A hybridoma cell line secreting quercetin monoclonal antibody and its application

Abstract

This invention relates to a hybridoma cell line secreting a quercetin monoclonal antibody and its application, belonging to the field of food safety immunoassay. The hybridoma cell line TDLX secreting the quercetin monoclonal antibody of this invention has been deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 46745. The quercetin monoclonal antibody obtained using this strain is used for the analysis and detection of quercetin residues in food safety testing. The quercetin monoclonal antibody cell line obtained by this invention can be used for immunoassay detection, exhibiting good detection sensitivity and specificity (IC50) for quercetin. 50 The value was 39.03 ng / mL, with a cross-linking rate of less than 5% to quercetin analogues. The cross-linking rate was calculated as follows: (IC50 of quercetin) 50 ICs of similar types 50 (×100%)

Description

Technical Field

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

[0002] Quercetin is a natural flavonoid compound widely found in plants. As a plant pigment, it is commonly found in apples, onions, grapes, broccoli, various berries, and tea. It is also recorded in traditional Chinese medicines such as Sophora japonica buds and Platycladus orientalis leaves. Modern research shows that quercetin has a wide range of physiological activities, with its core functions including potent antioxidant and anti-inflammatory effects, as well as potential anti-cancer, cardiovascular, and neuroprotective effects. Therefore, it is often used as a dietary supplement. In nature, quercetin usually exists in the form of glycosides bound to sugars. Human absorption of quercetin is limited, but its safety profile is relatively high.

[0003] For the detection of quercetin pesticide residues, high-performance liquid chromatography (HPLC), gas chromatography (GC), and GC-MS / MS are commonly used. However, these methods have drawbacks such as complex sample pretreatment and long detection times, making them unsuitable for rapid detection of large numbers of samples. To protect the interests of consumers, it is necessary to develop a highly efficient and rapid detection method for quercetin. Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive, and rapid detection method. It requires simple sample pretreatment, involves few purification steps, has a large analytical capacity, low detection cost, and is easy to operate, making it suitable for rapid on-site detection of large numbers of samples. Therefore, it has been widely used in pesticide residue analysis. However, the prerequisite for using ELISA to detect quercetin is obtaining a monoclonal antibody with high specificity and sensitivity to quercetin. Therefore, finding a method to prepare a monoclonal antibody with high specificity and sensitivity to quercetin is crucial. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes a quercetin monoclonal antibody and its applications. The quercetin monoclonal antibody secreted by this hybridoma cell line exhibits good specificity and detection sensitivity (IC50) for quercetin. 50 (with a value of 39.03 ng / mL), it can be used to establish an immunological detection method for quercetin to detect quercetin residues in food.

[0005] This invention is achieved through the following technical solution:

[0006] The first objective of this invention is to provide a hybridoma cell line that secretes a quercetin monoclonal antibody, which was deposited on November 12, 2025, 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. 46745.

[0007] A second objective of this invention is to provide a quercetin monoclonal antibody secreted by the hybridoma cell line.

[0008] The above-mentioned method for preparing quercetin monoclonal antibody is as follows: BALB / c mice are injected intraperitoneally with paraffin oil, followed by intraperitoneal injection of hybridoma cell line. Ascites fluid is collected after injection, purified, and the obtained monoclonal antibody is stored at low temperature.

[0009] In one embodiment of the present invention, the quercetin monoclonal antibody is obtained by immunizing animals with quercetin complete antigen.

[0010] In one embodiment of the present invention, the quercetin complete antigen is obtained by conjugating the quercetin hapten with a carrier protein.

[0011] In one embodiment of the present invention, the structural formula of the quercetin hapten is as follows:

[0012] .

[0013] In one embodiment of the present invention, the carrier protein includes keyhole hemocyanin and / or chicken ovalbumin.

[0014] A third object of the present invention is to provide a composition comprising the hybridoma cell line and / or the quercetin monoclonal antibody.

[0015] A fourth object of the present invention is to provide a kit comprising one or more of the hybridoma cell line, the quercetin monoclonal antibody, and the composition described herein.

[0016] A fifth object of the present invention is to provide a test strip comprising one or more of the hybridoma cell line, the quercetin monoclonal antibody, and the composition described herein.

[0017] A sixth object of the present invention is to provide the application of the hybridoma cell line, the quercetin monoclonal antibody, the composition, the kit, or the test strip in the detection of quercetin; the application does not relate to the diagnosis and treatment of diseases.

[0018] A seventh objective of this invention is to provide a method for preparing the hybridoma cell line that secretes quercetin monoclonal antibody, comprising the following steps:

[0019] (1) Use quercetin hapten to prepare quercetin complete antigen, and prepare the obtained quercetin complete antigen into antigen-containing Freund's adjuvant and antigen-incomplete Freund's adjuvant.

[0020] (2) The Freund's adjuvant was injected subcutaneously into the back of BALB / c mice for multiple immunizations. The first immunization used complete Freund's adjuvant, and the booster immunization used incomplete Freund's adjuvant.

[0021] (3) Blood was collected from mice that had undergone the above immunization process. The serum immune titer and immunosuppressive capacity of the mice were detected by indirect ELISA. Mice with high serum quercetin antibody content were screened to obtain immunity.

[0022] (4) The selected mice were given a final booster immunization with incomplete Freund's adjuvant, and then sprint immunization was performed by intraperitoneal injection. The sprint immunization was performed using complete quercetin antigen without Freund's adjuvant.

[0023] (5) Fusing spleen cells and myeloma cells from BALB / c mice after sprint immunization, culturing the fused cells in a culture medium, detecting positive cell pores using indirect ELISA, and further determining the inhibitory effect of positive cell pores using indirect competitive ELISA, subcloning the positive cell pores with the best inhibition using limiting dilution method, and finally screening out hybridoma cell lines that can secrete quercetin monoclonal antibodies.

[0024] In step (1), the molecular formula of the quercetin hapten is as follows:

[0025] ;

[0026] In step (1), the molecular formula of the quercetin complete antigen is as follows:

[0027] .

[0028] In one embodiment of the present invention, in steps (2) and (4), there is a one-month interval between the first immunization and the booster immunization, a 21-day interval between booster immunizations, and an 18-21-day interval between booster immunizations and the final immunization.

[0029] In one embodiment of the present invention, in steps (2) and (4), the initial immunization dose is 100 μg / animal, the booster immunization dose is 50 μg / animal, and the sprint immunization dose is 25 μg / animal.

[0030] In one embodiment of the present invention, in steps (2) and (4), the immunization process includes one initial immunization, four booster immunizations and one sprint immunization;

[0031] In one embodiment of the present invention, in step (3), blood is collected on the 7th day after the end of the 3rd immunization process.

[0032] In one embodiment of the present invention, in step (5), cell fusion is performed 3 days after the end of the sprint immunization.

[0033] In one embodiment of the present invention, in step (5), cell fusion is performed by the polyethylene glycol (PEG4000) method.

[0034] In one embodiment of the present invention, in step (5), the culture medium is RPMI-1640 medium.

[0035] In one embodiment of the present invention, in step (5), the number of subcloning operations is 3.

[0036] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0037] This invention provides a hybridoma cell line that secretes a monoclonal antibody against quercetin and its applications. The quercetin monoclonal antibody obtained by this invention can be used for immunoassay detection, exhibiting good detection sensitivity and specificity (IC50) for quercetin. 50 The value was 39.03 ng / mL, with a cross-linking rate of less than 10% to quercetin analogues. The cross-linking rate was calculated as follows: (IC50 of quercetin) 50 ICs of similar types 50 (×100%)

[0038] Preservation of biological material samples:

[0039] A hybridoma cell line, TDLX, secreting a monoclonal antibody against quercetin, has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. It has been classified as a monoclonal cell line, deposited on November 12, 2025, with accession number CGMCC No. 46745. Attached Figure Description

[0040] 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.

[0041] Figure 1 This is the standard curve of the inhibition of quercetin by the quercetin monoclonal antibody of this invention. Detailed Implementation

[0042] 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.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0044] The culture media involved in the following examples are as follows:

[0045] 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.

[0046] The reagents involved in the following examples are as follows:

[0047] 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.

[0048] 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, pH adjusted to 7.2-7.4 with NaOH or HCl, and then brought to a final volume of 1000 mL.

[0049] PBST: PBS containing 0.05% Tween 20;

[0050] Antibody dilution solution: PBS with 0.1% gelatin added.

[0051] TMB colorimetric solution: Solution A: Na2HPO4 .12H₂O 18.43 g, citric acid 9.33 g, 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 5:1 ratio to obtain the TMB colorimetric solution, and mix fresh before use.

[0052] The detection methods involved in the following embodiments are as follows:

[0053] Quercetin inhibition rate detection method: 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, quercetin standards were diluted to concentrations of 0, 1.37, 4.12, 12.35, 37.04, 111.11, 333.33, and 1000 ng / mL, following the ic-ELISA procedure. Finally, graphs were plotted using OriginPro 8.5 (results are shown in the figure). Figure 1 As shown), the standard inhibition curve of quercetin was obtained, and the IC50 was calculated. 50 .

[0054] Example 1: Synthesis of quercetin hapten

[0055] Since small molecules of quercetin are not immunogenic and cannot stimulate an immune response in mice to produce antibodies, it is necessary to use protein conjugation technology to couple quercetin to proteins to give it immunogenicity. Commonly used active groups in protein conjugation technology include amino, carboxyl, hydroxyl, and thiol groups. Given that the quercetin molecule contains hydroxyl groups, the original quercetin drug can be used directly as a hapten to prepare a complete antigen using the CDI method.

[0056] The quercetin hapten of this invention, namely its technical grade, CAS number 117-39-5, was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number Q4951-10G, and its structure is as follows:

[0057] .

[0058] Example 2: Synthesis of Quercetin Complete Antigen

[0059] Weigh 8.16 mg of quercetin hapten (HPS-OH) and 43.78 mg of N,N'-carbonyldiimidazole (CDI), dissolve them in 360 μL of N,N-dimethylformamide (DMF), and react with stirring at 60°C for 1–2 h (referred to as solution A). Take 6 mg of KLH, dilute it to 3 mg / mL with 0.01 M carbonate buffer (CBS) (referred to as solution B), and then slowly add solution A dropwise to solution B. React overnight at room temperature. Then dialyze with 0.01 M PBS solution to remove unreacted small molecule hapten, obtaining the complete antigen HPS-CDI-KLH, which is identified by ultraviolet absorption scanning.

[0060] Example 3: Synthesis of quercetin coating agent

[0061] 2.42 mg of quercetin hapten (HPS-OH) and 12.97 mg of N,N'-carbonyldiimidazole (CDI) were dissolved in 100 μL of anhydrous N,N-dimethylformamide (DMF) and reacted at 60 °C for 1–2 h to obtain solution A. 4 mg of chicken ovalbumin (OVA) was diluted with 1 mL of 0.01 mmol / L carbonate buffer (CBS) to obtain solution B. Solution A was slowly added dropwise to solution B to obtain the reaction solution. The reaction solution was dialyzed with PBS to remove unreacted small molecule hapten, yielding the coating antigen (HPS-CDI-OVA).

[0062] Example 4: Preparation of hybridoma cell lines secreting quercetin monoclonal antibodies

[0063] 1. Obtaining animal immunization: The complete quercetin antigen was emulsified with an equal amount of Freund's adjuvant and then administered to BALB / c mice via subcutaneous injection at multiple sites on the back of the neck (except for sprint immunization). The initial immunization used complete Freund's adjuvant at a dose of 100 μg / mouse; subsequent booster immunizations used incomplete Freund's adjuvant at half the dose (50 μg / mouse); sprint immunizations did not use adjuvant, but were directly diluted 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 the sprint immunization and the final booster immunization was 18-21 days. The immunization effect in mice was observed using an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), i.e., the titer and inhibition of mouse serum were detected.

[0064] 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:

[0065] 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 rubber 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.

[0066] b. Collection of SP 2 / 0 cells: 7-10 days before fusion, SP 2 / 0 tumor cells are 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 is 1-4 × 10⁻⁶ cells / year. 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.

[0067] c. Fusion process (7 min): Min 1, add 1 mL of PEG 1500 to the cells dropwise from slow to fast; Min 2, let stand; Min 3 and Min 4, add 1 mL of RPMI-1640 medium dropwise over 1 min; Min 5 and Min 6, add 2 mL of RPMI-1640 medium dropwise over 1 min; Min 7, add 1 mL of RPMI-1640 medium dropwise every 10 s; then incubate at 37℃ for 5 min; centrifuge (800 rpm, 8 min), 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℃ in a 5% CO2 incubator;

[0068] 3. Cell screening and cell line establishment: On the 3rd day of cell fusion, the fused cells were screened with RPMI-1640 medium with a half-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.

[0069] The screening process consists of two steps: First, positive cell wells are selected using ic-ELISA; second, quercetin is used as a standard, and the inhibitory effect on positive cells is determined using ic-ELISA.

[0070] Cell wells that showed good inhibition of quercetin standards were selected, and subcloning was performed using the limiting dilution method. The same method was used for detection seven days later.

[0071] Three subclonings were performed using the method described above, ultimately yielding the quercetin monoclonal antibody cell line TDLX.

[0072] Example 5: Preparation and Identification of Quercetin Monoclonal Antibody

[0073] 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 Quercetin hybridoma cells were used to collect ascites fluid starting from day 7. The ascites fluid was then purified for antibody treatment using the caprylic acid-saturated ammonium sulfate method.

[0074] 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℃.

[0075] The IC50 of quercetin monoclonal antibody was measured using an indirect competitive ELISA. 50 The value was 39.03 ng / mL, indicating good sensitivity to quercetin and suitable for quercetin immunoassay.

[0076] The cross-linking rate with quercetin was 100%, and the cross-linking rate with piracetam was <5%. Cross-linking rate = (IC50 of quercetin) / (IC50 of quercetin) 50 ICs of similar types 50 The cross-reactivity ratio (×100%) indicates that this antibody has high sensitivity and specificity to quercetin, as shown in Table 1.

[0077] Table 1. IC50 of monoclonal antibodies against quercetin and its analogues 50 and cross-reactivity rate

[0078]

[0079] Example 6: Application of quercetin monoclonal antibody

[0080] Monoclonal antibodies prepared from hybridoma cell lines via in vivo ascites fluid were used in a quercetin ELISA addition and recovery assay. The specific steps are as follows:

[0081] (1) Coat a 96-well microplate with 0.1 μg / mL of the coating stock diluted with carbonate buffer (CBS), 100 μL per well, at 37℃ for 2 h. Wash the plate three times with PBST washing buffer, 200 μL per well each time, for 3 min each time, and pat dry.

[0082] (2) Block with CBS containing 0.2% gelatin, 200 μL per well, block at 37℃ for 2 h, wash the plate three times with PBST washing solution, 200 μL per well each time, 3 min each time, and pat dry;

[0083] (3) Prepare quercetin standard solutions of 0, 1.37 ng / mL, 4.12 ng / mL, 12.35 ng / mL, 37.04 ng / mL, 111.11 ng / mL, 333.33 ng / mL and 1000 ng / mL using phosphate-buffered saline (PBS). Add the standard solutions and the extracts of the samples to be tested to the pre-blocked microplates, 50 μL per well, and repeat the test for each sample in 3 wells. Add 50 μL of anti-quercetin monoclonal antibody to each well. After reacting at 37°C for 0.5 h, wash the plate and pat dry.

[0084] (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℃ for 0.5 h, then wash and dry the plate.

[0085] (5) Add 100 μL of TMB colorimetric solution to each well, develop the color at 37℃ for 15 min, then add 50 μL of 2M H2SO4 stop solution to each well, and measure the absorbance at 450 nm.

[0086] The standard curve of quercetin inhibition by monoclonal antibody is shown below. Figure 1 As shown, the IC50 of quercetin monoclonal antibody was determined using ic-ELISA. 50 The value was 39.03 ng / mL, indicating that the antibody has good sensitivity to quercetin and can be used for the immunoassay detection of quercetin.

[0087] 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 against quercetin, characterized in that, The hybridoma cell line was deposited on November 12, 2025, 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. 46745.

2. A quercetin monoclonal antibody, characterized in that, Produced by the hybridoma cell line of claim 1.

3. The quercetin monoclonal antibody according to claim 1, characterized in that, The quercetin monoclonal antibody was obtained by immunizing animals with the quercetin complete antigen.

4. The quercetin monoclonal antibody according to claim 3, characterized in that, The quercetin complete antigen is obtained by conjugating the quercetin hapten with a carrier protein.

5. The quercetin monoclonal antibody according to claim 4, characterized in that, The structural formula of the quercetin hapten is shown below: 。 6. The quercetin monoclonal antibody according to claim 4, characterized in that, The carrier proteins include keyhole hemocyanin and / or chicken ovalbumin.

7. A composition, characterized in that, The composition comprises the hybridoma cell line of claim 1 and / or the quercetin monoclonal antibody of any one of claims 2-6.

8. A reagent kit, characterized in that, The kit comprises one or more of the hybridoma cell line of claim 1, the quercetin monoclonal antibody of any one of claims 2-6, and the composition of claim 7.

9. A test strip, characterized in that, The test strip comprises one or more of the hybridoma cell line of claim 1, the quercetin monoclonal antibody of any one of claims 2-6, and the composition of claim 7.

10. The use of the hybridoma cell line of claim 1, the quercetin monoclonal antibody of any one of claims 2-6, the composition of claim 7, the kit of claim 8, or the test strip of claim 9 in the detection of quercetin; the use does not relate to the diagnosis and treatment of disease.

Images