JP3709078B2 - Method and kit for measuring diacetylpolyamine - Google Patents

Description

DASPM ：ジアセチルスペルミン
AcSPM ：アセチルスペルミン
N1SPD ：Ｎ１アセチルスペルミジン
N8SPD ：Ｎ８アセチルスペルミジン
SPM ：スペルミン
【表２】

DASPD ：ジアセチルスペルミジン
AcPUT ：アセチルプトレッシン
N8SPD ：Ｎ８アセチルスペルミジン
【図面の簡単な説明】
【図１】図１は、ウシ血清アミンオキシダーゼの活性がある酵素溶液と９Ｇ９固相化プレートおよび西洋ワサビペルオキシダーゼ標識化モノアセチルスペルミンを用いて行ったイムノアッセイにおける各種ポリアミンに対する反応曲線を示したものである。
【図２】図２は、ウシ血清アミンオキシダーゼの活性がない酵素溶液と９Ｇ９固相化プレートおよび西洋ワサビペルオキシダーゼ標識化モノアセチルスペルミンを用いて行ったイムノアッセイにおける各種ポリアミンに対する反応曲線を示したものである。
【図３】図３は、ウシ血清アミンオキシダーゼの活性がある酵素溶液と１Ｄ８固相化プレートおよび西洋ワサビペルオキシダーゼ標識化Ｎ⁸ −アセチルスペルミジンを用いて行ったイムノアッセイにおける各種ポリアミンに対する反応曲線を示したものである。
【図４】図４は、ウシ血清アミンオキシダーゼの活性がない酵素溶液と１Ｄ８固相化プレートおよび西洋ワサビペルオキシダーゼ標識化Ｎ⁸ −アセチルスペルミジンを用いて行ったイムノアッセイにおける各種ポリアミンに対する反応曲線を示したものである。[0001]
[Technical field to which the invention belongs]
The present invention relates to a method for measuring diacetylpolyamine in a sample by immunoassay and a kit used for the method.
[0002]
[Prior art]
Since the polyamine concentration in the urine sample of cancer patients is higher than the concentration in the urine sample of healthy subjects, polyamine was expected as a diagnostic marker for cancer, but many false positives and false negatives were observed, Polyamine has not been adopted in clinical tests as a diagnostic marker for cancer.
However, recently, diacetyl isomers of polyamines such as diacetylspermine and diacetylspermidine (diacetylpolyamine) have been reported to be useful as cancer diagnosis, therapeutic effect determination and markers (J. Cancer Res. Clin. Oncol. 121: 317-319 (1995), J. Cancer Res. Clin. Oncol., 123: 539-545 (1997)).
[0003]
[Problems to be solved by the invention]
Conventionally, high-performance liquid chromatography (HPLC) has been the mainstream for measuring polyamines such as polyamines and diacetylpolyamines, but many sample samples cannot be processed in a short period of time, and the maintenance and accuracy management of measuring instruments are complicated. However, this method is not always satisfactory.
[0004]
Various immunoassays have been studied in place of such HPLC methods, but many urine samples contain impurities that are structurally similar to the target diacetylpolyamine. It was difficult to obtain by the method. For this reason, the antibody used for immunoassay can be used only after the substrate specificity is improved by affinity purification (Biochemistry, Vol. 69, No. 7, 898 (1997), J. Biochem. , 121 1134-1138 (1997)), such a labor of affinity purification has become a bottleneck in actual use in clinical tests.
[0005]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventors have determined that when diacetylpolyamine is measured by immunoassay, polyamines and monoacetylpolyamines having a problem of cross-reactivity are first graded by an enzyme such as amine oxidase. It was found that the cross-reactivity can be reduced by modifying the amino group, and that the target diacetylpolyamine can be measured specifically and with high sensitivity, thereby completing the present invention.
That is, the present invention is a method for measuring diacetylpolyamine in a sample by an immunoassay, wherein the sample to be measured is treated with an enzyme capable of modifying a primary amine, and a method for measuring diacetylpolyamine and use thereof It relates to the kit.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
The sample used in the method of the present invention is not particularly limited as long as it contains a diacetylpolyamine, particularly diacetylspermine and / or diacetylspermidine. Specific examples of such samples include urine and serum, and urine samples are particularly suitable for use.
[0007]
The present invention is characterized in that a sample used in such an immunoassay is treated with an enzyme capable of modifying a primary amine.
Enzymes capable of modifying primary amines are particularly limited as long as they are enzymes capable of modifying (for example, oxidation, transamination, dehydrogenation, etc.) primary substances in polyamines and monoacetylpolyamines, which are structurally similar to diacetylpolyamines. Not. As specific examples of such enzymes, amine oxidase, aminotransferase, or dehydrogenase may be used alone or in combination.
[0008]
The usage form of the enzyme may be any form such as a purified enzyme, a crudely purified enzyme, or an unpurified enzyme. For example, when amine oxidase is used as an enzyme, animal serum, particularly a preparation from bovine serum or bovine serum itself can be used.
The enzyme treatment can be performed by adding an enzyme to the sample and incubating at 10 to 40 ° C. for about 10 minutes to 4 hours with stirring as necessary. The enzyme treatment is preferably performed before the immunoassay, but may be performed simultaneously with the immunoassay.
[0009]
Measurement of the diacetylpolyamine in the enzyme-treated sample by an immunoassay can be performed by a known method or a modification thereof. That is, the antibody to be used may be either a monoclonal antibody or a polyclonal antibody as long as it can bind to free diacetylpolyamine and measure them. In particular, the present invention is characterized by the enzymatic treatment of a sample, and it is not necessary to use an antibody specific only for diacetylpolyamine, and it is somewhat different from contaminants structurally similar to diacetylpolyamine (for example, polyamine, monoacetylpolyamine, etc.). Even antibodies having cross-reactivity of can be used in the present invention. The preparation of such an antibody has already been reported and can be carried out according to the known method or by improving it (Biochemistry, Vol. 69, No. 7, 898 (1997), J. Chem. Biochem., 121 1134-1138 (1997)).
[0010]
The antibody to be used may be the antibody itself, but an active fragment can also be used. The active fragments may be various fragments such as F (ab ′) ₂ , Fab ′, Fab, etc., as long as the measurement functions are not lost.
These active fragments can be prepared by applying a known method such as papain, pepsin, trypsin treatment, etc. to the purified antibody (for example, “Immunobiochemical Research Method (Secondary Biochemistry Experiment Course 5)”). , Japan Biochemical Society, page 89 (1986)).
[0011]
The antibody or the active fragment thereof thus prepared is modified into a form suitable for the measurement method selected as necessary and used in the present invention.
Each modification of the antibody can be performed according to a conventional method. For example, when preparing a solid-phase antibody, the carrier material that can be used is not particularly limited as long as it has a high binding property to the antibody. For example, polyvinyl chloride, polystyrene, styrene-divinylbenzene copolymer , Synthetic organic polymer compounds such as styrene-maleic anhydride copolymer, nylon, polyvinyl alcohol, polyacrylamide, polyacrylonitrile, polypropylene, dextran derivatives (sephadex, etc.), agarose gel (sepharose, biogel, etc.), cellulose (paper) Inorganic polymer compounds such as glass, silica gel, silicone, etc., which are introduced with functional groups such as amino groups, aminoalkyl groups, carboxyl groups, acyl groups, and hydroxyl groups. It does not matter.
[0012]
Examples of the shape of the carrier include a microtiter plate, a flat plate such as a disk, a particle such as a bead, a tube such as a test tube and a tube, other fibers, and a membrane, and may be appropriately selected depending on the measurement method. Can do.
Immobilization of the antibody on the solid phase carrier is carried out by a known method such as physical adsorption method, ion binding method, covalent bond method, inclusion method (for example, “Immobilized enzyme” edited by Ichiro Chibata, March 20, 1975, ( (See Kodansha issue).
The solid phase reagent thus obtained may be subjected to a blocking treatment using a normal blocking agent such as BSA, sugar or skim milk in order to suppress nonspecific binding.
[0013]
Also, when preparing a labeled antibody or labeled antigen, as the labels used, radioisotopes (such as ^{32 P, 3 H, 14 C} , 125 I), enzymes (beta-galactosidase, peroxidase, alkaline phosphatase, etc. ), Coenzyme / prosthetic groups (FAD, FMN, ATP, biotin, heme, etc.), fluorescent dyes (fluorescein derivatives, rhodamine derivatives, etc.), metal particles (gold, silver, platinum, etc.), etc. can be used.
The labeling of the antibody or antigen may be performed by a known method suitable for the selected labeling agent (for example, “Second Life Chemistry Laboratory Lecture 5 Immunobiochemical Research Method”, Tokyo Chemical Doujin, (1986) pages 102-112. See). It is also possible to label an antibody or antigen via avidin-biotin or the like.
[0014]
The measurement procedure employed in the immunoassay may be any known procedure such as a competition method or an aggregation method. For the specific operation method of the measurement procedure employed, the following documents can be referred to, for example.
(a) Hiroshi Irie “Continuing Radioimmunoassay” (Kodansha Co., Ltd., issued on May 1, 1979)
(b) Edited by Eiji Ishikawa et al. “Enzyme Immunoassay” (2nd edition) (Medical School, published on December 15, 1982)
(c) Clinical Pathology Special Issue No. 53 “Immunoassay for Clinical Testing -Technology and Applications-” (Clinical Pathology Publications, published in 1983)
(d) “Biotechnology Encyclopedia” (CMC Inc., issued on October 9, 1986)
[0015]
(e) JP-B-6-43998, JP-A-55-15100, JP-A-52-148620, and JP-A-54-91296
(f) `` Methods in ENZYMOLOGY Vol.70 '' (Immunochemical techniques (Part A))
(g) Methods in ENZYMOLOGY Vol.73 (Immunochemical techniques (Part B))
(h) "Methods in ENZYMOLOGY Vol.74" (Immunochemical techniques (Part C))
(i) "Methods in ENZYMOLOGY Vol.84" (Immunochemical techniques (Part D: Selected Immunoassay))
(j) `` Methods in ENZYMOLOGY Vol.92 '' (Immunochemical techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods))
[(F) to (j) are issued by Academic Press]
[0016]
Specifically, among the known measurement procedures, the competitive method will be described as an example. For the immobilized anti-diacetylpolyamine antibody, free diacetylpolyamine in the enzyme-treated sample and a certain concentration of labeling are used. Competing reaction with diacetylpolyamine, separating the solid phase from the liquid phase (BF separation), and then measuring the amount of labeling in either phase enables detection or quantification of free diacetylpolyamine in the sample. .
[0017]
The kit of the present invention contains at least an anti-diacetylpolyamine antibody and an enzyme capable of modifying a primary amine as constituent reagents.
Taking the above competition method as an example and specifically explaining the constituent reagents of the kit for carrying out, the following may be exemplified.
(1) Solid-phased anti-diacetylpolyamine antibody (2) Labeled diacetylpolyamine (3) Enzyme capable of modifying primary amine
In this constituent reagent, when horseradish peroxidase is used as a label, for example, the following constitution can be adopted.
(1) Solid-phased anti-diacetylpolyamine antibody (2) Horseradish peroxidase-labeled diacetylpolyamine (3) Enzyme capable of modifying primary amine In addition to the above reagents, if necessary, a coloring reagent, a reaction stopping reagent, a standard antigen It is possible to appropriately select from reagents such as a reagent and a sample dilution reagent according to the situation, and use it as a constituent reagent for measurement. The enzyme capable of modifying the primary amine can be contained in the sample dilution reagent.
[0019]
【The invention's effect】
The inventor immunized several tens or more mice and tested the specificity of the antiserum against diacetylspermine. When the reactivity with diacetylspermine was 1, the cross-reactivity with N ¹ -acetylspermidine was observed. It was not possible to prepare antisera with a specificity lower than 1/300. This is because diacetylspermine and N ¹ -acetylspermidine are structurally similar compounds and low molecular weight compounds, so there is a limit to obtaining an antibody having specificity only for diacetylspermine by immunochemical techniques. It is thought that there is.
[0020]
Therefore, when such an antibody with low specificity is used, for example, even if the cross-reactivity of the anti-diacetylspermine antibody used in the immunoassay with N ¹ -acetylspermidine is about 1/300 of that of diacetylspermine, it is healthy. In human urine, about 30 times as much N ¹ -acetylspermidine as diacetylspermine is present, so about 10% of the measured value is derived from N ¹ -acetylspermidine, which is preferable as a method for measuring diacetylspermine. Not that.
However, if N ¹ -acetylspermidine is modified with an enzyme, the cross-reactivity with the antibody can be reduced to 1/1000 or less, and measurement with sufficient specificity can be performed without affinity purification of the antibody. It became possible for the first time to provide a system.
[0021]
Similarly, antibodies against diacetyl spermidine N ⁸ - cross-reactivity to acetyl spermidine had 1/20. Since approximately 8 times as much N ⁸ -acetylspermidine as diacetylspermidine is present in the urine of a healthy person, about 40% of the measured value was derived from N ⁸ -acetylspermidine, which was not preferable. By modifying ⁸ -acetylspermidine, the cross-reactivity with antibodies can be reduced to 1/100 or less, and a measurement system with sufficient specificity can be provided without affinity purification of the antibodies. Became possible for the first time.
[0022]
【Example】
EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated concretely, this invention is not limited at all by this.
Example 1
<Method>
(1) Preparation of anti-diacetylspermine antibody and anti-diacetylspermidine antibody BSA introduced with sulfhydryl group by SATA (N-succinimidyl-S-acetylthioacetate) and hydroxylamine, and maleimide group by EMCS (N- (ε-maleimidocaproyloxy) succinimide) Mice were immunized by a conventional method using a conjugate of monoacetylspermine introduced with a acetylene as an immunogen. When conjugated by this method, the primary amino group of monoacetylspermine becomes an acid amide bond, which is structurally close to a diacetyl form. Booster immunization 4-6 times every 2 weeks, cell fusion of the isolated mouse spleen cells and myeloma cells P3X63Ag8.653 (ATCC CRL-1580), and the resulting hybridoma specifically recognizes diacetylspermine Hybridoma 9G9 producing antibody was selected.
Further, instead of monoacetylspermine, N ⁸ -acetylspermidine was used to produce an immunogen, and hybridoma 1D8 producing a monoclonal antibody that recognizes diacetylspermidine was selected.
Monoclonal antibodies were produced from the obtained hybridomas by conventional methods, purified by various chromatographic methods, and subjected to the following experiments.
[0023]
(2) Preparation of horseradish peroxidase-labeled monoacetylspermine and horseradish peroxidase-labeled monoacetylspermidine Conjugate horseradish peroxidase and monoacetylspermine or N ⁸ -acetylspermidine in the same manner as the immunogen preparation method described above. Then, it was subjected to the following experiment.
(3) Preparation of primary amine-modifying enzyme solution Bovine serum amine oxidase is used as an enzyme for modifying contaminating cross-reactive substances, and 1M tricine sodium hydroxide buffer (pH 8. 8) One volume was added to obtain a primary amine-modifying enzyme solution. In addition, the following experiment was carried out using a solution obtained by inactivating this enzyme by adding semicarbazide, an inhibitor of bovine serum amine oxidase to 10 mM, to this solution (inactivated enzyme solution for control experiment). It was used for.
[0024]
(4) Immobilization of antibody and blocking 9G9 antibody solution or 1D8 antibody solution (10 μg / ml: prepared with phosphate buffered saline (pH 7.4)) in a 96-well plate (NUNC Immuno module C8 Maxisorp) at 100 μl / Wells were dispensed and reacted overnight at 4 ° C. The antibody solution is removed by aspiration, washed with phosphate buffered saline (pH 7.4), and a blocking solution (5% sucrose-0.5% skim milk (Difco)) is dispensed in an amount of 300 μl / well. After incubation for a period of time, the blocking solution was removed and dried for the following experiment.
[0025]
(5) Measurement procedure 50 μl / well of the measurement sample is dispensed onto the antibody-immobilized plate prepared in (4) above. Next, the primary amine-modifying enzyme solution prepared in (3) or the inactivated enzyme solution for the control experiment was added to the horseradish peroxidase-labeled monoacetylspermine prepared in (2) at 40 ng / For the 1D8 antibody-immobilized plate, 100 μl / well of a solution obtained by adding horseradish peroxidase-labeled N ⁸ -acetylspermidine prepared in (2) to 400 ng / ml was added, and the mixture was stirred at room temperature. For 2 hours. After the reaction, the wells are washed with phosphate buffered saline (pH 7.4), and TMBZ (3,3 ′, 5,5 ′,-Tetramethyl-benzidine) solution is dispensed at 100 μl / well for color development. Finally, 100 μl / well of 1N sulfuric acid is added to stop the reaction, and the absorbance at 450 nm is measured.
[0026]
<Result>
(1) Reaction curve In accordance with the above measurement procedure, reaction curves were prepared using various polyamine solutions. The results for the 9G9 solid phase plate are shown in FIG. 1, and the results for the 1D8 solid phase plate are shown in FIG. In addition, FIG. 2 and FIG. 4 show the respective reaction curves prepared using the control enzyme inactivating enzyme solution in which bovine serum amine oxidase is inactivated as a control experiment. Abbreviations are as follows.
DASPM: Diacetylspermine AcSpm: spermine AcPUT: acetyl putrescine N1SPD: N ¹ - acetyl spermidine N8SPD: N ⁸ - acetyl spermidine SPM: spermine [0027]
(2) Cross-reactivity From the results shown in FIGS. 1 and 2, Table 1 shows the cross-reactivity and specificity improvement magnification of various polyamines when the reactivity to diacetylspermine is 100%. From this result, it became clear that treatment with bovine serum amine oxidase reduces the cross-reactivity with antibodies of polyamines other than diacetylspermine and can provide a highly specific measurement system for diacetylspermine.
Similarly, from the results of FIGS. 3 to 4, Table 2 shows the cross-reactivity and specificity improvement magnification of various polyamines when the reactivity to diacetylspermidine is 100%. From this result, it became clear that treatment with bovine serum amine oxidase reduces the cross-reactivity with antibodies of polyamines other than diacetylspermidine, and can provide a highly specific measurement system for diacetylspermidine.
The specificity improvement factor was calculated as follows.
Specificity improvement magnification = A / B
A: Reciprocal of cross-reactivity in the absence of semicarbazide B: Reciprocal of cross-reactivity in the presence of semicarbazide
[Table 1]

DASPM: Diacetylspermine
AcSPM: Acetylspermine
N1SPD: N1 acetylspermidine
N8SPD: N8 acetylspermidine
SPM: Spermine [Table 2]

DASPD: Diacetylspermidine
AcPUT: Acetylputrescine
N8SPD: N8 Acetylspermidine [Brief description of the drawings]
FIG. 1 shows reaction curves for various polyamines in an immunoassay performed using an enzyme solution having an activity of bovine serum amine oxidase, a 9G9-immobilized plate and horseradish peroxidase-labeled monoacetylspermine. is there.
FIG. 2 shows reaction curves for various polyamines in an immunoassay performed using an enzyme solution having no activity of bovine serum amine oxidase, a 9G9-immobilized plate and horseradish peroxidase-labeled monoacetylspermine. is there.
FIG. 3 shows reaction curves for various polyamines in an immunoassay performed using an enzyme solution having the activity of bovine serum amine oxidase, a 1D8-immobilized plate, and horseradish peroxidase-labeled N ⁸ -acetylspermidine. Is.
FIG. 4 shows reaction curves for various polyamines in an immunoassay performed using an enzyme solution having no activity of bovine serum amine oxidase, a 1D8-immobilized plate and horseradish peroxidase-labeled N ⁸ -acetylspermidine. Is.

Claims (10)

A method for measuring diacetylpolyamine in a method for measuring diacetylpolyamine in a sample by immunoassay, characterized in that a sample treated with an enzyme capable of modifying a primary amine is used as a sample to be measured. The method according to claim 1, wherein the diacetylpolyamine is diacetylspermine and / or diacetylspermidine. The method according to claim 1, wherein the enzyme capable of modifying a primary amine is an amine oxidase, an aminotransferase and / or a dehydrogenase. The method according to claim 1, wherein the enzyme capable of modifying a primary amine is an amine oxidase derived from animal serum. The method of claim 1, wherein the sample is incubated with animal serum. The kit for performing the method of Claim 1 which contains the enzyme which can modify an anti- diacetylpolyamine antibody and a primary amine at least as a constituent reagent. The kit according to claim 6, wherein the enzyme capable of modifying a primary amine is an amine oxidase, an aminotransferase and / or a dehydrogenase. The kit according to claim 6, wherein the enzyme capable of modifying a primary amine is an amine oxidase derived from animal serum. The kit according to claim 6, wherein the enzyme is supplied in the form of a purified enzyme. The kit according to claim 6, wherein the enzyme is supplied in the form of animal serum.

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