JP2007256129A - Diagnostic method for tumor of non-human mammal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a tumor diagnostic method and a tumor diagnostic kit for non-human mammals. <P>SOLUTION: In this tumor diagnostic method for the non-human mammal for measuring an acetyl polyamine amount in a sample derived from the non-human mammal, the sample is urine or serum, the non-human mammal is a dog or a cat, an acetyl polyamine is diacetyl spermine or diacetyl supermidine, and a detection method for the acetyl polyamine is an immunoassay measuring method that uses an antibody to the acetyl polyamine. The kit also discloses the tumor diagnostic kit for the non-human mammal using the method. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for diagnosing a tumor in a non-human animal using an antibody against polyamine.

Polyamine is a general term for alkylamines having two or more amino groups. In human body, putrescine (H ₂ N (CH ₂ ) ₄ NH ₂ ), cadaverine (H ₂ N (CH ₂ ) ₅ NH ₂ ), spermidine (H ₂ N (CH ₂ ) ₄ NH (CH ₂ ) ₃ There are four types of NH ₂ ) and spermine (H ₂ N (CH ₂ ) ₃ NH (CH ₂ ) ₄ NH (CH ₂ ) ₃ NH ₂ ) and their acetylated forms.

In recent years, two diacetylpolyamines, N ¹ , N ⁸ -diacetylspermidine (hereinafter referred to as “DiAcSpd”) and N ¹ , N ¹² -diacetylspermine (hereinafter referred to as “DiAcSpm”), although in small quantities Was 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).

Recently, a monoclonal antibody against DiAcSpm was produced by Fujiwara et al. (Patent Document 1). These antibodies, 50% binding inhibition activity of the immune response to DiAcSpm is N ¹ - only been able to achieve 100 times the 50% binding inhibition activity of the immune response to acetyl spermidine (hereinafter referred to as "N ¹ -AcSpd"). Further N ¹ is a mono-acetyl form of spermine - not only achieved 20 times the 50% binding inhibition activity of the immune response to spermine (hereinafter referred to as "N ¹ -AcSpm"). Therefore, in order to increase the measurement sensitivity of DiAcSpm, it is necessary to develop an antibody specific to DiAcSpm with low cross-reactivity to N ¹ -AcSpd and N ⁸ -acetylspermidine (hereinafter referred to as “N ⁸ -AcSpd”). is there.

  By the way, in recent years, many households keep pets and dogs and cats. However, even dogs and cats often suffer from illness, and it is not uncommon to get cancer.

Conventionally, when cancer of dogs and cats was discovered, it was often too late, and therefore early detection of cancer for pets was desired.
JP-A-11-75839 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)

  An object of the present invention is to provide a method for diagnosing a tumor in a non-human mammal using an antibody against a polyamine.

  As a result of intensive studies to solve the above problems, the present inventors have reacted a sample collected from a non-human mammal such as a dog or a cat with an antibody against polyamine. The present inventors have found that a tumor can be detected and have completed the present invention.

That is, the present invention is as follows.
(1) A method for diagnosing a tumor of a non-human mammal, comprising detecting acetylpolyamine in a sample derived from the non-human mammal.

Examples of the sample include urine or serum. In the present invention, the non-human mammal to be diagnosed is a companion animal such as a dog or a cat.
In the present invention, examples of the urinary acetylpolyamine include diacetylspermine and diacetylspermidine. The acetylpolyamine can be detected by an immunoassay using an antibody against acetylpolyamine. Here, the antibody against acetylpolyamine is, for example, an antibody against diacetylspermine or an antibody against diacetylspermidine, and may be either a polyclonal antibody or a monoclonal antibody.

  Monoclonal antibodies include those having the following properties.

(a) Monoclonal antibody against diacetylspermine:
Inhibition of the immune reaction by diacetylspermine in the sample in a reaction system in which the sample diacetylspermine or the sample N ¹ -acetylspermidine is present so that the immune reaction between the diacetylspermine and the anti-diacetylspermine-specific monoclonal antibody is inhibited The measurement condition is such that the activity is at least 1000 times that of the inhibitory activity of the immune reaction by N ¹ -acetylspermidine in the sample.

  The monoclonal antibody is produced, for example, by a cell line having the deposit number FERM P-20090.

(b) Monoclonal antibody against diacetylspermidine:
50% inhibition of the immune reaction by the sample diacetylspermidine in the reaction system in which the sample diacetylspermidine or the sample N ⁸ -acetylspermidine or acetylputrescine is present so that the immune reaction between the diacetylspermidine and the monoclonal antibody is inhibited The measurement condition is such that the activity is at least 180 times or more compared with the inhibitory activity of the immune reaction by the specimen N ⁸ -acetylspermidine or acetylputrescine.

The monoclonal antibody is produced, for example, by a cell line having the accession number FERM AP-20668.
(2) A kit for diagnosing a tumor of a non-human mammal, comprising an antibody against acetylpolyamine.
Examples of the non-human mammal to be used for the kit of the present invention and the antibody contained in the kit include those described in (1) above.

  According to the method of the present invention, polyamine can be detected easily and with high sensitivity by reacting urine or serum of a pet (pet) such as a dog or cat with an antibody against polyamine as a measurement sample. Since this detection result shows a correlation with cancer, the method and kit of the present invention are useful for diagnosis of tumors in non-human mammals.

The present invention utilizes polyamines in non-human animals as tumor markers. Specifically, the present invention uses antibodies against polyamines for diagnosis of tumors in non-human mammals.
1. Polyamines as tumor markers In the present invention, in order to use polyamines such as DiAcSpm and DiAcSpd as tumor markers, it is important to detect polyamines contained in a measurement sample with high sensitivity. As the detection means, in the present invention, for example, an antibody against DiAcSpm or DiAcSpd can be employed.

  The present invention establishes a technique for developing a polyamine-specific antibody and a kit for measuring a urinary polyamine, and provides a method for utilizing polyamine as a tumor marker for non-human mammals.

  Polyamine is a general term for alkylamines having two or more amino groups, and there are four polyamines present in the human body and their acetylated forms. In recent years, polyamines have been found to be present in high concentrations in the urine of cancer patients.

  However, it was not clear whether such polyamines could be cancer markers in non-human mammals such as pets.

  Thus, as a result of establishing an antibody with high specificity for polyamine and applying it to a non-human mammal, the present inventor has found that a tumor can also be detected in a non-human mammal.

The polyamine that serves as a tumor marker in the present invention is not particularly limited, but putrescine (H ₂ N (CH ₂ ) ₄ NH ₂ ), cadaverine (H ₂ N (CH ₂ ) ₅ NH ₂ ), 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 ) and their acetylated form Among them, two kinds of diacetylpolyamines, N ¹ , N ⁸ -diacetylspermidine (referred to as “DiAcSpd”) and N ¹ , N ¹² -diacetylspermine (referred to as “DiAcSpm”) are preferable.

2. Production of antibodies against polyamines Hereinafter, methods for producing antibodies against polyamines such as DiAcSpm and DiAcSpd will be described.

In the present invention, the “antibody” means an entire antibody molecule capable of binding to an antigen polyamine or an active fragment having antigen-antibody reaction activity. The antibody molecule may be a polyclonal antibody or a monoclonal antibody. Examples of the active fragment include Fab, F (ab ′) ₂ fragment, Fv, recombinant Fv body, single chain Fv and the like.

  The antibodies (polyclonal and monoclonal antibodies and active fragments) of the present invention can be produced by any of a variety of methods. Methods for producing such antibodies are well known in the art.

Hereinafter, although experiment and an Example are concretely demonstrated about antibody preparation, it is not limited to these.
(1) Preparation of antigen
Since DiAcSpm and DiAcSpd are low molecular weight alkylamines, antigen-specific antibodies cannot be obtained by directly immunizing mice. Therefore, to prepare an antibody against DiAcSpm, bovine serum albumin, which is a carrier protein, and N ¹ -AcSpm are acylamide-bonded to produce an immune antigen having a large number of DiAcSpm-like substances as side chains. In order to produce an antibody against DiAcSpd, 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.

  When an antibody against DiAcSpm is prepared, an immunizing antigen can be prepared according to a known method (Hiramatsu, K. et al., J. Biochem., 124, 231-236 (1998)). First, BSA, which is a carrier protein, is reacted with acetylmercaptosuccinic anhydride (hereinafter referred to as “AMS”) to produce an AMS-BSA complex which is a reaction product. Next, AcSpm is acylamide-bonded to AMS-BSA via GMBS (N- (4-Maleimidobutyryloxy) succinimide), which is a bivalent cross-linking reagent, to produce immune antigen AcSpm-GMB-AMS-BSA.

On the other hand, in order to produce an antibody against DiAcSpd, an immune antigen can be produced 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 referred to as “AMS”) to produce an AMS-BSA complex which is a reaction product. Next, N ⁸ -AcSpd is acylamide-bonded to AMS-BSA via GMBS (N- (4-Maleimidobutyryloxy) succinimide), a bivalent cross-linking reagent, and immunizing antigen N ⁸ -AcSpd -GMB-AMS-BSA Is made.
(2) Preparation of polyclonal antibody against polyamine The antigen prepared as described above is administered to a mammal. The mammal is not particularly limited, and examples thereof include rats, mice, rabbits, and the like, and rabbits are preferable.

  The dose of the antigen per animal is 5 to 2 mg when no adjuvant is used, and 5 to 2 mg when an adjuvant is used. Examples of adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization is performed mainly by injecting intravenously, subcutaneously or intraperitoneally. 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 2 to 5 weeks.

  Then, 6 to 60 days after the final immunization, the antibody titer is measured by enzyme immunoassay (ELISA (enzyme-linked immunosorbent assay) or EIA (enzyme immunoassay)), radioimmunoassay (RIA), etc. On the day when the maximum antibody titer is shown, blood is collected to obtain antiserum.

  Thereafter, the reactivity of polyclonal antibodies in the antiserum against these proteins is measured by ELISA or the like using BSA or the like.

  As described above, DiAcSpm and DiAcSpd are present in a small amount in a mixture of Spd, Spm and the like. Therefore, in the present invention, antibodies that react with DiAcSpm or DiAcSpd are selected with higher accuracy.

That is, in the case of obtaining an antibody against DiAcSpm, the antibody is highly reactive to DiAcSpm and has (i) a cross-reactivity with N ¹ -AcSpd of 0.1% or less and / or (ii) present in urine. The total interference of the measurement results due to the cross-reaction with the DiAcSpm-like substance is 5% or less (preferably 3% or less). In the case of obtaining an antibody against DiAcSpd, it is an antibody showing strong reactivity to DiAcSpd, and (i) cross-reactivity with N ¹ -AcSpm is 0.1% or less, and / or (ii) DiAcSpd present in urine A substance having a total interference of 5% or less (preferably 3% or less) of the measurement results due to cross-reaction with similar substances is selected.

  In the present invention, the antibody titer is increased by 2 or more at the absorbance level due to immunization, and then 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, It is also possible to obtain an antibody with high specificity for polyamine by leaving the animal preferably for 4 to 6 months, more preferably 6 months. In that case, immunization is again performed one or more times at intervals of several weeks, and spleen cells are removed several days after the last immunization day, preferably 3-5 days later. In addition, when an antigen is injected subcutaneously into the footpad of an animal, the number of immunizations is 1 and the regional lymph nodes are removed 7 to 13 days later, preferably 8 to 10 days later. The interval between blood collections is 1 to 4 weeks, preferably 1 to 2 weeks after immunization.

  “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.
(3) Production of monoclonal antibodies against polyamines
(i) Collection of antibody-producing cells The antigen produced as described above is administered to mammals such as rats, mice, rabbits and the like. The dose of the antigen per animal is 500 to 200 μg when no adjuvant is used, and 500 to 200 μg when an adjuvant is used. Examples of adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization is performed mainly by injecting intravenously, subcutaneously or intraperitoneally. 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 2 to 5 weeks. Then, antibody-producing cells are collected 1 to 60 days after the last immunization day, preferably 1 to 14 days later. Examples of antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells, etc., but spleen cells or local lymph node cells are preferred.

(ii) Cell fusion Cell fusion between antibody-producing cells and myeloma cells is performed to obtain hybridomas. 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 has drug selectivity and cannot survive in a HAT selection medium (including hypoxanthine, aminopterin, and thymidine) in an unfused state, but can survive only in a state fused with antibody-producing cells. Those having the following are preferred. Examples of myeloma cells include mouse myeloma cell lines such as P3X63-Ag.8.U1 (P3U1) and NS-I.

Next, the myeloma cell and the antibody-producing cell are fused. Cell fusion is performed in animal cell culture media such as serum-free DMEM and RPMI-1640 media, and 1 × 10 ⁶ to 1 × 10 ⁷ antibody-producing cells and 2 × 10 ⁵ to 2 × 10 ^6. Per ml of myeloma cells (cell ratio of antibody-producing cells to myeloma cells is preferably 5: 1), and a fusion reaction is performed in the presence of a cell fusion promoter. As the cell fusion promoter, polyethylene glycol having an average molecular weight of 1000 to 6000 daltons can be used. Alternatively, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation).

(iii) Selection and cloning of hybridoma The target hybridoma is selected from the cells after cell fusion treatment. As a method, the cell suspension is appropriately diluted with, for example, fetal bovine serum-containing RPMI-1640 medium, then spread on a microtiter plate, a selective medium is added to each well, and then the selective medium is appropriately replaced and cultured. I do. As a result, cells that grow from about 14 days after the start of culture in the selective medium can be obtained as hybridomas.

  Next, the culture supernatant of the hybridoma that has grown is screened for the presence of antibodies that react with polyamines (eg, DiAcSpm). Hybridoma screening is not particularly limited, and may be performed according to ordinary methods. For example, a part of the culture supernatant contained in a well grown as a hybridoma can be collected and screened by enzyme immunoassay, radioimmunoassay or the like.

Cloning of fused cells is performed by the limiting dilution method or the like. In this case as well, as described in the section of the polyclonal antibody, when an antibody against DiAcSpm is obtained, it is an antibody showing strong reactivity to DiAcSpm, and (i) the cross-reactivity with N ¹ -AcSpd is 0.1. And / or (ii) a hybridoma that produces an antibody that has a total of 5% or less (preferably 3% or less) of interference with measurement results due to cross-reaction with a DiAcSpm-like substance present in urine. (An antibody against DiAcSpd is the same as described above).

(iv) Collection of monoclonal antibody As a method of collecting a monoclonal antibody from an established hybridoma, a normal cell culture method or ascites formation method can be employed.

In the cell culture method, the hybridoma is cultured in an animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium, or serum-free medium under normal culture conditions (for example, 37 ° C., 5% CO ₂ concentration). Cultivate for 7 to 14 days and obtain antibody from the culture supernatant.

In the case of the ascites formation method, about 1 × 10 ⁷ hybridomas are administered into the abdominal cavity of a mammal derived from a myeloma cell and the same strain, and the hybridomas are proliferated in large quantities. And ascitic fluid is collected after 1-2 weeks.

  When antibody purification is required in the antibody collection method, known methods such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, affinity chromatography are appropriately selected, or a combination thereof is used. Can be purified.

  The monoclonal antibody used in the present invention can also be obtained by adopting a method different from the conventional method for immunizing animals. That is, after immunization with an antigen and the antibody titer rising to an absorbance level of 2 or more, the immunized animal is allowed to stand for a while, and eventually the antibody titer is measured at an absorbance level of 0.05 to 1, preferably 0.05 to 0.5, more preferably. After the level drops to 0.05, antibody-producing cells can be prepared after further immunization.

  The increase level of the antibody titer is 2 or more in the absorbance level. The “leaving” period is the same as described above.

  By providing a high-affinity monoclonal antibody in the present invention, it is possible to select measurement conditions under which the concentration of DiAcSpm that inhibits the immune reaction of immobilized DiAcSpm by 50% is 1 nM or less. As a result, DiAcSpm contained in a trace amount in a urine sample containing a large amount of similar structural substances can be accurately quantified by enzyme immunoassay while avoiding interference by the similar structural substances.

  Further, according to the present invention, a DiAcSpd-specific monoclonal antibody is provided, and by this monoclonal antibody, the measurement conditions under which the concentration of DiAcSpd (competitive DiAcSpd) that inhibits the immune reaction of DiAcSpd by 50% is 200 nM or less, for example, 50 nM or less. It becomes possible to select. 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.

  In the present invention, a cell line (hybridoma) that produces a monoclonal antibody against DiAcSpm is referred to as “Anti-DiAcSpm hybridoma CN647” and, on June 18, 2004, the National Institute of Advanced Industrial Science and Technology (AIST) 305-8566 Deposited at 1-1-1 Higashi 1-1-1 Tsukuba City, Ibaraki Prefecture. The accession number is “FERM P-20090”.

In addition, the cell line (hybridoma) that produces monoclonal antibodies against DiAcSpd is called “Anti-DiAcSpd-MAb 19C10” and, on September 27, 2005, the National Institute of Advanced Industrial Science and Technology (AIST) -8566 Deposited at Higashi 1-1-1 Higashi 1-1-1 Tsukuba, Ibaraki. The receipt number indicating the receipt number (described in the receipt) is “FERM AP-20668”.
(4) Properties of monoclonal antibodies
(4-1) Anti-DiAcSpm monoclonal antibody The monoclonal antibody of the present invention is an antigen competitive inhibition assay system, that is, an immune reaction between immobilized diacetylspermine and a specific monoclonal antibody against the diacetylspermine is inhibited. In the reaction system in which the sample diacetylspermine or the sample diacetylspermidine is present, the following properties are provided.

(i) The inhibitory activity of the immune reaction by DiAcSpm in the sample satisfies a measurement condition that is at least 1000 times that of the inhibitory activity of the immune reaction by N ¹ -AcSpd in the sample.

  Here, a system is considered in which DiAcSpm is immobilized on the bottom of the plate and immobilized, and a monoclonal antibody against DiAcSpm is added thereto to react DiAcSpm with the antibody. In a reaction system in which no competing substance is present, the monoclonal antibody reacts with the immobilized DiAcSpm. To this reaction system, free DiAcSpm is added as a specimen. This free DiAcSpm reacts with the antibody in competition with the immobilized DiAcSpm. When the binding between the solid-phase DiAcSpm and the antibody is inhibited by 50%, that is, when the competitive DiAcSpm (free DiAcSpm) is added, the amount of binding between the monoclonal antibody and the solid-phase DiAcSpm is The reciprocal of the amount or concentration of the free DiAcSpm when it decreases to 50% compared to the amount of binding between the monoclonal antibody and the solid phase DiAcSpm when not present can be defined as “inhibitory activity” of the immune reaction.

For each of free DiAcSpm and N ¹ -AcSpd, the above inhibitory activity was measured, and when using free DiAcSpm, the inhibitory activity when using N ¹ -AcSpd that is liberated in urine in large amounts An antibody that satisfies the reaction conditions such that the inhibitory activity when using N ⁸ -AcSpd, which is 1000 times more than that of N ⁸ -AcSpd, which is liberated in the urine, is 100 times or more higher than that of the present invention. As an antibody. This means that the monoclonal antibody of the present invention does not cross-react with N ¹ -AcSpd etc. and is specific for DiAcSpm.

  (ii) The concentration of DiAcSpm in the sample when satisfying the above condition (i) (concentration in the reaction system in the plate) is 2 nM or less, preferably 0.1 to 2 nM.

  The antibody of the present invention having the above properties has an affinity at least 1000 times that of the monoclonal antibody described in JP-A-11-75839. In addition, the immunization method used to obtain the antibody in the present invention is a method in which high-affinity antibody-producing B cells are memorized in vivo and the memory B cells are efficiently activated. This means that the antibody of the present invention has a high affinity for DiAcSpm.

(4-2) Anti-DiAcSpd Monoclonal Antibody The monoclonal antibody of the present invention is an antigen competitive inhibition assay system, i.e., a sample diacetylspermidine so that the immune reaction between diacetylspermidine and a specific monoclonal antibody against the diacetylspermidine is inhibited. The reaction system to be present 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.

(i) The 50% inhibitory activity of the immune reaction by DiAcSpd in the sample has at least 180 times the inhibitory activity compared to the 50% inhibitory activity of the immune reaction by N ⁸ -AcSpd and AcPut in the sample .

  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. To this reaction system, free DiAcSpd is added 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 competitive DiAcSpd (free DiAcSpd) is added, the amount of binding between the monoclonal antibody and the solid-phase DiAcSpd is Inhibitory activity can be defined as the reciprocal of the amount or concentration of the free DiAcSpd when it is reduced to 50% compared to the amount of binding between the monoclonal antibody and the solid phase DiAcSpd when not present.

The above inhibitory activity was measured for each of free DiAcSpd and N ⁸ -AcSpd and AcPut, and N ⁸ -AcSpd or AcPut that was released in the urine in a large amount when the free DiAcSpd was used was used. An antibody that satisfies the reaction condition such that it is 180 times or more compared to the inhibitory activity can be selected as the antibody of the present invention. 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 sample when the above conditions are satisfied is 200 nM or less, preferably 10 to 200 nM, more preferably 50 nM or less.

(ii) The cross-reactivity with N ⁸ -AcSpd, which is approximately 8 times more in the urine as a similar structural substance of DiAcSpd, is 0.7% or less, preferably 0.5% or less.

(iii) The total cross-reactivity of DiAcSpd-like structural substances present in urine is 20% or less, preferably 18% or less, more preferably 12% or less.

4). Tumor Diagnosis Method Since polyamine can be used as a clinical marker (tumor marker) for cancer, the antibody of the present invention is reacted with a biological sample of a non-human mammal to detect (measure) DiAcSpm or DiAcSpd in the biological sample. Or quantifying), it is possible to diagnose whether or not the non-human mammal has a tumor using the detection result as an index.

  Detection of DiAcSpm or DiAcSpd can be performed by any method known as a hapten immunoassay that is generally performed, and is not particularly limited.

Although it does not specifically limit as a tumor, For example, at least 1 sort (s) chosen from the following groups is mentioned. The type of tumor to be detected may be one type, or two or more types may be combined.
(1) Oral cavity, nose, pharyngeal tongue cancer, gingival cancer, malignant lymphoma, maxillary cancer, nasal cancer, nasal cavity cancer, laryngeal cancer, pharyngeal cancer
(2) Cranial nervous system Brain tumor, glioma, meningioma
(3) Thyroid, endocrine organs Thyroid cancer (thyroid papillary carcinoma, follicular thyroid cancer, medullary thyroid carcinoma), adrenal tumor
(4) Respiratory lung cancer (squamous cell carcinoma, adenocarcinoma, alveolar cell carcinoma, large cell undifferentiated cancer, small cell undifferentiated cancer, carcinoid)
(5) Breast Breast cancer, breast tumor, Paget's disease, breast sarcoma
(6) Blood, lymph gland Acute myeloid leukemia, acute promedullary leukemia, acute myeloid monocytic leukemia, acute monocytic leukemia, acute lymphocytic leukemia, acute undifferentiated leukemia, chronic myelogenous leukemia, chronic lymphoid Leukemia, malignant lymphoma (lymphosarcoma, reticulosarcoma, Hodgkin's disease), multiple myeloma, primary macroglobulinemia, lymphoma
(7) Gastrointestinal esophageal cancer, gastric cancer, gastric / intestinal malignant lymphoma, pancreatic cancer, biliary tract cancer, gallbladder cancer, small intestine or duodenal cancer, colon cancer, anal cancer, liver cancer
(8) Female genitals Uterine cancer, ovarian cancer, uterine sarcoma (leiomyosarcoma, rhabdomyosarcoma, lymphosarcoma, reticulosarcoma)
(9) Urinary tract malignant tumor (prostate cancer, kidney cancer, bladder cancer, testicular tumor, urethral cancer)
(10) Motor organs Rhabdomyosarcoma, fibrosarcoma, osteosarcoma, chondrosarcoma, histiocytic sarcoma, synovial sarcoma, myxosarcoma, liposarcoma, Ewing sarcoma, multiple myeloma
(11) Skin Skin cancer, epithelial cancer (squamous cell carcinoma, transitional cell carcinoma, etc.), cutaneous Bowen's disease, cutaneous Paget's disease, cutaneous malignant melanoma
(12) Others Angioperiosteum, mastocytoma Non-human mammals to be detected or diagnosed include any mammals such as pets, livestock, and racehorses. For example, pets such as dogs and cats ( It is preferably a pet.

  A biological sample is collected from a non-human mammal suspected of having cancer, and a DiAcSpm or DiAcSpd measurement sample is prepared. Examples of biological samples include blood, urine, tissue, etc. Urine is preferred because it is easy to handle and places less burden on animals. Moreover, blood is preferable in consideration of early diagnosis of cancer although it is in a very small amount.

  Next, the measurement sample and the antibody are reacted. The measurement of polyamine can be performed by ELISA generally performed. To measure by ELISA, first, an antigen (DiAcSpm or DiAcSpd) is coated on a microplate. On the other hand, after the polyamine and the anti-polyamine-specific antibody in the biological sample and standard solution are reacted in advance, this reaction solution is plated on a plate. The unreacted antibody binds to the polyamine on the plate. Then, an HRP-labeled anti-rabbit IgG antibody that is a secondary antibody is added to the plate and reacted. Finally, the polyamine 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 the present invention, the state of the tumor can be evaluated using the detection result as an index. If the detection result exceeds the specified reference value, the polyamine is positive, and if the detection result is less than the specified reference value, the polyamine is negative. Can be evaluated.

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 and evaluating 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.

5). Kit containing antibody against polyamine In the present invention, a monoclonal antibody against polyamine can be used as a detection kit or reagent for the polyamine. The kit of the present invention can be used for the diagnosis of the tumor.

  Conventionally, when measuring polyamines as a general biochemical test, several types of polyamines in urine are measured as a group of similar structures, and there is no examination of the relationship between each type of similar structure and various pathologies. It was equal. Therefore, among the polyamine measurement methods, a method has been established to distinguish and measure the amount of polyamine in urine, but in particular, DiAcSpm or DiAcSpd, which is a kind of polyamine, becomes extremely high at the onset of cancer and at the time of recurrence after treatment. It has been confirmed that This indicates that if a method for easily and accurately measuring diacetylpolyamine can be developed, a great demand is expected in cancer therapy in non-human mammals as a novel tumor marker. Further, since the antibody of the present invention can detect a trace amount of polyamine with high sensitivity, it can be expected to measure polyamine in serum and detect an abnormality at an early stage, which has been extremely difficult in the past.

  In the tumor diagnosis kit of the present invention, the standard region can be set to 0.1 to 2 nM by adjusting the solid-phased antigen concentration to a low concentration side. The concentration of the immobilized antigen is 0.1 to 1 μg / mL, preferably 0.07 μg / mL. As a result, sufficient sensitivity and measurement accuracy can be achieved for measuring polyamines in urine.

  Measurement accuracy is an index that shows how much each measurement value varies when a single assay is performed by dividing the same sample into multiple tubes or wells. Is expressed as a coefficient of variation (CV), that is, a ratio (%) of a standard deviation to an average value. 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.

  The performance of the kit of the present invention is as follows. Minimum detection actual measurement value: (0.1 nM), specimen measurement detection sensitivity: 0.4 nM (0.1 × 4). The simultaneous reproducibility is 10% or less, preferably around 5%, and the daily reproducibility is 10% or less, preferably about 8-10%. All reproducibility is CV = 10% or less. As for the influence of coexisting substances, polyamine measurement is not affected in conjugated bilirubin, glucose, hemoglobin, and ascorbic acid.

  In the present invention, when an antibody against polyamine is used as a tumor diagnostic kit, the 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.

  In addition to the antibody of the present invention, the kit of the present invention includes an antigen-immobilized microplate, a polyamine standard (STD), an antibody dilution solution, an HRP-labeled anti-rabbit IgG antibody, an OPD (orthophenylenediamine) tablet, At least one selected from a substrate solution, a reaction stop solution, a concentrated washing solution, and the like can be included. In addition to HRP (peroxidase), alkaline phosphatase, malate dehydrase, α-glucosidase, α-galactosidase and the like can also be used as the labeling enzyme.

  As the reaction medium, a buffer solution which gives optimum conditions for the reaction or is useful for stabilizing the reaction product, a stabilizer for the reactant, and the like are also included.

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

  The immunizing antigen was prepared as follows (Hiramatsu, K. et al., J. Biochem., 124, 231-236 (1998)).

  DiAcSpm is a low molecular weight alkylamine, and an antibody specific for DiAcSpm cannot be obtained by directly immunizing rabbits. Thus, the carrier protein bovine serum albumin (hereinafter abbreviated as BSA) and AcSpm were acylamide-bonded to produce an immune antigen AcSpm-GMB-BSA having many DiAcSpm-like structures as side chains. First, BSA serving as a carrier protein was reacted with S-acetylmercaptosuccinic acid to produce S-acetylmercaptosuccinic acid-BSA (hereinafter abbreviated as AMS-BSA) as a reaction product. Furthermore, AcSpm was acylamide-bonded to AMS-BSA via GMBS, which is a bivalent cross-linking reagent, to prepare immune antigen AcSpm-GMB-BSA.

  Immunization was performed by a method of immunizing subcutaneously on the back of an emulsion in which equal amounts of an immunizing antigen and an adjuvant (primary immunization: complete adjuvant, booster immunization: incomplete adjuvant) were mixed. Immunization was performed every 2 weeks under the conditions of primary immunization 1 mg / fowl and booster immunization 0.3 mg / fowl. In addition, partial blood sampling was performed on the seventh day after the third immunization, and the antibody titer in the antiserum was checked by ELISA.

  Of the 19 antisera obtained, 3 types of anti-DiAcSpm polyclonal antisera (JPW9, NZW2, and NZW9) that showed high antibody titers yielded specific antibodies that can be used in urinary DiAcSpm measurement systems. It was examined by stepwise affinity purification. For affinity purification, stepwise affinity purification was performed using DiAcSpm columns and DiAcSpd columns in which carboxytoyopearl was derivatized with AcSpm and N8-AcSpd (Hiramatsu, K. et al., J. Biochem. , 124, 231-236 (1998)).

As a result, the antiserum of NZW2 was an antibody whose cross-reactivity with N ¹ -AcSpd present in the urine as a similar substance to DiAcSpm was about 0.1% or less. In addition, the total interference with the measurement result due to the cross-reaction with the DiAcSpm-like substance present in urine was 2.9%, and the cross-ability of 5% or less could be achieved (Table 1).

Preparation of Monoclonal Antibody Specific to Diacetylspermine (hereinafter DiAcSpm) (1) Preparation of Antigen As an immunizing antigen, AcSpm-GMB-AMS-BSA was prepared according to Example 1.
(2) Evaluation of antigen Antigen was evaluated using a polyclonal antibody specific to DiAcSpm. Regarding the six types of antigens prepared in (1), the activity of inhibiting the reaction of the anti-DiAcSpm polyclonal antibody against the immobilized AcSpm-HMC-peptide was examined. The activity was measured as in Experimental Example 1 below.

  As a result, No. 1 and No. 6 antigens had the highest inhibitory activity, followed by No. 5 with the highest inhibitory activity (FIG. 1). Since these antigens are relatively optimal antigens as DiAcSpm-like structures, they were selected as immune antigens.

Experimental Example 1 (Antigen Evaluation Method)
96-well microtiter prepared with 0.07μg / mL in PBS (pH 7.0) using monoacetylspermine as a solid-phased antigen (AcSpm-HMC-peptide) having a structure similar to DiAcSpm by acylamide bond 50 μL was added to each well of the plate and left at 25 ° C. for 1.5 hours. 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. . Next, the plate was frozen at −30 ° C. overnight, and the blocking solution was frozen and lyophilized to produce a lyophilized plate. Add 70 μL of 10 μg / mL anti-DiAcSpm polyclonal antibody to each dilution plate and 70 μL of immunological antigen AcSpm-GMB-AMS-BSA stepwise prepared for each concentration and add to each well, and leave at 25 ° C for 1 hour. The pre-reaction was performed.

Next, 300 μL of PBST was added to each well of the freeze-dried plate, allowed to stand at 25 ° C. for 30 minutes, and then washed once with PBST. Thereto, 50 μL of the pre-reacted reaction solution was added to each well and allowed to stand at 25 ° C. for 1 hour. Next, after washing 3 times with PBST, 50 μL of HRP-labeled anti-rabbit IgG antibody (Bio-Rad) diluted 80-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 0.012% hydrogen peroxide is added to each well. After standing at 25 ° C. for 9 minutes, the absorbance at 492 nm was measured with an ELISA reader.
(3) Immunization to mice Antigens used for immunization of mice were performed using the 3 antigens selected in (2). Mice were immunized by two methods.

  The first method (referred to as immunization method 1) was performed as follows. That is, an immune antigen prepared to 2 mg / mL was mixed with FCA in an equal amount, and an emulsion formed was administered subcutaneously to the back of the mouse 100 μL at a time, and then 1 mg / mL of the immune antigen and FIA were administered at intervals of 2 weeks. 100 μL of the mixture that was mixed in volume and formed an emulsion was administered subcutaneously to the back of the mouse. Administer the antigen three times in total, confirm the antibody titer by ELISA, and for those with a high antibody titer, 100 mg each of the 1 mg / mL immunizing antigen and IFA emulsion was administered intraperitoneally to the mouse, Three days later, the spleen was removed for cell fusion.

The second method (referred to as immunization method 2) was performed as follows. That is, until the third dose of antigen in the first method is the same, but the mouse is left until the antibody titer falls in the latter half of the year, and again the 1 mg / mL immunizing antigen and IFA emulsion are subcutaneously administered to the back of the mouse. Two weeks later, an emulsion of 1 mg / mL immunizing antigen and IFA was finally administered intraperitoneally to the mice, and three days later, the spleen was removed for cell fusion.
(4) Preparation of spleen cells and cell fusion The extracted spleen cells were ground to prepare spleen cells containing anti-DiAcSpm 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 a HAT medium, and the cells were seeded in each well of a 96-well culture plate so that the number of spleen cells was 0.5 × 10 ⁵ cells / well. When the colonies in each well were confirmed on the 10th to 12th day after cell fusion, both immunization methods 1 and 2 had a colony positive rate of 95% or more.
(5) Screening for antibody-producing positive clones After cell fusion, the culture supernatant on the 12th day was collected, and antibody-producing positive clones were screened as in Experimental Example 2 below.

  In immunization method 1, 24 out of 1320 wells were BSA negative and DiAcSpm positive. In immunization method 2, 247 out of 2880 wells were BSA negative and DiAcSpm positive. The cells in the wells where these were selected were transferred to 24-well plates, cultured for 1-2 days, and screened again. Finally, 16 wells in immunization method 1 and 208 wells in immunization method 2 were positive for DiAcSpm. there were.

Experimental Example 2 (Antibody screening method)
Antibody screening method Add 50 μL of BSA prepared at 1 mg / mL with a freeze-dried plate and PBS (pH 7.0) prepared in the same manner as in Experimental Example 1 to each well of a 96-well microtiter plate, and overnight at 25 ° C. What was left still was used for the following. 300 μL of PBST was added to each well of the freeze-dried plate and the BSA-immobilized plate, allowed to stand at 25 ° C. for 30 minutes, and then washed once with PBST. 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 (KPL) diluted 80-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 0.012% hydrogen peroxide is added to each well. After standing at 25 ° C. for 9 minutes, the absorbance at 492 nm was measured with an ELISA reader.
(6) Cross-reactivity test of antibody-producing positive clones Among the above-mentioned DiAcSpm-positive wells, 51 clones with a relatively high antibody titer were present in the urine in many previous studies and were relatively easy to cross-react. A possible cross-reactivity test for N ¹ -AcSpd was performed by the method of Experimental Example 3 below.

As a result, N ¹ -AcSpd 50% reaction inhibitory activity was approximately 800 times that of DiAcSpm, 2 wells and 3000 times were 1 well, and the others were 0 to 200 times. It was.

Experimental example 3 (cross reaction test)
The antibody titer in the diluted culture supernatant was measured, and the dilution factor at which the absorbance at 492 nm was 1 was set. 70 μL of culture supernatant diluted at the dilution rate set above on the dilution plate and DiAcSpm solution prepared stepwise to each concentration and its similar structure substances (N ¹ -AcSpm, N ¹ -AcSpd, DiAcSpd, etc.) at each concentration 70 μL prepared in a stepwise manner was mixed and added to each well, and allowed to stand at 25 ° C. for 1 hour for pre-reaction. Next, 300 μL of PBST was added to each well of the freeze-dried plate prepared by the same method as in (2) above, left still at 25 ° C. for 30 minutes, and then washed once with PBST. Thereto, 50 μL of the pre-reacted reaction solution was added to each well and allowed to stand at 25 ° C. for 1 hour. Next, after washing 3 times with PBST, 50 μL of HRP-labeled anti-mouse IgG antibody (KPL) diluted 80-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 0.012% hydrogen peroxide is added to each well. After standing at 25 ° C. for 9 minutes, the absorbance at 492 nm was measured with an ELISA reader.
(7) Cloning
One well with high specificity for DiAcSpm was 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. Five days later, when single colonies were confirmed, single colonies were confirmed in 17 wells in two 96-well culture plates. Ten days later, when the antibody titer against DiAcSpm in the culture supernatant was measured by the method of Experimental Example 2, 3 wells were positive. The three wells were further cultured, and a cross-reactivity test for AcSpm, N ¹ -AcSpd, N ⁸ -AcSpd, DiAcSpd, and AcPut was performed in the same manner as the cross-reactivity test described in (6) above. As a result, 3 to about 1000 times the 50% inhibitory activity of 50% inhibition activity DiAcSpm the wells both is N ¹ -AcSpd, about 70 times the AcSpm, about 100,000 times DiAcSpd, approximately 500,000 N ⁸ -AcSpd and AcPut The antibody was more than doubled and sufficiently fulfilled its purpose (FIG. 2). One of them was established clone CN647.

The established clone CN647 is referred to as “Anti-DiAcSpm hybridoma CN647” and is deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Accession Number: “FERM P-20090”).
(7) Purification of antibody The desired monoclonal antibody was purified from the established clone CN647 by the following method. First, the established clone CN647 was suspended in a commercially available serum-free medium (Hybridoma SFM: Invitrogen) to prepare 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 2 weeks. After culturing, the culture supernatant was collected. After collection, the culture supernatant was applied to a protein G column and eluted with glycine buffer (pH 3) to purify the monoclonal antibody. The purified antibody retained activity. Furthermore, when the purified antibody was typed, it was found to be κ of IgG1.

Preparation immunogen Preparation (1) antigen-specific monoclonal antibodies to diacetyl spermidine (hereinafter DiAcSpd) is prepared according to Example 2 to give N ⁸ -AcSpd -GMB-AMS-BSA is immunogenic .
(2) Immunization to mice Immunization to mice was performed according to the method of Example 2.
(3) Preparation of spleen cells and cell fusion Cell fusion was performed according to Example 2.
(4) Screening for antibody-producing positive clones After cell fusion, the culture supernatant on the 12th day was collected, and screening for antibody-producing positive clones was performed according to Example 2. As a result, finally, 75 wells were positive for DiAcSpd in the first immunization method and 90 wells in the second immunization method.
(5) Cross-reactivity test of antibody-producing positive clones With regard to the above-mentioned DiAcSpd positive well 165 clones, 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. As a result, N ⁸ -AcSpd 50% reaction inhibitory activity was about 180 times or more that of DiAcSpd, and there were 2 wells, and the others were 0 to 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 and confirmed to be positive. 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. As a result, in both clones, the 50% inhibitory activity of DiAcSpd was about 180 times or more of the 50% inhibitory activity of N ⁸ -AcSpd, and the antibodies sufficiently provided with the target specificity in the present invention. The clones were established clones 19C10 (FIG. 3) and 15C5, respectively (FIG. 4).

  The established clone 19C10 is referred to as “Anti-DiAcSpd-MAb 19C10” and is deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (receipt number (description) “FERM AP-20668” ).

Diagnosis of cancer in non-human mammals
As a DiAcSpm measurement method, a competitive ELISA method using AcSpm-HMC-peptide as a solid-phased antigen was adopted. As the primary antibody, NZW2 having the lowest urinary cross-reactivity among the polyclonal antibodies prepared in Example 1 was used, and the secondary antibody was a commercially available goat-derived HRP-labeled anti-rabbit IgG antibody (BIO-RAD). did. Since the normal concentration of immobilized antigen is about 100 nM in DiAcSpm in urine, the standard region is assumed to be 6.25 nM to 200 nM, and the solid phase concentration 0.07 μg / mL that draws the most ideal competition curve in this region is assumed. Asked. The concentration of the anti-DiAcSpm antibody was determined to be 50% of the maximum reaction efficiency at a solid phase concentration of 0.07 μg / ml, resulting in 0.02 μg / ml. As for the secondary antibody goat-derived HRP-labeled anti-rabbit IgG antibody, the dilution factor of 2000 was the most sensitive. Therefore, 2000 times the goat-derived HRP-labeled anti-mouse IgG antibody was used as the condition for the secondary antibody.
The samples used for the measurement were raw urine, x10-fold diluted urine and x100-fold diluted urine, 2 Well = n = 1, and each diluted urine was measured at n = 1.

  Table 2 shows a list of types, sexes, and diseases of dogs (including some cats) used.

  In Table 2, “mix” means hybrid, “spay” means ovarian removal (female castration), and “cast” means testicular removal (male castration).

  The results are shown in FIG.

  As shown in FIG. 5, the DiAcSpm concentration of the dog (cat) urine sample is the average value of the control + 2SD value of 1215 nM / g · creatinine, and the value exceeding the reference value among the disease samples is 35.3 of the disease sample. %Met.

  From the above, it has become possible to diagnose cancer of non-human mammals by using an anti-DiAcSpm polyclonal antibody.

The figure which shows evaluation of the immunizing antigen by the light absorbency in 492nm. The figure which shows the binding inhibitory activity of each substance in a CN647 strain culture supernatant. 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 measurement result of DiAcSpm in the urine sample of a cancer disease dog and a control dog.

Claims (17)

  A method for diagnosing a tumor of a non-human mammal, which comprises detecting acetylpolyamine in a sample derived from the non-human mammal.   The method of claim 1, wherein the sample is urine or serum.   The method according to claim 1, wherein the non-human mammal is a dog or a cat.   The method according to claim 1, wherein the acetylpolyamine is diacetylspermine or diacetylspermidine.   The method according to claim 1, wherein the method for detecting acetylpolyamine is an immunoassay using an antibody against acetylpolyamine.   The method according to claim 5, wherein the antibody against acetylpolyamine is an antibody against diacetylspermine or an antibody against diacetylspermidine. The method according to claim 6, wherein the antibody against diacetylspermine is a monoclonal antibody having the following properties:
Inhibition of the immune reaction by diacetylspermine in the sample in a reaction system in which the sample diacetylspermine or the sample N ¹ -acetylspermidine is present so that the immune reaction between the diacetylspermine and the anti-diacetylspermine-specific monoclonal antibody is inhibited The measurement condition is such that the activity is at least 1000 times that of the inhibitory activity of the immune reaction by N ¹ -acetylspermidine in the sample. The method according to claim 6, wherein the antibody against diacetylspermidine is a monoclonal antibody having the following properties:
Diacetyl spermidine and the monoclonal antibody analyte diacetyl spermidine or analyte as immune response is inhibited in the N ⁸ - in a reaction system the presence of acetyl spermidine or acetyl putrescine, the inhibitory activity of the immune response by the subject diacetyl spermidine The measurement condition is at least 180 times higher than the inhibitory activity of the immune reaction by the sample N ⁸ -acetylspermidine or acetylputrescine.   The method according to claim 6, wherein the antibody against diacetylspermine is a monoclonal antibody produced by a cell line having the accession number FERM P-20090. The method according to claim 6, wherein the antibody against diacetylspermidine is a monoclonal antibody produced by a cell line having the accession number FERM AP-20668.
  A kit for diagnosis of a tumor of a non-human mammal, comprising an antibody against acetylpolyamine.   The kit according to claim 11, wherein the non-human mammal is a dog or a cat.   The kit according to claim 11, wherein the antibody against acetylpolyamine is an antibody against diacetylspermine or an antibody against diacetylspermidine. The kit according to claim 13, wherein the antibody against diacetylspermine is a monoclonal antibody having the following properties:
Inhibition of the immune reaction by diacetylspermine in the sample in a reaction system in which the sample diacetylspermine or the sample N ¹ -acetylspermidine is present so that the immune reaction between the diacetylspermine and the anti-diacetylspermine-specific monoclonal antibody is inhibited The measurement condition is such that the activity is at least 1000 times that of the inhibitory activity of the immune reaction by N ¹ -acetylspermidine in the sample. The kit according to claim 13, wherein the antibody against diacetylspermidine is a monoclonal antibody having the following properties:
Diacetyl spermidine and the monoclonal antibody analyte diacetyl spermidine or analyte as immune response is inhibited in the N ⁸ - in a reaction system the presence of acetyl spermidine or acetyl putrescine, the inhibitory activity of the immune response by the subject diacetyl spermidine The measurement condition is at least 180 times higher than the inhibitory activity of the immune reaction by the sample N ⁸ -acetylspermidine or acetylputrescine.   The kit according to claim 13, wherein the antibody against diacetylspermine is a monoclonal antibody produced by a cell line having the deposit number FERM P-20090. 14. The kit according to claim 13, wherein the antibody against diacetylspermidine is a monoclonal antibody produced by a cell line having the accession number FERM AP-20668.