Monoclonal antibody recognizing Red 2G, hybridoma cell line secreting the antibody and application thereof

Abstract

The present invention belongs to the technical field of chemical synthesis, and specifically relates to a method for synthesizing 5-acetamido-3-azo-4-naphthol-2,7-disulfonic acid and its application. The present invention uses p-aminobenzoic acid and 1-amino-8-naphthol-3,6-disulfonic acid as raw materials, and synthesizes the carboxylation product of Red 2G (R2G) 5-acetamido-3-azo-4-naphthol-2,7-disulfonic acid (R2G-COOH) through diazotization, acetylation, coupling and other steps. The synthesis method of the present invention is simple, has little harm to the environment and human body, the raw materials are cheap and easy to obtain, the reaction conditions are mild, the product purity is high, and it can be used as a hapten. After coupling with the carrier protein BSA and immunizing mice, a monoclonal antibody with high sensitivity and strong specificity is prepared, and the IC of Red 2G is high. 50 Reaching 0.1µg / L, it can be used for rapid detection of red 2G residues in food and has high practical value.

Description

Technical Field

[0001] The present invention belongs to the fields of chemical synthesis technology and immunology technology, and particularly relates to a monoclonal antibody recognizing Red 2G, a hybridoma cell line secreting the antibody and applications thereof. Background Art

[0002] Red 2G, also known as Acid Red 1, Food Red 10, and 8-acetamido-2-phenylamino-1-naphthol-3,6-disulfonic acid disodium salt, is commonly used as a food colorant in cooked meats, sausages, jams, alcoholic beverages, eggs, and dairy products due to its low price and strong coloring power. Consuming Red 2G can cause poisoning in humans, and long-term consumption can even cause cancer.

[0003] Currently, Red 2G is mostly detected using instrumental methods, including high-performance liquid chromatography (HPLC), ultrahigh performance liquid chromatography (UPLC), liquid chromatography-mass spectrometry (LC-MS), and ultraviolet-visible spectroscopy (UV-Vis). However, HPLC and UPLC have disadvantages such as poor resistance to interference in complex matrices, expensive equipment, a limited variety of detectors, a lack of universal detectors, and time-consuming sample pretreatment. LC-MS is highly efficient and accurate, but the coupled instrument is expensive and far less widely available than HPLC. UV-visible spectroscopy is susceptible to interference from other optically active additives and flavorings, leading to false positives. Enzyme-linked immunosorbent assay (ELISA) offers advantages such as high sensitivity, strong specificity, simple operation, and low instrumentation costs.

[0004] At present, there are only two literature reports on the preparation of Red 2G monoclonal antibodies and their immunological detection methods at home and abroad. The invention patent application with application publication number CN 102798713 A discloses a detection kit for Food Red 10 (Red 2G) and its preparation method, and discloses the preparation of an artificial antigen for Food Red 10 (Red 2G). The hydroxyl group of the biphenol in the Red 2G structure is converted into a methanesulfonate group, and a hapten capable of coupling with protein is synthesized under the action of anhydrous ethanol and liquid ammonia. The hapten is coupled to bovine serum albumin (BSA) to synthesize an artificial antigen. The monoclonal antibody prepared from this antigen has an IC of 0.05 for Red 2G. 50The sensitivity of the monoclonal antibody prepared by this patent to Red 2G is low, and the preparation process of haptens and artificial antigens is relatively complicated and time-consuming. The use of volatile gases such as dichloromethane and liquid ammonia in the synthesis process can cause harm to the human body. The invention patent application with application publication number CN114032215 A discloses a hybridoma cell line that secretes Red 2G monoclonal antibodies and its application, and discloses a method for synthesizing Red 2G artificial antigens. Diazotized p-aminobenzenesulfonic acid is directly coupled with 1-amino-8-naphthol-3,6-disulfonic acid to make the Red 2G structure contain carboxyl groups, and then connected to BSA to synthesize artificial antigens. The IC of the monoclonal antibody prepared with this antigen to Red 2G is 0. 50 2μg / L. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for synthesizing 5-acetamido-3-azo-4-naphthol-2,7-disulfonic acid (R2G-COOH), a carboxylation product of Red 2G. The synthetic steps are simple and effective.

[0006] The present invention also provides a method for preparing a complete antigen by using R2G-COOH as a hapten, and obtaining a monoclonal antibody with high sensitivity and strong specificity by immunizing mice, cell fusion and screening. 50 Reach 0.1μg / L.

[0007] The present invention is achieved through the following technical solutions:

[0008] 1. The present invention provides a method for synthesizing R2G-COOH, and the specific preparation steps are as follows:

[0009] (1) Synthesis of p-diazoaminobenzoic acid

[0010] Dissolve p-aminobenzoic acid in ultrapure water, stir evenly at 0-5°C, add 30% hydrochloric acid, cool to 4°C, and add NaNO2 solution dropwise every 10s within 5-10min. The liquid will quickly turn blue when tested with Congo red paper and starch potassium iodide paper, indicating that the reaction is complete. At this time, the liquid is yellow. Stop adding NaNO2 solution, maintain the temperature at 0-5°C and continue stirring for 2h. The obtained solution is referred to as liquid A and is stored at 0-5°C for future use.

[0011] (2) Synthesis of 1-acetylamino-8-naphthol-3,6-disulfonic acid

[0012] Mix 1-amino-8-naphthol-3,6-disulfonic acid (H acid, gray powder) and water, stir at 20°C for 10 minutes, then add acetylation catalyst - acetic anhydride, heat to 30°C, react for 30 minutes, turn into a brown-black liquid, cool, adjust the pH value to 9-10 with Na2CO3, filter, and obtain 1-acetylamino-8-naphthol-3,6-disulfonic acid, referred to as liquid B, and store at 0-5°C for use.

[0013] (3) Synthesis of R2G-COOH

[0014] The solution A obtained in step (1) is added to the solution B obtained in step (2) at 0-5°C within 5-10 minutes. The optimal molar ratio of solution A to solution B is (1-1.5):1. The pH value is controlled at about 8. The mixture is stirred at 0-5°C for 1 hour. After the reaction is completed, sodium chloride is added, and the mixture is heated to 65°C and stirred for 30 minutes until there is no crystal at the bottom of the flask and the mixture becomes viscous. The mixture is cooled to 30°C, filtered, and the filter cake is collected.

[0015] (4) Purification of R2G-COOH

[0016] The filter cake obtained in step (3) was dissolved in hot water, heated to 65°C, stirred for 30 min, cooled to 40°C, and then filtered. The filter cake was collected and vacuum dried overnight to obtain the product R2G-COOH.

[0017] 2. The present invention provides the use of R2G-COOH in the field of immunology to prepare monoclonal antibodies that recognize Red 2G, which is carried out in the following manner:

[0018] (1) R2G-COOH was used as a hapten and coupled with a carrier protein to synthesize an immunogen and a coating. The carrier protein of the immunogen was bovine serum albumin (BSA). The immunogen feed ratio (R2G-COOH:BSA) was 100:1, i.e., the molar ratio was 15.5.

[0019] The carrier protein of the coating agent is ovalbumin (OVA), and the coating agent is set at different feed ratios (R2G-COOH:OVA) of 25:1, 50:1, 75:1, 100:1, and 125:1, among which 50:1 is the best feed ratio and the best coupling ratio is 15.4.

[0020] (2) The immunogen was injected subcutaneously at the back of mice at different doses. The first immunization was performed with two immunization doses of 50 μg / mouse and 100 μg / mouse. The immunogen was mixed with Freund's complete adjuvant and injected subcutaneously at multiple points. After an interval of 3 weeks, the same immunization dose was emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple points. The immunization interval was 2 weeks. Blood was collected by tail cutting 6 to 8 days after the first booster immunization. The titer and specificity of mouse serum were detected by indirect competitive ELISA. Mice with high titer and strong specificity were selected for cell fusion. Studies have shown that the titer and sensitivity of mouse serum at an immunization dose of 50 μg / mouse are better than those at 100 μg / mouse.

[0021] (3) Cell fusion and screening:

[0022] Mouse immune spleen cells and SP 2 / 0 myeloma cells were fused using polyethylene glycol (PEG4000), and hybridoma cells were cultured in HAT selection medium. Positive cell wells were screened by indirect competitive ELISA, and the cell wells with the highest titer and the best inhibitory effect were selected for subcloning. The cells were cultured in HT selection medium and detected by indirect competitive ELISA after 5 days. After four consecutive subclones, the hybridoma cell line 2G6 (deposit number CCTCCNO: C2022335) capable of secreting Red 2G-specific antibodies was obtained.

[0023] (5) Preparation of monoclonal antibodies:

[0024] Each mouse was pretreated by intraperitoneal injection of 0.5 mL of liquid paraffin. After 7 days, each mouse was intraperitoneally injected with 0.5 mL of hybridoma cell suspension (1×10 cells) in logarithmic growth phase. 6 cell / mL. After 7 days, observe the mice daily for ascites production. When the abdomen becomes noticeably distended, disinfect the abdominal skin with an alcohol cotton ball and collect the ascites. Centrifuge the collected ascites at 8000 rpm for 10 minutes, remove the surface oil layer, and aspirate the supernatant to obtain the monoclonal antibody. Add glycerol, mix thoroughly, and aliquot for storage at -20°C.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The p-aminobenzoic acid, hydrochloric acid, NaNO2, 1-amino-8-naphthol-3,6-disulfonic acid, acetic anhydride, sodium chloride, etc. used in the synthesis of the carboxylation product of Red 2G of the present invention, 5-acetamido-3-naphthol-4-sulfonic acid, are all commonly used raw materials and reagents and are easily available.

[0027] 2. The raw materials used in the synthesis of the carboxylation product of Red 2G of the present invention, 5-acetamido-3-benzoyl azo-4-naphthol-2,7-disulfonic acid, are relatively safe, have low toxicity, and cause little pollution to the environment.

[0028] 3. The reaction equipment and reaction conditions in the synthesis of the carboxylation product of Red 2G, 5-acetamido-3-benzoyl azo-4-naphthol-2,7-disulfonic acid, are easy to implement, and the experimental instruments used, such as the flask, constant temperature water bath, suction filtration bottle, pH meter, etc., are low in cost and simple to operate.

[0029] 4. The application process of the synthetic product R2G-COOH of the present invention is simple and effective, and is suitable for the field of immunological analysis.

[0030] 5. The present invention successfully immunized mice by screening different immune doses, obtained mice with high titer and good inhibitory effect for cell fusion, and screened out hybridoma cell line 2G6 (deposit number CCTCC NO: C2022335). The monoclonal antibody secreted by this cell line has good specificity and detection sensitivity (IC) for red 2G. 50 0.1μg / L), which has practical application value in immunological detection methods.

[0031] 6. The monoclonal antibody secreted by the hybridoma cell line 2G6 with the deposit number CCTCC NO: C2022335 obtained in the present invention has a sensitivity to Red 2G that is 20 times that of the invention patent application with the publication number CN114032215 A and 9514 times that of the invention patent application with the publication number CN 102798713 A (see Table 1).

[0032] Table 1. Positive effects of the present invention compared with the prior art

[0033] Comparing Technologies Hapten synthesis raw materials Coupling method Carrier protein <![CDATA[IC 50 (μg / L)]]> CN 102798713A Food Red 10 (Red 2G) EDC BSA / OVA 951.4 CN 114032215A p-Aminobenzenesulfonic acid, 1-amino-8-naphthol-3,6-disulfonic acid EDC BSA / OVA 2.0 The present invention p-Aminobenzoic acid, 1-acetylamino-8-naphthol-3,6-disulfonic acid EDC BSA / OVA 0.1 BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 UV scanning spectrum of R2G-COOH synthesized in the present invention (PBS is the solvent).

[0035] Figure 2 The infrared scanning spectrum (KBr) of R2G-COOH synthesized by the present invention.

[0036] Figure 3 R2G-COOH synthesized by the present invention 1 HNMR spectrum (tritiated water).

[0037] Figure 4 R2G-COOH synthesized by the present invention 13 CNMR spectrum (tritiated water).

[0038] Figure 5 Mass spectrum of R2G-COOH synthesized in the present invention (methanol as solvent).

[0039] Figure 6The chemical structural formula of R2G-COOH synthesized by the present invention.

[0040] Figure 7 Standard curve of drug inhibition of Red 2G by monoclonal antibodies.

[0041] Preservation Information

[0042] Deposit date: October 28, 2022;

[0043] Name of depository: China Center for Type Culture Collection;

[0044] Deposit number: CCTCC NO:C2022335;

[0045] Address of depository: Wuhan University, Wuhan, China;

[0046] Classification and nomenclature: hybridoma cell line 2G6. DETAILED DESCRIPTION

[0047] The present invention is described in more detail below through specific implementation methods to facilitate understanding of the technical solution of the present invention, but is not intended to limit the scope of protection of the present invention.

[0048] Example 1:

[0049] A chemical synthesis method of R2G-COOH, synthesis route:

[0050]

[0051] (1) Reaction solution A: Synthesis of p-diazoaminobenzoic acid

[0052] Add p-aminobenzoic acid (0.889 g, 6.0 mmol) to a beaker, dissolve in 3.5 mL of ultrapure water, add 1.8 mL of 30% hydrochloric acid, cool to 4°C, add 30% NaNO2 dropwise every 10 seconds within 10 minutes, and stop adding NaNO2 solution when Congo red test paper and starch potassium iodide test paper quickly turn blue. Stir for 2 hours. The obtained solution is referred to as solution A and is stored at 4°C for future use.

[0053]

[0054] (2) Reaction solution B: H acid acetylation

[0055] Add 1.375 g of 1-amino-8-naphthol-3,6-disulfonic acid (H acid, gray powder) to a flask and dissolve it in 40 mL of ultrapure water. Stir at 20°C for 10 min, then add 4 mL of acetic anhydride, raise the temperature to 30°C, and react for 30 min. It will turn into a brown-black liquid. After cooling, adjust the pH to 9 with Na2CO3, remove the generated acetic acid, and filter. The resulting solution is referred to as solution B and is stored at 4°C for later use.

[0056]

[0057] (3) Coupling: Add the solution A obtained in step (1) to the solution B obtained in step (2) at 4°C for 5 min. The molar ratio of solution A to solution B is 1:1. A dark red substance with foam is generated. The pH value is controlled at about 8. Stir at 4°C for 1 h. After the reaction is completed, sodium chloride is added. Heat to 65°C and stir for 30 min until there is no crystal at the bottom of the flask and the solution becomes viscous. Cool to 30°C, filter, and collect the filter cake.

[0058] (4) Purification: Add 20 mL of hot water and a small amount of sodium chloride to the filter cake obtained in step (3), heat to 65°C, stir for 30 min, filter at 40°C, and vacuum dry overnight to obtain about 1.06 g of product R2G-COOH.

[0059] The product prepared by the present invention was tested by ultraviolet, infrared, nuclear magnetic resonance and mass spectrometry, and the results were as follows:

[0060] Figure 1 The figure shows the UV scanning spectrum of R2G-COOH (Aglient 8453 model). PBS was used as the solvent. The peak at 311 nm may be due to the influence of the substituents on the benzene ring, resulting in a certain shift in the characteristic peak (the characteristic peak of benzene is at 254 nm). The peak at 232 nm should be attributed to the absorption of the aromatic ring. The maximum characteristic absorption peak in the visible region is around 510 nm, which is caused by the electronic transition of the entire conjugated system of the azo group and the aromatic ring, which is consistent with the characteristics of this substance.

[0061] Figure 2 The infrared scanning spectrum of R2G-COOH (VERTEX70, Fourier transform infrared spectrometer), potassium bromide (KBr) tablet, can be seen from the figure that the corresponding relationship between the main absorption peaks and each group is: 3439cm -1 Corresponding to the stretching vibration of NH, 1 700~1 680cm -1 is the vibration absorption peak of the carboxyl group at the para position of the benzene ring, 1603 cm -1 Vibration absorption of C=O on acetyl group on Red 2G, 1557~1492cm -1 Corresponding to the skeletal vibration of the benzene ring, 1 497 cm -1Corresponding to -N=N- stretching vibration, 1 324cm -1 It should be CN stretching vibration, 1 052cm -1 and 986cm -1 Corresponding to -SO 3- The symmetric and asymmetric stretching vibrations of S=O in the group are 897~688cm -1 It corresponds to the CH bending vibration on the benzene ring, which is consistent with the characteristics of this substance.

[0062] Figure 3 Shown is R2G-COOH 1 The HNMR spectrum (Bruker Avance III 400MHz, Germany) used deuterated water as the solvent, and the active hydrogen would produce deuterated hydroxyl, carboxyl, amino, and sulfonic acid groups, but no peaks would appear. In the hydrogen spectrum, the maximum chemical shift at δ8.68 (s, 1H) belongs to number 2; 7.61 (s, 1H) is one of the hydrogens at 3 or 4, and the other one coincides with the hydrogen of the para-substituted benzene ring at 7.54 (d, J = 8.3 Hz, 3H); 7.77 (d, J = 8.4 Hz, 2H) is the hydrogen at position 6 of the para-substituted benzene ring, and the hydrogen at position 5 is at 7.54 (d, J = 8.3 Hz, 3H); the methyl group of the acetyl group is at 2.19 (s, 3H), and there are a total of 10 hydrogens, which is consistent with the characteristics of this substance.

[0063] Figure 4 Shown is R2G-COOH 13 The CNMR spectrum (Bruker Avance III 400MHz, Germany) uses deuterated water as the solvent. There are a total of ten carbon peaks in the carbon spectrum, of which the carbonyl peaks 7 and 8 at δ179.95 and 172.94 are aromatic carbons, and most of them are quaternary carbons, which cannot be completely assigned. Among them, it can be clearly seen that 130.55 and 117.23 are assigned to carbons 5 and 6 of the para-substituted benzene ring, respectively; the methyl carbon 1 of the acetyl group is at 24.84; the number of remaining carbons is 12 in total, and the chemical shifts of some quaternary carbons may overlap, forming one peak; therefore, the carbon spectrum contains a total of 19 carbons, which is consistent with the characteristics of this substance.

[0064] Figure 5 The figure shows the mass spectrum of R2G-COOH (IT-TOF-MS, Shimadzu). Methanol was used as solvent and the synthesized product was subjected to positive ion Na + Mode, NH4 + Mode, H + Bombardment in mode and negative ion H - bombardment mode, 532.0068 is the [M+Na] of R2G-COOH +The theoretical value of the peak in the positive ion Na+ mode is 532.0091, and the measured value is 532.0068; 527.0496 is the [M+NH4] of R2G-COOH + Peak, positive ion NH4 + The theoretical value in the HPLC mode was 527.0537, and the measured value was 527.0496; 510.0242 was the [M+H] of R2G-COOH. + Peak, positive ion H + The theoretical value in the HPLC mode was 510.0272, and the measured value was 510.0242; 507.9921 was the [M+H] value of R2G-COOH. - Peak, negative ion H - The theoretical value in this mode is 508.0126, and the measured value is 507.9921, indicating that the molecular weight of the synthesized compound is consistent with the theoretical value.

[0065] In summary, it is determined that R2G-COOH is successfully synthesized. The chemical structure of the synthesized R2G-COOH is as follows Figure 6 shown.

[0066] Example 2: Preparation of immunogens and coatings

[0067] (1) Preparation of immunogen: Weigh 15.3 mg (0.03 mmol) of R2G-COOH and dissolve it in 1 mL of PBS (pH 4-6). Add 3.5 mg (0.03 mmol) of N-hydroxysuccinimide (NHS) and 5.8 mg (0.03 mmol) of carbodiimide (EDC). Stir and react at room temperature in the dark for 6 h. After the reaction is complete, filter to obtain the supernatant A. Dissolve 20 mg (0.0003 mmol) of BSA in 2 mL of PBS (R2G-COOH:BSA = 100:1, coupling ratio of 15.5) to obtain solution B. Slowly add solution A dropwise to solution B, stir and react overnight at low temperature, and dialyze against PBS at 4°C for 5 days, changing the dialysate three times a day. After dialysis, centrifuge at 8000 r / min for 10 min. Keep the supernatant to obtain R2G-COOH-BSA and store at -20°C for later use.

[0068] (2) Preparation of coating agents with different feed ratios: 7.64 mg (0.015 mmol), 15.3 mg (0.03 mmol), 22.90 mg (0.045 mmol), 30.55 mg (0.060 mmol), and 38.18 mg (0.075 mmol) of R2G-COOH were weighed and dissolved in 1 mL of PBS (pH = 5). N-hydroxysuccinimide (NHS) and carbodiimide (EDC) were added and stirred at room temperature in the dark overnight. After the reaction was complete, the supernatant solution C was obtained by filtration. 27 mg (0.0006 mmol) of OVA was dissolved in 5 mL of PBS to obtain solution D. Solution C was slowly added dropwise to solution D to obtain feed ratios R2G-COOH:OVA of 25:1, 50:1, 75:1, 100:1, and 125:1, and coupling ratios of 15.7, 15.4, 15.1, 14.9, and 13.6, respectively.

[0069] Example 3: Screening of hybridoma cell lines

[0070] (1) Mouse immunization

[0071] SPF-grade female Balb / c mice, 6 to 8 weeks old, were used for immunization. The first immunization consisted of an equal volume of Freund's complete adjuvant emulsified with the immunogen, administered subcutaneously at multiple points on the back of the neck. A booster immunization was administered 21 days later, using an equal volume of Freund's incomplete adjuvant emulsified with the immunogen, administered subcutaneously on the back of the neck. Each booster immunization was performed every 14 days. Except for the first immunization, blood was collected from the tail vein of the mice on days 6 to 8 after each booster immunization. The blood was allowed to settle overnight at 4°C and centrifuged at 8000 rpm for 5 minutes. Serum was isolated and assayed for serum antibody titer and specificity using an indirect competitive ELISA. The effects of different immunization doses on serum titer were compared, and mice with high titer and strong specificity were selected for a boost immunization. A double dose of the immunogen was injected directly into the peritoneal cavity.

[0072] Table 2. Immunization schedule

[0073]

[0074] (2) Preparation of hybridoma cells: In the three-immune serum titer test, the titer of mouse No. 2 in group A was 1:25600, and the inhibition rate reached 71.2%. A dose of 100 μg was injected into the mouse peritoneal cavity for shock immunization. After two consecutive shock immunizations, cell fusion was performed one day after the interval according to the limiting dilution method.

[0075] Here are the steps:

[0076] a. Preparation of Myeloma Cells: Frozen SP2 / 0 myeloma cells (SP2 / 0 myeloma cells were obtained from our laboratory) were removed from a liquid nitrogen tank and revived. After incubation, they were placed in a 37°C, 5% CO2 incubator. When the cells had grown to the bottom of the well and were in good condition, the cells were harvested and the density was adjusted to 1 × 10 6 Balb / c mice were inoculated subcutaneously at multiple points on the back of the neck with a 0.5 mL injection. After one week, the mice were observed daily for tumor growth. When the tumors grew to approximately 3 cm in diameter, the mice were anesthetized with ether and sacrificed by cervical dislocation. The mice were then immersed in 75% alcohol for 5 minutes. In a clean bench, the mice were positioned with their backs facing upwards. The dorsal skin was opened with ophthalmic scissors. After carefully dissecting the tumor capsule, the tumor tissue was removed with curved ophthalmic forceps and transferred to a glass homogenizer. 10 mL of RPMI-1640 basal medium was added, and the cells were thoroughly ground. Another 10 mL of RPMI-1640 basal medium was added, and the mixture was mixed. The tubes were allowed to stand for 2 minutes. 18 mL of the supernatant cell suspension was transferred to a 50 mL centrifuge tube. 10 mL of RPMI-1640 basal medium was added to the glass homogenizer and transferred again. The tubes were centrifuged at 1200 rpm for 5 minutes, the supernatant discarded, and the cells resuspended in 15 mL of RPMI-1640 basal medium. Slowly drip the cell suspension into a 50 mL centrifuge tube containing 15 mL of human lymphocyte separation medium. After the liquid layers are separated, centrifuge at 1700 rpm for 10 min. Use a pipette to aspirate the upper red cell suspension, then transfer the middle white myeloma cell suspension to another 50 mL centrifuge tube. Resuspend the cells in 10 mL of RPMI-1640 basal medium, count them with a hemocytometer, and set aside for use.

[0077] b. Preparation of feeder cells: An unimmunized Balb / c mouse was anesthetized with ether and sacrificed by cervical dislocation. The mouse was then immersed in 75% alcohol for 5 minutes. Under sterile conditions, the mouse was fixed with the abdomen facing upward. Using ophthalmic scissors, the entire abdominal skin and peritoneum were cut open to expose the spleen. The spleen was bluntly dissected and transferred to a glass homogenizer. 5 mL of RPMI-1640 basal medium was added and thoroughly ground. 10 mL was then added and the mixture was filtered through a 40 μm cell sieve into a 50 mL centrifuge tube. Centrifuge at 1200 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in 15 mL of RPMI-1640 basal medium. Connective tissue was removed and the mixture was centrifuged at 1200 rpm for 5 minutes. The supernatant was discarded and the cells were transferred to complete RPMI-1640 medium supplemented with 2% HAT.

[0078] c. Preparation of immune spleen cells: The method is the same as step b.

[0079] d. Cell fusion: Myeloma cells and immune spleen cells were mixed at a ratio of 1:5 to 1:10 in a 50 mL centrifuge tube. 15 mL of RPMI-1640 basal medium was added and mixed thoroughly. The tube was centrifuged at 1500 rpm for 10 minutes. The supernatant was discarded, and the water was drained. The bottom of the tube was gently tapped to loosen and evenly distribute the cells into a paste. The tube was then placed in a 37°C water bath. Over 1 minute, 0.8 mL to 1 mL of PEG preheated to 37°C was added dropwise while stirring. Gently stir for 1 minute to allow the PEG to fully contact the cells. Then, RPMI-1640 basal medium preheated to 37°C was slowly added, first slowly and then rapidly, to a total of 40 mL to terminate the fusion reaction. After gently mixing the cells, the tube was centrifuged at 800 rpm for 5 minutes. The supernatant was discarded and the tube was transferred to complete medium containing feeder cells. Gently stir to ensure even distribution of the cells. 200 μL of the fused cell suspension was plated per well of a 96-well cell culture plate and cultured at 37°C in a 5% CO2 incubator. The day of fusion was designated as day 0. Avoid moving cells for the first three days. On day 4, add 100 μL of 2% HAT complete medium to each well and observe colony growth. On day 5, aspirate 100 μL of supernatant from each well and add 100 μL of 2% HAT complete medium to continue monitoring fused cells.

[0080] (3) Screening and subcloning of positive hybridoma cell lines: According to the growth of cells, when the cells grow to about 1 / 5 of the bottom of the well, the titer and inhibition of the cell supernatant are detected (usually on the 7th to 9th day). Take the cell culture supernatant and screen it using the indirect competitive ELISA method, set the "0" well and the drug well (the concentration of R2G is set to 50μg / L when screening the drug for the first time, and the drug concentration is gradually reduced during cloning). Compared with the OD value of the "0" well, the wells whose OD value of the drug well can be significantly inhibited are judged as positive wells. Select several strong positive wells and perform subcloning using the limiting dilution method. The remaining positive wells are transferred to 24-well cell culture plates for expansion culture. The day of subcloning is day 0, and the indirect competitive ELISA test is directly performed on the 5th day. After 4 subclonings, the Red 2G monoclonal antibody cell line 2G6 was screened out and sent to the China Center for Type Culture Collection (CCTCC) located in Wuhan University, Wuhan City, Hubei Province for preservation on October 28, 2022. The preservation number is CCTCC NO: C2022335 and it is named hybridoma cell line 2G6.

[0081] Example 5: Preparation and detection of monoclonal antibodies

[0082] Each mouse was pretreated by intraperitoneal injection of 0.5 mL of liquid paraffin. Seven days later, each mouse was intraperitoneally injected with 0.5 mL of a hybridoma cell suspension (1×10 cells) in the logarithmic growth phase. 6cell / mL. After 7 days, the mice were observed daily for ascites production. When the abdomen of the mouse was noticeably distended, the ascites was collected and centrifuged at 8000 rpm for 10 minutes. The surface fat was removed, the supernatant was aspirated, and an equal volume of glycerol was added. The solution was stored at -20°C. The monoclonal antibody obtained in the present invention was subtyped according to the operating instructions of the mouse monoclonal antibody rapid ELISA subtype detection kit provided by Beijing Biolong Immunotechnology Co., Ltd., and the result was mouse IgG1 subtype.

[0083] (1) ELISA procedure

[0084] a. Array titration method: Dilute the coating agent in carbonate buffer (CBS) to a series of concentrations. Then, add 100 μL of each concentration of coating agent to each well of the ELISA plate, coating one column at a time. Incubate the plate in a humidified chamber at 4°C overnight. Discard the coating solution, pat dry, and add 250 μL of wash solution to each well. Let stand for 30 seconds, then discard the wash solution and pat dry. Repeat this process three times. Add 250 μL of blocking solution to each well and block the plate in a humidified chamber at 37°C for 1 hour. Discard the blocking solution, wash three times, and pat dry. Add 50 μL of PBS to each well. Then, dilute the serum or antibody to a series of concentrations and add 50 μL to each well of the ELISA plate. Set up negative and blank controls. Incubate the plate in a humidified chamber at 37°C for 40 minutes. Discard the solution, wash three times, and pat dry. Dilute HRP-conjugated goat anti-mouse secondary antibody in PBS to a working concentration of 1:6000. Add 100 μL per well of the ELISA plate and incubate in a 37°C wet chamber for 40 minutes. Discard the liquid in the wells, wash three times, and pat dry. Add 100 μL of substrate per well of the ELISA plate and incubate in a 37°C wet chamber for 15 minutes. Add 50 μL of stop solution per well. Measure the OD using a microplate reader. 450 Select the coating concentration and antibody dilution corresponding to the well with an OD value of around 2.0 and a significant difference from the OD value of the adjacent wells as the working concentration.

[0085] b. Indirect ELISA: Dilute the coating substrate with CBS to the working concentration determined by square array titration, add 100 μL / well, and incubate in a humidified chamber at 4°C for 10-16 hours (or at 37°C for 2-2.5 hours). Dilute the serum or antibody with PBS to the working concentration. Add 50 μL of PBS to each well, followed by 50 μL of serum or antibody. The remaining steps are the same as for the square array titration method.

[0086] c. Indirect competitive ELISA: The procedures of indirect competitive ELISA are basically the same as those of indirect ELISA. The only difference is that when adding serum or antibody, 50 μL of drug or sample should be added first, followed by 50 μL of diluted serum or antibody, and incubated in a 37°C wet box for 40 minutes.

[0087] (2) Monoclonal antibody sensitivity test

[0088] The working concentration of antigen and antibody was preliminarily determined by matrix titration method to preliminarily determine the dilution of coating source. The IC value of antibody was screened out when the coating source feed ratio was 50:1, the coupling ratio was 15.4, and the coating concentration was 80μg / L. 50 A series of antibody concentration gradients were designed for indirect competitive ELISA, and the OD value was around 2.0. IC 50 The antibody dilution corresponding to the lower value is the optimal primary antibody dilution. The optimal coating concentration is 80 μg / L, the optimal primary antibody dilution is 1∶54000, the secondary antibody dilution is 1∶6000, and the IC 50 The concentration of red 2G was 0.1 μg / L, which proved that the antibody had high sensitivity to red 2G and could be used in immunoassay detection.

[0089] (3) Monoclonal antibody specificity detection

[0090] The cross-reactivity rate of this antibody to Red 2G is 100%, of which the cross-reactivity rate to New Red is 27.5%, the cross-reactivity rate to Acid Fuchsin 6B is 23.3%, the cross-reactivity rate to Amaranth, Acid Red 26, Acid Red 33, and H acid is less than 5%, and the cross-reactivity rate to Acid Red 37, Carmine, Sudan Red 1, and p-aminobenzoic acid is less than 0.01%.

[0091] Table 3. Cross-reactivity of 2G6 monoclonal antibody to acid red compounds

[0092] Drug name <![CDATA[IC 50 (μg / L)]]> Cross-reaction rate (%) Linear range (μg / L) Red 2G 0.14 100 0.05～0.8 New Red 0.51 27.5 0.05～0.8 Acid Fuchsin 6B 0.60 23.3 0.05～0.8 Amaranth 5.46 2.56 0.05～0.8 Acid Red 26 30.92 0.45 0.39～50 Acid Red 33 12.23 1.14 0.39～50 H acid 226.99 0.06 7.81～1000 Acid Red 37 >1000 - - Carmine >1000 - - Sudan I >1000 - - p-Aminobenzoic acid >1000 - -

[0093] The embodiments described above are only preferred embodiments of the present invention and do not limit the scope of implementation of the present invention. Therefore, any equivalent changes or modifications made according to the structure, characteristics and principles described in the patent scope of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A hybridoma cell line secreting a monoclonal antibody, characterized in that: The monoclonal antibody recognizes Red 2G, and the hybridoma cell line is named hybridoma cell line 2G6, with a deposit number of CCTCC NO: C2022335.

2. Use of the hybridoma cell line according to claim 1 in the preparation of a monoclonal antibody that recognizes Red 2G.

3. A monoclonal antibody that recognizes Red 2G, characterized in that The monoclonal antibody is secreted by the hybridoma cell line according to claim 1.

4. Use of the hybridoma cell line according to claim 1 or the monoclonal antibody according to claim 2 in detecting Red 2G, wherein the detection is not for the purpose of diagnosing or treating a disease.

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