Hybridoma cell strain, anti-dibutyl phthalate monoclonal antibody generated by hybridoma cell strain and application of monoclonal antibody

Abstract

The invention provides a hybridoma cell strain, an anti-dibutyl phthalate monoclonal antibody generated by the hybridoma cell strain and application of the anti-dibutyl phthalate monoclonal antibody, and belongs to the technical field of monoclonal antibody preparation, and the preservation number of the hybridoma cell strain DBP-2C7 for generating the anti-dibutyl phthalate monoclonal antibody is CCTCC NO: C2024274. The anti-dibutyl phthalate monoclonal antibody is high in titer, the subtype of the anti-dibutyl phthalate monoclonal antibody is IgG1, the half inhibition ratio IC50 of the anti-dibutyl phthalate monoclonal antibody to dibutyl phthalate is 24.3 [mu] g / L, the cross reaction rate of the anti-dibutyl phthalate monoclonal antibody to dibutyl phthalate is 100.00%, and the cross reaction rate of the anti-dibutyl phthalate monoclonal antibody to structural analogues is less than 1%. The anti-dibutyl phthalate monoclonal antibody generated by the hybridoma cell strain DBP-2C7 disclosed by the invention can achieve the purpose of rapidly and sensitively detecting dibutyl phthalate in food and environment.

Description

Technical Field

[0001] The invention belongs to the technical field of monoclonal antibody preparation, and particularly relates to a hybridoma cell line and an anti-dibutyl phthalate monoclonal antibody produced by the hybridoma cell line and application thereof. Background Art

[0002] Phthalic acid esters (PAEs) are a class of widely distributed industrial chemicals. PAEs have good compatibility with polymers, can reduce intermolecular forces, and effectively reduce the viscosity of polyvinyl chloride (PVC), thus giving rigid PVC softness, flexibility, transparency and long life. It has a large output, a wide variety of types, and a wide range of applications. Dibutyl phthalate (DBP), with a molecular formula of C 16 H 22 O4, with a molecular weight of 278.34, is the most widely used PAEs plasticizer. It is a colorless oily liquid that is not easily soluble in water but soluble in most organic solvents such as ethanol and ether. DBP is often added as a plasticizer to polyethylene, textile lubricants, printing inks, children's toys, medical devices, cosmetics, nutritional supplements, cellulose esters and special adhesives.

[0003] Since DBP is not chemically bonded to the plastic matrix, but connected by hydrogen bonds or van der Waals forces, DBP will gradually be released from plastic products over time, temperature, pH value, etc., and enter the environment and organisms, causing potential harm. DBP mainly enters the human body through inhalation, skin contact, ingestion and other routes. Sometimes medical injection can be a highly dangerous exposure method. DBP can simulate natural hormones in the human body, interfere with hormone action, and affect human physiological regulation. It is considered to be an environmental endocrine disruptor, also known as environmental hormones. Long-term DBP exposure can lead to male genital dysplasia, testicular atrophy and spermatogenesis disorders. Long-term accumulation in the body can lead to deformities or cancer. High molecular weight PAEs or their monoesters are the main allergens that cause allergic reactions such as asthma and rash in adults, and it has been found that exposure to PAEs such as DBP and 2-ethylhexyl phthalate (DEHP) during pregnancy is the main factor causing allergic reactions in infants and young children. Studies have reported that DBP exhibits neurobehavioral toxicity in mouse models.

[0004] PAEs have been included in the list of "non-food substances that may be illegally added to food". DBP has been included in the routine monitoring projects of surface water in many urban drinking water sources. For the detection of DBP, gas chromatography, gas chromatography-mass spectrometry, liquid chromatography, liquid chromatography-mass spectrometry, etc. are currently mainly used. The pre-treatment process of these instrument detection methods is relatively cumbersome, and the instruments are expensive, time-consuming, and the operation process is complicated. They are not suitable for on-site analysis of samples and the detection of large quantities of samples. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a hybridoma cell line and an anti-dibutyl phthalate monoclonal antibody produced by the hybridoma cell line and its application, which can achieve high-sensitivity and high-specificity detection of dibutyl phthalate through an immunoassay method.

[0006] The invention provides a hybridoma cell strain DBP-2C7 producing an anti-dibutyl phthalate monoclonal antibody. The deposit number of the hybridoma cell strain DBP-2C7 is CCTCC NO: C2024274.

[0007] The invention provides an anti-dibutyl phthalate monoclonal antibody produced by the hybridoma cell line DBP-2C7.

[0008] Preferably, the anti-dibutyl phthalate monoclonal antibody is of IgG1 subtype.

[0009] The present invention provides the use of the hybridoma cell line DBP-2C7 or the anti-dibutyl phthalate monoclonal antibody in detecting dibutyl phthalate.

[0010] Preferably, the detection adopts an indirect competitive ELISA method.

[0011] The invention provides a kit for detecting dibutyl phthalate, comprising the anti-dibutyl phthalate monoclonal antibody.

[0012] Preferably, the anti-dibutyl phthalate monoclonal antibody is used as a separate working solution; the concentration of the anti-dibutyl phthalate monoclonal antibody is 1:32000.

[0013] Preferably, the kit further comprises an ELISA plate coated with a DBP coating source, wherein the DBP coating source is obtained by coupling a hapten and a carrier protein; and the hapten is dibutyl 4-aminophthalate.

[0014] Preferably, the carrier protein is bovine serum albumin.

[0015] Preferably, the kit further comprises a dibutyl phthalate standard and an enzyme-labeled secondary antibody.

[0016] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a hybridoma cell line DBP-2C7 that produces anti-dibutyl phthalate monoclonal antibodies, the titer of the anti-dibutyl phthalate monoclonal antibodies produced is 1:1024000, the subtype is IgG1, and the affinity constant Ka=4.2×10 8 L / mol, half inhibition rate IC of dibutyl phthalate 50 The cross-reaction rate with dibutyl phthalate is 100.00%, and the cross-reaction rate with structural analogs is less than 1%. The hybridoma cell line DBP-2C7 of the present invention and the anti-dibutyl phthalate monoclonal antibody produced by the hybridoma cell line DBP-2C7 and the anti-dibutyl phthalate monoclonal antibody produced by the hybridoma cell line DBP-2C7 can be used for the detection of dibutyl phthalate; the application of ... BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the original gel electrophoresis identification image of dibutyl phthalate DBP coated in Example 1 of the present invention;

[0018] Figure 2 This is a UV scanning identification diagram of the dibutyl phthalate immunogen prepared in Example 1 of the present invention, wherein 1 is KLH, 2 is DBAP, and 3 is DBAP-KLH;

[0019] Figure 3 This is a UV scanning identification diagram of the dibutyl phthalate coating prepared in Example 1 of the present invention, wherein 1 is BSA, 2 is DBAP, and 3 is DBAP-BSA;

[0020] Figure 4 This is a graph showing the titer of the monoclonal antibody against dibutyl phthalate prepared in Example 1 of the present invention;

[0021] Figure 5 This is a diagram for determining subtypes of the anti-dibutyl phthalate monoclonal antibody prepared in Example 2 of the present invention;

[0022] Figure 6 This is a graph showing the affinity determination of the anti-dibutyl phthalate monoclonal antibody prepared in Example 2 of the present invention;

[0023] Figure 7 This is a competition curve diagram of the anti-dibutyl phthalate monoclonal antibody prepared in Example 2 of the present invention.

[0024] Biological Deposit Description

[0025] The hybridoma cell line DBP-2C7 of anti-dibutyl phthalate monoclonal antibody is deposited in China Center for Type Culture Collection, deposit address: Wuhan University, Wuhan, China, deposit date is October 24, 2024, and the deposit number is CCTCC NO: C2024274. DETAILED DESCRIPTION

[0026] The invention provides a hybridoma cell strain DBP-2C7 producing an anti-dibutyl phthalate monoclonal antibody. The deposit number of the hybridoma cell strain DBP-2C7 is CCTCC NO: C2024274.

[0027] In the present invention, the hybridoma cell line is preferably obtained by immunizing an animal with an artificially prepared immunogen; the artificially prepared immunogen is preferably obtained by coupling a hapten and a protein carrier, the hapten is preferably 4-aminophthalic acid dibutyl ester, and the protein carrier is preferably hemocyanin; the present invention has no special restrictions on the specific method of the coupling, and a conventional coupling method in the art can be used. The present invention has no special restrictions on the method of immunizing an animal with the immunogen, and a conventional method in the art can be used.

[0028] The present invention also provides an anti-dibutyl phthalate monoclonal antibody produced by the hybridoma cell line DBP-2C7. In the present invention, the anti-dibutyl phthalate monoclonal antibody has a titer of 1:1024000, a subtype of IgG1, and an affinity constant Ka=4.2×10 8 L / mol, half inhibition rate IC of dibutyl phthalate 50 The cross-reaction rate with dibutyl phthalate was 100.00%, and the cross-reaction rate with structural analogues was less than 1%.

[0029] The present invention also provides the use of the hybridoma cell line DBP-2C7 or the anti-dibutyl phthalate monoclonal antibody in detecting dibutyl phthalate. The present invention has no special restrictions on the source of the sample to be detected in the application, including but not limited to the detection sample derived from food or environment. In the present invention, the detection preferably adopts the indirect competitive ELISA method, and the present invention has no special restrictions on the specific steps of the indirect competitive ELISA method, and the conventional steps in the art can be used.

[0030] The invention provides a kit for detecting dibutyl phthalate, comprising the anti-dibutyl phthalate monoclonal antibody.

[0031] In the present invention, the kit preferably also includes an ELISA plate coated with a DBP coating source, wherein the coating source is obtained by coupling a hapten and a carrier protein; the hapten is preferably 4-amino dibutyl phthalate; and the carrier protein is preferably bovine serum albumin. In the present invention, the kit preferably also includes a dibutyl phthalate standard and an enzyme-labeled secondary antibody. In the present invention, the concentration of the dibutyl phthalate standard is preferably 250, 125, 62.5, 31.25, 15.625 μg / L; the enzyme-labeled secondary antibody is preferably a horseradish peroxidase-labeled goat anti-mouse secondary antibody.

[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] Example 1

[0034] Preparation of artificial antigen of dibutyl phthalate

[0035] (1) Preparation of immunogen

[0036] Weigh 3 mg of dibutyl 4-aminophthalate (DBAP) and dissolve it in DMF (100 μL), then add it to pre-cooled 1 mol / L hydrochloric acid (1 mL), and stir it in an ice bath; add 1 mol / L NaNO2 solution dropwise until the starch potassium iodide test paper turns dark purple, stir it in the dark for 30 minutes, and then add urea until the test paper no longer shows color; adjust the pH value to 9.0-9.5 with sodium hydroxide solution, dissolve 20 mg of hemocyanin (KLH) in PBS (1 mL), and slowly add it to the above reaction solution, add 1 mol / L sodium hydroxide solution, and maintain the pH value of the solution at 9.0-9.5, and the solution gradually turns orange-red. Stir the reaction at 4°C in the dark for 3 hours, dialyze with PBS for 3 days, and change the solution twice a day to obtain the immune antigen (DBAP-KLH).

[0037] (2) Preparation of coating

[0038] The preparation process of the coating agent is the same as that of the immunogen, except that hemocyanin is replaced with bovine serum albumin (BSA) to obtain the coating agent DBAP-BSA.

[0039] Identification of the prepared dibutyl phthalate artificial antigen:

[0040] Protein content determination

[0041] The hapten was coupled to the carrier protein by diazotization reaction to obtain the synthetic antigens DBAP-KLH and DBAP-BSA. The antigen protein content was determined by BCA method. The BSA standard curve was y=0.0001x+0.3074(R2 =0.9868), the protein contents of DBAP-KLH and DBAP-BSA were 0.64 mg / mL and 22.8 mg / mL, respectively.

[0042] SDS-PAGE identification

[0043] According to the conventional SDS-PAGE method, the concentration of separation gel was 12%, the concentration of concentrated gel was 5%, the voltage was 90V until the protein band entered the separation gel, and the voltage was adjusted to 150V to continue electrophoresis until bromophenol blue reached the bottom of the gel. After staining and decolorization, the results are shown in Figure 1 , the DBAP-BSA and BSA bands were located around 67 kDa, and the DBAP-BSA band was wider than the BSA band.

[0044] UV spectrophotometry

[0045] DBAP, DBAP-KLH, DBAP-BSA, KLH and BSA were diluted to 1 mg / L, adjusted to zero with PBS (0.01 mol / L, pH 7.4), and scanned at an ultraviolet wavelength of 200-600 nm to obtain 2 sets of UV spectra. Figure 2 , Figure 3 It can be seen that there are obvious differences in the absorption peaks of artificially synthesized antigens, carrier proteins and haptens. Figure 2 This is the UV spectrum scanning result of DBAP-KLH. KLH has UV absorption peaks at 235nm and 278nm, DBAP has UV absorption peaks at 238nm and 290nm, and the synthetic DBAP-KLH has an absorption peak at 219nm-229nm, which is similar to DBAP, and a broader absorption peak at 335nm-350nm, which is significantly different from DBAP and KLH. It is its own characteristic absorption peak, which is produced by the action of the hapten and carrier protein conjugate. It is preliminarily determined that the artificially synthesized DBAP-KLH is successfully synthesized. Figure 3 This is the UV spectrum scanning result of DBAP-BSA. BSA has UV absorption peaks at 238nm and 280nm, DBAP has UV absorption peaks at 238nm and 290nm. Similar to DBAP-KLH, the synthesized DBAP-BSA has absorption peaks at 232nm, 280nm and 335nm. It is preliminarily determined that the synthesized DBAP-BSA is successfully synthesized.

[0046] Example 2

[0047] Preparation of monoclonal antibody against dibutyl phthalate

[0048] (1) Animal immunization

[0049] Three female Balb / c mice aged 6-8 weeks (purchased from Hebei Medical University) were selected, and the DBAP-KLH prepared in Example 1 was used as an immunogen, with an immunization dose of 40 μg / mouse, and immunization was boosted every 14 days. Blood was collected by tail cutting 7 days after the third immunization, and the mouse serum antibody titer was detected by indirect ELISA, and the serum antibody inhibition rate was determined by competitive ELISA, and the best immune mice were selected for fusion.

[0050] (2) Cell fusion

[0051] Mouse myeloma cells and immune mouse spleen cells were fused with PEG, and fused hybridoma cells were obtained by selective culture in HAT and HT medium. Cells with high positive and low inhibition rate were selected from the cell culture supernatant by ELISA, and the positive wells were subcloned by limiting dilution method until a single hybridoma cell line secreting anti-dibutyl phthalate antibodies was established.

[0052] (3) Preparation and purification of monoclonal antibodies against dibutyl phthalate

[0053] The number of cells of the hybridoma cell line to be screened was adjusted to 10 6 / ml, and injected into the peritoneal cavity of Balb / c female mice sensitized with paraffin oil, 1 mL per mouse. The obtained ascites was purified by caprylic acid-ammonium sulfate precipitation method, divided into small tubes, and stored at -20°C to obtain the anti-dibutyl phthalate monoclonal antibody DBP-2C7.

[0054] Characterization of the prepared anti-dibutyl phthalate monoclonal antibody

[0055] (1) Potency determination

[0056] Dilute DBAP-BSA to 0.5 μg / mL with coating buffer, add 100 μl / well to the wells of the ELISA plate, and place at 4°C overnight. Wash the ELISA plate 3 times with a plate washer and spin dry. Prepare 10% calf serum solution with washing buffer, 200 μL per well, and incubate at 37°C for 1 hour. Wash the plate. Dilute the antibody to be tested with buffer to 1:1000, 1:2000...1:1024000, with the well without antibody as the blank control, 100 μL / well, incubate at 37°C in the dark for 45 minutes. Wash the plate. Dilute the goat anti-mouse secondary antibody at 1:10000 with buffer containing 10% calf serum, 100 μL / well, incubate at 37°C in the dark for 30 minutes. Wash the plate. Add substrate colorimetric solution, 100 μL / well, and incubate at 37°C in the dark for 15 minutes. Add stop solution, 50 μL / well, read at 450nm on a microplate reader, the A value of the negative control well is N, and the positive wells to be tested are P. P / N ≥ 2.1 is used as the titer determination result. Figure 4, the negative control well N was measured to be 0.066, and P was 0.161 when the antibody was diluted 1024000 times, so the titer of the anti-dibutyl phthalate monoclonal antibody was 1:1024000.

[0057] (2) Subtype determination

[0058] The determination was performed using the mouse immunoglobulin subtype kit from Sigma. DBAP-BSA was diluted to 1 μg / mL with coating buffer and coated overnight at 4°C. After washing the plate, block for 1 hour. Wash the plate, add 100 μL / well of appropriately diluted monoclonal antibody DBP-2C7, and incubate at 37°C for 45 minutes. Wash the plate. Add 100 μL / well of 6 subtype reagents (1:1000) including IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM, and incubate at 37°C for 30 minutes. Wash the plate. Add 100 μL / well of HRP-labeled rabbit anti-goat secondary antibody diluted 1:5000, and incubate at 37°C for 30 minutes. Wash the plate. Add substrate colorimetric solution, 100 μL / well, and incubate at 37°C in the dark for 15 minutes. Add 50 μL / well of stop solution and read at 450 nm on an enzyme reader. The results are as follows. Figure 5 As shown, the A450 nm value of monoclonal antibody DBP-2C7 with IgG1 subtype reagent is the highest, and no color is developed with other subtype reagents. Therefore, monoclonal antibody DBP-2C7 belongs to IgG1 subtype.

[0059] (3) Affinity determination

[0060] The affinity constant (Ka) was determined by indirect ELISA. The coating buffer was used to dilute the coating source to concentrations of 1, 0.5, 0.25, and 0.125 μg / mL. The monoclonal antibody DBP-2C7 was diluted in multiples starting at 100 μg / mL, and four coating source concentrations were added respectively. The other steps were the same as the determination of the monoclonal antibody titer. The absorbance value at a wavelength of 450 nm (A450nm) was measured. Ka was calculated according to the following formula

[0061] Ka=(n-1) / 2(nAb'-Ab)

[0062] Where: Ab is the antibody concentration that produces half the absorbance value when the antigen concentration is Ag; Ab' is the antibody concentration that produces half the absorbance value when the antigen concentration is Ag'; n is the dilution multiple between Ag and Ag'. The affinity determination results are shown in Figure 6 , calculated affinity constant Ka = 4.2 × 10 8 L / mol.

[0063] (4) Inhibition rate determination

[0064] The inhibition rate of dibutyl phthalate monoclonal antibody against different concentrations of dibutyl phthalate was determined by competitive ELISA. The antigen DBAP-BSA was diluted with coating solution to a concentration of 0.5 μg / mL, 100 μL / well, and placed at 4°C overnight; the plate was washed and blocked for 1 hour. The plate was washed, and the monoclonal antibody and dibutyl phthalate standard were added: the mother solution of dibutyl phthalate standard was diluted with PBS to 250, 125, 62.5, 31.25, 15.625, and 0 μg / L, and 50 μL was added to each well, and then 50 μL / well of the monoclonal antibody diluted 1:32000 was added, and incubated at 37°C in the dark for 45 minutes. The plate was washed. The goat anti-mouse secondary antibody was diluted 1:10000 with a buffer containing 10% calf serum, 100 μL / well, and incubated at 37°C in the dark for 30 minutes. The plate was washed. Add substrate colorimetric solution, 100 μL / well, incubate at 37°C in the dark for 15 min. Add stop solution, 50 μL / well, read at 450 nm on a microplate reader. The inhibition percentage is B / B0 (B represents the A of different concentrations of dibutyl phthalate standard solution). 450 , B0 represents the A of the zero concentration standard solution 450 ) is the ordinate, and the Lg value of dibutyl phthalate concentration is the abscissa, and the competition curve of dibutyl phthalate antibody is drawn. Figure 7 The monoclonal antibody DBP-2C7 has a good linear relationship in the range of 15.6μg / L to 250μg / L, R 2 =0.9934,IC 50 It is 24.3μg / L, and is highly sensitive to dibutyl phthalate.

[0065] (5) Specificity determination

[0066] Structural analogs of dibutyl phthalate such as dimethyl phthalate and diethyl phthalate were selected for cross-reaction determination to evaluate the specificity of dibutyl phthalate antibodies. The smaller the cross-reaction rate (CR), the higher the specificity of the antibody to the target. The CR is calculated according to formula (1):

[0067]

[0068] In formula (1):

[0069] CR: cross-reaction rate, %;

[0070] IC 50 (DBP): The DBP concentration corresponding to 50% inhibition rate, μg / L;

[0071] IC 50 (Structural analogues): The concentration of DBP structural analogues corresponding to an inhibition rate of 50%, μg / L.

[0072] The cross-reaction results are shown in Table 1. The cross-reaction rate of dibutyl phthalate antibody with the hapten 4-aminodibutyl phthalate is 28.6%, and the cross-reaction rate with several other structural analogs is less than 1%, with good specificity.

[0073] Table 1 Cross-reaction results

[0074]

[0075] Example 3

[0076] The anti-dibutyl phthalate monoclonal antibody prepared in Example 1 was used in an enzyme-linked immunosorbent assay kit. The detection principle of the kit in the present invention is an indirect competitive ELISA method.

[0077] Dilute the antigen DBAP-BSA with coating solution to a concentration of 0.5 μg / mL, 100 μL / well, and place at 4°C overnight. Remove the strips as needed and place them on the ELISA plate; add dibutyl phthalate standards (250, 125, 62.5, 31.25, 15.625, 0 μg / L) or 50 μL of the sample to be tested and 50 μL of anti-dibutyl phthalate monoclonal antibody working solution (1:32000) to the wells of the ELISA plate in sequence, gently shake to mix, and incubate at 37°C in the dark for 45 minutes. Wash 4 times with 250 μL / well of washing working solution, and pat dry; add 100 μL of horseradish peroxidase-labeled goat anti-mouse secondary antibody working solution, and incubate at 37°C in the dark for 30 min; wash and pat dry, mix color development solution A and color development solution B in a 1:1 ratio, add 100 μL / well for color development, and incubate at 37°C in the dark for 15 min; add 50 μL / well of stop solution to terminate the reaction, and measure with a dual wavelength of 450nm / 630nm at 450nm using an enzyme reader, and quantify or quantify the sample according to the standard curve.

[0078] Result determination: (a) Quantitative analysis: Calculate the average absorbance value (B) of dibutyl phthalate standards of different concentrations and the sample to be tested, divide it by the absorbance value (B0) of the 0μg / L standard and multiply it by 100%, which is the percentage absorbance value, percentage absorbance value = (B / B0) × 100%. Draw a standard curve with the percentage absorbance value of the standard as the ordinate and the logarithm of the concentration (μg / L) of the dibutyl phthalate standard as the abscissa. Substitute the percentage absorbance value of the sample to be tested into the standard curve, read the concentration corresponding to the sample from the standard curve, and multiply it by the corresponding dilution multiple to obtain the actual concentration of dibutyl phthalate in the sample to be tested. (b) Qualitative analysis: Compare the average absorbance value of the sample to be tested with the absorbance value of the standard to obtain the concentration range of dibutyl phthalate in the sample. The test range of this embodiment is 7.8 μg / L to 250 μg / L, y=-0.3322x+0.9772, IC 50 The value is 27.32μg / L.

[0079] Example 4

[0080] Pretreatment of water and beverage samples: Accurately transfer 5 mL of sample into a test tube, add 5 mL of n-hexane, shake thoroughly for 2 min to mix and extract, blow dry 2 mL of supernatant with nitrogen, and re-dissolve with 400 μL PBS for later use.

[0081] The pre-treated samples prepared above were detected using the kit and detection method described in Example 3.

[0082] It can be seen from the above examples that the hybridoma cell line DBP-2C7 provided by the present invention produces a high titer of anti-dibutyl phthalate monoclonal antibody, a cross-reaction rate with dibutyl phthalate of 100.00%, and a cross-reaction rate with structural analogs of less than 1%. It is shown that the hybridoma cell line DBP-2C7 of the present invention and the anti-dibutyl phthalate monoclonal antibody produced by it can be used for the detection of dibutyl phthalate, and have good controllability and repeatability; it is applied to the preparation of an enzyme-linked immunosorbent assay kit for detecting dibutyl phthalate, which can achieve the purpose of quickly and sensitively detecting dibutyl phthalate in food and the environment. The quality of the anti-dibutyl phthalate monoclonal antibody provided by the present invention has strong controllability and repeatability, and can achieve specific and sensitive immunoassay of dibutyl phthalate.

[0083] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A hybridoma cell line DBP-2C7 producing anti-dibutyl phthalate monoclonal antibodies, characterized in that: The deposit number of the hybridoma cell line DBP-2C7 is CCTCC NO: C2024274.

2. The anti-dibutyl phthalate monoclonal antibody produced by the hybridoma cell line DBP-2C7 according to claim 1.

3. The anti-dibutyl phthalate monoclonal antibody according to claim 2, characterized in that: The anti-dibutyl phthalate monoclonal antibody is of IgG1 subtype.

4. Use of the hybridoma cell line DBP-2C7 according to claim 1 or the anti-dibutyl phthalate monoclonal antibody according to claim 2 in detecting dibutyl phthalate.

5. The use according to claim 4, characterized in that: The detection adopts the indirect competitive ELISA method.

6. A kit for detecting dibutyl phthalate, characterized in that: The invention comprises the anti-dibutyl phthalate monoclonal antibody according to claim 2.

7. The kit according to claim 6, characterized in that The anti-dibutyl phthalate monoclonal antibody is used as a separate working solution; the concentration of the anti-dibutyl phthalate monoclonal antibody is 1:32000.

8. The kit according to claim 6, characterized in that The kit also includes an ELISA plate coated with a DBP coating source, wherein the DBP coating source is obtained by coupling a hapten and a carrier protein; the hapten is dibutyl 4-aminophthalate.

9. The kit according to claim 8, characterized in that The carrier protein is bovine serum albumin.

10. The kit according to claim 7, characterized in that The kit also includes a dibutyl phthalate standard and an enzyme-labeled secondary antibody.

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