Hybridoma cell strain capable of secreting anthraquinone compound monoclonal antibody and application of hybridoma cell strain

Abstract

The invention relates to a hybridoma cell strain capable of secreting an anthraquinone compound monoclonal antibody and application of the hybridoma cell strain, and belongs to the technical field of immunodetection. A hybridoma cell strain is prepared, the hybridoma cell strain secretes monoclonal antibodies of various anthraquinone compounds including 9, 10-anthradione, emodin, aloe-emodin, rhein, chrysophanol and 1, 2-dihydroxy anthraquinone, the monoclonal antibodies are preserved in the China General Microbiological Culture Collection Center (CGMCC) on April 17, 2025, and the preservation number of the monoclonal antibodies is CGMCC. The preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) NO.46503. The obtained monoclonal antibody has good detection sensitivity and broad-spectrum specificity to anthraquinone compounds, can be applied to preparation of anthraquinone compound detection products, provides a brand new tool for immunological detection of anthraquinone compound residues in the food industry, and has practical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunoassay, in particular to a hybridoma cell strain secreting monoclone antibody of anthraquinone and application thereof. BACKGROUND

[0002] Anthraquinone, chemical formula is C 14 H8O2, molecular weight is 208.21, CAS number is 84-65-1, is a synthetic natural dye, some anthraquinones exist in plants such as rhubarb, cassia seed, senna leaf and other Chinese herbal medicines, and usually exist in the form of glycosides (anthraquinone glycosides), which are one of the effective components of these plants to exert pharmacological effects such as purgation and antibiosis. Some anthraquinones have been used as pesticide intermediates or bactericides, and if used irregularly, they can be left in crops, and long-term intake can be toxic to the human body. Industrial emissions in the environment, such as dye production wastewater, may contain anthraquinones, which pollute crops through soil and water sources, and therefore, the residual amount needs to be detected and controlled to ensure food safety.

[0003] At present, there are few studies on the analysis method for determining the content of anthraquinone, mainly including instrument methods such as gas chromatography-tandem mass spectrometry (GC-MS / MS), high performance liquid chromatography (HPLC-UV), liquid chromatography-tandem mass spectrometry (LC-MS / MS) and the like. These detection methods have the disadvantages of time-consuming, high cost, complicated operation steps and inability to conduct on-site rapid detection, and therefore, it is of great significance to establish a rapid and simple detection method for anthraquinone and anthraquinone compounds. Enzyme-linked immunosorbent assay (ELISA) is a highly efficient, sensitive and rapid detection method, which is suitable for on-site rapid detection of a large number of samples, and provides a new detection approach for anthraquinone and anthraquinone compounds. SUMMARY

[0004] To solve the above technical problems, the present application uses an anthraquinone compound hapten to prepare an anthraquinone compound complete antigen, and through a mouse immunization reaction and cell fusion step, a hybridoma cell strain secreting monoclone antibody of anthraquinone is screened and obtained, which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO.46503.

[0005] The first object of the present application is to provide a hybridoma cell strain secreting monoclone antibody of anthraquinone, and the preservation number of the hybridoma cell strain XDL is CGMCC NO.46503.

[0006] Further, the anthraquinone compound includes 9,10-anthracenedione, emodin, aloe emodin, rhein, chrysophanol and 1,2-dihydroxyanthraquinone.

[0007] Further, the preparation method of the hybridoma cell strain comprises the following steps:

[0008] Step S1, obtaining an anthraquinone compound hapten, preparing an anthraquinone compound complete antigen, and preparing the obtained anthraquinone compound complete antigen into antigen-containing Freund's complete adjuvant and antigen-containing Freund's incomplete adjuvant;

[0009] Step S2, first immunization, booster immunization and priming immunization are performed on the immunized animal, the first immunization adopts the antigen-containing Freund's complete adjuvant in S1, the booster immunization adopts the antigen-containing Freund's incomplete adjuvant in S1, and the priming immunization adopts the anthraquinone compound complete antigen in S1;

[0010] Step S3, the spleen cells and myeloma cells of the immunized animal after the priming immunization in S2 are fused to obtain the hybridoma cell strain of the anthraquinone compound monoclonal antibody.

[0011] Further, the structural formula of the anthraquinone compound hapten is as follows:

[0012]

[0013] Further, in S1, the preparation method of the anthraquinone compound complete antigen comprises the following steps: the anthraquinone compound hapten, N-hydroxysuccinimide and 1-ethylcarbodiimide hydrochloride are mixed and dissolved in a solvent to obtain a mixed solution, and then the mixed solution is added to a carrier protein solution to react to obtain the anthraquinone compound complete antigen.

[0014] In an embodiment of the present application, the carrier protein is chicken ovalbumin (OVA) and / or keyhole limpet hemocyanin (KLH).

[0015] In an embodiment of the present application, the solvent is N,N-dimethylformamide (DMF).

[0016] In an embodiment of the present application, in S1, the antigen-containing Freund's complete adjuvant is an emulsion of equal volumes of Freund's complete adjuvant and the anthraquinone compound complete antigen.

[0017] In an embodiment of the present application, in S1, the antigen-containing Freund's incomplete adjuvant is an emulsion of equal volumes of Freund's incomplete adjuvant and the anthraquinone compound complete antigen.

[0018] In an embodiment of the present application, in S1, the whole immunization process comprises one first immunization, 3-5 booster immunizations and one priming immunization.

[0019] In one embodiment of the present application, in step S2, the interval between the first immunization and the booster immunization during the whole immunization process is 28-31 days, the interval between the booster immunizations is 20-22 days, and the interval between the booster immunization and the boost-up immunization is 18-21 days.

[0020] In one embodiment of the present application, in step S2, the first immunization and the booster immunization are performed by subcutaneous injection on the back of the immunized animal, and the boost-up immunization is performed by intraperitoneal injection.

[0021] In one embodiment of the present application, in step S2, the immunized animal is bled during the booster immunization process, and the serum titer and inhibition rate are detected by indirect competitive enzyme-linked immunosorbent assay (icELISA) to screen the immunized animal with high titer and good inhibition.

[0022] In one embodiment of the present application, the bleeding is performed 6-8 days after the end of the booster immunization process.

[0023] In one embodiment of the present application, in step S3, the cell fusion is performed by culturing the fused cells in HAT medium, detecting the positive cell wells by icELISA, further determining the inhibition effect of the positive cell wells by icELISA, subcloning the positive cell wells with the best inhibition by limited dilution method, and obtaining the hybridoma cell strain.

[0024] In one embodiment of the present application, the HAT medium is RPMI-1640 medium.

[0025] In one embodiment of the present application, the subcloning is performed 2-4 times.

[0026] In one embodiment of the present application, in step S3, the cell fusion is performed by polyethylene glycol (PEG1500) method.

[0027] In one embodiment of the present application, in step S3, the cell fusion is performed 2-4 days after the end of the boost-up immunization.

[0028] The second object of the present application is to provide the use of the above-mentioned hybridoma cell strain in the preparation of a monoclonal antibody of an anthraquinone compound.

[0029] The third object of the present application is to provide a monoclonal antibody of an anthraquinone compound, characterized in that the monoclonal antibody of the anthraquinone compound is secreted by the above-mentioned hybridoma cell strain.

[0030] Further, the preparation method of the anthraquinone compound monoclonal antibody comprises injecting sterile paraffin oil into the abdominal cavity of an immunized animal, then injecting the above hybridoma cell strain into the abdominal cavity, collecting ascites after injection, purifying the ascites, and obtaining the anthraquinone compound monoclonal antibody for low-temperature preservation.

[0031] The fourth object of the present application is to provide a composition comprising the above hybridoma cell strain or the above anthraquinone compound monoclonal antibody.

[0032] The fifth object of the present application is to provide the use of the above hybridoma cell strain, the above anthraquinone compound monoclonal antibody or the above composition in the preparation of an anthraquinone compound detection product.

[0033] Further, the anthraquinone compound detection product comprises a reagent, a test strip, a kit or a biochip.

[0034] Further, the biochip is a protein chip.

[0035] The sixth object of the present application is to provide a test strip containing the above hybridoma cell strain, the above anthraquinone compound monoclonal antibody or the above composition.

[0036] Further, the test strip further comprises a sample pad, a conjugate pad, a water-absorbing pad and a fiber membrane, and the conjugate pad is coated with the anthraquinone compound monoclonal antibody.

[0037] Further, the anthraquinone compound monoclonal antibody is labeled by colloidal gold or fluorescent microspheres.

[0038] The seventh object of the present application is to provide a biochip containing the above hybridoma cell strain, the above anthraquinone compound monoclonal antibody or the above composition.

[0039] The eighth object of the present application is to provide a kit containing the above hybridoma cell strain, the above anthraquinone compound monoclonal antibody or the above composition.

[0040] Further, the kit is an enzyme-linked immunosorbent assay kit, a fluorescent immunosorbent assay kit or a chemiluminescent immunosorbent assay kit.

[0041] The beneficial effects of the present application are as follows:

[0042] The present application prepares a hybridoma cell strain secreting an anthraquinone compound monoclonal antibody, which has broad-spectrum specificity and detection sensitivity for the anthraquinone compound, and the cross rate of the anthraquinone compound monoclonal antibody for anthracene compounds is less than 1%, and the IC 50The value is 3.433 ng / mL, and the IC of emodin, aloe emodin, rhein, chrysophanol and other anthraquinone compounds is less than 5 ng / mL. 50 The value is less than 5 ng / mL, which can realize rapid detection of anthraquinone compound residues in food, and provides a new tool for detection of anthraquinone compounds.

[0043] Biological material preservation

[0044] The hybridoma cell strain XDL secreting the anthraquinone compound monoclonal antibody is preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Haidian District, Beijing, China Institute of Microbiology, and classified as a monoclonal cell strain, with a preservation date of April 17, 2025 and a preservation number of CGMCC NO. 46503. BRIEF DESCRIPTION OF DRAWINGS

[0045] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, in which:

[0046] Figure 1 The anthraquinone compound monoclonal antibody in the embodiments of the present application is used to draw a standard curve for inhibiting anthraquinone compounds. DETAILED DESCRIPTION

[0047] The present application will be further described below in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the present application and implement it, but the embodiments are not limiting to the present application.

[0048] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0049] (1) The culture medium involved in the following examples is as follows:

[0050] RPMI-1640 medium (mg / L): L-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cystine dihydrochloride 65.15, L-glutamic acid 20, glycine 10, L-histidine 15, L-hydroxyproline 20, L-isoleucine 50, L-leucine 50, L-lysine hydrochloride 40, L-methionine 15, L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5, L-tyrosine 23.19, L-valine 20, p-aminobenzoic acid 1, calcium nitrate 100, magnesium sulfate anhydrous 48.84, sodium phosphate monobasic anhydrous 676.13, potassium chloride 400, sodium chloride 6000, glucose 2000, reduced glutathione 1, phenol red 5, L-glutamine 300, biotin 0.2, calcium D-pantothenate 0.25, folic acid 1, i-inositol 35, nicotinamide 1, choline chloride 3, pyridoxine hydrochloride 1, riboflavin 0.2, thiamine hydrochloride 1, vitamin B12 0.005, sodium bicarbonate 2000.

[0051] (2) The reagents involved in the following examples are as follows:

[0052] Carbonate buffer solution (CBS): weigh Na2CO3 1.59 g and NaHCO3 2.93 g, respectively, dissolve in a small amount of double distilled water, mix, add double distilled water to about 800 mL, mix well, adjust the pH value to 9.6, add double distilled water to 1000 mL, store at 4°C for standby;

[0053] Phosphate buffer solution (PBS): 8.00 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, 2.9 g Na2HPO4·12H2O, dissolved in 800 mL of pure water, adjusted to pH 7.2-7.4 with NaOH or HCl, and diluted to 1000 mL with ultrapure water;

[0054] Washing solution (PBST): 1000 mL of 0.01 mol / L pH 7.4 PBS solution, add 0.5 mL of Tween-20;

[0055] PBST: PBS containing 0.05% Tween-20;

[0056] Antibody diluent: washing solution containing 0.1% gelatin;

[0057] TMB developing solution: A solution: Na2HPO4·12H2O 18.43 g, citric acid 9.33 g, pure water to 1000 mL; B solution: 60 mg TMB dissolved in 100 mL ethylene glycol. A and B solutions are mixed in a volume ratio of 5:1 to form TMB developing solution, which is mixed fresh.

[0058] (3) The detection method involved in the following examples is as follows:

[0059] Anthraquinone inhibition rate detection method: The most appropriate antigen and antibody concentration in icELISA is selected by chessboard test. The antigen is diluted to 1, 0.3, 0.1 and 0.03 μg / mL with carbonate buffer solution (CBS), and the antibody is diluted to 1, 0.3, 0.1 and 0.03 μg / mL with antibody diluent. After selecting the optimal working point, the anthraquinone standard is diluted to 100, 33.33, 11.11, 3.70, 1.23, 0.41, and 0.14 ng / mL, and the icELISA operation steps are followed. Finally, the Origin2024 is used for plotting to obtain the anthraquinone standard inhibition curve, and the IC 50 .

[0060] Example

[0061] I. Preparation of complete antigen

[0062] Weigh 13.2 mg of anthracene compound hapten AQ-COOH, dissolve in 200 μL of DMF, add 6.8 mg of N-hydroxysuccinimide under stirring at room temperature, react for 15 min, then add 13.2 mg of 1-ethylcarbodiimide hydrochloride, react at room temperature for 6 h, and the obtained mixture is called A liquid; then weigh 6 mg of keyhole limpet hemocyanin (KLH) and dissolve in 2 mL of carbonate buffer solution, which is called B liquid; slowly drop A liquid into B liquid under stirring, react at room temperature for 24 h, dialyze with phosphate buffer PBS for 3 days, and obtain the conjugate AQ-COOH-KLH, which is stored at -20℃ for standby.

[0063] The structure of the hapten AQ-COOH is as follows:

[0064]

[0065] The structure of the hapten AQ-COOH-KLH is as follows:

[0066]

[0067] II. Preparation of coating antigen

[0068] Take 4.9 mg of anthraquinone hapten AQ-COOH, dissolve in 200 μL of DMF, add 4.3 mg of N-hydroxysuccinimide under stirring at room temperature, react for 30 min, then add 5.8 mg of 1-ethylcarbodiimide hydrochloride, react for 4 h, and the obtained mixture is referred to as A liquid; then take 6 mg of chicken egg white protein (OVA) and dissolve in 2 mL of carbonate buffer, referred to as B liquid; slowly drop A liquid into B liquid, stir and react at room temperature for 12 h, dialyze with 0.01 mol / L phosphate buffer PBS for 3 days, and the obtained conjugate AQ-COOH-OVA is stored at -20℃ for standby use;

[0069] The structure of the hapten AQ-COOH-OVA is shown as follows:

[0070]

[0071] Three, immunization of mice

[0072] Healthy 6-8 week old BALB / c mice are selected for immunization. After mixing and emulsifying the anthraquinone immunogen with an equal amount of Freund's adjuvant, the BALB / c mice are immunized by subcutaneous injection on the back. Complete Freund's adjuvant is used for the first immunization, and incomplete Freund's adjuvant is used thereafter. The interval between the first immunization and the second booster immunization is 28 days, and the interval between multiple booster immunizations is 21 days. Blood is collected 7 days after the third immunization (5 μL of mouse tail blood + 995 μL of antibody diluent = antiserum), and the mouse serum titer and inhibition are determined using icELISA. Mice with high titer and good inhibition are selected for a boost immunization 21 days after the fifth immunization, and the boost immunization is performed by intraperitoneal injection, with the requirement that the dose is halved and does not contain any adjuvant.

[0073] Four, cell fusion

[0074] Three days after the boost immunization, cell fusion is performed according to the conventional PEG (polyethylene glycol, molecular weight 1500) method, and the specific steps are as follows:

[0075] (1) After the mice are sacrificed by cervical dislocation with the aid of carbon dioxide, they are immediately sterilized in 75% alcohol for about 5 min, and the spleen is removed under sterile conditions, ground with a syringe rubber head, and passed through a 200-mesh cell screen to obtain a spleen cell suspension, which is collected and centrifuged (1200 rpm, 8 min). The spleen cells are washed three times with RPMI-1640 medium, and after the last centrifugation, the spleen cells are diluted to a certain volume, counted, and reserved for standby use;

[0076] (2) Collection of murine myeloma SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. The number of SP2 / 0 tumor cells was required to reach (1-4) x 10 7 , to ensure that the SP2 / 0 tumor cells were in the logarithmic growth phase before fusion. At the time of fusion, the tumor cells were collected and suspended in RPMI-1640 base medium for cell counting;

[0077] (3) Fusion process 7 min. At the 1st min, 1 mL of PEG 1500 was added to the cells from slow to fast; at the 2nd min, it was left standing. At the 3rd and 4th min, 1 mL of RPMI-1640 medium was added within 1 min; at the 5th and 6th min, 2 mL of RPMI-1640 medium was added within 1 min; at the 7th min, 1 mL of RPMI-1640 medium was added every 10 seconds. Then it was incubated at 37°C for 5-8 min. Centrifugation (800 rpm, 8 min), discard the supernatant, resuspend in RPMI-1640 screening medium containing 20% fetal bovine serum, 2% 50x HAT, add 200 μL / well to a 96-well cell plate, and incubate in a 37°C, 5% CO2 incubator.

[0078] (4) Cell screening and cell strain establishment: On the 3rd day after cell fusion, the fusion cells were subjected to RPMI-1640 screening medium semi-replacement, on the 5th day, they were subjected to full replacement with RPMI-1640 transition medium containing 20% fetal bovine serum, 1% 100x HT, and on the 7th day, the cell supernatant was taken for screening. The screening was divided into two steps: first, positive cell wells were screened out using icELISA, and second, the positive cells were subjected to inhibition effect determination using icELISA with anthraquinone compound 9,10-anthracenedione as a standard. Cell wells with good inhibition to the anthraquinone compound standard were selected, and subcloning was performed using limited dilution method, which was detected repeatedly three times using the same method to obtain cell strains.

[0079] Test Example

[0080] I. Preparation and identification of monoclonal antibodies

[0081] Take 6-8 week old BALB / c mice, inject 0.5 mL of sterile paraffin oil into the abdominal cavity of each mouse; 7 days later, inject 1 x 10 6Hybridoma cells, from the 7th day, collect ascites, and purify ascites by octanoic acid-ammonium sulfate method. In the partial acid condition, normal octanoic acid can precipitate other impurities in the ascites except IgG immunoglobulin, then centrifugal, and discard the precipitate; then use the same amount of saturated ammonium sulfate solution to precipitate IgG type monoclonal antibody, centrifugal, discard the supernatant, dissolve with 0.01M PBS solution (pH=7.4) after, dialysis desalination, finally get the purified monoclonal antibody to be stored at -20℃.

[0082] Using indirect competitive ELISA method, the IC 50 of the monoclonal antibody to 9,10-anthracenedione is determined to be 3.433 ng / mL, and the IC 50 of the antibody to other natural anthraquinones is verified. 50 The cross-reactivity of the antibody to 9,10-anthracenedione, emodin, aloe emodin, rhein, chrysophanol, 1,2-dihydroxyanthraquinone is 100%, 92.3%, 84.3%, 71.5%, 83.9% and 72.1% respectively, and the cross-reactivity of the antibody to other anthracycline compounds daunorubicin, doxorubicin, epirubicin, pirarubicin, aclarubicin is less than 1%. 50 According to the cross-reactivity value, it can be seen that the antibody has high sensitivity and specificity to anthraquinones, and the specific values are shown in Table 1:

[0083] Table 1 IC 50 and cross-reactivity of monoclonal antibody to anthraquinones

[0084]

[0085] II. Application of Antibody

[0086] The monoclonal antibody prepared by hybridoma cell line through in vivo ascites is applied to the addition recovery test of anthraquinones, and the specific steps are as follows:

[0087] (1) Coating: the coating agent AQ-COOH-OVA is diluted by 100 μL / well from 1 μg / mL, 37°C for 2h;

[0088] (2) Washing: pour out the solution in the plate, and wash with washing solution for 3 times, 3min each time;

[0089] (3) Blocking: after drying, add 200 μL / well of blocking solution, 37°C for 2h. After washing, dry and reserve;

[0090] (4) Add sample: dilute the antiserum (antibodies are diluted to the corresponding multiple after the mouse tail blood is collected, and the antiserum is obtained) from 1:1000 by multiple dilution, and add to the coated wells of each dilution, 100 μL / well, 37°C for 30 min; after washing thoroughly, add HRP-goat anti-mouse IgG diluted by 1:3000, 100 μL / well, 37°C for 30 min;

[0091] (5) Color development: take out the enzyme-labeled plate, wash thoroughly, and add 100 μL of TMB color developing liquid to each well, 37°C for 15 min in the dark;

[0092] (6) Termination and determination: add 50 μL of termination liquid to each well to terminate the reaction, and then determine the OD 450 value of each well by using an enzyme-labeled instrument.

[0093] The inhibition standard curve of the monoclonal antibody of the anthraquinone compound to the anthraquinone compound is shown in Figure 1 The IC 50 of the monoclonal antibody to 9,10-anthracenedione is 3.433 ng / mL, which shows that the monoclonal antibody has good sensitivity to the anthraquinone compound and can be used for the immunoassay detection of the anthraquinone compound.

[0094] For the recovery rate, the food matrix is detected by liquid chromatography tandem mass spectrometry (LC-MS / MS), and the 9,10-anthracenedione residues in the two samples are not detected by LC-MS / MS detection, so the recovery rate is determined by adding 9,10-anthracenedione. As shown in Table 2, the results of the indirect competitive ELISA are similar to those of the LC-MS / MS, which shows that the method for detecting 9,10-anthracenedione based on the 9,10-anthracenedione monoclonal antibody is relatively accurate, and can be used for the immunoassay detection of 9,10-anthracenedione residues in tea and grape food samples. The results are shown in Table 2.

[0095] Table 2 Recovery rate of monoclonal antibody to 9,10-anthracenedione in tea and grape

[0096]

[0097] Obviously, the above examples are only examples for clearly illustrating, and are not limitation to the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments are not required to be exhausted, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A hybridoma cell strain secreting a monoclonal antibody to an anthraquinone compound, characterized by: The preservation number of the hybridoma cell strain is CGMCC NO. 46503.

2. The hybridoma cell line according to claim 1, characterized in that: The anthraquinones include 9,10-anthracenedione, emodin, aloe emodin, rhein, chrysophanol and 1,2-dihydroxyanthraquinone.

3. Use of the hybridoma cell strain of claim 1 or 2 in the preparation of an anthraquinone monoclonal antibody.

4. A monoclonal antibody of an anthraquinone compound, characterized by: The anthraquinone monoclonal antibody is secreted by the hybridoma cell strain of claim 1 or 2.

5. A composition characterized in that: The composition comprises the hybridoma cell strain of claim 1 or 2 or the anthraquinone monoclonal antibody of claim 4.

6. Use of the hybridoma cell strain of claim 1 or 2, the anthraquinone monoclonal antibody of claim 4 or the composition of claim 5 in the preparation of an anthraquinone detection product.

7. A test strip, characterized by: The test strip contains the hybridoma cell strain of claim 1 or 2, the anthraquinone monoclonal antibody of claim 4 or the composition of claim 5.

8. The test strip of claim 7, wherein: The test strip further comprises a sample pad, a conjugate pad, an absorbent pad and a fiber membrane, and the conjugate pad is coated with the anthraquinone monoclonal antibody.

9. A biochip, characterized by: The biochip contains the hybridoma cell strain of claim 1 or 2, the anthraquinone monoclonal antibody of claim 4 or the composition of claim 5.

10. A kit characterized in that: The kit contains the hybridoma cell strain of claim 1 or 2, the anthraquinone monoclonal antibody of claim 4 or the composition of claim 5.

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