protein or synthetic polymer) can be used, which can be immobilized on N ¹ , N ¹² -2Ac-Spm or N ¹ -Ac. −
It can be manufactured in the same manner as the method described in the method of producing Spm. The monoclonal antibodies of the present invention include, for example, monoclonal antibodies 0520, 4914 and 8624.
These are respectively ACSPM-1 (Deposited by the National Institute of Advanced Industrial Science and Technology, Biotechnology and Industrial Technology Research Institute, FERM P-
16297), ACSPM-2 (Deposited by the National Institute of Advanced Industrial Science and Technology, FERM P-16298) and ACSPM-3 (Deposited by the National Institute of Advanced Industrial Science and Technology, FERMP-16299) It can be obtained by culturing the strain.

The assay of the present invention can be performed by any method known as a hapten immunoassay, and is not particularly limited. For example, if the binding inhibition method, for example, as in Experimental Example 2, N ^1, N ¹² -2Ac-
A method for detecting the inhibition of an immune reaction between an antibody and an immobilized hapten by Spm by various means as a decrease in the amount of antibody bound to the immobilized hapten, and an example of using latex as a solid phase carrier is hapten. A method for detecting the agglutination reaction of an immobilized latex reagent by an antibody as inhibition by N ¹ , N ¹² -2Ac-Spm, and conversely, by using a latex reagent immobilized with an antibody and adding a polyepitopic polymorphized to a multi-epitope. Latex agglutination reaction was performed using N ¹ , N ¹² -2A
There is a method of inhibiting with c-Spm. In the case of the competitive method, N ¹ , N ¹² -2A
After a c-Spm and a labeled N ¹ , N ¹² -2Ac-Spm or a labeled N ¹ -Ac-Spm are subjected to a competitive reaction, after BF separation, the labeled N ¹ , N ¹² − 2Ac
The label of -Spm or labeled N ¹ -Ac-Spm may be appropriately detected by each method.

The measurement conditions for carrying out the measurement method of the present invention are not particularly limited, but conventionally known measurement conditions utilizing an immune reaction can be used. For example, a sample and a reagent (solid or labeled N ¹ , N ¹² -2Ac-Spm or solid or labeled N ¹ -Ac-Spm and anti-N ¹ , N ¹² -2Ac)
-Spm monoclonal antibody) is usually performed at 0 to 45 ° C, preferably 10 to 40 ° C. In performing the measurement of the present invention, anti-N ¹ , N ¹² -2
The Ac-Spm monoclonal antibody is used after being immobilized or labeled as necessary.

In carrying out the measurement method of the present invention, the immobilized or labeled N ¹ , N ¹² -2Ac-Spm or the immobilized or labeled N ¹ -Ac-Spm includes anti-N ¹ , N
Types of spacers were used to create the immunogen used to create a ¹² -2Ac-Spm monoclonal ^{antibodies, N 1, N 12 -2Ac-} Spm or N ¹ -Ac-S
It is desirable to use one prepared by changing at least one of the bonding position to pm or the chemical bonding method.

In the present invention, immobilized or labeled N ¹ , N ¹² -2Ac-Spm or immobilized or labeled N ¹ -Ac-Spm and an anti-N ¹ , N ¹² -2Ac-Spm monoclonal antibody N ¹ , where the immune reaction with
The concentration of N ¹² -2Ac-Spm is such that N ¹ and N ¹² -2A reduce the signal obtained by reacting both under measurement conditions by half.
can be obtained by determining the concentration of c-Spm,
This can be a measure of the sensitivity of the reaction system.

In the present invention, the specimens include urine, serum,
Although various substances such as plasma can be used, urine is particularly preferable.

[0021]

EXAMPLES The present invention will be described more specifically with reference to Experimental Examples and Examples, but the present invention is not limited to these.

Experimental Example 1 Antibody Screening Method 96-well microtiter plate (Nunc Im
To each well of munoplate II), N ¹ as an antigen - human serum albumin conjugate of spermine (N ¹ -A
c-Spm-GMBS-HSA) 100 μL of 10 mM Tris / HCl buffer (pH 8.5) containing 15 μg / mL
And left at 40 ° C. for 20 minutes to coat.
Next, the coating solution is discarded, and 0.1% Tween 2 is added.
Saline buffered with 10 mM phosphoric acid (PBS
After washing with T), 200 μL of a saline solution (pH 7.3) buffered with 50 mM Tris-hydrochloric acid containing 1% of skim milk
Was subjected to a blocking treatment. Discard the blocking solution and PB
After washing with ST, the culture supernatant of hybridoma 6 was added to each well.
0 μL and 20 μL of a saline solution buffered with 10 mM phosphoric acid containing 20 mg / mL of BSA were added and allowed to react at 4 ° C. overnight. After washing the wells with PBST, the wells were washed with PBST for 20 minutes.
Horseradish peroxidase (HR
P) Label-Goat anti-mouse IgG solution (Kappel) 50
μL was added and reacted at 37 ° C. for 40 minutes. The activity of the bound enzyme was determined by adding o-phenylenediamine 0.5 mg / mL and hydrogen peroxide 0.01 to each well washed with PBST.
0.1 M citrate phosphate buffer containing 2% (pH 5.
3) 100 μL was added, and a color reaction was performed at room temperature for 9 minutes.
ELISA plate reader (SLT-Lab Instruments
Was measured as an increase in absorbance at 492 nm.

[0023] Experimental Example 2 ELISA binding inhibition method [N ^1, N ¹² - Diacetylspermine human serum albumin conjugate ^{(N 1, N 12 -2Ac-} Spm-GA-HSA)
Preparation of ¹ mL of 0.083 M glutaraldehyde solution was added to 0.5 mL of a 1 M sodium acetate solution containing 6.5 mg of N ¹ , N ¹² -diacetylspermine (N ¹ , N ¹² -2Ac-Spm), and the mixture was stirred. It was left for 30 seconds. Further, 0.5 mL of a 1 M sodium acetate solution containing 10.5 mg of human serum albumin (HSA) was added and stirred,
The reaction was performed at room temperature for 30 minutes. Furthermore, after adding 5 mg of sodium borohydride to this reaction solution and reacting at room temperature for 10 minutes, 300 mL of a 10 mM sodium acetate solution was added.
Then, dialysis was performed for 3 hours and 45 minutes while exchanging the solution four times. [N ¹ - Preparation of human serum albumin complex of acetyl spermidine ^{(N 1 -Ac-Spd-GA} -HSA)] 3
mg of N ¹ -acetylspermidine (N ¹ -Ac-Sp
0.5 ml of a 1 M sodium acetate solution containing d).
1 mL of a 021 M glutaraldehyde solution was added, stirred, and left for 30 seconds. Further, 0.5 mL of a 1 M sodium acetate solution containing 6 mg of HSA was added, stirred, and reacted at room temperature for 30 minutes. Further, 2.5 mg of sodium borohydride was added to the reaction solution, and the mixture was reacted at room temperature for 10 minutes. Then, 300 mL of a 10 mM sodium acetate solution was added.
Then, dialysis was performed for 3 hours while exchanging the solution four times.

[0024] To each well of the Measurement Method] 96-well microtiter plates (Nunc Nunc Immunoplate II), N ¹ as an antigen - human serum albumin conjugate of spermine ^{(N 1 -2Ac-Spm-GMBS} -H
SA), N ¹ - human serum albumin conjugate of acetyl spermidine ^{(N 1 -Ac-Spd-GA} -HSA), or, N ^1, N ¹² - human serum albumin conjugate of Diacetylspermine (N ^1, N ¹² - Ac-Spm-GA-HSA)
10 mM Tris / HCl buffer containing 15 μg / mL (p
H8.5) 200 μL was added and left at 40 ° C. for 20 minutes for coating. Next, discard the coating liquid and
After washing with BST, blocking treatment was performed with 200 μL of 50 mM Tris / HCl buffer (pH 7.3) containing skim milk 1%. After discarding the blocking solution and washing with PBST, 50 μL of the culture supernatant of each of the three hybridoma strains diluted 100-fold with PBST, spermine (Spm), N ¹ -acetylspermidine (N ^{1 -Ac-Spd), N 1} - spermine ^{(N 1 -Ac-Spm),} N 1, N 12 - diacetylspermine ^{(N 1, N 12 -2Ac-} Spm) or the like, and diluted to various concentrations with PBST 50 μL of the solution, and
Allowed to react for hours. After washing the wells with PBST,
50 μL of horseradish peroxidase-labeled goat anti-mouse IgG solution diluted 2000-fold with T
The reaction was performed at 40 ° C. for 40 minutes. The activity of the bound enzyme is PB
Each well washed with ST contains 0.5 mg / mL of o-phenylenediamine and 0.012% of hydrogen peroxide.
100 μL of 1M citrate phosphate buffer (pH 5.3) was added, and a color reaction was performed at room temperature for 5 minutes, and 492 was performed using an ELISA plate reader (SLT-Lab Instruments).
Measured as increase in absorbance in nm.

[0025] Example ¹ N 1, N ¹² - Creating a monoclonal antibody against Diacetylspermine [N ¹ - Preparation of BSA conjugates of spermine (antigen)] S- acetyl mercaptosuccinic anhydride 50 mg (A
MS) in 1 mL of tetrahydrofuran solution
mg of bovine serum albumin (BSA) in 5 mL of a 0.1 M phosphate buffer (pH 7.0), and stirred.
The reaction was carried out at room temperature for 1 hour while maintaining the pH at 7.0 with a sodium hydroxide solution. This reaction solution was prepared by swelling with 5 mM phosphate buffer (pH 6.8).
The mixture was applied to 75 columns (2 cm × 100 cm) and eluted with 5 mM phosphate buffer (pH 6.8). The derivatized BSA fractions were collected and lyophilized to give AMS-converted BSA.
(AMS-BSA) 180 mg was obtained. SH to BSA
The number of introduced groups was 17 ± 0.5. Next, 10.4m
0.1 g of N ¹ -acetylspermine trihydrochloride
M phosphate buffer (pH 6.9)
(4-maleimidobutyloxy) succinimide (GMB
0.5 mL of a tetrahydrofuran solution containing S) is added,
The reaction was carried out at room temperature for 100 minutes while maintaining the pH at around 7, to prepare a GMBS-modified N ¹ -acetylspermine solution.

On the other hand, to 0.2 mL of a 0.1 M phosphate buffer (pH 6.9) containing 17.3 mg of AMS-BSA,
0.5μ hydroxylamine solution (pH 7.0) 50μ
L, and allowed to stand at room temperature for 10 minutes.
2 mL of phosphate buffer (pH 6.9) was added. This solution was mixed with the GMBS-modified N ¹ -acetylspermine solution prepared above and stirred with a vortex mixer.
Sephadex G-100 column (2.2 cm x 43 cm) equilibrated with Tris / hydrochloric acid buffer (pH 7.5)
And eluted with the same buffer. Collected protein fraction absorbed at 280 nm, N ¹ - acetyl spermine BSA
A complex (N ¹ -Ac-Spm-GMBS-BSA) was obtained. In a similar manner, human serum albumin (HS
N ¹ corresponding to A) - HSA complex of spermine ^{(N 1 -Ac-Spm-GMBS} -HSA) were also prepared.

[Immunity] N ¹ -A emulsified with Complete Freund's adjuvant was injected into female BALB / c mice.
100 μg of c-Spm-GMBS-BSA antigen was administered intraperitoneally. Further, 100 μg of N ¹ -Ac-Spm-
The GMBS-BSA antigen was diluted with a saline solution (PBS) buffered with 10 mM phosphoric acid, and boosted four times every two weeks. [Cell fusion] Four days after the fifth final sensitization,
The mouse spleen cells were removed, and this and myeloma cell P3
/ NS-1 / Ag4-1 in the presence of 40% polyethylene glycol 1500 (Boehringer) in the presence of Shu.
Cell fusion was performed according to the method of Iman et al. The fused cells were then cultured in HAT medium using a 96-well culture plate (Corning) at a cell density of 10 5 cells per well. Ten to twenty days after cell fusion, cell growth was observed in 708 of 960 wells (74%).

[Selection of Cells] The antibody titer in the culture supernatant of each well was searched by the "antibody screening method" of Experimental Example 1 to obtain three cells positive for immunoreactivity. These are cloned by the limiting dilution method, and cells that continuously produce antibodies, AC
Three strains of SPM-1, ACSPM-2 and ACSPM-3 were established. Furthermore, in order to confirm the reactivity of these three strains to the antigen site, GM which may be a by-product during antigen production
BA-HSA (GMSA with spacer on AMS-HSA)
Into which the antigen N ¹ -Ac of Experimental Example 1 was prepared.
-Instead of Spm-GMBS-HSA, GMBA-H
SA, AMS-HSA (in which an SH group is introduced into HSA) or a plate coated with HSA of the carrier protein itself was also prepared, and the ACSPM-1 strain, which was diluted twice, was prepared.
The reactivity of the ACSPM-2 strain and the ACSPM-3 strain with the culture supernatant was confirmed. The results are shown in FIGS. All culture supernatants were N ¹ -Ac-Spm-GMB
Reacts only with S-HSA, GMBA-HSA, AMS-
It did not react with HSA or HSA at all. [Clone Cell Subtype] The subtype of the immunoglobulin produced by each clone cell was determined using a mouse monoclonal Sub-isotyping kit (Zymed Code No. 97-
6550). As a result, the 0520 antibody (ACSPM-1 strain) and the 4914 antibody (ACSPM-2
Strain) was IgG ₁ , and the 8624 antibody (ACSPM-3 strain) was IgG _2b .

Example 2 Specificity of Monoclonal Antibody The specificity of the prepared monoclonal antibody was measured by a system for detecting the extent to which the substance to be measured inhibits the binding of the antibody to the immobilized antigen. Was evaluated. That is, according to the method of Experimental Example 2, three antigens, N ¹ -Ac-
Spm-GMBS-HSA, N ¹ -Ac-Spd-GA
^{-HSA, N 1, N 12 -2Ac} -Spm-GA-HSA
Was evaluated using a plate on which each was immobilized, to what extent each concentration of polyamines inhibited the reaction between the antibody and the immobilized antigen. N ¹ -Ac- corresponding to immunogen
The system in which Spm-GMBS-HSA was immobilized was not inhibited at all by any of the polyamines examined, and was unsuitable as a measurement system. On the other hand, N ¹ -Ac-Spd
-GA-HSA, ^{or, N 1, N 12 -2Ac-} Spm-G
In the system in which A-HSA was immobilized, each component examined, Sp
m, N ¹ -Ac-Spd, N ¹ -Ac-Spm, N ¹ , N ¹²
The binding inhibition curves (calibration curves) shown in FIGS. 4 to 9 were obtained by -2Ac-Spm. 0520, 4914, 86
Each of the 24 antibodies had N ¹ -Ac-Spm and N ¹ , N ¹²
Strongly reacted with -2Ac-Spm, very weakly reacted (inhibited) with N ¹ -Ac-Spd, and did not react (inhibited) with Spm at all. From the figure, the measurement sensitivity when polyamines were measured using these antibodies was determined to be 50% binding inhibition concentration (EC ₅₀
Value), and the results are shown in Table 1.

FIG. 4 shows N¹, N¹²-2Ac-Spm-
0520 by GA-HSA immobilized ELISA binding inhibition method
FIG. 5 shows the specificity of the antibody.¹, N¹²-2Ac-Spm-
4914 in GA-HSA immobilized ELISA binding inhibition method
FIG. 6 shows the specificity of the antibody. ¹, N¹²-2Ac-Spm-
8624 by GA-HSA immobilized ELISA binding inhibition method
FIG. 7 shows the specificity of the antibody.¹-Ac-Spd-GA-
0520 Antibody by HSA Immobilized ELISA Binding Inhibition Method
The specificity is shown in FIG.¹-Ac-Spd-GA-HSA
Specificity of 4914 antibody in solid-phase ELISA binding inhibition method
And FIG. ¹-Ac-Spd-GA-HSA immobilization
Shows the specificity of the 8624 antibody in ELISA binding inhibition
It is a thing.

All of the antibodies 0520, 4914 and 8624 were made of N ¹ -Ac-Spm and N ¹ , N ¹² -2Ac-Sp.
m specific. Hiramatsu et al. (J. Biochem. 1)
17p107-112 (1995)) and G. et al. A. van
den Berg et al. (Clin. Chem. 32 (1
0) p1930-1937 (1986))
The concentration of N ¹ -Ac-Spm in urine is N ¹ , N ¹² -2Ac-
From significantly lower than Spm, in this measuring method, it is possible to obtain a measurement that reflects the substantially N ^1, N ¹² -2Ac-Spm concentration in urine.

[0032]

[Table 1]

Example 3 96-well microtiter plate (Nunc Im manufactured by Nunc)
munoplate) as antigens N ¹ , N ¹² -2A
1 containing 16.1 μg / mL of c-Spm-GA-HSA
150 μL of 0 mM Tris / hydrochloric acid buffer (pH 8.5) was added thereto, and left at 40 ° C. for 30 minutes to perform coating. Then, after washing with 300 μL of PBST, 300 μM of 50 mM Tris / HCl buffer (pH 7.2) containing 1% of skim milk
1 to perform a blocking treatment. After washing with PBST,
50 μL of a 25-fold dilution of the culture supernatant of ACSPM-1 in PBST, and Put (putrescine), Ac-Put, Orn (ornithine), Cad
(Cadaverine), Spd, N ¹ -Ac-Spd, N ⁸ -A
^{c-Spd, N 1, N} 8 -2Ac-Spd, Spm, N 1 -
50 μL of a solution obtained by diluting Ac-Spm, N ¹ , N ¹² -2Ac-Spm to various concentrations with PBST was added, and reacted at 4 ° C. overnight. After each well was washed with PBST, the well was washed with PBST for 20 minutes.
100 μL of goat anti-mouse IgG (H & L) -biotin (AMERICA QUALEX) diluted 1:00
, And reacted at room temperature for 1.5 hours. After washing with PBST,
100 μL of HRP-streptavidin (Funakoshi) diluted 3000 times with PBST was added, and reacted at room temperature for 30 minutes. The activity of the bound enzyme was determined by adding 0.5 mg / m o-phenylenediamine to each well washed with PBST.
0.1M citric acid containing L and 0.012% of hydrogen peroxide
100 μL of a phosphate buffer (pH 5.3) was added, and the mixture was reacted at room temperature for 6 minutes. An ELISA plate reader (SLT-
Lab Instruments) as an increase in absorbance at 492 nm. The results are shown in FIG. FIG.
As apparent from FIG. 0, according to this reaction system, N ¹ , N ¹²
It can be seen that the specificity for -2Ac-Spm has been improved.

Example 4 Specificity of Monoclonal Antibody in Optimized ELISA Binding Inhibition Method The measurement method of Experimental Example 2 was improved, and the antibody specificity and measurement sensitivity were confirmed again. To each well of a 96-well microtiter plates (Nunc Nunc Immunoplate II), N ¹ as an antigen - human serum albumin conjugate of spermine ^{(N 1 -Ac-Spm-GA} -HSA), or,
N ^1, N ¹² - human serum albumin conjugate of Diacetylspermine ^{(N 1, N 12 -2Ac-} Spm-GA-HSA) 1
10 mM Tris / HCl buffer containing 5 μg / mL (pH
8.5) 150 μL was added and left at 40 ° C. for 30 minutes to perform coating. Next, discard the coating liquid and
After washing with ST, add 300 μL of a buffer (pH 7.4) with 50 mM Tris / hydrochloric acid containing skim milk 1%, and add 37 ° C.
For 1 hour.

After discarding the blocking solution and washing with PBST, the culture supernatant of each of the three hybridoma strains was added to each well at the magnification shown in Table 2 (200 to 20,000 times).
A liquid 25μL diluted in PBST to, spermine of the measuring object ^{(Spm), N 1, N} 8 - diacetyl spermidine ^{(N 1, N 8 -2Ac-} Spd), N 1 - acetyl spermidine (N ¹ -Ac-Spd ), N ¹ - spermine ^{(N 1 -Ac-Spm),} N 1, N 12 - diacetylspermine ^{(N 1, N 12 -2Ac-} Spm) was added a solution 75μL diluted to each concentration with PBST, room temperature For 3 hours. After washing the wells with PBST, a biotin-labeled goat anti-mouse IgG antibody solution 5
0 μL was added, and the mixture was reacted at room temperature for 1 hour. After the wells were washed again with PBST, 50 μL of a horseradish peroxidase-labeled streptavidin solution diluted 3000-fold with PBST was added, and reacted at room temperature for 30 minutes.

The activity of the bound enzyme was determined by adding 0.5 mg / m o-phenylenediamine to each well washed with PBST.
L and 100 μL of 0.1 M citrate phosphate buffer (pH 5.3) containing 0.012% of hydrogen peroxide, and allowed to undergo a color development reaction at room temperature for 5 minutes, followed by an ELISA plate reader (SL
(T-Lab Instruments) as an increase in absorbance at 492 nm. The results are summarized in Table 2. By improving the measurement system and increasing the dilution ratio of the antibody, N
^1, N ¹² measuring sensitivity and specificity of the immobilized system -Ac-Spm-GA-HSA was significantly improved. That is, N ¹ , N ¹²
The measurement sensitivity of -Ac-Spm was as follows:
914 antibody is improved by 22 times, 8624 antibody is improved by 70 times,
The sensitivity compared at the 50% binding inhibitory concentration was 0.06 to 0.
2 μM. In addition, the specificity was determined as N ¹ , N ¹² -Ac-Sp
When expressed as the ratio of the 50% binding inhibitory activity by m to the 50% binding inhibitory activity by N ¹ -Ac-Spd, 05
20 antibodies 48-fold, 4914 antibodies 117-fold, 8624
The antibody was 45-fold, all of which improved from before the improvement.

[0037]

[Table 2]

Example 5 4914 Monoclonal Antibody
Specificity The 4914 model with the highest measurement sensitivity and specificity in Example 4
Using a noclonal antibody, putressi by the method of Example 3
(Put), acetyl putrescine (Ac-Pu)
t), L-ornithine (Orn), cadaverine (Ca
d), spermidine (Spd), N⁸-Acetyl spelling
Midin (N⁸-Ac-Spd), N¹-Acetyl spermi
Gin (N¹-Ac-Spd), N¹, N⁸-Diacetylspec
Lumidine (N¹, N⁸-2Ac-Spd), spermine
(Spm), N¹-Acetyl spermine (N ¹-Ac-S
pm) or N¹, N¹²-Diacetylspermine (N¹, N¹²−
2Ac-Spm) was measured and 4914 monoclonal
The specificity of the nal antibody was examined. As shown in FIG.
The measurement system is N¹, N¹²-2Ac-Spm specific, N¹
What is -Ac-Spm?¹, N¹²-2Ac-Spm of 24
%, N¹-Ac-Spd is 0.85%, N¹, N⁸-2A
0.6% for c-Spd, 0.1% for Spm, and others
Hardly reacted with the polyamines.

Example 6 N ¹ and N ¹² -2Ac in urine of healthy subjects
-Measurement of Spm According to the method of Example 5, 16 urine samples from healthy subjects (8 males,
The dilution series of 8 females) were measured, and N ¹ , N ¹² -2A in urine was measured.
The c-Spm concentration was determined. A part of the measurement example is shown in FIG. The average values ± SD for each of 8 male and 8 female cases were 0.34 ± 0.16 and 0.39 ± 0.14 μM / g-creatinine, respectively, and the overall average value was 0.36 μM / g-creatinine. . Hiramatsu et al.'S report (J. Bioche
m. 117p107-112 (1995))
N ¹ , N ¹² -2Ac-Spm in urine: N ¹ -Ac-
Spm: N ¹ -Ac-Spd: N ¹ , N ⁸ -2Ac-Spd
Is 3.2%: 1.0%: 86.2%: 9.6.
% Is reported. According to the specificity of this measurement system, the influence of polyamine components other than N ¹ , N ¹² -2Ac-Spm is considered to be minor, and N ¹ , N ¹² -2Ac in urine is considered to be minor.
-Spm is considered to be measured almost exactly.

[0040]

The monoclonal antibody which reacts with N ¹ , N ¹² -diacetylspermine according to the present invention can be used for detecting N 2 in a sample such as urine.
¹ , N ¹² -diacetylspermine can be measured,
Useful for diagnosing cancer.

[Brief description of the drawings]

FIG. 1 Specificity of the culture supernatant of the ACSPM-1 strain for immunogen

FIG. 2 Specificity of the culture supernatant of ACSPM-2 strain for immunogen

FIG. 3 Specificity of ACSPM-3 strain culture supernatant for immunogen

[4] N ^1, the specificity of the 0520 antibody with ^{N 12 -2Ac-Spm-GA-} HSA immobilized ELISA binding inhibition method

[5] N ^1, the specificity of the 4914 antibody with ^{N 12 -2Ac-Spm-GA-} HSA immobilized ELISA binding inhibition method

[6] N ^1, the specificity of the 8624 antibody with ^{N 12 -2Ac-Spm-GA-} HSA immobilized ELISA binding inhibition method

FIG. 7: N ¹ -Ac-Spd-GA-HSA immobilized EL
Specificity of 0520 antibody in ISA binding inhibition method

FIG. 8: N ¹ -Ac-Spd-GA-HSA immobilized EL
Specificity of 4914 antibody in ISA binding inhibition method

FIG. 9: N ¹ -Ac-Spd-GA-HSA immobilized EL
Specificity of 8624 antibody in ISA binding inhibition method

[10] N ^1, the specificity of the 0520 antibody with ^{N 12 -2Ac-Spm-GA-} HSA immobilized ELISA binding inhibition method

FIG. 11: Specificity of antibody 4914 by N ¹ , N ¹² -2Ac-Spm-GA-HSA immobilized ELISA binding inhibition method

The concentration of N ^1, N ¹² -2Ac-Spm in FIG. 12 N ^1, a calibration curve of the N ¹² -2Ac-Spm and urine specimens

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // (C12P 21/08 C12R 1:91)

Claims (9)

[Claims] 1. A sample using an immunoreaction between immobilized or labeled N ¹ , N ¹² -diacetylspermine or immobilized or labeled N ¹ -acetylspermine and an anti-N ¹ , N ¹² -diacetylspermine monoclonal antibody. N ¹ , N ¹² −
When a measurement system for diacetylspermine is set up, N ¹ , N
¹² - diacetylspermine inhibitory activity by the immune response N ¹ - anti N ¹ of the measurement conditions is 20 times more inhibitory activity it is possible to select of the immune response by acetyl spermidine, N ¹² - diacetylspermine monochrome Nal antibody. Wherein immobilized or labeled N ^1, N ¹² - Diacetylspermine or immobilized or labeled N ¹ - N ^1, N ¹² where immunoreaction with spermine is inhibited 50% -
2. The monoclonal antibody according to claim 1, wherein measurement conditions under which the concentration of diacetylspermine is 20 μM or less can be selected. 3. The monoclonal antibody according to claim 1, wherein the sample is a urine sample. 4. The monoclonal antibody 0520, 4914 or 8624. 5. A cell line that produces the monoclonal antibody according to claim 1, 2, 3, or 4. 6. An immunoreaction between immobilized or labeled N ¹ , N ¹² -diacetylspermine or immobilized or labeled N ¹ -acetylspermine and an anti-N ¹ , N ¹² -diacetylspermine monoclonal antibody. N ¹ , N ¹² − in the sample
When diacetylspermine is measured, anti-N ¹ , in which the inhibitory activity of the immune response by N ¹ , N ¹² -diacetylspermine under the measurement conditions is at least 20 times the inhibitory activity of the immune response by N ¹ -acetylspermidine, A method for measuring N ¹ , N ¹² -diacetylspermine, comprising using an N ¹² -diacetylspermine monoclonal antibody. 7. Immunization of an anti-N ¹ , N ¹² -diacetylspermine monoclonal antibody with immobilized or labeled N ¹ , N ¹² -diacetylspermine or immobilized or labeled N ¹ -acetylspermine under measurement conditions. 50% reaction
When the inhibitory N ¹ , N ¹² -diacetylspermine concentration is 20
Anti N ¹ serving as μM or less, N ¹² - Measurement of claim 6 wherein the diacetylspermine monoclonal antibodies. 8. An anti-N ¹ , N ¹² -diacetylspermine monoclonal antibody which is a monoclonal antibody 0520,4914.
Or the measuring method according to claim 7, which is 8624. 9. The method according to claim 6, wherein the sample is a urine sample.