Hybridoma cell strain capable of secreting broad-spectrum monoclonal antibody of biguanide drugs and application of hybridoma cell strain

Abstract

The invention relates to a hybridoma cell strain secreting a biguanide drug broad-spectrum monoclonal antibody and application of the hybridoma cell strain, and belongs to the field of food safety immunodetection. The hybridoma cell strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on November 12, 2025, the preservation address is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.46731. The biguanide drug broad-spectrum monoclonal antibody secreted by the hybridoma cell strain has excellent affinity and sensitivity to metformin, phenformin and metformin, IC50 of the biguanide drug broad-spectrum monoclonal antibody to the phenformin reaches 1 ng / mL, IC50 of the metformin reaches 20 ng / mL, IC50 of the metformin reaches 200 ng / mL, and the biguanide drug broad-spectrum monoclonal antibody can be used for preparing a broad-spectrum immunodetection product of biguanide drugs and has good application prospects. And an efficient detection method and means are provided for multi-residue detection of biguanide drugs in food.

Description

Technical Field

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

[0002] Diabetes has become a persistent and widespread global public health challenge, a trend particularly pronounced in developing countries. Statistics show that in 2021, the prevalence of diabetes among people aged 20-79 worldwide reached 10.5%, affecting approximately 536.6 million people. More worryingly, according to the International Diabetes Federation's projections, this rate will climb to 12.2% by 2045, with the number of affected individuals increasing to 783.2 million. With the continued increase in the disease burden, the prevention and control of diabetes has received widespread attention globally, especially among middle-aged and elderly populations, where awareness of disease prevention is generally strengthening. Against this backdrop, my country's health supplement industry has entered a period of rapid development, with the market for hypoglycemic products performing particularly well. However, regulators have found that some manufacturers illegally add chemical hypoglycemic ingredients to their products in pursuit of hypoglycemic effects. Common illegal additives include hypoglycemic drugs such as phenformin hydrochloride, metformin, and buformin. While these chemical drugs have therapeutic value when used in regulated medical settings, their illegal addition to health supplements can trigger severe hypoglycemic reactions and each has specific toxic side effects. Among them, phenformin hydrochloride and buformin have been withdrawn from the market in most countries around the world due to their potential to cause fatal lactic acidosis.

[0003] Among commonly illegally added drugs, the complex situation of biguanide drugs requires special attention. Metformin, as a first-line oral hypoglycemic agent for the global treatment of type 2 diabetes, exerts its hypoglycemic effect by inhibiting hepatic glycogen output and improving peripheral insulin sensitivity. It has a relatively high safety profile and a relatively low risk of lactic acidosis, making it a cornerstone drug in clinical diabetes treatment. However, in stark contrast, phenformin and buformin, also belonging to the biguanide class, are first-generation biguanide drugs. Although they have significant hypoglycemic effects, they are banned in most countries worldwide due to the high risk of fatal lactic acidosis. These withdrawn drugs are now mainly used as historical case studies in drug safety research, but due to their strong hypoglycemic effect and low production cost, their addition to illegal health products still occurs frequently, continuing to pose a potential threat to public health. It is worth noting that the current problem of illegal additives in hypoglycemic health products exhibits new characteristics. On the one hand, illegal additive activities are more covert, and the added ingredients are more complex; on the other hand, consumers' awareness of the safety of health products remains insufficient. These factors have increased the difficulty of regulation, making the establishment of rapid, accurate, and efficient testing methods particularly urgent.

[0004] Therefore, developing analytical methods that can simultaneously detect multiple biguanide drugs and other common hypoglycemic components will not only help strengthen market supervision and combat illegal additives, but also provide strong technical support for ensuring consumer medication safety, which is of great significance for maintaining public health and promoting the healthy development of the health product industry.

[0005] In recent years, the detection of biguanide hypoglycemic drugs has mainly relied on instrumental analysis. Ju Yingying et al. (Simultaneous detection of 18 major hypoglycemic components in hypoglycemic health products by high performance liquid chromatography [J]. Practical Preventive Medicine, 2025, 32(02):242-246.) used high performance liquid chromatography to detect 18 hypoglycemic drugs, including three biguanide drugs, in health foods, and showed good linearity in the concentration range of 0.76-46.99 μg / ml. Guan Dong et al. (Detection and ecological risk assessment of 15 commonly used drug active ingredients in water bodies [J]. Journal of Environmental Engineering, 2024, 18(11):3186-3196.) established a method for detecting commonly used drug residues in water bodies by high performance liquid chromatography-tandem mass spectrometry. The detection limit of this method was 0.03-0.32 ng / L, and the quantitation limit was 0.10-1.07 ng / L. These methods are complex to operate and require cumbersome sample processing, making them unsuitable for testing large quantities of samples in actual production. There is an urgent need to establish a simple, highly sensitive, and specific detection method. Enzyme-linked immunosorbent assay (ELISA) is a highly efficient, sensitive, and rapid detection method, and it is increasingly being used in food safety testing. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes broad-spectrum monoclonal antibodies against biguanide drugs and its applications. The product prepared by this invention is used for research on immunoassay methods for biguanide drugs, providing essential artificial antigens for future research. The antibodies prepared from this cell line exhibit good specificity and detection sensitivity against metformin, phenformin, and butylformin, and can be used to establish immunological detection methods for biguanide drugs.

[0007] This invention is achieved through the following technical solution: The first objective of this invention is to provide a hybridoma cell line that secretes a broad-spectrum monoclonal antibody against biguanide drugs. This 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. 46731.

[0008] In one embodiment of the present invention, the biguanide drug is one or more of metformin, phenformin, and buformin.

[0009] A second objective of this invention is to provide a broad-spectrum monoclonal antibody against biguanide drugs, produced by the hybridoma cell line.

[0010] In one embodiment of the present invention, the biguanide broad-spectrum monoclonal antibody is obtained by immunizing animals with a complete biguanide antigen.

[0011] In one embodiment of the present invention, the complete biguanide drug antigen is obtained by conjugating a biguanide drug hapten with a carrier protein.

[0012] In one embodiment of the present invention, the structural formula of the biguanide drug hapten is as follows: ; And / or, the carrier protein includes keyhole hemocyanin and / or chicken oocyte albumin.

[0013] A third object of the present invention is to provide a composition comprising the hybridoma cell line described above and / or the broad-spectrum monoclonal antibody against the biguanide drug described above.

[0014] A fourth object of the present invention is to provide a kit comprising one or more of the hybridoma cell line, the biguanide broad-spectrum monoclonal antibody, and the composition thereof.

[0015] A fifth objective of the present invention is to provide a test strip comprising one or more of the aforementioned hybridoma cell line, the aforementioned biguanide broad-spectrum monoclonal antibody, and the aforementioned composition.

[0016] A sixth objective of this invention is to provide the application of the hybridoma cell line, the broad-spectrum monoclonal antibody against biguanide drugs, the composition, the kit, or the test strip in the detection of biguanide drugs; the application does not involve the diagnosis or treatment of diseases.

[0017] In one embodiment of the present invention, the hybridoma cell line and the broad-spectrum monoclonal antibody against biguanides can simultaneously detect metformin, phenformin, and buformin, exhibiting excellent affinity and sensitivity for metformin, phenformin, and buformin, and a high IC50 value for phenformin. 50 The IC50 of buprofen reached 1 ng / mL. 50 The IC50 of metformin reached 20 ng / mL. 50 It reached 200 ng / mL.

[0018] This invention also provides a method for preparing a hybridoma cell line that secretes broad-spectrum monoclonal antibodies against biguanide drugs, comprising the following steps: S1. Prepare a complete biguanide drug antigen from a biguanide drug hapten, and use the complete biguanide drug antigen to immunize animals; S2. Blood samples are collected from immunized animals to screen for serum immunogenicity and immunosuppressive capacity. S3. The spleen cells and myeloma cells of the selected immunized animals are fused and cultured to obtain the hybridoma cell line that secretes the broad-spectrum monoclonal antibody of biguanide drugs.

[0019] Further, in step S1, the animal immunization process includes primary immunization, booster immunization and sprint immunization. Primary immunization uses complete antigen and complete Freund's adjuvant, booster immunization uses complete antigen and incomplete Freund's adjuvant, and sprint immunization uses complete antigen.

[0020] In one embodiment of the present invention, the animal is a mouse.

[0021] In one embodiment of the present invention, the structure of the biguanide hapten in step S1 is as follows: .

[0022] In one embodiment of the present invention, the biguanide drug hapten is obtained by the following preparation method: reacting bis(triphenylphosphine)palladium(II) chloride with methyl acrylate to obtain the biguanide drug hapten.

[0023] In one embodiment of the present invention, in step S1, the complete biguanide drug antigen is obtained by conjugating a biguanide drug hapten with a carrier protein; the carrier protein includes bovine serum albumin (BSA), ovalbumin (OVA), etc.

[0024] In one embodiment of the present invention, the biguanide drug hapten is conjugated to a carrier protein via a carbodiimide method. The specific preparation method includes the following steps: (1) Activate the above-mentioned biguanide drug hapten to obtain an activated solution; (2) Add the activation solution obtained in step (1) above into the carrier protein solution and react to obtain the complete antigen of biguanide drug.

[0025] In one embodiment of the present invention, in step (1), the activation is: dissolving the biguanide drug hapten with N,N-dimethylformamide, and adding 1-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for reaction.

[0026] In one embodiment of the present invention, in step (2), the carrier protein solution is obtained by dissolving the carrier protein in a carbonate buffer solution.

[0027] In one embodiment of the present invention, the carbonate buffer solution has a concentration of 0.01 mol / L to 0.5 mol / L, preferably 0.05 mol / L; and a pH of 8.0 to 10.0, preferably 9.6.

[0028] In one embodiment of the present invention, in step (2), the solution after the reaction is dialyzed and separated to obtain the complete antigen of biguanide drugs.

[0029] In one embodiment of the present invention, serum immune titer and immunosuppressive capacity are detected by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA).

[0030] In one embodiment of the present invention, the biguanide broad-spectrum detection product further includes a coating antigen, which is obtained by conjugating a biguanide hapten with ovalbumin.

[0031] In one embodiment of the present invention, the biguanide broad-spectrum detection kit is used for the residue analysis and detection of biguanide drugs in food (such as health food) safety testing.

[0032] The technical solution of the present invention has the following advantages over the prior art: This invention provides a hybridoma cell line that secretes a broad-spectrum monoclonal antibody against biguanide drugs and its applications. The biguanide drug hapten synthesis procedure provided by this invention is simple and efficient, and can be fully utilized in immunoassays, offering a convenient approach for future research. The complete antigen prepared based on this hapten, the hybridoma cell line obtained by immunizing mice with the complete antigen, and the monoclonal antibody secreted by it exhibit good specificity and detection sensitivity (IC50 of the biguanide drug monoclonal antibody against phenformin). 50 The IC50 of butylguanidine is 1 ng / mL. 50 The IC50 of metformin is 20 ng / mL. 50 With a concentration of 200 ng / mL, it can detect multiple residues of biguanide drugs, providing an immunoassay method and raw materials for the immunoassay of illegally added biguanide drug residues in health foods, and has practical application value.

[0033] Preservation of biological materials A hybridoma cell line, THF, secreting a broad-spectrum monoclonal antibody against biguanide drugs, 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. 46731. Attached Figure Description

[0034] 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: Figure 1 The standard inhibition curve of the biguanide broad-spectrum monoclonal antibody prepared in the embodiments of the present invention is shown. Detailed Implementation

[0035] 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.

[0036] This invention uses a product with a carboxyl group obtained by reacting bis(triphenylphosphine)palladium(II) chloride with methyl acrylate as a hapten. The hapten is conjugated to a carrier protein using the carbodiimide method, and the success of the conjugation is determined by ultraviolet spectrophotometry. For the initial immunization, BALB / c mice are immunized with a mixture of 100 μg of the complete biguanide antigen and an equal volume of complete Freund's adjuvant via subcutaneous injection at multiple sites on the neck and back. Multiple booster immunizations (50 μg / mouse) are performed using incomplete Freund's adjuvant. The final immunization is performed by intraperitoneal injection of 25 μg of the complete biguanide antigen (diluted with physiological saline and without adjuvant). High titer and low IC50 are selected. 50 Mouse spleen cells were fused with SP2 / 0 myeloma cells using the PEG4000 method. Hybrid cells from the three cell lines were selected using selective culture medium. Cells were then screened using an indirect competitive enzyme-linked immunosorbent assay (ELISA) and subjected to four subcloning processes to finally obtain a monoclonal antibody hybridoma cell line. The monoclonal antibody secreted by this cell line exhibits good broad-spectrum activity and detection sensitivity against biguanide drugs (IC50 of the biguanide monoclonal antibody against phenformin). 50 The IC50 of butylguanidine is 1 ng / mL. 50 The IC50 of metformin is 20 ng / mL. 50 This invention, with a concentration of 200 ng / mL, enables the detection of biguanide drugs, providing a raw material for the immunoassay of biguanide drug residues in health foods and possessing practical application value. Furthermore, this invention successfully synthesized an artificial antigen for biguanide drugs; the synthesis steps are simple and effective, and it can be fully utilized in immunoassays, providing essential artificial antigens for future research.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0038] (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.

[0039] (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. Phosphate buffer (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 with ultrapure water. Washing buffer (PBST): Add 0.5 mL of Tween-20 to 1000 mL of 0.01 mol / L pH 7.4 PBS solution; PBST: PBS containing 0.05% Tween-20; Antibody diluent: a washing solution containing 0.1% gelatin; 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.

[0040] (3) The detection methods involved in the following embodiments are as follows: Method for detecting biguanide inhibition rate: The optimal antigen and antibody concentrations for icELISA were selected using a checkerboard assay. The antigen was diluted to 1 μg / mL, 0.3 μg / mL, 0.1 μg / mL, and 0.03 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 1 μg / mL, 0.3 μg / mL, 0.1 μg / mL, and 0.03 μg / mL with antibody dilution buffer. After selecting the optimal operating point, biguanide standards were diluted to concentrations of 3600 ng / mL, 1800 ng / mL, 600 ng / mL, 200 ng / mL, 66.67 ng / mL, 22.22 ng / mL, and 7.41 ng / mL. Following the icELISA procedure, the biguanide standard inhibition curve was obtained using Origin 2024, and the IC50 was calculated. 50 .

[0041] Example 1 Synthesis of hapten BYSH-2 The product containing a carboxyl group, namely the hapten BYSH-2, is obtained by reacting bis(triphenylphosphine)palladium(II) chloride with methyl acrylate.

[0042] The synthesis route is as follows:

[0043] The specific steps are as follows: K₂CO₃ (2.764 g, 20 mmol), bis(triphenylphosphine)palladium(II) chloride (140.1 mg, 0.2 mmol), and methyl acrylate (1.8 mL, 20 mmol) were added to a DMF (50 mL) solution of compound 1 (3.0 g, 10 mmol). The mixture was stirred at 120 °C for 16 h. The mixture was diluted with H₂O (50 mL) and extracted with EA (3 x 80 mL). The bound organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography (PE / EA = 10 / 1) to give compound 2 (2.53 g, 79.7% yield).

[0044] Pd / C (253 mg, 10%) was added to a MeOH solution (30 mL) of compound 2 (2.53 g, 8.29 mmol). The mixture was stirred at 20 °C for 16 h under H2. The mixture was recovered by filtration, and the filtrate was concentrated under vacuum to give crude compound 3 (2.2 g, yield 86.4%).

[0045] TFA (10 mL) was added to a solution of compound 3 (2.2 g, 7.16 mmol) in DCM (30 mL). The mixture was stirred at 20 °C for 3 h. The solution was concentrated under vacuum to give crude compound 4 (1.3 g, 87.7% yield). TMSOTf (1.4 g, 6.27 mmol) was added to a solution of compound 4 (1.3 g, 6.27 mmol) in 1,2-dichloroethane (30 mL). The mixture was stirred at room temperature for 30 min. Dicyandiamide (0.53 g, 6.27 mmol) was added to the reaction solution, and the mixture was stirred at 80 °C for 16 h. After the reaction was complete, the product was kept at room temperature, and 12N HCl (0.5 mL, 6.27 mmol) was added and stirred for 1 h. The resulting solid was filtered and washed with 10 mL of dichloromethane; the solid was product 5 (1.5 g, 72.8% yield). A solution of 5 (1.5 g, 4.58 mmol) in 2N HCl (30 mL) was prepared. The mixture was stirred at 80 °C for 16 hours. The solution was filtered to obtain a solid as the crude product. The crude oil was purified by reverse flash chromatography (ACN / H2O = 5 / 95 to 95 / 5) to give the crude oil. The crude oil was further purified by Prep-HPLC to give the product BYSH-2 (450 mg, yield 35.4%).

[0046] Example 2 Preparation of complete antigen The hapten BYSH-2 is coupled to a carrier protein using the carbodiimide method to obtain an artificial antigen for biguanide drugs. Specifically, the hapten BYSH-2 prepared in Example 1 is coupled to bovine serum albumin (BSA) to obtain the immunogen BYSH-2-EDC-BSA; or coupled to chicken ovalbumin (OVA) to obtain the coating antigen BYSH-2-EDC-OVA.

[0047] The immunogen BYSH-2-EDC-BSA is prepared as follows: a. Weigh 0.826 mg of the hapten BYSH-2, 3.162 mg of N-hydroxysuccinimide, and 1.0 mg of 1-ethylcarbodiimide hydrochloride prepared in Example 1, and dissolve them in 300 μL of N,N-dimethylformamide (referred to as solution A). Stir the mixture at room temperature for 4-6 h. Weigh 6.0 mg of BSA (BYSH-2 to BSA molar ratio of 20:1), add 2 mL of carbonate buffer solution (the dissolved BSA protein is referred to as solution B), and add solution A dropwise to solution B at room temperature. Adjust the pH of the mixture to 8-9 with 1 M NaOH solution, and react at room temperature overnight to obtain the conjugate BYSH-2-EDC-BSA.

[0048] b. Dialysis: Cut an 8 cm dialysis bag, boil it in boiling water for 3 minutes and cool it, then store it in deionized water at 4℃ for later use; put the conjugate BYSH-2-EDC-BSA / OVA into the dialysis bag and dialyze it in 0.01 mol / L PBS, changing it every 8 hours, and dialyze for 3 days to obtain the complete antigen BYSH-2-EDC-BSA / OVA, which should be taken out and stored at -20℃.

[0049] The preparation method of the coating antigen BYSH-2-EDC-OVA is similar to that of the immunogen BYSH-2-EDC-BSA, except that chicken ovalbumin OVA is used instead of bovine serum albumin BSA.

[0050] Example 3 Immunization in mice For the initial immunization, BALB / c mice were immunized with a mixture of 100 μg of the complete biguanide antigen and an equal volume of complete Freund's adjuvant, emulsified, and injected subcutaneously at multiple sites on the neck and back. Four weeks later, a booster immunization was performed with half the dose of the complete antigen (50 μg / mouse), emulsified with incomplete Freund's adjuvant. Subsequent booster immunizations were administered at 3-week intervals. For the final sprint immunization, the dose was again halved (25 μg / mouse), and the complete antigen was diluted with physiological saline and injected intraperitoneally. After the third immunization, tail-disconnected blood samples were collected for testing. Serum titers and IC50 values ​​were determined using an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). 50 Choose high-performance ICs 50 Low-grade mice were fused; Example 4 Cell Fusion and Screening (1) Three days after the sprint immunization, cell fusion was performed according to the conventional PEG 4000 (polyethylene glycol) method. The specific steps are as follows: a. Collection of SP2 / 0 tumor cells: 7-10 days before fusion, culture SP2 / 0 tumor cells 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. During fusion, tumor cells are collected, suspended in RPMI-1640 basal culture medium, and cell counting is performed. b. After euthanizing mice by cervical dislocation, immediately sterilize them in 75% alcohol for about 5 minutes. Aseptically remove the spleen, gently grind it with a syringe tip, and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect 50 mL of the suspension in a sterile centrifuge tube, centrifuge at 1200 r / min for 8 minutes, wash the spleen cells with RPMI-1640 medium, remove any large tissue impurities, and repeat the process three times. After the final centrifugation, dilute the spleen cells to a specific volume, count them, and set aside for later use. c. Fusion process (7 min): At min 1, add 1 mL of PEG 4000 dropwise to the cells, gradually increasing the speed. At min 2, allow the centrifuge tube to stand and hold it firmly with both hands. At min 3 and min 4, add 1 mL of RPMI-1640 medium dropwise every 1 min. At min 5 and min 6, add 1 mL of RPMI-1640 medium dropwise every 30 s. At min 7, add 1 mL of RPMI-1640 medium dropwise every 10 s. Then incubate at 37°C for 5 min. Centrifuge at 800 r / min for 10 min, discard the supernatant, gently break up the cells in the centrifuge tube, and add RPMI-1640 selective medium (HAT medium) containing 20% ​​fetal bovine serum and 2% 50×HAT to the medium. Add 200 μL / well to a 96-well cell plate and incubate at 37°C in a 5% CO2 incubator.

[0051] (2) Cell screening and cell line establishment: On day 3 after cell fusion, the fused cells were partially replaced with HAT medium; on day 5, the medium was completely replaced with RPMI-1640 transition medium (HT medium) containing 20% ​​fetal bovine serum and 1% 100×HT; on day 7, the cell supernatant was collected for screening. The screening was carried out in two steps: first, positive cell wells were screened using ic-ELISA; second, biguanide drug standards were selected, and the inhibitory effect on positive cells was determined using ic-ELISA. Cell wells that showed good inhibition of biguanide drug standards were selected, and subcloning was performed using the limiting dilution method. The same method was used for detection seven days later. Subcloning was performed four times according to the above method to finally obtain the biguanide drug monoclonal antibody cell line LBEM.

[0052] Example 5: Preparation and Identification of Monoclonal Antibodies 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 2 × 10⁻⁶ g of paraffin oil. 6Biguanide hybridoma cells were used, and ascites fluid was collected starting from day 7. The ascites fluid was then purified for antibody using the caprylic acid-saturated ammonium sulfate method. Under slightly acidic conditions, caprylic acid precipitates other proteins in the ascites fluid besides IgG immunoglobulins. The precipitate was then discarded by centrifugation. IgG-type monoclonal antibodies were then precipitated with an equal volume of saturated ammonium sulfate solution, centrifuged, and the supernatant was discarded. The precipitate was dissolved in 0.01 M PBS solution (pH 7.4), dialyzed to desalt, and finally the purified monoclonal antibodies were stored at -20°C.

[0053] The IC50 of monoclonal antibodies against biguanide drugs was determined using the icELISA method. 50 The concentrations were phenformin 1 ng / mL, buformin 20 ng / mL, and metformin 200 ng / mL.

[0054] Antibody application (1) Coating: The original coating BYSH-2-EDC-OVA was diluted 3-fold from 1 µg / mL with 0.05 M (pH 9.6) carbonate buffer, 100 μL / well, and reacted at 37℃ for 2 h.

[0055] (2) Washing: Pour out the solution in the plate and wash with washing solution 3 times, 3 min each time.

[0056] (3) Sealing: After patting dry, add 200 μL / well sealing solution and react at 37℃ for 2 h. Wash and dry for later use.

[0057] (4) Sample addition: The antiserum (antiserum obtained by diluting the blood from the tail of mice with antibody diluent) was serially diluted from 1:1000 and added to each well of the coating at 100 μL / well. The reaction was carried out at 37℃ for 30 min. After thorough washing, HRP-goat anti-mouse IgG diluted at 1:3000 was added at 100 μL / well. The reaction was carried out at 37℃ for 30 min.

[0058] (5) 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. (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.

[0059] IC50 of monoclonal antibody biguanide drugs was determined using ic-ELISA. 50 The values ​​were: phenformin 1 ng / mL, buformin 20 ng / mL, and metformin 200 ng / mL, indicating good sensitivity to biguanide drugs and suitable for immunoassay detection of biguanide drugs.

[0060] 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 that secretes a broad-spectrum monoclonal antibody against biguanide drugs, 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. 46731.

2. The hybridoma cell line according to claim 1, characterized in that, The biguanide drug is one or more of metformin, phenformin, and buformin.

3. A broad-spectrum monoclonal antibody against biguanide drugs, characterized in that, Produced by the hybridoma cell line described in claim 1 or 2.

4. The broad-spectrum monoclonal antibody against biguanides according to claim 3, characterized in that, The biguanide broad-spectrum monoclonal antibody was obtained by immunizing animals with the complete biguanide antigen.

5. The broad-spectrum monoclonal antibody against biguanides according to claim 4, characterized in that, The complete biguanide antigen is obtained by conjugating a biguanide hapten with a carrier protein.

6. The broad-spectrum monoclonal antibody against biguanides according to claim 5, characterized in that, The structural formula of the biguanide hapten is as follows: ； And / or, the carrier protein includes keyhole hemocyanin and / or chicken oocyte albumin.

7. A composition, characterized in that, The composition comprises the hybridoma cell line of claim 1 or 2 and / or the biguanide broad-spectrum monoclonal antibody of any one of claims 3-6.

8. A reagent kit, characterized in that, The kit comprises one or more of the hybridoma cell lines of claim 1 or 2, the biguanide broad-spectrum monoclonal antibody of any one of claims 3-6, and the composition of claim 7.

9. A test strip, characterized in that, The test strip comprises one or more of the hybridoma cell lines of claim 1 or 2, the biguanide broad-spectrum monoclonal antibody of any one of claims 3-6, and the composition of claim 7.

10. The use of the hybridoma cell line of claim 1 or 2, the broad-spectrum monoclonal antibody against biguanide drugs of any one of claims 3-6, the composition of claim 7, the kit of claim 8, or the test strip of claim 9 in the detection of biguanide drugs; the use does not relate to the diagnosis and treatment of diseases.

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