Hybridoma cell strain DBP-8A5 and anti-dibutyl phthalate monoclonal antibody generated by hybridoma cell strain DBP-8A5

Abstract

The invention relates to a hybridoma cell strain DBP-8A5 and an anti-dibutyl phthalate monoclonal antibody generated by the hybridoma cell strain DBP-8A5. The hybridoma cell strain DBP-8A5 (CCTCC NO: C202360) provided by the invention can be used for preparing a high-sensitivity dibutyl phthalate (DBP) monoclonal antibody, and the affinity of the antibody measured by an enzyme linked immunosorbent assay (ELISA) method is 1.72 * 10 < 7 > L / mol; the anti-dibutyl phthalate monoclonal antibody provided by the invention is high in sensitivity and good in specificity, the 50% inhibition concentration IC50 to dibutyl phthalate is 2.06 ng / mL, and the anti-dibutyl phthalate monoclonal antibody has no obvious cross to common phthalate plasticizers and is good in specificity.

Description

Technical Field

[0001] The invention relates to a hybridoma cell line DBP-8A5 and an anti-dibutyl phthalate monoclonal antibody produced by the hybridoma cell line. Background Art

[0002] Plasticizers, also known as plasticizers, are widely used in industrial production to increase the plasticity of resin molecules. Dibutyl phthalate (DBP) is a commonly used plasticizer. It is a colorless, oily, viscous liquid that is easily soluble in oils and fats, difficult to dissolve in water, and difficult to volatilize at room temperature. DBP is added to plastic products, and the combination is not stable. Over time, it will gradually separate from the mother body and enter the surrounding environment. DBP has endocrine disrupting properties and is classified as reproductive toxicity Class 1B. Even at extremely low concentration levels, it can interfere with the endocrine system of humans and animals and affect normal hormone levels in the body. In addition to strong reproductive developmental toxicity, long-term and large-scale intake of DBP may increase the risk of liver cancer and gastric cancer. People are exposed to plastic products almost every day, and DBP pollution is inevitable.

[0003] The existing DBP detection methods are mainly based on laboratory confirmatory detection technology using large instruments. Immunological detection methods are based on antigen-antibody specific recognition, and use enzyme, fluorescence and other labeling technologies to convert immune signals into detectable signals, thereby qualitatively or quantitatively displaying the content of the target. This type of method has the advantages of simple sample processing, high sensitivity, strong specificity, and short experimental cycle. The sensitivity and specificity of the antibody directly determine the key to the accuracy of the immunoassay. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a hybridoma cell line DBP-8A5, an anti-dibutyl phthalate (DBP) monoclonal antibody produced by the hybridoma cell line, and applications thereof. The anti-dibutyl phthalate monoclonal antibody provided by the present invention has high sensitivity and good specificity.

[0005] The first aspect of the present invention provides a hybridoma cell line DBP-8A5, which has been deposited in the China Center for Type Culture Collection (CCTCC) on March 16, 2023, and the deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: C202360. It has the gene sequence of the anti-dibutyl phthalate monoclonal antibody heavy chain variable region shown in SEQ ID NO: 1 in the coding sequence table and the gene sequence of the anti-dibutyl phthalate monoclonal antibody light chain variable region shown in SEQ ID NO: 2 in the coding sequence table.

[0006] The second aspect of the present invention provides an anti-dibutyl phthalate monoclonal antibody, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:1 in the sequence list; and the light chain variable region has the amino acid sequence shown in SEQ ID NO:2 in the sequence list. It is secreted and produced by the hybridoma cell line DBP-8A5 with a deposit number of CCTCC NO:C202360. The anti-dibutyl phthalate monoclonal antibody provided by the present invention has high sensitivity and high specificity.

[0007] The third aspect of the present invention provides the use of anti-dibutyl phthalate monoclonal antibodies in the determination of dibutyl phthalate content.

[0008] A fourth aspect of the present invention provides a product for detecting dibutyl phthalate comprising the above-mentioned anti-dibutyl phthalate monoclonal antibody.

[0009] According to the above scheme, the products are enzyme-linked immunosorbent assay kits, colloidal gold test strip kits, fluorescent test strip kits, immunoaffinity columns and other products.

[0010] The hybridoma cell line DBP-8A5 of the present invention is obtained by adopting a two-step screening method, and the specific steps are: BALB / c mice are immunized with dibutyl phthalate complete antigen DBP-BSA for 3-4 times, and then the complete antigen DBP-BSA with a dose twice that of the previous immunization is used for booster immunization, cell fusion is performed after 3 days, and after culturing with HAT semi-solid culture medium for 1-2 weeks, a single cell colony is transferred to a 96-well cell culture plate for culture, and when the cells grow to 1 / 10 of the well, an indirect competitive ELISA method is used for screening, a dibutyl phthalate standard is used as a competitor, wells with higher absorbance values ​​and inhibition rates are selected, and subcloning is performed 3-4 times by a limiting dilution method, and finally the hybridoma cell line DBP-8A5 is screened.

[0011] The present invention further provides a method for preparing an anti-dibutyl phthalate monoclonal antibody, comprising the following steps: injecting the hybridoma cell line DBP-8A5 obtained above into the abdomen of a BALB / c mouse that has been treated with Freund's incomplete adjuvant in advance, collecting ascites into a centrifuge tube after it grows out, and purifying the ascites by an octanoic acid ammonium sulfate method to obtain the anti-dibutyl phthalate monoclonal antibody.

[0012] According to the above scheme, the purification method described is the octanoic acid-ammonium sulfate method, and the specific steps are: filter the mouse ascites with double-layer filter paper, centrifuge at 4°C, 10000r / min for more than 10min, mix the obtained supernatant with 2-4 times the volume of acetate buffer, add octanoic acid dropwise under stirring, the volume of octanoic acid required for each milliliter of ascites is 30-35μL, mix at room temperature for 30-60min, and let stand at 4°C for more than 2h. 12000r / min, centrifuge at 4℃ for more than 30min, discard the precipitate, filter the supernatant with double filter paper, add 1 / 10 volume of filtrate (0.1mol / L and pH 7.4) of phosphate buffer, adjust the pH of the mixture to 7.4 with sodium hydroxide (2mol / L) solution, slowly add under ice bath condition, the final solution ammonium sulfate concentration is 0.277g / mL, stand at 4℃ for more than 2h, centrifuge at 12000r / min, 4℃ for 30min, discard the supernatant, resuspend the obtained precipitate with 1 / 10 volume of original ascites phosphate buffer (0.01mol / L, pH 7.4), place in dialysis bag and dialyze with 0.01mol / LPBS for three days, take out the antibody solution in the dialysis bag, centrifuge, collect the supernatant, discard the precipitate, pre-freeze at -70℃ and freeze dry in a freeze dryer. Collect the freeze-dried powder, which is the purified anti-dibutyl phthalate monoclonal antibody.

[0013] The acetate buffer is obtained by adding water to 0.29g sodium acetate and 0.141mL acetic acid to make the volume to 100mL; the 0.01mol / L phosphate buffer is obtained by adding water to 0.29g disodium hydrogen phosphate dodecahydrate, 0.8g sodium chloride, 0.02g potassium dihydrogen phosphate, and 0.02g potassium chloride to make the volume to 100mL; the 0.1mol / L phosphate buffer is obtained by adding water to 2.9g disodium hydrogen phosphate dodecahydrate, 8g sodium chloride, 0.2g potassium dihydrogen phosphate, and 0.2g potassium chloride to make the volume to 100mL.

[0014] Beneficial effects of the present invention:

[0015] (1) The hybridoma cell line DBP-8A5 provided by the present invention can be used to prepare anti-dibutyl phthalate monoclonal antibodies, and the antibody titer measured by enzyme-linked immunosorbent assay (ELISA) can reach 4×10 4 .

[0016] (2) The anti-dibutyl phthalate monoclonal antibody provided by the present invention has high sensitivity and good specificity, and the sensitivity (50% inhibition concentration of dibutyl phthalate (DBP), IC 50) was 2.06 ng / mL; there was no obvious cross-reaction with common phthalate plasticizers: the cross-reaction rates with BBP and DiBP were 11.3% and 5.32%, respectively, and the cross-reaction rates with DEHP, DiNP, and DMP were less than 0.01%; the antibody affinity measured by enzyme-linked immunosorbent assay (ELISA) was 1.72×10 7 L / mol.

[0017] (3) The anti-dibutyl phthalate monoclonal antibody provided by the present invention can be applied to the determination of dibutyl phthalate content. DETAILED DESCRIPTION

[0018] Example 1: Screening of hybridoma cell line DBP-8A5

[0019] 1. Animal immunization

[0020] BALB / c mice aged 6-7 weeks were immunized with complete dibutyl phthalate antigen DBP-BSA prepared in the laboratory. For the first immunization, the complete dibutyl phthalate antigen was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple points on the back of the mouse's neck. The second immunization was carried out 4 weeks later, and the complete dibutyl phthalate antigen was emulsified with an equal volume of Freund's incomplete adjuvant and injected intraperitoneally into the mouse. The third immunization was 4 weeks apart from the second immunization, and the immunization method was the same. The fourth immunization was carried out 3 weeks after the third immunization, and the immunization method was the same as the second immunization, also intraperitoneal injection. The 4 immunization doses were the same, all 200 μg per mouse. 8 to 10 days after each of the first 3 immunizations, blood was collected from the tail vein, serum was separated, and the serum titer of the mice was detected by indirect ELISA. 8 days after the third immunization, the tail was cut off and blood was collected, and mice with relatively high titer and sensitivity were selected for the final booster immunization, and the immunization dose was twice that of the previous one.

[0021] 2. Cell Fusion

[0022] Three days after booster immunization, 50% by weight of PEG with a molecular weight of 1500 was used as a fusion agent, and cell fusion was performed according to conventional methods. The specific steps were aseptically dislocated the mouse, the spleen was removed, the spleen was crushed with a homogenizer, the spleen cells were separated with a filter, and the cells were mixed with mouse myeloma cells SP2 / 0 at a ratio of 5:1, centrifuged, and the mixed cells were resuspended with RPMI-1640 basic culture medium, centrifuged, and the supernatant was discarded. 1-2mL of 50% PEG was added, and the total time was 1 minute. 10-20mL of RPMI-1640 basic culture medium was added to the adherent cells, centrifuged, and the supernatant was discarded. The fused cells at the bottom of the tube were resuspended with 20mL of complete cell culture medium containing 1% HAT, and the suspended cells were added to 80mL of semi-solid culture medium, mixed and added to a 6-well cell culture plate, 1.5mL / well, and placed in a 37°C carbon dioxide incubator for culture. The complete cell culture medium containing 1% HAT contains 20% (volume percentage) fetal bovine serum, 75% (volume percentage) RPMI-1640 basic culture medium, 1% (weight percentage) L-glutamine, 1% (volume percentage) HEPES, 1% (volume percentage) double antibody (10,000 units per milliliter of penicillin and 10,000 micrograms per milliliter of streptomycin), 2% (volume percentage) growth factor (HFCS) and 1% (weight percentage) hypoxanthine-aminopterin-thymidine nucleoside, i.e., HAT and methylcellulose, which are purchased from Sigma-Aldrich.

[0023] Cell line screening and cloning

[0024] 2-3 weeks after cell fusion, when the cell colonies grow to be visible to the naked eye, use a micropipette to pick out the clones from the culture medium, transfer them to a 96-well cell culture plate and culture them in HAT liquid. When the cells grow to 2 / 3 of the bottom of the well, aspirate the culture supernatant for detection. A two-step screening method is used. The first step is to use the indirect ELISA method to screen out the positive wells that are resistant to dibutyl phthalate but not to the carrier protein BSA; the second step is to use the indirect competitive ELISA method to detect the positive wells screened in the first step, using dibutyl phthalate as the competitor, and selecting the wells with higher absorbance and sensitivity (higher absorbance means that the wells with a competitor of 0, i.e., the positive control wells, have a higher final measured value, and higher sensitivity means that the competitor concentration when the inhibition rate is 50% is also IC 50 The value is small), and the subcloning is performed by limiting dilution method. After subcloning, the same two-step method is used for detection. After repeating the subcloning 3-4 times, the hybridoma cell line DBP-8A5 is obtained. The hybridoma cell line has been deposited in China Center for Type Culture Collection (CCTCC) on March 16, 2023, and the deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: C202360.

[0025] Example 2: Determination of the variable region sequence of the anti-dibutyl phthalate monoclonal antibody hybridoma cell line DBP-8A5

[0026] (1) Extracting total RNA: Using the total RNA extraction kit of Tiangen Company and following the instructions, extract the total RNA of the hybridoma cell line DBP-8A5;

[0027] (2) cDNA synthesis: Using the total RNA obtained in step 1 as a template and oligo(dT)15 as a primer, the cDNA was synthesized according to SuperScript TM -2II reverse transcriptase instructions were used for reverse transcription to synthesize the first strand of cDNA; primer oligo(dT)15 was purchased from Invitrogen;

[0028] (3) PCR method for cloning variable region genes: Primers were designed based on the conserved sites of mouse antibody gene sequences in GENBANK, and CDNA was used as a template to amplify antibody heavy chain and light chain variable region genes. The PCR program was: 94°C for 30s, 58°C for 45s, 72°C for 1min, 30 cycles of amplification, and finally 72°C for 10min. After the PCR products were separated by 1% (weight percent) agarose gel electrophoresis, the DNA fragments were purified and recovered using a kit, connected to the vector pMD18-T, transformed into E. coli DH5α competent cells, and the positive clones were picked and sent to Shanghai Sunny Biotechnology Co., Ltd. for sequencing. The sequences of the primers are: the heavy chain variable region primers are 5'-CAG GTS MAR CTG MAG GAG TCW G-3' (22mer) and 5'-CAG GGG CCA GTGGAT AGA CAG ATG GGG G-3' (28mer), where S, M, R and W are merged bases, M = A / C, R = A / G, S = G / C, W = A / T; the light chain variable region primers are 5'-GAC ATC AAG ATG ACC CAG TCT CCA-3' (24mer) and 5'-CCGTTT TAT TTC CAG CTT GGT CCC-3' (24mer).

[0029] The obtained gene sequence results: the heavy chain variable region consists of 120 amino acids, the sequence is shown in SEQ ID NO: 1. The light chain variable region consists of 107 amino acids, the sequence is shown in SEQ ID NO: 2.

[0030] Example 3: Preparation, purification, subtype and characterization of anti-dibutyl phthalate monoclonal antibodies

[0031] The anti-dibutyl phthalate monoclonal antibody hybridoma cell line DBP-8A5 obtained in Example 1 was injected into BALB / c mice treated with Freund's incomplete adjuvant in advance, and the ascites of the mice was collected. The antibody was purified by the octanoic acid-ammonium sulfate method. The specific operation was as follows: the mouse ascites was filtered with a double-layer filter paper, centrifuged at 4°C and 12000r / min for more than 15min, the supernatant was aspirated, and the obtained ascites supernatant was mixed with 4 times the volume of acetate buffer, and octanoic acid was slowly added under stirring, the volume of octanoic acid required per milliliter of ascites was 30-35μL, mixed at room temperature for 30-60min, and allowed to stand at 4°C for more than 2h. 12000r / min, centrifuge at 4℃ for more than 30min, discard the precipitate, filter the obtained supernatant with double-layer filter paper, add 1 / 10 volume of the filtrate with a molar concentration of 0.1mol / L and a pH of 7.4 phosphate buffer, adjust the pH of the mixture to 7.4 with 2mol / L sodium hydroxide solution, slowly add ammonium sulfate in an ice bath to a final concentration of 0.277g / mL ammonium sulfate, let stand at 4℃ for more than 2h, then centrifuge at 12000r / min, 4℃ for more than 30min, discard the supernatant, resuspend the obtained precipitate with 1 / 10 volume of the original ascites water volume with a molar concentration of 0.01mol / L and a pH of 7.4 phosphate buffer, put it into a dialysis bag, dialyze it with 0.01mol / L PBS for two days, and then dialyze it with PB for two days, take out the protein solution in the dialysis bag, centrifuge, collect the supernatant, discard the precipitate, pre-freeze it at -70℃, and then put it into a freeze dryer for freeze drying. Collecting the lyophilized powder, which is the purified anti-dibutyl phthalate monoclonal antibody;

[0032] The acetate buffer is obtained by adding water to 0.29g sodium acetate and 0.141mL acetic acid to make the volume to 100mL; the 0.01mol / L phosphate buffer is obtained by adding water to 0.8g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, and 0.02g potassium dihydrogen phosphate to make the volume to 100mL; the 0.1mol / L phosphate buffer is obtained by adding water to 8g sodium chloride, 2.9g disodium hydrogen phosphate dodecahydrate, 0.2g potassium chloride, and 0.2g potassium dihydrogen phosphate to make the volume to 100mL.

[0033] The subtype of the anti-dibutyl phthalate monoclonal antibody secreted by the hybridoma cell line DBP-8A5 was identified as IgG2a using a commercially available subtype identification kit.

[0034] The antibody titer purified from mouse ascites was measured by conventional non-competitive enzyme-linked immunosorbent assay (ELISA) to be 4×10 4 , i.e. antibody dilution 4×10 3 The result of the solution test was positive when the concentration was 2.06 ng / mL. The sensitivity of the dibutyl phthalate was 2.06 ng / mL by conventional indirect competitive ELISA.

[0035] The specificity of the antibody can be evaluated by the cross-reaction rate. The DBP-8A5 monoclonal antibody was determined by the indirect competitive ELISA method. A series of standard solutions of DBP and other phthalates DEHP, DiBP, DiNP, BBP, and DMP were prepared and added to the ELISA plate with equal volumes of antibodies. The plates were incubated for 1 hour. The other steps were the same as the indirect competitive ELISA method. The competition inhibition curve was drawn with the concentration of the above-mentioned phthalate standards as the horizontal axis and the OD value B / B0 at 450nm measured by the microplate reader as the vertical axis. The IC 50 The cross-reaction rate is determined by the ratio of the values. The calculation formula is as follows:

[0036] CR%=(IC 50DBP / IC 50其他邻苯二甲酸酯 )×100.

[0037] Results: The cross-reaction rates of BBP and DiBP were 11.3% and 5.32% respectively, and the cross-reaction rate with other common phthalate plasticizers was less than 0.01, with good specificity.

[0038] The affinity of DBP-8A5 was determined by indirect non-competitive ELISA. DBP-OVA was coated on the ELISA plate at a concentration of 1.0, 0.5, 0.25, and 0.125 μg / mL, 100 μL / well, 37°C, 2 h; after blocking with blocking solution for 1 h, the antibody diluted with PBS (dilution factor 1:2) was added to the ELISA plate, and the remaining steps were the same as the indirect non-competitive ELISA method. The OD 450 The value is the ordinate, and the logarithm of the antibody concentration (mol / L) is the abscissa, and 4 S-shaped curves of 4 concentrations are made. Find the maximum OD value at the top of each S curve, that is, ODmax, and find the antibody concentration corresponding to the 50% ODmax value of each curve. The 4 concentrations are randomly grouped in pairs, and the affinity constant of the antibody is calculated according to the formula Ka=(n-1) / 2(n[Ab']t-[Ab]t), where [Ab']t and [Ab]t are the antibody concentrations corresponding to the two 50% maximum OD values ​​in each group, and n is the multiple of the coated antigen concentration in each group (including 1:2, 1:4, and 1:8 ratios), and a total of 6 Ka values ​​are obtained. The average of the six Ka values ​​obtained shows that the affinity of the anti-dibutyl phthalate mouse ascites antibody enzyme-linked immunosorbent assay (ELISA) method can reach 1.72×10 7 L / moL.

[0039] Example 4: Establishment of the above-mentioned dibutyl phthalate ic-ELISA detection method:

[0040] Treatment of edible oil samples: The edible oil sample treatment method refers to GB5009.271-2016, weigh 1g of soybean oil (accurate to 0.0001g) in a 10mL ground-mouth centrifuge tube with a stopper, add 4mL of acetonitrile and 200uL of n-hexane, vortex for 1min, then ultrasonically extract for 20min, centrifuge at 4000r / min for 5min, collect the supernatant, add 2mL of acetonitrile to the residue, repeat the extraction step twice, combine the three supernatants, blow dry with nitrogen, and reconstitute with 40% methanol-PBS.

[0041] The specific operation steps are as follows: Coat the complete dibutyl phthalate antigen on a 96-well ELISA plate at 4°C overnight. Wash the plate three times with PBST, add 4% skim milk powder and block at 37°C for one hour. Add 50ul of dibutyl phthalate monoclonal antibody of appropriate working concentration and gradient diluted dibutyl phthalate standard solution, and incubate at 37°C for one hour. Wash the plate three times with PBST, add HRP-labeled secondary antibody, wash the plate three times with PBST, stop color development, and read the OD value with an ELISA reader. The antigen dosage is 1μg / mL, the antibody concentration is 0.2ng, the salt ion concentration is 10mM, the pH is 7.4, the Tween concentration in the antibody diluent is 0.025%, and the sensitivity of the anti-dibutyl phthalate monoclonal antibody of the present invention detected by ic-ELISA (50% inhibition concentration for dibutyl phthalate (DBP), IC 50 ) is 2.06 ng / mL. The content of dibutyl phthalate in the sample solution to be tested is obtained based on the relationship curve between the obtained OD value and the concentration of dibutyl phthalate.

[0042] In order to evaluate the accuracy of the established dibutyl phthalate ic-ELISA (indirect competitive ELISA) method, a matrix standard curve was established, and a spike recovery experiment was performed on blank edible oil samples. The edible oil sample processing method refers to GB5009.271-2016, weigh 1g of soybean oil (accurate to 0.0001g) in a 10mL stoppered ground-mouth centrifuge tube, add 4mL acetonitrile and 200uL n-hexane, vortex for 1min, then ultrasonically extract for 20min, centrifuge at 4000r / min for 5min, collect the supernatant, add 2mL acetonitrile to the residue, repeat the extraction step twice, combine the three supernatants, blow dry with nitrogen, and reconstitute with 40% methanol-PBS. The DBP standard was diluted from 2500ng / mL downward in a gradient manner, and a series of concentrations of DBP standard were added to the matrix solution. In addition, three concentrations of dibutyl phthalate standard 40 ng / mL, 200 ng / mL, and 600 ng / mL were added to the sample diluent, and the ic-ELISA method established above was used to detect the content of the spiked samples. The calculated spiked recoveries of the samples were 78.16%, 86.81%, and 93.32%, respectively.

[0043] The established ic-ELISA method was used to detect the five edible oil samples collected and compared with gas chromatography-tandem mass spectrometry for verification. The test results showed that the test results of ic-ELISA and GC-MS were highly correlated. 2 =0.9878.

[0044] The above method is also applicable to immunoassay methods (including ELISA, test strips, gas pressure method, electrochemical method, etc.) established by replacing other markers (colloidal gold, time-resolved fluorescence, up-conversion fluorescent materials, biomimetic catalytic enzymes, etc.) of horseradish peroxidase.

[0045] Example 5: DBP immunoaffinity column preparation

[0046] 1. Matrix Preparation

[0047] Weigh the required 1g CNBr-activated Sepharose lyophilized matrix powder (each gram of lyophilized matrix powder can form a final volume of 3.5mL swelling matrix) and dissolve it in 1mmol / L HCl. The matrix will swell immediately and then be placed in a sintered glass filter and washed with 1mmol / L HCl for 15min.

[0048] 2. Ligand (antibody) coupling

[0049] a Use coupling buffer 0.2 mol / L NaHCO 3 Dissolve the DBP antibody to be coupled at pH 8.3 to a concentration of 15 mg / mL. Place the dissolved antibody in an ice bath for temporary storage. Add the coupling buffer containing the antibody to a fully sealable container with a lid. Quickly transfer the CNBr-activated Sepharose to the antibody solution. Mix thoroughly for 2-4 hours at room temperature (20-25°C).

[0050] b Calculation of coupling rate: Centrifuge at 2,000 rpm to centrifuge the sepharose to the bottom of the tube, transfer the supernatant to a new centrifuge tube, and measure the protein content of the supernatant. The coupling rate is calculated to be 98.5%. Take the sepharose that has been centrifuged to the bottom of the tube and wash it with coupling buffer to remove excess ligand.

[0051] c Blocking: Transfer the matrix to 0.1 mol / L Tris-HCl buffer and let stand at room temperature for 2-4 hours to block all residual active groups.

[0052] d. To remove the excess ligands that were not coupled after coupling, wash the matrix for 3-5 cycles with buffers of pH 4 and 8, i.e., 0.1mol / L acetic acid / sodium acetate buffer and 0.1mol / L Tris-HCl buffer, respectively. The amount of each buffer used is 3-5 times the volume of the matrix. Each washing cycle step: first wash with 0.1mol / L acetic acid / sodium acetate buffer, then wash with 0.1mol / L Tris-HCl buffer. e. Wash with 3-5 times the volume of the gel in 0.01% NaN3-PBS, and store with 0.01% NaN3-PBS.

[0053] 3. Column filling Use binding buffer to prepare slurry, mix in a ratio of 75% sedimentation matrix and 25% phosphate buffer (pH 7.0). Pour the slurry into the column in a continuous operation. Using a glass rod leaning against the inner wall of the column to fill the column will help reduce the generation of bubbles. After filling the column, close the opening at the lower end of the affinity column and remove the top part of the affinity column. Carefully add pH 7.0 PBS buffer to the remaining part of the affinity column to form an upward curved liquid surface at the top of the affinity column. Insert the top sieve plate into the affinity column at a certain angle to ensure that there is no air under the sieve plate. Lock the sieve plate in the appropriate position on the surface of the matrix, open the opening below the affinity column, pass the column with 3-5 times the column bed volume of sterile filtered 0.01% NaN3-PBS, and store it with 0.01% NaN3-PBS. At this point, the affinity column has been filled and balanced and can be used directly.

[0054] Table 1. Cross-reactivity of DBP-8A5 with other phthalates.

[0055]

Claims

1. A hybridoma cell line DBP-8A5, characterized in that: The hybridoma cell line was deposited in the China Center for Type Culture Collection (CCTCC) on March 16, 2023, and the deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: C202360.

2. An anti-dibutyl phthalate monoclonal antibody, characterized in that: The heavy chain variable region of the anti-dibutyl phthalate monoclonal antibody has the amino acid sequence shown in SEQ ID NO: 1 in the sequence list; the light chain variable region has the amino acid sequence shown in SEQ ID NO: 2 in the sequence list.

3. Use of the anti-dibutyl phthalate monoclonal antibody according to claim 2 in the determination of dibutyl phthalate content.

4. A product for detecting dibutyl phthalate comprising the anti-dibutyl phthalate monoclonal antibody according to claim 2.

5. The product for detecting dibutyl phthalate according to claim 4, characterized in that: The product is an enzyme-linked immunosorbent assay kit, a colloidal gold test strip kit, a fluorescent test strip kit or an immunoaffinity column.

6. The method for preparing the anti-dibutyl phthalate monoclonal antibody according to claim 2, characterized in that: The hybridoma cell line DBP-8A5 according to claim 1 is injected into BALB / c mice treated with Freund's incomplete adjuvant in advance, and the ascites of the mice is collected and purified to obtain anti-dibutyl phthalate monoclonal antibodies.

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