JP5028020B2 - Highly specific monoclonal antibody against N1, N8-diacetylspermidine - Google Patents

Description

  The present invention relates to a monoclonal antibody against diacetylspermidine.

Polyamine is a general term for alkylamines having two or more amino groups, and putrescine (H ₂ N (CH ₂ ) ₄ NH ₂ ), cadaverine (H ₂ N (CH ₂ ) ₅ NH ₂ ) in the human body. , Spermidine (H ₂ N (CH ₂ ) ₄ NH (CH ₂ ) ₃ NH ₂ ) and spermine (H ₂ N (CH ₂ ) ₃ NH (CH ₂ ) ₄ NH (CH ₂ ) ₃ NH ₂ ) It was known that polyamines and their acetylated bodies exist.

In recent years, N ¹ , N ⁸ -diacetylspermidine (hereinafter also referred to as “diacetylspermidine” and “DiAcSpd”) and N ¹ , N ¹² -diacetylspermine (hereinafter also referred to as “DiAcSpm”), although in small quantities Two diacetylpolyamines were found excreted in the urine. In healthy people's urine, these components only account for 1.4% and 0.6% of the total polyamines respectively, but in cancer patients, the rate of increase is significantly higher compared to other polyamine components, In addition, it has been shown to be a substance exhibiting characteristics as a tumor marker (Non-patent Documents 1 and 2).

  DiAcSpd and DiAcSpm were initially quantified by a method combining a fractionation measurement system by HPLC and a detection system by an enzymatic method (Non-patent Document 3), but development of a simple measurement method has been promoted. Regarding the measurement, an ELISA method using a specific antibody has been developed (Non-patent Document 4).

In recent years, a monoclonal antibody against DiAcSpd has been produced by Ukioki et al. (Patent Document 1). These antibodies have a binding inhibitory activity by DiAcSpd for the immune reaction between the antibody and DiAcSpd of about 20 times that of N ⁸ -acetylspermidine (hereinafter also referred to as “N ⁸ -AcSpd”). N ⁸ -AcSpd in healthy human urine is approximately 8 times the amount of DiAcSpd, so about 40% of the measured value is derived from N ⁸ -AcSpd, which is the most suitable method for measuring DiAcSpd. I can not say. Hagioki et al. Improved the effect on the measurement of N ⁸ -AcSpd to about 1/100 by treating the sample with a primary amine-modifying enzyme. However, it is expected that the process of enzyme treatment in the measurement process will hinder the development of a simple measurement system. Therefore, in order to provide a simpler measurement system, a monoclonal antibody specific for DiAcSpd having low cross-reactivity with N ⁸ -AcSpd is required.
Japanese Unexamined Patent Publication No. 2000-74917 Sugimoto, M. et al., J. Cancer Res. Clin. Oncol., 121, 317-319 (1995) Hiramatsu, K. et al., J. Cancer Res. Clin. Oncol., 123, 539-545 (1997) Hiramatsu, K. et al., J. Biochem., 117, 107-112 (1995) Hiramatsu, K. et al., J. Biochem., 124, 231-236 (1998) Shunichiro Kubota: Japanese Clinical Practice, 53, Special Issue, pp. 501-505 (1995)

  An object of the present invention is to provide a specific monoclonal antibody against DiAcSpd and a cell line producing the antibody. Another object of the present invention is to provide a DiAcSpd detection method and a DiAcSpd detection reagent characterized by reacting the antibody with a biological sample as a previous step without requiring an enzyme treatment.

The present inventor has intensively studied to solve the above problems. Then, after immunizing the animal with the antigen to increase the antibody titer, the animal is allowed to stand until the antibody titer decreases, and then the antibody-producing cells are prepared, or the GANP transgenic non-human mammal is immunized with the antigen. As a result, the present inventors succeeded in obtaining a monoclonal antibody whose binding inhibitory activity by DiAcSpd against the immune reaction between DiAcSpd and an antibody is at least 180 times that of N ⁸ -acetylspermidine. It came to be completed.

That is, the present invention is as follows.
(1) A monoclonal antibody against diacetylspermidine, in which a sample diacetylspermidine or a sample N ⁸ -acetylspermidine or acetylputrescine is present so that an immune reaction between diacetylspermidine and the monoclonal antibody is inhibited, The monoclonal antibody that satisfies the measurement condition in which the inhibition reaction of the immune reaction by the sample diacetylspermidine is at least 180 times or more compared to the inhibition activity of the immune reaction by the sample N ⁸ -acetylspermidine or acetylputrescine .
(2) The monoclonal antibody according to (1) above, wherein the concentration of the sample diacetylspermidine in the reaction system is 200 nM or less.
(3) A monoclonal antibody against diacetylspermidine produced by a cell line with the accession number FERM P-20668.
(4) Accession No. (receipt number) is produced from a cell strain under FERM P-20847 (FERM AP- 20847), a monoclonal antibody against diacetyl spermidine.
(5) A cell line that produces the monoclonal antibody according to (1) above.
(6) A cell line producing a monoclonal antibody against diacetylspermidine, whose accession number is FERM P-20668.
(7) A cell line producing a monoclonal antibody against diacetylspermidine, whose deposit number ( reception number ) is FERM P-20847 ( FERM AP-20847 ) .
(8) A reagent for detecting diacetylspermidine, comprising the monoclonal antibody according to any one of (1) to (4) above.
(9) A method for detecting diacetylspermidine, comprising detecting diacetylspermidine by reacting the monoclonal antibody according to any one of (1) to (4) above and a biological sample.

  Here, the biological sample includes urine or serum.

When the monoclonal antibody of (1) above is prepared by immunizing a GANP transgenic non-human mammal with an antigen, the inhibitory activity by diacetylspermidine is the inhibitory activity by N ⁸ -acetylspermidine or acetylputrescine. An antibody that satisfies the above measurement conditions can be produced that is at least 180 times or more, preferably 1500 times or more, more preferably 1700 times or more. At this time, the concentration of the sample diacetylspermidine in the reaction system is, for example, 200 nM or less, preferably 50 nM or less.

Such a monoclonal antibody is, for example, a monoclonal antibody against diacetylspermidine described in (4) above, and is produced by a cell line whose accession number described in (7) is FERM P- 20847.

The present invention also relates to the following.
(10) A method for evaluating the state of a tumor using the result detected by the method according to (9) above as an index.
(11) A tumor diagnostic agent comprising the monoclonal antibody according to any one of (1) to (4) above.
(12) A method for producing a monoclonal antibody, comprising the following steps:
(a) leaving the animal at the stage where the antibody titer of the immunized animal has increased,
(b) Re-immunize when the antibody titer drops to a level of 0.05-1 in the absorbance level, or re-immunize after standing for 2-6 months,
(c) collecting antibody-producing cells from the reimmunized animal;
(d) preparing a fused cell line of the collected antibody-producing cells and myeloma cells,
(e) producing said monoclonal antibody from the resulting cell line.
(13) A method for producing an anti-DiAcSpd antibody, comprising immunizing a GANP transgenic non-human mammal with an antigen and collecting the antibody from the obtained animal.

  The present invention provides a specific antibody against DiAcSpd and a cell line that produces the antibody. In addition, the present invention provides a method for detecting DiAcSpd and a reagent for detecting diacetylspermidine, which comprises reacting the antibody with a biological sample without requiring enzyme treatment as a pretreatment. .

  The monoclonal antibody that reacts with DiAcSpd of the present invention can measure DiAcSpd in a sample such as urine with high sensitivity and specificity, and is preferably useful for diagnosis of cancer.

1. Summary of the Invention The antibody of the present invention comprises the following (i) to (iii):
(i) When the immune reaction between DiAcSpd and anti-DiAcSpd monoclonal antibody is inhibited by 50%, the concentration of DiAcSpd as a competitor is 200 nM or less, preferably 50 nM or less,
(ii) The cross-reactivity with N ⁸ -AcSpd (Ki _DiAcSpd / Ki _"N ⁸ _-AcSpd" ) present in the urine about 8 times more as a similar structure substance of _DiAcSpd is 0.7% or less, preferably 0.06% or less, as well as
(iii) The total cross-reactivity of DiAcSpd-like structural substances present in urine has at least one property selected from the group consisting of 20% or less, preferably 4% or less.

DiAcSpd is a kind of polyamine. Polyamine is a general term for low molecular weight, specifically alkylamines having two or more amino groups, and one of four polyamines existing in the human body. , An acetyl form of spermidine (H ₂ N (CH ₂ ) ₃ NH (CH ₂ ) ₄ NH ₂ ). Recently, DiAcSpd has been found to be present in high concentrations in the urine of cancer patients.

However, biological samples, especially urine, contain a large amount of DiAcSpd-like structural substances typified by N ¹ -AcSpd and N ⁸ -AcSpd, so that DiAcSpd can be quantified or specifically detected. It is necessary to consider interference caused by similar structural materials. That is, to establish a measurement kit for accurately quantifying DiAcSpd by enzyme immunization, it is most important to obtain a monoclonal antibody having a high degree of specificity for DiAcSpd. The present invention has been completed to solve such problems.

  The monoclonal antibody of the present invention can be obtained by adopting a method different from the conventional method for immunizing animals. That is, after immunizing an antigen and leaving the immunized animal at a stage where the antibody titer has risen to an absorbance level of 2 or higher, leave the immunized animal for a while, and eventually the antibody titer is at an absorbance level of 0.05 to 1, preferably 0.05 to 0.5, more preferably Is characterized by further immunization after lowering to a level of 0.05, and then preparing antibody-producing cells.

  The increase level of the antibody titer is 2 or more in the absorbance level.

  “Left” means keeping an animal without immunization, and the period is 2 to 6 months, preferably 4 to 6 months, more preferably 6 months.

  The decreasing level of the antibody titer is 0.05 to 1, preferably 0.05 to 0.5, more preferably 0.05 in terms of absorbance level.

  Alternatively, the monoclonal antibody of the present invention can be obtained by using a GANP transgenic non-human mammal as the animal to be immunized.

  As a method for obtaining the monoclonal antibody of the present invention (hereinafter also referred to as “anti-DiAcSpd antibody”), first, an immunized animal such as a GANP transgenic non-human mammal is immunized with an acetylspermidine derivative of bovine serum albumin. Antibody-producing cells (eg, B cells) are collected from the cells, and the antibody-producing cells and myeloma cells are fused to produce a hybridoma (fusion cell line). And the target monoclonal antibody can be obtained by extract | collecting the antibody produced from this hybridoma.

  The anti-DiAcSpd antibody is a so-called hapten antibody. When such a hapten antibody is produced, the molecular structure design of the hapten-carrier complex has a great influence on the performance of the specific antibody. Antibodies produced using spermine conjugated to BSA with glutaraldehyde as a hapten have been reported to be more reactive to spermine and spermidine in competitive ELISA than to acetylpolyamine. Therefore, the presence of an acylamide bond in the hapten-carrier complex is particularly effective in producing an antibody that reacts preferentially with acetylpolyamines such as DiAcSpd.

Here, if the specificity to DiAcSpd is ensured, a polyclonal antibody against DiAcSpd can be used for quantification or detection of DiAcSpd. However, polyclonal antibodies often contain a lot of antibodies that are highly cross-reactive with other similar structural substances such as acetylpolyamine. In this case, antibodies with high specificity to DiAcSpd are obtained through several purification steps. Must be prepared. In particular, due to the nature of the hapten-carrier complex used for immunization, it is inevitable that antibodies that are highly cross-reactive with N ⁸ -AcSpd and acetylputrescine (hereinafter also referred to as “AcPut”) are easily generated. is there. In order to obtain antibodies with high specificity to DiAcSpd in a situation where antibodies with various specificities are likely to be produced, monoclonal antibodies are prepared in order to obtain stable, easily purifiable antibodies. This is very important.

According to the present invention, a DiAcSpd-specific monoclonal antibody is provided. With this monoclonal antibody, it is possible to select measurement conditions that give a concentration of DiAcSpd that inhibits the immune reaction between DiAcSpd and the antibody by 50% (specimen DiAcSpd) of 200 nM or less, preferably 100 nM or less, more preferably 50 nM or less. become. As a result, DiAcSpd contained in a trace amount in a urine sample containing a large amount of a similar structural substance can be accurately quantified by an enzyme immunoassay while avoiding interference by the similar structural substance.

2. Antigen preparation
Since DiAcSpd is a low molecular weight alkylamine, an antibody specific for DiAcSpd cannot be obtained by directly immunizing mice. Thus, bovine serum albumin, which is a carrier protein, and N ⁸ -AcSpd are acylamide-bonded to produce an immune antigen having many DiAcSpd-like substances as side chains.

In the present invention, an immunizing antigen can be prepared according to a known method (Kunio Fujiwara. Et al., Journal of Immunological Methods, 61, 217-226 (1983)). First, BSA, which is a carrier protein, is reacted with acetylmercaptosuccinic anhydride (hereinafter also referred to as “AMS”) to produce an AMS-BSA complex which is a reaction product. Furthermore, N ⁸ -AcSpd is acylamide-bonded to AMS-BSA via GMBS (N- (4-Maleimidobutyryloxy) succinimide), a bivalent cross-linking reagent, to produce immune antigen N ⁸ -AcSpd -GMB-AMS-BSA To do. However, the production of the antigen is not limited to this.

  As the carrier protein, for example, KLH (Keyhole Limpet Hemocyanin), OVA (Ovalbumin) and the like can be used in addition to BSA. Those skilled in the art can prepare immunizing antigens by known methods.

Further, as described above, in order to produce an antibody specific for acetylpolyamine, it is preferable that an acylamide bond is present in the hapten-carrier complex.

3. Antigen immunization and antibody titer measurement Examples of animals to be immunized include mammals such as mice, rats, and rabbits. Moreover, it can also be used for the animal which immunizes a GANP transgenic non-human mammal (WO2004 / 040971), for example, a GANP transgenic mouse. The total dose of the antigen per animal is 10 to 2000 μg. When immunizing an antigen, an adjuvant and an antigen solution are generally mixed. Examples of the adjuvant include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization is performed mainly by injecting intravenously, subcutaneously, intraperitoneally, intramuscularly, subcutaneously into the footpad, or the like. The immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 5 times, at intervals of several days to several weeks, preferably at intervals of 2 to 3 weeks.

  After the antibody titer has increased by 2 or more at the absorbance level due to immunization, the antibody titer is decreased to 0.05 to 1, preferably 0.05 to 0.5, more preferably 0.05 to the absorbance level, for 2 to 6 months, preferably 4 to 6 Leave the animal for months, more preferably 6 months. In addition, the dilution rate of the serum which shows the said light absorbency level is 27,000 times, for example.

  However, when immunizing a GANP transgenic non-human mammal, it is not necessary to reduce the absorbance level, and the final immunization may be performed according to the immunization interval of a general monoclonal antibody production method.

  The antibody titer can be examined using blood collected from the immunized animal. The collected blood is preferably centrifuged at a low temperature after blood collection, and quickly centrifuged to separate serum. The obtained serum can be serially diluted, and the antibody titer can be measured by enzyme immunoassay (ELISA (enzyme-linked immunosorbent assay) or EIA (enzyme immunoassay)), radioimmunoassay (RIA (radioimmuno assay)), etc. it can. When the antibody titer is measured by ELISA or EIA, the absorbance can be measured with a spectrophotometer. The absorbance level shown in the present invention is a straight line of the sigmoid curves obtained when the antibody titer is measured by ELISA shown in <Experimental Example 1> described below for serum obtained from a collected blood by a known method. The absorbance at the midpoint of the characteristic part. Specifically, for example, the midpoint of the linear part of the absorbance indicated by the S-shaped curve (sigmoid curve) indicated by a x3 dilution series (7 concentrations) from serum diluted 1000 times, for example, The absorbance of the serum diluted 27,000 times. If OD = 1.0, the absorbance level means “OD = 1.0 at a dilution factor of 27,000 times (optical path length 10 mm)”.

  As a result of the measurement, a mouse having a high antibody titer against DiAcSpd is subjected to final immunization. However, the immunity of the antigen and the measurement of the antibody titer are not limited to the above measurement methods.

Thereafter, immunocompetent cells (such as spleen cells) are removed several days after the final immunization day, preferably 3 to 5 days later. In addition, when an antigen is injected subcutaneously into the footpad of an animal, the number of final immunization is one, 7-13 days after immunization, preferably 8-10 days later, immunocompetent cells such as spleen cells or regional lymph Remove the knot. The interval between blood collections is 1 to 4 weeks, preferably 1 to 2 weeks after immunization.

4). Preparation of antibody against DiAcSpd A method for producing an antibody against DiAcSpd will be described below, but is not limited thereto.
(1) Preparation of antibody-producing cells Antibody-producing cells are prepared from spleen cells or the like of the immunized GANP transgenic non-human mammal or the like, or regional lymph nodes. Examples of lymph nodes include inguinal lymph nodes and mediastinal lymph nodes. Although it is not necessary to separate the antibody-producing cells from the collected cell population, it is desirable to separate only antibody-producing cells from the cell population. In preparing antibody-producing cells, it is preferable to remove tissue debris and erythrocytes as much as possible. As a method of removing erythrocytes, a method of using a commercially available erythrocyte removal solution or a method of preparing and using a neutral buffer prepared with ammonium chloride and Tris is preferably employed. The prepared antibody-producing cells may deteriorate if the next operation is not started immediately after preparation, so if it takes time until the next operation after preparation, it should be left on ice. preferable.
(2) Cell fusion Cell fusion is an operation performed to fuse the antibody-producing cells and myeloma cells (myeloma cells) to produce cells (hybridomas) that continue to grow semipermanently while producing antibodies. . As myeloma cells to be fused with antibody-producing cells, generally available cell lines of animals such as mice can be used. The cell line to be used is preferably one that has the property that it cannot survive in a HAT selective medium (medium containing hypoxanthine, thymidine, and aminopterin) and can survive only in a state fused with antibody-producing cells. Examples of myeloma cells include P3X63-Ag.8.U1 (P3U1) and P3 / NS I / 1-Ag4-1 (NS I).

Cell fusion is a commercially available medium such as DMEM or RPMI1640 medium that does not contain fetal calf serum (FCS), etc., and 1 × 10 ⁶ to 1 × 10 ⁷ cells / mL of spleen cells and / or lymph node cells 1 × 10 ⁵ to 1 × 10 ⁶ cells / mL myeloma cells are mixed (the ratio of spleen cells and / or lymph node cells to myeloma cells is preferably 5: 1) in the presence of a cell fusion promoter. Perform fusion. As the cell fusion promoter, polyethylene glycol having an average molecular weight of 200 to 20,000 daltons can be used.

  Moreover, it can also fuse | melt using the commercially available cell fusion apparatus using electrical stimulation (for example, electroporation). Furthermore, cells can be fused using Sendai virus. A person skilled in the art can fuse the antibody-producing cells and myeloma cells using a known cell fusion method.

After the fusion, for example, dilute the cells with HAT medium made with 10-20% (preferably 20%) FCS-containing RPMI1640 medium, and then inoculate 0.5- 3 x 10 ⁵ cells into each well of a 96-well culture plate. Incubate in a CO ₂ incubator.

(3) Establishment of hybridoma Next, a hybridoma producing the target antibody is selected from the cells after cell fusion treatment. Ten to 14 days after cell fusion, cells selected in HAT medium as described above form colonies. The culture supernatant of each well of the colony-positive 96-well culture plate is collected and the antibody titer against DiAcSpd is confirmed. As a confirmation method, enzyme immunoassay (ELISA), radioimmunoassay (RIA) or the like is performed. At this time, since the antibody produced from the cell includes an antibody against BSA which is a carrier protein, BSA antibody positive wells having a high antibody titer against BSA can be excluded by measuring the antibody titer against BSA. After confirming antibody production positive wells against DiAcSpd, transfer the cells to 24-well or 12-well culture plates.

  Here, the medium is preferably replaced with an HT medium (a medium containing hypoxanthine and thymidine) excluding aminopterin. The reason for this is that aminopterin is a substance that inhibits cell DNA replication, so if aminopterin remains in the cell even if aminopterin in the medium is removed, the cell does not exist in the absence of hypoxanthine and thymidine. This is because there is no DNA replication inside. After culturing in HT medium for a while, the antibody titer in the culture supernatant is confirmed again. Since the hybridoma is unstable because it is a fused cell, and there is a high possibility that antibody production will soon disappear, it is preferable to confirm the antibody titer for the second time. As described above, since it is necessary to obtain a hybridoma having high specificity for DiAcSpd in the present invention, what is important here is cross-reaction with other DiAcSpd-like substances at the culture supernatant level. It is to confirm sex by ELISA or RIA.

In the present invention, examples of the DiAcSpd-like substance include N ⁸ -AcSpd, AcPut, putrescine, cadaverine, spermidine, spermine, and DiAcSpm.

The cells in the final selected well are cloned to make a single cell. For cloning, for example, after appropriately diluting the cell suspension with RPMI1640 medium containing 10 to 20% FCS (preferably 20%), seed the cells so that 0.3 to 2 cells are added to each well of a 96-well culture plate. . In order to increase the probability that the number of cells in each well of the 96-well culture plate is one cell, it is preferable to seed one cell in each well. After cell seeding, the culture supernatant of the colony-positive well is collected 7 to 10 days later. At this time, it is preferable to confirm that it is a single colony after 3 to 5 days. The collected culture supernatant is checked for antibody titer. Again, clones are selected that have high specificity for DiAcSpd and low cross-reactivity to DiAcSpd analogs. Further, the number of cells in the selected well is increased to some extent to establish a hybridoma strain. Cloning may be performed several times as necessary.

(4) Preparation of monoclonal antibody DiAcSpd-specific monoclonal antibody is purified from the established hybridoma strain and prepared by the following method. That is, a method of preparing an antibody from a culture supernatant cultured in a medium with reduced serum concentration, a method of preparing an antibody from a culture supernatant cultured in a commercially available serum-free medium, or injecting a hybridoma into the abdominal cavity of an animal. There is a method of collecting ascites and preparing antibodies from the ascites. The culture supernatant is collected from cells prepared at 0.1-4 × 10 ⁵ cells / mL and cultured for 1-2 weeks. In the case of ascites, 0.1 to 1 × 10 ⁷ hybridomas are administered into the abdominal cavity of a myeloma cell-derived mammal and the same type of animal, and the hybridomas are proliferated in large quantities. And ascitic fluid is collected after 1-2 weeks.

Examples of the culture method include a method using a culture flask, a method using a spinner flask, a method using a shaker flask, and a method using a bioreactor. Antibody purification methods include protein G affinity column or protein A affinity column purification method, DiAcSpd affinity column purification method, ammonium sulfate salting-out fraction purification method using gel filtration chromatography, ion exchange chromatography purification method There are methods and the like, and these known methods can be appropriately selected or purified by combining them. When purifying mouse IgG ₁ using a protein A affinity column, it is effective to use a buffer or the like in which binding conditions are optimized, and those skilled in the art appropriately select the optimal conditions, Can be purified.

  The cell line (hybridoma) producing the monoclonal antibody of the present invention is called “Anti-DiAcSpd MAb 19C10”, and is dated September 27, 2005, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (〒305-8566 Deposited at Chuo 6th 1-1-1 Higashi Tsukuba City, Ibaraki Prefecture. The deposit number (described on the deposit certificate) is “FERM P-20668”. “FERM P-20668” is a clone established as “19C10” in Example 1.

A cell line (hybridoma) that produces the monoclonal antibody of the present invention is called “Anti DiAcSpd MAb Clone: 20D7”, and on March 17, 2006, the National Institute of Advanced Industrial Science and Technology (AIST) Deposited with 6th Central 1-1-1 Tsukuba City, Ibaraki 305-8566. Accession number indicating the acceptance number (described in the contract document) is a "FERM P -20847". “FERM P- 20847” is a cell line prepared using the spleen of an immunized GANP transgenic mouse, and is a clone established as “20D7” in Example 2 described later.

(5) Properties of Monoclonal Antibody The monoclonal antibody of the present invention is an antigen competitive inhibition assay system, that is, a reaction in which a competitive substance is present so that an immune reaction between diacetylspermidine and a specific monoclonal antibody against the diacetylspermidine is inhibited. The system has at least one property selected from the group consisting of the following (i) to (iii). The competitive inhibition assay system is preferably a system using solid-phased diacetylspermidine.

Examples of competing substances in the antigen competitive inhibition assay system include diacetylspermidine, N ⁸ -AcSpd, and AcPut. One of these may be used as the competitive substance, or a mixture of a plurality of kinds may be used. When mixing, for example, each concentration may be set based on the abundance ratio in human urine. In the specification, these substances added to the reaction system as competing substances may be indicated with the word “specimen”.

(i) The inhibitory activity of the immune reaction by DiAcSpd in the reaction system is at least 180 times or more, preferably 1500 times or more compared to the inhibitory activity of the immune reaction by N ⁸ -AcSpd and AcPut in the reaction system, More preferably, it has an inhibitory activity of 1700 times or more.

  Here, a system is considered in which DiAcSpd is immobilized on the bottom of the plate and immobilized, a monoclonal antibody against DiAcSpd is added thereto, and DiAcSpd reacts with the antibody. In a reaction system in which no competing substance exists, the monoclonal antibody reacts with the immobilized DiAcSpd. In this reaction system, free DiAcSpd is present as a specimen. This free DiAcSpd reacts with the antibody by competing with the immobilized DiAcSpd. When the binding between the solid-phase DiAcSpd and the antibody is inhibited by 50%, that is, when the sample DiAcSpd is added, the amount of binding between the monoclonal antibody and the solid-phase DiAcSpd is the above when there is no free sample DiAcSpd. The reciprocal of the amount of the sample DiAcSpd or the reciprocal of the concentration (50% inhibitory concentration, IC50) when the amount is reduced to 50% compared to the amount of binding between the monoclonal antibody and the solid phase DiAcSpd is “inhibitory activity” or “ It can be defined as “binding inhibition activity”.

For each of the free specimens DiAcSpd and N ⁸ -AcSpd and AcPut, the above inhibitory activity was measured, and when using free DiAcSpd, N ⁸ -AcSpd or AcPut, which is largely released in urine, was used. An antibody that satisfies the reaction condition such that it is 180 times or more, preferably 1500 times or more, more preferably 1700 times or more compared to the inhibitory activity of the present invention can be selected as the antibody of the present invention. In other words, in the above reaction system, the antibody of the present invention has a 50% inhibitory concentration of the sample DiAcSpd of 1/180 or less compared to the 50% inhibitory concentration of the sample N ⁸ -AcSpd or AcPut, It can also be said that the antibody satisfies the reaction condition such that it is preferably 1/500 or less, more preferably 1/700 or less. This means that the monoclonal antibody of the present invention has low cross-reactivity with N ⁸ -AcSpd, AcPut and the like, and is specific for DiAcSpd.

  The concentration of DiAcSpd in the reaction system when the above conditions are satisfied is 200 nM or less, preferably 100 nM or less, more preferably 50 nM or less. Alternatively, the concentration of DiAcSpd is preferably 10 to 200 nM or less, preferably 10 to 100 nM.

(ii) The cross-reactivity with N ⁸ -AcSpd present in the urine as a similar structural substance of DiAcSpd is 0.7% or less, preferably 0.06% or less.

  (iii) The total cross-reactivity with respect to the DiAcSpd-like structural substance present in urine is 20% or less, preferably 18% or less, more preferably 12% or less, and most preferably 4% or less.

  In the present invention, “cross-reactivity” means the reactivity of an anti-DiAcSpd antibody to a substance other than DiAcSpd.

In (ii) and (iii), the cross-reactivity of the anti-DiAcSpd antibody to a DiAcSpd-like structural substance can be calculated as follows.
(a) The absorbance in the competitive ELISA results graph for each polyamine, with the vertical axis representing absorbance and the horizontal axis representing competitor concentration (analyte concentration), is changed to absorbance (%). Specifically, the absorbance / competitor graph is re-created with the value (%) calculated by “(each absorbance−average absorbance of blank wells) ÷ (average absorbance of assay control−average absorbance of blank wells) × 100”. To do.
(b) In the sigmoid curve (sigmoid curve) obtained in each polyamine graph of (a), select the absorbance in the linear region containing the value of 50% inhibitory activity, and obtain a logarithmic approximation curve that passes through those absorbances. .
(c) Based on the logarithmic approximation curve obtained in (b), Y = 50, that is, the analyte concentration at which the inhibitory activity is −50% (50%) (Ki: the concentration of competitor that inhibits the reaction by 50% ( μM)). The analyte concentration at this time is derived by the EXP function that returns the inverse of the LAN function.
(d) Cross reactivity (%) is calculated using Ki calculated in (c). At this time, the value is calculated assuming that the crossing property of DiAcSpd is 100%. Cross reactivity is calculated with other polyamines against DiAcSpd. Specifically, “Ki _DiAcSpd / Ki” S ”(%) = Ki _DiAcSpd ÷“ each competitor Ki × 100 (%) ”(S is a polyamine).
(e) In some cases, the sum of cross-reactivity is finally determined based on the urinary ratio of each polyamine to DiAcSpd. Specifically, the sum of the cross-reactivity is shown as the sum of values obtained by multiplying the urinary abundance ratio for DiAcSpd shown in Table 1 or Table 2 by cross-reactivity, and it is expressed as “Ki _DiAcSpd / Ki” S ”× DiAcSpd. It is the sum of the values derived from the formula “ratio of polyamines in urine (%)”.

5. Tumor detection method
DiAcSpd can be used as a cancer clinical marker (tumor marker), so the antibody of the present invention is reacted with a biological sample, and DiAcSpd is measured in the biological sample, and the tumor is detected using the measurement result as an index. can do. DiAcSpd can be measured by any method (for example, ELISA, EIA) known as a hapten immunoassay that is generally performed, and is not particularly limited.

  The tumor is not particularly limited, but for example, brain tumor, esophageal cancer, pharyngeal cancer, tongue cancer, lung cancer, gastric cancer, small intestine or duodenal cancer, colon cancer, urinary tract malignant tumor (eg prostate cancer, renal cancer, bladder) Cancer, testicular cancer), liver cancer, prostate cancer, breast cancer, uterine cancer, ovarian cancer, thyroid cancer, gallbladder cancer, biliary tract cancer, sarcoma (eg, osteosarcoma, muscle species, etc.) and at least one selected from the group consisting of melanoma Is mentioned. The type of cancer to be detected may be one, or two or more types may be combined.

  A biological sample is collected from a cancer patient, a patient suspected of having cancer, or a medical examination recipient, and a DiAcSpd measurement sample is prepared. Examples of the biological sample include blood, urine, tissue and the like. It is preferable that blood is collected and centrifuged to separate serum, and the obtained serum is used. Moreover, urine is more preferable in terms of easy handling and less burden on the patient.

  Next, the measurement sample is reacted with the antibody of the present invention. DiAcSpd can be detected by a commonly used ELISA. Here, for convenience of explanation, a mouse-derived antibody is used as the antibody of the present invention.

To measure by ELISA, first, an antigen (DiAcSpd) is coated on a microplate. On the other hand, after DiAcSpd in a biological sample (specimen) or standard solution is reacted with an anti-DiAcSpd specific antibody in advance, this reaction solution is added to a well on the plate. The unreacted antibody binds to DiAcSpd on the plate. Then, after washing the plate, an HRP-labeled anti-mouse IgG antibody, which is a secondary antibody, is added to the plate and reacted with the antibody bound to DiAcSpd on the plate. Finally, DiAcSpd in the biological sample is quantified by a color reaction catalyzed by HRP. In addition to HRP (peroxidase), alkaline phosphatase, malate dehydrase, α-glucosidase, α-galactosidase, and the like can be used as the labeling enzyme used in the secondary antibody. In a competitive assay, as the amount of DiAcSpd in a biological sample increases, values such as color development amount, fluorescence amount, and luminescence amount are measured to be smaller.

6). Tumor Evaluation Method In the present invention, the state of a tumor can be evaluated using the detection result obtained by the detection method shown in 5 above as an index. If the detection result exceeds the predetermined reference value, DiAcSpd is positive, and if the detection result is less than the predetermined reference value, DiAcSpd is negative.If it is positive, it is determined that there is a possibility of developing cancer, and the state of the tumor is determined. Can be evaluated. The predetermined reference value is appropriately set depending on the type of tumor.

The state of a tumor means the presence or absence or progression of a tumor, and includes the presence or absence of onset of cancer, the progression of cancer, the malignancy of cancer, the presence or absence of cancer metastasis, and the presence or absence of cancer recurrence. In the above evaluation, one of these tumor states may be selected, or a plurality may be selected in appropriate combination. To evaluate the presence or absence of cancer, it is determined whether or not the patient is afflicted with cancer. Cancer malignancy is an indicator of how far the cancer has progressed, and it is evaluated by classifying the stage (Stage), or by classifying so-called early cancer and advanced cancer It is also possible. Cancer metastasis is evaluated by whether or not a neoplasm has appeared at a site distant from the location of the primary lesion. Recurrence is assessed by whether the cancer reappears after an intermittent period or remission.

7). Kits and Reagents Containing the Antibody of the Present Invention In the present invention, a monoclonal antibody against DiAcSpd can be used as a detection kit or reagent for DiAcSpd. The kit or reagent of the present invention can be used for the detection of the tumor.

  Conventionally, when measuring polyamines as a general biochemical test, several types of polyamines in urine are measured together as similar structures, and there is no examination of the relationship between each similar structure and various pathologies. It was equal. To date, several methods have been established for measuring the amount of polyamine in the urine by distinguishing the structures among polyamine measurement methods. In particular, DiAcSpd, a type of polyamine, does not increase in patients with benign diseases and malignant diseases. It has been confirmed that the value is high only in patients, and that the value is high upon recurrence after treatment. This means that if a simple and accurate method for measuring diacetylpolyamines such as DiAcSpd can be developed, DiAcSpd can be used as a novel tumor marker in the clinical practice of cancer treatment, and the examination of tumors using a DiAcSpd detection reagent is significant. It shows that demand is expected.

  The present invention provides a measurement system for DiAcSpd by a competitive ELISA method using an anti-DiAcSpd antibody as a DiAcSpd detection system.

In this measurement system, N ⁸ -acetylspermine can be used as a solid-phased antigen and a DiAcSpd-like structure (N ⁸ -AcSpd-EMC-AMS-BSA) can be used by an acylamide bond. This solid-phased antigen can be prepared according to the method of immune antigen. First, BSA, which is a carrier protein, is reacted with AMS to produce an AMS-BSA complex, which is a reaction product. Furthermore, N ⁸ -AcSpd is acylamide-bonded to AMS-BSA via EMCS (N- (6-Maleimidocaproyloxy) succinimide), a bivalent cross-linking reagent, and immobilized antigen N ⁸ -AcSpd-EMC-AMS-BSA Is made. However, the production of the antigen is not limited to this.

In the present invention, DiAcSpd or N ⁸ -AcSpd-EMC-AMS-BSA may be used as the antigen used at the time of animal immunization, the screening antigen for hybridoma, and the plate-immobilized antigen for detecting DiAcSpd. Good. This is because N ⁸ -AcSpd and BSA are structurally similar to DiAcSpd when bound using a bivalent cross-linking reagent.

  Although the kit or reagent of the present invention can specifically measure DiAcSpd, it is preferably required to be reproducible for use in clinical practice of cancer. Therefore, the present invention provides preferable DiAcSpd measurement conditions when using the kit of the present invention as a tumor diagnostic agent.

  When the kit or reagent of the present invention is used as a tumor diagnostic agent, the average urinary DiAcSpd excretion in healthy subjects is 301 nmol / g creatinine (Hiramatsu, K. et al., J. Biochem., 117, 107-112 ( 1995)), the standard region contained in the kit is preferably set to 600 nM to 9.375 nM. The concentration of the immobilized antigen is 0.01 to 0.05 μg / mL, preferably 0.03 μg / mL. As a result, sufficient sensitivity and measurement accuracy can be achieved for measuring urinary DiAcSpd.

  Measurement accuracy is an index that shows how much each measured value varies when a single assay is performed with multiple test tubes or wells divided into the same sample. Is expressed as a coefficient of variation (CV), that is, as a ratio (%) of the standard deviation to the average value of the measurement. In the present invention, this coefficient of variation (CV) is called reproducibility. The reproducibility is 15% or less, preferably 10% or less, and more preferably 5% or less.

  Although the preferable performance of the kit or reagent of the present invention is not limited, the minimum detection range is 18.75 nM, and the analyte measurement detection sensitivity is 37.5 nM (18.75 × 2). The simultaneous reproducibility is 10% or less, preferably around 5%, and the daily reproducibility is 10% or less, preferably around 5%. Any reproducibility is preferably CV = 10% or less.

  When the monoclonal antibody of the present invention is used as a diagnostic agent, the monoclonal antibody can be combined with another solvent or solute to form a composition. For example, distilled water, pH buffer reagent, salt, protein, surfactant and the like can be combined. Monoclonal antibodies can be used after enzyme labeling. As a labeling enzyme, in addition to HRP (horseradish peroxidase), alkaline phosphatase, malate dehydrase, α-glucosidase, α-galactosidase, colloidal gold, and the like can be used.

In the kit of the present invention, in addition to the antibody of the present invention, the above solvent, solute, enzyme labeling reagent, antigen-immobilized microplate, DiAcSpd standard (standard), antibody dilution solution, OPD (orthophenylenediamine) tablet , Substrate solution, reaction stop solution, concentrated washing solution, instructions and the like. In addition, a reaction medium such as a buffer solution that gives optimum conditions for the reaction, a buffer solution that is useful for stabilizing the reaction product, and a stabilizer for the reaction material may be included in the kit of the present invention.

Hereinafter, the present invention will be described more specifically with reference to examples and experimental examples. However, the present invention is not limited to these examples and experimental examples.

Preparation of monoclonal antibody specific for diacetylspermidine (hereinafter referred to as DiAcSpd) (1) Preparation of antigen An immune antigen was prepared according to a known method as follows (Kunio Fujiwara. Et al., Journal of Immunological Methods, 61, 217). -226 (1983)). DiAcSpd is a low molecular weight alkylamine, and an antibody specific for DiAcSpd cannot be obtained even if the mouse is directly immunized. Therefore, bovine serum albumin (hereinafter referred to as “BSA”), which is a carrier protein, and N ⁸ -AcSpd are acylamide-linked, and an immune antigen N ⁸ -AcSpd -GMB-AMS-BSA having many DiAcSpd-like structures as side chains is obtained. It produced as follows.

First, BSA serving as a carrier protein was reacted with AMS and purified with a Sephadex G-25 gel filtration column to prepare AMS-BSA. On the other hand, GMB-N ⁸ -AcSpd was prepared in order to bind N ⁸ -AcSpd to AMS-BSA via GMBS, which is a bivalent crosslinking reagent. Furthermore, AMS-BSA and GMB-N ⁸ -AcSpd were reacted to prepare N ⁸ -AcSpd -GMB-AMS-BSA, which is an immunizing antigen.
(2) Immunization to mice Immunization to mice was performed by two methods. An outline of the immunization method is shown in FIG. The first method was performed by the following method. That is, immunity 1: Immunized antigen prepared at 2 mg / mL was mixed with FCA in an equal amount, and 100 μL of the emulsion formed was administered subcutaneously to the back of the mouse, and then 1 mg / mL of immunizing antigen was administered every 2 weeks. 100 μL of an equal amount mixed with FIA to form an emulsion was subcutaneously administered to the back of the mouse. Antigen titer was administered 3 times in total, and antibody titer was confirmed by ELISA. For those with high antibody titer, 100 μL of 1 mg / mL of immunizing antigen was finally administered intraperitoneally to mice, and 3 days later for cell fusion The spleen was removed. The second method was performed as follows. In other words, immunization 2: the same procedure up to the third administration of antigen in the first method, but the mice were left until the antibody titer decreased in the latter half of the year, and then an emulsion of 1 mg / mL of immunizing antigen and IFA was added to the back of the mice. Two weeks later, 100 μL of 1 mg / mL immunoantigen was finally administered intraperitoneally to the mice, and 3 days later, the spleen was removed for cell fusion.
(3) Preparation and cell fusion of spleen cells The extracted spleen was ground to prepare spleen cells containing anti-DiAcSpd antibody-producing cells. In both immunization methods, approximately 1 × 10 ⁸ spleen cells per mouse could be prepared. On the other hand, P3U1, which is a myeloma cell, was cultured, and P3U1 having a viable cell ratio of 95% or more was prepared on the day of cell fusion. These spleen cells and P3U1 were mixed at 5: 1, and cell fusion was performed with polyethylene glycol having a molecular weight of 1,450 at a concentration of 50%. After the fusion, the cells were washed with a medium and suspended in HAT medium, and cells were seeded at 1 × 10 ⁵ cells / well in each well of a 96-well culture plate.
(4) Screening of antibody production positive wells After cell fusion, the culture supernatant on the 12th day was collected, and antibody production positive wells were screened by the method of Experimental Example 1 described later. In the immunization 1 method, 115 out of 3872 wells were positive for DiAcSpd. In the immunization 2 method, 114 out of 2584 wells were positive for DiAcSpd. Cells in these selected wells were transferred to a 24-well plate, cultured for 1-2 days, and screened again using the method of Experimental Example 1. In the method, 90 wells were positive for DiAcSpd.
(5) Cross-reactivity test of antibody-producing positive wells Regarding the above-mentioned 165 clones of DiAcSpd positive wells, cross-reactivity to N ⁸ -AcSpd, which is present in urine and is considered to be relatively easy to cross-react in previous studies. The test was performed by the method of Experimental Example 2 described later. As a result, there were two wells in which the 50% inhibitory concentration of N ⁸ -AcSpd was about 180 times or more of the 50% inhibitory concentration of DiAcSpd, and the other was 0-100 times.
(6) Cloning
Two wells with high specificity for DiAcSpd were cloned by limiting dilution. That is, cells were prepared at 2.5 cells / mL with RPMI medium containing 20% FCS, and 200 μL was added to each well of two 96-well culture plates. Ten days later, the antibody titer against DiAcSpd in the culture supernatant was measured by the method of Experimental Example 1 to confirm that it was positive, and clones derived from each well were obtained. Then further cultured, AcSpm in Experimental Example 2 ^{method, N 1 -AcSpd, N 8 -AcSpd} , DiAcSpd, was subjected to cross-reactivity test for AcPut, N ⁸ both 2 clones inhibitory activity of DiAcSpd is -AcSpd or AcPut This antibody was about 180 times or more of the inhibitory activity and sufficiently provided with the target specificity in the present invention. The respective established clones were designated as “19C10” and “15C5” (FIGS. 2 and 3).

In FIG. 2, the 50% inhibitory concentration of DiAcSpd was about 0.037 μM, and the 50% inhibitory concentration of N ⁸ -AcSpd was about 8.08 μM. As a result of comparing each inhibitory activity, the difference was about 218 times. .

In FIG. 3, the 50% inhibitory concentration of DiAcSpd is about 0.026 μM, and the 50% inhibitory concentration of N ⁸ -AcSpd is about 5.169 μM. As a result of comparing each inhibitory activity, the difference is about 198 times. there were.
(7) Basic Data of Established Clones Under the conditions of Experimental Example 2, the antibodies produced by the established clones were examined for cross-reactivity with DiAcSpd-like structural substances in urine samples. That is, the 50% inhibitory concentrations of DiAcSpd, N ⁸ -AcSpd, AcPut, N ¹ -AcSpd, and AcSpm were examined, and the cross-reactivity of other similar structural substances to DiAcSpd in urine was calculated. As a result, in the established clone 19C10, the cross-reactivity to N ⁸ -AcSpd is 3.668%, other AcPut is 13.655%, N ¹ -AcSpd is 0.078%, AcSpm is 0.000%, and the total cross-reactivity is 17.401% there were. On the other hand, in the established clone 15C5, the cross-reactivity to N ⁸ -AcSpd in urine is 4.029%, other AcPut is 7.608%, N ¹ -AcSpd is 0.113%, AcSpm is 0.000%, and the total cross-reactivity is 11.750. % (Table 1).

    Table 1 Cross-reactivity of urinary DiAcSpd-like structural substances to DiAcSpd

From the above, the monoclonal antibody of the present invention has extremely high specificity for DiAcSpd and extremely low cross-reactivity, so that it is extremely useful as compared with conventionally known anti-DiAcSpd antibodies.

<Experimental example 1>
Antibody screening methods
To each well of a 96-well microtiter plate, 50 μL each of N ⁸ -AcSpd -HMC-peptide prepared to 0.1 μg / mL with PBS (pH 7.0) was added as a solid-phased antigen and left at 25 ° C. for 1 hour. . Next, after washing 3 times with PBS (pH 7.0) (PBST) containing 0.05% Tween20, 200 μL of PBST (blocking solution) containing 0.5% gelatin was added to each well and allowed to stand at 25 ° C. for 1 hour. . After washing, 50 μL of the culture supernatant was added to each well as the stock solution and allowed to stand at 25 ° C. for 40 minutes. Next, after washing 3 times with PBST, 50 μL each of HRP-labeled anti-mouse IgG antibody (ZYMED) diluted 2500-fold was added to each well and allowed to stand at 25 ° C. for 40 minutes. Next, after washing 3 times with PBST, 100 μL of o-phenylenediamine solution prepared to 0.5 mg / mL with 0.1 M citrate phosphate buffer (pH 5.0) containing 0.02% hydrogen peroxide is added to each well. After standing at 25 ° C. for 10 minutes, 100 μL of 1M sulfuric acid solution was added to each well to stop the color reaction. The absorbance at 490 nm was then measured with an ELISA reader.

<Experimental example 2>
Cross-reactivity test The antibody titer in the diluted culture supernatant was measured by the method of Experimental Example 1. As a result, the dilution factor at which the absorbance at 490 nm was 1 was set (FIGS. 4 and 5). 70 μL of culture supernatant diluted at the dilution rate set above on the dilution plate, DiAcSpd solution prepared stepwise for each concentration, or similar structural substances (N ⁸ -AcSpd, AcSpm, N ¹ -AcSpd, DiAcSpm, etc.) 70 μL prepared stepwise to each concentration was added to each well, and the mixture was allowed to stand at 25 ° C. for 1 hour for pre-reaction. Next, 50 μL of the reaction solution pre-reacted in each well of the plate subjected to the blocking treatment in the same manner as in Experimental Example 1 was added to each well and allowed to stand at 25 ° C. for 1 hour. Next, after washing 3 times with PBST, 50 μL each of HRP-labeled anti-mouse IgG antibody (ZYMED) diluted 2500-fold was added to each well and allowed to stand at 25 ° C. for 1 hour. Next, after washing 3 times with PBST, 100 μL of o-phenylenediamine solution prepared to 0.5 mg / mL with 0.1 M citrate phosphate buffer (pH 5.0) containing 0.02% hydrogen peroxide is added to each well. After standing at 25 ° C. for 10 minutes, 100 μL of 1M sulfuric acid solution was added to each well to stop the color reaction. The absorbance at 490 nm was then measured with an ELISA reader.

Preparation of Monoclonal Antibody Specific to Diacetylspermidine (hereinafter DiAcSpd) (1) Preparation of Antigen An immune antigen N ⁸ -AcSpd-GMB-AMS-BSA was prepared according to the same method as in Example 1 (1).
(2) Immunization to GANP transgenic mice Immunization to mice was performed by the following method. 100 μL of the immunizing antigen prepared at 2 mg / mL mixed with FCA and the emulsion formed is administered subcutaneously to the back of the mouse in 100 μL aliquots, and then 1 mg / mL of immunizing antigen and FIA are mixed at equal intervals every 2 weeks. Then, 100 μL of the emulsion formed was administered subcutaneously to the back of the mouse. The antigen was administered three times in total, and the antibody titer was confirmed by ELISA. For those with high antibody titers, 100 μL of 1 mg / mL of immunizing antigen was finally administered intraperitoneally to the mice, and 3 days later The spleen was removed for fusion.
(3) Preparation and cell fusion of spleen cells The extracted spleen was ground to prepare spleen cells containing anti-DiAcSpd antibody-producing cells. Approximately 2 × 10 ⁸ spleen cells could be prepared per mouse. Preparation of myeloma cells P3U1 and cell fusion between spleen cells and P3U1 were performed in the same manner as described in Example 1 (3).
(4) Screening of antibody production positive wells After cell fusion, the culture supernatant on the 12th day was collected, and antibody production positive wells were screened by the method of Experimental Example 1. 110 out of 2208 wells were positive for DiAcSpd. Cells in these selected wells were transferred to a 24-well plate, cultured for 1-2 days, and screened again by the method of Experimental Example 1. Finally, 97 wells were positive for DiAcSpd.
(5) Cross-reactivity test of antibody production positive well As with Example 1, the cross-reactivity test with respect to N ⁸ -AcSpd was performed by the method of Experimental Example 2 for the above-mentioned DiAcSpd positive well 97 clone. As a result, there were 4 wells in which the 50% inhibitory concentration of N ⁸ -AcSpd was about 1700 times or more of the 50% inhibitory concentration of DiAcSpd, and the others were 100-1500 times.
(6) Cloning
Four wells having high specificity for DiAcSpd were cloned by limiting dilution in the same manner as in Example 1 (6). That is, after culturing at 2.5 cells / mL, the antibody titer against DiAcSpd in the culture supernatant was measured by the method of Experimental Example 1 to confirm that it was positive, and clones derived from each well were obtained. Thereafter, the cells were further cultured, and cross-reactivity tests for AcSpm, N ¹ -AcSpd, N ⁸ -AcSpd, DiAcSpd, and AcPut were performed by the method of Experimental Example 2. In result, 3 clones of about 1500 times more inhibitory activity of DiAcSpd of inhibitory activity of AcPut, is approximately 1700-fold more inhibitory activity of N ⁸ -AcSpd, antibodies replete with specificity of interest in the present invention there were. The three established clones were designated as “5D1”, “15D4”, and “20D7”, respectively.
(7) Purification of antibody The target monoclonal antibody was purified from the above three clones by the following method. First, established clones were suspended in a commercially available serum-free medium (Hybridoma SFM: Invitrogen) and prepared to 4 × 10 ⁵ cells / mL. 50 mL of the cell suspension was placed in a T225 flask and cultured in a 37 ° C., 5.0% CO ₂ environment for about one week. After culturing, the culture supernatant was collected. The collected culture supernatant was applied to a protein G column and eluted with glycine buffer (pH 3.0) to purify the monoclonal antibody. Thereafter, a cross-reactivity test for AcSpm, N ¹ -AcSpd, N ⁸ -AcSpd, DiAcSpd, AcPut, and DiAcSpm was performed again by the method of Experimental Example 2. As a result, the same results as in the culture supernatant were obtained (FIGS. 6, 7 and 8).

In the purified antibody derived from the 5D1 clone, the 50% inhibitory concentration of DiAcSpd was about 0.011 μM, and the 50% inhibitory concentration of N ⁸ -AcSpd was about 19.29 μM. As a result of comparing each inhibitory activity, the difference was about 1756. Doubled (Figure 6).

In the purified antibody derived from the 15D4 clone, the 50% inhibitory concentration of DiAcSpd was about 0.058 μM, and the 50% inhibitory concentration of N ⁸ -AcSpd was about 135.568 μM. It was about 2340 times (FIG. 7).

Furthermore, in the purified antibody derived from the 20D7 clone, the 50% inhibitory concentration of DiAcSpd is about 0.025 μM, and the 50% inhibitory concentration of N ⁸ -AcSpd is about 75.561 μM. As a result of comparing each inhibitory activity, the difference is about It was 3000 times (FIG. 8).
(8) Basic data of established clones In the same manner as in Example 1 (7), urinary cross-reactivity of antibodies produced by established clones was examined. As a result, in the established clone 5D1, the cross-reactivity to N ⁸ -AcSpd in the urine is 0.474%, the other AcPut is 2.409%, N ¹ -AcSpd is 0.173%, and AcSpm is 0.000%. It was 3.057%. In addition, in the established clone 15D4, the cross-reactivity to N ⁸ -AcSpd in urine is 0.343%, the other AcPut is 1.723%, N ¹ -AcSpd is 0.130%, AcSpm is 0.000%, and the total cross-reactivity is 2.196. %Met. Furthermore, in the established clone 20D7, the cross-reactivity to N ⁸ -AcSpd is 0.262%, the other AcPut is 1.402% in urine, N ¹ -AcSpd is 0.162%, AcSpm is 0.000%, and the total cross-reactivity is 1.827. % (Table 2).

    Table 2 Cross-reactivity of urinary DiAcSpd-like structural substances to DiAcSpd

From the above, the monoclonal antibody of the present invention is extremely useful compared to conventionally known anti-DiAcSpd antibodies because of its extremely high specificity to DiAcSpd and no cross-reactivity.

Measurement of DiAcSpd using kit (1) Establishment of kit conditions
As the DiAcSpd measurement kit, a competitive ELISA method using N ⁸ -AcSpd-EMC-AMS-BSA as the immobilized antigen was adopted. Among the monoclonal antibodies prepared in Example 2, the 20D7 clone having the lowest urinary cross-reactivity was used as the antibody. The antibody was a commercially available HRP labeling reagent Peroxidase Labeling Kit-SH (DOJINDO), and the anti-DiAcSpd antibody obtained in Example 2 (7) was used as the HRP-labeled antibody. The standard region was set to 600 nM to 9.375 nM, and the immobilized antigen concentration was 0.03 μg / mL as a result of the examination. The concentration of HRP-labeled anti-DiAcSpd monoclonal antibody used was determined to be 40% of the maximum reaction efficiency under N ⁸ -AcSpd-EMC-AMS-BSA immobilization conditions of 0.03 μg / mL. It was to be used at double dilution (FIG. 9).

  When the reproducibility of the standard curve was confirmed under these conditions, a good curve with reproducibility was obtained at 600 to 9.375 nM (FIG. 10, Table 3).

    Table 3 Standard curve reproducibility

(2) Basic data of kit In addition, in order to evaluate the precision and performance of the DiAcSpd measurement ELISA, two different types of control samples (Sample A and Sample B) were used, and the daily reproducibility was N = 10, and the values were reproduced daily. Sex was evaluated at N = 10. For intraday reproducibility, two different concentrations of urine samples were used, and during each day, each concentration sample was duplicated (n = 2 well = 1), and the DiAcSpd concentration measurement test was performed 10 times (n = 10) or more. For daily reproducibility, two different concentrations of urine samples were used, and each was duplicated (n = 2well = 1), and the DiAcSpd concentration measurement test was performed 10 times or more (10 days or more).

  As a result, intraday reproducibility was variability coefficient (CV) = 5.76% for sample A, CV = 2.58% for sample B, and daily reproducibility was CV = 6.76% for sample A and CV = 4.25% for sample B. It was. From this, it was found that this kit is sufficiently reliable in reproducibility (Table 4).

    Table 4 Intraday and daily reproducibility

  Next, an addition recovery test was conducted. The addition recovery test is a test for evaluating the accuracy of the measurement system. For example, in the DiAcSpd measurement system to be prepared, two types are prepared, one in which a known concentration of DiAcSpd (competitive substance) is added to the sample to be measured and the other in which no sample is added. If both concentrations are compared and the concentration difference of the obtained DiAcSpd matches the actual addition amount, it can be evaluated that the measurement system is accurate from the result.

  In addition to the DiAcSpd for preparing a standard curve, the addition and recovery test conducted in this example was performed using an ELISA method, and (A) a mixture of an equal volume of a buffer diluted with a known concentration of DiAcSpd and a urine sample (B Three types were prepared: an equal mixture of a known concentration (200 nM) of DiAcSpd solution and a urine specimen, and (C) an equal mixture of a DiAcSpd solution of known concentration (200 nM) and a dilution buffer. Each test was performed in duplicate (n = 2 well = 1), each with n = 3 in 3 concentrations (urine sample x 2-fold dilution, x 4-fold dilution, x 8-fold dilution). The DiAcSpd concentration in each measurement sample of (A) to (C) was calculated. The recovery rate (%) was calculated by ((B)-(A)) ÷ C × 100 using the calculated DiAcSpd concentration.

  As a result of the addition recovery test, it was found that the recovery rate of 96.3% to 108.4% of the urine sample was obtained at a dilution of 2 or more (Table 5). This revealed that DiAcSpd can be measured with high accuracy using the antibody of the present invention. It was also revealed that the DiAcSpd measurement kit using the antibody of the present invention has high accuracy.

    Table 5 Additive recovery test

  The performance of the kit of the present invention was 9.375 nM, preferably 18.75 nM, and analyte measurement detection sensitivity: 37.5 nM (18.75 × 2).

Furthermore, a dilution test was performed using two types of urine samples having different DiAcSpd concentrations. In the dilution test, two types of urine samples were prepared so as to have different concentrations in the standard measurement region (it is desirable to use two types of high concentration side and low concentration side). Next, the urine sample was diluted appropriately (for example, 6 concentrations) with the concentration of each prepared urine sample as the Top concentration, and the sample was measured using the sample as a duplicate (n = 2 well = 1). A graph was created with the vertical axis: DiAcSpd concentration and the horizontal axis: dilution rate, and the points obtained at the end were connected by a linear approximation curve to calculate the R ² value.

As a result, a good dilution curve was obtained for any specimen (FIG. 11). From this, it was shown that the ELISA kit of the present invention can measure DiAcSpd in urine with high accuracy.

The figure which shows the schematic of an immunization schedule and a method. The figure which shows the binding inhibitory activity of each substance in a 19C10 stock | strain culture supernatant. The figure which shows the binding inhibitory activity of each substance in a 15C5 stock | cultivation culture supernatant. The figure which shows the result of the dilution test which becomes 1 by the light absorbency (OD490nm) in 19C10 stock | strain culture supernatant. The figure which shows the result of the dilution test which becomes 1 by the light absorbency (OD490nm) in a 15C5 stock | cultivation culture supernatant. The figure which shows the binding inhibitory activity of each substance in 5D1 purified antibody. The figure which shows the binding inhibitory activity of each substance in 15D4 purified antibody. The figure which shows the binding inhibitory activity of each substance in 20D7 purified antibody. The figure which shows the examination result of the density | concentration in HRP labeled 20D7 antibody. The figure which shows the standard curve of a DiAcSpd measuring kit. The figure which shows the result of the dilution test by 2 types of urine samples from which DiAcSpd density | concentration differs.

Claims (8)

A monoclonal antibody against diacetyl spermidine, analyte diacetyl spermidine or analyte N ⁸ as immune reaction with diacetyl spermidine and the monoclonal antibody is inhibited - in a reaction system the presence of acetyl spermidine or acetyl putrescine, the analyte diacetyl The monoclonal antibody, wherein an inhibitory activity of the immune reaction by spermidine is at least 1700 times higher than an inhibitory activity of the immune reaction by the specimen N ⁸ -acetylspermidine or acetylputrescine.   The monoclonal antibody according to claim 1, wherein the concentration of the sample diacetylspermidine in the reaction system is 200 nM or less.   A monoclonal antibody against diacetylspermidine produced by a cell line with the accession number FERM P-20847.   A cell line producing the monoclonal antibody according to claim 1.   A cell line producing a monoclonal antibody against diacetylspermidine, whose accession number is FERM P-20847.   The reagent for a diacetyl spermidine detection containing the monoclonal antibody of any one of Claims 1-3.   A method for detecting diacetylspermidine, comprising reacting the monoclonal antibody according to any one of claims 1 to 3 with a biological sample to detect diacetylspermidine.   The method according to claim 7, wherein the biological sample is urine or serum.