TW581810B - Monoclonal antibody against 7-amino-flunitrazepam, hybridoma producing the antibody and the use thereof - Google Patents

Abstract

The present invention discloses the development of a monoclonal antibody specific against the major metabolite of flunitrazepam (FM2), 7-amino-flunitrazepam, and a diagnostic kit and a method for detecting the drug abuse of FM2 by using this antibody.

Description

581810 V. Description of the invention (1) Field of the invention The present invention relates to a single antibody specific to the main metabolite of FM2 (flunitrazepam), 7-amino-FM2 (7-amino-flunitrazepam), which produces fusion antibodies of the antibody , The kit containing this single antibody, and the use of this fusion tumor, antibody and kit to detect the abuse of FM2. BACKGROUND OF THE INVENTION FM2 (flunitrazepam) is a sedative sedative that belongs to the benzodiazepines (BZD) group. Due to its lower side effects and higher safety than barbiturates, these drugs have gradually replaced barbie women. Acid salt has become the most widely used sleeping sedative. Among them, FM2 (Flunitrazepam), Diazepam, and Triaz〇lam, commonly known as small whiteboard, are more commonly abused. Long-term use of these drugs can cause symptoms such as drug resistance, dependence, dependence, and drowsiness, instability, inattention, memory and I, and loss of power. Sudden discontinuation of the drug can also cause withdrawal symptoms. Patients with excessive oral use of BZDj mostly have excessive muscle relaxation and deep sleep and sleep states, which rarely cause death. However, if used with alcohol or other central nervous systems, the risk is greatly increased. Many abusers are responsible for Mental = accidental or fatal due to inhaled vomit. After taking FM2, the substance is 7-amino-FM2. I know whether to take FM2. That is, it enters the blood and undergoes metabolism. It is mainly excreted in the urine, so it can be detected in urine. FM2 is commonly known as "rape pills". It is abused by domestic adolescents as a strong drug. And suicide tools are the focus of prosecutions. ‘There is no in vitro test reagent sensitive to FM2 metabolites at home and abroad’, so if 7

Page 5 581810 V. Description of the invention (2) The FM2 must be checked to determine whether or not to take FM2. It must be checked by GC / MS. General drug screening is divided into two major steps: initial screening and further confirmation (see Drug 〇f Abuse Testing Market, Theta Corp. iqqi \ 4+ I, ^; of which, the initial screening needs to have high sensitivity It is easy to use and suitable for handling the characteristics of a large number of samples. The positive samples after screening are then subjected to methods with high specificity and accuracy, such as gas chromatography / mass spectrometry (CHn.

Cl ^ m · 37/9: 1 59 5- 1 60 1 (1 9 9 1)) to confirm that the analytical methods currently available for screening drug abuse include thin layers for preliminary screening ^ ( TLC) and immunoassay) are used for further accurate detection of high-performance liquid chromatography (HPLC) and gas-mass chromatography (GC / MS). Among them, they are developed using multiple or single antibodies. Immunity reagents are good. In addition to having high specificity and sensitivity, they are easy to use and fast in response, and the sample does not need to undergo any pretreatment steps, which is very useful for large i screening. However, there is currently no immunoreagent kit for FM 2 metabolites, 7-amino-FM 2. Therefore, the inventor has developed a single antibody with 7% amino-f% 2 specificity. , And developed the test reagents. Summary of the Invention In view of this, the main object of the present invention is to provide a monoclonal antibody specific for 7-amine-FM2. Another object of the present invention is to provide a fusion tumor that secretes the monoclonal antibody. Another object of the present invention is to provide a method for detecting the content of FM2 metabolite, 7-amino-FM2, including the following steps: (a) providing a monoclonal antibody specific for anti-7-amino-FM2; (b) Provide a tool for signal generation, which

Page 6 581810, description of the invention (3) = finely combined with 7-amino-FM2 to generate a signal; (c) the single body is adhered to a support tool to form an antibody-support Conjugate_a 'and (d) make the test sample or 7-amine-FM2 standard solution compete with the anti-7-amine and FMj Λ conjugates, and bind with the antibody-supporting conjugate, and / or The signal generated by the signal generation tool. Still another object of the present invention is to provide a kit for detecting FM2 metabolites, ^ -amino-FM2, including ... (1) a sample contact area; (2) an indicator area including a single plant of the present invention Antibodies; (3) a reaction test zone; and (4) a liquid recovery zone. In order to make the above and other objects, features, and advantages of the present invention more comprehensible, 'the preferred embodiment is described below in conjunction with the accompanying drawings, and is described in detail as follows:' A brief description of the drawings. FIG. 1 is the present invention. Test results of FM2 chromatographic immunoassay tablets at different concentrations of 7-amino-FM 2. DETAILED DESCRIPTION OF THE INVENTION The present invention uses a β-cell fusion method to prepare a β-cell fusion tumor specific to 7-amino-FM2, a major metabolite of FM2. The method is to use a known cell fusion agent, such as polyethylene glycol (peg), to fuse a mouse myeloma cell line with a B lymphocyte that produces an anti-7-amino-FM2 antibody, and use AT to screen the fusion tumor cell line. Then, indirect enzyme-linked immunosorbent assay (ELISA) and competitive EL ISA were used to analyze the specificity of antibodies in the fusion tumor culture medium, and then a single fusion tumor cell line specific for 7-amino-FM2 was selected. The fusion tumor cell line was then injected into mice 581810 V. Description of the invention (4) Ascites was produced in the abdominal cavity, and this type of enzyme immunoassay test piece (One Stick) was used to detect the specificity of 7-1 in the urine ^ Whether the sensitivity and sensitivity can produce specific antibodies, especially if the molecular weight is less than 1000 d. Due to its small molecular weight, it will not stimulate the immune system molecular hapten and large molecular weight with the carrier's immunogenicity when the immunogen exists alone. Assist the semi-invention to use 7-amino-FM2 as an immunogen to prepare anti-7-amine 7-amino-FM2 derivatives used for monoclonal antibody formulation and develop direct competition

Step Chromatographic Dip Amino-FM2. Sensitivity depends on the characteristics of the antibody, and the design and manufacture of the pathogen is critical. Swallowed drugs, such as 7-amino-FM2, are hapten, that is, the immune response is generally generated. Generally, a small immunogenic carrier protein is required to bind, and the antigen generates immunity. A conjugate of a derivative and a carrier protein. Monoclonal antibody to F-M2. All the following general formulae of the present invention: Η2-& 02C—RfC, Ο

〇 F where κ and dagger can be inserted as long-chain (CH2) n or benzyl as needed, where n = 1 to 6; and-heart ~ (〇0)-(: 112-1- can be restored as needed Into C-C H2-R2-. In a preferred embodiment of the present invention, the 7-amino-FM2 derivative is a 7-amino-FM2 derivative with no substituents inserted into the dagger 581810 5. Description of the invention (5). As mentioned above, suitable carrier proteins include, but are not limited to, albumin, globulin, hemocyanin, or ferritin. In a preferred embodiment of the present invention, a 7-amine group is used. -A conjugate of F M 2 derivative (wherein R! And & have no substituents inserted) and bovine serum albumin (BSA) as the immunogen to generate a specific monoclonal antibody. In another comparison In a preferred embodiment, the fusion tumor cell is a mouse myeloma cell F0 cell line and an anti-7-amino-FM2 derivative (wherein R1 and ^ have no substituents inserted)-bovine serum diprotein murine B The cell line is obtained by fusion. The details are as described in the following examples. The fusion tumor prepared according to the present invention can secrete and produce anti-7 ~ amine groups- The light and heavy chain variable peptides of the FM2 monoclonal antibody, that is, the fusion tumor can secrete and produce heavy chain variable peptides and light chain peptides that are specific to anti-7-amino-FM2 Monoclonal antibody against variable peptides. In order to effectively detect the presence of FM2, the present invention provides a method for detecting the content of FM2 metabolites, 7-amino_FM2, including the following steps: (a) providing a specific anti-7- Monoclonal antibody to amine-FM2; (b) Provide a signal generating tool that can be operatively combined with 7-amino-FM2 to generate a signal; (c) Adhere the single antibody to a support tool To form an antibody-object conjugate, and (d) compete the test sample or the 7-amino-FM2 standard solution: anti-7-amino-FM2 flood number conjugate and compete with the antibody-support conjugate Combination and measure the signal generated by the signal generating tool. Two suitable flood signal generating tools are well known to those skilled in the art and can be selected according to the needs, including radioimmunoassay, fluorescent immunoassay

581810 V. Description of the invention (6) Test, cold light label (such as ^ substance) or enzyme (for example, cold light label or chemical cold light labeling of glycosidase). Suitable = hydrogenase, phospholipase, or galactose, which is composed of polyethylene, poly] tools including a microplate, microspheres, and paper fixing materials. The protein of ethylene, nitrocellulose or Nylon is based on the above-mentioned formula FM2 metabolites, 7-amino group ~ FM2 of the present invention; the state is to provide a detection area; (2) the indicator area is to cover (1) Sample contact test ... and ⑷ liquid hair :: single antibody; ⑶ reactive or solid-phase immunoassay tablets; this set can be used for chromatography for the purpose of rapid analysis. Select a chromatographic immunoassay.誃 烚 二 二 目的 乂 目的 疋 疋 乂 谪 舻 疋 谪 舻 谪 舻 姑 姑 姑 姑 Xi Xi 々 '^ test film 检验 You can provide capillarity and fluid? The material is composed of a negative carrier material, such as #, but: in: chemical fiber film, dragon film, glass fiber film, etc. Suitable agents include, for example, Arihiko | Multi-Cut Eyes, Spoon Inspection, Poison # 7 Ratio, Color Gift Gum Granules, such as Polyethylene, Polyethylene, Polyethylene, Acrylic, Polyacryl, etc. The materials of the material, metal colloidal solutions, such as tannin solution, these raw materials are commercial products. And wood glue is, grain liquid, the suction piece ..., contact the sample to be tested, move the attached sample mouth to the other end according to the capillary phenomenon, so that the test piece. The indicator area is close to the sample contact area. This area is a combination of a dry antibody = 7-amino-FM2 and a highly stable indicator that can be read with the naked eye. The test area is located above the '= two, and the area is fixed with a color-developing reactant. When the amine group is 2: 2, the two: the original conjugate, and the anti-7-amino-FM2 antibody is an indicator.

PBS TMB BSA V. Description of the invention (7) It can show a signal change that can be interpreted by the naked eye. In the reaction test, the spleen & & μ, the degree of 7-amino-FM2 urine, and the end of the contact area and the pre-existing Hanshi, then the sample was in contact. Liquor wets upwards, and dissolves the anti-pyridine-bed / night lye, together with the amine group-FM2 without a single finger, it will wash: 4L, kiss right urine, and test the area 7-Amine-FM2 on the carrier—Anti-7-Amine-FM2 with limited competition on the carrier— "MZ carrier" 1 person said ▲ Shan Cairi did not conjugate, so the base-FM2 contains more , Reading, ρ & ~ σ Master ^ ^ & Ruan sex signal is weaker. Urine pan Φ s can be determined from the presence or absence of the test and its strength. 4) Does the ice shoe f contain 7-amino-FM2? The present invention will be further exemplified by the following examples. These examples are merely examples and are not intended to be limiting. The abbreviations of the English abbreviations appearing before and after this manual are: HAT ·· Hypoxanthine / Aminopterin / thymidine.

Tg: burdock globulin. FCS ·· Fetal bovine serum. DMS0: Jiya Feng. Dish buffer solution. Tetramethyl benzidine Bovine serum albumin. It is known that the standard capillary phenomenon contains 7-antigen in 7-antigen interactions and the description of its content. However, the present invention is determined as follows

58l8l 5. Description of the invention (8) Preparation of 2 albumin: 14 mg of 7-amino-FM2 derivative (where & and dagger have no substituents inserted) are dissolved in 2.8 ml of DMSO; 2.55 ml of the solution was added to 20 ml of bovine serum albumin (100 mg / 0.05 M sodium carbonate solution), and the reaction was stirred in an ice bath. After overnight, the reaction was fully dialyzed in 50 mM dish acid buffer pH 7-4, and then frozen crystals were stored. (2) Born from a fusion tumor, bovine serum albumin conjugate (7-amino-FM2-BSA, with a molar ratio of 30) and Freunds adjuvant (after complete adjuvant with the first immunization, no Complete adjuvant) 1: 1 (v / v) emulsion was injected subcutaneously into BALB / C mice (each used 125 mg of 7-amino-FM2-BSA) once a week for 4 times in a row. It was then reinforced once every four weeks (40 mg of 7-amino-FM2-BSA each) for a total of 5 times. Three days before cell fusion, an additional intravenous injection (without adjuvant) was performed. Three days later, spleen cells were fused with murine myeloma cell line FO (ATCC CRL 1 646) with 50% polyethylene glycol-400 (PEG 400) at a ratio of 1: 3. After fusion, the cells were suspended in RPMI medium (HAT, 20% FCS), diluted to 1 x 105 FO cells, and seeded into 96-well culture plates (0.2 ml / well). After 10 days, the cell culture supernatant was tested in an ELISA plate to which a 7-amino-FM2-bovine thyroid protein complex (7-amino-FM2-Tg, a molar ratio of 30) was adsorbed. Fusion tumors specific for 7-amino-FM2 were selected and singulated by limiting dilution method. This method was used to select single cell fusion tumors 490 3-1 7.2 'and test the secreted monoclonal antibodies. It was found to be ig (ji, Kappa light bond. This fusion tumor (containing 10% DMS0 and 90% FCS) Medium) can be stored

Page 12 581810 V. Description of the invention (9) In a 70t and liquid nitrogen, and can use standard mammalian cell culture technology (RPMI 1 640 with 10% fetal bovine serum supplemented with 200 Glycine (Gin) and 50-mercapto-mercaptoethanol) were cultured. This fusion tumor was deposited at the Strain Preservation and Research Center of the Food Industry Development Institute (Hsinchu, Taiwan) on January 11, 1988. The deposit number is c 960092. 0: Detection of 7-amino-FM2 layer Duplicate immunoassay; ^ U.M2 (, 49 0 3--1 7. ^ 21)-^ Preparation method of the complex In a 15 ml small centrifuge tube, gradually add 1.0 ml of red homogeneous mixed latex (Carboxy-modified polystyrene, 10% solids, 0.17 fm, Seradyn, USA ·), 6.132 ml of borate buffer (50 mM, PΗ8.5 ·), 5.0 mg 4903-1 7.21 antibody. After mixing well, put on a rotary shaker and react at room temperature for 8 hours. The supernatant was then removed by centrifugation at 12,000 xg for 20 minutes at 4 ° C. The pellets were resuspended in 7 ml of 50 mM borate buffer (Ph. 8 · 5). After centrifugation at 12,000 x g, the supernatant was removed. After repeating the washing twice, the antibody-latex particles (pe 1 1 et) were suspended in 7 ml of 10% BSA-borate buffer solution (50%, pH 8 · 5) and placed in a rotary shaker. The reaction was carried out at room temperature for 2 hours. Then, 0.35 g of Trehalose was added, and the solution was stirred and dissolved with a shaker, and then stored at 40 ° C. When used, add 0.75 ml of anti-7-amino-FM2 red latex conjugate plus 50 mM borate buffer (pH 5% with 5% trehalose-1% BSA-〇 · 〇〇 05 8.5), at 20. 〇The drying room with constant temperature and 30% humidity

581810 V. Description of the invention (10) The flow of the latex antibody conjugate is dried on the glass fiber membrane. Preparation of Chromatographic Immunoassay Test Sheet Nitrocellulose fibers (Schleider & Schnell) with a pore size of 5 / zm were cut into thin sheets with a length of 30 cm x 2.0 cm. At the top of the wide side (2.0 cm), about 0. 7.5 cm (at the control area) and 1. 25 cm (at the test area), use spray guns (36 psi). Pressure) rabbit anti-mouse IgG antibody (0.8 mg / ml) and 7-amino-FM2-BSA (0.3 mg / ml) were sprayed on the two places to form a line with a width of about 0.8 mm. After air-drying at room temperature for 30 minutes, it was then placed in blocking buffer (0.25% kusuin, 0.01% Triton X-1〇〇, 3% trehalose-PB (20 mM)). After blocking at room temperature for 30 minutes, take it out and dry it in a constant temperature and humidity room at 2.0%, 30% RH for 30 minutes. Cut out a plastic backboard with a length and width of 30 cm x 7 cm, with glue on one side. Attach the prepared bowl fiber membrane from 4.2 cm to 6.2 cm from the top of the wide side (7 cm). From the top to 3 cm, attach 30 cm X 2.4 cm Filter paper (Whatman). At the bottom of the sample contact area, attach a piece of 30 cm X 1 cm anti-7-amine-FM2 —red latex conjugate conditions' and then cover a layer of 4 · 2 cm X 3 0 along the lower edge of the back plate. Fold the glass fiber membrane in half centimeters. After drying overnight in a constant temperature and humidity room at 20 ° C and 30% RH humidity, cut into 0 · 8 cm x 7 cm thin pieces. During the test, soak the sample contact area at the end in about 200 microliters of urine containing 7-amino-FM2 at 50, 100, 200, 300, 50 0, and 1000 ng / ml, and one without 7-amino-F M2 Pee As a control group, 3 - 5 minutes, if the control area and test areas are a red signal,

Page 14 V. Description of the invention (11) ---- ^ should be negative, indicating that the sample does not contain the 7-amino-FM2 to be detected; only the control area shows a red signal, and the test area does not appear A red signal, "Positive reaction" indicates that the sample contains 7-amino-FM2 to be detected. The test results are shown in Figure 1, which shows that the sensitivity of this test piece is 300 ng / ml. In the specificity test experiment of the present invention, the prepared test piece and 132 commonly used drugs are tested at 100 μg / ml (formulated in artificial urine) for 4 parenchyma reactions; in addition, the present invention further uses benzene Diazepam (BZD) drugs were tested for specificity. Only desmethyldiazepam and flurazepam had cross-reactions at 100 μg / ml. Only dizepam had cross-reactions at 10 μg / ml. Cross reactions occur. The results are shown in Tables 1 and 2. V. Description of the invention (12) Table 1: Test results of cross-reactivity specificity of various drugs and the FM 2 test kit of the present invention

581810 V. Description of the invention (13) epinephrine except for phnctcrminc negative Dclorazepam except for erythromycin except phenylephrine dangerous Diazepam odor ethyl-p-amino- benzoate dangerous phenylethylamine negative Estazolam negative EDTA disodium salt negative phenylpropanolam ine negative Flurazethylmorpham Negative prednisone negative Flunitrazepam (FM2) negative fenoprofen negative procainamide negative Lorazepam negative gentamycin negative procaine negative Lormetazepam negative D-(+)-glucose negative promethazine negative Medazepam negative homotropine negative propoxyphene negative ampuppy negative pyroxypyramid negative pyramid penicillin G-negative Prazepam-negative haloperidol-negative prozosin-negative Temazepam-negative b-hydroxy-phenethyl amine-negative quinine-negative indomethacin-negative ranitidine-negative isoxsuprine-negative salicylic acid-negative 1. Cross-reaction tests were conducted with 132 drugs (100 μg / ml) Very light Analyzing by Yin and Yang have two drugs (Flurazepam, desmethyldiazepam); no coloring reaction, a positive result is determined by one species pharmaceutical (Diazepam). 2. This reagent does not cross-react with other drugs of abuse such as morphine, methamphetamine 'cocaine, etc. 581810 V. Description of the invention (14) Table 2: Test result of cross-reactivity specificity Drug 100 μg / ml 10 μg / ml 1.0 μg / ml Judgment and judgment Alprazepam negative negative Bromazepam negative negative negative Chlordiazepoxide negative negative negative Chorzepate negative negative negative Clonazepam negative negative negative Delorazepam negative negative negative Diazepam negative positive negative desmethyldiazepam negative negative negative Estazolam negative negative negative Flurazepam Facial negative negative Flunitrazepam (FM2) negative negative negative Lorazepam negative negative negative Lormetazepam negative negative negative MedazePam negative negative Negative negative Oxazepam negative negative negative Prazepam negative negative negative Temazepam negative negative negative

Note: Drugs that cross-react with this reagent are desmethyldiazepam (100 μg / ml), flurazepam (100 μg / ml), and diazepam (10 μg / ml).

Although the present invention has been disclosed in the preferred embodiment as above, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and retouches without departing from the spirit and scope of the present invention. The scope of protection shall be determined by the scope of the attached patent application.

Page 18

Claims (1)

581810 _3 .u: j is the revised claw. 6. Scope of patent application of 1st _________________ 1. A fusion tumor 4903- 1 7.2 1. It produces antibodies to 7-amino-FM2, a metabolite of FM2. 2. The fusion tumor according to item 1 of the scope of the patent application, which is a cell line in which osteomyeloma cells are fused with B cells producing anti-7-amino-FM2 antibodies. 3. The fusion tumor according to item 2 of the scope of the patent application, wherein the B cell line is obtained from an animal immunized with a 7-amino-FM2 derivative and a carrier protein conjugate. 4. The fusion tumor according to item 3 of the scope of the patent application, wherein the 7-amino-FM2 derivative has the following general formula: h2 S -N02C -R2 ^ C " c -Ri -hn o Among them, 1 ~ 2 can be optionally inserted with long-chain (CH2) n or phenyl, where 'n = 1 to 6; and -R!-(C = 0) -CH2-optionally reduced to- R ^ CHrCH. 5. The fusion tumor according to item 4 of the scope of the patent application, wherein the 7-amino-FM2 derivative is a 7-amino-FM2 derivative with no substituents inserted in the heart and the heart. 6. The fusion tumor according to item 3 of the scope of patent application, wherein 0296-5421TWFl.ptc Page 19 581810 ------- Case No. 89111403 Rev. 6, Application for a Patent ^ ~ '^-ΐ: The quality is selected from albumin, globulin, hemocyanin and iron A group of proteins. 7. The fusion tumor described in item 6 of the scope of the patent application can secrete a light chain variable region peptide of a monoclonal antibody against 7-amino-FM2. 8 · According to the fusion tumor described in item 6 of the scope of the patent application, it can secrete a heavy chain variable region polypeptide that produces a single antibody against 7-amino-FM2. 9 · According to the fusion tumor described in item 6 of the scope of the patent application, it can secrete and produce a single strain including a heavy chain variable region polypeptide specific to anti-7-amino-FM2 and a light chain variable region polypeptide. antibody. 10 · —A method for detecting the content of 7-amino-FM2 of FM2 metabolite, including the following steps: (a) providing a monoclonal antibody specific for anti-7-amino-FM2, wherein the anti-7-amino-FM2 The single antibody is produced by the fusion tumor 490 3- 1 7. 2 1; (b) Provide a signal generation tool, which can be operatively combined with 7-amino-FM2 to generate a signal; (c) Adhering the monoclonal antibody to a support tool to form an antibody-support co-profile; and (d) Make the test sample or 7-amino-FM2 standard solution compete with the anti-7-amino-FM2 signal conjugate, and bind with the antibody-supporting conjugate, and measure the signal generated by the signal generation tool . 11. The method according to item 10 of the scope of the patent application, wherein the means for generating the signal is selected from the group consisting of a radioimmunoassay, a fluorescent immunoassay, a cold light marker and an enzyme. 1 2 · The method according to item 11 of the scope of patent application, wherein the cold 0296-5421TWl.ptc Page 20 581810 ^ ° Her object is selected from biological cold light markers or chemical cold light markers. 1 3. The method according to item 丨 丨 in the scope of the patent application, wherein the enzyme is modified and selected from the group consisting of catalase, alkaline phosphatase and / 5-galactosidase. 1 4. The method according to item 10 of the scope of the patent application, wherein the supporting tool comprises a micro-drop plate, a micro-sphere, and a protein fixing material selected from the group consisting of polyethylene, polystyrene, petrified fiber, or nylon. Paper. 1 5 · According to the method described in the scope of the patent application No. 丨 0, wherein the A fused tumor is a cell line in which myeloma cells are fused with B cells producing anti-7-amino M2 antibodies. 16. The method according to item 5 of the scope of the patent application, wherein the B cell line is obtained from an animal immunized with a 7-amino-FM2 derivative and a carrier protein conjugate. 1 7. The method according to item 6 of the scope of the patent application, wherein the 7-amino-FM2 derivative has the following general formula: η2 H r S -N〇2C —R2〆, C ——ΗΝ Ο Among them, h and dagger can be inserted as long-chain (CH2) n or benzyl as needed, where n = 1 to 6; and-&-( 〇〇)-CH2_R2 — can be reduced to 〇296-5421TWFl.ptc Page 21 581810 _____ 篆 Dai 89111403 VI. Application scope 1-CH2-R2 1 8 · According to the method described in item 17 of the scope of patent application 'wherein the amine-FM2 derivative is & and no substituent is inserted 7-Amino-FM2 derivative. 19. The method according to item 16 of the scope of the patent application, wherein the carrier protein is selected from the group consisting of albumin, globulin, hemocyanin, and ferritin. 20 · The method according to item 10 of the scope of the patent application, wherein the fusion tumor cell is a murine B cell strain consisting of a mouse myeloma cell F0 cell line and an anti-amido-FM2 derivative-bovine serum albumin Fusion. 21-The method according to item 20 of the scope of patent application 'is cell fusion using polyethylene glycol. 2 2 · A kit for detecting F Μ 2 metabolites 7 -amino-FM 2 includes' (1) sample contact area; (2) indicator area, which includes items 7 to 9 according to the scope of patent application The monoclonal antibody of any one of (3) a reaction test area; and (4) a liquid recovery area. 2 3 · The kit according to item 22 of the scope of the patent application, wherein the remote kit is a chromatographic immunoassay tablet. 24. According to the item set described in Item 23 of the scope of the patent application, wherein the test piece is composed of a porous carrier material that can provide capillary phenomenon and absorb liquid. 25. The kit according to item 24 of the scope of the patent application, wherein the test sheet is selected from the group consisting of filter paper, nitrocellulose membrane, nylon film and glass fiber membrane ", ~ T 〇 Amendment 26. The set according to item 22 of the scope of application for patent, wherein the indicator is selected from the group consisting of colored milk particles, metal colloidal solution and dye colloidal solution. 27. The kit according to item 26 of the scope of the patent application, wherein the colored milk particles include polymers of polyethylene, polyethylene fbenzene, polystyrene-acrylic acid, and polyacryl. The set according to item 26 of the scope of the patent application, wherein the metal knee fluid * includes a capsule solution. 29. The kit according to item 22 of the scope of the patent application, wherein the reaction f-region includes a 7-amino-FM2 derivative and a carrier protein conjugate. # & The set according to item 29 of the scope of the application for patent, wherein the carrier protein is negatively selected from the group consisting of albumin, globulin, hemocyanin, and iron protein. 3 ^ · A method for detecting 7-amino-FM2 in urine, which is a method for detecting urine by using a kit according to any of the items in the scope of patent application, and interpreting the result