A hybridoma cell line and anti-4-chlorophenoxyacetic acid monoclonal antibody produced by the same and application of the antibody

Abstract

The present invention provides a hybridoma cell line and an anti-4-chlorophenoxyacetic acid monoclonal antibody and an application of the antibody produced by the hybridoma cell line, belonging to the technical field of immunology and pesticide residue analysis. The hybridoma cell line 4-CPA-3C8 provided by the present invention is deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, with a deposit date of July 27, 2022, and a deposit number of CCTCCNO: C2022235; the anti-4-chlorophenoxyacetic acid monoclonal antibody is produced by the hybridoma cell line 4-CPA-3C8. The anti-4-chlorophenoxyacetic acid monoclonal antibody provided by the present invention can be used to prepare an enzyme-linked immunosorbent assay kit, a colloidal gold chromatography test strip and an evanescent wave fluorescent immunosensor for detecting 4-chlorophenoxyacetic acid, so as to achieve the purpose of quickly and sensitively detecting 4-chlorophenoxyacetic acid drugs in food and the environment.

Description

Technical Field

[0001] The invention relates to the technical field of immunology and plant growth regulator residue analysis, and in particular to a hybridoma cell line and an anti-4-chlorophenoxyacetic acid monoclonal antibody produced by the hybridoma cell line and the application of the antibody. Background Art

[0002] 4-chlorophenoxyacetic acid (4-CPA, commonly known as anti-drop agent) has a chemical formula of C8H7ClO3. It belongs to the category of auxin among the synthetic plant growth regulators. It has the functions of inhibiting the decomposition of chlorophyll, nucleic acid, and protein in plants, keeping green and preventing aging; it also has multiple functions such as transporting amino acids, auxins, inorganic salts, etc. to the treated parts. 4-Chlorophenoxyacetic acid has good efficacy and low price. It is widely used in the cultivation of rootless bean sprouts, but the substance will remain and accumulate in the human body. Long-term consumption will cause cumulative harm to the human body. For example, it causes children to develop early, changes in women's physiology, and has carcinogenic and teratogenic effects on the human body.

[0003] In view of such hazards, relevant departments at home and abroad are also constantly increasing the management and monitoring of such drug residues in food and the environment, and have invested a lot of technology, funds and manpower to develop and apply such drug residue analysis technology. At present, the methods used to detect 4-chlorophenoxyacetic acid mainly include spectrophotometry, high performance liquid chromatography and its combination technology. Although these methods can accurately quantify, they are expensive, take a long time to detect, and require professional personnel to operate, so they cannot achieve real on-site rapid detection. The analytical methods for rapid screening of 4-chlorophenoxyacetic acid are still immature. To explore the reasons, the acquisition of highly specific and highly sensitive excellent functional biomaterials is the main bottleneck restricting its development. Therefore, the preparation of highly specific and highly sensitive anti-4-chlorophenoxyacetic acid monoclonal antibodies and the acquisition of excellent biological functional materials have great theoretical and practical significance for solving the core problems behind the bottleneck of small molecule drug rapid detection technology and establishing 4-chlorophenoxyacetic acid on-site instant detection technology. Summary of the invention

[0004] The object of the present invention is to provide a hybridoma cell line and an anti-4-chlorophenoxyacetic acid monoclonal antibody produced by the hybridoma cell line and the application of the antibody. The quality of the anti-4-chlorophenoxyacetic acid monoclonal antibody provided has strong controllability and repeatability. Through preliminary analysis and identification of the obtained antibody characteristics, experimental basis and basic raw materials are provided for the further establishment and application of a specific and sensitive 4-chlorophenoxyacetic acid immunoassay method. It can be used to prepare an enzyme-linked immunosorbent assay kit, colloidal gold chromatography test strips and evanescent wave fluorescence immunosensor for detecting 4-chlorophenoxyacetic acid, so as to achieve the purpose of quickly and sensitively detecting 4-chlorophenoxyacetic acid drugs in food and the environment.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a hybridoma cell line 4-CPA-3C8, which is deposited in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, with a deposit date of July 27, 2022, and a deposit number of CCTCCNO: C2022235.

[0007] The present invention also provides an anti-4-chlorophenoxyacetic acid monoclonal antibody produced by the hybridoma cell line 4-CPA-3C8.

[0008] The present invention also provides an application of the anti-4-chlorophenoxyacetic acid monoclonal antibody in preparing a detection product for detecting 4-chlorophenoxyacetic acid.

[0009] Preferably, the detection products include enzyme-linked immunosorbent assay kit, colloidal gold chromatography test strips, and evanescent wave fluorescence immunosensor.

[0010] The present invention also provides an enzyme-linked immunosorbent assay kit for detecting 4-chlorophenoxyacetic acid. The enzyme-linked immunosorbent assay kit comprises the anti-4-chlorophenoxyacetic acid monoclonal antibody.

[0011] The present invention also provides a colloidal gold chromatography test strip for detecting 4-chlorophenoxyacetic acid, wherein the colloidal gold chromatography test strip comprises the anti-4-chlorophenoxyacetic acid monoclonal antibody.

[0012] The present invention also provides an evanescent wave fluorescence immunosensor for detecting 4-chlorophenoxyacetic acid. The evanescent wave fluorescence immunosensor comprises the anti-4-chlorophenoxyacetic acid monoclonal antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a gel electrophoresis identification diagram of the 4-chlorophenoxyacetic acid artificial antigen prepared in Example 1 of the present invention;

[0014] Figure 2 This is a UV scanning identification diagram of the 4-chlorophenoxyacetic acid immunogen prepared in Example 1 of the present invention;

[0015] Figure 3 This is a diagram for determining subtypes of the anti-4-chlorophenoxyacetic acid monoclonal antibody prepared in Example 2 of the present invention;

[0016] Figure 4 This is a graph showing the affinity determination of the anti-4-chlorophenoxyacetic acid monoclonal antibody prepared in Example 2 of the present invention;

[0017] Figure 5 This is a graph showing the inhibition rate of the anti-4-chlorophenoxyacetic acid monoclonal antibody prepared in Example 2 of the present invention.

[0018] Collection Instructions

[0019] The hybridoma cell line 4-CPA-3C8 was deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China. The deposit date is July 27, 2022, and the deposit number is CCTCC NO: C2022235. DETAILED DESCRIPTION

[0020] 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.

[0021] Example 1

[0022] Preparation of artificial antigen of 4-chlorophenoxyacetic acid

[0023] (1) Preparation of immunogen

[0024] Using the carbodiimide method, 6 mg of 4-CPA standard was weighed and dissolved in 1 ml of anhydrous dioxane, 200 μL of 0.1 mol / L NaOH was added dropwise, and the mixture was shaken to fully dissolve. 6 mg of carrier protein BSA was weighed and dissolved in 1 ml of PBS, and 8 mg of EDC was dissolved in 1 ml of distilled water. The mixture of 4-CPA and carrier protein was shaken at room temperature for 18 hours, centrifuged at 4000 r / min for 10 minutes, and the supernatant was added dropwise to the shaken EDC solution. After addition, the mixture was shaken for 5 hours and centrifuged at 4000 r / min for 10 minutes. The reaction solution was placed in a dialysis bag and dialyzed with PBS for 3 days. The solution was changed 3 times a day and collected and stored at -20°C for use.

[0025] (2) Preparation of coating

[0026] The preparation process of the coating source is the same as that of the immunogen, except that bovine serum albumin is replaced with ovalbumin to obtain the coating source 4-CPA-OVA.

[0027] Identification of the prepared 4-chlorophenoxyacetic acid artificial antigen:

[0028] SDS-PAGE identification

[0029] SDS-PAGE was used to identify the immunogen 4-CPA-BSA and the coating 4-CPA-OVA. According to the conventional SDS-PAGE method, 12% separation gel and 5% concentration gel were prepared, and the voltage was 30V until the protein band entered the separation gel, and then the voltage was adjusted to 90V and continued electrophoresis for about 2 hours. After staining and decolorization, the results are shown in Figure 1 The coupling product 4-CPA-BSA was significantly different from BSA, with a wide bandwidth and tailing, indicating that the coupling of 4-CPA-BSA and 4-CPA-OVA was successful.

[0030] UV spectrophotometry

[0031] 4-Chlorophenoxyacetic acid and BSA were diluted to 1 mg / ml with PBS, the protein concentration of BSA was determined, 4-CPA-BSA was diluted to the same concentration as BSA, and UV scanning was performed at a wavelength of 280 nm. The results are shown in Figure 2 The maximum absorption peaks of BSA and 4-CPA are at 281 and 278. After coupling, the peaks of BSA and 4-CPA are superimposed at 268.5, indicating that the coupling is successful. According to the Lambert-Beer law, the coupling ratio of 4-CPA-BSA is calculated to be about 5.32:1.

[0032] Example 2

[0033] Preparation of monoclonal antibody against 4-chlorophenoxyacetic acid

[0034] (1) Animal immunization

[0035] The immunogen 4-CPA-BSA prepared in Example 1 was used to immunize female Balb / c mice aged 6 to 8 weeks, and the immunization was performed once every 2 weeks. The immunization process is shown in Table 1. After three immunizations, the tail was cut off and blood was collected to determine the titer and inhibition rate. The mice with the best results were selected for booster immunization, and the mice with the best titer results were selected as fusion mice.

[0036] Table 1 Immunization process chart

[0037] Immunization time Immunization times Immune site Immunization dose Remark 0 First immunization Tail vein injection 10μg / each 2 weeks Second immunization Tail vein injection 10μg / each 4 weeks The third immunization Tail vein injection 10μg / each Determination of titer and inhibition rate 6 weeks Strengthen immunity Tail vein injection 10μg / each

[0038] (2) Cell fusion

[0039] The fusion mice selected in step (1) were eyeballed and bled, and the spleens were removed under sterile conditions after being killed by cervical dislocation. Splenocytes were prepared and fused with SP2 / 0 cells at a ratio of 5:1 by PEG. The fused cell suspension was added to a 96-well plate already plated with feeder cells and cultured in an incubator at 37°C and 5% CO2.

[0040] (3) Screening of positive hybridoma cell lines

[0041] After fusion, the cells were checked for contamination the next day, and HT medium was used on the 10th day after fusion. Three days after the medium change, positive wells were screened by indirect ELISA and indirect competitive ELISA, and wells with strong positive, high inhibition rate, single clone, and good cell state were selected. Subcloning was performed by limiting dilution method, and expansion culture was performed at the same time until a monoclonal cell line secreting anti-4-chlorophenoxyacetic acid antibodies was established. It was preserved in the China Center for Type Culture Collection and named hybridoma cell line 4-CPA-3C8, with the preservation number CCTCC NO: C2022235.

[0042] (4) Preparation and purification of anti-4-chlorophenoxyacetic acid monoclonal antibodies

[0043] Using the method of inducing ascites in mice, healthy Balb / c female mice were selected and 0.5 mL of paraffin oil was injected into each mouse. After 7 days, 10 cloned positive hybridoma cells were adjusted. 6 / mL, 1 mL was injected intraperitoneally into each mouse, and the ascites was collected 8 days later, the fat was discarded by centrifugation, purified by caprylic acid-ammonium sulfate, freeze-dried and stored at -20°C to obtain the purified anti-4-chlorophenoxyacetic acid monoclonal antibody.

[0044] Characterization of the prepared anti-4-chlorophenoxyacetic acid monoclonal antibody

[0045] 1. Potency determination

[0046] The titer was determined by conventional indirect ELISA. The coated original 4-CPA-OVA was diluted to 5 μg / mL with carbonate buffer (CBS), and 100 μL / well was added to the wells of the ELISA plate and placed at 4°C overnight. The ELISA plate was washed 3 times with a Thermo plate washer and dried. A 10% calf serum solution was prepared with the washing solution, and 200 μL / well was added to the wells of the ELISA plate and incubated at 37°C for 1 hour. Wash the plate. Dilute the purified anti-4-chlorophenoxyacetic acid monoclonal antibody with buffer to 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000, 1:1024000, 1:2048000, with buffer as negative control, 100 μL / well, incubate at 37℃ in dark for 45 min. Wash the plate. Dilute the horseradish peroxidase-labeled goat anti-mouse secondary antibody with buffer containing 10% calf serum at 1:10000, 100 μL / well, incubate at 37℃ in dark for 30 min. Wash the plate. Add substrate colorimetric solution, 100 μL / well, incubate at 37℃ in dark for 15 min. 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 result. Results The titer of the anti-4-chlorophenoxyacetic acid monoclonal antibody 4-CPA-3C8 reached 1:2048000 at a concentration of 1 mg / mL.

[0047] (2) Subtype determination

[0048] The subtype determination was performed using a mouse monoclonal antibody subtype identification kit purchased from Sigma. The anti-4-chlorophenoxyacetic acid monoclonal antibody secreted by hybridoma cells had obvious differences in color development with secondary antibodies of different subtypes, among which the A450nm value with the IgG1 secondary antibody was the highest, and the A450nm value with the IgG 2a The secondary antibody showed weak color, while the IgG 2b, IgG3, IgA and IgM secondary antibodies showed almost no color. Therefore, the antibody type secreted by the hybridoma cells was mainly IgG1. Figure 3 Therefore, the subtype of the anti-4-chlorophenoxyacetic acid monoclonal antibody of the present invention is IgG1.

[0049] (3) Affinity determination

[0050] The affinity constant (Ka) was determined by non-competitive ELISA. The specific steps are as follows: Coating: Dilute the original 4-CPA-OVA with carbonate buffer to a concentration of 1, 0.5, 0.25, and 0.125 μg / mL, and coat 100 μL / well of a 96-well ELISA plate, overnight at 4°C; pour off the coating solution, wash 3 times, and pat dry. Blocking: Add 200 μL of a washing solution containing 10% calf serum to each well, incubate at 37°C for 1h; pour off the blocking solution, wash 3 times, and pat dry. Add monoclonal antibody (i.e., the anti-4-chlorophenoxyacetic acid monoclonal antibody prepared in this example): Use the washing solution to dilute the monoclonal antibody in multiples starting at 100 μg / mL, add 100 μL to each well, and keep warm and moist at 37°C for 45 minutes; pour off the primary antibody, wash 3 times, and pat dry. Add enzyme-labeled secondary antibody: Add 100μL of 1:10000-fold diluted HRP enzyme-labeled goat anti-mouse IgG to each well and place at 37℃ for 30min; remove enzyme-labeled secondary antibody, wash 3 times, and pat dry. Color development: Add 100μL of substrate color development solution to each well and react at 37℃ in the dark for 15min. Stop: Add 50μL of stop solution to each well to stop the reaction. Detection: Measure the absorbance value at a wavelength of 450nm (A450nm). Calculate Ka according to the following formula,

[0051] Ka = (n-1) / 2 (nAb'-Ab);

[0052] 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 constant of 4-chlorophenoxyacetic acid monoclonal antibody was determined by non-competitive enzyme-linked immunosorbent assay. The results are as follows: Figure 4 As shown, the affinity constant Ka is calculated to be 3.5×10 8 L / mol.

[0053] (4) Inhibition rate determination

[0054] The inhibition rate was determined by indirect competitive ELISA. The specific steps are as follows: Coating: dilute the original 4-CPA-OVA with carbonate buffer to a concentration of 5 μg / mL, 100 μL / well of a 96-well ELISA plate, overnight at 4°C; pour off the coating solution, wash 3 times, and pat dry; Blocking: add 200 μL of washing solution containing 10% calf serum to each well, incubate at 37°C for 1 hour; pour off the blocking solution, wash 3 times, and pat dry; Add monoclonal antibody and 4-chlorophenoxyacetic acid standard: dilute the 4-chlorophenoxyacetic acid standard stock solution with PBS to 2.0, 1.0, 0.50, 0.25, 0.125, 0 μg / L, add 50 μL to each well, and then add a certain dilution multiple (1 :64000 times) monoclonal antibody, 50μL per well, set up blank control well (add 100μL PBS) and negative control well (add 50μL monoclonal antibody + 50μL PBS), keep warm and moisturize at 37℃ for 45min; pour off the primary antibody, wash 3 times, and pat dry; add enzyme-labeled secondary antibody: add 100μL of 1:10000 diluted HRP enzyme-labeled goat anti-mouse IgG to each well, place at 37℃ for 30min; pour off the enzyme-labeled secondary antibody, wash 3 times, and pat dry; color development: add 100μL substrate color development solution to each well, react at 37℃ in the dark for 15min; stop: add 50μL stop solution to each well to stop the reaction;

[0055] Detection: Measure the absorbance value (A450) at a wavelength of 450 nm.

[0056] The inhibition curve of the monoclonal antibody was plotted with the absorbance percentage B / B0% (B represents the A450 of the 4-chlorophenoxyacetic acid standard solution of different concentrations, and B0 represents the A450 of the 4-chlorophenoxyacetic acid standard solution of zero concentration) as the ordinate and the logarithm of the 4-chlorophenoxyacetic acid standard solution of different concentrations [Lg(4-CPA)] as the abscissa. The curve is shown in FIG. Figure 5 The half inhibition rate IC of the monoclonal antibody to 4-chlorophenoxyacetic acid is 50 It is 0.54μg / L.

[0057] (5) Specificity determination

[0058] The cross-reactivity of the anti-4-chlorophenoxyacetic acid monoclonal antibody with structural analogs such as 6-BA and indoleacetic acid was detected by competitive ELISA, and the specificity of the anti-4-chlorophenoxyacetic acid monoclonal antibody was determined according to the cross reaction rate (CR). CR = (IC50 of 4-CPA / IC50 of each structural analog) × 100%, and the results are shown in Table 2. The CR (%) of the antibody with 4-chlorophenoxyacetic acid is 100%, and the CR (%) with other structural analogs is less than 0.01%.

[0059] Table 2 Cross-reaction results of anti-4-chlorophenoxyacetic acid monoclonal antibodies

[0060] Cross-reactants <![CDATA[IC 50 / μg / L]]> Cross-reaction rate (%) 4-Chlorophenoxyacetic acid 0.54 100.00 6-Benzyl adenine <![CDATA[>10 6 ]]> ＜0.01 Gibberellins <![CDATA[>10 6 ]]> ＜0.01 Indoleacetic acid <![CDATA[>10 6 ]]> ＜0.01 Indolebutyric acid <![CDATA[>10 6 ]]> ＜0.01 Naphthaleneacetic acid <![CDATA[>10 6 ]]> ＜0.01

[0061] Example 3

[0062] The anti-4-chlorophenoxyacetic acid monoclonal antibody of the present invention is used in an enzyme-linked immunosorbent assay kit, and the detection principle of the kit of the present invention is an indirect competitive ELISA method.

[0063] Coat the 4-chlorophenoxyacetic acid coating material on a microplate, remove the strips as needed, and place them on an ELISA plate; mix the anti-4-chlorophenoxyacetic acid monoclonal antibody working solution and the enzyme-labeled anti-antibody working solution at a volume ratio of 5:1; add 50 μL of 4-chlorophenoxyacetic acid standard or sample to be tested diluted according to a concentration gradient, 50 μL of a mixture of the anti-4-chlorophenoxyacetic acid monoclonal antibody working solution and the enzyme-labeled anti-antibody working solution to the wells of the ELISA plate coated with the coating material 4-CPA-OVA, and gently shake to mix. , incubate at 37℃ in dark for 30min; spin dry the liquid in the wells, wash 4 times with 250μL / well of washing working solution (20× concentrated washing solution diluted 20 times with deionized water), and pat dry; mix color development solution A and color development solution B in a 1:1 ratio, add 100μL / well for color development, and incubate at 25℃ in dark for 15min; add 50μL of stop solution to terminate the reaction, and measure with dual wavelength 450nm / 630nm at 450nm of the microplate reader, and quantify or qualitatively analyze the samples according to the standard curve.

[0064] Result determination: (a) Quantitative analysis: Calculate the average absorbance values ​​of 4-chlorophenoxyacetic acid standards of different concentrations and the sample to be tested respectively. The absorbance value (B) of the 4-chlorophenoxyacetic acid standards of different concentrations is divided by the absorbance value (B0) of the 4-chlorophenoxyacetic acid standard of 0 concentration and then multiplied by 100%, which is the percentage absorbance value. Percentage absorbance value = (B / B0) × 100%. Draw a standard curve with the percentage absorbance value as the ordinate and the logarithm of the concentration of the 4-chlorophenoxyacetic acid standard as the abscissa. Substitute the percentage absorbance value of the sample to be tested into the standard curve to calculate the concentration of 4-chlorophenoxyacetic acid in the sample to be tested, and then multiply it by the dilution multiple to obtain the actual content of 4-chlorophenoxyacetic acid 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 4-chlorophenoxyacetic acid in the sample to be tested. The test range of this embodiment is 2 μg / L to 10 μg / mL.

[0065] The results of ELISA determination of bean sprout samples are shown in Table 3.

[0066] Table 3 4-chlorophenoxyacetic acid concentration determination results in bean sprouts

[0067]

[0068] Example 4

[0069] The anti-4-chlorophenoxyacetic acid monoclonal antibody of the invention is applied to detection by a colloidal gold chromatography test strip method.

[0070] Reaction principle: 4-chlorophenoxyacetic acid is semi-quantitatively detected by the competition method. The 4-chlorophenoxyacetic acid in the sample first binds to the gold particle-labeled antibody 4-CPA-3C8 during its upward movement along the test strip. The coating precursor fixed on the NC membrane competes with the 4-chlorophenoxyacetic acid in the sample for binding to the gold-labeled antibody at the same time. The color intensity of the T position (detection line) is inversely proportional to the content of residual 4-chlorophenoxyacetic acid in the sample. If there is no residual 4-chlorophenoxyacetic acid in the sample, the gold-labeled antibody will react with the coating precursor and the T line of the test strip will develop color.

[0071] Detection: Take out the 4-chlorophenoxyacetic acid colloidal gold chromatography test strip, insert the sample end into the sample solution to be tested, the insertion depth should not exceed the marked line, take out the test strip after about 15 seconds, place it horizontally, observe and determine the test result after 3 minutes, and the result will be invalid after 10 minutes.

[0072] Result determination: A brown-red line is displayed at the corresponding quality control area C position (quality control line) on the coating membrane, and no brown-red line is displayed at the detection area T position, indicating that the test result is positive, indicating that the sample solution to be tested contains 4-chlorophenoxyacetic acid; two brown-red lines are displayed at the T and C positions on the coating membrane, indicating that the result is negative, indicating that the sample to be tested does not contain 4-chlorophenoxyacetic acid; when the quality control area C does not show a brown-red band, the test paper is judged invalid regardless of whether the detection area T shows a brown-red band or not.

[0073] Example 5

[0074] The anti-4-chlorophenoxyacetic acid monoclonal antibody of the invention is applied to detection by an evanescent wave fluorescence immunosensor method.

[0075] Reaction principle: The evanescent wave fluorescence analysis principle is combined with the bioaffinity reaction principle, that is, the fluorescently labeled anti-4-chlorophenoxyacetic acid monoclonal antibody competes with the sample to be tested and the 4-chlorophenoxyacetic acid on the surface of the optical fiber biosensor. After the excitation light is introduced into the optical fiber probe, an evanescent wave is generated, which makes the fluorescent labeled antibody bound to the surface emit light. The fluorescence collected by the photodetector is processed and the results are automatically displayed.

[0076] The instrument includes: a flow injection system, a fiber optic biosensor, an optical path system, and a signal processing system. The flow injection system is used to transport buffer solutions, samples to be tested, and regeneration solutions. The surface of the fiber optic biosensor is modified with an artificial antigen of 4-chlorophenoxyacetic acid by a fiber optic probe to achieve specific detection of the drug. The fiber optic probe modification uses N, N′-disuccinimidyl carbonate (DSC) to replace glutaraldehyde and covalently fix the 4-chlorophenoxyacetic acid artificial antigen (4-CPA-BSA) to the surface of the fiber optic biosensor. The optical path system uses an optical switch to control the excitation light to alternately enter the mode fiber at intervals of 1 second. After the mode fiber introduces the excitation light into the fiber optic probe, an evanescent wave is generated on the surface of the fiber optic, causing the fluorescent labeled antibody bound to the surface to emit light. The photodetector detects the collected fluorescence at intervals of 1 second, and the data is processed by the signal processing system to automatically display the results.

[0077] Detection: Take 50 μL of 4-chlorophenoxyacetic acid solution of different concentrations or the sample to be tested and 50 μL of Cy5.5 fluorescently labeled anti-4-chlorophenoxyacetic acid monoclonal antibody and mix them in 0.5 mL brown centrifuge tubes. After shaking and mixing on an oscillator, place them in the dark to avoid light and react for 6 minutes. After the reaction, perform instrument detection. According to the pre-set program, first, pass 10mM PBS buffer solution into the optical fiber reaction tank in the optical fiber biosensor to clean the surface optical fiber (30s) to restore the baseline to a stable state. After that, the system program automatically draws the mixed solutions of Cy5.5 fluorescently labeled anti-4-chlorophenoxyacetic acid monoclonal antibody and 4-chlorophenoxyacetic acid solution of different concentrations or the mixed solutions of the sample to be tested into the optical fiber reaction tank. Then stabilize the reaction for 4 minutes, at which time the detection signal is observed to gradually rise and is recorded by the computer. Then pump the regeneration solution (0.5% (pH 1.9) SDS solution) into the optical fiber reaction tank for 3 minutes to regenerate and strip the fluorescently labeled antibody that has been bound to the optical fiber surface. Finally, 10 mM PBS buffer solution was passed through for 1 min to restore the pH value of the optical fiber surface, and the detection was completed.

[0078] Result determination: Taking the fluorescence signal (e0) of the blank sample (PBS) as the benchmark, the signal value (e) obtained by reacting different concentrations of 4-chlorophenoxyacetic acid solutions with Cy5.5 fluorescently labeled anti-4-chlorophenoxyacetic acid monoclonal antibodies was compared with it to obtain the standardized value (e / e0). The Logistic function was used for fitting to establish the functional relationship between the standardized value (e / e0) and the concentration of 4-chlorophenoxyacetic acid, which is the standard detection curve. By comparing the average signal value of the sample to be tested with the signal value of the standard, the concentration of 4-chlorophenoxyacetic acid in the sample to be tested can be obtained.

[0079] In the present invention, the samples to be tested used in Examples 3 to 5 are plant growth regulators extracted from bean sprouts after pretreatment: 10.0 g of the crushed bean sprout sample is weighed in a 50 mL centrifuge tube, 20 mL of acetonitrile and 40 μL of formic acid are added, vortexed for 1 min, ultrasonically extracted for 30 min, centrifuged at 8000 r / min for 5 min, the supernatant is transferred to another 50 mL centrifuge tube, 3.0 g of NaCl is added, vortexed; centrifuged at 8000 r / min for 5 min, the acetonitrile layer is sucked out, dehydrated with anhydrous Na2SO4, collected in a round-bottom flask, vacuum concentrated to dryness in a 50°C water bath, 2 mL of methanol is added to the round-bottom flask for ultrasonic dissolution. Take 1 mL of the above methanol sample solution, add it to the test tube and mix it, let it stand for 5 min, mix it, centrifuge it at 10000 r / min for 2 min, and take the supernatant.

[0080] The test results of bean sprout samples determined by the evanescent wave immunofluorescence technique are shown in Table 4.

[0081] Table 4 Detection results of bean sprout samples by evanescent wave immunofluorescence technique

[0082]

[0083]

[0084]

[0085] 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 4-CPA-3C8, characterized in that: The hybridoma cell line is deposited in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, on July 27, 2022, with the deposit number CCTCCNO: C2022235; The hybridoma cell line 4-CPA-3C8 secretes anti-4-chlorophenoxyacetic acid monoclonal antibodies.

2. An anti-4-chlorophenoxyacetic acid monoclonal antibody, characterized in that: Produced by the hybridoma cell line 4-CPA-3C8 according to claim 1.

3. Use of the anti-4-chlorophenoxyacetic acid monoclonal antibody according to claim 1 in preparing a detection product for detecting 4-chlorophenoxyacetic acid.

4. The use according to claim 3, characterized in that: The detection products include enzyme-linked immunosorbent assay kit, colloidal gold chromatography test strips, and evanescent wave fluorescence immunosensor.

5. An enzyme-linked immunosorbent assay kit for detecting 4-chlorophenoxyacetic acid, characterized in that: The enzyme-linked immunosorbent assay kit comprises the anti-4-chlorophenoxyacetic acid monoclonal antibody according to claim 2.

6. A colloidal gold chromatography test strip for detecting 4-chlorophenoxyacetic acid, characterized in that: The colloidal gold chromatography test strip comprises the anti-4-chlorophenoxyacetic acid monoclonal antibody according to claim 2.

7. An evanescent wave fluorescence immunosensor for detecting 4-chlorophenoxyacetic acid, characterized in that: The evanescent wave fluorescent immunosensor comprises the anti-4-chlorophenoxyacetic acid monoclonal antibody according to claim 2.

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