A secreting dihexyl phthalate monoclonal antibody hybridoma cell strain and application thereof

Abstract

The present application relates to a kind of secreting phthalic acid dihexyl ester monoclonal antibody hybridoma cell strain and its application, belong to immune detection technical field.The monoclonal antibody secreted by the hybridoma cell strain of the present application has good sensitivity to phthalic acid dihexyl ester, wherein the IC 50 of phthalic acid dihexyl ester is 1.96 μg / mL, there is no cross reaction to related analogues such as phthalic acid diallyl ester, phthalic acid dibutyl ester, phthalic acid dimethyl ester, phthalic acid diethyl ester, phthalic acid diisononyl ester, etc., so that low concentration phthalic acid dihexyl ester can be accurately detected.

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 dihexyl phthalate and its application. Background Technology

[0002] Dihexyl phthalate (DHXP), chemically known as 1,2-dihexyl phthalate, belongs to the phthalate ester class of endocrine disruptors. It is mainly used as a plasticizer for polyvinyl chloride (PVC) plastics, and as an additive in coatings and adhesives. It is widely found in food packaging, building materials, children's toys, and indoor dust.

[0003] Currently, the main methods for determining the content of dihexyl phthalate (DHB) include gas chromatography-FID (GC-FID), gas chromatography-mass spectrometry (GC-MS), and high-performance liquid chromatography (HPLC-UV). These methods require specialized equipment and operators, and are costly, making them unsuitable for real-time, batch testing. Enzyme-linked immunosorbent assay (ELISA) technology offers a new solution for DHB detection. This technology, with its high sensitivity and fast detection speed, can achieve trace detection of DHB, and its operation is relatively simple, greatly improving the flexibility and timeliness of detection. It is expected to solve the problems of traditional detection methods, promote the development of DHB detection technology, and provide more efficient technical support for the rapid screening of this type of residue in the environment and food. However, the prerequisite for using ELISA to simultaneously detect DHB is obtaining a monoclonal antibody with high sensitivity and specificity for DHB, which is currently lacking in existing technologies. Summary of the Invention

[0004] The technical problem to be solved by this invention is to overcome the lack of a monoclonal antibody in the prior art that simultaneously possesses high sensitivity and specificity to dihexyl phthalate. A hybridoma cell line secreting a monoclonal antibody against dihexyl phthalate is provided, along with its applications.

[0005] The monoclonal antibody secreted by the hybridoma cell line of the present invention has good sensitivity to dihexyl phthalate, wherein the IC50 value for dihexyl phthalate is [not specified]. 50 The concentration is 1.96 μg / mL, and it has no cross-reactivity with related analogues such as diallyl phthalate, dibutyl phthalate, dimethyl phthalate, diethyl phthalate, and diisononyl phthalate. Therefore, it can accurately detect low concentrations of dihexyl phthalate.

[0006] The technical solution of this invention is as follows: The first objective of this invention is to provide a hybridoma cell line, named cell line XXET, classified as a monoclonal cell line, which was deposited on April 24, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46828.

[0007] A second objective of this invention is to provide a method for preparing the aforementioned hybridoma cell line, wherein the hybridoma cell line is obtained by immunizing animals with a complete antigen prepared from a hapten, and the structural formula of the hapten is shown in Formula I. (Formula I).

[0008] Furthermore, the complete antigen is the hapten coupled with a carrier protein, and the carrier protein is keyhole hemocyanin.

[0009] A third object of the present invention is to provide a monoclonal antibody produced from the aforementioned hybridoma cell line, the monoclonal antibody specifically recognizing dihexyl phthalate.

[0010] The fourth objective of this invention is to provide a method for preparing the aforementioned monoclonal antibody, wherein the aforementioned hybridoma cell line is inoculated into the peritoneal cavity of mice, ascites fluid is collected, and then purified to obtain the monoclonal antibody.

[0011] A fifth object of the present invention is to provide a detection product for dihexyl phthalate, the detection product comprising the aforementioned monoclonal antibody.

[0012] Furthermore, the test product also includes coating antigens.

[0013] Furthermore, the coating is prepared by a hapten-coupled carrier protein as shown in Formula I, wherein the carrier protein is chicken ovalbumin.

[0014] The sixth object of the present invention is to provide the application of the aforementioned hybridoma cell line or the aforementioned monoclonal antibody in the preparation of a detection product for detecting dihexyl phthalate.

[0015] A seventh object of the present invention is to provide the use of the aforementioned hybridoma cell line, the aforementioned monoclonal antibody, or the aforementioned detection product in the detection of dihexyl phthalate, wherein the use does not involve the diagnosis or treatment of disease.

[0016] The beneficial effects of this invention are: This invention marks the first isolation of a hybridoma cell line capable of accurately detecting low concentrations of dihexyl phthalate (DHB). Specifically, the monoclonal antibody secreted by this hybridoma cell line exhibits good sensitivity to DHB, with an IC50 value of [missing information - likely related to DHB detection]. 50The concentration was 1.96 μg / mL. It showed no cross-reactivity with related analogues such as diallyl phthalate, dibutyl phthalate, dimethyl phthalate, dihexyl phthalate, and diisononyl phthalate, thus enabling the detection of low concentrations of dihexyl phthalate.

[0017] Information on the preservation of biological materials

[0018] The cell line XXET, classified as a monoclonal cell line, was deposited on April 24, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46828. The address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code: 100101. Attached Figure Description

[0019] Figure 1 This is the synthetic route for the hapten of the present invention.

[0020] Figure 2 This is the standard curve of the inhibition of dihexyl phthalate by the monoclonal antibody of the present invention. Detailed Implementation

[0021] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0023] (1) The culture media involved in the following examples are as follows: RPMI-1640 medium (mg / L): L-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cysteine ​​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, para-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, nicotinamide 1, choline chloride 3, pyridoxine hydrochloride 1, riboflavin 0.2, thiamine hydrochloride 1, vitamin B12 0.005, sodium bicarbonate 2000, solvent: water.

[0024] (2) The reagents involved in the following examples are as follows: Carbonate buffer (CBS): Weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them separately in a small amount of double-distilled water and mix them together. Add double-distilled water to about 800 mL and mix well. Adjust the pH to 9.6 and add double-distilled water to a final volume of 1000 mL. Store at 4°C for later use.

[0025] 4M NaOH solution containing 20% ​​methanol: Weigh 16 g of NaOH, dissolve it in 80 mL of double-distilled water, then add 20 mL of methanol and mix well for later use.

[0026] Phosphate-buffered saline (PBS): Dissolve 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4, and 2.9g Na2HPO4·12H2O in 800 mL of pure water. Adjust the pH to 7.2-7.4 with NaOH or HCl, and bring the volume to 1000 mL with ultrapure water.

[0027] Washing buffer (PBST): Add 0.5 mL of Tween-20 to 1000 mL of 0.01 mol / L pH 7.4 PBS solution.

[0028] PBST: PBS containing 0.05% Tween-20.

[0029] Antibody diluent: Washing solution containing 0.1% gelatin.

[0030] TMB colorimetric solution: Solution A: 18.43 g Na2HPO4·12H2O, 9.33 g citric acid, diluted to 1000 mL with pure water; Solution B: 60 mg TMB dissolved in 100 mL ethylene glycol. Mix solutions A and B in a volume ratio of 5:1 to obtain the TMB colorimetric solution. Mix fresh before use.

[0031] PBS buffer containing 10% DMF: Measure 10 mL of N,N-dimethylformamide (DMF), add 90 mL of PBS buffer solution, and mix well before use.

[0032] Example 1: Preparation of Hapten In the synthesis of the hapten, 4-bromophthalic acid (5 g, 20.4 mmol) was dissolved in hexanol (12.51 g, 122.4 mmol), and thionyl chloride (7.28 g, 61.2 mmol) was added dropwise at 0 °C. After the addition was complete, the reaction mixture was stirred at 80 °C for 4 hours. The mixture was then diluted with ethyl acetate (200 mL), washed successively with water (100 mL × 3) and saturated sodium bicarbonate solution (100 mL), dried, and concentrated to obtain the product 4-bromophthalic acid dihexyl ester. 4-bromophthalic acid dihexyl ester (1.98 g, 4.8 mmol), palladium acetate (50 mg, 0.21 mmol), DPPP (170 mg, 0.42 mmol), and triethylamine (1.27 g, 12.6 mmol) were dissolved in MeCN (35 mL) / H2O (3 mL) and stirred at 80 °C for 20 hours under CO2 balloon protection. The reaction mixture was concentrated to approximately 10 mL and the pH was adjusted to 2 with 1N HCl. The residue was diluted with water (50 mL) and extracted with ethyl acetate (50 mL × 2). The combined organic phases were dried and concentrated. The resulting compound of formula I is the DHXP hapten (CAS: 142288-22-0), and the reaction route is as follows: Figure 1 As shown.

[0033] Example 2: Preparation of Immunogen Weigh 6.873 mg of the hapten shown in Formula I, dissolve it in 600 μL of N,N-dimethylformamide, add 7.9 mg of N-hydroxysuccinimide under stirring at room temperature and react for 15 min, then add 13.4 mg of 1-ethylcarbodiimide hydrochloride and react at room temperature for 6 h. The resulting mixture is called solution A.

[0034] Then weigh 5 mg of keyhole hemocyanin (KLH) and dissolve it in 2 mL of carbonate buffer, which is called solution B.

[0035] Solution A was slowly added dropwise to solution B, and the mixture was stirred and reacted at room temperature for 24 h. After dialysis with phosphate-buffered saline (PBS) for 3 days, the conjugate dihexyl phthalate-KLH was obtained and used as an immunogen. It was then frozen at -20°C for later use.

[0036] Example 3: Preparation of the coating agent Weigh 3.79 mg of the hapten shown in Formula I, dissolve it in 600 μL of N,N-dimethylformamide, add 7.7 mg of N-hydroxysuccinimide under stirring at room temperature and react for 15 min, then add 9.2 mg of 1-ethylcarbodiimide hydrochloride and react for 4 h. The resulting mixture is called solution A.

[0037] Then weigh 5 mg of ovalbumin (OVA) and dissolve it in 2 mL of carbonate buffer, which is called solution B.

[0038] Solution A was slowly added dropwise to solution B, and the mixture was stirred and reacted at room temperature for 24 h. The mixture was then dialyzed against 0.01 mol / L phosphate buffered PBS for 3 days to obtain the conjugate dihexyl phthalate-OVA, which was used as the coating agent and stored at -20℃ for later use.

[0039] Example 4: Preparation of hybridoma cell lines 1. Immunization of mice: Healthy BALB / c mice aged 6-8 weeks were selected for immunization. Dihexyl phthalate-KLH immunogen prepared in Example 2 was mixed with an equal amount of Freund's adjuvant, emulsified, and then injected subcutaneously into the back of BALB / c mice.

[0040] The adjuvant used for the first immunization was complete Freund's adjuvant (manufacturer: SIGMA-ALDRICH, specification: F5581-10ML), and the dosage was a mixture of immunogen and adjuvant containing 100 µg of immunogen per mouse.

[0041] The adjuvant used for subsequent booster immunization was incomplete Freund's adjuvant (manufacturer: SIGMA-ALDRICH, specification: F5506-10ML), and the dosage was a mixture of immunogen and adjuvant containing 50 µg of immunogen per mouse.

[0042] The interval between the first and second booster immunizations was 28 days, and the interval between subsequent booster immunizations was 21 days. Seven days after the third immunization, blood was collected from mice. 5 μL of blood was collected from the mouse tail and mixed with 995 μL of antibody dilution buffer to obtain antiserum. The titer and inhibition of the mouse antiserum were determined using icELISA, as detailed below: The coating agent was diluted to 1, 0.3, and 0.1 μg / mL with carbonate buffer (CBS), and the antiserum was diluted to 1:1000, 1:3000, 1:9000, and 1:27000 with antibody diluent to prepare ELISA plates for detecting mouse serum.

[0043] Dihexyl phthalate standard was diluted to 20 μg / mL with PBS solution containing 10% DMF. The titer and inhibition rate of mouse antiserum were detected according to the icELISA procedure. The concentration of standard in subsequent tests was half that of the previous test.

[0044] Select mice with high titers and good inhibition, and perform a booster immunization 21 days after the fifth immunization by intraperitoneal injection. The booster immunization dose should be half of the booster immunization dose, i.e., 25 µg of immunogen, and should not contain any adjuvants.

[0045] 2. Cell fusion: Three days after the sprint immunization, cell fusion was performed using the standard PEG (polyethylene glycol, molecular weight 1500) method. The specific steps are as follows: a. After euthanizing the mice by enucleation and cervical dislocation-assisted carbon dioxide euthanasia, immediately disinfect the mice in 75% alcohol for about 5 minutes. Aseptically remove the spleens of the mice, grind them moderately with the rubber tip of a syringe and pass them through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect the suspension and centrifuge (1200 rpm, 8 min). 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 them aside for later use.

[0046] b. Collection of murine myeloma SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells (National Biomedical Experimental Cell Resource Bank, resource number 3101MOUTCM18) 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 should reach (1-4) × 10⁻⁶. 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion, tumor cells are collected during fusion, suspended in RPMI-1640 basal culture medium, and cell counting is performed.

[0047] c. Fusion process (7 min): At min 1, add 1 mL of PEG 1500 dropwise to the mixed solution of tumor cells from step b and spleen cells from step a, gradually increasing the speed. At min 2, allow to stand. At min 3 and min 4, add 1 mL of RPMI-1640 medium dropwise over 1 min. At min 5 and min 6, add 2 mL of RPMI-1640 medium dropwise over 1 min. At min 7, add 1 mL of RPMI-1640 medium dropwise every 10 seconds. Then incubate at 37°C for 5-8 min. Centrifuge (800 rpm, 8 min), 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.

[0048] 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.

[0049] The screening process consists of two steps: First, positive cell wells are selected using icELISA. Second, dihexyl phthalate is used as a standard, and the inhibitory effect on positive cells is determined using icELISA.

[0050] The specific procedures are as follows: Dilute the coating agent to 0.3 μg / mL with carbonate buffer (CBS), and dilute the cell supernatant to 1:2, 1:4, and 1:8 with antibody diluent. Dilute the dihexyl phthalate standard to 2 μg / mL, and detect the titer and inhibition rate of the cell supernatant according to the icELISA procedure.

[0051] Cell wells that showed good inhibition against dihexyl phthalate standards were selected, and subcloning was performed using the limiting dilution method. The same method was used for detection, and the process was repeated three times to obtain the cell line, which is the hybridoma cell line with the preservation number CGMCC No. 46828.

[0052] Example 5: Preparation and Identification of Monoclonal Antibodies BALB / c mice aged 6-8 weeks were injected intraperitoneally with 0.5 mL of sterile paraffin oil; 7 days later, each mouse was injected intraperitoneally with 1×10 6 Ascites fluid was collected from the hybridoma cell line with accession number CGMCC No. 46828 starting from day 7, and the ascites fluid was purified by the caprylic acid-ammonium sulfate method.

[0053] 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 antibody is dissolved in 0.01M PBS solution (pH 7.4), dialyzed to desalt, and finally purified monoclonal antibodies are obtained and stored at -20℃.

[0054] The IC50 of the monoclonal antibody against dihexyl phthalate was determined using the icELISA method. 50 The concentration was 1.96 μg / mL, and its IC50 for functional analogues was verified. 50 and cross-reactivity rate, with a cross-reactivity of less than 1% for related analogues, where cross-reactivity = (IC50 of dihexyl phthalate) 50 ICs of similar types 50 The cross-reactivity ratio (CRR) is calculated as 100%. Based on the CRR values, it can be seen that this antibody has a high sensitivity to dihexyl phthalate, as shown in Table 1.

[0055] The detection procedure is as follows: The coating 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. Dihexyl phthalate standard was diluted to 1.96 μg / mL, and other phthalic acid analogs were diluted to 10 mg / mL. The specificity of the antibody for dihexyl phthalate was compared according to the icELISA procedure.

[0056] Table 1. Sensitivity and specificity of monoclonal antibodies against dihexyl phthalate.

[0057] Antibody application The monoclonal antibody prepared in vivo using hybridoma cell lines via ascites in the aforementioned steps was applied to the addition and recovery assay of dihexyl phthalate. The specific steps are as follows: a. Coating: The coated dihexyl phthalate-OVA prepared in Example 3 was serially diluted from 1 µg / mL with 0.05 M pH 9.6 carbonate buffer, 100 μL / well, and reacted at 37°C for 2 h; b. Washing: Pour off the solution in the plate and wash with washing solution 3 times, 3 minutes each time; c. Sealing: After patting dry, add 200 μL / well sealing solution and react at 37℃ for 2 h. Wash and dry for later use. d. Sample loading: The monoclonal antibody obtained above is serially diluted from 1:1000 and added to each well of the coating at each dilution, 100 μL / well, and reacted at 37℃ for 30 min; after thorough washing, add HRP-goat anti-mouse IgG diluted 1:3000, 100 μL / well, and react at 37℃ for 30 min. 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 min. 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.

[0058] The standard curve of the inhibition of dihexyl phthalate by the monoclonal antibody against dihexyl phthalate is as follows: Figure 2 As shown, the results indicate that the obtained antibody has an IC50 value of 50% for dihexyl phthalate. 50 The detection range was 0.98–3.92 μg / mL, and the limit of detection was 0.65 μg / mL. The IC50 of the monoclonal antibody against dihexyl phthalate was determined by icELISA. 50 The concentration was 1.96 μg / mL, indicating good sensitivity to dihexyl phthalate, making it suitable for immunoassay detection of dihexyl phthalate.

[0059] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A hybridoma cell line, characterized in that, The hybridoma cell line was named cell line XXET, classified as a monoclonal cell line, and was deposited at the China General Microbiological Culture Collection Center on April 24, 2026, with accession number CGMCC No. 46828.

2. The method for preparing the hybridoma cell line according to claim 1, characterized in that, The hybridoma cell line was obtained by immunizing animals with a complete antigen prepared from a hapten, the structural formula of which is shown in Formula I. (Formula I).

3. The preparation method according to claim 2, characterized in that, The complete antigen is the hapten coupled with a carrier protein, and the carrier protein is keyhole hemocyanin.

4. A monoclonal antibody, characterized in that, The monoclonal antibody is produced by the hybridoma cell line of claim 1, and the monoclonal antibody specifically recognizes dihexyl phthalate.

5. The method for preparing the monoclonal antibody according to claim 4, characterized in that, The hybridoma cell line described in claim 1 was inoculated into the peritoneal cavity of mice, and the ascites fluid was collected and purified to obtain monoclonal antibodies.

6. A detection product for dihexyl phthalate, characterized in that, The detection product includes the monoclonal antibody as described in claim 4.

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

8. The testing product according to claim 7, characterized in that, The coating is prepared by conjugating a hapten to a carrier protein as shown in Formula I, wherein the carrier protein is chicken ovalbumin.

9. The use of the hybridoma cell line of claim 1 or the monoclonal antibody of claim 4 in the preparation of a detection product for detecting dihexyl phthalate.

10. The use of the hybridoma cell line of claim 1, the monoclonal antibody of claim 4, or the detection product of claim 6 in the detection of dihexyl phthalate, wherein the use does not involve the diagnosis or treatment of a disease.

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