A hybridoma cell strain secreting a monoclonal antibody of lanreotide and its use

Abstract

The present application relates to a kind of hybridoma cell strains secreting monoclone antibody of ganciclovir and its application, belong to the field of immunodetection technique.The present application is by synthesizing ganciclovir hapten, preparation ganciclovir complete antigen and coating antigen, after cell fusion and screening, obtain ganciclovir monoclone antibody cell strain CLM-5D8, number is CGMCC NO.45924, preserved in China Microbial Culture Collection Center for General Microorganism.The hybridoma cell strain secretes ganciclovir monoclone antibody, has good detection sensitivity and detection specificity to ganciclovir, the IC 50 Value of ganciclovir is 2.03ng / mL, the cross reactivity of ganciclovir structural analog and functional analog is less than 1%, establishes solid foundation for ganciclovir immunological detection method, can be applied to the quantitative detection of ganciclovir.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to a hybridoma cell line that secretes a monoclonal antibody against cyproterone and its applications. Background Technology

[0002] Climbazole is a common imidazole fungicide. It is widely used as an active antifungal ingredient in personal care products, such as anti-dandruff shampoos, face creams, and foot care products. Because climbazole often carries chlorophenol residues, its presence in aquatic environments poses a potential risk to aquatic organisms, and studies have shown that it exhibits high ecotoxicity against green algae and duckweed.

[0003] Currently, the main analytical methods for detecting clonidine content in cosmetics and clonidine residues in aquatic environments are high-performance liquid chromatography (HPLC) and gas chromatography (GC). However, these methods are time-consuming, cumbersome, and costly, making them unsuitable for rapid on-site testing of large numbers of samples. According to the industry standard QB / T 4128-2010 "Determination of Clomidazole (Clonidine) in Cosmetics by High-Performance Liquid Chromatography," the detection limit for clonidine content in cosmetics using HPLC is 0.001%, and the quantitation limit is 0.01%. Therefore, establishing an efficient, rapid, and convenient detection method for clonidine is of great significance.

[0004] Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive, and rapid detection method. Its sample pretreatment is simple, the detection cost is low, and the operation is straightforward, making it well-suited for rapid on-site testing of large numbers of samples. This provides a new detection route for ganbaosu. However, obtaining a monoclonal antibody with high specificity and sensitivity to ganbaosu is a prerequisite for using ELISA to detect it. Therefore, the preparation of such a monoclonal antibody is crucial. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes a monoclonal antibody against galbacin. By synthesizing a galbacin hapten, a complete galbacin antigen and a coating antigen are prepared. After cell fusion and screening, a galbacin monoclonal antibody cell line CLM-5D8 is obtained, numbered CGMCCNO.45924, and is deposited at the China General Microbiological Culture Collection Center.

[0006] The first objective of this invention is to provide a hybridoma cell line that secretes a monoclonal antibody against cyproterone, the hybridoma cell line having the accession number CGMCC NO.45924.

[0007] Furthermore, the method for preparing the above-mentioned hybridoma cell line includes the following steps:

[0008] Step S1: Prepare ganbaosu hapten, prepare ganbaosu complete antigen using the obtained ganbaosu hapten, combine the obtained ganbaosu complete antigen with complete Freund's adjuvant to obtain antigen-containing complete Freund's adjuvant, emulsify the obtained ganbaosu complete antigen and incomplete Freund's adjuvant to obtain antigen-containing incomplete Freund's adjuvant.

[0009] Step S2: The immunized animals are first immunized with the complete Freund's adjuvant containing the antigen obtained in step S1.

[0010] Step S3: The immunized animals in Step S2 are boosted with the incomplete Freund's adjuvant containing antigen obtained in Step S1, and then sprinted with the complete antigen of Ganbaosu.

[0011] Step S4: Take spleen cells and myeloma cells from the immunized animals that underwent sprint immunization in step S3 and fuse them to obtain the hybridoma cell line.

[0012] The ganbaosu hapten is CLM-COOH, with the following structural formula:

[0013]

[0014] The complete antigen of Ganbaosu is CLM-COOH-KLH, with the following structural formula:

[0015]

[0016] A second objective of this invention is to provide the application of the above-mentioned hybridoma cell line in the preparation of ganbaosu monoclonal antibody.

[0017] A third objective of this invention is to provide a galbaurin monoclonal antibody, which is obtained from the above-mentioned hybridoma cell line.

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

[0019] A fifth object of the present invention is to provide a chip containing the above-mentioned hybridoma cell line, the above-mentioned ganbaosu monoclonal antibody, or the above-mentioned composition.

[0020] A sixth object of the present invention is to provide a test strip containing the above-mentioned hybridoma cell line, the above-mentioned ganbaosu monoclonal antibody, or the above-mentioned composition.

[0021] Furthermore, the test strip also includes a sample pad, a colloidal gold pad, an absorbent pad, and a cellulose acetate pad, wherein the colloidal gold pad is coated with colloidal gold-labeled ganbaosu monoclonal antibody.

[0022] A seventh object of the present invention is to provide a kit containing the above-described hybridoma cell line, the above-described ganbaosu monoclonal antibody, or the above-described composition.

[0023] Furthermore, the kit also includes an ELISA plate, galbac-coated antigen, galbac standard solution, enzyme-labeled secondary antibody, and colorimetric solution.

[0024] An eighth object of the present invention is to provide the application of the above-mentioned ganbaosu monoclonal antibody, the above-mentioned composition, the above-mentioned chip, the above-mentioned test strip or the above-mentioned kit in the detection of ganbaosu.

[0025] The beneficial effects of this invention are:

[0026] The hybridoma cell line provided by this invention secretes a monoclonal antibody against galbacin, which exhibits good detection sensitivity and specificity for galbacin, and has an IC50 value of [missing value]. 50 The value was 2.03 ng / mL, and the cross-reactivity with both structural and functional analogues of ganbaosu was less than 1%, which laid a solid foundation for the immunological detection method of ganbaosu and can be applied to the quantitative detection of ganbaosu.

[0027] Preservation of biological materials

[0028] The hybridoma cell line CLM-5D8, which secretes the monoclonal antibody cystatin, is deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. It is classified as a monoclonal cell line, deposited on April 18, 2024, with accession number CGMCC No. 45924. Attached Figure Description

[0029] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0030] Figure 1 This is a standard curve showing the inhibition of ganbaosu monoclonal antibody against ganbaosu in an embodiment of the present invention. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

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

[0033] Carbonate buffer (CBS): Weigh 1.59g of Na2CO3 and 2.93g of NaHCO3, dissolve them separately in a small amount of double-distilled water and mix them together. Add double-distilled water to about 800mL and mix well. Adjust the pH to 9.6 and add double-distilled water to make up to 1000mL. Store at 4℃ for later use.

[0034] Phosphate-buffered saline (PBS): 8g NaCl, 0.2g KCl, 0.2g KH2PO4, 2.9g Na2HPO4·12H2O, dissolved in 800mL pure water, pH adjusted to 7.2-7.4 with NaOH or HCl, and then brought to a final volume of 1000mL.

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

[0036] Antibody dilution solution: PBS with 0.1% gelatin added.

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

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

[0039] Method for detecting the inhibition rate of ganbaosu: The optimal antigen and antibody concentrations for ic-ELISA were selected using a checkerboard assay. 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 dilution buffer. After selecting the optimal operating point, ganbaosu standard was diluted to concentrations of 0, 0.14, 0.41, 1.23, 3.7, 11.11, 33.3, and 100 ng / mL. Following the ic-ELISA procedure, the inhibition curves of the ganbaosu standard were plotted using OriginPro 9, and the IC50 was calculated. 50 value.

[0040] Example 1: Synthesis of ganbaosu hapten

[0041] Since galopyrine small molecules are not immunogenic and cannot stimulate mice to produce an immune response and thus produce antibodies, galopyrine needs to be coupled to proteins through protein coupling technology to acquire immunogenicity. Commonly used active groups in protein coupling technology include amino, carboxyl, hydroxyl, and thiol groups. Since galopyrine does not contain amino, carboxyl, or hydroxyl groups in its molecular structure, it is necessary to derive carboxyl groups from its structure.

[0042] The structure of the derived ganbaosu hapten of this invention is as follows:

[0043]

[0044] The derivation process is as follows:

[0045] Dissolve 100 mg of glycyrrhizin in 150 μL of pyridine and stir at 25 °C. Add 130 mg of carboxymethyl hydroxylamine hemihydrochloride while stirring. Stir the mixture in a 60 °C water bath for 4 hours. Remove the mixture and allow it to stand overnight at room temperature in the dark. Dry under nitrogen at 40 °C. Redissolve the mixture in 6 mL of ethyl acetate and extract with 2 mL of pure water to remove excess carboxymethyl hydroxylamine hemihydrochloride. Repeat this process three times. Dry the organic phase under nitrogen to obtain the product CLM-COOH.

[0046] Example 2: Synthesis of Ganbaosu Complete Antigen

[0047] Weigh 3.4 mg of CLM-COOH and dissolve it in 200 μL of anhydrous N,N-dimethylformamide. Add 4.2 mg of N-hydroxysuccinimide while stirring at room temperature and react for 30 minutes. Then add 7.4 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and react at room temperature for 12 hours. The resulting mixture is called solution A. Then weigh 8 mg of keyhole hemocyanin (KLH) and dissolve it in 2 mL of carbonate buffer, which is called solution B. Slowly add solution A to solution B and react at room temperature for 12 hours while stirring. Dialyze the mixture with phosphate-buffered saline (PBS) for 3 days to obtain CLM-COOH-KLH, thus obtaining the complete antigen CLM-COOH-KLH. Identify the complete antigen CLM-COOH-KLH by ultraviolet absorption scanning. The structural formula of the complete antigen of CLM-COOH-KLH is shown below:

[0048]

[0049] Example 3: Synthesis of glycyrrhizin-coated precursor

[0050] Weigh 2.6 mg of CLM-COOH and dissolve it in 200 μL of anhydrous N,N-dimethylformamide. Add 3.5 mg of N-hydroxysuccinimide while stirring at room temperature and react for 30 minutes. Then add 6.3 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and react at room temperature for 12 hours. The resulting mixture is called solution A. Then weigh 8 mg of ovalbumin (OVA) and dissolve it in 2 mL of carbonate buffer, which is called solution B. Slowly add solution A to solution B and react at room temperature for 12 hours while stirring. Then dialyze the solution against phosphate-buffered saline (PBS) for 3 days to obtain CLM-COOH-OVA, which is then stored at -20°C for later use.

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

[0052] 1. Mouse Immunization: BALB / c mice were immunized by subcutaneous injection at multiple points on the neck and back of the neck with a mixture of complete ganbaosu antigen and an equal volume of Freund's adjuvant (except for sprint immunization). The initial immunization used complete Freund's adjuvant at a dose of 100 μg / mouse. For multiple booster immunizations, incomplete Freund's adjuvant was used at half the dose (50 μg / mouse). For sprint immunization, no adjuvant was used; the adjuvant was diluted directly with physiological saline and injected intraperitoneally at a dose halved (25 μg / mouse). The interval between the initial and second booster immunizations was one month, the interval between multiple booster immunizations was 21 days, and the interval between sprint immunization and the final booster immunization was 18-21 days. The immunization effect was observed using an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), specifically by detecting the serum titer and inhibition.

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

[0054] a. After euthanizing the mouse by tail dislocation and cervical dislocation, immediately disinfect the mouse in 75% alcohol for about 5 minutes. Under aseptic conditions, remove the spleen and grind it moderately with the tip of a syringe and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect the suspension, centrifuge (1200 rpm, 8 min), wash the spleen cells three times with RPMI-1640 medium, and after the last centrifugation, dilute the spleen cells to a certain volume, count them, and set them aside for later use.

[0055] b. Collection of 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 should reach (1-4)×10⁻⁶ cells before fusion. 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 for cell counting during fusion.

[0056] c. Fusion process over 7 minutes: In the first minute, add 1 mL of PEG 1500 to the cells dropwise from slow to fast; in the second minute, let stand; in the third and fourth minutes, add 1 mL of RPMI-1640 medium dropwise over 1 minute; in the fifth and sixth minutes, add 2 mL of RPMI-1640 medium dropwise over 1 minute; in the seventh minute, add 1 mL of RPMI-1640 medium dropwise every 10 seconds; then incubate at 37°C for 5 minutes; centrifuge (800 rpm, 8 minutes), discard the supernatant, resuspend in RPMI-1640 selection medium containing 20% ​​fetal bovine serum and 2% 50×HAT, add 200 μL / well to a 96-well cell plate, and incubate at 37°C in a 5% CO2 incubator;

[0057] 3. Cell screening and cell line establishment: On the 3rd day of cell fusion, the fused cells were screened with RPMI-1640 medium with half medium replacement. On the 5th day, the medium was completely replaced with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 1% 100×HT. On the 7th day, the cell supernatant was collected for screening.

[0058] The screening process consists of two steps: First, positive cell wells are selected using ic-ELISA; second, cyproterone is used as a standard, and the inhibitory effect on positive cells is determined using ic-ELISA.

[0059] Cell wells that showed good inhibition of the cyproterone standard were selected, and subcloning was performed using the limiting dilution method. The same method was used to detect the cells after seven days.

[0060] Three subclonings were performed using the method described above, and the CLM-5D8 monoclonal antibody cell line of Ganbaosu was finally obtained.

[0061] Example 5: Preparation and Identification of Ganbaosu Monoclonal Antibody

[0062] 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 Ganbaosu hybridoma cells were used to collect ascites fluid starting from day 7. The ascites fluid was then purified for antibody using the caprylic acid-saturated ammonium sulfate method.

[0063] Under slightly acidic conditions, octanoic acid can precipitate other proteins in the ascites fluid besides IgG immunoglobulin. After centrifugation, the precipitate is discarded. Then, an equal volume of saturated ammonium sulfate solution is used to precipitate IgG-type monoclonal antibodies. After centrifugation, the supernatant is discarded. The antibodies are dissolved in 0.01M PBS solution (pH=7.4), dialyzed to desalt, and finally purified monoclonal antibodies are obtained and stored at -20℃.

[0064] The IC50 of the ganbaosu monoclonal antibody was measured using an indirect competitive ELISA. 50 The value was 2.03 ng / mL, and its IC50 for analogues was verified. 50 The cross-reactivity rate is less than 1% for structural and functional analogues of ganbaosu, where the cross-reactivity rate = (IC50 of ganbaosu) / (IC50 of ganbaosu). 50 ICs of similar types 50 The cross-reactivity rate (×100%) indicates that this antibody has high sensitivity and specificity for galacin, as shown in Table 1. The cross-reactivity rate demonstrates good sensitivity and specificity for galacin, making it suitable for immunoassay detection of galacin.

[0065] Table 1 shows the IC50 values ​​of monoclonal antibodies against cyproconazole, functional analogs triclosan, triclocarban, benzylchlorophen, and structural analogs fluconazole, clotrimazole, and miconazole.50 and cross-reactivity rate:

[0066] Table 1. Results of Specificity Detection of Ganbaosu Monoclonal Antibody

[0067] <![CDATA[IC 50 (ng / mL)]]> Cross-reactivity rate (%) Ganbaosu 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 Ganbaosu Monoclonal Antibody

[0069] The monoclonal antibody prepared from the hybridoma cell line CLM-5D8 via in vivo ascites fluid was used in the ELISA addition and recovery assay of ganbaosu. The specific steps are as follows:

[0070] (1) Coat a 96-well microplate with 0.3 μg / mL of the coating stock diluted with carbonate buffer (CBS), 100 μL per well. After coating at 37°C for 2 hours, wash the plate three times with PBST washing buffer, 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, block at 37°C for 2 hours, wash the plate three times with PBST washing solution, 200 μL per well each time, 3 minutes each time, and pat dry;

[0072] (3) Prepare 0, 0.04, 0.12, 0.37, 1.11, 3.33, 10 and 30 ng / mL of cyproterone standard solutions using phosphate-buffered saline (PBS). Add the standard solutions and the extracts of the samples to be tested to the sealed microplates, 50 μL per well, and repeat each sample in 3 wells. Then add 50 μL of anti-cyproterone monoclonal antibody diluted 1:32000 to each well. After reacting 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 with PBS containing 0.1% gelatin to each well, react at 37°C for 30 minutes, then wash the plate and pat dry.

[0074] (5) Add 100 μL of TMB colorimetric solution to each well, develop the color at 37℃ for 15 minutes, then add 50 μL of 2M H2SO4 stop solution to each well and measure the absorbance at 450 nm.

[0075] The standard curve of inhibition of ganbaosu monoclonal antibody against ganbaosu is as follows: Figure 1 As shown, the IC50 of the cytokine monoclonal antibody was determined using ic-ELISA. 50 The value was 2.03 ng / mL, indicating that the antibody has good sensitivity to galbacin and can be used for the immunoassay detection of galbacin.

[0076] 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 secreting a monoclonal antibody to galanin, characterized in that: The preservation number of the hybridoma cell strain is CGMCC NO. 45924.

2. Use of the hybridoma cell strain of claim 1 in the preparation of a galantamine monoclonal antibody.

3. A monoclonal antibody to mannosyl-oxidase, characterized by: The galantamine monoclonal antibody is secreted by the hybridoma cell strain of claim 1.

4. A composition characterized in that: The composition comprises the hybridoma cell strain of claim 1 and / or the galantamine monoclonal antibody of claim 3.

5. A chip, characterized by: The chip contains the hybridoma cell strain of claim 1, the galantamine monoclonal antibody of claim 3 or the composition of claim 4.

6. A test strip, characterized by: The test strip contains the hybridoma cell strain of claim 1, the galantamine monoclonal antibody of claim 3 or the composition of claim 4.

7. The test strip of claim 6, wherein: The test strip further comprises a sample pad, a colloidal gold pad, a water absorption pad and an acetic acid fiber pad, and the colloidal gold pad is coated with the galantamine monoclonal antibody labeled with colloidal gold.

8. A kit characterized in that: The kit contains the hybridoma cell strain of claim 1, the galantamine monoclonal antibody of claim 3 or the composition of claim 4.

9. The kit of claim 8, wherein: The kit further comprises an enzyme-labeled plate, a galantamine coated antigen, a galantamine standard solution, an enzyme-labeled secondary antibody and a color developing solution.

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

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