Hybridoma cell line 24DB-8E7 and the anti-2,4-dichlorophenoxybutyric acid antibody produced thereby

Abstract

The application discloses a hybridoma cell strain 24DB-8E7 and a monoclonal antibody against 2,4-dichlorophenoxybutyric acid generated by the hybridoma cell strain. The hybridoma cell strain 24DB-8E7 has been preserved in the China General Microbiological Culture Collection Center on June 3, 2024, with a preservation number of CGMCC No.45935 and a preservation address of No.3, Beichen West Road, Chaoyang District, Beijing. The monoclonal antibody against 2,4-dichlorophenoxybutyric acid secreted by the hybridoma cell strain 24DB-8E7 has high affinity, good specificity and detection sensitivity for 2,4-dichlorophenoxybutyric acid.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection, in particular to a hybridoma cell strain 24DB-8E7 and an anti-2,4-dichlorophenoxybutyric acid monoclonal antibody produced by the hybridoma cell strain. BACKGROUND

[0002] 2,4-dichlorophenoxybutyric acid (2,4-DB), CAS No. 94-82-6, commonly known as 2,4-dibutyl acid, is one of the important varieties of chlorophenoxy pesticides, often used as a herbicide, and also used as a plant growth regulator. Studies have shown that 2,4-DB can be transferred to animals or humans through the food chain, causing damage to the body tissues, and long-term intake will lead to accumulation in the body, causing chronic toxicity risk, and the US Environmental Protection Agency (IRIS) has classified it as a systemic poison (acute class III). Different countries have maximum residue limits (MRL) for 2,4-DB, such as Australia and the United States, which are 0.02 and 0.2 mg / kg, respectively. The latest OEKO-TEX certification Standard 100 stipulates that the total amount of pesticide residues and the amount of residues in baby products should be less than 1.0 and 0.5 mg / kg, respectively, and China's GB / T 18885-2002 uses the same definition for 2,4-DB. Therefore, it is important to establish a rapid detection method 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 chromatography-mass spectrometry, etc. Although these precise instrument analysis methods show 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, low cost, etc., and is suitable for the detection of large quantities of samples, especially the immunoassay lateral flow chromatography test paper technology, which has the advantages of extremely short detection time, easy use and portability, and has been widely used in the rapid detection of various substances in recent years.

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

[0005] The present application relates to the technical field of detection, in particular to a hybridoma cell strain 24DB-8E7 and an anti-2,4-dichlorophenoxybutyric acid monoclonal antibody produced by the hybridoma cell strain.

[0006] To achieve the above-mentioned purpose, the technical solutions adopted by the present application are as follows:

[0007] The first aspect of the present application provides a hybridoma cell strain 24DB-8E7, which has been preserved in the China General Microbiological Culture Collection Center on June 3, 2024, with a preservation number of CGMCC No. 45935 and a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing.

[0008] The second aspect of the present application provides an application of the above-mentioned hybridoma cell strain 24DB-8E7 in the preparation of an anti-2,4-dichlorophenoxybutyric acid monoclonal antibody.

[0009] The third aspect of the present application provides an anti-2,4-dichlorophenoxybutyric acid monoclonal antibody, which is secreted by the above-mentioned hybridoma cell strain 24DB-8E7.

[0010] The fourth aspect of the present application provides a preparation method of the above-mentioned anti-2,4-dichlorophenoxybutyric acid monoclonal antibody, which is injecting the hybridoma cell strain 24DB-8E7 into the abdominal cavity of a liquid paraffin sensitized Balb / c female mouse, extracting the ascites when the mouse abdomen is swollen and has a tense feeling, and obtaining the anti-2,4-dichlorophenoxybutyric acid monoclonal antibody through separation and purification.

[0011] The fifth aspect of the present application provides an application of the above-mentioned anti-2,4-dichlorophenoxybutyric acid monoclonal antibody in the detection of 2,4-dichlorophenoxybutyric acid.

[0012] The sixth aspect of the present application provides an application of the above-mentioned anti-2,4-dichlorophenoxybutyric acid monoclonal antibody in the preparation of a 2,4-dichlorophenoxybutyric acid detection kit.

[0013] The seventh aspect of the present application provides a 2,4-dichlorophenoxybutyric acid detection kit, which comprises the anti-2,4-dichlorophenoxybutyric acid monoclonal antibody according to claim 3.

[0014] The present application has the following advantages:

[0015] The anti-2,4-dichlorophenoxybutyric acid monoclonal antibody secreted by the hybridoma cell strain 24DB-8E7 of the present application can specifically bind to 2,4-dichlorophenoxybutyric acid, with a titer of more than 2.56x10 5 , and an affinity constant Kaff of 1.15x10 9 L / mol; the IC 50 of 2,4-dichlorophenoxybutyric acid is 17.48 ng / mL, the IC 90 of 2,4-dichlorophenoxybutyric acid is 2.46 ng / mL, and the detection limit (LOD) is 2.46 ng / mL. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Figure 2: Chromosome analysis of hybridoma cell line 24DB-8E7.

[0017] Figure 2 Figure 5: Subtype identification of anti-2,4-Dichlorophenoxybutyric acid monoclonal antibody.

[0018] Figure 3 Figure 6: Identification of purification results of anti-2,4-Dichlorophenoxybutyric acid monoclonal antibody. Lane M: Maker; Lanes 1 and 2: ascites protein; Lanes 3 and 4: monoclonal antibody after purification.

[0019] Figure 4 Figure 7: Titer of purification results of anti-2,4-Dichlorophenoxybutyric acid monoclonal antibody.

[0020] Figure 5 Figure 8: Affinity analysis of anti-2,4-Dichlorophenoxybutyric acid monoclonal antibody.

[0021] Figure 6 Figure 9: Specificity analysis of anti-2,4-Dichlorophenoxybutyric acid monoclonal antibody.

[0022] Figure 7 Figure 10: Cross-reactivity analysis of anti-2,4-Dichlorophenoxybutyric acid monoclonal antibody.

[0023] Figure 8 Figure 11: Indirect competitive ELISA standard curve of anti-2,4-Dichlorophenoxybutyric acid monoclonal antibody.

[0024] Figure 9 Figure 12: Linear part of indirect competitive ELISA standard curve of anti-2,4-Dichlorophenoxybutyric acid monoclonal antibody. DETAILED DESCRIPTION

[0025] In order to make the content of the present application more convenient to understand, the technical solutions of the present application will be further described below in combination with specific embodiments, but the present application is not limited to this.

[0026] Example 1: Preparation of hybridoma cell line 24DB-8E7

[0027] 1. Preparation of artificial complete antigen

[0028] Take 1.2 mg 2,4-DB standard dissolved in 0.5 mL 1,4-dioxane, 1.5 mg NHS, 4.5 mg EDC-HCl were added in 0.5 mL 0.1 M MES buffer (pH 5.5) in turn, then mixed the above solution incubated on a shaker at room temperature for 30 min to obtain the hapten activation solution; the obtained hapten activation solution was slowly added to 20 mL 10×PBS (pH 7.4) which had been dissolved 10 mg bovine serum albumin (BSA), low speed stirring in the dark 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, the dialysis fluid was changed every 4 h during the period, after dialysis, concentrated to obtain the complete antigen, recorded as 24DB-BSA.

[0029] Take 1.2 mg 2,4-DB standard dissolved in 0.5 mL 1,4-dioxane, 1.5 mg NHS, 4.5 mg EDC-HCl were added in 0.5 mL 0.1 M MES buffer (pH 5.5) in turn, then mixed the above solution incubated on a shaker at room temperature for 30 min to obtain the hapten activation solution; the obtained hapten activation solution was slowly added to 20 mL 10×PBS (pH 7.4) which had been dissolved 10 mg bovine serum albumin (BSA), low speed stirring in the dark 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, the dialysis fluid was changed every 4 h during the period, after dialysis, concentrated to obtain the complete antigen, recorded as 24DB-BSA.

[0030] Take 1.2 mg 2,4-DB standard dissolved in 0.5 mL 1,4-dioxane, 1.5 mg NHS, 4.5 mg EDC-HCl were added in 0.5 mL 0.1 M MES buffer (pH 5.5) in turn, then mixed the above solution incubated on a shaker at room temperature for 30 min to obtain the hapten activation solution; the obtained hapten activation solution was slowly added to 20 mL 10×PBS (pH 7.4) which had been dissolved 10 mg bovine serum albumin (BSA), low speed stirring in the dark 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, the dialysis fluid was changed every 4 h during the period, after dialysis, concentrated to obtain the complete antigen, recorded as 24DB-BSA.

[0031] 2、Animal immunization

[0032] Select 6 weeks old BalB / c female mice, take the complete antigen 24DB-KLH and equal volume of Freund's complete adjuvant mixed, ultrasonic emulsification complete, with each 100 μg / 500 μL dose using mouse neck back subcutaneous injection. First boost interval 21 days, take the same antigen and equal volume of Freund's incomplete adjuvant mixed, according to each 40 μg / 500 μL boost. The rest of the boost interval is 14 days, the dose and injection as above. From the fourth immunization, within 5-7 days after each immunization using indirect ELISA to monitor the serum titer of mice. Until the serum titer of mice is more than 1:128k, with 100 μg / 500 μL dose intraperitoneal injection for impact immunization.

[0033] 3、Cell fusion

[0034] The mouse spleen cells were taken 3-4 days after impact immunization, 50% PEG1450 was used as a fusion agent, and the spleen cells were mixed with SP2 / 0 at a cell number ratio of 5:1 in 20 mL of fresh 1640 incomplete culture medium. After gentle mixing, centrifugation at 1000 rpm for 7 min, the cells were retained and gently tapped into a paste. After 37°C water bath for 5 min, 1 mL of 37°C preheated 50% PEG1450 was added at a constant speed within 1 min, followed by standing for 0.5 min, then slowly adding 1 mL of 1640 incomplete culture solution within 1 min, and then adding 1640 incomplete culture solution to 40 mL; centrifugation at 1000 rpm for 10 min, discard the supernatant, transfer the bottom cells to 95 mL of complete culture medium containing 2% HAT (1640 culture medium containing 20% FBS), mix well and plate to the 96-well plate pre-coated with feeder cells, 200 μL per well; replace the complete culture medium containing 1% HT (1640 culture medium containing 20% FBS) after 14 days of fusion.

[0035] 4、Cell strain screening and subcloning

[0036] When the cells grow to 5-7 days after fusion, 100 μL of supernatant is taken from each well, and 120 μL of complete culture medium containing 1% HAT (1640 culture medium containing 2% FBS) is added. After 9-12 days, indirect ELISA method is used for screening, and positive wells are selected. The wells with high absorbance value are selected for subcloning by limiting dilution method. After repeated subcloning for 3-4 times, until the positive rate is 100%, the high affinity specific hybridoma cell strain 24DB-8E7 is obtained.

[0037] 5、Chromosome analysis

[0038] The cells in logarithmic growth phase were resuspended after 6h treatment with 0.1% colchicine, centrifuged at 1000r / min for 10min, the supernatant was discarded, 5mL 0.075mol / L KCl was added to resuspend the cells, and the cells were allowed to stand for 20min; 1mL fresh fixing solution (methanol: glacial acetic acid = 3:1) was added, mixed well, and allowed to stand for 5min, then centrifuged at 1000r / min for 10min, and the supernatant was discarded; 5mL fixing solution was added to resuspend the cells, and the cells were allowed to stand at 4°C overnight; centrifuged at 1000r / min for 10min, the supernatant was discarded, 300μL fixing solution was added, mixed well, 1-2 drops of cell suspension were dropped on a pre-cooled glass slide, dried, and then subjected to Giemsa staining. The results are shown in Figure 1 The number of chromosomes of the hybridoma cell strain 24DB-8E7 in metaphase was calculated to be 106±6, which was close to the sum of the chromosome numbers of the two parents, proving that the hybridoma cell strain 24DB-8E7 was formed by fusion of myeloma cells and spleen cells.

[0039] 6. Antibody subtype identification

[0040] The supernatant antibody secreted by the hybridoma cell strain 24DB-8E7 against 2,4-dichlorophenoxybutyric acid was identified by using a commercially available subtype determination kit, and the results are shown in Figure 2 The antibody was IgG1 subtype antibody.

[0041] Biological material preservation information: the hybridoma cell strain (Mus musculus) 24DB-8E7 was preserved in the China General Microbiological Culture Collection Center on June 3, 2024, with the preservation number of CGMCC No.45935, and the preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing.

[0042] Example 2: Preparation and property identification of the monoclonal antibody against 2,4-dichlorophenoxybutyric acid

[0043] 1. Preparation and purification of ascites

[0044] 1) Preparation of ascites: the hybridoma cell strain 24DB-8E7 in logarithmic growth phase was injected into the abdominal cavity of a 12-week-old Balb / c female mouse sensitized with liquid paraffin at 1×10 6 cells / only mouse, and the ascites was extracted when the mouse had a tense feeling due to abdominal distension, centrifuged at 2500r / min for 5min, and the supernatant was obtained by centrifugation at 12000r / min for 20min at 4°C. The supernatant was divided into three layers (from bottom to top: aggregates, middle layer containing a large amount of antibodies, and lipid layer), and the middle layer was taken.

[0045] 2) Antibody purification: crude extraction with caprylic acid-ammonium sulfate method and fine extraction with Protein G affinity chromatography were used for purification, and 10% SDS-PAGE gel electrophoresis was used to detect and verify the purity of the antibody, as shown in Figure 3 , the purified antibody had bands at 25 kDa and 50 kDa, corresponding to the light chain and heavy chain of IgG antibody, respectively, and the background band was clean, indicating good purification effect. After dialysis with ultrapure water, freeze-drying, and collection of freeze-dried powder, anti-2,4-dichlorophenoxybutyric acid monoclonal antibody was obtained and stored in a -80°C refrigerator.

[0046] 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), and the results are shown in Figure 4 , the titer of ascites was more than 2.56 x 10 5 , and the titer of purified anti-2,4-dichlorophenoxybutyric acid monoclonal antibody was more than 2.56 x 10 5 , indicating that the purified anti-2,4-dichlorophenoxybutyric acid monoclonal antibody still maintained high activity.

[0047] 2. Characterization of monoclonal antibody

[0048] 1) Affinity determination: according to Beatty's method, indirect ELISA was used to determine the affinity constant Kaff of the monoclonal antibody. 24DB-BSA was diluted to 10 μg / mL, 5 μg / mL, and 2.5 μg / mL with coating buffer to coat the enzyme-labeled plate, and the rest of the steps were performed according to the conventional indirect ELISA operation. Analysis was performed using Origin 8.0, as shown in Figure 5 , the affinity constant of anti-2,4-dichlorophenoxybutyric acid monoclonal antibody was calculated according to the following formula, and the calculation result was 1.15 x 10 9 L / mol, which was a high-affinity antibody.

[0049]

[0050] In the formula: [Ab] represents the concentration of the antibody at IC 50 when the concentration of the antigen is [Ag];

[0051] [Ab]t represents the concentration of the antibody at IC 50 when the concentration of the antigen is [Ag]t;

[0052] n = [Ag] / [Ag]t

[0053] 2) Specificity analysis

[0054] The 4-chlorophenoxyacetic acid conjugated BSA complete antigen (4C-BSA), 4-chlorophenoxyacetic acid conjugated OVA complete antigen (4C-OVA), 2,4-dichlorophenoxyacetic acid conjugated BSA complete antigen (24D-BSA), 2,4-dichlorophenoxyacetic acid conjugated OVA complete antigen (24D-OVA), 2,4-dichlorophenoxybutyric acid conjugated BSA complete antigen (24DB-BSA), 2,4-dichlorophenoxybutyric acid conjugated OVA complete antigen (24DB-OVA), diclofenac acid conjugated BSA complete antigen (DCF-BSA), bovine serum albumin (BSA), ovalbumin (OVA) and PBSM were diluted to 5 μg / mL, 10 different coating antigens were used to coat the enzyme-labeled plate, and the determination was carried out by using the conventional indirect ELISA method, and the experimental results are shown in Figure 6 As shown in the figure, the anti-2,4-dichlorophenoxybutyric acid monoclonal antibody strongly reacts with the two complete antigens 24DB-BSA and 24DB-OVA of 2,4-DB, but does not react with other coating antigens.

[0055] 3) Cross-reactivity analysis

[0056] The original 24DB-BSA was used to coat the enzyme-labeled plate at 5 μg / mL, and 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-benzyl purine (6-BA), 6-benzyl adenosine (6-BAS), gibberellin (GA3), diclofenac (DCF), PBSM and other standard substances were used as competitive antigens, and various standard substances were diluted by 500 ng / mL, the anti-2,4-dichlorophenoxybutyric acid monoclonal antibody was diluted by 1:64K, and the cross-reactivity was identified by using the conventional indirect competitive ELISA method, and the results are shown in Figure 7 As shown in the figure, the anti-2,4-dichlorophenoxybutyric acid monoclonal antibody can specifically recognize 2,4-DB, but has no cross-reaction with other substances.

[0057] 4) Antibody sensitivity determination

[0058] The sensitivity of the anti-2,4-dichlorophenoxybutyric acid monoclonal antibody to 2,4-DB was identified by using the conventional indirect competitive ELISA method, and the standard competition curve is 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, wherein IC 50 =17.48 ng / mL, IC 90 =2.46 ng / mL, and the IC20 IC 80 between them showed good linear relationship, the linear equation was y=1.2995-0.64463x, R 2 =0.99836, as shown in the linear range was 5.32-57.86ng / mL, the detection limit (LOD) was 2.46ng / mL. Figure 9

[0059] The above only describes the preferred embodiments of the present application, any equivalent changes and modifications made according to the scope of the present application should be included in the scope of the present application.​

Claims

1. A hybridoma cell line 24DB-8E7 secreting monoclonal antibody against 2,4-dichlorophenoxybutyric acid, characterized by: It was preserved in China General Microbiological Culture Collection Center on June 3, 2024, with a preservation number of CGMCC No. 45935, and a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing.

2. Use of the hybridoma cell strain 24DB-8E7 of claim 1 in the preparation of an anti-2,4-dichlorophenoxybutyric acid monoclonal antibody.

3. An anti-2,4-dichlorophenoxybutyric acid monoclonal antibody, characterized in that: The anti-2,4-dichlorophenoxybutyric acid monoclonal antibody is secreted by the hybridoma cell strain 24DB-8E7 of claim 1.

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

5. A 2,4-dichlorophenoxybutyric acid test kit, characterized by: The kit comprises the anti-2,4-dichlorophenoxybutyric acid monoclonal antibody of claim 3.

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