Hybridoma cell strain secreting anti-dimethyl phthalate monoclonal antibody and application of hybridoma cell strain

Abstract

The invention discloses a hybridoma cell strain capable of secreting an anti-dimethyl phthalate monoclonal antibody and application of the hybridoma cell strain, and belongs to the field of food safety immunodetection. The hybridoma cell strain 3F7 capable of secreting the dimethyl phthalate cloned antibody is preserved in the China General Microbiological Culture Collection Center (CGMCC for short), the preservation date is 20250417, and the preservation number is 46516. The anti-dimethyl phthalate monoclonal antibody obtained by secretion of the cell strain is used for analyzing and detecting residual dimethyl phthalate in food safety detection. The anti-dimethyl phthalate monoclonal antibody cell strain obtained by the invention can be used for immunoassay detection, and has better detection sensitivity and specificity (the IC50 value is 26.6 ng / mL) on dimethyl phthalate. The achievement of the invention can be used for preparing an immunodetection kit and a colloidal gold test strip of dimethyl phthalate, and provides a powerful detection method and means for detecting the residual quantity of dimethyl phthalate in food such as fruit juice, milk, white spirit and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to a hybridoma cell strain secreting anti-dimethyl phthalate monoclonal antibody and its application, and belongs to the field of food safety immune detection. BACKGROUND

[0002] Dimethyl phthalate (DMP) is a plasticizer with strong solubility to various resins, which can be compatible with various cellulose resins, rubber, and vinyl resins. It has good film-forming property, adhesion, and water resistance. It is usually used in combination with diethyl phthalate for the production of cellulose acetate film, varnish, transparent paper, and molding powder, and a small amount is used in the production of nitrocellulose. Dimethyl phthalate has good compatibility with plastics and good plasticizing effect, so it is mainly used in plastic products as a plasticizer. However, they are easily migrated to the environment because they are combined with plastic media by non-covalent bonds, and dimethyl phthalate has become a ubiquitous environmental pollutant worldwide due to its good stability.

[0003] At present, the detection method for dimethyl phthalate residues is mainly instrument analysis method, including gas chromatography, gas chromatography-mass spectrometry, etc. These methods have reliable results and high sensitivity, and there are related technical standards for reference. However, due to the need for expensive instruments, specialized operators, and complex sample pretreatment, high cost, and long time, they cannot better meet the requirements of rapid and simple on-site detection. Therefore, it is of great significance to establish a rapid and simple detection method for dimethyl phthalate. Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive, and rapid detection method, which is suitable for on-site rapid detection of a large number of samples, and provides a new detection approach for dimethyl phthalate. SUMMARY

[0004] The purpose of the present application is to provide a hybridoma cell strain secreting anti-dimethyl phthalate monoclonal antibody; another purpose of the present application is to provide an anti-dimethyl phthalate monoclonal antibody; another purpose of the present application is to provide a composition; another purpose of the present application is to provide the application of the anti-dimethyl phthalate monoclonal antibody and the composition.

[0005] Technical solution: the hybridoma cell strain secreting anti-dimethyl phthalate monoclonal antibody of the present application, the hybridoma cell strain has been preserved in China General Microbiological Culture Collection Center CGMCC, the preservation number is CGMCC No. 46516, the preservation date is April 17, 2025, and the classification and naming is monoclonal cell strain.

[0006] Further, the preparation method of the hybridoma cell strain comprises the following steps:

[0007] S1, a dimethyl phthalate hapten is prepared into a complete antigen, and an animal is immunized by using the complete antigen;

[0008] S2, blood is collected from the immunized animal, and the serum immune titer and immune inhibition ability of the immunized animal are screened;

[0009] S3, the spleen cells and myeloma cells of the immunized animal screened in step S2 are fused and cultured to obtain the hybridoma cell strain secreting the dimethyl phthalate monoclonal antibody;

[0010] The dimethyl phthalate hapten is selected from the following structure:

[0011]

[0012] Further, the dimethyl phthalate hapten is prepared by the following steps: 4-nitrophthalic acid is reacted with methanol by esterification to obtain 4-nitrophthalic acid dimethyl ester, and then the nitro group is reduced to obtain 4-amino phthalic acid dimethyl ester, i.e. the hapten.

[0013] Further, the dimethyl phthalate complete antigen is obtained by coupling the dimethyl phthalate hapten with a carrier protein.

[0014] Further, the carrier protein is KLH (keyhole limpet hemocyanin) or BSA (bovine serum albumin).

[0015] As a preferred embodiment, the application provides a preparation method of the hybridoma cell strain 3F7 secreting the anti-dimethyl phthalate monoclonal antibody, which comprises the following steps:

[0016] (1) Preparation of dimethyl phthalate hapten:

[0017] First, 4-nitrophthalic acid is reacted with methanol by esterification to obtain 4-nitrophthalic acid dimethyl ester, and then the nitro group is reduced to obtain 4-amino phthalic acid dimethyl ester, i.e. the hapten;

[0018] (2) Preparation of dimethyl phthalate complete antigen: the dimethyl phthalate hapten is coupled with the carrier protein KLH by the glutaraldehyde method to prepare the immunogen DMP-GA-KLH, and the dimethyl phthalate hapten is coupled with the carrier protein BSA by the diazotization method to prepare the coating antigen DMP-ZD-BSA;

[0019] (3) Immunization of mice: the immunogen DMP-GA-KLH was mixed with an equal amount of Freund's adjuvant and emulsified, and then the BALB / c mice were immunized by subcutaneous injection on the back; the complete Freund's adjuvant was used for the first immunization, and the incomplete Freund's adjuvant was used for the multiple booster immunization; the interval between the first immunization and the second booster immunization was 28 days, the interval between the multiple booster immunizations was 21 days, and the last time, the DMP-GA-KLH complete antigen (without adjuvant) was used for the boost immunization; the serum titer and inhibition were detected by indirect competitive enzyme-linked immunoassay (ic-ELISA);

[0020] (4) Cell fusion and cell strain establishment: the mouse spleen cells and mouse myeloma cells were fused by polyethylene glycol (PEG 4000) method, and cultured by HAT medium; the positive cell wells were detected by ic-ELISA, and the inhibition effect of the positive cell wells was further determined by ic-ELISA; the positive cell wells with the best inhibition effect were subcloned by limited dilution method for three times, and finally, the hybridoma cell strain 3F7 was obtained by screening.

[0021] (5) Identification of the properties of the hybridoma cell strain: the sensitivity and specificity were determined by ic-ELISA.

[0022] On the other hand, the present application provides an anti-dimethyl phthalate monoclonal antibody, which is secreted by the hybridoma cell strain described above.

[0023] On the other hand, the present application provides an immunological detection kit or colloidal gold test strip, which contains the anti-dimethyl phthalate monoclonal antibody described above.

[0024] On the other hand, the present application provides the use of the anti-dimethyl phthalate monoclonal antibody described above or the immunological detection kit or colloidal gold test strip described above in the detection of dimethyl phthalate.

[0025] Further, the present application is used for the detection of the residual amount of dimethyl phthalate in food.

[0026] Advantages: compared with the prior art, the present application has the following remarkable advantages: the monoclonal antibody secreted by the hybridoma cell strain 3F7 provided by the present application has good specificity and detection sensitivity (IC 50 all of which are 26.6 ng / mL) for dimethyl phthalate, and can realize the detection of the residual amount of dimethyl phthalate in fruit juice, milk and other food, and provide raw materials for the immunological detection of dimethyl phthalate residues in food, and has practical application value.

[0027] The biological material preservation: a hybridoma cell strain 3F7 secreting an anti-dimethyl phthalate monoclonal antibody has been preserved in the China General Microbiological Culture Collection Center (CGMCC), with a preservation number of CGMCC No. 46516, a preservation address of No. 1, Beichen West Road, Hua-yuan District, Beijing, and a preservation date of April 17, 2025, and is classified as a monoclonal cell strain. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 A standard curve for inhibition of dimethyl phthalate by the monoclonal antibody 3F7. DETAILED DESCRIPTION

[0029] The technical solutions of the present application are further described below in combination with the drawings.

[0030] The present application obtains the monoclonal antibody hybridoma cell strain 3F7 with high sensitivity to dimethyl phthalate by immunizing mice with dimethyl phthalate complete antigen, cell fusion, HAT selective medium culture, and ic-ELISA screening of cell supernatant.

[0031] Example 1: Synthesis of hapten The synthesis of hapten is shown in the following reaction equation:

[0032]

[0033] The specific steps are as follows:

[0034] Preparation of dimethyl phthalate hapten 4-amino dimethyl phthalate:

[0035] First, 0.12 mol of 4-nitro phthalic acid was weighed into a 100 mL three-necked flask, 0.1 mol of methanol was added, 6 mL of benzene was added as a solvent, magnetic stirring was performed, 1.6 mL of concentrated sulfuric acid was slowly added, heating was performed to 120℃, reaction was performed for 12 h, organic solvent was distilled off under reduced pressure, the oily residue was added to cold water and washed with 10% Na2CO3 solution until the lower water layer was colorless, and the upper layer was a dark red oily crude product, which was recrystallized with anhydrous ethanol to obtain 4-nitro dimethyl phthalate. Second, 7 mmol of reduced iron powder was weighed into a 50 mL round-bottom flask, 2 mL of water and 1 mmol of ammonium chloride were added, and water bath heating was performed for 15 min. 4-nitro dimethyl phthalate 1.4 mmol was taken, dissolved in 5 mL of methanol, and added to the above reaction liquid in three portions, and reflux reaction was performed for 5 h. The iron powder was filtered while hot, the filtrate was distilled under reduced pressure, the orange-red residue obtained was dissolved in a small amount of hot methanol, and 1.17 mmol of 4-amino dimethyl phthalate was precipitated at low temperature as a hapten for standby use.

[0036] Preparation of coating antigen

[0037] Dissolve 5 mg of hapten 4-amino-phthalic acid dimethyl ester in 1 ml DMF, add 0.2 ml of 1 mol / L dilute hydrochloric acid to make the solution pH 2-3, and pre-cool in a refrigerator at 4°C for 30 min. Then transfer the mixture into an ice bath at 4°C, and add 0.1 ml of 10% sodium nitrite solution drop by drop under slow stirring, and monitor with starch-iodine paper until the paper turns blue-gray; activate for 1 h under an ice bath at 4°C to obtain solution A. Dissolve 20 mg of BSA in 4 ml of 0.1 mol / L carbonate solution at pH 9.6 to obtain solution B; then slowly add solution A drop by drop into solution B, and adjust the pH to 9-10 with 1 mol / L sodium hydroxide solution during the addition, and react at 4°C for 5-6 h. After dialysis, the coating antigen DMP-ZD-BSA is obtained.

[0038] Preparation of immunogen

[0039] Dissolve 5.3 mg of hapten 4-amino-phthalic acid dimethyl ester in 1 ml DMF, and add 86.8 μl of glutaraldehyde solution (10-fold dilution) drop by drop. After activation at room temperature for 30 min, add the activated solution drop by drop into 30 mg of KLH (dissolved in 6 ml of 0.1 mol / L PBS solution at pH 7.4), and react at room temperature for 3-4 h. Dialyze against 0.01M PBS solution at pH 7.4 to obtain the immunogen DMP-GA-KLH.

[0040] Preparation of cloned antibody

[0041] 1. Immunization of mice: Healthy 6-8 week old BALB / c mice are selected for immunization. The dimethyl phthalate complete antigen (DMP-ZD-BSA) prepared in Example 2 is mixed with an equal amount of Freund's adjuvant, and then emulsified by subcutaneous injection on the back of the BALB / c mice. Complete Freund's adjuvant is used for the first immunization, and incomplete Freund's adjuvant is used thereafter. The interval between the first immunization and the second booster immunization is 28 days, and the interval between the multiple booster immunizations is 21 days. Blood is collected 7 days after the third immunization (5 μL of mouse tail blood + 995 μL of antibody diluent = antiserum), and the mouse serum titer and inhibition are determined using ic-ELISA. Mice with high titer and good inhibition are selected, and a boost immunization is performed 21 days after the fifth immunization by intraperitoneal injection, with the requirement that the boost dose is halved and does not contain any adjuvant.

[0042] 2. Cell fusion: Three days after the boost immunization, cell fusion is performed according to the conventional PEG (polyethylene glycol, molecular weight 4000) method, with the following specific steps:

[0043] (1) Enucleation blood, cervical dislocation method after mice were immediately put into 75% alcohol disinfection, soak for 5 min or so, aseptic operation to remove the spleen of mice, with a syringe rubber head moderate grinding and through 200 mesh cell screen to get spleen cell suspension, collection, and centrifugation (1200 rpm, 8 min), washing the spleen cells three times with RPMI-1640 medium, the last time after centrifugation, the spleen cells were diluted to a certain volume, count, standby;

[0044] (2) collection of mouse myeloma SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells were cultured with 10% FBS (fetal bovine serum) RPMI-1640 medium in a 5% CO2 incubator. Before fusion, the number of SP2 / 0 tumor cells is required to reach 1-4 x 10 7 , to ensure that the SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. At the time of fusion, the tumor cells were collected and suspended in RPMI-1640 base culture medium for cell counting;

[0045] (3) fusion process 7 min. 1 min, 1 mL of PEG 1500 was added slowly to the cells; 2 min, stand. 3 min and 4 min, 1 mL of RPMI-1640 medium was added within 1 min; 5 min and 6 min, 2 mL of RPMI-1640 medium was added within 1 min; 7 min, 1 mL of RPMI-1640 medium was added every 10 s, then 37°C water bath for 5 min, centrifugation (800 rpm, 8 min), discard the supernatant, resuspended in RPMI-1640 screening medium containing 20% fetal bovine serum, 2% 50x HAT, 200 μL / well to 96-well cell plate, placed in a 37°C, 5% CO2 incubator.

[0046] 3. Cell screening and cell strain establishment: On the 3rd day of cell fusion, the fusion cells were subjected to RPMI-1640 screening medium semi-replacement, on the 5th day, the cells were subjected to full replacement with RPMI-1640 transition medium containing 20% fetal bovine serum, 1% 100x HT, and on the 7th day, the cell supernatant was collected for screening. The screening was divided into two steps: first, positive cell wells were screened out by ic-ELISA, and second, DMP was used as a standard for inhibitory effect determination of positive cells by ic-ELISA. Cell wells with good inhibition to DMP standard were selected, and subcloning was performed by limiting dilution, and detection was performed by the same method. Three times were repeated to obtain cell strain 3F7.

[0047] 4. Preparation and identification of monoclonal antibody: 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil per mouse; 7 days later, each mouse was injected intraperitoneally with 1 x 10 6Hybridoma cells, from the seventh day, collect ascites, and purify the ascites by the caprylic acid-ammonium sulfate method. In the slightly acidic condition, n-caprylic acid can precipitate other impurities in the ascites except IgG immunoglobulin, then centrifugal, and discard the precipitate; then use an equal amount of saturated ammonium sulfate solution to precipitate IgG type monoclonal antibody, centrifugal, discard the supernatant, and then dissolve in 0.01M PBS solution (pH 7.4), dialysis and desalination, and finally obtain the purified monoclonal antibody stored at -20℃.

[0048] Using the indirect competitive ELISA method, the IC 50 of the monoclonal antibody to DMP is determined to be 26.6 ng / mL, and the IC 50 and cross-reactivity thereof are verified, as shown in Table 1.

[0049] Table 1 IC 50 and cross-reactivity of monoclonal antibody 3F7 to dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dinonyl phthalate, butyl benzyl phthalate, diisobutyl phthalate, di(2-ethylhexyl) phthalate, and diisononyl phthalate

[0050]

[0051] Example 5 Preparation of Cloned Antibody

[0052] The difference between this example and Example 4 is the immunization step of the mouse, and the rest of the steps are the same.

[0053] Immunization of the mouse: the immunogen DMP-GA-KLH is mixed and emulsified with an equal amount of Freund's adjuvant, and then the BALB / c mouse is immunized by subcutaneous injection on the back; the complete Freund's adjuvant is used for the first immunization, and the incomplete Freund's adjuvant is used for multiple booster immunizations, the interval between the first immunization and the second booster immunization is 28 days, the interval between multiple booster immunizations is 21 days, and the last time is a DMP-GA-KLH complete antigen (without adjuvant) boost immunization; the serum titer and inhibition are detected by indirect competitive enzyme-linked immunization (ic-ELISA).

[0054] Example 6 Application of Antibody

[0055] The monoclonal antibody prepared by the hybridoma cell strain 3F7 of Example 4 is applied to the DMP addition recovery test, and the specific steps are as follows:

[0056] (1) Coating: the coating agent DMP-BSA is diluted by 2 times from 1 μg / mL, 100 μL / well, and reacted at 37℃ for 2 h.

[0057] (2) Washing: The solution in the plate was poured off and washed with washing solution for 3 times, 3 min each time.

[0058] (3) Blocking: After being patted dry, 200 μL / well of blocking solution was added and reacted at 37°C for 2 h. After washing, it was dried and stored for use.

[0059] (4) Sample adding: The antiserum (after the tail of mouse was cut, the corresponding multiple dilution of the antibody was diluted to be the antiserum) was diluted by multiple dilution from 1:1000 and added to the coated wells of each dilution, 100 μL / well, reacted at 37°C for 30 min; after being washed thoroughly, HRP-goat anti-mouse IgG diluted by 1:3000 was added, 100 μL / well, reacted at 37°C for 30 min.

[0060] (5) Color developing: The enzyme-labeled plate was taken out and washed thoroughly, 100 μL of TMB color developing solution was added to each well, reacted at 37°C for 15 min in the dark.

[0061] (6) Termination and determination: 50 μL of termination solution was added to each well to terminate the reaction, and then the OD450 value of each well was determined by an enzyme-labeled instrument.

[0062] Determination of the IC of the monoclonal antibody to DMP by ic-ELISA 50 was 30.13 ng / mL, which indicated that the monoclonal antibody had good sensitivity to DMP and could be used for the determination of DMP by immunoassay.

[0063] The inhibition standard curve of the 3F7 monoclonal antibody to DMP is shown in Fig. 2. Figure 1

[0064] Solution preparation:

[0065] Carbonate buffer solution (CBS): 1.59 g of Na2CO3 and 2.93 g of NaHCO3 were weighed, respectively dissolved in a small amount of double distilled water, mixed, double distilled water was added to about 800 mL, mixed, the pH value was adjusted to 9.6, double distilled water was added to constant volume to 1000 mL, and stored at 4°C for standby.

[0066] Phosphate buffer solution (PBS): 8.0 g of NaCl, 0.2 g of KCl, 0.2 g of KH2PO4, and 2.9 g of Na2HPO4·12H2O were dissolved in 800 mL of pure water, the pH was adjusted to 7.2-7.4 with NaOH or HCl, and the constant volume was added to 1000 mL;

[0067] Washing solution (PBST): 0.5 mL of Tween-20 was added to 1000 mL of 0.01 mol / L PBS solution with pH 7.4;

[0068] PBST: PBS containing 0.05% Tween-20;

[0069] ​Antibody dilution: washing buffer containing 0.1% gelatin;

[0070] TMB developing solution: A solution: Na2HPO4.12H2O 18.43g, citric acid 9.33g, and pure water to 1000mL; B solution: 60mg TMB is dissolved in 100mL ethylene glycol. A, B solution mixed by volume ratio 5:1 is TMB.

[0071] Developing solution, mix immediately before use.

[0072] To sum up, although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A hybridoma cell line that secretes a monoclonal antibody against dimethyl phthalate, characterized in that, The hybridoma cell line has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNo.46516, deposit date of April 17, 2025, and classified as a monoclonal cell line.

2. The hybridoma cell line secreting anti-dimethyl phthalate monoclonal antibody according to claim 1, characterized in that, The method for preparing hybridoma cell lines includes the following steps: S1. Prepare a complete antigen from the dimethyl phthalate hapten, and use the complete antigen to immunize animals. S2. Blood samples are collected from immunized animals to screen for serum immunogenicity and immunosuppressive capacity. S3. The spleen cells and myeloma cells of the immunized animals selected in step S2 are fused and cultured to obtain the hybridoma cell line that secretes dimethyl phthalate monoclonal antibody. The dimethyl phthalate hapten is selected from the following structures:

3. The hybridoma cell line secreting anti-dimethyl phthalate monoclonal antibody according to claim 2, characterized in that, The dimethyl phthalate hapten is prepared by the following steps: 4-nitrophthalic acid and methanol are esterified to obtain dimethyl 4-nitrophthalate, and then the nitro group is reduced to obtain dimethyl 4-aminophthalate, which is the hapten.

4. The hybridoma cell line secreting anti-dimethyl phthalate monoclonal antibody according to claim 2, characterized in that, The dimethyl phthalate complete antigen is obtained by conjugating the dimethyl phthalate hapten with a carrier protein.

5. The hybridoma cell line secreting anti-dimethyl phthalate monoclonal antibody according to claim 2, characterized in that, The carrier protein is keyhole hemocyanin or bovine serum albumin.

6. A monoclonal antibody against dimethyl phthalate, characterized in that, The anti-dimethyl phthalate monoclonal antibody is obtained by secretion from the hybridoma cell line described in claim 1.

7. An immunoassay kit or colloidal gold test strip, characterized in that, The immunoassay kit or colloidal gold test strip contains the anti-dimethyl phthalate monoclonal antibody as described in claim 6.

8. The use of the anti-dimethyl phthalate monoclonal antibody as described in claim 6 or the immunoassay kit or colloidal gold test strip as described in claim 7 in the detection of dimethyl phthalate.

9. The application according to claim 8, characterized in that, Used for the detection of dimethyl phthalate residues in food.

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