Hybridoma cell strain 24DB-8E7 and 2, 4-dichlorophenoxybutyric acid antibody generated by hybridoma cell strain 24DB-8E7

Abstract

The invention discloses a hybridoma cell strain 24DB-8E7 and an anti-2, 4-dichlorophenoxybutyric acid monoclonal antibody generated by the hybridoma cell strain 24DB-8E7. The hybridoma cell strain 24DB-8E7 is preserved in the China General Microbiological Culture Collection Center on June 3, 2024, the preservation number is CGMCC No.45935, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The 2, 4-dichlorphenoxybutyric acid monoclonal antibody secreted by the hybridoma cell strain 24DB-8E7 has high affinity, and has good specificity and detection sensitivity to 2, 4-dichlorphenoxybutyric acid.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to a hybridoma cell line 24DB-8E7 and an anti-2,4-dichlorophenoxybutyric acid monoclonal antibody produced by the hybridoma cell line. Background Art

[0002] 2,4-Dichlorophenoxybutyric acid (2,4-DB), CAS No. 94-82-6, commonly known as 2,4-D butyric acid, is one of the important varieties of chlorophenoxy pesticides. It is often used as a herbicide and a plant growth regulator. Studies have shown that 2,4-DB can be transferred to animals or humans through the food chain, causing tissue damage. Long-term intake will lead to accumulation in the body, causing chronic toxicity risks. The US Environmental Protection Agency (IRIS) has classified it as a systemic poison (acute Class III). Different countries have maximum residue limits (MRLs) for 2,4-DB, such as 0.02 and 0.2 mg / kg for 2,4-DB in Australia and the United States, respectively. The latest OEKO-TEX certification Standard 100 stipulates that the total amount of pesticide residues and the residues in baby products are below 1.0 and 0.5 mg / kg, respectively. my country's GB / T18885-2002 uses the same definition for 2,4-DB. Therefore, establishing a rapid detection method is extremely important for food safety and import and export trade.

[0003] At present, the main detection methods for 2,4-DB include gas chromatography, high performance liquid chromatography, gas chromatography-mass spectrometry, liquid-mass spectrometry, etc. Although these precision instrumental analysis methods have shown good sensitivity, accuracy and reproducibility, they are difficult to meet the needs of large-scale on-site rapid detection due to their high cost, complex operation and expensive equipment. Immunoassay has the advantages of good specificity, high sensitivity, simple operation technology and low cost. It is suitable for the detection of large quantities of samples, especially the immune lateral flow chromatography test paper technology, which has the advantages of extremely short detection time, easy use and easy to carry. It has been widely used in the rapid detection of various substances in recent years.

[0004] High-affinity specific monoclonal antibodies are widely used in immunoassays due to their strong specificity, sustainable production and stable performance. However, there are very few immunological studies on 2,4-DB at home and abroad, and antibodies as core reagents of immunoassays are still very rare. Summary of the invention

[0005] The object of the present invention is to provide a hybridoma cell line 24DB-8E7 and an anti-2,4-dichlorophenoxybutyric acid monoclonal antibody produced by the hybridoma cell line.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides a hybridoma cell line 24DB-8E7, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 3, 2024, with a deposit number of CGMCC No. 45935, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The second aspect of the present invention provides the use of the hybridoma cell line 24DB-8E7 in the preparation of anti-2,4-dichlorophenoxybutyric acid monoclonal antibodies.

[0008] The third aspect of the present invention provides an anti-2,4-dichlorophenoxybutyric acid monoclonal antibody, which is secreted and produced by the above-mentioned hybridoma cell line 24DB-8E7.

[0009] The fourth aspect of the present invention provides a method for preparing the above-mentioned anti-2,4-dichlorophenoxybutyric acid monoclonal antibody, which comprises injecting the hybridoma cell line 24DB-8E7 into the abdominal cavity of Balb / c female mice sensitized with liquid paraffin, extracting ascites when the abdomen of the mouse is swollen and tense, and obtaining the anti-2,4-dichlorophenoxybutyric acid monoclonal antibody through separation and purification.

[0010] The fifth aspect of the present invention provides the use of the above-mentioned anti-2,4-dichlorophenoxybutyric acid monoclonal antibody in detecting 2,4-dichlorophenoxybutyric acid.

[0011] The sixth aspect of the present invention provides the use of the above-mentioned anti-2,4-dichlorophenoxybutyric acid monoclonal antibody in the preparation of a 2,4-dichlorophenoxybutyric acid detection kit.

[0012] A seventh aspect of the present invention provides a 2,4-dichlorophenoxybutyric acid detection kit, the kit comprising the anti-2,4-dichlorophenoxybutyric acid monoclonal antibody according to claim 3.

[0013] The significant advantages of the present invention are: The anti-2,4-dichlorophenoxybutyric acid monoclonal antibody secreted by the hybridoma cell line 24DB-8E7 of the present invention can specifically bind to 2,4-dichlorophenoxybutyric acid, and the titer exceeds 2.56×10 5 , the affinity constant Kaff is 1.15×10 9 L / mol; measured by indirect competitive ELISA, IC 50 The IC value of 2,4-dichlorophenoxybutyric acid was 17.48 ng / mL. 90 It was 2.46 ng / mL, and the limit of detection (LOD) was 2.46 ng / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1: Chromosome analysis of hybridoma cell line 24DB-8E7.

[0015] Figure 2 :Isolation diagram of anti-2,4-dichlorophenoxybutyric acid monoclonal antibody.

[0016] Figure 3 : Identification diagram of the purification results of anti-2,4-dichlorophenoxybutyric acid monoclonal antibody. Lane M: Maker; Lanes 1 and 2: Ascites protein; Lanes 3 and 4: Purified monoclonal antibody.

[0017] Figure 4 : Anti-2,4-dichlorophenoxybutyric acid monoclonal antibody purification results titer diagram.

[0018] Figure 5 : Affinity analysis diagram of anti-2,4-dichlorophenoxybutyric acid monoclonal antibody.

[0019] Figure 6 : Specificity analysis diagram of anti-2,4-dichlorophenoxybutyric acid monoclonal antibody.

[0020] Figure 7 : Cross-reactivity analysis diagram of anti-2,4-dichlorophenoxybutyric acid monoclonal antibodies.

[0021] Figure 8 : Standard curve of indirect competitive ELISA of anti-2,4-dichlorophenoxybutyric acid monoclonal antibody.

[0022] Fig. 9 : The straight line part of the indirect competition ELISA standard curve of anti-2,4-dichlorophenoxybutyric acid monoclonal antibody. DETAILED DESCRIPTION

[0023] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0024] Example 1: Preparation of hybridoma cell line 24DB-8E7 1. Preparation of artificial complete antigen 1.2 mg of 2,4-dichlorophenoxybutyric acid (2,4-DB) standard was dissolved in 0.5 mL of 1,4-dioxane, 1.5 mg of NHS and 4.5 mg of EDC-HCl were added to 0.5 mL of 0.1 M MES buffer (pH 5.5) in sequence, and then the above solutions were mixed and incubated on a shaker at room temperature for 30 min to obtain a hapten activation solution; the obtained hapten activation solution was slowly added dropwise to 20 mL of 10×PBS (pH 7.4) in which 10 mg of bovine serum albumin (BSA) was dissolved, and the mixture was stirred at a low speed and reacted in the dark for 6 h; the obtained reaction solution was centrifuged at 12000 r / min for 30 min, and dialyzed with 1×PBS (pH 7.4) for 3 days, during which the dialyzate was replaced every 4 h, and concentrated after dialysis to obtain a complete antigen, which was recorded as 24DB-BSA.

[0025] 1.2 mg of 2,4-DB standard was dissolved in 0.5 mL of 1,4-dioxane, 1.5 mg of NHS and 4.5 mg of EDC-HCl were added to 0.5 mL of 0.1 M MES buffer (pH 5.5) in sequence, and then the above solutions were mixed and incubated on a shaker at room temperature for 30 min to obtain a hapten activation solution; the obtained hapten activation solution was slowly added dropwise to 20 mL of 10×PBS (pH 7.4) in which 10 mg of hemocyanin (KLH) was dissolved, and the reaction was stirred at a low speed and protected from light for 6 h; the obtained reaction solution was centrifuged at 12000 r / min for 30 min, dialyzed with 1×PBS (pH 7.4) for 3 days, during which the dialyzate was replaced every 4 h, and concentrated after dialysis to obtain a complete antigen, which was recorded as 24DB-KLH.

[0026] 1.2 mg of 2,4-DB standard was dissolved in 0.5 mL of 1,4-dioxane, 1.5 mg of NHS and 4.5 mg of EDC-HCl were added to 0.5 mL of 0.1 M MES buffer (pH 5.5) in sequence, and then the above solutions were mixed and incubated on a shaker at room temperature for 30 min to obtain a hapten activation solution; the obtained hapten activation solution was slowly added dropwise to 20 mL of 10×PBS (pH 7.4) in which 10 mg of ovalbumin (OVA) had been dissolved, and the mixture was stirred at a low speed and reacted in the dark for 6 h; the obtained reaction solution was centrifuged at 12000 r / min for 30 min, and dialyzed with 1×PBS (pH 7.4) for 3 days, during which the dialyzate was replaced every 4 h, and concentrated after dialysis to obtain a complete antigen, which was recorded as 24DB-OVA.

[0027] 2. Animal immunization Six-week-old BalB / c female mice were selected, and the complete antigen 24DB-KLH was mixed with an equal volume of Freund's complete adjuvant. After complete ultrasonic emulsification, the mouse neck was subcutaneously injected at multiple points at a dose of 100μg / 500μL per mouse. The first booster immunization was 21 days apart, and the same antigen was mixed with an equal volume of Freund's incomplete adjuvant, and boosted at 40μg / 500μL per mouse. The intervals for the remaining booster immunizations were 14 days, and the dosages were the same as the injections. Starting from the fourth immunization, indirect ELISA was used to monitor the mouse serum titer within 5 to 7 days after each immunization. When the mouse serum titer exceeded 1:128k, a shock immunization was performed by intraperitoneal injection at a dose of 100μg / 500μL.

[0028] 3. Cell fusion On the 3rd to 4th day after the impact immunization, the spleen cells of the mice were taken, and 50% PEG1450 was used as the fusion agent. The spleen cells and SP2 / 0 were mixed in 20 mL of fresh 1640 incomplete medium at a cell number ratio of 5:1. After gentle mixing, the mixture was centrifuged at 1000 rpm for 7 min. The cells were retained and tapped into a paste. After 5 min of water bath at 37°C, 1 mL of 50% PEG1450 preheated at 37°C was uniformly added within 1 min, and then allowed to stand for 0.5 min, and then 1 mL of PEG1450 was slowly added within 1 min. 1640 incomplete culture medium, then add 1640 incomplete culture medium to 40mL; centrifuge at 1000rpm for 10min, discard the supernatant, transfer the bottom cells into 95mL complete culture medium containing 2% HAT (1640 culture medium containing 20% ​​FBS), mix well and plate on a 96-well plate pre-plated with feeder cells, 200μL per well; replace with complete culture medium containing 1% HT (1640 culture medium containing 20% ​​FBS) after 14 days of fusion.

[0029] 4. Cell line screening and subcloning When the cells grow to fusion on the 5th to 7th day, aspirate 100 μL of supernatant from each well and add 120 μL of complete medium containing 1% HAT (1640 medium containing 2% FBS). On the 9th to 12th day, screen the positive wells by indirect ELISA method; select the wells with higher absorbance values ​​and perform subcloning by limiting dilution method; repeat subcloning 3 to 4 times until the positive rate is 100%, and obtain the high-affinity and specific hybridoma cell line 24DB-8E7.

[0030] 5. Chromosome analysis Treat the cells in logarithmic growth phase with 0.1% colchicine for 6 hours, resuspend them, centrifuge at 1000r / min for 10min, discard the supernatant, add 5mL 0.075mol / L KCl to resuspend the cells, let stand for 20min; add 1mL fresh fixative (methanol: glacial acetic acid = 3:1), mix well and let stand for 5min, centrifuge at 1000r / min for 10min, discard the supernatant; add 5mL fixative, resuspend, let stand at 4℃ overnight; centrifuge at 1000r / min for 10min, discard the supernatant, add 300μL fixative, mix well, add 1-2 drops of cell suspension on the pre-cooled slide, dry, and stain with Giemsa. The results are as follows: Figure 1 As shown, the chromosome number of the hybridoma cell line 24DB-8E7 in metaphase was calculated to be 106±6, and the chromosome number of the hybridoma cell line 24DB-8E7 was close to the sum of the chromosome numbers of the two parents, proving that the hybridoma cell line 24DB-8E7 was formed by the fusion of myeloma cells and spleen cells.

[0031] 6. Antibody subtype identification The anti-2,4-dichlorophenoxybutyric acid supernatant antibodies secreted by the hybridoma cell line 24DB-8E7 were identified using a commercially available isotype determination kit. Figure 2 As shown, the antibody is an IgG1 subtype antibody.

[0032] Biomaterial preservation information: Hybridoma cell line (Mus musculus) 24DB-8E7 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 3, 2024, with the deposit number CGMCC No.45935, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0033] Example 2: Preparation and characterization of anti-2,4-dichlorophenoxybutyric acid monoclonal antibodies 1. Preparation and purification of ascites 1) Preparation of ascites: Hybridoma cell line 24DB-8E7 in logarithmic growth phase was cultured at 1×10 6 cells / mouse were injected into the abdominal cavity of 12-week-old Balb / c female mice sensitized with liquid paraffin. When the abdomen of the mouse was swollen and tense, the ascites was extracted and centrifuged at 2500r / min for 5min to remove the cells. The supernatant was taken and centrifuged at 4℃ and 12000r / min for 20min. After centrifugation, it was divided into three layers (from bottom to top: aggregates, middle layer containing a large amount of antibodies, lipid layer), and the middle layer was taken.

[0034] 2) Antibody purification: The antibody was purified by caprylic acid-ammonium sulfate method and protein G affinity chromatography, and the purity of the antibody was verified by 10% SDS-PAGE gel electrophoresis. Figure 3As shown, the purified antibody has bands at 25kDa and 50kDa, corresponding to the light chain and heavy chain of the IgG antibody, respectively, and the background bands are relatively clean, indicating that the purification effect is good. After complete dialysis with ultrapure water, freeze-drying, and collecting the lyophilized powder, the anti-2,4-dichlorophenoxybutyric acid monoclonal antibody is obtained and placed in a -80℃ refrigerator for use.

[0035] 3) Titer determination: The titer of ascites and the titer of purified anti-2,4-dichlorophenoxybutyric acid monoclonal antibody were determined by conventional non-competitive enzyme-linked immunosorbent assay (ELISA). Figure 4 As shown, the titer of ascites exceeded 2.56×10 5 After purification, the titer of anti-2,4-dichlorophenoxybutyric acid monoclonal antibody exceeded 2.56×10 5 , indicating that the anti-2,4-dichlorophenoxybutyric acid monoclonal antibody still maintains high activity after purification.

[0036] 2. Characterization of monoclonal antibodies 1) Affinity determination: According to the Beatty method, the affinity constant Kaff of the monoclonal antibody was determined by indirect ELISA. 24DB-BSA was diluted to 10 μg / mL, 5 μg / mL, and 2.5 μg / mL with coating buffer and coated on the ELISA plate. The remaining steps were performed according to the conventional indirect ELISA operation. Origin 8.0 was used for analysis. Figure 5 As shown, the affinity constant of the anti-2,4-dichlorophenoxybutyric acid monoclonal antibody was calculated according to the following formula, and the result was 1.15×10 9 L / mol, which is a high affinity antibody.

[0037] Where: [Ab] means the antigen concentration is [Ag], at IC 50 The antibody concentration at [Ab]t means the antigen concentration is [Ag]t. 50 The antibody concentration at n=[Ag] / [Ag]t 2) Specificity analysis 4-Chlorophenoxyacetic acid coupled to BSA complete antigen (4C-BSA), 4-Chlorophenoxyacetic acid coupled to OVA complete antigen (4C-OVA), 2,4-dichlorophenoxyacetic acid coupled to BSA complete antigen (24D-BSA), 2,4-dichlorophenoxyacetic acid coupled to OVA complete antigen (24D-OVA), 2,4-dichlorophenoxybutyric acid coupled to BSA complete antigen (24DB-BSA), 2,4-dichlorophenoxybutyric acid coupled to OVA complete antigen (24DB-OVA), diclofenac coupled to BSA complete antigen (DCF-BSA), bovine serum albumin (BSA), ovalbumin (OVA) and PBSM were diluted to a final concentration of 5 μg / mL. A total of 10 different coating antigens were coated on the ELISA plate and determined by the conventional indirect ELISA method. The experimental results are shown in the figure. Figure 6 As shown, the anti-2,4-DB monoclonal antibody reacted strongly with two complete antigens of 2,4-DB, 24DB-BSA and 24DB-OVA, but did not react with other coating antigens.

[0038] 3) Cross-reactivity analysis The original 24DB-BSA was coated on the ELISA plate at 5 μg / mL, and the standards such as 2,4-dichlorophenoxybutyric acid (2,4-DB), 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chlorophenoxyacetic acid (4-CPA), 4-(P-chlorophenoxy)butyric acid (4-PCB), 4-phenoxybutyric acid (4-PBA), 6-benzylpurine (6-BA), 6-benzyladenosine (6-BAS), gibberellin (GA3), diclofenac (DCF), PBSM were used as competitive antigens. Various standards were diluted to 500 ng / mL, and the anti-2,4-dichlorophenoxybutyric acid monoclonal antibody was diluted at 1:64K. The conventional indirect competitive ELISA method was used for cross-reactivity identification. The results are as follows: Figure 7 As shown, the anti-2,4-dichlorophenoxybutyric acid monoclonal antibody can recognize 2,4-DB with high specificity, but has no cross-reaction with several other substances.

[0039] 4) Antibody sensitivity determination The sensitivity of anti-2,4-dichlorophenoxybutyric acid monoclonal antibody to 2,4-DB was determined by conventional indirect competitive ELISA. The results of the standard competition curve are shown in Figure 8 , the curve equation is y = 0.02377 + (0.9843–0.02377) / [1 + (x / 17.47787) ^ 1.26184], R 2 =0.99976, where IC 50 =17.48 ng / mL, IC 90 =2.46 ng / mL, at IC 20 ~IC 80There is a good linear relationship between them. The linear equation of the linear part is y=1.2995-0.64463x, R 2 =0.99836, such as Fig. 9 As shown, the linear range was 5.32-57.86 ng / mL, and the limit of detection (LOD) was 2.46 ng / mL.

[0040] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A hybridoma cell line 24DB-8E7, characterized in that: It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 3, 2024, with the deposit number CGMCC No.45935, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the hybridoma cell line 24DB-8E7 according to claim 1 in the preparation of anti-2,4-dichlorophenoxybutyric acid monoclonal antibodies.

3. An anti-2,4-dichlorophenoxybutyric acid monoclonal antibody, characterized in that: The hybridoma cell line 24DB-8E7 according to claim 1 is secreted and produced.

4. The method for preparing the anti-2,4-dichlorophenoxybutyric acid monoclonal antibody according to claim 3, characterized in that: The hybridoma cell line 24DB-8E7 was injected into the abdominal cavity of Balb / c female mice sensitized with liquid paraffin. When the abdomen of the mouse was swollen and tense, ascites was extracted and anti-2,4-dichlorophenoxybutyric acid monoclonal antibody was obtained through separation and purification.

5. Use of the anti-2,4-dichlorophenoxybutyric acid monoclonal antibody according to claim 3 in detecting 2,4-dichlorophenoxybutyric acid.

6. Use of the anti-2,4-dichlorophenoxybutyric acid monoclonal antibody according to claim 3 in the preparation of a 2,4-dichlorophenoxybutyric acid detection kit.

7. A 2,4-dichlorophenoxybutyric acid detection kit, characterized in that: The kit comprises the anti-2,4-dichlorophenoxybutyric acid monoclonal antibody as claimed in claim 3.

Images