Hybridoma cell strain secreting monoclonal antibody of baclofen and application thereof

Abstract

The present application relates to a hybridoma cell strain secreting benzyl chlorophenol monoclonal antibody and its application, and belongs to the technical field of immune detection. The present application synthesizes benzyl chlorophenol hapten, prepares benzyl chlorophenol complete antigen, and through the steps of mouse immune response and cell fusion, a hybridoma cell strain secreting benzyl chlorophenol monoclonal antibody is screened and named CBP-3D4, which is preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC NO.46222. The monoclonal antibody secreted by the hybridoma cell strain has good sensitivity and specificity, the IC 50 value of the monoclonal antibody secreted by the hybridoma cell strain for benzyl chlorophenol is 97.741 ng / mL, the cross reactivity of the monoclonal antibody for various benzyl chlorophenol functional analogs is less than 1%, and the monoclonal antibody can be applied to the preparation of benzyl chlorophenol detection products to realize the rapid quantification of benzyl chlorophenol.

Description

TECHNICAL FIELD

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

[0002] Clorene (Clorene, 4-Chloro-2-benzyl phenol, CBP), also known as 2-benzyl-4-chlorophenol, 2-benzyl chlorophenol, 4-chloro-2-(phenylmethyl) phenol, chlorobenzene, etc., has broad-spectrum antibacterial activity, and can be used to manufacture various disinfectants, antiseptics and bactericides, and can be used for disinfection in environments such as homes and public places. For example, it can be added to products such as cleaning agents and disinfectant sprays to disinfect object surfaces, effectively killing a variety of bacteria, viruses and fungi and other microorganisms, and reducing the risk of infection. In some places with high hygiene requirements, such as hospitals, laboratories and food processing plants, the disinfecting effect of clorene is particularly important.

[0003] In the production process of personal care products such as cosmetics, clorene may be added as a preservative or bactericide. However, excessive clorene may pose a risk to human health, including skin irritation, eye damage, and potential carcinogenicity. In addition, the presence of clorene in water bodies, soil and air may cause damage to the ecosystem and affect the survival and reproduction of animals and plants. Therefore, detecting the concentration of clorene in personal care products and the environment is crucial for assessing its potential risks to human health and taking appropriate environmental protection measures.

[0004] The analysis method for determining the content of clorene is currently less studied, mainly including 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) methods. These methods often require the use of large instruments and complex and tedious operation steps, and have high requirements for the professional ability of the detection personnel. Therefore, it is of great significance to establish a rapid and simple method for detecting clorene. SUMMARY

[0005] To solve the above technical problems, the present application synthesizes a clorene hapten, prepares a clorene complete antigen, and through a mouse immunization reaction and cell fusion step, a hybridoma cell strain secreting a monoclonal antibody of clorene is screened, named CBP-3D4, and preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC NO.46222.

[0006] The first object of the present application is to provide a hybridoma cell strain secreting a monoclonal antibody of clorene, and the preservation number of the hybridoma cell strain is CGMCC NO.46222.

[0007] Further, the preparation method of the hybridoma cell strain comprises the following steps:

[0008] Step S1, preparing a benzochlorophenol hapten, using the obtained benzochlorophenol hapten to prepare a benzochlorophenol immunogen, emulsifying the obtained benzochlorophenol immunogen and complete Freund's adjuvant to obtain an antigen-containing complete Freund's adjuvant, and emulsifying the obtained benzochlorophenol complete antigen and incomplete Freund's adjuvant to obtain an antigen-containing incomplete Freund's adjuvant;

[0009] Step S2, using the antigen-containing complete Freund's adjuvant obtained in step S1 to immunize an animal for the first time;

[0010] Step S3, using the antigen-containing incomplete Freund's adjuvant obtained in step S1 to immunize the immunized animal in step S2 for a booster immunization, and using a benzochlorophenol immunogen for a priming immunization;

[0011] Step S4, taking the spleen cells and myeloma cells of the immunized animal in step S3 for a priming immunization to perform cell fusion, to obtain the hybridoma cell strain;

[0012] The benzochlorophenol hapten is CBP-COOH, and the structural formula is:

[0013] The benzochlorophenol complete antigen is CBP-COOH-KLH, and the structural formula is:

[0014]

[0015] The second object of the present application is to provide the use of the above-mentioned hybridoma cell strain in the preparation of a benzochlorophenol monoclonal antibody.

[0016] The third object of the present application is to provide a benzochlorophenol monoclonal antibody, which is secreted by the above-mentioned hybridoma cell strain.

[0017] The fourth object of the present application is to provide a composition comprising the above-mentioned hybridoma cell strain and / or the above-mentioned monoclonal antibody.

[0018] The fifth object of the present application is to provide the use of the above-mentioned hybridoma cell strain, the above-mentioned benzochlorophenol monoclonal antibody, or the above-mentioned composition in the preparation of a benzochlorophenol detection product.

[0019] Further, the benzochlorophenol detection product comprises a reagent, a test strip, a kit, or a biochip.

[0020] The sixth object of the present application is to provide a test strip comprising the above-mentioned hybridoma cell strain, the above-mentioned monoclonal antibody, or the above-mentioned composition.

[0021] Furthermore, the test strip also includes a sample pad, a conjugate pad, an absorbent pad, and a cellulose acetate membrane. The conjugate pad is coated with a benzylchlorophenol monoclonal antibody, which is labeled with colloidal gold or fluorescent microspheres.

[0022] A seventh object of the present invention is to provide a kit, characterized in that the kit contains the above-mentioned hybridoma cell line, the above-mentioned benzylchlorophene monoclonal antibody, or the above-mentioned composition.

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

[0024] In one embodiment of the present invention, the enzyme-labeled secondary antibody is labeled with horseradish peroxidase.

[0025] In one embodiment of the present invention, the color developing solution is a TMB color developing solution.

[0026] The beneficial effects of this invention are:

[0027] The monoclonal antibody secreted by the hybridoma cell line provided by this invention has good sensitivity and specificity, and an IC50 value of [missing information - likely related to benzylchlorophenol]. 50 The concentration was 97.741 ng / mL, and the cross-reactivity with various benzylchlorophenol functional analogs was less than 1%. This hybridoma cell line and its secreted monoclonal antibody can be used to prepare various benzylchlorophenol detection products, such as test strips, kits, and biochips, enabling rapid quantification of benzylchlorophenol and meeting detection needs in different scenarios. In addition, the large-scale preparation of monoclonal antibodies using hybridoma cell lines can reduce production costs and improve the cost-effectiveness of detection products, showing broad application prospects.

[0028] Preservation of biological materials

[0029] The hybridoma cell line CBP-3D4, which secretes benzylchlorophene monoclonal antibody, 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 October 31, 2024, with accession number CGMCC No. 46222. Attached Figure Description

[0030] 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...

[0031] Figure 1 This is a standard curve showing the inhibition of benzylchlorophenol by the monoclonal antibody in an embodiment of the present invention. Detailed Implementation

[0032] The present application will be further described with reference to the drawings and specific examples so that those skilled in the art can better understand the present application and implement it.

[0033] The culture media involved in the following examples are as follows:

[0034] 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, magnesium sulfate anhydrous 48.84, sodium phosphate monobasic 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.

[0035] The reagents involved in the following examples are as follows:

[0036] Carbonate buffer solution (CBS): weigh Na2CO3 1.59 g and NaHCO3 2.93 g, respectively, dissolve in a small amount of double distilled water, mix, add double distilled water to about 800 mL, mix well, adjust the pH value to 9.6, add double distilled water to 1000 mL, store at 4°C for standby;

[0037] Phosphate buffer solution (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, and made up to 1000 mL with ultrapure water;

[0038] Washing solution (PBST): 0.5 mL of Tween-20 was added to 1000 mL of 0.01 mol / L PBS solution with pH=7.4;

[0039] PBST: PBS containing 0.05% Tween-20;

[0040] Antibody diluent: washing solution containing 0.1% gelatin;

[0041] TMB color developing solution: A solution: Na2HPO4·12H2O 18.43 g, citric acid 9.33 g, and pure water to 1000 mL; B solution: 60 mg TMB dissolved in 100 mL ethylene glycol. A and B solutions mixed in a volume ratio of 5:1 to form TMB color developing solution, which is mixed immediately before use.

[0042] The detection method involved in the following examples is as follows:

[0043] Benzochlorophenol 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 solution (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 benzochlorophenol standard was diluted to 1800, 600, 300, 100, 33.33, 11.11, and 3.70 ng / mL, and the ic-ELISA operation steps were followed. Finally, the Origin2024 was used to plot the benzochlorophenol standard inhibition curve, and the IC 50 .

[0044] Example 1: Synthesis of complete antigen

[0045] 1. Synthesis of hapten

[0046] 400 mg of benzochlorophenol was dissolved in 500 μL of N,N-dimethylformamide, and the system was clarified. Then 812 mg of bromohexanoic acid and 624 mg of potassium carbonate were added in sequence, and the reaction was stirred at 70°C for 6 hours. After the reaction was completed, the temperature was lowered to 25°C, and the product was filtered, washed, and dried to obtain the benzochlorophenol hapten CBP-COOH. The reaction process for synthesizing the hapten is shown below:

[0047]

[0048] 2. Synthesis of complete antigen

[0049] (1) Preparation of immunogen benzochlorophenol-KLH: 3.4 mg of CBP-COOH prepared was dissolved in 200 μL of DMF, and 4.89 mg of N-hydroxysuccinimide (NHS) was added under stirring at room temperature for 30 minutes. Then 7.24 mg of 1-ethylcarbodiimide hydrochloride (EDC) was added, and the reaction was carried out at room temperature for 4 hours to obtain a mixture, which was denoted as A solution. Then 8 mg of hemocyanin (KLH) was dissolved in 2 mL of carbonate buffer solution to obtain B solution. A solution was slowly added dropwise into B solution, and the reaction was carried out at room temperature for 12 hours under stirring. The product was dialyzed against 0.01 mol / L phosphate buffer PBS for 3 days to obtain the conjugate spirolactone and its metabolite-BSA, which was stored at -20°C for later use;

[0050]

[0051] (2) Preparation of coated benzoic acid chlorophenol-OVA: 6.12 mg of the prepared benzoic acid chlorophenol hapten CBP-COOH was weighed and dissolved in 200 μL of DMF. After 30 minutes of stirring at room temperature, 9.39 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was added, and the mixture was reacted at room temperature for 4 hours. The resulting mixture was referred to as A liquid. Then, 8 mg of chicken egg albumin (OVA) was weighed and dissolved in 2 mL of carbonate buffer to obtain B liquid. A liquid was slowly added to B liquid, and the mixture was stirred and reacted at room temperature for 12 hours. The product was dialyzed against PBS for 3 days to obtain the conjugate CBP-COOH-OVA, which was stored at -20°C for later use. The reaction process is as follows:

[0052]

[0053] Example 2: Preparation of hybridoma cell lines

[0054] 1. Mouse immunization

[0055] Healthy 6-8 week old BALB / c mice were selected for immunization. The mice were immunized by subcutaneous injection of benzoic acid chlorophenol complete antigen mixed with an equal amount of Freund's adjuvant. 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 ic-ELISA. Mice with high titers and good inhibition were selected for a fifth immunization, and the mice were given a booster immunization by intraperitoneal injection 21 days after the fifth immunization. The dose was reduced by half and no adjuvant was used.

[0056] 2. Cell fusion

[0057] Three days after the booster immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 1500) method, and the specific steps are as follows:

[0058] (1) After the mouse was sacrificed by cervical dislocation, it was immediately sterilized in 75% alcohol for about 5 minutes. The mouse's spleen was removed and ground with a syringe rubber head, and the spleen cell suspension was obtained by passing through a 200-mesh cell screen. The suspension was collected and centrifuged (1200 rpm, 8 minutes). The spleen cells were washed three times with RPMI-1640 medium, and then diluted to a certain volume, counted, and stored for later use.

[0059] (2) Collection of murine 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 required number of SP2 / 0 tumor cells before fusion was (1-4) × 10⁻⁶. 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. During fusion, tumor cells are collected, suspended in RPMI-1640 basal culture medium, and cell counting is performed.

[0060] (3) Fusion process: 7 minutes. At minute 1, add 1 mL of PEG 1500 dropwise to the cells, gradually increasing the speed. At minute 2, allow to stand. At minutes 3 and 4, add 1 mL of RPMI-1640 medium dropwise over 1 minute. At minutes 5 and 6, add 2 mL of RPMI-1640 medium dropwise over 1 minute. At minute 7, 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, and 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.

[0061] 3. Cell selection and cell line establishment

[0062] On day 3 of cell fusion, the fused cells underwent a half-medium replacement 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. The screening process consisted of two steps: first, positive cell wells were selected using ic-ELISA; second, benzylchlorophenol was used as a standard, and its inhibitory effect on positive cells was determined using ic-ELISA. Cell wells showing good inhibition against both benzylchlorophenol and standard were selected for subcloning using the limiting dilution method. The same method was repeated three times to obtain cell lines.

[0063] Example 3: Preparation and identification of benzylchlorophene monoclonal antibody

[0064] 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 6Hybridoma cells, from the 7th day, collect ascites, and purify the ascites by octanoic acid-ammonium sulfate method. In the slightly acidic condition, normal octanoic acid can precipitate other impurities in the ascites except IgG immunoglobulin, then centrifugal, and discard the precipitate; then use the same amount of saturated ammonium sulfate solution to precipitate IgG type monoclonal antibody, centrifugal, discard the supernatant, dissolve with 0.01M PBS solution (pH = 7.4) after washing, dialysis desalination, and finally obtain the purified monoclonal antibody stored at -20℃.

[0065] Using indirect competitive ELISA method, the IC 50 of the monoclonal antibody to benzochlorophenol is 97.741 ng / mL, and the IC 50 and cross reactivity of the analogs are verified, and the cross reactivity of the related bactericide analogs is less than 1%, wherein the cross reactivity = (IC 50 of benzochlorophenol / IC 50 of the analog) x 100%, according to the cross reactivity value, it can be seen that the antibody has high sensitivity and specificity to benzochlorophenol, and the specific values are shown in Table 1.

[0066] Table 1: IC 50 and cross reactivity of monoclonal antibody to benzochlorophenol and functional analogs

[0067] IC 50 (ng / mL) Cross-reactivity (%) Benzochlorophenol 97.741 100 Triclosan >970 <1 Triclocarban >970 <1 Dichlorophen >970 <1 Bromochlorophenol >970 <1 Hexachlorophene >970 <1 Chloroxylenol >970 <1

[0068] Example 4: Application of benzochlorophenol monoclonal antibody

[0069] The monoclonal antibody prepared by hybridoma cell line through in vivo ascites is applied to the addition recovery test of benzochlorophenol, and the specific steps are as follows:

[0070] (1) Coating: the coating original benzochlorophenol-OVA is diluted by 100 μL / well from 1 μg / mL, 37℃ reaction for 2 hours.

[0071] (2) Washing: pour out the solution in the plate, and wash with washing solution for 3 times, 3 minutes each time.

[0072] (3) Blocking: after drying, add 200 μL / well of blocking solution, 37℃ reaction for 2 hours. After washing, dry and reserve.

[0073] (4) Sample adding: dilute the antiserum (after taking blood from the tail of mice, dilute the corresponding multiple with the antibody diluent, which is the antiserum) by 1000 times, and add to the coating wells of each dilution, 100 μL / well, 37℃ reaction for 30 minutes; after washing, add HRP-goat anti-mouse IgG diluted by 3000 times, 100 μL / well, 37℃ reaction for 30 minutes.

[0074] (5) Color development: Take out 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.

[0075] (6) 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.

[0076] The standard curve of the inhibition of benzylchlorophenol by the monoclonal antibody is as follows: Figure 1 As shown, the IC50 of the monoclonal antibody against benzylchlorophenol was determined by ic-ELISA. 50 The concentration was 97.741 ng / mL, indicating good sensitivity to benzylchlorophenol, and it can be used for the immunoassay detection of benzylchlorophenol.

[0077] 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 monoclonal antibodies to benzochlorophenol, characterized in that: The preservation number of the hybridoma cell strain is CGMCC NO. 46222.

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

3. A monoclonal antibody to clopidol, characterized in that: The monoclonal antibody of bercylchlorophenol 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 monoclonal antibody of claim 3.

5. Use of the hybridoma cell strain of claim 1, the monoclonal antibody of bercylchlorophenol of claim 3 or the composition of claim 4 in the preparation of a detection product of bercylchlorophenol.

6. Use according to claim 5, characterized in that: The detection product of bercylchlorophenol comprises a reagent, a test strip, a kit or a biochip.

7. A test strip, characterized by: The test strip contains the monoclonal antibody of bercylchlorophenol of claim 3.

8. The test strip of claim 7, wherein: The test strip further comprises a sample pad, a conjugate pad, an absorbent pad and an acetate membrane, the conjugate pad is coated with the monoclonal antibody of bercylchlorophenol, and the monoclonal antibody of bercylchlorophenol is labeled with colloidal gold or fluorescent microspheres.

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

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

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