Hybridoma cell strain capable of secreting climbazole monoclonal antibody and application of hybridoma cell strain

Abstract

The invention relates to a hybridoma cell strain capable of secreting a climbazole monoclonal antibody and application of the hybridoma cell strain, and belongs to the technical field of immunodetection. The climbazole monoclonal antibody cell strain CLM-5D8 is obtained by synthesizing a climbazole hapten, preparing a climbazole complete antigen and a climbazole coating antigen and carrying out cell fusion and screening, the number of the climbazole monoclonal antibody cell strain CLM-5D8 is CGMCC NO.45924, and the climbazole monoclonal antibody cell strain CLM-5D8 is preserved in the China General Microbiological Culture Collection Center. The hybridoma cell strain secretes the climbazole monoclonal antibody, the climbazole monoclonal antibody has good detection sensitivity and detection specificity to climbazole, the IC50 value to climbazole is 2.03 ng / mL, the cross reaction rates to climbazole structural analogues and functional analogues are both smaller than 1%, a solid foundation is established for a climbazole immunological detection method, and the application prospect is broad. The method can be applied to quantitative detection of climbazole.

Description

Technical Field

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

[0002] Climbazole is a common imidazole fungicide. It is widely used as an active antifungal ingredient in personal care products, such as anti-dandruff shampoo, face cream, foot care products and other detergents. Since climbazole usually contains chlorophenol residues, the presence of climbazole in the aquatic environment will pose a potential risk to aquatic organisms, and studies have shown that it exhibits high ecotoxicity to green algae and duckweed.

[0003] At present, the main methods for the detection of climbazole content in cosmetics and the analysis of climbazole residues in water environments are instrumental methods such as high performance liquid chromatography and gas chromatography. However, these detection methods are time-consuming, cumbersome to operate, and costly, and are not suitable for rapid detection of a large number of samples on site. According to the industry standard QB / T 4128-2010 "High Performance Liquid Chromatography for the Determination of Climbazole (Climbazole) in Cosmetics", the detection limit of climbazole content in cosmetics using high performance liquid chromatography is 0.001%, and the quantitative limit is 0.01%. Therefore, it is of great significance to establish an efficient, rapid, and convenient detection method for climbazole.

[0004] Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive and rapid detection method, and the sample pre-treatment method is simple, the detection cost is low, and the operation is simple. It is very suitable for on-site rapid detection of a large number of samples, providing a new detection method for climbazole detection. The premise of using ELISA to detect climbazole is to obtain a monoclonal antibody with high specificity and high sensitivity to climbazole. Therefore, it is also very important to prepare a monoclonal antibody with high specificity and high sensitivity to climbazole. Summary of the invention

[0005] To solve the above technical problems, the present invention provides a hybridoma cell line secreting climbazole monoclonal antibody, and prepares climbazole complete antigen and coating antigen by synthesizing climbazole hapten. After cell fusion and screening, the climbazole monoclonal antibody cell line CLM-5D8 is obtained, which is numbered CGMCCNO.45924 and preserved in the General Microbiology Center of China National Committee for the Collection of Microbiological Cultures.

[0006] The first object of the present invention is to provide a hybridoma cell strain that secretes climbazole monoclonal antibody, and the deposit number of the hybridoma cell strain is CGMCC NO.45924.

[0007] Furthermore, the method for preparing the hybridoma cell line comprises the following steps:

[0008] Step S1, preparing climbazole hapten, using the obtained climbazole hapten to prepare climbazole complete antigen, mixing the obtained climbazole complete antigen with complete Freund's adjuvant to obtain antigen-containing complete Freund's adjuvant, and emulsifying the obtained climbazole complete antigen with incomplete Freund's adjuvant to obtain antigen-containing incomplete Freund's adjuvant;

[0009] Step S2, using the antigen-containing complete Freund's adjuvant obtained in step S1 to perform the first immunization on the immunized animal;

[0010] Step S3, performing booster immunization on the immunized animal after immunization in step S2 using the incomplete Freund's adjuvant containing the antigen obtained in step S1, and performing sprint immunization using climbazole complete antigen;

[0011] 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;

[0012] Wherein, the climbazole hapten is CLM-COOH, and the structural formula is:

[0013]

[0014] The climbazole complete antigen is CLM-COOH-KLH, and the structural formula is:

[0015]

[0016] The second object of the present invention is to provide the use of the above hybridoma cell line in the preparation of climbazole monoclonal antibody.

[0017] The third object of the present invention is to provide a climbazole monoclonal antibody, which is secreted by the above hybridoma cell line.

[0018] The fourth object of the present invention is to provide a composition comprising the above hybridoma cell line and / or the above climbazole monoclonal antibody.

[0019] The fifth object of the present invention is to provide a chip, wherein the chip contains the above-mentioned hybridoma cell line, the above-mentioned climbazole monoclonal antibody or the above-mentioned composition.

[0020] The sixth object of the present invention is to provide a test strip, wherein the test strip contains the above-mentioned hybridoma cell line, the above-mentioned climbazole monoclonal antibody or the above-mentioned composition.

[0021] Furthermore, the test strip also includes a sample pad, a colloidal gold pad, a water-absorbing pad and an acetate fiber pad, and the colloidal gold pad is coated with climbazole monoclonal antibodies labeled with colloidal gold.

[0022] The seventh object of the present invention is to provide a kit, which contains the above-mentioned hybridoma cell line, the above-mentioned climbazole monoclonal antibody or the above-mentioned composition.

[0023] Furthermore, the kit also includes an ELISA plate, a climbazole-coated antigen, a climbazole standard solution, an enzyme-labeled secondary antibody and a color developing solution.

[0024] The eighth object of the present invention is to provide the use of the above-mentioned climbazole monoclonal antibody, the above-mentioned composition, the above-mentioned chip, the above-mentioned test strip or the above-mentioned kit in detecting climbazole.

[0025] Beneficial effects of the present invention:

[0026] The hybridoma cell line provided by the present invention secretes a climbazole monoclonal antibody, which has good detection sensitivity and detection specificity for climbazole, and the IC 50 The value was 2.03 ng / mL, and the cross-reaction rates to the structural analogs and functional analogs of climbazole were both less than 1%, which established a solid foundation for the immunological detection method of climbazole and can be applied to the quantitative detection of climbazole.

[0027] Biomaterial Deposit

[0028] The hybridoma cell line CLM-5D8 secreting climbazole monoclonal antibody is deposited in the General Microbiology Center CGMCC of the China Microbiological Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named as monoclonal cell line, the deposit date is April 18, 2024, and the deposit number is CGMCC No.45924. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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

[0030] Figure 1 This is the standard curve of the inhibition of climbazole by the climbazole monoclonal antibody in the embodiment of the present invention. DETAILED DESCRIPTION

[0031] 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.

[0032] The reagents involved in the following examples are as follows:

[0033] Carbonate buffer (CBS): Weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them in a small amount of double distilled water, mix them, add double distilled water to about 800 mL, adjust the pH to 9.6, add double distilled water to 1000 mL, and store at 4°C for later use;

[0034] Phosphate buffered saline (PBS): 8 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;

[0035] PBST: PBS containing 0.05% Tween 20;

[0036] Antibody diluent: PBS with 0.1% gelatin.

[0037] TMB colorimetric solution: Solution A: 18.43 g Na2HPO4·12H2O, 9.33 g citric acid, dilute to 1000 mL with pure water; Solution B: 60 mg TMB dissolved in 100 mL ethylene glycol. Mixing solution A and solution B in a volume ratio of 5:1 is the TMB colorimetric solution.

[0038] The detection methods involved in the following embodiments are as follows:

[0039] Climbazole 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 optimal working point, the climbazole standard was diluted to 0, 0.14, 0.41, 1.23, 3.7, 11.11, 33.3 and 100 ng / mL, and the ic-ELISA operation steps were followed. Finally, OriginPro 9 was used to draw the graph to obtain the climbazole standard inhibition curve and calculate the IC 50 value.

[0040] Example 1: Synthesis of Climbazole Hapten

[0041] Since the small molecule of climbazole is not immunogenic and cannot stimulate mice to produce an immune response and then produce antibodies, it is necessary to couple climbazole to the protein through protein coupling technology to make it immunogenic; the active groups commonly used in protein coupling technology are amino, carboxyl, hydroxyl, thiol, etc. Since the molecular structure of climbazole does not contain amino, carboxyl, and hydroxyl groups, it is necessary to derive carboxyl groups from its structure.

[0042] The structure of the climbazole hapten derived from the present invention is as follows:

[0043]

[0044] The derivation process is as follows:

[0045] 100 mg of climbazole was dissolved in 150 μL of pyridine, stirred and dissolved at 25°C, 130 mg of carboxymethylhydroxylamine hemihydrochloride was added under stirring, the mixture was stirred and reacted in a 60°C water bath for 4 hours, taken out and kept in dark overnight at room temperature, dried with nitrogen at 40°C, 6 mL of ethyl acetate was added for redissolution, and the excess carboxymethylhydroxylamine hemihydrochloride was removed by extraction with 2 mL of pure water, and the operation was repeated 3 times. The organic phase was dried with nitrogen to obtain the product CLM-COOH.

[0046] Example 2: Synthesis of Climbazole Complete Antigen

[0047] 3.4 mg CLM-COOH was weighed and dissolved in 200 μL anhydrous N, N-dimethylformamide, 4.2 mg N-hydroxysuccinimide was added under stirring at room temperature, and the mixture was reacted for 30 minutes, followed by adding 7.4 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and reacted at room temperature for 12 hours. The resulting mixture was called liquid A; then 8 mg keyhole limpet hemocyanin (KLH) was weighed and dissolved in 2 mL carbonate buffer, which was called liquid B; liquid A was slowly dripped into liquid B, and the mixture was reacted at room temperature for 12 hours under stirring, and dialyzed with phosphate buffer PBS for 3 days to obtain CLM-COOH-KLH, and the complete antigen CLM-COOH-KLH was obtained, and was identified by ultraviolet absorption scanning method. The structural formula of the complete antigen of climbazole is shown as follows:

[0048]

[0049] Example 3: Synthesis of Climbazole Coating

[0050] 2.6 mg CLM-COOH was weighed and dissolved in 200 μL anhydrous N, N-dimethylformamide, 3.5 mg N-hydroxysuccinimide was added under stirring at room temperature, and the mixture was reacted for 30 minutes. Subsequently, 6.3 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was added and the mixture was reacted at room temperature for 12 hours. The resulting mixture was called liquid A. Then 8 mg chicken ovalbumin (OVA) was weighed and dissolved in 2 mL carbonate buffer, which was called liquid B. Liquid A was slowly dripped into liquid B, and the mixture was reacted at room temperature for 12 hours under stirring. Subsequently, the mixture was dialyzed with phosphate buffer PBS for 3 days to obtain CLM-COOH-OVA, which was frozen at -20°C for later use.

[0051] Example 4: Preparation of hybridoma cell lines secreting climbazole monoclonal antibodies

[0052] 1. Mouse immunization: After the complete climbazole antigen was mixed and emulsified with an equal amount of Freund's adjuvant, BALB / c mice were immunized by multiple subcutaneous injections at the back of the neck (except for sprint immunization); the first immunization used complete Freund's adjuvant, the dose was 100μg / mouse; multiple booster immunizations used incomplete Freund's adjuvant and the dose was halved to 50μg / mouse; sprint immunizations did not use adjuvants, directly diluted with physiological saline and injected intraperitoneally, and the dose was halved again to 25μg / mouse; the first immunization and the second booster immunization were separated by one month, the multiple booster immunizations were separated by 21 days, and the sprint immunization and the last booster immunization were separated by 18-21 days; the immune effect of mice was observed by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), that is, the titer and inhibition of mouse serum were 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, SP2 / 0 tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. Before fusion, the number of SP2 / 0 tumor cells should reach (1-4)×10 7 , ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. When fusion occurs, tumor cells are collected, suspended in RPMI-1640 basal culture medium, and cell counts are performed;

[0056] c. Fusion process 7 minutes: In the first minute, 1 mL of PEG 1500 was added to the cells from slow to fast speed; in the second minute, it was allowed to stand; in the third and fourth minutes, 1 mL of RPMI-1640 medium was added within 1 minute; in the fifth and sixth minutes, 2 mL of RPMI-1640 medium was added within 1 minute; in the seventh minute, 1 mL of RPMI-1640 medium was added every 10 seconds; then 37°C water bath was performed for 5 minutes; centrifugation (800 rpm, 8 minutes), 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 96-well cell plates at 200 μL / 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 changed 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 using the ic-ELISA method, and the second step was to use climbazole as the standard and use the ic-ELISA method to measure the inhibitory effect on the positive cells;

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

[0060] Subcloning was performed three times according to the above method, and finally the climbazole monoclonal antibody cell line CLM-5D8 was obtained.

[0061] Example 5: Preparation and identification of climbazole monoclonal antibody

[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 Climbazole hybridoma cells, ascites was collected from the seventh day, and the ascites was purified for antibodies 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] IC of climbazole monoclonal antibody was measured using indirect competitive ELISA 50 The value was 2.03ng / mL, and its IC 50 and cross-reaction rate, the cross-reaction rate of climbazole structure and function analogs is less than 1%, wherein the cross-reaction rate = (IC 50 / Analog IC 50 )×100%. According to the cross-reaction rate value, it can be seen that the antibody has high sensitivity and specificity for climbazole, as shown in Table 1. According to the cross-reaction rate value, it is shown that the antibody has good sensitivity and specificity for climbazole and can be used for climbazole immunoassay detection.

[0065] Table 1 shows the IC values ​​of monoclonal antibodies against climbazole, functional analogs triclosan, triclocarban, benzylchlorophenol, and structural analogs fluconazole, clotrimazole, and miconazole.50 And cross-reaction rate:

[0066] Table 1 Climbazole monoclonal antibody specificity test results

[0067] <![CDATA[IC 50 (ng / mL)]]> Cross-reaction rate (%) Climbazole 2.03 100 Triclosan >200 <1 Triclocarban >200 <1 Benzylchlorophenol >200 <1 Fluconazole >200 <1 Clotrimazole >200 <1 Miconazole >200 <1

[0068] Example 6: Application of Climbazole Monoclonal Antibody

[0069] The monoclonal antibody prepared from the hybridoma cell line CLM-5D8 in vivo by ascites was applied to the ELISA addition recovery test of climbazole. The specific steps are as follows:

[0070] (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 hours, wash the plate three times with PBST solution, 200 μL per well each time, for 3 minutes each time, and pat dry.

[0071] (2) Block with CBS containing 0.2% gelatin, 200 μL per well, at 37°C for 2 h, wash the plate three times with PBST, 200 μL per well each time, for 3 min each time, and pat dry;

[0072] (3) Prepare 0, 0.04, 0.12, 0.37, 1.11, 3.33, 10 and 30 ng / mL climbazole standard solutions with phosphate buffered saline (PBS), add the standard solutions and the sample extracts to be tested to the blocked ELISA plate, 50 μL per well, repeat 3 wells for each sample, then add 50 μL of anti-climbazole monoclonal antibody diluted 1:32000 to each well, react at 37°C for 30 minutes, wash the plate and pat dry;

[0073] (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 30 minutes, wash the plate and pat dry;

[0074] (5) Add 100 μL of TMB colorimetric solution to each well. After color development at 37°C for 15 min, add 50 μL of 2 M H2SO4 stop solution to each well and measure the absorbance at 450 nm.

[0075] The standard curve of the inhibition of climbazole by the climbazole monoclonal antibody is shown in Figure 1 As shown, the IC of climbazole monoclonal antibody was determined by IC-ELISA 50 The value was 2.03 ng / mL, indicating that the antibody had good sensitivity to climbazole and could be used for immunoassay detection of climbazole.

[0076] 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 climbazole monoclonal antibody, characterized in that: The deposit number of the hybridoma cell line is CGMCC NO.45924.

2. Use of the hybridoma cell line according to claim 1 in the preparation of climbazole monoclonal antibodies.

3. A climbazole monoclonal antibody, characterized in that: The climbazole monoclonal antibody is secreted by the hybridoma cell line according to claim 1.

4. A composition, characterized in that: The composition comprises the hybridoma cell line according to claim 1 and / or the climbazole monoclonal antibody according to claim 3.

5. A chip, characterized in that: The chip contains the hybridoma cell line according to claim 1, the climbazole monoclonal antibody according to claim 3 or the composition according to claim 4.

6. A test strip, characterized in that: The test strip contains the hybridoma cell line according to claim 1, the climbazole monoclonal antibody according to claim 3 or the composition according to claim 4.

7. The test strip according to claim 6, characterized in that: The test strip also includes a sample pad, a colloidal gold pad, a water-absorbing pad and an acetate fiber pad, and the colloidal gold pad is coated with climbazole monoclonal antibody marked with colloidal gold.

8. A kit, characterized in that: The kit contains the hybridoma cell line according to claim 1, the climbazole monoclonal antibody according to claim 3 or the composition according to claim 4.

9. The kit according to claim 8, characterized in that: The kit also includes an ELISA plate, a climbazole-coated antigen, a climbazole standard solution, an enzyme-labeled secondary antibody and a color developing solution.

10. Use of the climbazole monoclonal antibody according to claim 3, the composition according to claim 4, the chip according to claim 5, the test strip according to any one of claims 6-7, or the kit according to any one of claims 8-9 in detecting climbazole.

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