A hapten for detecting paclobutrazol, an artificial antigen and application thereof

Abstract

This invention discloses a hapten, an artificial antigen, and their applications for detecting paclobutrazol. The paclobutrazol hapten prepared by this invention retains the characteristic structure of paclobutrazol to the greatest extent possible. The paclobutrazol antibody prepared using the paclobutrazol hapten of this invention exhibits high potency and specificity, with a half-maximum inhibitory concentration (WMC) of 0.31 ng / mL and a linear range of 0.03–19.63 ng / mL. Furthermore, it shows no cross-reactivity with other structural and functional analogs such as uniconazole and hexaconazole. This 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 also establishes a more efficient immunoassay method, capable of meeting the requirements for large-scale on-site sample testing and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, specifically to a hapten and artificial antigen for detecting paclobutrazol, and their applications. Background Technology

[0002] Paclobutrazol (PBZ) is a triazole plant growth regulator and broad-spectrum fungicide developed by ICI in the UK in the 1970s and began to be used in the early 1980s. Because paclobutrazol can delay plant growth, inhibit stem elongation, improve fruit quality, and increase yield, it is widely used in agricultural production. However, like other pesticides, paclobutrazol can cause harm to health if the dosage is exceeded. Paclobutrazol powder has a mild to moderate irritant effect on the skin and eyes, and it is easily adsorbed into the soil, with its residues polluting nearby water bodies, potentially endangering human and animal health and affecting soil microbial activity. Studies have found that after 96 hours of exposure to Pseudomonas aeruginosa, the degradation products of paclobutrazol become mutagenic. Other studies have shown that certain doses of paclobutrazol can damage male reproductive organs in animals, reduce offspring weight and reproductive index, damage the liver, and may be carcinogenic.

[0003] Currently, countries worldwide have established strict maximum residue limits (MRLs) for paclobutrazol in agricultural products. For example, the United States and South Korea require "not detectable" MRLs for paclobutrazol in various vegetables; the European Union sets the MRL for paclobutrazol in food at 0.02 mg / mL; and my country's national standard GB2763-2012 also specifies the MRL for paclobutrazol in food as 0.2–0.5 mg / kg. Therefore, to strengthen the supervision of pesticide residues in agricultural products, it is necessary to establish an accurate, portable, and rapid detection method for paclobutrazol residues.

[0004] The detection principle of immunoassay is based on the specific recognition and binding of antigens and antibodies. It uses signal amplification molecules, colloidal gold, or fluorescent substances to label antigens or antibodies, and uses instruments such as enzyme-linked immunosorbent assay (ELISA) readers or fluorescence spectrometers to perform qualitative and quantitative detection of the analytes in the sample. It has greater advantages in on-site detection, with advantages such as high efficiency and speed, high sensitivity, strong specificity, and convenient detection. It is also inexpensive and requires less skill from operators.

[0005] Currently, various paclobutrazol haptens and artificial antigens have been developed for paclobutrazol, but the potency of antibodies obtained using these paclobutrazol haptens and artificial antigens is not high. In on-site testing, highly potent antibodies can reduce the dosage per use, lower production costs, and also capture the target antigen in low-concentration samples, reducing false negatives and ensuring the reliability of test results. Antibody potency is measured using IC50. 50 The value is evaluated, IC50 The lower the value, the higher the antibody potency. Chinese invention patents CN104327183A, CN104356237A, and CN105348206A disclose paclobutrazol antibodies obtained using paclobutrazol artificial antigens, with IC50 values... 50 The values ​​were 13.13 ng / mL, 325.07 ng / mL, and 17.8 ng / mL, respectively, indicating low antibody potency. In practical testing, this not only resulted in high costs but also compromised the reliability of the results. Therefore, developing a paclobutrazol hapten and artificial antigen capable of producing highly potent antibodies is of great significance. Summary of the Invention

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

[0007] The first objective of this invention is to provide a paclobutrazol hapten.

[0008] A second objective of this invention is to provide the application of the above-mentioned paclobutrazol hapten in the preparation of paclobutrazol artificial antigens.

[0009] A third objective of this invention is to provide a paclobutrazol artificial antigen.

[0010] A fourth objective of this invention is to provide the application of the above-described paclobutrazol artificial antigen in the preparation of paclobutrazol antibodies.

[0011] The fifth objective of this invention is to provide a paclobutrazol artificial antigen combination.

[0012] The sixth objective of this invention is to provide a paclobutrazol antibody.

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

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

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

[0016] This invention claims protection for the following:

[0017] A paclobutrazol hapten, the structural formula of which is shown in formula (I),

[0018]

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

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

[0021]

[0022] Z represents the carrier protein.

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

[0024] The above-mentioned paclobutrazol artificial antigen is used in the preparation of paclobutrazol antibodies.

[0025] The above-mentioned application of paclobutrazol artificial antigen in the detection of paclobutrazol is for the purpose of non-disease treatment diagnosis.

[0026] A paclobutrazol artificial antigen combination comprising an immunogen and a coating antigen, wherein the immunogen and the coating antigen are both the aforementioned 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 oocyte albumin.

[0028] A paclobutrazol antibody was prepared using the aforementioned paclobutrazol artificial antigen as an immunogen.

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

[0030] The above-mentioned combination of paclobutrazol artificial antigens and / or paclobutrazol antibodies are used in the preparation of reagents and / or kits for detecting paclobutrazol.

[0031] A kit for detecting paclobutrazol, the kit comprising the above-described combination of paclobutrazol artificial antigens.

[0032] Preferably, the kit further comprises an enzyme-labeled plate, paclobutrazol standards, and / or substrate chromogenic solution.

[0033] More preferably, the substrate colorimetric solution contains urea peroxide and / or 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] More preferably, the terminating solution is an H2SO4 solution with a volume fraction of 8% to 12%.

[0036] Most preferably, the terminating solution is a 10% (v / v) H2SO4 solution.

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

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

[0039] More preferably, the blocking solution is a 0.1 mol / L to 0.3 mol / L phosphate buffer containing 1% to 3% casein by mass, with a pH of 7.1 to 7.5.

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

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

[0042] More preferably, the enzyme conjugate diluent is a 0.1 mol / L to 0.3 mol / L phosphate buffer.

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

[0044] A method for detecting paclobutrazol, using the aforementioned combination of artificial antigens, wherein the detection is for the purpose of non-disease treatment diagnosis.

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

[0046] This invention discloses a hapten, an artificial antigen, and their applications for detecting paclobutrazol. The paclobutrazol hapten prepared by this invention retains the characteristic structure of paclobutrazol to the greatest extent. The paclobutrazol polyclonal antibody prepared using the paclobutrazol hapten of this invention has high potency and specificity, with a half-maximum inhibitory concentration (WMC) of 0.31 ng / mL for paclobutrazol and a linear range of 0.03–19.63 ng / mL. Furthermore, it shows no cross-reactivity with other structural and functional analogs such as uniconazole and hexaconazole.

[0047] This invention simplifies the synthesis steps of paclobutrazol haptens, providing a simple and low-cost method for preparing paclobutrazol artificial antigens and their antibodies. It offers a novel paclobutrazol hapten with a high degree of overlap in its scaffold structure with the analyte paclobutrazol, effectively improving the immunogenicity of the paclobutrazol hapten-carrier protein conjugate. The paclobutrazol artificial antigen obtained by conjugating the carrier protein has a significant structural difference from the hapten, creating substantial steric hindrance and further enhancing antibody affinity. Simultaneously, this invention establishes a more efficient immunoassay method, capable of meeting the requirements for large-scale on-site sample testing and possessing broad application prospects. Attached Figure Description

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

[0049] Figure 2 The image shows the results of ultraviolet full-wavelength scanning identification of PBZ-4C, PBZ-4C-BSA, and BSA.

[0050] Figure 3 The image shows the results of ultraviolet full-wavelength scanning identification of PBZ-4C, PBZ-4C-OVA, and OVA.

[0051] Figure 4 This is a standard curve for an indirect competitive ELISA used to detect paclobutrazol.

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

[0053] Figure 6 The result interpretation diagram for the paclobutrazol colloidal gold immunochromatographic test strip is as follows: A: Negative, B: Positive, C: Invalid, D: Invalid. Detailed Implementation

[0054] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

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

[0056] Example 1: Synthesis and Identification of Paclobutrazol Hapten

[0057] I. Experimental Methods

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

[0059] The synthetic route of paclobutrazol hapten PBZ-4C is as follows: Figure 1 As shown, 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)pentane-3-ol as the starting material, the starting material and sodium hydroxide in a molar ratio of 1:2 were fully dissolved in dimethyl sulfoxide. Then, mercaptobutyric acid in a molar ratio of 1:1.5 was added, and the entire system was stirred at 70 °C for 12 h.

[0061] The reaction was detected by TLC using dichloromethane-petroleum ether (5:4, v / v) as the developing solvent. After the reaction, 20 mL of distilled water was added to dissolve the product, and a small amount of 3M NaOH solution was added to adjust the pH to approximately 10.0. Then, ethyl acetate was added, and after thorough shaking, the mixture was extracted 2–3 times. The ethyl acetate layer was discarded, and the aqueous layer was collected. A small amount of 3M HCl solution was added to the aqueous layer to adjust the pH to approximately 3.0, and ethyl acetate was added. After thorough shaking, the mixture was extracted 2–3 times, and the aqueous layer was discarded. The combined ethyl acetate layers were collected. 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 the concentrated product. The concentrated product was dissolved in ethyl acetate, and TLC was performed using dichloromethane-petroleum ether (5:4, v / v) as the developing solvent. The product was then purified by column chromatography to obtain the paclobutrazol hapten PBZ-4C.

[0062] The paclobutrazol hapten PBZ-4C was identified by proton nuclear magnetic resonance (NMR) and mass spectrometry.

[0063] II. Experimental Results

[0064] The 1H NMR spectrum of the paclobutrazol hapten PBZ-4C is 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,2 H), 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 spectrometry results for the paclobutrazol hapten PBZ-4C were: MS: 377.2, ESI: 4.5. + [MH] + 378.6.

[0066] The mass spectrometry and NMR results show 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 was named systematically 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 Methods

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

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

[0074] Slowly add 200 μL of solution A dropwise to 1 mL of solution B, and react at 4°C for 12 h. After the reaction is complete, transfer the reaction solution to a dialysis bag and dialyze with PBS buffer for 3 days, 3 times a day. After dialysis, collect the solution from the dialysis bag, which is the paclobutrazol artificial antigen PBZ-4C-BSA. Aliquot the solution into centrifuge tubes and store at -20°C for later use.

[0075] The PBS buffer formula is as follows: Na2HPO4·12H2O 2.90g, NaCl 8.50g, KCl 0.20g, KH2PO4 0.20g, and distilled water is added to bring the volume to 1000mL.

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

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

[0078] II. Experimental Results

[0079] The results of ultraviolet full-wavelength scanning identification are as follows: Figure 2 and Figure 3 As shown, comparing the highest absorbance values ​​of BSA and OVA before and after coupling reveals that the absorption curves of PBZ-4C-BSA and PBZ-4C-OVA are significantly different from those of BSA and OVA, respectively. At 280 nm, the absorption curves of PBZ-4C-BSA and PBZ-4C-OVA show varying degrees of shift from the absorption curves of BSA and OVA, respectively.

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

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

[0082]

[0083] Where Z represents 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 (structural formula as shown in formula (II), where Z is bovine serum albumin) prepared in Example 2 was mixed and emulsified with an immune adjuvant (Frederick complete adjuvant for primary immunization and Frederick incomplete adjuvant for booster immunization) at a volume ratio of 1:1 and set aside for later use.

[0087] For the initial immunization, two healthy 6-week-old New Zealand white rabbits (one male and one female) were injected subcutaneously at multiple points on their backs. Each rabbit received an immunization dose of 500 μg of immune protein. Booster immunizations were then administered every three weeks for a total of four booster immunizations.

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

[0089] 2. Acquisition of polyclonal antibodies

[0090] One week after the fifth immunization, blood was collected from the heart of the rabbits. The rabbit blood was incubated at 37°C for 2 hours and then left overnight (12 hours) at 4°C. The next day, the supernatant was collected and centrifuged at 3000 r / min for 10 minutes at 4°C to remove the precipitate. The supernatant was obtained, which was 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 (structural formula shown in formula (II), where Z is bovine serum albumin) prepared in Example 2 was used as an immunogen. It was diluted to 50 μL with PBS at a dose of 50 μg equivalent per animal and mixed with 50 μL of Freund's adjuvant in a 2.5 mL syringe. The mixture was emulsified with an emulsifier until the emulsion did not diffuse on the water surface during the test. It was then set aside for later use.

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

[0095] Table 1. Freund's adjuvant immunization regimen for Balb / C mice

[0096] Immune phase Immunization time Dosage (μg / animal) Injection method Freund's adjuvant First immunization 4 weeks 50 Multiple subcutaneous injections in the abdomen and back Freund's complete adjuvant Strengthen immunity 3 weeks 50 Multiple subcutaneous injections in the abdomen and back Freund's incomplete adjuvant Strengthen immunity 3 weeks 50 Multiple subcutaneous injections in the abdomen and back Freund's incomplete adjuvant Strengthen immunity 3 weeks 50 Multiple subcutaneous injections in the abdomen and back Freund's incomplete adjuvant Strengthen immunity 3 weeks 50 Multiple subcutaneous injections in the abdomen and back Freund's incomplete adjuvant Sprint Immunity 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 mice, centrifuged, and antiserum was obtained. The antibody titer and inhibition rate of the antiserum were examined using icELISA. The results are shown in Table 2.

[0099] Table 2. Antibody titers and inhibition rates of monoclonal antibodies.

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

[0101] 3. Cell fusion

[0102] (1) Myeloma cell resuscitation and collection

[0103] Myeloma cells were removed from the -80°C freezer and quickly placed in a 37°C water bath to thaw. The thawed cells were centrifuged at 1000 rpm for 7 min and the cell cryopreservation solution was discarded. The basal culture medium was aspirated with a pipette to disperse the cells. After dispersion, the cells were centrifuged at 1000 rpm for 7 min and the basal culture medium was discarded. The cells were then aspirated with a pipette and mixed with the complete culture medium before being placed in a 9 cm culture dish for scale-up culture.

[0104] On the day of cell fusion, the supernatant of myeloma cells in the culture dish was removed. 25 mL of fresh basal culture medium was added using a bent pipette, and the adherent myeloma cells were repeatedly blown up in a specific direction until the bottom of the culture plate became clear and no longer blurred. All culture medium was collected, the plate was sealed, and centrifuged at 1000 rpm for 8 minutes. After centrifugation, the supernatant was discarded, yielding the myeloma cells.

[0105] (2) Strengthen immunity

[0106] Three days before cell fusion, mice were given a booster immunization at a concentration of 1 mg / mL and a volume of 100 μL, without the need for adjuvants.

[0107] (3) Obtaining immune spleen cells

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

[0109] The immunized mice were euthanized by removing their eyeballs, and blood was collected. After soaking in 75% ethanol for 4 minutes, the blood was transferred to a clean bench, and the spleen was removed.

[0110] Place the removed spleen in a disposable cell grinding mesh in a culture dish. First, use a syringe to draw up culture medium and inject it into the spleen. Repeat the injection to blow out the cells until the spleen changes color from red to transparent. Then, gently grind the spleen on the cell grinding mesh with the plunger of the syringe and rinse the mesh with culture medium.

[0111] Collect all the culture medium, seal it, centrifuge at 1000 r / min for 7 min, discard the supernatant, and you will get immune spleen cells.

[0112] (4) Cell fusion

[0113] Myeloma cells and immune spleen cells were mixed, added to 25 mL of basal culture medium, sealed, and centrifuged at 1000 r / min for 7 min. The supernatant was then discarded.

[0114] Gently loosen the precipitated cells with your fingers. Place the centrifuge tube in 37°C warm water. Using a pipette tip, add 1 mL of preheated 37°C polyethylene glycol (PEG2000) and slowly add it to the precipitated cells over 1 minute, gently stirring after each drop to ensure even mixing. Let stand for 30 seconds. Preheat the basal medium and add 1.5 mL of basal medium over 1 minute, stirring gently along the wall. Continue adding basal medium until a total of 20 mL is reached over 5 minutes, stirring gently up and down. Seal the centrifuge tube and centrifuge at 1000 rpm for 7 minutes. Discard the supernatant and add the precipitate to 200 mL of complete culture medium. Gently aspirate the liquid using a bent pipette and gently stir the cells. Spread 2 mL of the complete culture medium containing confluent cells into 24-well plates and incubate at 37°C in a 5% CO2 cell culture incubator.

[0115] (5) Hybridoma cell screening and identification

[0116] Starting from day 7 of fusion cell culture, cell growth and culture medium status should be observed daily.

[0117] Table 3 Monoclonal cell culture and screening

[0118]

[0119]

[0120] As shown in Table 3, on day 10, or when cell growth was too rapid, the medium was partially replaced with HAT medium; on day 13, the medium was completely replaced with HAT medium; on day 15, the medium was partially replaced with HT medium, and the cell supernatant was subjected to the first icELISA test to determine whether cell fusion had occurred and the degree of fusion; on day 17, the medium was completely replaced with HT medium, and the second icELISA test was performed; on day 19, the medium was partially replaced with HT medium, and the third icELISA test was performed. The results of the first two supernatant tests were compared, and wells with high titers were selected for limiting dilution. When performing icELISA on the limitingly diluted cell supernatant, titer and inhibition rate were used as indicators. After selecting positive wells, the next round of limiting dilution was performed until every well in every plate was positive and the titer and inhibition rate were similar. At this point, the hybridoma cell line was successfully established and promptly cryopreserved. The hybridoma cells obtained from the expanded culture were collected and used for animal immunization to obtain ascites fluid. After identification and purification, paclobutrazol monoclonal antibody was obtained.

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

[0122] The paclobutrazol polyclonal antibody prepared in Example 3, and the paclobutrazol artificial antigen PBZ-4C-OVA (structural formula shown in formula (II), where Z is chicken ovalbumin) prepared in Example 2, were used as coating antigens. The optimal combination of immunogen and coating antigen was selected by measuring serum titers and inhibition rates obtained through an indirect competitive ELISA method. The specific operating steps are as follows:

[0123] 1. Paclobutrazol artificial antigen PBZ-4C-BSA (structural formula as shown in formula (II), where Z is bovine serum albumin) and PBZ-4C-OVA (structural formula as shown in formula (II), where Z is chicken ovalbumin) were diluted to a concentration of 250 ng / mL with coating buffer (0.05 M carbonate buffer, pH 9.6), 100 μL / well was used to coat 96-well microplates, and incubated overnight at 37°C. The coating buffer was discarded, and the plates were washed twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)).

[0124] 2. Add 120 μL of blocking buffer (pH 7.3, 0.2 mol / L phosphate buffer containing 2% casein) to each well, block at 37°C for 3 h, discard the blocking buffer, plate, and dry in an oven at 37°C for later use.

[0125] 3. The paclobutrazol polyclonal antibody prepared in Example 3 was diluted with PBST at concentrations of 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000, respectively. Blank control wells (using PBST) were also prepared. The 1 mg / mL paclobutrazol standard was diluted 1000 times with PBST to a concentration of 1 μg / mL.

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

[0127] 5. For the inhibition column, first add 50 μL of paclobutrazol standard to each well, then add 50 μL of paclobutrazol polyclonal antibody at different dilution ratios to each well sequentially. Do not add paclobutrazol polyclonal antibody to the last well, and replace it with 50 μL of PBST.

[0128] Incubate at 6.37℃ for 40 minutes, wash 5 times, and then plate.

[0129] 7. Add goat anti-rabbit secondary antibody-HRP (5000-fold dilution), incubate at 37℃ for 30 minutes, wash 5 times, and plate.

[0130] 8. Add the color developing solution and incubate at 37°C for 10 minutes;

[0131] 9. Terminate the reaction by adding 10% (v / v) H₂SO₄ solution and read the OD value at 450 nm; calculate the titer and inhibition rate. The titer is OD₂O₅. 450 The dilution factor of paclobutrazol polyclonal antibody corresponding to approximately 1.0 is given by: Inhibition rate = (OD value of titer - OD value of inhibition) / OD value of inhibition × 100%.

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

[0133] Table 4. Screening results of immunogens and coating agents.

[0134] serial number Immunogen Envelope valence 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 shown in Table 4, the antisera 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) all exhibited certain titers, and the resulting antisera all showed varying degrees of inhibitory effects on the target analyte paclobutrazol. Among them, the combination of immunogen and coating agent number 2, with an antisera titer of 1:128000 and an inhibition rate of 85.82%, represents the optimal combination. Under this combination, the paclobutrazol polyclonal antibody not only recognized the target analyte paclobutrazol but also demonstrated good antibody sensitivity. Therefore, the combination of immunogen and coating agent number 2 is the optimal combination, namely, using PBZ-4C-BSA (structural formula shown in formula (II), where Z is bovine serum albumin) as the immunogen and PBZ-4C-OVA (structural formula shown in formula (II), where Z is chicken ovalbumin) as the coating agent.

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

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

[0138] 1. The paclobutrazol artificial antigen PBZ-4C-OVA (structural formula shown in formula (II), where Z is chicken ovalbumin) prepared in Example 2 was used as the coating antigen. It was diluted to 1 μg / mL with coating buffer (0.05M carbonate buffer, pH 9.6), and 100 μL / well was used to coat a 96-well microplate. The plate was incubated overnight (12 h) at 37°C.

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

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

[0141] 4. Discard the sealing solution, tap the plate, dry at 37℃ for 30 minutes, then remove and pack in a self-sealing bag for later use;

[0142] 5. Dilute the paclobutrazol polyclonal antibody prepared in Example 3 with PBST at a ratio of 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 solution to each row (four sets in parallel), then add 50 μL / well of 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℃ for 30 min, wash 5 times, and pat dry;

[0145] 8. Add 100 μL of colorimetric reagent to each well and develop the color for 10 min;

[0146] 9. Terminate the reaction by adding 50 μL of 10% (v / v) H₂SO₄ solution, and read the OD value at 450 nm. Plot the ELISA standard curve based on the OD values:

[0147] Plot B / B0 as the ordinate (B represents the absorbance OD of paclobutrazol standards at different concentrations). 450 B0 is the absorbance value (OD) of the blank control well. 450 The logarithm of the standard concentration is used as the abscissa. A logistic function is used for curve fitting to obtain the formula for the standard curve, and the standard curve is then prepared. (Example:) Figure 4 As shown. The half-maximal inhibitory concentration (IC50) of the paclobutrazol polyclonal antibody was calculated based on the standard curve. 50 The effective concentration 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] I. Experimental Methods

[0150] The specificity of paclobutrazol polyclonal antibodies was determined by cross-reactivity experiments with paclobutrazol and its analogues. Antibody specificity was expressed as cross-reactivity rate (CR); the lower the CR, the stronger the specificity. Paclobutrazol and its analogues were serially diluted and measured using an indirect competitive ELISA method, following the same procedure as in Example 6, to obtain the IC50 values ​​for each analogue. 50 The cross-reactivity ratio (CR) of paclobutrazol is calculated using the following formula:

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

[0152] II. Experimental Results

[0153] Table 5 shows the cross-reactivity results of paclobutrazol and its analogues. It can be seen that the cross-reactivity rate of paclobutrazol polyclonal antibodies to paclobutrazol is 100%, IC50... 50 The concentration was 0.31 ng / mL, and the cross-reactivity with paclobutrazol, hexaconazole, tebuconazole, and triadimefon was <0.01%, indicating that the polyclonal antibody used to detect paclobutrazol has high specificity and can effectively eliminate the interference of its analogues on the detection of paclobutrazol, and can be used specifically for the detection of paclobutrazol.

[0154] Table 5. Cross-reactivity results of paclobutrazol and its analogues

[0155] name <![CDATA[IC 50 (ng / mL)]]> CR (100%) Paclobutrazol 0.31 100％ uniconazole NR NR Hexaconazole NR NR Tebuconazole NR NR Triazolone NR NR

[0156] Example 8: An ELISA kit for detecting paclobutrazol

[0157] 1. Composition

[0158] (1) ELISA plate coated with coating antigen

[0159] The enzyme-labeled plate was prepared by the following method:

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

[0161] (2) Standard products

[0162] Eleven paclobutrazol standard solutions of different concentrations were prepared, with concentrations of 10000 ng / mL, 2000 ng / mL, 400 ng / mL, 80 ng / mL, 16 ng / mL, 3.2 ng / mL, 0.64 ng / mL, 0.128 ng / mL, 0.0256 ng / mL, 0.00512 ng / mL, and 0.001024 ng / mL.

[0163] (3) Antibodies

[0164] Example 3: A polyclonal antibody prepared using paclobutrazol artificial antigen PBZ-4C-BSA (structural formula as shown in formula (II), where Z is bovine serum albumin) as an immunogen; or Example 4: A monoclonal antibody prepared using paclobutrazol artificial antigen PBZ-4C-BSA (structural formula as shown in formula (II), where Z is bovine serum albumin) as an immunogen.

[0165] (4) Enzyme conjugate concentrate

[0166] Horseradish peroxidase-labeled goat anti-rabbit antibody or goat anti-mouse antibody;

[0167] (5) Substrate and colorimetric solution

[0168] It consists of solution A and solution B, where solution A is urea peroxide and solution B is tetramethylbenzidine;

[0169] (6) Termination solution

[0170] A 10% (v / v) H2SO4 solution;

[0171] (7) Washing liquid

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

[0173] (8) Enzyme conjugate diluent

[0174] 0.2 mol / L phosphate buffer.

[0175] 2. How to use

[0176] (1) Sample testing

[0177] Number the microwells corresponding to the test samples and standards sequentially, and prepare two parallel wells for each test sample and standard. Record the positions of the standard wells and sample wells. Dilute the enzyme conjugate concentrate with enzyme conjugate diluent at a volume ratio of 1:10 (i.e., add one part enzyme conjugate concentrate to 10 parts enzyme conjugate diluent, prepare fresh before use) to obtain the enzyme conjugate working solution.

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

[0179] Shake off the liquid in the well and add 250 μL of washing solution per well. Wash thoroughly 4-5 times, with 10-second intervals between each wash. Discard the washing solution in the well and pat dry with absorbent paper (any air bubbles not removed after patting can be popped with an unused pipette tip).

[0180] Add 50 μL of substrate chromogenic solution A to each well, then add 50 μL of substrate chromogenic solution B to each well. Gently vortex to mix, cover with a cover plate membrane, and incubate at 25°C in the dark for 10 min. Add 50 μL of stop solution to each well, gently vortex to mix, set the microplate reader to 450 nm, and measure the OD value of each well.

[0181] (2) Plotting the standard curve

[0182] ELISA standard curves were plotted based on the OD values ​​of paclobutrazol standards at 450 nm, using the following method:

[0183] Plot B / B0 as the ordinate (B represents the absorbance OD of paclobutrazol standards at different concentrations). 450 B0 is the absorbance value (OD) of the blank control well. 450 The logarithm of the standard concentration is used as the x-axis. The Logistic function is used for curve fitting to obtain the formula for the standard curve, and the standard curve is prepared.

[0184] (3) Calculation of sample concentration

[0185] OD of the sample to be tested 450 Substituting the average value into the formula of the standard curve above, we obtain the concentration of the sample to be tested. Multiplying it by the corresponding dilution factor gives the actual concentration of paclobutrazol in the sample.

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

[0187] 1. Preparation of gold-labeled antibody and gold-labeled conjugate pad

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

[0189] First, the pH of the colloidal gold was adjusted to 8.5 with 0.2 mol K2CO3 solution. 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 used for labeling to obtain the gold-labeled antibody, which was stored at 4°C.

[0190] like Figure 5 As shown, the colloidal gold rapid test strip is composed of an NC membrane (nitrocellulose membrane), a gold label conjugate pad, a sample pad, an absorbent pad, and a PVC base plate stacked together.

[0191] Using an XYZ-3000 three-dimensional spraying apparatus, a 4% BSA solution was sprayed onto glass wool at a rate of 8 μL / cm. The mixture was then dried in a drying oven at 42°C for 50 min. Next, a gold-labeled antibody was sprayed onto the glass wool at a rate of 6 μL / cm. After drying in a drying oven at 42°C for 50 min, the gold-labeled conjugate pad was obtained and then vacuum dried for storage.

[0192] 2. Cellulose membrane coated with conjugated goat anti-rabbit antigen

[0193] Using an XYZ-3000 three-dimensional spray membrane instrument, a coating agent (PBZ-4C-OVA (structural formula as shown in formula (II), where Z is chicken oocyte albumin)) with a concentration of 1 mg / mL was sprayed at a rate of 1.2 μL / cm onto the right side of the cellulose membrane in a direction perpendicular to the long side of the cellulose membrane, as a detection line (i.e., T line).

[0194] Using an XYZ-3000 three-dimensional spray membrane apparatus, goat anti-rabbit IgG with a concentration of 120 μg / L was sprayed at a rate of 1.2 μL / cm onto the left side of the cellulose membrane in a direction perpendicular to the long side, serving as the control line (i.e., line C). The two lines were spaced 8 mm apart, thus obtaining a cellulose membrane coated with goat anti-rabbit conjugated antigen.

[0195] 3. Assembly of rapid test strips

[0196] Attach the cellulose membrane to the center of the PVC base plate; attach the absorbent pad to the left side of the cellulose membrane with a 1mm overlap; attach the gold conjugate pad to the right side of the cellulose membrane with a 1mm overlap; attach the sample pad to the right side of the gold conjugate pad with a 2mm overlap, thus obtaining the colloidal gold test strip. Cut the colloidal gold test strip into 3.05mm wide pieces using a cutting machine.

[0197] 4. Preparation of the test solution

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

[0199] 5. Test strip detection and interpretation

[0200] The test solution is added to the sample pad and left to stand. The test solution carries the analyte and the gold-labeled antibody in the gold-labeled conjugate pad to diffuse towards the cellulose membrane through the siphon effect and eventually penetrates into the absorbent pad.

[0201] Judgment result: such as Figure 6 As shown in A to D, the gold-labeled antibody binds to the invisible control line (i.e., line C) on the cellulose membrane, making the control line (i.e., line C) appear red, indicating a valid test result. If the control line (i.e., line C) does not appear, it indicates an invalid test result.

[0202] If the sample contains paclobutrazol, it 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 detection line (the conjugate of hapten and carrier protein) on the cellulose membrane. This causes the invisible detection line (i.e., the T line) to be unspotted or very weakly spotted, indicating a positive or weakly positive result for the sample. If the sample does not contain paclobutrazol, the gold-labeled antibody will show a clear red line when it encounters the invisible detection line (i.e., the T line) during its upward movement, indicating a negative result for the sample.

[0203] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A paclobutrazol artificial antigen combination, characterized in that, It comprises an immunogen and a coating antigen, both of which are derived from a paclobutrazol hapten conjugate carrier protein, the structural formula of which is shown in formula (II). Formula (II); Wherein Z is a carrier protein, the carrier protein on the immunogen is bovine serum albumin, and the carrier protein on the coating antigen is chicken oocyte albumin.

2. The use of the paclobutrazol artificial antigen combination according to claim 1 in the preparation of reagents and / or kits for detecting paclobutrazol.

3. A kit for detecting paclobutrazol, characterized in that, The kit comprises the paclobutrazol artificial antigen combination as described in claim 1.

4. A method for detecting paclobutrazol, characterized in that, The detection is performed using the artificial antigen combination as described in claim 1, wherein the detection is for the purpose of non-disease treatment diagnosis.

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