A hybridoma cell line secreting monoclonal antibodies against three ochratoxins and its application

Abstract

The present invention discloses a hybridoma cell line A7G8 secreting a broad-spectrum monoclonal antibody against three ochratoxins, which has been deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee, with the deposit number of CGMCC No. 45336. The broad-spectrum monoclonal antibody against three ochratoxins secreted by this cell line has high sensitivity and good specificity, and the 50% inhibitory concentration IC 50 of ochratoxin A, ochratoxin B, and ochratoxin C are 0.37 ng / mL, 0.23 ng / mL, and 2.24 ng / mL respectively, and the cross-reaction rates with aflatoxin B1, vomitoxin, zearalenone, and fumonisin B1 are all less than 0.037%. The immunochromatographic strip detection method developed based on this antibody has high sensitivity, simple operation, and strong anti-interference ability against complex matrices, and is suitable for rapid on-site large-scale screening of ochratoxins in complex matrices of traditional Chinese medicines.

Description

Technical Field

[0001] The present invention relates to a hybridoma cell line secreting broad-spectrum monoclonal antibodies against three ochratoxins and an application thereof, which is suitable for on-site rapid screening of three mycotoxins including ochratoxin A, ochratoxin B and ochratoxin C, and belongs to the field of rapid detection of mycotoxins. Background Art

[0002] Ochratoxins are mainly produced by some species of Penicillium and Aspergillus and are an extremely important class of mycotoxins among the more than 400 known mycotoxins. The ochratoxin family is mainly composed of ochratoxin A (OTA), ochratoxin B (OTB) and ochratoxin C (OTC). OTB is a dechlorinated derivative of OTA, while OTC is an ethyl ester derivative of OTA. Among them, OTA is the most common and has been detected in various foods, such as grapes, grains, coffee, tea, milk, fish, poultry, eggs, beans and animal feed. In addition, co-contamination of foods with multiple ochratoxins has also been reported, such as the simultaneous detection of OTA and OTB in sorghum and the simultaneous detection of OTA, OTB and OTC in wine. In addition to contaminated foods, OTA has also been frequently detected in herbal medicines in recent years, which has attracted great attention worldwide.

[0003] Studies have shown that ochratoxins have multiple toxic effects, including nephrotoxicity, hepatotoxicity, teratogenicity, mutagenicity, immunotoxicity, and neurotoxicity. OTA is classified as a Class 2B carcinogen by the International Agency for Research on Cancer, the toxicity of OTC is similar to or weaker than OTA, and the toxicity of OTB is lower than OTA and OTC. Given that ochratoxins are present in trace amounts and are ubiquitous in food and herbal medicines, and that the three ochratoxins can be converted into each other, it is urgent to establish a convenient, rapid, and sensitive method for the simultaneous detection of the three ochratoxins.

[0004] Traditional methods for determining ochratoxins include thin layer chromatography (TLC), high performance liquid chromatography-fluorescence detector (HPLC-FLD) and liquid chromatography-tandem mass spectrometry (LC-MS / MS). These traditional methods have high sensitivity, accuracy and reproducibility, but they are complicated to operate, expensive equipment, high cost, time-consuming steps, and are not suitable for rapid on-site screening of a large number of samples. In recent years, immunoassays based on antibody-antigen recognition have become a new method for rapid and sensitive detection of ochratoxins. Antibodies are the core element in the development of immunoassays, which determine the sensitivity and specificity of their detection of analytes. At present, most of the reported anti-ochratoxin monoclonal antibodies can only detect one type of ochratoxin, and have a low cross-reaction rate with the other two toxins. Therefore, it is particularly necessary to prepare a broad-spectrum monoclonal antibody against three types of ochratoxins. Summary of the invention

[0005] The purpose of the present invention is to provide a hybridoma cell line that secretes broad-spectrum monoclonal antibodies against three ochratoxins, and to develop an immunochromatographic test strip detection method based on the monoclonal antibodies secreted by the cell line, which can be used for high-sensitivity, rapid and simultaneous detection of three ochratoxins in a complex matrix of traditional Chinese medicine. The method has the advantages of convenient operation, low cost, short time, and visible to the naked eye, and is suitable for large-scale on-site screening of ochratoxins, and has broad application prospects.

[0006] The present invention obtains a hybridoma cell line A7G8 that can produce broad-spectrum monoclonal antibodies that specifically recognize three ochratoxins. This cell line has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration (CGMCC) with a deposit number of CGMCC No.45336 and a deposit date of December 8, 2022. In addition, the present invention establishes an immunochromatographic test strip detection method for on-site high-sensitivity rapid screening of three ochratoxins by synthesizing coated antigens.

[0007] Technical solution:

[0008] 1. The hybridoma cell line A7G8 resistant to three ochratoxins has a deposit number of CGMCC No.45336.

[0009] 2. A broad-spectrum monoclonal antibody against three ochratoxins, secreted by a hybridoma cell line with a deposit number of CGMCC No.45336.

[0010] 3. The basic preparation and application of the A7G8 hybridoma cell line provided by the present invention comprises the following steps:

[0011] (1) Synthesis and identification of artificial antigens:

[0012] Dissolve 1.14 mg of NHS, 2.0 mg of OTA and 4.09 mg of DCC in 1.0 mL of anhydrous tetrahydrofuran, shake slowly overnight at room temperature in the dark, centrifuge at 8000 r / min for 15 min, take the supernatant and blow dry with nitrogen, add 0.5 mL of N, N-dimethylformamide to dissolve, and the activated product is obtained. Weigh 15 mg of BSA and 10 g of OVA and dissolve them in 2 mL of 0.13 mol / L NaHCO3 solution, slowly add the activated product to the BSA and OVA solutions, and shake slowly at room temperature in the dark for 4 h. The reaction product is dialyzed in 0.01 mol / L PBS solution for 72 h, centrifuged to remove the precipitate, and the immune antigen OTA-BSA and the coating antigen OTA-OVA are obtained;

[0013] (2) Mouse immunization:

[0014] The prepared immune antigen OTA-BSA was used to immunize 6-week-old Balb / c female mice. Each mouse was injected with the immunogen intraperitoneally and subcutaneously on the back at a dose of 100 μg / time. The immunization was performed once every two weeks for a total of four times. The orbital blood of the mice was collected and the serum titer and inhibition rate were detected by indirect ELISA method.

[0015] (3) Cell fusion and cell line screening:

[0016] The immune mice with the highest serum titer and the best inhibition were screened, and 100 μg of immunogen was injected intraperitoneally for shock immunization three days before cell fusion. The spleen cells of the immune mice were fused with the cultured Sp2 / 0 myeloma cells at a ratio of 3:1 to 10:1 using the PEG method. The obtained hybridoma cells were cultured in a 96-well culture plate at 37°C in a carbon dioxide incubator, and the supernatant was detected by indirect non-competitive ELISA for screening after 10 days. The positive cell line with the best inhibition was screened and subcloned four times, and finally the hybridoma cell line A7G8 was obtained. The hybridoma cell line was sent to the General Microbiology Center of the China Microbiological Culture Collection Administration, and the collection number is CGMCC No.45336;

[0017] (4) Antibody preparation and application

[0018] The hybridoma cell line CGMCC No.45336 was injected into the peritoneal cavity of mice to prepare ascites, and the ascites was collected 10 days later. The ascites was purified and freeze-dried to obtain monoclonal antibodies. Immunochromatographic test strips were prepared based on the broad-spectrum monoclonal antibodies against three ochratoxins secreted by the hybridoma cell line CGMCC No.45336 for the rapid detection of ochratoxins in complex matrices of traditional Chinese medicine.

[0019] The beneficial effects of the present invention are:

[0020] The present invention immunizes Balb / c female mice with a synthetic OTA-BSA artificial antigen to prepare a hybridoma cell line that secretes broad-spectrum monoclonal antibodies against three ochratoxins, prepares highly sensitive broad-spectrum monoclonal antibodies against three ochratoxins based on the cell line, and establishes an immunochromatographic test strip detection method using the OTA-OVA artificial antigen as a detection antigen, which can simultaneously detect the three mycotoxins OTA, OTB and OTC with high sensitivity. The method has high sensitivity, convenient operation, low cost and short time, and can be used for high-sensitivity, on-site rapid and large-scale screening of ochratoxins in food and traditional Chinese medicine.

[0021] Storage Instructions:

[0022] Reference biomaterial strain: A7G8

[0023] Scientific description: Mouse hybridoma cell line

[0024] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0025] Abbreviation of depository institution: CGMCC

[0026] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0027] Date of deposit: December 8, 2022

[0028] Registration number of the Tibet Tibetan Affairs Center: CGMCC No.45336 BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 UV scanning image of carrier protein and artificial antigen

[0030] Figure 2 Standard curve for ELISA detection of three ochratoxins

[0031] Figure 3 The test strip test result judgment diagram DETAILED DESCRIPTION

[0032] The present invention is further described by the following examples. The following examples are not intended to be limiting in any way.

[0033] Solution preparation:

[0034] Coating buffer: 1.465 g NaHCO3, 0.75 g Na2CO3, ultrapure water to 500 mL;

[0035] Sample diluent (0.01 mol / L PBS buffer, pH 7.4): 4.0 g NaCl, 0.1 g KH2PO4, 1.48 g Na2HPO4·12H2O, 0.1 g KCl, ultrapure water to 500 mL;

[0036] Washing solution: 0.5mL Tween-20 plus 500mL sample diluent and mix well;

[0037] TMB colorimetric solution: Solution A: 2.55g citric acid, 9.215g Na2HPO4·12H2O, 0.5mL Tween-20, ultrapure water to 500mL; Solution B: 1.03g citric acid dissolved in 96mL water, 70mg TMB dissolved in 4mL DMSO, mix the two and store at 4℃. Before use, take equal volumes of solution A and solution B and mix them, add 0.1% H2O2;

[0038] Stop solution: 9 mL of concentrated hydrochloric acid mixed with 99 mL of ultrapure water;

[0039] 0.002M borate buffer: 0.21g Na2BO7·10H2O and 0.11g H3BO3, dilute to 1L with ultrapure water.

[0040] Example 1: Screening of hybridoma cell line A7G8

[0041] 1. Synthesis and identification of artificial antigens

[0042] Dissolve 1.14 mg of NHS, 2.0 mg of OTA and 4.09 mg of DCC in 1.0 mL of anhydrous tetrahydrofuran, shake slowly overnight at room temperature in the dark, centrifuge at 8000 r / min for 15 min, take the supernatant and blow dry with nitrogen, add 0.5 mL of N, N-dimethylformamide to dissolve, and the activated product is obtained. Weigh 15 mg of BSA and 10 g of OVA and dissolve them in 2 mL of 0.13 mol / L NaHCO3 solution, slowly add the activated product to the BSA and OVA solutions, and shake slowly at room temperature in the dark for 4 h. The reaction product is dialyzed in 0.01 mol / L PBS solution for 72 h, and the solution is changed every 8 h. Centrifuge to remove the precipitate, and the immune antigen OTA-BSA and the coating antigen OTA-OVA are obtained, which are packaged and stored at -20 ° C for standby use.

[0043] Take the above-synthesized artificial antigen for UV scanning, the result is as follows Figure 1 The carrier protein and OTA have characteristic absorption peaks at 280 and 380 nm respectively, while the synthesized immunogen OTA-BSA has characteristic absorption peaks of both the carrier protein and OTA, indicating that the antigen was successfully prepared.

[0044] 2. Animal Immunization

[0045] OTA-BSA was used as an immunogen to immunize 6-week-old Balb / c female mice. Each mouse was injected with the immunogen intraperitoneally and subcutaneously on the back at 100 μg / time. The immunogen was immunized once every two weeks for a total of four times. For the first immunization, an equal volume of the immunogen and Freund's complete adjuvant were used for emulsification. For the next three immunizations, an equal volume of the immunogen and Freund's incomplete adjuvant were used for emulsification. One week after the third immunization, the mouse orbital blood was collected and the serum titer was detected by indirect ELISA.

[0046] 3. Screening of hybridoma cells

[0047] The immune mice with the highest serum titer and the best inhibition were screened, and 100 μg of immunogen was injected intraperitoneally for shock immunization three days before cell fusion. The spleen cells of the immune mice were fused with the cultured Sp2 / 0 myeloma cells at a ratio of 3:1 to 10:1 using the PEG method. The obtained hybridoma cells were cultured in a 96-well culture plate at 37°C in a carbon dioxide incubator. After 10 days, the supernatant was detected by indirect non-competitive ELISA for screening. The basic operation process is as follows:

[0048] 1) Dilute the coating antigen OTA-OVA 4000 times with the coating solution, add 100 μL / well to a 96-well ELISA plate, coat at 37°C for 4 hours, and wash the plate 4 times with washing solution;

[0049] 2) Add 50 μL of sample diluent and 50 ng / mL OTA standard to the blank well and inhibition well, respectively, then add 50 μL of hybridoma cell culture medium to the blank well and inhibition well, incubate at 37°C for 45 min, and wash the plate 4 times;

[0050] 3) Add 100 μL of goat anti-mouse enzyme-labeled secondary antibody IgG-HRP diluted 2000 times with plate washing buffer to each well, incubate at 37°C for 30 min, and wash the plate 4 times;

[0051] 4) Add 100 μL of freshly prepared TMB colorimetric solution to each well and develop the color at 37°C in the dark for 10 min;

[0052] 5) Add 50 μL of stop solution to each well and measure the OD value at 450 nm using an ELISA reader.

[0053] The best positive cell line was screened and subcloned four times, and finally the hybridoma cell line A7G8 was obtained. The subtype of the monoclonal antibody secreted by the hybridoma cell line A7G8 was detected by a commercially available monoclonal antibody typing kit as IgG1. The hybridoma cells in the positive wells were sent to the General Microbiology Center of the China Microbiological Culture Collection Administration for preservation, and the preservation number is CGMCC No.45336.

[0054] Example 2: Preparation, purification and characterization of broad-spectrum monoclonal antibodies against three ochratoxins

[0055] Five 6-week-old Balb / c female mice were desensitized by intraperitoneal injection of 0.5 mL / mouse Freund's incomplete adjuvant. Three days later, 0.2 mL / mouse (about 10 5 ~10 6 45336 hybridoma cells were injected into the mouse. After 10 days, the abdomen of the mouse was significantly enlarged, and the ascites in the mouse peritoneal cavity was collected and purified by the caprylic acid-saturated ammonium sulfate method. The obtained precipitate was re-dissolved with 0.01 mol / L PBS solution, and the solution was transferred to a 4KD dialysis bag and dialyzed in 0.01 mol / L PBS solution at 4°C for 72 hours, with the solution changed every 8 hours. The purified solution was transferred to a 2 mL centrifuge tube for freeze-drying and stored at -80°C.

[0056] The indirect non-competitive ELISA method was used to evaluate the affinity, sensitivity and specificity of monoclonal antibodies. The basic operating procedures are as follows:

[0057] 1) Dilute the coating antigen OTA-OVA 16000 times with the coating solution, add 100 μL / well to a 96-well ELISA plate, coat at 37°C for 4 hours, and wash the plate 4 times with washing solution;

[0058] 2) Add 200 μL of blocking solution (2% BSA) to each well, block at 37°C for 1 h, and wash the plate 4 times;

[0059] 3) Add 100 μL of sample diluent to the control wells, 50 μL of sample diluent to the blank wells, and 50 μL of standards with concentrations of 1000, 250, 50, 12.5, 3.125, 0.781, 0.391, 0.195, 0.098, and 0.024 ng / mL to the inhibition wells; then add 50 μL of 8000-fold diluted monoclonal antibody to the blank wells and inhibition wells, incubate at 37°C for 45 min, and wash the plate 4 times;

[0060] 4) Add 100 μL of goat anti-mouse enzyme-labeled secondary antibody IgG-HRP diluted 2000 times with plate washing buffer to each well, incubate at 37°C for 30 min, and wash the plate 4 times;

[0061] 5) Add 100 μL of freshly prepared TMB colorimetric solution to each well and develop the color at 37°C in the dark for 10 min;

[0062] 6) Add 50 μL of stop solution to each well and measure the OD value at 450 nm using an ELISA reader.

[0063] K of monoclonal antibody D The value is 1.77×10 8 L / mol, which is a high affinity antibody. Based on the 50% inhibition concentration (IC 50 ) was used to evaluate the sensitivity of the monoclonal antibody. The specificity of the monoclonal antibody was evaluated by evaluating the cross-reactivity (CR) of OTA and other mycotoxins (including OTB, OTC, AFB1, ZEN, FB1 and DON). CR (%) = (IC 50 IC of other analytes 50 )×100%. As shown in Table 1, the IC values ​​of the monoclonal antibodies against ochratoxin A, ochratoxin B, and ochratoxin C 50 The cross-reaction rates with aflatoxin B1, vomitoxin, zearalenone and fumonisin B1 were all less than 0.037%, indicating that the prepared monoclonal antibodies can be used for the detection of three ochratoxins with high sensitivity and specificity.

[0064] Table 1 Cross-reactions of monoclonal antibodies with OTA and its analogs

[0065]

[0066]

[0067] Example 3: Application of broad-spectrum monoclonal antibodies against three ochratoxins

[0068] The broad-spectrum monoclonal antibodies against three ochratoxins secreted by the hybridoma cell line A7G8 were used to prepare immunochromatographic test strips for rapid screening of ochratoxins in complex matrices of traditional Chinese medicine. The operation process is as follows:

[0069] (1) Preparation of colloidal gold-labeled monoclonal antibodies

[0070] Colloidal gold was prepared by the trisodium citrate reduction chloroauric acid method, with an average particle size of 20 nm. It was stored at 4°C in the dark for later use. 2 μL of 0.1M K2CO3 solution was added to 1mL of colloidal gold solution to adjust the pH to 8.4. 16 μg of monoclonal antibody was added to the colloidal gold solution and reacted in the dark for 1 hour. 50 μL of 10% BSA solution was added and incubated for 0.5 hours. The mixture was centrifuged at 12,000 r / min for 25 minutes at 4°C, and the precipitate was resuspended in 1mL of 0.002M borate buffer. Centrifuge again using the above method, discard the supernatant, add 100 μL of resuspension solution (0.01M PBS solution containing 0.01% PEG-2000, 1% BSA, and 7.5% sucrose) and resuspend to obtain a colloidal gold labeled monoclonal antibody solution, which was stored at 4°C.

[0071] (2) Immunochromatographic test strip assembly

[0072] The test strip consists of a PVC base, a nitrocellulose (NC) membrane, a sample pad and an absorbent pad. The NC membrane is pasted on the PVC base, and 0.5 mg / ml goat anti-mouse secondary antibody (IgG) and 0.08 mg / mL coated antigen OTA-OVA are sprayed on the NC membrane to form a quality control (C) line and a test (T) line, respectively, and dried at 37°C for 3 hours. The sample pad and the absorbent pad are pasted on the PVC base. The assembled card is cut into test strips of about 3 mm wide and stored in a desiccator for later use.

[0073] (3) Sample pretreatment

[0074] 1 g of sample powder and 0.2 g of sodium chloride were accurately weighed into a 15 mL centrifuge tube, 5 mL of methanol was added, and the mixture was rotated for 30 seconds and then ultrasonically extracted for 15 minutes. The mixture was centrifuged at 7000 rpm for 5 minutes, and the supernatant was diluted 6 times with 0.01 M PBS. The above method was used to extract 20 batches of Astragalus and 15 batches of dried ginger.

[0075] (4) Determination of test strip test results

[0076] Mix 2 μL colloidal gold-labeled monoclonal antibody with 100 μL sample solution, draw 80 μL of the mixture and add it to the sample pad. The solution migrates to the absorbent pad by capillary action, and the test result is determined after 15 minutes. Figure 3 As shown in the figure, if both the T line and the C line are colored, it means that the concentration of ochratoxin in the sample is lower than the detection limit, and the result is negative; if the C line is colored and the T line is colorless, it means that the concentration of ochratoxin in the sample is equal to or higher than the detection limit, and the result is positive; if the C line does not appear, it means that the operation process is incorrect or the test strip has expired, and the result is invalid.

[0077] (5) Actual sample testing

[0078] According to the above steps, 20 batches of astragalus and 15 batches of dried ginger were tested. The T line and C line of 15 batches of dried ginger samples were colored, and the results were negative. Among the 20 batches of astragalus samples, 3 batches of C line were colored and T line was colorless or nearly colorless, and the results were positive, and the content of ochratoxin in the sample solution was equal to or higher than 1ng / mL. After conversion, the content of ochratoxin in the 3 batches of samples was equal to or higher than 30μg / kg.

Claims

1. A hybridoma cell line A7G8 secreting broad-spectrum monoclonal antibodies against three ochratoxins, wherein the deposit number of the hybridoma cell line A7G8 is CGMCC No.45336.

2. A broad-spectrum monoclonal antibody against three ochratoxins, characterized in that: The antibody is produced by secretion of the hybridoma cell line A7G8 secreting broad-spectrum monoclonal antibodies against three ochratoxins as described in claim 1.

Images