Hapten and artificial antigen for detecting paclobutrazol and application of hapten and artificial antigen

Abstract

The invention discloses a hapten and an artificial antigen for detecting paclobutrazol and application of the hapten and the artificial antigen. The paclobutrazol hapten prepared by the invention retains the characteristic structure of paclobutrazol to the greatest extent, a paclobutrazol antibody prepared by using the paclobutrazol hapten disclosed by the invention has relatively high potency and specificity, the semi-inhibitory concentration of the paclobutrazol hapten to paclobutrazol is 0.31 ng / mL, the linear range is 0.03-19.63 ng / mL, and the paclobutrazol hapten can be used for preparing a paclobutrazol antibody. No cross reaction exists on other structural and functional analogues such as uniconazole and hexaconazole. The synthetic steps of the paclobutrazol hapten are simplified, the method for preparing the paclobutrazol artificial antigen and the paclobutrazol antibody is simple and low in cost, meanwhile, a more efficient immunoassay method is established, the requirement for on-site detection of large-batch samples can be met, and the paclobutrazol artificial antigen and the paclobutrazol antibody have wide application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and specifically, to a hapten, an artificial antigen for detecting paclobutrazol, and their applications. Background Art

[0002] Paclobutrazol (PBZ) is a triazole plant growth regulator and broad-spectrum fungicide developed by ICI Company in the UK in the 1970s and began to be applied in the early 1980s. Because paclobutrazol can delay plant growth, inhibit stem elongation, improve fruit quality and increase its yield, it is widely used in agricultural production. However, like other pesticides, if the dose exceeds a certain level, paclobutrazol will cause certain harm to human health. Paclobutrazol powder has a mild to moderate irritating effect on the skin and eyes, and paclobutrazol is easily adsorbed in the soil, and its residues will pollute the nearby water bodies, which may endanger the health of humans and animals and affect the activities of soil microorganisms. Some studies have found that after 96 hours of action by Pseudomonas, the degradation products of paclobutrazol will produce mutagenicity. Other studies have shown that a certain dose of paclobutrazol can damage the male reproductive organs of animals, reduce the body weight and reproductive index of offspring, damage the liver, and may become a carcinogen.

[0003] At present, countries around the world have strictly regulated the maximum residue limits of paclobutrazol in agricultural products. For example, the United States, South Korea and other countries require that the residue limit of paclobutrazol in various vegetables be "not detectable"; the European Union has stipulated that the maximum residue limit of paclobutrazol in food is 0.02 mg / mL; the national standard GB2763-2012 in China has also stipulated that the residue limit standard of paclobutrazol in food is 0.2 - 0.5 mg / kg. Therefore, in order to strengthen the supervision of pesticide residues in agricultural products, it is necessary to establish an accurate and portable rapid detection method for paclobutrazol residues.

[0004] The detection principle of immunoassay is to utilize the specific recognition and binding of antigen and antibody, label the antigen or antibody with signal amplification molecules such as enzymes, colloidal gold or fluorescent substances, and use instruments such as enzyme-labeled instruments or fluorescence detectors to qualitatively and quantitatively detect the analyte in the sample. It has greater advantages in on-site detection, with the advantages of high efficiency, high sensitivity, strong specificity, convenient detection, etc., and has low cost and low requirements for the skills of operators.

[0005] At present, a variety of paclobutrazol haptens and artificial antigens have been developed for paclobutrazol, but the potency of the antibodies obtained using these paclobutrazol haptens and artificial antigens is not high. During the on-site detection process, high-potency antibodies can reduce the single-use dose, lower the production cost. At the same time, high-potency antibodies can also capture the target antigen in low-concentration samples, reduce false negatives, and ensure the reliability of the detection results. The potency of the antibody is evaluated using the IC 50 value, and the IC50 The lower the value, the higher the potency of the antibody. Chinese Invention Patents CN104327183A, CN104356237A, and CN105348206A respectively disclose paclobutrazol antibodies obtained using paclobutrazol artificial antigens, with IC 50 values of 13.13 ng / mL, 325.07 ng / mL, and 17.8 ng / mL respectively. The potency of the antibodies is low, and in the actual detection process, it is not only costly but also unable to guarantee the reliability of the detection results. Therefore, it is of great significance to develop a paclobutrazol hapten and artificial antigen that can obtain antibodies with high potency. Summary of the Invention

[0006] To overcome the above-mentioned defects and deficiencies in the prior art, the present invention provides a hapten, artificial antigen for detecting paclobutrazol, and their applications.

[0007] The first object of the present invention is to provide a paclobutrazol hapten.

[0008] The second object of the present invention is to provide the application of the above-mentioned paclobutrazol hapten in the preparation of paclobutrazol artificial antigen.

[0009] The third object of the present invention is to provide a paclobutrazol artificial antigen.

[0010] The fourth object of the present invention is to provide the application of the above-mentioned paclobutrazol artificial antigen in the preparation of paclobutrazol antibody.

[0011] The fifth object of the present invention is to provide a paclobutrazol artificial antigen combination.

[0012] The sixth object of the present invention is to provide a paclobutrazol antibody.

[0013] The seventh object of the present invention is to provide the application of the above-mentioned paclobutrazol artificial antigen combination and / or paclobutrazol antibody in the preparation of reagents and / or test kits for detecting paclobutrazol.

[0014] The eighth object of the present invention is to provide a test kit for detecting paclobutrazol.

[0015] The ninth object of the present invention is to provide a method for detecting paclobutrazol.

[0016] The present invention claims protection for the following:

[0017] A paclobutrazol hapten, whose structural formula is shown in formula (Ⅰ),

[0018]

[0019] The application of the above-mentioned paclobutrazol hapten in the preparation of paclobutrazol artificial antigen.

[0020] A paclobutrazol artificial antigen is obtained by conjugating the above-mentioned paclobutrazol hapten with a carrier protein, and its structural formula is shown in Formula (II),

[0021]

[0022] wherein Z is the carrier protein.

[0023] Preferably, the carrier protein is bovine serum albumin or chicken ovalbumin.

[0024] Application of the above-mentioned paclobutrazol artificial antigen in the preparation of paclobutrazol antibodies.

[0025] Application of the above-mentioned paclobutrazol artificial antigen in the detection of paclobutrazol, and the detection is for non-disease treatment diagnosis purposes.

[0026] A paclobutrazol artificial antigen combination, comprising an immunogen and a coating antigen, and both the immunogen and the coating antigen are the above-mentioned paclobutrazol artificial antigens.

[0027] Preferably, the carrier protein on the immunogen is bovine serum albumin, and the carrier protein on the coating antigen is chicken ovalbumin.

[0028] A paclobutrazol antibody is prepared by using the above-mentioned paclobutrazol artificial antigen as an immunogen.

[0029] Preferably, the carrier protein on the immunogen is bovine serum albumin.

[0030] Application of the above-mentioned paclobutrazol artificial antigen combination and / or paclobutrazol antibody in the preparation of reagents and / or kits for detecting paclobutrazol.

[0031] A kit for detecting paclobutrazol, and the kit contains the above-mentioned paclobutrazol artificial antigen combination.

[0032] Preferably, the kit further comprises an enzyme-linked immunosorbent assay (ELISA) plate, a paclobutrazol standard and / or a substrate chromogenic solution.

[0033] More preferably, the substrate chromogenic solution contains urea peroxide and / or 3,3′,5,5′-tetramethylbenzidine.

[0034] More preferably, the kit further comprises a stop solution, a washing solution, a blocking solution, an enzyme conjugate concentrate and / or an enzyme conjugate diluent.

[0035] Further preferably, the stop solution is an H2SO4 solution with a volume fraction of 8% - 12%.

[0036] Most preferably, the stop solution is an H2SO4 solution with a volume fraction of 10%.

[0037] Further preferably, the washing solution is a 0.1 mol / L - 0.3 mol / L phosphate buffer solution containing Tween-20 with a volume fraction of 0.05% - 0.10% and sodium azide preservative with a mass fraction of 0.01% - 0.03%, and the pH value is 7.2 - 7.6.

[0038] Most preferably, the washing solution is a 0.2 mol / L phosphate buffer solution containing Tween-20 with a volume fraction of 0.06% and sodium azide preservative with a mass fraction of 0.02%, and the pH value is 7.4.

[0039] Further preferably, the blocking solution is a 0.1 mol / L - 0.3 mol / L phosphate buffer solution containing casein with a mass fraction of 1% - 3%, and the pH value is 7.1 - 7.5.

[0040] Most preferably, the blocking solution is a 0.2 mol / L phosphate buffer solution containing casein with a mass fraction of 2%, and the pH value is 7.3.

[0041] Further preferably, the enzyme conjugate concentrate is horseradish peroxidase-labeled goat anti-rabbit antibody or goat anti-mouse antibody.

[0042] Further preferably, the enzyme conjugate diluent is a 0.1 mol / L - 0.3 mol / L phosphate buffer solution.

[0043] Most preferably, the enzyme conjugate diluent is a 0.2 mol / L phosphate buffer solution.

[0044] A method for detecting paclobutrazol, which is detected using the above artificial antigen combination, and the detection is for non-disease treatment diagnosis purposes.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] The present invention discloses a hapten, artificial antigen for detecting paclobutrazol and its application. The paclobutrazol hapten prepared by the present invention retains the characteristic structure of paclobutrazol to the greatest extent. The polyclonal antibody of paclobutrazol prepared using the paclobutrazol hapten of the present invention has high potency and specificity. Its half-inhibitory concentration for paclobutrazol is 0.31 ng / mL, the linear range is 0.03 - 19.63 ng / mL, and there is no cross-reaction with other structural and functional analogues such as uniconazole and hexaconazole.

[0047] The present invention simplifies the synthesis steps of the paclobutrazol hapten, and the method for preparing the paclobutrazol artificial antigen and its antibody is simple and low-cost. It provides a new paclobutrazol hapten with a high degree of overlap in the backbone structure with the analyte paclobutrazol, effectively improving the immunogenicity of the paclobutrazol hapten-carrier protein conjugate. The paclobutrazol artificial antigen obtained by conjugating with the carrier protein has a large structural difference from the hapten, forming a large steric hindrance and further improving the antibody affinity. At the same time, the present invention also establishes a more efficient immunoassay method, which can meet the requirements of on-site detection of a large number of samples and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is a synthetic route diagram of the paclobutrazol hapten PBZ-4C.

[0049] Figure 2 It is a diagram of the ultraviolet full-wavelength scanning identification results of PBZ-4C, PBZ-4C-BSA, and BSA.

[0050] Figure 3 It is a diagram of the ultraviolet full-wavelength scanning identification results of PBZ-4C, PBZ-4C-OVA, and OVA.

[0051] Figure 4 It is a standard curve diagram of the indirect competitive ELISA for the antibody used to detect paclobutrazol.

[0052] Figure 5 It is a schematic side structure diagram of the paclobutrazol colloidal gold immunochromatographic test strip.

[0053] Figure 6 It is a result determination diagram of the paclobutrazol colloidal gold immunochromatographic test strip; A: negative, B: positive, C: invalid, D: invalid. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0054] The following specific examples are used to further illustrate the present invention, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0055] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0056] Example 1 Synthesis and Identification of Paclobutrazol Hapten

[0057] I. Experimental Method

[0058] 1. Synthesis of the paclobutrazol hapten PBZ-4C

[0059] The synthesis route of the paclobutrazol hapten PBZ-4C is as Figure 1 shown, and the specific steps are as follows:

[0060] Using 500 mg of (2R,3R)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl)pentan-3-ol as the raw material, the raw material and sodium hydroxide with a molar ratio of 1:2 to the raw material are fully dissolved in dimethyl sulfoxide, and then mercaptobutyric acid with a molar ratio of 1:1.5 to the raw material is added, and the whole system is stirred and reacted at 70 °C for 12 h.

[0061] The reaction was monitored by TLC, and the developing agent was dichloromethane - petroleum ether (5:4, v / v). After the reaction was completed, 20 mL of distilled water was added for dissolution, and a small amount of 3M NaOH solution was added to adjust the pH to about 10.0, then ethyl acetate was added. After thorough shaking, extraction was carried out 2 - 3 times, and the ethyl acetate layer was discarded, and the aqueous layer was taken. A small amount of 3M HCl solution was added to the aqueous layer to adjust the pH to about 3.0, ethyl acetate was added. After thorough shaking, extraction was carried out 2 - 3 times, the aqueous layer was discarded, and the ethyl acetate layers were collected and combined. 20 g of anhydrous sodium sulfate was added to the ethyl acetate layer to remove water, and the solvent was removed by rotary evaporation to obtain a concentrated product. The concentrated product was dissolved in ethyl acetate, TLC monitoring was carried out using dichloromethane - petroleum ether (5:4, v / v) as the developing agent, and then purification was carried out by column chromatography to obtain the paclobutrazol hapten PBZ-4C.

[0062] The paclobutrazol hapten PBZ-4C was identified by nuclear magnetic resonance hydrogen spectrum and mass spectrum.

[0063] II. Experimental Results

[0064] The nuclear magnetic resonance hydrogen spectrum results of the paclobutrazol hapten PBZ-4C were as follows: 1 H NMR(600MHz,Chloroform-d)δ8.17(s,0H),8.02(s,1H),7.29 - 7.24(m,2H),7.09(dt,J=8.4,1.1Hz,2H),4.66(q,J=7.3Hz,1H),3.93 - 3.85(m,2H),3.23 - 3.16(m,1H),3.14(dt,J=14.3,6.2Hz,1H),3.09 - 3.01(m,2H),2.44 - 2.31(m,2H),2.04(dq,J=13.3,6.8Hz,1H),1.92(dp,J=13.5,6.7Hz,1H),0.97(s,9H).

[0065] The mass spectrum results of the paclobutrazol hapten PBZ-4C were: MS: 377.2, ESI + [M - H] + : 378.6.

[0066] It can be seen from the results of mass spectrometry and nuclear magnetic resonance that the paclobutrazol hapten PBZ-4C was successfully prepared, and its structural formula is shown in Formula (I):

[0067]

[0068] The paclobutrazol hapten PBZ-4C is named by the systematic nomenclature method as: 4-((4-((2R,3R)-3-hydroxy-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl)pentyl)phenyl)thio)butyric acid.

[0069] Example 2 Synthesis and Identification of Paclobutrazol Artificial Antigen

[0070] I. Experimental Method

[0071] The paclobutrazol hapten PBZ-4C prepared in Example 1 (structural formula shown in Formula (I)) was coupled with bovine serum albumin (BSA) and chicken ovalbumin (OVA) by the active ester method. The specific steps are as follows:

[0072] 1 mol of the paclobutrazol hapten PBZ-4C (structural formula shown in Formula (I)) was dissolved in 200 μL of N,N-dimethylformamide (DMF) together with 0.8 mol of N-hydroxysuccinimide (NHS) and 1.9 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and stirred in the dark at room temperature of 25 °C for 4 h to obtain the activated solution of the paclobutrazol hapten PBZ-4C, denoted as Solution A;

[0073] 10 mg of bovine serum albumin (BSA) was added to 1 mL of PBS buffer (0.01 mol / L, pH = 7.4) and completely dissolved, denoted as Solution B.

[0074] 200 μL of Solution A was slowly added dropwise to 1 mL of Solution B, and the reaction was carried out at 4 °C for 12 h. After the reaction was completed, the reaction solution was filled into a dialysis bag and dialyzed with PBS buffer for 3 days, 3 times a day. After dialysis, the solution in the dialysis bag was collected to obtain the paclobutrazol artificial antigen PBZ-4C-BSA, which was sub-packed in centrifuge tubes and stored at -20 °C for use.

[0075] Among them, the formula of the PBS buffer: 2.90 g of Na2HPO4·12H2O, 8.50 g of NaCl, 0.20 g of KCl, 0.20 g of KH2PO4, and distilled water was added to a constant volume of 1000 mL.

[0076] The preparation method of the paclobutrazol artificial antigen PBZ-4C-OVA is similar to the synthesis method of the above paclobutrazol artificial antigen PBZ-4C-BSA, except that OVA is used instead of BSA, and the paclobutrazol hapten PBZ-4C (the structural formula is shown in formula (Ⅰ)) prepared in Example 1 is used to synthesize the paclobutrazol artificial antigen PBZ-4C-OVA.

[0077] BSA, OVA, the paclobutrazol hapten PBZ-4C (the structural formula is shown in formula (Ⅰ)), the paclobutrazol artificial antigens PBZ-4C-BSA and PBZ-4C-OVA were respectively identified by the ultraviolet full wavelength method (150 - 400 nm).

[0078] II. Experimental Results

[0079] The identification results of the ultraviolet full wavelength scanning are as Figure 2 and Figure 3 shown. By comparing the highest absorbance values of BSA and OVA before and after coupling, it can be seen that the absorption curve of PBZ-4C-BSA is significantly different from the BSA absorption curve, the absorption curve of PBZ-4C-OVA is significantly different from the OVA absorption curve, and the absorption curves of PBZ-4C-BSA and PBZ-4C-OVA show different degrees of deviation from the BSA and OVA absorption curves at 280 nm respectively.

[0080] Thus, it can be seen that the paclobutrazol hapten PBZ-4C (the structural formula is shown in formula (Ⅰ)) is successfully coupled with BSA and OVA respectively, and the paclobutrazol artificial antigens PBZ-4C-BSA and PBZ-4C-OVA are successfully prepared.

[0081] The structural formula of the paclobutrazol artificial antigen is shown in formula (Ⅱ):

[0082]

[0083] wherein, Z is the carrier protein OVA or BSA.

[0084] Example 3 Preparation of Polyclonal Antibodies for Detecting Paclobutrazol

[0085] 1. Animal Immunization

[0086] The paclobutrazol artificial antigen PBZ-4C-BSA (the structural formula is shown in formula (Ⅱ), wherein Z is bovine serum albumin) prepared in Example 2 was mixed and emulsified evenly with an immune adjuvant (Freund's complete adjuvant for primary immunization and Freund's incomplete adjuvant for booster immunization) according to a volume ratio of 1:1 for standby.

[0087] At the first immunization, two healthy 6-week-old New Zealand white rabbits (one male and one female) were subcutaneously injected at multiple points on the back, with an immunization dose of 500 μg of immunoprotein for each rabbit. Subsequently, booster immunizations were carried out every three weeks for a total of four times.

[0088] One week after the third immunization, blood was collected from the ear vein of the rabbits. After centrifugation, the supernatant was taken and stored at -20 °C for ELISA detection of the immunization effect.

[0089] 2. Obtaining polyclonal antibodies

[0090] One week after the fifth immunization, blood was collected from the hearts of the rabbits. The obtained rabbit blood was incubated at 37 °C for 2 h and then left overnight (12 h) at 4 °C. The next day, the upper clear liquid was taken and centrifuged at 3000 r / min for 10 min at 4 °C to remove the precipitate, obtaining the supernatant, i.e., the paclobutrazol polyclonal antibody. It was aliquoted, labeled, and stored at -20 °C.

[0091] Example 4 Preparation of monoclonal antibodies for detecting paclobutrazol

[0092] 1. Animal immunization

[0093] The paclobutrazol artificial antigen PBZ-4C-BSA prepared in Example 2 (the structural formula is shown in Formula (Ⅱ), where Z is bovine serum albumin) was used as the immunogen, diluted to 50 μL with PBS at 50 μg equivalent / only, and mixed with 50 μL of Freund's adjuvant in a 2.5 mL syringe. It was emulsified with an emulsifier until the emulsion did not spread in the water surface test and was reserved.

[0094] Balb / C female mice were used as immunized animals and were injected at multiple points on the abdomen and back (100 μL / only) according to the immunization adjuvant and immunization cycle shown in Table 1.

[0095] Table 1 Immunization protocol of Balb / C mice with Freund's adjuvant

[0096] Immune stage Immune time Dose (μg / animal) Injection method Freund's adjuvant Primary immunization 4 weeks 50 Multiple subcutaneous injections in the abdomen and back Freund's complete adjuvant Booster immunization 3 weeks 50 Multiple subcutaneous injections in the abdomen and back Freund's incomplete adjuvant Booster immunization 3 weeks 50 Multiple subcutaneous injections in the abdomen and back Freund's incomplete adjuvant Booster immunization 3 weeks 50 Multiple subcutaneous injections in the abdomen and back Freund's incomplete adjuvant Booster immunization 3 weeks 50 Multiple subcutaneous injections in the abdomen and back Freund's incomplete adjuvant Boost immunization 3 days 50 Multiple subcutaneous injections in the abdomen and back Freund's incomplete adjuvant

[0097] 2. Detection of antibody production in animals

[0098] One week after the third immunization (i.e., the second booster immunization) and each subsequent immunization, blood was collected from the tail vein of the mice, centrifuged, and antiserum was obtained. The antibody titer and inhibition rate of the antiserum were examined by icELISA. The results are shown in Table 2.

[0099] Table 2 Antibody titer and inhibition rate of monoclonal antibodies

[0100] Immunogen Coating antigen Antiserum titer Inhibition rate PBZ-4C-BSA PBZ-4C-OVA 16K 80.31％

[0101] 3. Cell fusion

[0102] (1) Resuscitation and collection of myeloma cells

[0103] Take out the myeloma cells from the -80°C refrigerator and quickly place them in a 37°C water bath for thawing. Centrifuge the thawed cells at 1000 r / min for 7 min and discard the cell cryopreservation solution. Use a pipette to aspirate the basal medium to disperse the cells. After dispersion, centrifuge at 1000 r / min for 7 min and discard the basal medium. Aspirate the cells with a pipette, mix them with the complete medium, and then place them in a 9-cm culture dish for expansion culture.

[0104] On the day of cell fusion, remove the supernatant of the myeloma cells in the culture dish, add 25 mL of fresh basal medium using a bent pipette, and repeatedly blow the adherent myeloma cells along a certain direction until the bottom of the culture plate is no longer blurred and becomes transparent. Collect all the medium, seal it, and centrifuge at 1000 r / min for 8 min. After centrifugation, discard the supernatant to obtain myeloma cells.

[0105] (2) Booster immunization

[0106] Three days before cell fusion, perform booster immunization on the mice. Injection concentration: 1 mg / mL, injection volume: 100 μL, no adjuvant is required.

[0107] (3) Obtaining immune spleen cells

[0108] Add 25 mL of basal medium to the culture dish and place a disposable cell grinding mesh in the culture dish for use.

[0109] Euthanize the immunized mice by removing their eyeballs and collect the blood. After soaking in 75% (v / v) alcohol for 4 min, transfer them to the laminar flow hood and take out the spleens.

[0110] Place the removed spleen in the disposable cell grinding mesh in the culture dish. First, use a syringe to aspirate the medium and inject it into the spleen, repeatedly inject to blow out the cells until the spleen color changes from red to transparent. Then, gently grind the spleen with the piston of the syringe on the cell grinding mesh and aspirate the medium to wash the sieve mesh.

[0111] Collect all the medium, seal it, and centrifuge at 1000 r / min for 7 min. Discard the supernatant to obtain immune spleen cells.

[0112] (4) Cell fusion

[0113] Mix the myeloma cells and immune spleen cells, add 25 mL of basal medium, seal it, and centrifuge at 1000 r / min for 7 min. Discard the supernatant.

[0114] Gently flick the precipitated cells loose with your finger. Place the centrifuge tube in warm water at 37°C. Use a pipette tip to aspirate 1 mL of polyethylene glycol (PEG2000) preheated to 37°C and slowly add the PEG2000 to the precipitated cells within 1 minute. Gently stir with the pipette tip after each drop of PEG2000 is added to mix evenly. Let it stand for 30 s, preheat the basal medium, and add 1.5 mL of the basal medium within 1 minute while gently stirring. Add it along the wall, then add more basal medium and bring the volume to 20 mL within 5 minutes while gently swirling up and down. Seal the centrifuge tube and centrifuge at 1000 rpm for 7 minutes. Pour off the supernatant, add the precipitate to 200 mL of complete medium, gently aspirate and dispense the liquid with a bent pipette, and gently stir the cells. Spread the complete medium containing the fused cells into a 24-well plate at 2 mL / well and continue culturing in a 37°C, 5% CO2 cell culture incubator.

[0115] (5) Screening and identification of hybridoma cells

[0116] Begin to observe the cell growth status and the medium condition every day starting from the 7th day of culturing the fused cells.

[0117] Table 3 Monoclonal cell culture and screening

[0118]

[0119]

[0120] As shown in Table 3, at day 10 or when the cells grow too fast, perform a half-medium change with HAT medium; perform a full-medium change with HAT medium at day 13; at day 15, perform a half-medium change with HT medium and conduct the first icELISA test on the cell supernatant to determine whether cell fusion has occurred and the degree of cell fusion; at day 17, perform a full-medium change with HT medium and conduct the second icELISA test; at day 19, perform a half-medium change with HT medium and conduct the third icELISA test. Compare the results of the previous two supernatant tests, pick out the wells with high titers for limiting dilution. When performing icELISA tests on the cell supernatants of the limited dilution, use the titer and inhibition rate as the measurement indicators. After selecting the positive wells, conduct the next round of limiting dilution until each well on each plate is positive and the detected titer and inhibition rate are similar. At this point, the hybridoma cell line is successfully established and cryopreserved in a timely manner. Expand the cultured hybridoma cells and collect the cells for animal immunization to obtain ascites. After identification and purification, the paclobutrazol monoclonal antibody is obtained.

[0121] Example 5 Optimization of the combination of paclobutrazol immunogen and coating antigen

[0122] Using the polyclonal antibody against paclobutrazol prepared in Example 3 and using the artificial antigen PBZ-4C-OVA of paclobutrazol prepared in Example 2 (the structural formula is shown in Formula (II), where Z is chicken ovalbumin) as the coating antigen, the combination of the best immunogen and coating antigen was selected by the serum titer and inhibition rate obtained by the indirect competitive ELISA method. The specific operation steps are as follows:

[0123] 1. Dilute the artificial antigens PBZ-4C-BSA (the structural formula is shown in Formula (II), where Z is bovine serum albumin) and PBZ-4C-OVA (the structural formula is shown in Formula (II), where Z is chicken ovalbumin) of paclobutrazol to a concentration of 250 ng / mL with the coating solution (0.05 M carbonate buffer solution, pH 9.6), coat a 96-well enzyme-linked immunosorbent assay (ELISA) plate with 100 μL per well, incubate overnight in a constant temperature water bath at 37 °C, discard the coating solution, and wash twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v));

[0124] 2. Add 120 μL of the blocking solution (0.2 mol / L phosphate buffer solution with a pH value of 7.3 and containing 2% casein by mass fraction) to each well, block at 37 °C for 3 h, discard the blocking solution, tap the plate, and dry it in an oven at 37 °C for standby;

[0125] 3. Dilute the polyclonal antibody against paclobutrazol prepared in Example 3 with PBST at 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000 respectively, and set a blank control well (replaced with PBST); dilute the 1 mg / mL paclobutrazol standard product 1000 times with PBST to a concentration of 1 μg / mL;

[0126] 4. For the titer column, first add 50 μL of PBST to each well, then add 50 μL of the polyclonal antibody against paclobutrazol at different dilution ratios to each well in sequence, and do not add antibody to the last well, replaced with 50 μL of PBST;

[0127] 5. For the inhibition column, first add 50 μL of the paclobutrazol standard product to each well, then add 50 μL of the polyclonal antibody against paclobutrazol at different dilution ratios to each well in sequence, and do not add the polyclonal antibody against paclobutrazol to the last well, replaced with 50 μL of PBST;

[0128] 6. Incubate at 37 °C for 40 min, wash 5 times, and tap the plate;

[0129] 7. Add goat anti-rabbit secondary antibody-HRP (diluted 5000 times), incubate at 37 °C for 30 min, wash 5 times, and tap the plate;

[0130] 8. Add the chromogenic solution and incubate at 37 °C for 10 min for color development;

[0131] 9. Add an H2SO4 solution with a volume fraction of 10% to terminate the reaction, and read the OD value at 450 nm; count the titer and inhibition rate. The titer is the dilution factor of the paclobutrazol polyclonal antibody corresponding to an OD of about 1.0. The inhibition rate = (OD value of the titer - OD value of the inhibition) / OD value of the inhibition × 100%. 450 The inhibition rate = (OD value of the titer - OD value of the inhibition) / OD value of the inhibition × 100%.

[0132] The combined results of different immunogens and coating antigens are shown in Table 4.

[0133] Table 4 Screening results of immunogens and coating antigens

[0134] Number Immunogen Coating antigen Potency Inhibition rate 1 PBZ-4C-BSA PBZ-4C-BSA 1:16000 65.32％ 2 PBZ-4C-BSA PBZ-4C-OVA 1:128000 85.82％

[0135] As can be seen from Table 4, the antiserum produced by immunizing New Zealand white rabbits with the paclobutrazol artificial antigen PBZ-4C-BSA (structural formula shown in Formula (II), where Z is bovine serum albumin) as the immunogen all have a certain titer, and the obtained antiserum all have different degrees of inhibitory effects on the target analyte paclobutrazol. Among them, the antiserum titer of 1:128000 and inhibition rate of 85.82% shown by the combination of the immunogen and coating antigen No. 2 are the best combination; under this combination, the paclobutrazol polyclonal antibody can not only recognize the target analyte paclobutrazol, but also the antibody sensitivity is good. Therefore, the combination of the immunogen and coating antigen No. 2 is the best combination, that is, PBZ-4C-BSA (structural formula shown in Formula (II), where Z is bovine serum albumin) is used as the immunogen, and PBZ-4C-OVA (structural formula shown in Formula (II), where Z is chicken ovalbumin) is used as the coating antigen.

[0136] Example 6 Establishment of an indirect competitive ELISA detection method for paclobutrazol

[0137] This example provides an indirect competitive ELISA method for detecting paclobutrazol, including the following steps:

[0138] 1. Use the paclobutrazol artificial antigen PBZ-4C-OVA (structural formula shown in Formula (II), where Z is chicken ovalbumin) prepared in Example 2 as the coating antigen, dilute it to 1 μg / mL with a coating solution (0.05 M carbonate buffer solution, pH 9.6), and coat a 96-well enzyme-linked immunosorbent assay (ELISA) plate at 100 μL / well and incubate overnight (12 h) at 37 °C;

[0139] 2. Discard the coating solution, wash 2 times, and pat dry;

[0140] 3. Add 120 μL of blocking solution (0.2 mol / L phosphate buffer solution with a pH value of 7.3 and containing 2% casein by mass fraction) to each well and block for 3 h at 37 °C;

[0141] 4. Discard the blocking solution, clap the plate, dry at 37℃ for 30min, take it out, and put it in a ziplock bag for later use;

[0142] 5. Dilute the paclobutrazol polyclonal antibody prepared in Example 3 with PBST at 1:16000, and dilute the paclobutrazol standard to 10000 ng / mL, 1000 ng / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.1 ng / mL, 0.01 ng / mL and 0.001 ng / mL;

[0143] 6. Add 50 μL of paclobutrazol standard dilution to each row (four parallel groups), then add 50 μL / well PBST, incubate at 37°C for 40 min, and wash 5 times;

[0144] 7. Add goat anti-rabbit secondary antibody-HRP (5000-fold dilution), incubate at 37°C for 30 min, wash 5 times, and pat dry;

[0145] 8. Add color developing solution, 100 μL per well, and develop color for 10 minutes;

[0146] 9. Add 50 μL of 10% H2SO4 solution to terminate the reaction, read the OD value at 450 nm, and draw the ELISA standard curve based on the OD value:

[0147] With B / B0 as the ordinate (B is the absorbance value OD of different concentrations of paclobutrazol standard 450 , B0 is the absorbance value OD of the blank control well 450 ), the logarithm of the standard concentration is the horizontal axis, and the logistic function is used for curve fitting to obtain the formula of the standard curve, and the standard curve is prepared, such as Figure 4 The half inhibitory concentration (IC) of the polyclonal antibody of paclobutrazol was calculated according to the standard curve. 50 ) was 0.31 ng / mL, and the linear range was 0.03~19.63 ng / mL.

[0148] Example 7 Specificity evaluation of paclobutrazol polyclonal antibody

[0149] 1. Experimental Methods

[0150] The specificity of the polyclonal antibody of paclobutrazol was determined by cross-reaction experiments with paclobutrazol and its analogs. The specificity of the antibody is represented by the cross-reaction rate (CR). The smaller the cross-reaction rate, the stronger the specificity. Paclobutrazol and its analogs were diluted in multiples and measured by indirect competitive ELISA method. The steps were the same as those in Example 6 to obtain the IC values ​​of each analog. 50 The cross-reaction rate (CR) of paclobutrazol was calculated according to the following formula:

[0151] CR(%)=IC50 (Paclobutrazol) / IC 50 (analog)×100%.

[0152] 2. Experimental Results

[0153] The cross-reaction results of paclobutrazol and its analogs are shown in Table 5. It can be seen that the cross-reaction rate of paclobutrazol polyclonal antibody to paclobutrazol is 100%, IC 50 The cross-reaction rates to paclobutrazol, hexaconazole, tebuconazole and triadimefon were all less than 0.01%, indicating that the polyclonal antibody used to detect paclobutrazol has strong specificity, can effectively eliminate the interference of its analogues on the detection of paclobutrazol, and can be specifically used for the detection of paclobutrazol.

[0154] Table 5 Cross-reaction results of paclobutrazol and its analogs

[0155] Name <![CDATA[IC 50 (ng / mL)]]> CR (100%) Paclobutrazol 0.31 100％ Uniconazole NR NR Hexaconazole NR NR Tebuconazole NR NR Triadimefon NR NR

[0156] Example 8 An ELISA kit for detecting paclobutrazol

[0157] 1. Composition

[0158] (1) ELISA plate coated with a coating agent

[0159] The ELISA plate is prepared by the following method:

[0160] The artificial paclobutrazol antigen PBZ-4C-OVA (structural formula as shown in formula (II), wherein Z is chicken ovalbumin) prepared in Example 2 was used as a coating source, diluted to 125 ng / mL with a coating buffer (0.05 M carbonate buffer solution, pH 9.6), 100 μL / well was added to the ELISA plate, incubated at 37° C. in the dark overnight, the liquid in the wells was removed, the wells were washed twice with a washing solution, each time for 30 s, and patted dry. Then, 120 μL of blocking solution (pH value of 7.3, 0.2 mol / L phosphate buffer containing 2% casein by mass) was added to each well, incubated at 37° C. in the dark for 3 h, the liquid in the wells was poured off, patted dry, and after drying, the wells were vacuum-sealed with aluminum film for storage;

[0161] (2) Standard products

[0162] 11 different concentrations of paclobutrazol standard solutions, the concentrations are 10000ng / mL, 2000ng / mL, 400ng / mL, 80ng / mL, 16ng / mL, 3.2ng / mL, 0.64ng / mL, 0.128ng / mL, 0.0256ng / mL, 0.00512ng / mL, 0.001024ng / mL;

[0163] (3) Antibodies

[0164] The polyclonal antibody prepared with the artificial antigen PBZ-4C-BSA of paclobutrazol (the structural formula is shown in formula (II), where Z is bovine serum albumin) as the immunogen in Example 3, or the monoclonal antibody prepared with the artificial antigen PBZ-4C-BSA of paclobutrazol (the structural formula is shown in formula (II), where Z is bovine serum albumin) as the immunogen in Example 4;

[0165] (4) Enzyme conjugate concentrate

[0166] Goat anti-rabbit antibody or goat anti-mouse antibody labeled with horseradish peroxidase;

[0167] (5) Substrate chromogenic solution

[0168] It consists of liquid A and liquid B. Liquid A is urea peroxide, and liquid B is tetramethylbenzidine;

[0169] (6) Stop solution

[0170] H2SO4 solution with a volume fraction of 10%;

[0171] (7) Washing solution

[0172] 0.2 mol / L phosphate buffer solution with a pH value of 7.4, containing 0.06% (volume fraction) of Tween-20 and 0.02% (mass fraction) of sodium azide preservative.

[0173] (8) Enzyme conjugate diluent

[0174] 0.2 mol / L phosphate buffer solution.

[0175] 2. Usage method

[0176] (1) Sample detection

[0177] Number the corresponding micro wells of the test samples and standards in sequence. Make 2 parallel wells for each test sample and standard, and record the positions of the standard wells and sample wells. Dilute the enzyme conjugate concentrate with the enzyme conjugate diluent at a volume ratio of 1:10 according to the required amount (that is, add 1 part of the enzyme conjugate concentrate to 10 parts of the enzyme conjugate diluent, and prepare it immediately before use) to obtain the enzyme conjugate working solution.

[0178] Add 50 μL of the standard or test sample to the corresponding micro well, then add 50 μL of the enzyme conjugate working solution, gently shake and mix well, cover with a cover film, and react in a dark environment at 25 °C for 30 min.

[0179] Discard the liquid in the well by centrifugation, and add 250 μL / well of the washing working solution. Wash thoroughly 4 - 5 times, with an interval of 10 s each time. Discard the washing solution in the plate wells, and pat dry with absorbent paper (the bubbles not removed after patting dry can be punctured with an unused pipette tip).

[0180] Add 50 μL / well of substrate colorimetric solution A, then add 50 μL / well of substrate colorimetric solution B, gently shake to mix, cover the plate with a cover film, and place it in a dark environment at 25°C for 10 minutes. Add 50 μL / well of stop solution, gently shake to mix, set the microplate reader at 450 nm, and measure the OD value of each well.

[0181] (2) Drawing of standard curve

[0182] The ELISA standard curve was drawn according to the OD value of the paclobutrazol standard at 450 nm. The specific method is as follows:

[0183] With B / B0 as the ordinate (B is the absorbance value OD of different concentrations of paclobutrazol standard 450 , B0 is the absorbance value OD of the blank control well 450 ), the logarithm of the standard concentration was taken as the horizontal axis, and the Logistic function was used for curve fitting to obtain the formula of the standard curve and prepare the standard curve.

[0184] (3) Calculation of sample concentration

[0185] The OD of the sample to be tested 450 Substitute the average value into the formula of the standard curve to obtain the concentration of the sample to be tested, and then multiply it by the corresponding dilution factor to obtain the actual concentration of paclobutrazol in the test sample.

[0186] Example 9 A colloidal gold test strip for detecting paclobutrazol

[0187] 1. Preparation of gold-labeled antibodies and gold-labeled conjugate pads

[0188] The colloidal gold suspension with an average diameter of 40 nm was prepared by reducing chloroauric acid with trisodium citrate.

[0189] First, the colloidal gold was adjusted to pH 8.5 with 0.2 mol K2CO3 solution, and then the antibody labeling amount was determined by the classical NaCl titration method. Finally, 20 μg of the paclobutrazol polyclonal antibody prepared in Example 3 and 1 mL of colloidal gold solution were selected for labeling to obtain the gold-labeled antibody, which was stored at 4°C.

[0190] like Figure 5 As shown in FIG. 1 , the colloidal gold rapid test strip is composed of a NC membrane (nitrocellulose membrane), a gold-labeled conjugate pad, a sample pad, a water-absorbing pad and a PVC bottom plate.

[0191] Use the XYZ-3000 three-dimensional spraying film instrument to spray a BSA solution with a mass fraction of 4% on glass wool at a rate of 8 μL / cm. Dry it in an oven at 42 °C for 50 min. Then spray the gold-labeled antibody on the glass wool at a rate of 6 μL / cm. After drying in an oven at 42 °C for 50 min, the gold-labeled conjugate pad is obtained and stored under vacuum drying.

[0192] 2. Coupled antigen, cellulose membrane coated with goat anti-rabbit

[0193] Use the XYZ-3000 three-dimensional spraying film instrument to spray the coating antigen with a concentration of 1 mg / mL (PBZ-4C-OVA (the structural formula is shown in formula (Ⅱ), where Z is chicken ovalbumin)) on the right side of the cellulose membrane at a rate of 1.2 μL / cm in a direction perpendicular to the long side of the cellulose membrane as the test line (i.e., the T line).

[0194] Use the XYZ-3000 three-dimensional spraying film instrument to spray goat anti-rabbit IgG with a concentration of 120 μg / L on the left side of the cellulose membrane at a rate of 1.2 μL / cm in a direction perpendicular to the long side of the cellulose membrane as the control line (i.e., the C line). The two lines are spaced 8 mm apart, and the cellulose membrane coated with coupled antigen and goat anti-rabbit is obtained.

[0195] 3. Assembly of the rapid test strip

[0196] Paste the cellulose membrane in the middle part of the PVC bottom plate; paste the absorbent pad on the left side of the cellulose membrane with an overlap of 1 mm; paste the gold-labeled conjugate pad on the right side of the cellulose membrane with an overlap of 1 mm; paste the sample pad on the right side of the gold-labeled conjugate pad with an overlap of 2 mm, and the colloidal gold test strip is obtained. Cut the colloidal gold test strip into 3.05 mm wide with a cutting machine.

[0197] 4. Preparation of the test solution

[0198] Absorb 0.5 mL of the test sample and mix it thoroughly with 9.5 mL of 0.2 mol / L phosphate buffer solution with a pH of 7.4. Vortex for 30 s to obtain the test solution.

[0199] 5. Test strip detection and judgment

[0200] Drop the test solution onto the sample pad and let it stand. The test solution drives the test substance and the gold-labeled antibody in the gold-labeled conjugate pad to diffuse towards the cellulose membrane through siphon action and finally penetrate into the absorbent pad.

[0201] Judgment result: As shown in A - D in Figure 6 When the gold-labeled antibody binds to the invisible quality control line (i.e., the C line) on the cellulose membrane, the quality control line (i.e., the C line) shows red, indicating that the test result is valid. If the quality control line (i.e., the C line) does not show color, it indicates that the test result is invalid.

[0202] If there is paclobutrazol in the sample, paclobutrazol binds to the gold-labeled antibody, thereby occupying the antigen-binding site on the gold-labeled antibody and preventing the gold-labeled antibody from binding to the invisible test line (the conjugate of hapten and carrier protein) on the cellulose membrane, resulting in the invisible test line (i.e., the T line) not showing color or showing very weak color, indicating that the test sample is positive or weakly positive; if there is no paclobutrazol in the sample, during the upward movement of the gold-labeled antibody, when it encounters the invisible test line (i.e., the T line), a clear red line appears, indicating that the test sample is negative.

[0203] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A paclobutrazol hapten, characterized in that: Its structural formula is shown in formula (I).

2. Use of the paclobutrazol hapten according to claim 1 in the preparation of paclobutrazol artificial antigen.

3. A paclobutrazol artificial antigen, characterized in that: It is obtained from the paclobutrazol hapten-coupled carrier protein according to claim 1, and its structural formula is shown in formula (II). Wherein Z is a carrier protein.

4. Use of the paclobutrazol artificial antigen according to claim 3 in the preparation of paclobutrazol antibodies.

5. A paclobutrazol artificial antigen combination, characterized in that: It comprises an immunogen and a coating agent, and both the immunogen and the coating agent are the artificial paclobutrazol antigen according to claim 3.

6. The paclobutrazol artificial antigen combination according to claim 5, characterized in that: The carrier protein on the immunogen is bovine serum albumin, and the carrier protein on the coating source is chicken ovalbumin.

7. A paclobutrazol antibody, characterized in that: The method is prepared by using the paclobutrazol artificial antigen described in claim 3 as an immunogen.

8. Use of the paclobutrazol artificial antigen combination according to any one of claims 5 to 6 and / or the paclobutrazol antibody according to claim 7 in the preparation of a reagent and / or a kit for detecting paclobutrazol.

9. A kit for detecting paclobutrazol, characterized in that: The kit comprises the paclobutrazol artificial antigen combination according to any one of claims 5 to 6.

10. A method for detecting paclobutrazol, characterized in that: The detection is performed using the artificial antigen combination described in any one of claims 5 to 6, wherein the detection is for the purpose of non-disease treatment diagnosis.

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