Hybridoma cell strain secreting clotrimazole monoclonal antibody and application thereof

Abstract

The invention provides a hybridoma cell strain secreting a clotrimazole monoclonal antibody and application of the hybridoma cell strain, and relates to the technical field of immunodetection. The monoclonal antibody secreted by the hybridoma cell strain has good sensitivity and specificity to clotrimazole, the IC50 value of clotrimazole is 7.386 ng / mL, and the monoclonal antibody has good sensitivity; moreover, the clotrimazole monoclonal antibody has no cross reaction on functional analogues of clotrimazole, such as miconazole, econazole, ketoconazole, bifonazole and sertaconazole, and has good specificity. Therefore, the clotrimazole monoclonal antibody secreted by the hybridoma cell strain disclosed by the invention can be used for accurately detecting low-concentration clotrimazole.

Description

Technical Field

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

[0002] Clotrimazole, also known as 1-[(2-chlorophenyl)benzhydryl]-1H-imidazole (CMZ), is a commonly used imidazole antifungal drug, which has important application value in clinical treatment and prevention and treatment of fungal infections in daily life. In animal husbandry and breeding, since animals are also attacked by various fungi, the use of clotrimazole to treat fungal infections in animals may cause drug residues in animal-derived foods (such as meat, dairy products, etc.). If there are clotrimazole residues in the water environment and soil, it will affect the survival and ecological environment of organisms. Therefore, it is very necessary to detect the content of clotrimazole in animal-derived foods and the environment.

[0003] There are few analytical methods for measuring the content of clotrimazole at present, mainly including instrumental methods such as ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS), gas chromatography-tandem mass spectrometry (GC-MS / MS) and high performance liquid chromatography (HPLC-UV). These detection methods have the disadvantages of being time-consuming, costly, complicated operation steps, and unable to perform rapid on-site detection. Therefore, it is of great significance to establish a fast and simple clotrimazole detection method. Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive and rapid detection method, which is suitable for rapid on-site detection of a large number of samples and provides a new detection approach for clotrimazole detection. However, the prior art lacks a monoclonal antibody with high sensitivity and high specificity for clotrimazole. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the problem in the prior art of lacking a monoclonal antibody with high sensitivity and high specificity to clotrimazole.

[0005] In order to solve the above technical problems, the present invention provides a hybridoma cell line secreting clotrimazole monoclonal antibody and its application. The monoclonal antibody secreted by the hybridoma cell line of the present invention has good sensitivity and specificity to clotrimazole, wherein the IC 50 The value is 7.386ng / mL, which has good sensitivity; and there is no cross reaction to the functional analogs of clotrimazole, such as miconazole, econazole, ketoconazole, bifonazole, sertaconazole, etc., which has good specificity. Therefore, the clotrimazole monoclonal antibody secreted by the hybridoma cell line of the present invention can accurately detect low concentrations of clotrimazole.

[0006] The first object of the present invention is to provide a hybridoma cell strain, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on October 31, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.46227.

[0007] Furthermore, the hybridoma cell line is obtained by immunizing an animal with a complete antigen prepared by a hapten, wherein the structural formula of the hapten is as shown in Formula I:

[0008]

[0009] Furthermore, the complete antigen is obtained by coupling the hapten to a carrier protein.

[0010] Furthermore, the carrier protein includes bovine serum albumin.

[0011] The second object of the present invention is to provide an application of the hybridoma cell line in detecting clotrimazole.

[0012] The third object of the present invention is to provide a monoclonal antibody secreted by the hybridoma cell line.

[0013] The fourth object of the present invention is to provide a use of the above monoclonal antibody in detecting clotrimazole.

[0014] A fifth object of the present invention is to provide a clotrimazole detection product, which comprises the above-mentioned monoclonal antibody.

[0015] Furthermore, the detection product also includes a coating source.

[0016] Furthermore, the coating agent is prepared by coupling a hapten to a carrier protein, wherein the carrier protein comprises chicken ovalbumin.

[0017] Beneficial effects of the present invention:

[0018] The monoclonal antibody secreted by the hybridoma cell line of the present invention has good sensitivity and specificity to clotrimazole, wherein the IC 50 The value is 7.386ng / mL, which has good sensitivity; and there is no cross reaction to the functional analogs of clotrimazole, such as miconazole, econazole, ketoconazole, bifonazole, sertaconazole, etc., which has good specificity. Therefore, the clotrimazole monoclonal antibody secreted by the hybridoma cell line of the present invention can accurately detect low concentrations of clotrimazole.

[0019] Biomaterial Deposit

[0020] The monoclonal cell line FEP was deposited in the General Microbiology Center of China Culture Collection Administration on October 31, 2024, with the deposit number CGMCC No.46227, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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

[0022] Figure 1 is the inhibition standard curve of clotrimazole by the clotrimazole monoclonal antibody of the present invention;

[0023] Figure 2 The invention discloses a process for preparing the clotrimazole hapten. DETAILED DESCRIPTION

[0024] 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.

[0025] The culture medium involved in the following examples is as follows:

[0026] 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, niacinamide 1, choline chloride 3, pyridoxine hydrochloride 1, riboflavin 0.2, thiamine hydrochloride 1, vitamin B12 0.005, sodium bicarbonate 2000.

[0027] The reagents involved in the following examples are as follows:

[0028] 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;

[0029] 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 with ultrapure water;

[0030] Washing solution (PBST): add 0.5 mL of Tween-20 to 1000 mL of 0.01 mol / L pH 7.4 PBS solution;

[0031] PBST: PBS containing 0.05% Tween-20;

[0032] Antibody diluent: washing solution containing 0.1% gelatin;

[0033] 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 volume ratio of 5:1 to obtain the TMB colorimetric solution, which should be mixed before use.

[0034] The detection methods involved in the following embodiments are as follows:

[0035] Clotrimazole inhibition rate detection method: The most suitable antigen and antibody concentrations in ic-ELISA were selected by chessboard test. The antigen was diluted to 1, 0.3, 0.1 and 0.03 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 1, 0.3, 0.1 and 0.03 μg / mL with antibody diluent. After selecting the optimal working point, the clotrimazole standard was diluted to 500, 166.67, 55.56, 18.52, 6.17, 2.06 and 0.69 ng / mL, and the icELISA operation steps were followed. Finally, Origin2024 was used to draw the graph to obtain the clotrimazole standard inhibition curve and calculate the IC 50 .

[0036] Example 1: Preparation of hapten

[0037] The preparation process of hapten Figure 2As shown, 1g of 1-(1-trityl-1H-imidazole-4-yl)ethanone and 2g of CMO were dissolved in 8mL of DMF and heated in a water bath at 70°C for 5h. The organic solvent was removed by rotary evaporation under reduced pressure to obtain a yellow precipitate. The precipitate was washed three times with deionized water and 1M HCl, and ethanol was added for recrystallization to obtain yellow needle-shaped crystals, that is, the clotrimazole hapten CMZ-COOH as shown in Formula 1 was obtained.

[0038]

[0039] Example 2: Preparation of complete antigen

[0040] 6.12 mg of clotrimazole hapten CMZ-COOH was weighed and dissolved in 400 μL DMF. 4.8 mg of N-hydroxysuccinimide was added under stirring at room temperature for 30 minutes, and then 7.9 mg of 1-ethylcarbodiimide hydrochloride was added and reacted at room temperature for 4 hours. The resulting mixture was called solution A. Then 8 mg of bovine serum albumin (BSA) was weighed and dissolved in 2 mL of carbonate buffer, which was called solution B. Solution A was slowly dripped into solution B, and the mixture was reacted at room temperature for 12 hours under stirring. The mixture was dialyzed with phosphate buffer PBS for 3 days to obtain the conjugate CMZ-COOH-BSA, which was frozen at -20°C for later use.

[0041] Example 3: Preparation of coating

[0042] Weigh 2.7 mg of the prepared CMZ-COOH and dissolve it in 400 μL DMF. Add 3.1 mg of N-hydroxysuccinimide under stirring at room temperature and react for 30 minutes. Then add 4.2 mg of 1-ethylcarbodiimide hydrochloride and react for 4 hours. The resulting mixture is called solution A. Then weigh 10 mg of chicken ovalbumin (OVA) and dissolve it in 2 mL of carbonate buffer, which is called solution B. Slowly drip solution A into solution B, react at room temperature for 12 hours under stirring, and dialyze with 0.01 mol / L phosphate buffer PBS for 3 days to obtain the conjugate CMZ-COOH-OVA, which is frozen at -20°C for later use.

[0043] Example 4: Preparation of hybridoma cell lines

[0044] 1. Immunization of mice: Select healthy 6-8 week old BALB / c mice for immunization. Take the clotrimazole immunogen and mix it with an equal amount of Freund's adjuvant for emulsification, and immunize the BALB / c mice by subcutaneous injection on the back. Complete Freund's adjuvant is used for the first immunization, and incomplete Freund's adjuvant is used thereafter. There is a 28-day interval between the first immunization and the second booster immunization, and a 21-day interval between multiple booster immunizations. Blood is collected 7 days after the third immunization (5 μL of blood collected from the mouse tail + 995 μL of antibody diluent = antiserum), and the mouse serum titer and inhibition are determined by icELISA. Mice with high titer and good inhibition are selected, and a sprint immunization is performed 21 days after the fifth immunization, and the sprint dose is required to be halved and does not contain any adjuvant.

[0045] 2. Cell fusion: Three days after the sprint immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 1500) method. The specific steps are as follows:

[0046] a. Remove the eyeballs to collect blood. After killing the mice by cervical dislocation, immediately put them in 75% alcohol for disinfection and soak for about 5 minutes. Remove the spleen of the mice by aseptic operation, 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, and centrifuge them (1200 rpm, 8 minutes). Wash the spleen cells three times with RPMI-1640 medium. After the last centrifugation, dilute the spleen cells to a certain volume, count them, and set aside;

[0047] b. Collecting mouse myeloma SP2 / 0 cells: 7-10 days before fusion, incubate SP2 / 0 cells in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. Before fusion, the number of SP2 / 0 cells should reach (1-4)×10 7 , to 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;

[0048] c. The fusion process takes 7 minutes. In the first minute, 1 mL of PEG 1500 is added to the cells from slow to fast speed; in the second minute, it is allowed to stand. In the third and fourth minutes, 1 mL of RPMI-1640 medium is added within 1 minute; in the fifth and sixth minutes, 2 mL of RPMI-1640 medium is added within 1 minute; in the seventh minute, 1 mL of RPMI-1640 medium is added every 10 seconds. Then, it is incubated at 37°C for 5-8 minutes. Centrifuge (800 rpm, 8 minutes), discard the supernatant, resuspend in RPMI-1640 screening culture medium containing 20% ​​fetal bovine serum and 2% 50×HAT, add to 96-well cell plates at 200 μL / well, and culture in a 37°C, 5% CO2 incubator.

[0049] 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. The screening was divided into two steps: the first step was to screen the positive cell wells with icELISA, and the second step was to select clotrimazole as the standard and use icELISA to measure the inhibitory effect of positive cells. The cell wells with good inhibition of the clotrimazole standard were selected, subcloned by limiting dilution method, and tested by the same method, repeated three times to obtain cell lines.

[0050] Example 5: Preparation and identification of monoclonal antibodies

[0051] 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 Hybridoma cells, ascites was collected from the 7th day, and the ascites was purified by the caprylic acid-ammonium sulfate method. Under slightly acidic conditions, caprylic acid can precipitate other miscellaneous proteins 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℃.

[0052] The IC of monoclonal antibodies against clotrimazole was determined using an indirect competitive ELISA method. 50 The IC value of the analogues was 7.386 ng / mL, and its IC value was verified. 50 and cross-reaction rate, the cross-reaction rate to related fungicide 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 to clotrimazole, as shown in Table 1.

[0053] Table 1 IC50 and cross-reaction rate of monoclonal antibodies to clotrimazole and its functional analogs:

[0054] <![CDATA[IC 50 (ng / mL)]]> Cross-reaction rate (%) Clotrimazole 7.386 100 Miconazole >112 <1 Econazole >112 <1 Ketoconazole >112 <1 Bifonazole >112 <1 Sertaconazole >112 <1

[0055] The monoclonal antibody prepared by hybridoma cell line through ascites in vivo was applied to the clotrimazole additive recovery test, and the specific steps were as follows:

[0056] a. Coating: The original clotrimazole-OVA was diluted serially from 1 μg / mL with 0.05M pH 9.6 carbonate buffer, 100 μL / well, and reacted at 37°C for 2 hours.

[0057] b. Washing: Pour off the solution in the plate and wash with washing solution 3 times, 3 minutes each time.

[0058] c. Blocking: After patting dry, add 200 μL / well blocking solution and react at 37°C for 2 hours. Wash and dry for later use.

[0059] d. Sample addition: dilute the antiserum (blood is collected by tail amputation of mice, and then diluted with antibody diluent to obtain the antiserum) from 1:1000 and add to the coated wells of each dilution, 100 μL / well, and react at 37°C for 30 minutes; after thorough washing, add 1:3000 diluted HRP-sheep anti-mouse IgG, 100 μL / well, and react at 37°C for 30 minutes.

[0060] e. Color development: Take out the ELISA plate, wash it thoroughly, add 100 μL of TMB color development solution to each well, and react at 37°C in the dark for 15 minutes.

[0061] f. Stop and measure: Add 50 μL of stop solution to each well to stop the reaction, and then measure the OD of each well with an ELISA reader. 450 value.

[0062] The standard curve of inhibition of clotrimazole by monoclonal antibody is shown in Figure 1 As shown, the I of monoclonal antibodies to clotrimazole was determined by icELISA. C50 It is 7.386 ng / mL, indicating that it has good sensitivity to clotrimazole and can be used for clotrimazole immunoassay detection.

[0063] 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, characterized in that The hybridoma cell line was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on October 31, 2024, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.46227.

2. The hybridoma cell line according to claim 1, characterized in that The hybridoma cell line is obtained by immunizing an animal with a complete antigen prepared by a hapten, wherein the structural formula of the hapten is shown in Formula I:

3. The hybridoma cell line according to claim 2, characterized in that The complete antigen is obtained by coupling the hapten to a carrier protein.

4. The hybridoma cell line according to claim 3, characterized in that The carrier protein includes bovine serum albumin.

5. Use of the hybridoma cell line according to any one of claims 1 to 4 in detecting clotrimazole.

6. A monoclonal antibody secreted by the hybridoma cell line according to any one of claims 1 to 4.

7. Use of the monoclonal antibody according to claim 6 in detecting clotrimazole.

8. A clotrimazole detection product, characterized in that: The detection product comprises the monoclonal antibody according to claim 6.

9. The detection product according to claim 8, characterized in that: The detection product also includes a coating source.

10. The detection product according to claim 9, characterized in that: The coating is prepared by coupling hapten with carrier protein, wherein the carrier protein includes chicken ovalbumin.

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