Hybridoma cell strain secreting nicloramine monoclonal antibody and application of hybridoma cell strain

Abstract

The invention relates to a hybridoma cell strain secreting a nicloramine monoclonal antibody and application of the hybridoma cell strain, and belongs to the technical field of immunochemistry. 4-amino-3, 5-dichlorobenzoic acid is used as a nicitramine hapten, a nicitramine complete antigen is prepared, a hybridoma cell strain is obtained through screening of cell fusion and indirect competitive enzyme-linked immunosorbent assay, a nicitramine monoclonal antibody secreted by the cell strain has good detection sensitivity to nicitramine, the IC50 value is 3.5 ng / mL, the detection sensitivity is high, and the detection sensitivity is high. The method can be used for establishing an immunological detection method of the nicloramine to detect the residue of the nicloramine in food.

Description

Technical Field

[0001] The invention relates to the technical field of immunochemistry, in particular to a hybridoma cell line secreting chlordiazepoxide monoclonal antibody and application thereof. Background Art

[0002] 2,6-Dichloro-4-nitroaniline, also known as 2,6-dichloro-4-nitroaniline, began to be sold as a fungicide in the late 1950s. As a broad-spectrum agricultural fungicide, it can prevent and control sclerotinia of sweet potatoes, kenaf, cucumbers, lettuce, cotton, tobacco, strawberries, potatoes, etc.; soft rot of sweet potatoes, cotton and peaches; late blight of potatoes and tomatoes; wilt of apricots, almonds and apples; smut of wheat; and blossom end rot of broad beans. 2,6-Dichloro-4-nitroaniline can be mixed with most insecticides, fungicides, Bordeaux mixture and lime sulfur.

[0003] For the detection of chloranil pesticide residues, high performance liquid chromatography, gas chromatography, gas or liquid chromatography-mass spectrometry are often used. However, these methods have the disadvantages of complex sample pretreatment and long detection time, and are not suitable for rapid detection of a large number of samples. In order to safeguard the interests of consumers, it is necessary to establish an efficient and rapid detection method for chloranil. Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive and rapid detection method. During the detection, the pretreatment of the sample is simple, the purification steps are few, the analysis capacity is large, the detection cost is low and the operation is simple. It is suitable for rapid on-site detection of a large number of samples, so it has been widely used in pesticide residue analysis. The premise of using enzyme-linked immunosorbent assay to detect chloranil is to obtain a monoclonal antibody with high specificity and high sensitivity to chloranil. Therefore, it is very critical to find a method for preparing a monoclonal antibody with high specificity and high sensitivity to chloranil. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a hybridoma cell line secreting a monoclonal antibody against chloranil and its application. The monoclonal antibody against chloranil secreted by the hybridoma cell line provided by the present invention has good specificity and detection sensitivity (IC 50 The value is 3.5ng / mL), which can be used to establish an immunological detection method for chlordiazepoxide and detect the residues of chlordiazepoxide in food.

[0005] The first object of the present invention is to provide a hybridoma cell line that secretes a monoclonal antibody to chloranil, which has been deposited in the General Microbiology Center (CGMCC) of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named as a monoclonal cell line. The deposit date is April 18, 2024, and the deposit number is CGMCC No.45917.

[0006] The second object of the present invention is to provide a method for preparing a hybridoma cell line that secretes a monoclonal antibody against chloranil. The chloranil hapten used in the preparation process is 4-amino-3,5-dichlorobenzoic acid, and the molecular structure is:

[0007]

[0008] Furthermore, the complete chlordiazepoxide antigen used in the preparation process is BJS-KLH, and the molecular structure is:

[0009]

[0010] Furthermore, the method for preparing the complete chlordiazepoxide antigen comprises the following steps:

[0011] 4-amino-3,5-dichlorobenzoic acid and N-hydroxysuccinimide (NHS) were weighed and dissolved in N,N-dimethylformamide (DMF), and stirred at room temperature to obtain a mixed solution A; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was weighed, fully dissolved in DMF, and added to the mixed solution A, and stirred at room temperature for 4-6 hours to obtain a mixed solution B; hemocyanin (KLH) was taken, diluted to 3 mg / mL with carbonate buffer, and then the mixed solution B was slowly added dropwise thereto, and reacted at room temperature overnight to obtain a reaction solution; the reaction solution was then dialyzed with phosphate buffer to remove unreacted small molecule haptens to obtain a complete chloranilamine antigen BJS-KLH.

[0012] Furthermore, the preparation method of the chlordiazepoxide-coated antigen comprises the following steps:

[0013] 4-amino-3,5-dichlorobenzoic acid and N-hydroxysuccinimide (NHS) were dissolved in anhydrous N,N-dimethylformamide (DMF), and the mixture was stirred at room temperature to obtain a mixed solution C; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was dissolved in anhydrous N,N-dimethylformamide, and then added to the mixed solution C, and the mixture was stirred at room temperature for reaction for 4-6 hours to obtain a mixed solution D; chicken ovalbumin was diluted to 3 mg / mL with carbonate buffer (CBS), and then the mixed solution D was slowly added dropwise thereto to react to obtain a reaction solution; the reaction solution was dialyzed with PBS solution to remove unreacted small molecule haptens to obtain a chloranilamine-coated antigen (BJS-OVA).

[0014] Furthermore, the method for preparing a hybridoma cell line secreting a monoclonal antibody against chlordiazepoxide comprises the following steps:

[0015] Step S1, using the chloranil hapten to prepare the chloranil complete antigen, mixing the obtained chloranil complete antigen with complete Freund's adjuvant to obtain the antigen-containing complete Freund's adjuvant, and mixing the obtained chloranil complete antigen with incomplete Freund's adjuvant to obtain the antigen-containing incomplete Freund's adjuvant;

[0016] Step S2, subcutaneously immunizing the immunized animal with the antigen-containing complete Freund's adjuvant obtained in step S1;

[0017] Step S3, the immunized animals immunized in step S2 are boosted with the incomplete Freund's adjuvant containing the antigen obtained in step S1, and sprint immunized with the complete chloranil antigen;

[0018] Step S4: taking spleen cells from the immunized animal subjected to sprint immunization in step S3 and performing cell fusion with myeloma cells.

[0019] Step S5, using the indirect ELISA method to screen out the positive cell wells, selecting chlordiazepoxide as the standard, using the indirect ELISA method to measure the inhibitory effect of the positive cells, using the limiting dilution method to perform subcloning, and finally screening out the hybridoma cell line that secretes the chlordiazepoxide monoclonal antibody.

[0020] The third object of the present invention is to provide a monoclonal antibody against chlordiazepoxide, which is secreted by the above hybridoma cell line.

[0021] Furthermore, the preparation method of the monoclonal antibody of chlordiazepoxide comprises the following steps:

[0022] 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil. Seven days later, 1×10 6 For chloranil hybridoma cells, ascites was collected from the 7th day, and antibodies were purified from the ascites by the caprylic acid-saturated ammonium sulfate method. The purified monoclonal antibodies were stored at -20°C.

[0023] The fourth object of the present invention is to provide a composition comprising the above hybridoma cell line and / or the above chloranil monoclonal antibody.

[0024] The fifth object of the present invention is to provide a test strip, which contains the hybridoma cell line, the chloranil monoclonal antibody or the combination.

[0025] The sixth object of the present invention is to provide a kit, which contains the above hybridoma cell line, the above chloranil monoclonal antibody or the above composition.

[0026] The seventh object of the present invention is to provide a chip, which contains the above hybridoma cell line, the above chloranil monoclonal antibody or the above composition.

[0027] The eighth object of the present invention is to provide the use of the above-mentioned chloranil monoclonal antibody, the above-mentioned composition, the above-mentioned test strip, the above-mentioned kit or the above-mentioned chip in the detection of chloranil.

[0028] The above technical solution of the present invention has the following advantages compared with the prior art:

[0029] The monoclonal antibody against chloranilamide secreted by the hybridoma cell line of the present invention has good specificity and detection sensitivity (IC 50 The value is 3.5ng / mL), which can be used to establish an immunological detection method for chlordiazepoxide and detect the residues of chlordiazepoxide in food.

[0030] Biomaterial Deposit

[0031] The hybridoma cell line secreting chlordiazepoxide monoclonal antibody is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and is classified and named as monoclonal cell line. The deposit date is April 18, 2024, and the deposit number is CGMCC No.45917. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0033] Figure 1 It is the inhibition standard curve of the chloranil monoclonal antibody of the present invention on chloranil. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0035] The culture medium involved in the embodiment is as follows:

[0036] 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. Anhydrous magnesium sulfate 48.84. Anhydrous sodium dihydrogen phosphate 676.13. Potassium chloride 400. Sodium chloride 6000. Glucose 2000. Reduced glutathione 1. Phenol red 5. L-glutamine 300. Biotin 0.2. D-calcium 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.

[0037] The reagents involved in the following examples are as follows:

[0038] Carbonate buffer (CBS): Weigh 1.59 g Na2CO3 and 2.93 g NaHCO3, dissolve them in a small amount of double distilled water respectively and mix them, add double distilled water to about 800 mL and mix well, adjust the pH to 9.6, add double distilled water to 1000 mL, and store at 4°C for later use.

[0039] Phosphate buffered saline (PBS): 8.00 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, 2.9 g Na2HPO4·12H2O, dissolved in 800 mL pure water, adjusted to pH 7.2-7.4 with NaOH or HCl, and made up to 1000 mL;

[0040] PBST: PBS containing 0.05% Tween 20;

[0041] Antibody diluent: PBS with 0.1% gelatin.

[0042] TMB colorimetric solution: Solution A: 18.43g Na2HPO4.12H2O, 9.33g citric acid, dilute to 1000mL with pure water; Solution B: 60mg TMB dissolved in 100mL ethylene glycol. Mix solution A and solution B in a ratio of 5:1 to obtain the TMB colorimetric solution, which can be mixed before use.

[0043] Chloroform hapten: The present invention selects 4-amino-3,5-dichlorobenzoic acid as the chloroform hapten, and the molecular structure is:

[0044]

[0045] The detection methods involved in the following embodiments are as follows:

[0046] Chlornitramine inhibition rate detection method: The most suitable antigen and antibody concentrations in ic-ELISA were selected by chessboard test. The antigen was diluted to 0.03, 0.1, 0.3 and 1 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 0.03, 0.1, 0.3 and 1 μg / mL with antibody diluent. After selecting the best working point, the chlornitramine standard was diluted to concentrations of 0, 0.1, 0.3, 1, 3, 9, 27 and 81 ng / mL, and the ic-ELISA operation steps were followed. Finally, OriginPro 8.5 was used to draw the graph (the results are shown in Figure 1 As shown), obtain the standard inhibition curve of chlordiazepoxide and calculate IC 50 .

[0047] Example 1: Synthesis of complete chloranilamide antigen

[0048] 5 mg 4-amino-3,5-dichlorobenzoic acid and 8.28 mg N-hydroxysuccinimide (NHS) were weighed, dissolved in 300 μL N,N-dimethylformamide (DMF), stirred at room temperature for 10 min to obtain mixed solution A; 13.75 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was weighed, fully dissolved in 100 μL DMF, added to mixed solution A, stirred at room temperature for 4-6 h to obtain mixed solution B. 6 mg hemocyanin (KLH) was diluted to 3 mg / mL with 0.01 M carbonate buffer (CBS), and then mixed solution B was slowly added dropwise thereto, reacted at room temperature overnight to obtain a reaction solution; then the reaction solution was dialyzed with 0.01 M PBS solution to remove unreacted small molecule haptens, and the complete chloranilamine antigen BJS-KLH was obtained, and identified by ultraviolet absorption scanning method.

[0049] Example 2: Synthesis of chloranil coated antigen

[0050] 3 mg 4-amino-3,5-dichlorobenzoic acid and 4.97 mg N-hydroxysuccinimide (NHS) were dissolved in 300 μL anhydrous N,N-dimethylformamide (DMF), and the mixture was stirred at room temperature for 10 min to obtain a mixed solution C; 8.25 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was dissolved in 100 μL anhydrous DMF, and then added to the mixed solution C, and the mixture was stirred at room temperature for 4-6 h to obtain a mixed solution D; 6 mg chicken ovalbumin (OVA) was diluted with 1 mL 0.01 M carbonate buffer (CBS), and then the mixed solution D was added dropwise thereto for reaction to obtain a reaction solution; the reaction solution was dialyzed with PBS solution to remove unreacted small molecule haptens to obtain a chloranilamine-coated antigen (BJS-OVA).

[0051] Example 3: Preparation of hybridoma cell lines secreting monoclonal antibodies to chloranil

[0052] 1. Animal immunization: complete Freund's adjuvant containing antigen is obtained by mixing complete Freund's adjuvant containing chloranil with an equal amount of complete Freund's adjuvant, and incomplete Freund's adjuvant containing antigen is obtained by mixing complete Freund's adjuvant containing chloranil with an equal amount of incomplete Freund's adjuvant, and BALB / c mice are immunized by multiple subcutaneous injections at the back of the neck (except for sprint immunization); the complete Freund's adjuvant containing antigen is used for the first immunization, and the dose is 100ug / mouse; the incomplete Freund's adjuvant containing antigen is used for multiple booster immunizations, and the dose is halved to 50ug / mouse; the sprint immunization does not use an adjuvant, and the complete antigen diluted with physiological saline is directly injected intraperitoneally, and the dose is further halved to 25ug / mouse; the interval between the first immunization and the second booster immunization is one month, the interval between multiple booster immunizations is 21 days, and the interval between the sprint immunization and the last booster immunization is 18-21 days; the immune effect of mice is observed by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), that is, the titer and inhibition of mouse serum are detected;

[0053] 2. Cell fusion: Three days after the sprint immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 4000) method. The specific steps are as follows:

[0054] a. Cut off the tail to collect blood. After killing the mice by cervical dislocation, immediately put them in 75% alcohol for disinfection and soak for about 5 minutes. Take out the spleen of the mice aseptically, grind them moderately with the rubber tip of the syringe and pass them through a 200-mesh cell sieve to obtain a spleen cell suspension, collect them, centrifuge them (1200rpm, 8min), wash the spleen cells three times with RPMI-1640 medium, dilute the spleen cells to a certain volume after the last centrifugation, count them, and set aside;

[0055] b. Collect SP2 / 0 cells: 7-10 days before fusion, culture SP2 / 0 tumor cells in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. Before fusion, the number of SP2 / 0 tumor cells per ml of culture medium should reach (1-4)×10 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion, tumor cells are collected and suspended in RPMI-1640 basal culture medium during fusion, and cell counting is performed;

[0056] c. Fusion process 7min: In the first minute, 1mL of PEG 1500 was added to the cells from slow to fast speed; in the second minute, the cells were allowed to stand; in the third and fourth minutes, 1mL of RPMI-1640 medium was added within 1min; in the fifth and sixth minutes, 2mL of RPMI-1640 medium was added within 1min; in the seventh minute, 1mL of RPMI-1640 medium was added every 10s; then the cells were incubated at 37°C for 5min; centrifuged (800rpm, 8min), the supernatant was discarded, and the cells were resuspended in RPMI-1640 screening culture medium containing 20% ​​fetal bovine serum and 2% 50×HAT, and added to a 96-well cell plate at 200μL / well, and cultured in a 37°C, 5% CO2 incubator;

[0057] 3. Cell screening and cell line establishment: On the third day of cell fusion, the fused cells were half-changed with RPMI-1640 screening medium, and on the fifth day, the medium was fully replaced with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 1% 100×HT. On the seventh day, the cell supernatant was taken for screening.

[0058] The screening was divided into two steps: the first step was to screen the positive cell wells by ic-ELISA method, and the second step was to use chlordiazepoxide as the standard and measure the inhibitory effect of positive cells by ic-ELISA method;

[0059] The cell wells with good inhibition of chlordiazepoxide standard were selected and subcloned by limiting dilution method, and tested by the same method 7 days later;

[0060] Subcloning was performed three times according to the above method, and finally a hybridoma cell line secreting chloranil monoclonal antibody was obtained.

[0061] Example 4: Preparation and identification of monoclonal antibodies against chloranilamide

[0062] 8-10 week old BALB / c mice were taken and each mouse was intraperitoneally injected with 1 mL of sterile paraffin oil. Seven days later, each mouse was intraperitoneally injected with 1×10 6 For chloranil hybridoma cells, ascites was collected from day 7 and the antibodies were purified by the caprylic acid-saturated ammonium sulfate method.

[0063] Under slightly acidic conditions, octanoic acid can precipitate other impurities in the ascites except IgG immunoglobulin, and then centrifuge and discard the precipitate; then use an equal amount of saturated ammonium sulfate solution to precipitate IgG type monoclonal antibodies, centrifuge, discard the supernatant, dissolve with 0.01M PBS solution (pH 7.4), dialyze and desalt, and finally obtain the purified monoclonal antibody and store it at -20°C.

[0064] Using indirect competitive ELISA, chloranil monoclonal antibody was used to detect chloranil and the IC of chloranil was determined. 50 The value was 3.5ng / mL, indicating that the detection sensitivity of chloranilamine was very good; at the same time, the chloranilamine monoclonal antibody was used to detect chloranilamine analogs (2,4-dichloroaniline, 4,5-dichloro-2-nitroaniline, 2-chloro-5-nitroaniline, 2-nitro-1,4-phenylenediamine), and the cross-rate was less than 10% (IC 50 >35ng / mL), indicating that the monoclonal antibody has good specificity, as shown in Table 1, and can therefore be used for chloranil immunoassay detection. (Crossover rate = (IC 50 / Analog IC 50 )×100%).

[0065] Table 1 Cross-reaction rate of monoclonal antibodies against chloranilamide

[0066] Aniline compounds <![CDATA[IC 50 Value (ng / ml)]]> Cross-reaction rate Chlornithine 3.5 100％ 2,4-Dichloroaniline >35 ＜10％ 4,5-Dichloro-2-nitroaniline >35 ＜10％ 2-Chloro-5-nitroaniline >35 ＜10％ 2-Nitro-1,4-phenylenediamine >35 ＜10％

[0067] Example 5: Application of monoclonal antibody against chloranilamide

[0068] The monoclonal antibody prepared by hybridoma cell line through ascites in vivo was applied to the ELISA spike recovery test of chlordiazepoxide. The specific steps are as follows:

[0069] (1) Coat a 96-well ELISA plate with 100 μL of the coating agent diluted with carbonate buffer (CBS) at a concentration of 0.3 μg / mL per well. After coating at 37°C for 2 h, wash the plate three times with PBST solution, 200 μL per well each time, for 3 min each time, and pat dry.

[0070] (2) Block with CBS containing 0.2% gelatin, 200 μL per well, block at 37°C for 2 h, wash the plate three times with PBST, 200 μL per well each time, 3 min each time, and pat dry;

[0071] (3) Prepare 0, 0.1, 0.3, 1, 3, 9, 27 and 81 ng / mL chloranil standard solutions with phosphate buffered saline (PBS), add the standard solutions and the sample extracts to the blocked ELISA plate, 50 μL per well, repeat 3 wells for each sample, then add 50 μL of anti-chloranil monoclonal antibody diluted 1:32000 to each well, react at 37°C for 0.5 h, wash the plate and pat dry;

[0072] (4) Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:3000 in PBS containing 0.1% gelatin to each well, react at 37°C for 0.5 h, wash the plate and pat dry;

[0073] (5) Add 100 μL of TMB colorimetric solution to each well, develop at 37°C for 15 min, then add 50 μL of 2 M H2SO4 stop solution to each well and measure the absorbance at 450 nm;

[0074] The standard curve of inhibition of chloranil by monoclonal antibody against chloranil is shown in Figure 1 As shown, the IC of the monoclonal antibody against chlordiazepoxide was determined by IC-ELISA. 50 The value is 3.5 ng / mL, indicating that the antibody has good sensitivity to chloranilamine and can be used for immunoassay detection of chloranilamine.

[0075] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A hybridoma cell line secreting a monoclonal antibody against chlordiazepoxide, characterized in that: The deposit number of the hybridoma cell line is CGMCC No.45917.

2. The method for preparing a hybridoma cell line secreting a monoclonal antibody against chlordiazepoxide according to claim 1, characterized in that: The molecular structure of the chloronitramine hapten used in the preparation process is:

3. The preparation method according to claim 2, characterized in that: The molecular structure of the complete chlordiazepoxide antigen used in the preparation process is:

4. The preparation method according to claim 2 or 3, characterized in that: The following steps are involved: Step S1, using the chloranil hapten to prepare the chloranil complete antigen, mixing the obtained chloranil complete antigen with complete Freund's adjuvant to obtain the antigen-containing complete Freund's adjuvant, and mixing the obtained chloranil complete antigen with incomplete Freund's adjuvant to obtain the antigen-containing incomplete Freund's adjuvant; Step S2, subcutaneously immunizing the immunized animal with the antigen-containing complete Freund's adjuvant obtained in step S1; Step S3, the immunized animals immunized in step S2 are boosted with the incomplete Freund's adjuvant containing the antigen obtained in step S1, and sprint immunized with the complete chloranil antigen; Step S4, taking the spleen cells of the immunized animal subjected to sprint immunization in step S3 and performing cell fusion with myeloma cells to obtain the hybridoma cell line secreting the monoclonal antibody against chlordiazepoxide.

5. A monoclonal antibody against chloranilamide, characterized in that: The monoclonal antibody against chlordiazepoxide is secreted by the hybridoma cell line according to claim 1.

6. A composition, characterized in that The composition comprises the hybridoma cell line according to claim 1 and / or the monoclonal antibody against chlordiazepoxide according to claim 5.

7. A test strip, characterized in that: The test strip contains the hybridoma cell line according to claim 1, the monoclonal antibody against chlordiazepoxide according to claim 5 or the composition according to claim 6.

8. A kit, characterized in that: The kit contains the hybridoma cell line according to claim 1, the monoclonal antibody against chloranilamide according to claim 5 or the composition according to claim 6.

9. A chip, characterized in that: The chip contains the hybridoma cell line according to claim 1, the monoclonal antibody against chlordiazepoxide according to claim 5 or the composition according to claim 6. Use of the monoclonal antibody against chloranil as claimed in claim 5, the composition as claimed in claim 6, the test strip as claimed in claim 7, the kit as claimed in claim 8 or the chip as claimed in claim 9 in detecting chloranil.

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