Hybridoma cell strain secreting diallyl phthalate monoclonal antibody and application of hybridoma cell strain

Abstract

The invention relates to a hybridoma cell strain capable of secreting a diallyl phthalate monoclonal antibody and application of the hybridoma cell strain, and belongs to the technical field of immunodetection. The monoclonal antibody secreted by the hybridoma cell strain has excellent affinity and sensitivity to diallyl phthalate, IC50 of the monoclonal antibody to the diallyl phthalate reaches 500 ng / mL, and the monoclonal antibody secreted by the hybridoma cell strain has excellent affinity and sensitivity to structural analogues of the diallyl phthalate. The invention relates to a method for preparing diisononyl phthalate, which is characterized in that the diisononyl phthalate, such as dimethyl phthalate, diethyl phthalate, dinonyl phthalate, butyl benzyl phthalate, diisobutyl phthalate, di (2-ethylhexyl) phthalate and diisononyl phthalate, are not subjected to cross reaction. Therefore, the monoclonal antibody provided by the invention can be used for preparing an immunodetection product of diallyl phthalate, and an efficient detection method and means are provided for residue detection of diallyl phthalate in food.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to a hybridoma cell line that secretes diallyl phthalate monoclonal antibody and its application. Background Technology

[0002] Diallyl phthalate (DAP) is an important organic compound that has been used since the mid-20th century. With the development of the petrochemical industry, its production technology has been continuously improved, and it has gradually become an important chemical raw material in the plastics and rubber industries. As a crosslinking agent, DAP promotes the formation of chemical bonds between polymer chains, resulting in a more rigid three-dimensional structure and improving the heat and chemical resistance of materials. It is commonly used in injection molding and extrusion molding processes. DAP has low toxicity but is lachrymatory and can irritate mucous membranes and skin, causing dermatitis. Furthermore, it is highly toxic to aquatic organisms and may have long-term adverse effects on the aquatic environment. Therefore, relevant safety regulations must be followed during its use and disposal to prevent release into the environment.

[0003] Currently, the main methods for detecting diallyl phthalate residues are instrumental analytical methods, including gas chromatography and gas chromatography-mass spectrometry (GC-MS). These methods are reliable and highly sensitive, and relevant technical standards are available for reference. However, due to the need for expensive instruments, specialized operators, and complex, costly, and time-consuming sample pretreatment, they cannot better meet the requirements for rapid and convenient on-site detection. Therefore, establishing a rapid and convenient method for detecting diallyl phthalate is of great significance. Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive, and rapid detection method suitable for rapid on-site detection of large numbers of samples, providing a new detection route for diallyl phthalate. ELISA detection requires a monoclonal antibody against diallyl phthalate, but current technologies lack such a monoclonal antibody. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the lack of a monoclonal antibody against diallyl phthalate in the prior art.

[0005] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes a monoclonal antibody against diallyl phthalate and its applications. Through multiple screenings, this invention has obtained a hybridoma cell line capable of secreting a monoclonal antibody against diallyl phthalate. The monoclonal antibody against diallyl phthalate secreted by this hybridoma cell line exhibits excellent affinity and sensitivity to diallyl phthalate, with an IC50 value of [missing information - likely related to IC50]. 50The concentration reaches 500 ng / mL, and there is no cross-reactivity with structural analogs of phthalates, such as dimethyl phthalate, diethyl phthalate, dinonyl phthalate, butyl benzyl phthalate, diisobutyl phthalate, di(2-ethylhexyl) phthalate, and diisononyl phthalate. Therefore, the monoclonal antibody of this invention can be used to prepare immunoassay products for diallyl phthalate, providing an efficient detection method and means for the detection of diallyl phthalate residues in food.

[0006] The first objective of this invention is to provide a hybridoma cell line, the proposed taxonomic name of which is a monoclonal antibody, and the accession number is CGMCC No. 46746.

[0007] Furthermore, the hybridoma cell line is obtained by immunizing animals with a complete antigen prepared from a hapten, wherein the structural formula of the hapten is shown in Formula I:

[0008] .

[0009] Furthermore, the complete antigen is obtained from the hapten coupled with a carrier protein.

[0010] Furthermore, the carrier protein includes keyhole hemocyanin.

[0011] A second objective of this invention is to provide an application of the above-mentioned hybridoma cell line in the detection of diallyl phthalate.

[0012] A third objective of this invention is to provide a monoclonal antibody secreted by the aforementioned hybridoma cell line.

[0013] A fourth objective of this invention is to provide an application of the above-mentioned monoclonal antibody in the detection of diallyl phthalate.

[0014] A fifth objective of this invention is to provide a detection product for diallyl phthalate, the detection product comprising the aforementioned monoclonal antibody.

[0015] Furthermore, the test product also includes a coating agent.

[0016] Furthermore, the coating is prepared from a hapten-coupled carrier protein; the carrier protein includes bovine serum albumin.

[0017] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0018] The monoclonal antibody secreted by the hybridoma cell line of the present invention exhibits good specificity and detection sensitivity against diallyl phthalate (IC50 for diallyl phthalate). 50With a concentration of 500 ng / mL and no cross-reactivity with structural analogs of diallyl phthalate, such as dimethyl phthalate, diethyl phthalate, dinonyl phthalate, butyl benzyl phthalate, diisobutyl phthalate, di(2-ethylhexyl) phthalate, and diisononyl phthalate, it can be used to detect diallyl phthalate residues, providing an immunoassay method and raw materials for the immunoassay of diallyl phthalate residues in food, and has practical application value.

[0019] Preservation of biological materials

[0020] The monoclonal cell line CTF was deposited on November 12, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46746, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0021] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0022] Figure 1 This is the standard inhibition curve of the diallyl phthalate monoclonal antibody of the present invention;

[0023] Figure 2 This is the synthetic route of a hapten. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0025] The culture media involved in the following examples are as follows:

[0026] RPMI-1640 medium (mg / L): L-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cysteine ​​dihydrochloride 65.15, L-glutamic acid 20, glycine 10, L-histidine 15, L-hydroxyproline 20, L-isoleucine 50, L-leucine 50, L-lysine hydrochloride 40, L-methionine 15, L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5. L-Tyrosine 23.19, L-Valine 20, Para-aminobenzoic acid 1, Calcium nitrate 100, Anhydrous magnesium sulfate 48.84, Anhydrous sodium dihydrogen phosphate 676.13, Potassium chloride 400, Sodium chloride 6000, Glucose 2000, Reduced glutathione 1, Phenol red 5, L-Glutamine 300, Biotin 0.2, D-Calcium pantothenate 0.25, Folic acid 1, I-Inositol 35, Nicotinamide 1, Choline chloride 3, Pyridoxine hydrochloride 1, Riboflavin 0.2, Thiamine hydrochloride 1, Vitamin B12 0.005, Sodium bicarbonate 2000.

[0027] Solution preparation:

[0028] Carbonate buffer (CBS): Weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them separately in a small amount of double-distilled water and mix them together. Add double-distilled water to about 800 mL and mix well. Adjust the pH to 9.6 and add double-distilled water to a final volume of 1000 mL. Store at 4°C for later use.

[0029] Phosphate-buffered saline (PBS): 8.0 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, 2.9 g Na2HPO4•12H2O, dissolved in 800 mL pure water, pH adjusted to 7.2-7.4 with NaOH or HCl, and then brought to a final volume of 1000 mL.

[0030] Washing buffer (PBST): Add 0.5 mL of Tween-20 to 1000 mL of 0.01 mol / L pH 7.4 PBS solution;

[0031] PBST: PBS containing 0.05% Tween-20;

[0032] Antibody dilution buffer: washing buffer containing 0.1% gelatin;

[0033] TMB colorimetric solution: Solution A: 18.43 g Na2HPO4•12H2O, 9.33 g citric acid, diluted to 1000 mL with pure water; Solution B: 60 mg TMB dissolved in 100 mL ethylene glycol. Mix solutions A and B in a volume ratio of 5:1 to obtain TMB.

[0034] The colorimetric reagent should be mixed and used immediately.

[0035] The detection methods involved in the following embodiments are as follows:

[0036] Method for detecting diallyl phthalate inhibition rate: The optimal antigen and antibody concentrations for ic-ELISA were selected using a checkerboard assay. The antigen was diluted to 0.1 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 0.03 μg / mL with antibody diluent. After selecting the optimal operating point, diallyl phthalate standards were diluted to eight concentrations (0, 50, 100, 200, 500, 1000, 2000, and 5000 ng / mL). Following the ic-ELISA procedure, the results were plotted using OriginPro 8.5 (see results below). Figure 1 As shown), the standard inhibition curve of diallyl phthalate was obtained, and the IC50 was calculated. 50 .

[0037] Example 1: Synthesis of Hapten

[0038] The esterification of 4-nitrophthalic acid with n-butanol yields dibutyl 4-nitrophthalate, followed by nitro reduction to give dibutyl 4-aminophthalate, thus producing the hapten. The reaction route is as follows: Figure 2 As shown.

[0039] Example 2: Synthesis of the coating agent

[0040] Dissolve 5 mg of hapten in 1 mL of DMF, add 0.2 mL of 1 mol / L dilute hydrochloric acid to adjust the pH to 2-3, and pre-cool at 4°C for 30 min. Then transfer the mixture to an ice bath at 4°C and add 0.1 mL of 10% sodium nitrite solution dropwise with slow stirring. The solution is tested with starch-potassium iodide test paper; the paper turns blue-gray. Activate at 4°C for 1 h to obtain solution A. Dissolve 20 mg of BSA in 4 mL of 0.1 mol / L pH 9.6 carbonate solution to obtain solution B. Then, slowly add solution A dropwise to solution B, adjusting the pH with 1 mol / L sodium hydroxide solution during the addition to maintain a pH of 9-10. React at 4°C for 5-6 h. After dialysis, the coated DAP-ZD-BSA is obtained.

[0041] Example 3: Synthesis of complete antigen

[0042] Dissolve 5.3 mg of hapten in 1 ml of DMF, add dropwise 86.8 µl of glutaraldehyde solution (10-fold dilution), activate at room temperature for 30 min, then add the activated solution dropwise to 30 mg of KLH (dissolved in 6 ml of 0.1 mol / L pH 7.4 PBS solution), react at room temperature for 3-4 h, and dialyze with 0.01 M pH 7.4 PBS solution to obtain the immunogen DAP-GA-KLH.

[0043] Example 4: Preparation of hybridoma cell lines

[0044] 1. Immunization of mice: Healthy 6-8 week old BALB / c mice were selected for immunization. Diallyl phthalate complete antigen was emulsified with an equal volume of Freund's adjuvant and administered subcutaneously via the back of the mouse. The first immunization used complete Freund's adjuvant, and subsequent immunizations used incomplete Freund's adjuvant. There was a 28-day interval between the first and second booster immunizations, and a 21-day interval between multiple booster immunizations. Seven days after the third immunization, blood was collected (5 μL of blood from tail amputation + 995 μL of antibody diluent = antiserum). The serum titer and inhibition were measured using ic-ELISA. Mice with high titers and good inhibition were selected for a sprint immunization 21 days after the fifth immunization, administered via intraperitoneal injection. The sprint dose was halved and contained no adjuvant.

[0045] 2. Cell fusion: Three days after the sprint immunization, cell fusion was performed using the standard PEG (polyethylene glycol, molecular weight 4000) method. The specific steps are as follows:

[0046] (1) After euthanizing the mice by removing the eyeballs and dislocating the cervical spine, the mice were immediately disinfected in 75% alcohol and soaked for about 5 minutes. The spleen of the mice was removed under aseptic conditions. The spleen cells were moderately ground with the rubber tip of a syringe and passed through a 200-mesh cell sieve to obtain a suspension of spleen cells. The suspension was collected and centrifuged (1200 rpm, 8 min). The spleen cells were washed three times with RPMI-1640 medium. After the last centrifugation, the spleen cells were diluted to a certain volume, counted, and set aside for later use.

[0047] (2) Collection of murine myeloma SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. The required number of SP2 / 0 tumor cells before fusion was 1-4 × 10⁻⁶. 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. During fusion, tumor cells are collected, suspended in RPMI-1640 basal culture medium, and cell counting is performed.

[0048] (3) Fusion process 7 min. At 1 min, 1 mL of PEG 1500 was added to the cells dropwise from slow to fast; at 2 min, the cells were left to stand. At 3 min and 4 min, 1 mL of RPMI-1640 medium was added dropwise over 1 min; at 5 min and 6 min, 2 mL of RPMI-1640 medium was added dropwise over 1 min; at 7 min, 1 mL of RPMI-1640 medium was added dropwise every 10 s, then the cells were incubated at 37°C for 5 min, centrifuged (800 rpm, 8 min), the supernatant was discarded, and the cells were resuspended in RPMI-1640 selection medium containing 20% ​​fetal bovine serum and 2% 50×HAT. The medium was added to 96-well cell plates at a rate of 200 μL / well and incubated at 37°C in a 5% CO2 incubator.

[0049] 3. Cell Screening and Cell Line Establishment: On day 3 of cell fusion, the fused cells were partially replaced with RPMI-1640 selection medium. On day 5, the medium was completely replaced with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 1% 100×HT. On day 7, the cell supernatant was collected for screening. Screening consisted of two steps: First, positive cell wells were selected using ic-ELISA. Second, DAP was used as a standard, and the inhibitory effect on positive cells was determined using ic-ELISA. Cell wells showing good inhibition of DAP standard were selected, and subcloning was performed using limiting dilution. The same method was used for detection. This process was repeated three times to obtain the cell line CTF.

[0050] Example 5: Preparation of Monoclonal Antibodies

[0051] 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil; 7 days later, each mouse was injected intraperitoneally with 1×10 6 Hybridoma cells were used, and ascites fluid was collected starting on day seven. The ascites fluid was purified using the caprylic acid-ammonium sulfate method. Under slightly acidic conditions, caprylic acid precipitates other proteins in the ascites fluid besides IgG immunoglobulins. The precipitate was then discarded by centrifugation. IgG-type monoclonal antibodies were then precipitated with an equal volume of saturated ammonium sulfate solution, centrifuged, and the supernatant was discarded. The precipitate was dissolved in 0.01M PBS solution (pH 7.4), dialyzed to desalt, and finally the purified monoclonal antibodies were stored at -20°C.

[0052] The IC50 of monoclonal antibodies against DAP was determined using an indirect competitive ELISA method. 50 The concentration was 500 ng / mL, and its IC50 for DMP and other substances was verified. 50 The cross-reactivity rate is shown in Table 1.

[0053] Table 1. Cross-reactivity of monoclonal antibody CTFs with diallyl phthalate and its structural analogues

[0054]

[0055] Example 6: Application of Monoclonal Antibodies

[0056] The monoclonal antibody prepared from the hybridoma cell line CTF via in vivo ascites fluid was used in the DAP spiking and recovery assay. The specific steps are as follows:

[0057] (1) Coating: The original DAP-BSA to be coated was serially diluted with 0.05 M pH 9.6 carbonate buffer starting from 1 µg / mL, 100 μL / well, and reacted at 37 ℃ for 2 h.

[0058] (2) Washing: Pour off the solution in the plate and wash with washing solution 3 times, 3 min each time.

[0059] (3) Sealing: After patting dry, add 200 μL / well sealing solution and react at 37℃ for 2 h. Wash and dry for later use.

[0060] (4) Sample addition: The antiserum (antiserum obtained by diluting the blood from the tail of mice with antibody diluent) was serially diluted from 1:1000 and added to each well of the coating at 100 μL / well. The reaction was carried out at 37℃ for 30 min. After thorough washing, HRP-goat anti-mouse IgG diluted at 1:3000 was added at 100 μL / well. The reaction was carried out at 37℃ for 30 min.

[0061] (5) Color development: Remove the microplate, wash it thoroughly, add 100 μL of TMB color development solution to each well, and react at 37 ℃ in the dark for 15 min.

[0062] (6) Termination and measurement: Add 50 μL of stop solution to each well to terminate the reaction, and then measure the OD of each well using a microplate reader. 450 value.

[0063] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A hybridoma cell line, characterized in that, The proposed taxonomic name for the hybridoma cell line is monoclonal antibody, and its accession number is CGMCC NO.46746.

2. The hybridoma cell line according to claim 1, characterized in that, The hybridoma cell line was obtained by immunizing animals with a complete antigen prepared from a hapten, wherein the structural formula of the hapten is shown in Formula I: 。 3. The hybridoma cell line according to claim 1, characterized in that, The complete antigen is obtained by conjugating the hapten to a carrier protein.

4. The hybridoma cell line according to claim 1, characterized in that, The carrier protein includes keyhole hemocyanin.

5. The use of the hybridoma cell line according to any one of claims 1-4 in the detection of diallyl phthalate.

6. A monoclonal antibody secreted by a hybridoma cell line according to any one of claims 1-4.

7. The use of the monoclonal antibody according to claim 6 in the detection of diallyl phthalate.

8. A detection product for diallyl phthalate, characterized in that, The detection product includes the monoclonal antibody as described in claim 6.

9. The testing product according to claim 8, characterized in that, The tested products also include coating agents.

10. The testing product according to claim 9, characterized in that, The coating is prepared from a hapten coupled with a carrier protein; the carrier protein includes bovine serum albumin.

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