A hybridoma cell strain secreting a monoclonal antibody of clotrimazole and its application

Abstract

The application provides a hybridoma cell strain secreting a monoclone antibody of clotrimazole and application thereof, and relates to the technical field of immune detection. The monoclone antibody secreted by the hybridoma cell strain has good sensitivity and specificity to clotrimazole, wherein the IC 50 value of clotrimazole is 7.386 ng / mL, and the monoclone antibody has good sensitivity; and the monoclone antibody has no cross reaction to functional analogues of clotrimazole, such as miconazole, econazole, ketoconazole, bifonazole and sertaconazole, and has good specificity. Therefore, the monoclone antibody of clotrimazole secreted by the hybridoma cell strain can accurately detect low-concentration clotrimazole.

Description

TECHNICAL FIELD

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

[0002] Clotrimazole, namely 1-[(2-chlorophenyl)diphenylmethyl]-1H-imidazole (CMZ), is a commonly used imidazole antifungal drug, which has important application value in clinical treatment and daily life for the prevention and treatment of fungal infection. In the livestock and breeding industry, animals can also be attacked by various fungi, and the use of clotrimazole to treat fungal infection diseases of animals may lead to drug residues in animal-derived foods (such as meat, dairy products, etc.). If clotrimazole residues exist in water environment and soil, it will affect the survival of organisms and the ecological environment. Therefore, it is necessary to detect the content of clotrimazole in animal-derived foods and environment.

[0003] The analysis method for determining the content of clotrimazole is currently less studied, mainly including instrument 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 time-consuming, high cost, complicated operation steps, and inability to conduct on-site rapid detection, etc. Therefore, it is of great significance to establish a rapid and simple detection method for clotrimazole. Enzyme-linked immunosorbent assay (ELISA) is an extremely 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 clotrimazole detection. However, there is a lack of a monoclone antibody with high sensitivity and high specificity for clotrimazole in the prior art. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to overcome the problem that there is a lack of a monoclone antibody with high sensitivity and high specificity for clotrimazole in the prior art.

[0005] To solve the above technical problem, the present application provides a hybridoma cell strain secreting monoclone antibody of clotrimazole and application thereof. The monoclone antibody secreted by the hybridoma cell strain of the present application has good sensitivity and specificity for clotrimazole, wherein the IC 50 value of clotrimazole is 7.386 ng / mL, which has good sensitivity; and there is no cross reaction for functional analogues of clotrimazole, such as miconazole, econazole, ketoconazole, bifonazole and sertaconazole, which has good specificity. Therefore, the monoclone antibody of clotrimazole secreted by the hybridoma cell strain of the present application can accurately detect low concentration of clotrimazole.

[0006] The first object of the present application is to provide a hybridoma cell strain, which is preserved in the China General Microbiological Culture Collection Center on October 31, 2024, and the address of the preservation center is No. 1, Beichen West Road, Haidian District, Beijing, and the preservation number is CGMCC No. 46227.

[0007] Further, the hybridoma cell strain is obtained by immunizing animals with a complete antigen prepared from a hapten, wherein the structure of the hapten is shown as Formula I:

[0008]

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

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

[0011] The second object of the present application is to provide an application of the above-mentioned hybridoma cell strain in detecting clotrimazole.

[0012] The third object of the present application is to provide a monoclonal antibody secreted by the above-mentioned hybridoma cell strain.

[0013] The fourth object of the present application is to provide an application of the above-mentioned monoclonal antibody in detecting clotrimazole.

[0014] The fifth object of the present application is to provide a detection product of clotrimazole, which includes the above-mentioned monoclonal antibody.

[0015] Further, the detection product further includes a coating agent.

[0016] Further, the coating agent is prepared by coupling a hapten with a carrier protein, wherein the carrier protein includes chicken egg white protein.

[0017] The beneficial effects of the present application are as follows:

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

[0019] Biological material preservation

[0020] A monoclonal cell strain FEP, which was preserved in China General Microbiological Culture Collection Center on October 31, 2024, has a preservation number of CGMCC No. 46227 and a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF DRAWINGS

[0021] 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

[0022] Figure 1 is a standard inhibition curve of the clotrimazole monoclonal antibody of the present application on clotrimazole;

[0023] Figure 2 is a preparation process of the hapten of clotrimazole of the present application. DETAILED DESCRIPTION

[0024] 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.

[0025] The culture media involved in the following examples are as follows:

[0026] RPMI-1640 culture 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 dihydrogen phosphate 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.

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

[0028] Carbonate buffer (CBS): Na2CO3 1.59 g, NaHCO3 2.93 g, respectively dissolved in a small amount of double distilled water, mixed, added with double distilled water to about 800 mL, mixed, pH value adjusted to 9.6, added with double distilled water to constant volume to 1000 mL, stored at 4℃ for standby;

[0029] Phosphate buffer (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, constant volume to 1000 mL with ultrapure water;

[0030] Washing solution (PBST): 0.5 mL of Tween-20 was added 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 developing solution: A solution: Na2HPO4·12H2O 18.43 g, citric acid 9.33 g, constant volume to 1000 mL with pure water; B solution: 60 mg TMB dissolved in 100 mL ethylene glycol. A and B solutions were mixed in a volume ratio of 5:1 to obtain TMB developing solution, which was mixed immediately before use.

[0034] The detection method involved in the following examples is as follows:

[0035] Clotrimazole inhibition rate detection method: The most appropriate 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 best 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, the Origin2024 was used to draw a graph to obtain the standard inhibition curve of clotrimazole, and the IC 50 .

[0036] Example 1: Preparation of hapten

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

[0038]

[0039] Example 2: Preparation of a complete antigen

[0040] Example 2: Preparation of a complete antigen

[0041] Example 3: Preparation of a coating antigen

[0042] Example 3: Preparation of a coating antigen

[0043] Example 4: Preparation of a hybridoma cell line

[0044] 1. Immunization of mice: Healthy 6-8 week old BALB / c mice were selected for immunization. After mixing and emulsifying the clotrimazole immunogen with an equal amount of Freund's adjuvant, the BALB / c mice were immunized by subcutaneous injection on the back. Complete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used thereafter. The interval between the first immunization and the second booster immunization was 28 days, and the interval between multiple booster immunizations was 21 days. Blood was 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 were determined using icELISA. Mice with high titers and good inhibition were selected for a fifth immunization, and a 21-day post-immunization boost was performed by intraperitoneal injection, with a requirement for a half dose and without any adjuvant.

[0045] 2. Cell fusion: Three days after the boost, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 1500) method, with the following specific steps:

[0046] a. After the mice were sacrificed by cervical dislocation, the eyes were immediately disinfected in 75% alcohol for about 5 minutes, and the spleen was removed under sterile conditions. The spleen was gently ground with a syringe rubber head and passed through a 200-mesh cell sieve to obtain a spleen cell suspension. The suspension was collected and centrifuged (1200 rpm, 8 minutes). The spleen cells were washed three times with RPMI-1640 medium, and after the last centrifugation, the spleen cells were diluted to a certain volume, counted, and stored for use;

[0047] b. Collection of mouse 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) × 10 7 before fusion to ensure that the SP2 / 0 tumor cells were in the logarithmic growth phase. At the time of fusion, the tumor cells were collected and suspended in RPMI-1640 base medium for cell counting;

[0048] c. Fusion process: 7 minutes. In the first minute, 1 mL of PEG 1500 was slowly added to the cells; in the second minute, the cells were left undisturbed. 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; and in the seventh minute, 1 mL of RPMI-1640 medium was added every 10 seconds. Then, the cells were incubated at 37°C for 5-8 minutes. Centrifugation (800 rpm, 8 minutes) was performed, the supernatant was discarded, and the cells were resuspended in RPMI-1640 medium containing 20% fetal bovine serum and 2% 50×HAT for screening. The cells were added to a 96-well cell plate at 200 μL / well and incubated at 37°C in a 5% CO2 incubator.

[0049] 3. Cell Screening and Cell Line Establishment: On day 3 of cell fusion, the fused cells were partially replaced with RPMI-1640 selection medium. On day 5, the medium was completely replaced with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 1% 100×HT. On day 7, the cell supernatant was collected for screening. Screening consisted of two steps: First, positive cell wells were selected using icELISA. Second, clotrimazole was used as a standard, and its inhibitory effect on positive cells was determined using icELISA. Cell wells showing good inhibition against the clotrimazole standard were selected, and subcloning was performed using the limiting dilution method. The same method was repeated three times to obtain the cell line.

[0050] Example 5: Preparation and Identification of Monoclonal Antibodies

[0051] 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil; 7 days later, each mouse was injected intraperitoneally with 1×10 6 Hybridoma cells were used, and ascites fluid was collected starting on day 7. The ascites fluid was purified using the caprylic acid-ammonium sulfate method. Under slightly acidic conditions, caprylic acid precipitates other proteins in the ascites fluid besides IgG immunoglobulins. The precipitate was then discarded by centrifugation. IgG-type monoclonal antibodies were then precipitated with an equal volume of saturated ammonium sulfate solution, centrifuged, and the supernatant was discarded. The precipitate was dissolved in 0.01M PBS solution (pH 7.4), dialyzed to desalt, and finally the purified monoclonal antibodies were stored at -20°C.

[0052] The IC50 of the monoclonal antibody against clotrimazole was determined using an indirect competitive ELISA method. 50 The concentration was 7.386 ng / mL, and its IC50 for analogues was verified. 50 and cross-reactivity rate, with a cross-reactivity rate of less than 1% to related fungicide analogues, where cross-reactivity rate = (IC50 of clotrimazole) / (IC50 of clotrimazole) 50 ICs of similar types 50 The cross-reactivity rate (CRR) is calculated as 100%. Based on the CRR values, it can be seen that this antibody has high sensitivity and specificity to clotrimazole, as shown in Table 1.

[0053] Table 1. IC50 and cross-reactivity of monoclonal antibodies against clotrimazole and its functional analogues:

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

[0055] The monoclonal antibody prepared from hybridoma cell lines via in vivo ascites fluid was used in a clotrimazole spiking and recovery assay. The specific steps are as follows:

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

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

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

[0059] d. Sample addition: Serially dilute the antiserum (antiserum obtained by diluting mouse tail blood with antibody diluent) starting from 1:1000 and add it to each well of the coated sample at 100 μL / well. Incubate at 37°C for 30 minutes. After thorough washing, add HRP-goat anti-mouse IgG diluted 1:3000 at 100 μL / well and incubate at 37°C for 30 minutes.

[0060] e. Color development: Remove the microplate, 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. Termination and Measurement: Add 50 μL of stop solution to each well to terminate the reaction, and then measure the OD of each well using a microplate reader. 450 value.

[0062] The standard curve of clotrimazole monoclonal antibody inhibition against clotrimazole is shown below. Figure 1 As shown, the I-reactivity of monoclonal antibody against clotrimazole was determined by icELISA. C50 The concentration was 7.386 ng / mL, indicating good sensitivity to clotrimazole, and it can be used for the immunoassay of clotrimazole.

[0063] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A hybridoma cell line, characterized in that, The hybridoma cell line was deposited on October 31, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46227.

2. The use of the hybridoma cell line described in claim 1 in the detection of clotrimazole.

3. A monoclonal antibody secreted by the hybridoma cell line of claim 1.

4. The use of the monoclonal antibody according to claim 3 in the detection of clotrimazole.

5. A detection product for clotrimazole, characterized in that, The detection product includes the monoclonal antibody as described in claim 3.

6. The testing product according to claim 5, characterized in that, The tested products also include coating agents.

7. The testing product according to claim 6, characterized in that, The coating is prepared from a hapten-coupled carrier protein, wherein the carrier protein includes chicken ovalbumin.

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