N-(naphthalene-1-yl) phenazine-1-formamide hapten, antigen, antibody, colloidal gold test strip and application

Abstract

The invention discloses an N-(naphthalene-1-yl) phenazine-1-formamide hapten, an antigen, an antibody, a colloidal gold test strip and application. The hapten has a molecular structural formula as shown in a formula I, and the molecular formula and the molecular weight of the hapten are C23H16N4O, 364.41.

Description

Technical Field

[0001] The invention belongs to the technical field of new agricultural compound immunoassay, and particularly relates to an N-(naphthalene-1-yl)phenazine-1-carboxamide hapten, antigen, antibody, colloidal gold test strip and application thereof. Background Art

[0002] Rice sheath blight is one of the three major diseases of rice. The pathogen has a wide range of hosts, is both parasitic and corrosive, has a wide nutrient zone, is highly resistant to stress, and is a soil-borne disease with strong concealment. It is very difficult to control when the disease occurs. In production, the commonly used prevention and control methods mainly include agricultural prevention, physical prevention, chemical prevention, biological prevention, etc., among which chemical prevention is the most effective measure and can be controlled in a short time. However, the long-term use of a single agent, such as Jinggangmycin, also has the risk of drug resistance. Therefore, it is urgent to explore new alternatives to develop potential fungicides. The present invention has been modified in the early stage to obtain a new fungicide compound N-(naphthalen-1-yl)phenazine-1-carboxamide (NNPCN), which has a strong inhibitory effect on sheath blight bacteria and has broad prospects for development into a candidate drug for rice sheath blight. The safety of this type of new fungicide compound to the environment is unknown, so it is very important to strengthen detection. At present, the detection of fungicides is mostly carried out by laboratory chromatographs, such as gas chromatography, liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, etc. However, the detection cost is high, the time is long, and there are many steps. The requirements for technicians and the environment are high, which limits its promotion and application, and it is difficult to popularize it at the grassroots level. The use of immunological detection methods not only has the advantages of strong specificity and high sensitivity, but also low cost, simple operation, fast time, and low dependence on instruments. It has become a hot spot for research in recent years. Based on this, no reports on NNPCN immunological detection have been found through literature research so far, and it itself is not a hapten. It will not produce antibodies when directly inoculated into animals. Considering the above points, it is necessary to design and develop a suitable NNPCN hapten, and thus establish a corresponding NNPCN rapid detection method, so as to realize immunological detection and then judge the NNPCN content in the environment, which has a high practical application value. Summary of the invention

[0003] The present invention aims to overcome the problems of the prior art and provide a N-(naphthalen-1-yl)phenazine-1-carboxamide (NNPCN) hapten, antigen, antibody, colloidal gold test strip and application. In order to achieve the above purpose, the technical solution provided by the present invention is:

[0004] One of the technical problems to be solved by the present invention is to provide a new bactericidal compound hapten. The molecular formula is C 23 H 15 N3O (molecular weight 349) has no active groups in its molecular structure that can be directly coupled to the carrier. It is necessary to first perform chemical modification at the appropriate position of its structure to introduce a suitable linker and an active group for coupling with the carrier protein.

[0005] The present invention first hydrolyzes phenazine-1-carboxamide under acidic conditions to obtain compound 1, then performs condensation acylation reaction on compound 1 and 6-nitronaphthalene-1-amine to obtain compound 2, and finally obtains the target product N-(naphthalene-1-yl)phenazine-1-carboxamide hapten (NNPCN-NH2, synthesis route shown in formula II) through oxidation-reduction reaction. The specific synthesis steps are as follows: phenazine-1-carboxamide (0.01 mol) and 10 mL of 30% hydrochloric acid are added to 10 mL of dimethyl sulfoxide, 2 mL of sodium nitrite aqueous solution (6 mol / L) is slowly added dropwise while stirring continuously, and stirred in an ice alcohol bath for 4 hours, and the reaction temperature is kept below 0°C during the entire reaction process. After the reaction, 60 mL of water is added thereto, stirred at low temperature until no solid dissolves, and then suction filtered, and the obtained solid is washed with water 4 times (25 mL each time) to obtain a light yellow solid compound 1. Compound 1 was placed in a 10 mL sample bottle and aniline (0.001 mol), 6-nitro-1-naphthylamine (0.001 mol), trifluoroacetic acid borate (0.002 mol) and 2 mL acetonitrile were added, and stirred at 80 ° C for 24 h on a constant temperature magnetic stirrer. Then 3 mL chloroform and 0.5 mL water were added to terminate the reaction to obtain a crude compound 2. Then 150 mg of ion exchange resins Amberlyst A-26, Amberlyst 15 and mberlylite IRA743 were added to the crude product in sequence, stirred at room temperature for 30 min and filtered, and solid magnesium sulfate and chloroform were added to the mixture in sequence to remove moisture and excess solid magnesium sulfate. Finally, rotary evaporation was performed to generate an orange-red solid compound 2. Compound 2 was dissolved in 10 mL ethanol, 5 mmol SnCl2 and 5 mL concentrated HCl were added in sequence, heated and stirred for 6 h, and the pH of the solution was adjusted to 7 after cooling. The resulting solution was extracted, dried, and distilled under reduced pressure to obtain a red solid product NNPCN-NH2 (C 23 H 16 N4O, 364.41), stored at 4°C in the dark.

[0006]

[0007] The second technical problem to be solved by the present invention is to provide an artificial antigen. The glutaraldehyde method is mainly used to couple NNPCN-NH2 with bovine serum albumin (BSA) and ovalbumin (OVA) to prepare NNPCN immune antigen (NNPCN-BSA) and coating antigen (NNPCN-OVA); SDS-PAGE detection and ultraviolet spectrum scanning show that the immune antigen and coating antigen bands are dragged and move backward, and the ultraviolet absorption peak of the immune antigen is 203nm and the ultraviolet absorption peak of the coating antigen is 198nm, which are different from the ultraviolet absorption peaks of their respective carrier proteins and NNPCN, indicating that the NNPCN immune antigen and coating antigen are successfully prepared.

[0008] The third technical problem to be solved by the present invention is to provide a new bactericidal compound NNPCN antibody. New Zealand male white rabbits were mainly immunized to obtain NNPCN rabbit serum polyclonal antibodies with titers greater than 100,000 before and after purification; the polyclonal antibodies were purified by ammonium sulfate precipitation method, and after SDS-PAGE detection, the results showed that the purified antibodies had clear bands near 25kDa and 50kDa, proving that the polyclonal antibodies were well purified.

[0009] The fourth technical problem to be solved by the present invention is to provide a rapid immune colloidal gold test strip. The colloidal gold test strip mainly consists of a PVC base plate, a sample pad, a colloidal gold pad, a T control line, a C control line, an NC film, and a water-absorbing pad, and can quickly detect residual NNPCN in the soil within 10-15 minutes.

[0010] The polyclonal antibody of the present invention can also be used to prepare other preparations for detecting N-(naphthalene-1-yl)phenazine-1-carboxamide.

[0011] Compared with the prior art, the present invention has the following advantages:

[0012] 1. The present invention synthesized the new bactericidal compound NNPCN hapten for the first time, clarified its structure and characteristics, and provided a material basis for further preparation of antibodies.

[0013] 2. For the first time, we successfully created a new bactericidal compound NNPCN artificial antigen and identified its effectiveness, laying the foundation for the subsequent preparation of NNPCN polyclonal antibodies.

[0014] 3. The present invention has developed a polyclonal antibody against a new bactericidal compound NNPCN, which has been tested to have good stability, high purity, excellent potency, and strong specificity.

[0015] 4. The present invention provides a colloidal gold immune test strip for detecting NNPCN, which enriches the NNPCN detection method.

[0016] This provides a new idea for rapid detection of NNPCN.

[0017] In summary, the present invention includes: synthesizing NNPCN-NH2 hapten, creating complete antigen, assembling polyclonal antibody, colloidal gold immunoassay, etc.; the NNPCN-NH2 hapten is connected to the active group amino group at a position away from the phenazine group on NNPCN by using condensation acylation of carboxylic acid and amino group, and the structure of NNPCN-NH2 is characterized by nuclear magnetic resonance and liquid chromatography-mass spectrometry to obtain pure NNPCN-NH2 hapten; NNPCN-NH2 is hydrated with bovine serum albumin (BSA) by glutaraldehyde method. albumin (BSA) and ovalbumin (OVA) were coupled to prepare NNPCN immune antigen (NNPCN-BSA) and coating antigen (NNPCN-OVA); SDS-PAGE detection and ultraviolet spectrum scanning showed that the immune antigen and coating antigen bands were dragged and moved backward, and the ultraviolet absorption peaks of the immune antigen at 203nm and the coating antigen at 198nm were different from their respective carrier proteins and NNPCN ultraviolet absorption peaks, indicating that the NNPCN immune antigen and coating antigen were successfully prepared; New Zealand male white rabbits were immunized to obtain NNPCN rabbit serum polyclonal antibodies with titers greater than 100,000 before and after purification; the polyclonal antibodies were purified by ammonium sulfate precipitation method, and the results of SDS-PAGE detection showed that the purified antibodies had clear bands near 25kDa and 50kDa, and the polyclonal antibodies with good purity were obtained; the IC of the polyclonal antibodies of the present invention to NNPCN 50 The value was 21.42 μg / mL, the cross-reaction rate of phenazine-1-carboxylic acid was 12.92%, there was no cross-reaction with other agents, and the specificity was good; therefore, the immunoassay method established by the antibody of the present invention can be applied to the rapid detection of NNPCN in the environment, and has good development potential and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the synthetic route of NNPCN-NH2;

[0019] Figure 2 It is the compound NNPCN-NH21HNMR verification;

[0020] Figure 3 LC-MS verification of compound NNPCN-NH2;

[0021] Figure 4 This is the synthesis route of NNPCN complete antigen and coating antigen; in the figure: R is BSA or OVA;

[0022] Figure 5The electrophoresis diagram of NNPCN immune antigen and coating antigen protein; in the figure: (A) NNPCN-BSA protein electrophoresis diagram; (B) NNPCN-OVA protein electrophoresis diagram; (1) Marker; (2-5) 2×BSA, 1×BSA, 2×NNPCN-BSA, 1×NNPCN-BSA; (6) Marker; (7-10) 1×OVA, 2×OVA, 2×NNPCN-OVA, 1×NNPCN-OVA;

[0023] Figure 6 The UV spectra of NNPCN immune antigen and coating antigen are scanned; in the figure: (A) UV spectra of NNPCN-BSA; (B) UV spectra of NNPCN-OVA;

[0024] Figure 7 This is the electrophoresis diagram of the purified NNPCN rabbit serum polyclonal antibody protein; in the figure: (1) Marker; (2) Purified rabbit serum antibody protein No. Ⅰ; (3) Purified rabbit serum antibody protein No. Ⅲ; (4) Unpurified rabbit serum antibody protein No. Ⅰ; (5) Unpurified rabbit serum antibody protein No. Ⅲ;

[0025] Figure 8 is the standard curve of NNPCN polyclonal antibody ic-ELISA;

[0026] Fig. 9 For the preparation of colloidal gold;

[0027] Fig.10 It is a colloidal gold chromatography immunoassay strip structure;

[0028] Fig.11 This is a schematic diagram of the colloidal gold result judgment;

[0029] Fig.12 This is the result of immunochromatographic colloidal gold test. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below in conjunction with specific tests and embodiments. The percentages involved in the tests and embodiments of the present invention are all mass percentages.

[0031] Example 1: Synthesis of N-(naphthalen-1-yl)phenazine-1-carboxamide immune hapten

[0032] (1) Test reagents

[0033] Anhydrous ethanol, Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd.; acetonitrile, Sinopharm Chemical Reagent Co., Ltd.; analytical grade hydrochloric acid, Sinopharm Chemical Reagent Co., Ltd.; sodium nitrite, Sinopharm Chemical Reagent Co., Ltd.; phenazine-1-carboxamide, Shanghai Qinba Chemical Co., Ltd.; 6-nitronaphthalene-1-amine, Henan Weitixi Chemical Technology Co., Ltd.; stannous chloride, Sinopharm Chemical Reagent Co., Ltd.; 1-naphthylamine, Shanghai MacLean Biochemical Technology Co., Ltd.; aniline, Merck; trifluoroacetic acid borate, Shanghai Aladdin Biochemical Technology Co., Ltd.; magnesium sulfate, Sinopharm Chemical Reagent Co., Ltd.; chloroform, Sinopharm Chemical Reagent Co., Ltd.

[0034] (2) Test instruments

[0035] ZNCL-S-5D multi-point intelligent magnetic stirrer, Henan Aibote Technology Development Co., Ltd.; RE-52A rotary evaporator, Shanghai Yarong Biochemical Equipment and Instrument Co., Ltd.; SHZ-D(Ⅲ) circulating water vacuum pump, Shanghai Qiangqiang Instrument and Equipment Co., Ltd.

[0036] (3) Synthesis and characterization of NNPCN-NH2

[0037] Synthesis of NNPCN-NH2: First, phenazine-1-carboxamide is hydrolyzed under acidic conditions to obtain compound 1, and then compound 1 is subjected to condensation acylation reaction with 6-nitronaphthalene-1-amine to obtain compound 2. The nitro group in compound 2 is subjected to redox reaction to finally obtain the target product NNPCN-NH2 ( Figure 1 ). The specific synthesis steps are as follows:

[0038] Phenazine-1-carboxamide (0.01 mol) and 10 mL of 30% hydrochloric acid were added to 10 mL of dimethyl sulfoxide, and 2 mL of an aqueous solution of sodium nitrite (6 mol / L) was slowly added dropwise while stirring continuously. The mixture was stirred in an ice-alcohol bath for 4 h. The reaction temperature was kept below 0°C during the entire reaction process. After the reaction, 60 mL of water was added, and the mixture was stirred at low temperature until no solid dissolved. The mixture was then filtered and the obtained solid was washed with water 4 times (25 mL each time) to obtain a light yellow solid compound 1.

[0039] Compound 1 was placed in a 10 mL sample bottle and aniline (0.001 mol), 6-nitro-1-naphthylamine (0.001 mol), trifluoroacetic acid borate (0.002 mol) and 2 mL acetonitrile were added, and stirred at 80 ° C for 24 h on a constant temperature magnetic stirrer. Then 3 mL chloroform and 0.5 mL water were added to terminate the reaction to obtain a crude product of compound 2. Then 150 mg of ion exchange resins Amberlyst A-26, Amberlyst 15 and mberlylite IRA743 were added to the crude product in sequence, stirred at room temperature for 30 min and filtered, and solid magnesium sulfate and chloroform were added to the mixture in sequence to remove moisture and excess solid magnesium sulfate. Finally, rotary evaporation was performed to generate orange-red solid compound 2.

[0040] Compound 2 was dissolved in 10 mL of ethanol, and 5 mmol SnCl2 and 5 mL of concentrated HCl were added in sequence. The mixture was heated and stirred for 6 h. After cooling, the pH of the solution was adjusted to 7. The resulting solution was extracted, dried, and distilled under reduced pressure to obtain a red solid product NNPCN-NH2, which was stored at 4 ° C in the dark. The obtained red solid product NNPCN-NH2 was characterized by 1HNMR and LC-MS.

[0041] (4) 1HNMR characterization of NNPCN-NH2

[0042] The purified product was subjected to proton spectroscopy ( Figure 2 )analyze, 1 HNMR (400MHz, DMSO-d6)δ H 12.90(s,1H),8.88(dd,J=7.1,1.5Hz,1H),8.51(dd,J=8.7,1.5Hz,1H),8.43–8.29(m,3H),8.21–8.06(m,3H),8.01(dd,J =7.2,1.3Hz,1H),7.39(ddd,J=15.5,8.3,7.1Hz,2H),7.18(dd,J=9.0,2.3Hz,1H),6.96(d,J=2.3Hz,1H),5.53(s,2H), where δ H =12.9 hydrogen signal is the signal carried by the amide group on NNPCN-NH2; δ H =5.53 hydrogen signal is the signal carried by -NH2 connected to the naphthyl group on NNPCN-NH2; δ HThe hydrogen signals at 6.96, 7.18, 7.39, 8.01, 8.06-8.21, 8.29-8.43, 8.51 and 8.88 are the signals carried by the phenazine ring or naphthalene ring on NNPCN-NH2. The above data are basically consistent with the compound NNPCN-NH2, which preliminarily proves that this structure is NNPCN-NH2.

[0043] (5) LC-MS characterization of NNPCN-NH2

[0044] The purified product was analyzed by LC-MS ( Figure 3 ), a chromatographic peak with high response value appeared at the retention time of 1.4min in the liquid chromatogram, and no chromatographic peak with high response value appeared at other times, indicating that the product purification result was good and the purity was very high; a mass peak with a mass-to-charge ratio of 365.15 appeared in the mass spectrum, which was consistent with the relative atomic mass of NNPCN-NH2, indicating that the purified product was NNPCN-NH2.

[0045] Example 2: Preparation of N-(naphthalen-1-yl)phenazine-1-carboxamide polyclonal antibody

[0046] (1) Test reagents

[0047] Methanol, Sinopharm Chemical Reagent Co., Ltd.; bovine serum albumin, Sinopharm Chemical Reagent Co., Ltd.; sodium bicarbonate, Tianjin Damao Chemical Reagent Factory; sodium dihydrogen phosphate, Sinopharm Chemical Reagent Co., Ltd.; disodium hydrogen phosphate dodecahydrate, Sinopharm Chemical Reagent Co., Ltd.; ethylenediaminetetraacetic acid disodium salt dihydrate, Sinopharm Chemical Reagent Co., Ltd.; 25% glutaraldehyde, biological grade, Sinopharm Chemical Reagent Co., Ltd.; glucose, Sinopharm Chemical Reagent Co., Ltd.; agar powder, Beijing Aoboxing Biotechnology Co., Ltd.; ammonium sulfate, Sinopharm Chemical Reagent Co., Ltd.; Freund's complete adjuvant, SIGMA; Freund's incomplete adjuvant, SIG MA; chicken ovalbumin (OVA) biological reagent, SIGMA; goat anti-rabbit HRP labeled secondary antibody, Solarbio; antibody diluent, Solarbio; TMB colorimetric solution, Solarbio; 5× protein loading buffer, Solarbio; colored pre-stained protein, biosharp; ultrafiltration tube, millipore; skim milk powder, Biofroxx; tris(hydroxymethyl)aminomethane, Biofroxx; glycine, Biofroxx; dialysis bag, biosharp; SDS-PAGE gel preparation test strip, Solarbio; 96-well ELISA plate, Costor;

[0048] (2) Test equipment

[0049] SCIENTZ-10N freeze dryer, Ningbo Xinzhi Biotechnology Co., Ltd.; PHS-25 pH meter, Shanghai Yidian Scientific Instrument Co., Ltd.; ZNCL-S-5D multi-point intelligent magnetic stirrer, Henan Aibote Technology Development Co., Ltd.; UVmini-1240 UV-visible spectrophotometer, Shimadzu Instruments Suzhou Co., Ltd.; DYY-7C electrophoresis instrument, Beijing Liuyi Biotechnology Co., Ltd.; 3K15 low-temperature high-speed centrifuge, SIGMA; ChampGel 6000 fully automatic gel imager, Beijing Saizhi Venture Technology Co., Ltd.; GC-100 dry thermostat, Hangzhou Youning Instrument Co., Ltd.; Multiskan FC fully automatic enzyme-linked immunosorbent assay analyzer, ThermoFisher Scientific; TS-2 decolorization shaker, Haimen Qilin Bell Instrument Manufacturing Co., Ltd.

[0050] (3) Commonly used solutions and experimental animals

[0051] Na2HPO4·12H2O mother solution (0.2M): Weigh 35.814g Na2HPO4·12H2O, dissolve it in 500mL pure water, and store it at room temperature for later use. NaH2PO4 mother solution (0.2M): Weigh 11.998g NaH2PO4, dissolve it in 500mL pure water, and store it at room temperature for later use. Phosphate buffer (PBS, 0.01M, pH=7.4): Mix 9.5mL NaH2PO4 mother solution and 40.5mL Na2HPO4·12H2O mother solution, dilute to 1000mL, and store it at room temperature for later use. EDTA (1mmol / L, pH=8): Dissolve 0.372g ethylenediaminetetraacetic acid disodium salt in 1000mL pure water, adjust the pH to 8 on a pH meter with 0.1M NaOH, and store it for later use. Dialysis bag: Cut the dialysis bag into 15cm small pieces, add to boiling 500mL EDTA containing 10g sodium bicarbonate for 10min, take out and wash with distilled water, then put into 500mL boiling EDTA for 10min, cool and store in 30% ethanol. Decolorizing solution: Mix 250mL anhydrous ethanol and 80mL acetic acid and dilute to 1L with distilled water, and store at room temperature. Staining solution: Add 1.25g Coomassie Brilliant Blue R-250 to 500mL decolorizing solution to obtain the staining solution. 5× electrophoresis buffer: Weigh 15g Tris-base, 94g glycine, 5g SDS, add 1L distilled water to dissolve and prepare, and store at room temperature. PBST: Add 1mL Tween 20 to 1L PBS buffer, mix well, and store at room temperature. Blocking solution: Dissolve 5g skim milk powder in 100mL PBST and store at 4℃. Sample diluent: Add 1 mL of Tween 20 and 1 g of melted gelatin to 1 L of PBS buffer, mix well, and store at 4°C. Sulfuric acid stop solution: Add 21.7 mL of 98% sulfuric acid to 178.3 mL of ultrapure water, mix well, and store at room temperature. Saturated ammonium sulfate solution: Weigh an excess of ammonium sulfate (≥200 g) and dissolve it in 200 mL of distilled water. Stir and heat the solution to above 80°C to completely dissolve. When the temperature drops to room temperature, ammonium sulfate crystals will precipitate. Take the supernatant for use and store at room temperature. New Zealand male white rabbits were purchased from Hunan Taiping Biotechnology Co., Ltd.

[0052] (4) Preparation of NNPCN immune antigen and coating antigen

[0053] Preparation of NNPCN immune antigen and coating antigen: The prepared NNPCN-NH2 and the amino groups on the macromolecular proteins (OVA, BSA) can react with the aldehyde groups on glutaraldehyde to form Schiff base, thereby preparing NNPCN immune antigen and coating antigen ( Figure 4). The specific synthesis operation is as follows: weigh 2.875 mg of NNPCN-NH2, dissolve it in 0.6 mL of methanol, add 1.2 mL of PBS, that is, liquid I; weigh 15 mg of macromolecular protein (BSA, OVA) and dissolve it in 3 mL of PBS, that is, liquid II; measure 0.2 mL of 25% glutaraldehyde and 0.8 mL of PBS, that is, liquid III. Mix liquid I and liquid III respectively, stir in a dark on a magnetic stirrer for 20 minutes, slowly add the dissolved liquid II, and continue to stir in a dark for 11 hours. Then put the reaction mixture into a dialysis bag and dialyze it in PBS at 4°C for 72 hours, changing the water once every 8 hours. The dialyzed products were placed in 50 kDa and 100 kDa ultrafiltration tubes respectively, centrifuged at 4000 rpm in a 4°C centrifuge for 10 minutes, and the filtered products in the lower layer of the ultrafiltration tube were freeze-dried for 12 hours. The freeze-dried white flocculent products were stored separately at -20°C, which are the NNPCN immune antigen and coating antigen.

[0054] (5) SDS-PAGE identification of NNPCN immune antigen and coating antigen

[0055] Prepare 10% separation gel, then inject the separation gel into the clamped glass plate, and add 1-2mL of anhydrous ethanol to remove bubbles and make the liquid surface flat. After 30 minutes, the separation gel solidifies and the excess alcohol is poured out, and then wiped dry with absorbent paper. Use 1.65mL of 30% gel-making solution; 1.25mL of 1.5MTris-HCl (pH=6.8); 50μL of 10% SDS; 2mL of ddH2O; 50μL of 10% PAGE gel coagulant; 5μL of PAGE gel accelerator. Prepare 5% concentrated gel, then inject the concentrated gel into the glass plate and insert the sample comb to make the loading hole. After 10 minutes, the concentrated gel solidifies, and the glass plate is placed in the electrophoresis tank filled with electrophoresis solution and the sample comb is removed. 0.332mL of 30% gel preparation solution; 0.25mL of 1MTris-HCl (pH=6.8); 20μL of 10% SDS; 1.368mL of ddH2O; 30μL of 10% PAGE gel coagulant; 3μL of PAGE gel accelerator. Dilute NNPCN-BSA with an appropriate amount of PBS to two concentrations of 1×NNPCN-BSA and 2×NNPCN-BSA; repeat this operation for dilution of NNPCN-OVA, BSA and OVA. Use a pipette to take 40μL of each and mix with 10μL of 5× protein loading buffer and place in a 100℃ metal bath for 3-5min to denature the protein. Cool to room temperature and centrifuge at low speed for 2min, take 10μL of supernatant and add it to the loading well of the electrophoresis tank (Marker 5μL). Electrophoresis at 120V for 1.5h. After the electrophoresis is completed, place the polyacrylamide gel in a staining box containing staining solution and let it stand for staining for 4h. The staining solution was recovered, and the destaining solution was added to the box, and the box was placed in a destaining shaker for destaining overnight. The polyacrylamide gel was taken out and photographed with a gel imager.

[0056] (6) Identification of NNPCN immune antigen and coating antigen by UV spectroscopy

[0057] NNPCN-BSA, NNPCN-OVA, NNPCN, BSA, OVA and NNPCN-NH2 were diluted to appropriate concentrations with PBS, 1 mL was taken into cuvettes, the baseline was calibrated with PBS, the UV spectra of the above solutions at a wavelength of 190-300 nm were scanned on a UV-visible spectrophotometer, and the results were statistically analyzed in Excel.

[0058] (7) Immunization regimen

[0059] Five 1.5kg male New Zealand white rabbits were purchased, of which four were used as immunization objects, numbered Ⅰ-Ⅳ; the fifth was a negative control. Before each titer test, 5mL of blood was collected from the ear vein, and the serum was collected by standing at 4℃ overnight as a negative control serum, which was stored at -20℃ for use. NNPCN-BSA was dissolved in 2mg / mL of normal saline, and equal amounts of Freund's complete adjuvant and Freund's incomplete adjuvant were mixed according to the amount of NNPCN-BSA injected, and vortexed to make an "oil-in-water" emulsion. The immunization scheme is shown in Table 1 below. The first immunization used NNPCN-BSA mixed with Freund's complete adjuvant, 1mL / rabbit, and the injection method was subcutaneous multiple injections. A booster immunization was performed every two weeks, using NNPCN-BSA mixed with Freund's incomplete adjuvant, 1.5mL / rabbit, and the injection method was subcutaneous multiple injections. Blood was collected from the ear vein on the 7th day of each booster immunization, and the serum was collected every other day for antibody titer detection. When the titer was greater than 10w, blood was collected from the heart on the 5th day of the next booster immunization, and the serum was collected by standing at 4°C overnight to prepare the unpurified NNPCN rabbit serum polyclonal antibody, which was packaged and stored at -20°C.

[0060] Table 1 NNPCN polyclonal antibody immunization scheme

[0061]

[0062] (8) Serum antibody titer determination

[0063] Dissolve NNPCN-OVA in carbonate buffer to prepare 2 μg / mL, and add 100 μL / well to the ELISA plate.

[0064] 4℃ coating

[0065] Overnight. Pat the coated ELISA plate dry, add blocking solution at 110μL / well, and block at 37℃ for 1h. Take it out of the oven and pat it dry, then put it in the oven to dry for 2h, put it in a ziplock bag with desiccant, and store it at 4℃. Take out the sample diluent and the blocked ELISA plate in advance and warm it up at room temperature, and dilute the serum from 1000 times with antibody diluent, 100μL / well. Negative serum is used as a control, incubate at 37℃ for 30min, and wash the plate three times with PBST after completion. Dilute the HRP-labeled goat anti-rabbit secondary antibody 5000 times with PBS, add 100μL / well to the ELISA plate, incubate at 37℃ for 30min, and wash the plate three times with PBST after completion. Take out the TMB colorimetric solution in advance and warm it up, add 50μL / well of TMB colorimetric solution to the ELISA plate, and incubate at 37℃ for 15min. Add sulfuric acid stop solution, 50μL / well, and read the absorbance at 450nm. When the positive-to-negative ratio is >2.1 and the positive value is >0.1, the highest ratio of serum dilution is the current serum antibody titer.

[0066] (9) Purification and titer concentration determination of NNPCN polyclonal antibodies

[0067] Purification of NNPCN polyclonal antibody: Ammonium sulfate precipitation method was used to purify NNPCN rabbit serum polyclonal antibody. Unpurified NNPCN rabbit serum polyclonal antibody was taken out of the refrigerator and thawed, centrifuged at 5000rpm for 10min, and the supernatant was taken. Then, an equal volume of PBS was taken and mixed thoroughly, and a saturated ammonium sulfate solution twice the volume of PBS was added dropwise and mixed thoroughly, and then placed at 4°C for 3h. Then, centrifuged at 5000rpm for 10min at 4°C, the supernatant was discarded, and an equal volume of PBS was added to the precipitate to re-dissolve. An equal volume of saturated ammonium sulfate solution was added dropwise to PBS again, mixed thoroughly, and placed at 4°C for 3h, and centrifuged at 5000rpm for 10min at 4°C. Finally, the supernatant was re-dissolved with an equal volume of PBS, placed in a dialysis bag, and dialyzed in PBS buffer for 72h, with the dialysate changed every 8h. After dialysis, purified NNPCN rabbit serum polyclonal antibody was obtained, and the antibody was taken out, aliquoted, and stored frozen at -20°C.

[0068] NNPCN polyclonal antibody titer and concentration detection: The purified antibody serum antibody titer was tested and the test results were recorded in Excel. 1 mL of purified NNPCN rabbit serum polyclonal antibody was taken into a cuvette, zeroed with PBS, and the absorbance of the polyclonal antibody at 260 nm and 280 nm was measured on a UV spectrophotometer. The protein concentration of the purified NNPCN rabbit serum polyclonal antibody was calculated by formula 1.

[0069] C (mg / mL) =1.45×A 280 -0.74×A 260 Formula 1

[0070] (10) SDS-PAGE detection of purified NNPCN rabbit serum polyclonal antibody

[0071] The operation method was the same as that of SDS-PAGE to identify NNPCN immune antigen and coating antigen. 10 μL of unpurified NNPCN rabbit serum polyclonal antibody and purified NNPCN rabbit serum polyclonal antibody (Marker 5 μL) were added to the loading wells respectively, and the polyclonal antibody purification results were detected by taking pictures with a gel imager.

[0072] (11) SDS-PAGE identification of NNPCN immune antigen and coating antigen

[0073] The gel was exposed and photographed by a gel imager to obtain the protein electrophoresis diagram of NNPCN-BSA and NNPCN-OVA ( Figure 5 ), it can be seen from the figure that the synthesized NNPCN-BSA and NNPCN-OVA have obvious dragging phenomenon compared with the bands of BSA and OVA, and both have band hysteresis phenomenon, indicating that the molecular weight of NNPCN-BSA and NNPCN-OVA is increased compared with BSA and OVA, which preliminarily proves that the NNPCN immune antigen and coating antigen are successfully synthesized.

[0074] (12) Identification of NNPCN immune antigen and coating antigen by UV spectroscopy

[0075] By scanning the UV spectrum of the compound ( Figure 6 ) to identify the synthesis of the substance, the maximum UV absorption wavelength of NNPCN-BSA is 203 nm ( Figure 6 A), compared with the maximum UV absorption wavelength of BSA at 218 nm and the maximum UV absorption wavelength of NNPCN-NH2 at 211 nm, there is an obvious blue shift; the maximum UV absorption wavelength of NNPCN-OVA is 198 nm ( Figure 6 B), compared with the maximum UV absorption wavelength of OVA at 208 nm and the maximum UV absorption wavelength of NNPCN-NH2 at 211 nm, there was also an obvious blue shift, which indicated that the spatial structure of the NNPCN immune antigen and the coating antigen had changed compared with the precursor compound, further proving that the NNPCN immune antigen and the coating antigen were successfully synthesized.

[0076] (13) Serum antibody titer determination

[0077] The indirect ELISA method was used to determine the NNPCN rabbit serum antibody titers of the four rabbits. The results of the last ELISA test are shown in Table 2. The rabbit serum antibody titers of rabbits No. Ⅰ and No. Ⅲ were the highest, both 1.0×10 5The titer results of rabbits II and IV were not high. Rabbit II died after the second booster immunization and could not be immunized further. The reason why the titer of rabbit IV was not high may be related to the differences in the immune systems of different rabbits. 450 The value does not change with the dilution multiple. In summary, it was decided to collect blood from the hearts of rabbits No. Ⅰ and No. Ⅲ to prepare NNPCN rabbit serum polyclonal antibodies.

[0078] Table 2 NNPCN rabbit serum antibody titer detection

[0079]

[0080] Note: Rabbit serum titer in the table is positive OD 450 Value / Negative OD 450 The maximum serum dilution factor with a value > 2.1 (i.e. P / N > 2.1).

[0081] (14) Purification and titer concentration determination of NNPCN polyclonal antibodies

[0082] Purified NNPCN rabbit serum polyclonal antibody titer detection: Purified rabbit serum antibodies No. Ⅰ and No. Ⅲ

[0083] The results of ELISA test are shown in Table 3. The P / N values ​​of No. Ⅰ and No. Ⅲ were 2.39 and 2.733, which were lower than those of No. Ⅰ and No. Ⅲ in Table 2. The negative OD 450 The value did not change with the dilution multiple, indicating that some impurity proteins in rabbit serum were removed during the purification process. This preliminarily proved that the ammonium sulfate precipitation method was effective in removing impurity proteins in rabbit serum.

[0084] Table 3 Detection of antibody titers of purified No. Ⅰ and No. Ⅲ NNPCN rabbit serum

[0085]

[0086] Note: Rabbit serum titer in the table is positive OD 450 Value / Negative OD 450 The maximum serum dilution factor with a value > 2.1 (i.e. P / N > 2.1).

[0087] Determination of the concentration of purified NNPCN rabbit serum polyclonal antibodies: By calculation, the concentrations of purified No. Ⅰ and No. Ⅲ NNPCN rabbit serum polyclonal antibodies were found to be 2.643 mg / mL and 2.211 mg / mL, respectively (Table 4).

[0088] Table 4 Concentration of purified No. Ⅰ and No. Ⅲ NNPCN rabbit serum polyclonal antibodies

[0089]

[0090] (15) SDS-PAGE detection of purified NNPCN rabbit serum polyclonal antibody

[0091] In order to determine the effect of ammonium sulfate precipitation to purify the protein, SDS-PAGE was used to detect the change in the molecular weight of the protein before and after purification ( Figure 7 ). The purified rabbit serum antibody proteins No. Ⅰ and No. Ⅲ in lanes 2 and 3 only have obvious bands at 25kDa and 50kDa, which is consistent with the molecular weight of most antibody light chains and heavy chains, but there are still some impurity bands in the purified antibodies, among which the impurity band of purified rabbit serum antibody No. Ⅰ in lane 2 is more obvious than the impurity band of purified rabbit serum antibody No. Ⅲ in lane 3; There are two obvious bands at 50-70kDa for No. Ⅰ and No. Ⅲ rabbit serum antibody proteins before purification in lanes 4 and 5, and the band at 55kDa is an impurity band, indicating that the ammonium sulfate precipitation method correctly removes most of the impurity proteins in rabbit serum, but some impurity proteins still exist. Purified rabbit serum antibody No. Ⅲ with fewer impurities will be used for subsequent experiments.

[0092] Example 3: Establishment of indirect competitive ELISA standard curve of N-(naphthalen-1-yl)phenazine-1-carboxamide polyclonal antibody

[0093] (1) Coating and sealing

[0094] Dilute the NNPCN coating source to 50 μg / mL with coating solution (pH 9.6, 0.01 mol / L carbonate buffer), 100 μL per well, and coat overnight at 4°C. Wash twice with phosphate-buffered Tween buffer (PBST), add 2% skim milk powder, 110 μL per well at 37°C for 2h, discard the blocking solution, dry at 37°C for 100min, seal and store at 4°C for later use.

[0095] (2) Establishment of standard curve

[0096] Add 50 μL NNPCN standard of different concentrations (0.1 μg / mL, 1 μg / mL, 10 μg / mL, 100 μg / mL, 1000 μg / mL) and 50 μL NNPCN polyclonal antibody of 2.0 μg / mL to each well of the coated ELISA plate, incubate at 37°C for 30 min, wash five times with PBST, pat dry the liquid in the well, add 1:5000 diluted enzyme-labeled secondary antibody (goat anti-rabbit), incubate at 37°C for 40 min, wash five times with PBST, pat dry the liquid in the well, add 50 μL TMB substrate, color at 37°C in the dark for 10 min; add 100 μL stop solution (2M H2SO4) to stop the reaction; read the absorbance value at 450 nm with an ELISA reader. The concentration of NNPCN standard is plotted against the horizontal axis, B / B0 (OD value of the microwell with NNPCN added)450 Value / OD of microwell without NNPCN 450 The indirect competition standard curve was established. Figure 8 It can be seen that the standard curve has good correlation, IC 50 The value is 21.42μg / mL.

[0097] (3) Cross-reaction

[0098] Based on the constructed ELISA, the IC of each drug was calculated using the indirect competitive ELISA method for phenazine-1-carboxylic acid, carbendazim, and procymidone. 50 The results showed that the cross-reaction rate of NNPCN with phenazine-1-carboxylic acid was 12.92%, and there was no cross-reaction with carbendazim and imazalil (Table 5).

[0099] Table 5 NNPCN-NH2 antibody cross-reactivity

[0100]

[0101] Example 4: N-(naphthalen-1-yl)phenazine-1-carboxamide colloidal gold test strip

[0102] (1) Test reagents

[0103] Sodium citrate, Sinopharm Chemical Reagent Co., Ltd.; chloroauric acid, Sinopharm Chemical Reagent Co., Ltd.; potassium carbonate, Sinopharm Chemical Reagent Co., Ltd.; bovine serum albumin, Sinopharm Chemical Reagent Co., Ltd.; Tween, Sinopharm Chemical Reagent Co., Ltd.; dimethyldichlorosilane, Sinopharm Chemical Reagent Co., Ltd.; chloroform, Sinopharm Chemical Reagent Co., Ltd.; potassium dihydrogen phosphate, Sinopharm Chemical Reagent Co., Ltd.; sodium dihydrogen phosphate, Sinopharm Chemical Reagent Co., Ltd., etc.

[0104] (2) Test instruments

[0105] ZNCL-S-5D multi-point intelligent magnetic stirrer, Henan Aibote Technology Development Co., Ltd.; ME204E electronic balance, Mettler-Toledo Instruments (Shanghai) Co., Ltd.; D1008E centrifuge, Dalong Xingchuang Testing Instruments (Beijing) Co., Ltd., etc.

[0106] (3) Preparation of colloidal gold solution

[0107] Soak the conical flask in chloroform silanization with 5% dimethyldichlorosilane for 1 minute, rinse, dry and set aside (can only be used for the preparation of colloidal gold with the same diameter). Take 99mL of ultrapure water and 1mL of 1% chloroauric acid solution respectively to dilute it into a 0.01% solution, place it in a three-shaped flask, heat it to boiling on a magnetic stirrer, and continue to stir at high temperature. Add 1mL of 1% sodium citrate solution and stir for 10 minutes. Heat and boil for 10 minutes and stop when the solution turns red. Cool to room temperature, add ultrapure water to 100mL to obtain a pure colloidal gold solution, and store it at 4°C away from light. The results showed that the appearance of the colloidal gold solution prepared by the sodium citrate reduction method was purple-red, clear, and bright ( Fig. 9 ).

[0108] (4) Preparation of polyclonal antibody-colloidal gold marker

[0109] NNPCN polyclonal antibody and colloidal gold were coupled. Under magnetic stirring conditions, 10 1.5mL centrifuge tubes were taken, 1mL of colloidal gold solution was added to each tube, and different pH values ​​were adjusted with 1-10μL K2CO3 (0.1M), 10μg NNPCN polyclonal antibody was added per milliliter of colloidal gold solution, and the reaction was allowed to stand for 20 minutes, and 10% NaCL was added to terminate the reaction. The color did not change, which was the optimal pH. Take 7 1.5mL centrifuge tubes, adjust the optimal pH, add 1-64μg antibody, take the first antibody amount that did not change color, and add 20% more on this basis to obtain the optimal antibody addition amount. Add polyclonal antibody to the colloidal gold solution, stir continuously for 20-30 minutes, let stand for 30 minutes, add 10% bovine serum albumin (BSA) solution to make its volume percentage in the colloidal gold solution 1%, and seal and stand for 10 minutes. Centrifuge at 10000 rpm at 4°C for 30 min, discard the supernatant, resuspend the precipitate with a coupling solution whose volume is 1 / 10 of the initial colloidal gold solution to prepare NNPCN polyclonal antibody-colloidal gold marker, and store at 4°C for future use;

[0110] (5) Coating original and goat anti-rabbit IgG coated cellulose membrane (NC membrane)

[0111] Use a film sprayer to draw the membrane. The coating antigen is coated at the test line (T line), with 4-5 concentrations of coating antigen and a fixed concentration of secondary antibody. The goat anti-rabbit IgG is coated at the quality control line (C line), with 4-5 concentrations of goat anti-rabbit IgG and a fixed concentration of coating antigen. The two lines are 5mm apart. After drawing the membrane, place it in a 37℃ oven to dry for 1h.

[0112] (6) Preparation of colloidal gold pad (binding pad)

[0113] The conjugate pad was soaked in 4% bovine serum albumin PBS solution for 60 min, and dried at 37°C for 3 h for later use. The gold-labeled antibody diluted 200 times was evenly sprayed on the conjugate pad using a film sprayer, and dried at 37°C for 3 h for later use.

[0114] (7)Colloidal gold test strip assembly

[0115] Take a dry cellulose membrane and stick it in the middle of the PVC plastic bottom plate. Stick the absorbent pad to the upper end of the cellulose membrane, overlapping with the cellulose membrane by 1mm. Stick the binding pad to the lower end of the cellulose membrane, overlapping with the cellulose membrane by 1mm. Stick the sample pad to the lower end of the binding pad, overlapping with the binding pad by 2mm. After the assembly is completed, cut the test plate into 5mm wide test strips with a strip cutter and store them in a dry condition away from light for later use ( Fig.10 ).

[0116] (8) Performance evaluation of NNPCN colloidal gold test strips

[0117] 8.1 Sensitivity

[0118] Prepare a series of NNPCN solutions (100, 80, 40, 20, 10, 1, 0 μg / mL) in PBS containing acetonitrile, take 100 μL of each solution and drop it onto the sample pad of the test strip, and observe the color development results within 8-10 minutes ( Fig.11 ). Result judgment basis: Positive: When the quality control area (C) shows a band and the detection area (T) does not show color or shows a weak color, it is judged as positive, that is, there is NNPCN in the sample, indicated by "+"; Negative: When both the quality control area and the detection area show a band or the color of the detection area is darker than that of the quality control area, it is judged as negative, that is, there is no NNPCN in the sample, indicated by "-"; Invalid: When the quality control area (C) does not show a band, the test paper is invalid or the operation is improper. The test results based on the above judgment basis show that when the NNPCN concentration is 100, 80, 40, 20, and 10μg / mL, the T line is clearly colored, which is positive. When NNPCN is 1 and 0μg / mL, respectively, the T line cannot be observed, which is negative. Therefore, the sensitivity of the NNPCN test strip is 1μg / mL.

[0119] 8.2 Specificity

[0120] Dilute the fungicides (carbendazim and imazalil) into a 1000μg / mL standard solution with PBS containing 10% acetonitrile, take 100μL of each and drop it onto the sample pad of the test strip, and observe the color development results within 8-10 minutes. The results showed that all test strips were negative, indicating that the NNPCN test strip had no cross-reaction with other pesticides and had strong specificity.

[0121] 8.3 Repeatability

[0122] Six test strips were drawn from the prepared test strips, and the negative and positive samples were tested, and the results were consistent with the expected results.

[0123] 8.4 Stability

[0124] The test strips were placed in a black ziplock bag and stored at room temperature for 30 days. The test strips were taken out and the sensitivity was measured. The results showed that the test strips had clear strips, indicating that the NNPCN test strips had good stability.

[0125] 8.5 Actual sample testing

[0126] Weigh 2g of soil blank sample into a 50mL centrifuge tube, add NNPCN to a final concentration of 0.1mg / kg, and let stand for 4h. Add 5mL of acetonitrile-formic acid (a small amount) mixed solution, shake for 1min, take 1mL of soil solution, dilute 3 times with PBS, shake well, take 100μL and add it dropwise to the sample pad of the test strip, and observe the results within 8-10min. The results are determined by comparing the color depth of the control line (C line) and the test line (T line). The results show that the test strip is positive, indicating that it can be used for rapid detection of NNPCN ( Fig.12 ).

Claims

1. A N-(naphthalene-1-yl)phenazine-1-carboxamide hapten, characterized in that: The hapten has a molecular structure shown in Formula I, and the molecular formula and molecular weight are: 23 H 16 N4O, 364.41, 2. The method for synthesizing a hapten according to claim 1, characterized in that: The synthesis method is as follows: First, phenazine-1-carboxamide is hydrolyzed under acidic conditions to obtain compound 1, and then compound 1 is subjected to condensation acylation reaction with 6-nitronaphthalene-1-amine to obtain compound 2. The nitro group in compound 2 is subjected to oxidation-reduction reaction to finally obtain the target product N-(naphthalene-1-yl)phenazine-1-carboxamide hapten; the route of the synthesis method is shown in Formula II:

3. The method for synthesizing a hapten according to claim 2, characterized in that: The synthesis method specifically comprises the following steps: (1) 0.01 mol of phenazine-1-carboxamide and 10 mL of 30% hydrochloric acid were added to 10 mL of dimethyl sulfoxide, and 2 mL of 6 mol / L sodium nitrite aqueous solution was slowly added dropwise while stirring continuously. The mixture was stirred in an ice-alcohol bath for 4 h. The reaction temperature was kept below 0° C. during the entire reaction process. After the reaction was completed, 60 mL of water was added, and the mixture was stirred at low temperature until no solid dissolved. The mixture was then filtered and the obtained solid was washed with water 4 times to obtain a light yellow solid compound 1. (2) Compound 1 was placed in a 10 mL sample bottle and 0.001 mol aniline, 0.001 mol 6-nitro-1-naphthylamine, 0.002 mol trifluoroacetic acid borate and 2 mL acetonitrile were added, and stirred at 80°C for 24 h on a constant temperature magnetic stirrer; then 3 mL chloroform and 0.5 mL water were added to terminate the reaction to obtain a crude product of compound 2; 150 mg each of ion exchange resins Amberlyst A-26, Amberlyst 15 and mberlylite IRA743 were added to the crude product in sequence, stirred at room temperature for 30 min and filtered, and solid magnesium sulfate and chloroform were added to the mixture in sequence to remove moisture and excess solid magnesium sulfate; finally, rotary evaporation was performed to generate an orange-red solid compound 2; compound 2 was dissolved in 10 mL ethanol, and 5 mmol SnCl2, 5mL concentrated HCl, heated and stirred for 6h, and after cooling, the pH of the solution was adjusted to 7. The resulting solution was extracted, dried, and distilled under reduced pressure to obtain a red solid product N-(naphthalene-1-yl)phenazine-1-carboxamide hapten, which was stored at 4°C in the dark.

4. An artificial N-(naphthalene-1-yl)phenazine-1-carboxamide antigen obtained by coupling the N-(naphthalene-1-yl)phenazine-1-carboxamide hapten according to claim 1 or 2 with a carrier protein.

5. The artificial antigen according to claim 4, comprising an immunizing antigen and a coating antigen.

6. A N-(naphthalene-1-yl)phenazine-1-carboxamide polyclonal antibody obtained based on the artificial antigen according to claim 5; the polyclonal antibody specifically recognizes N-(naphthalene-1-yl)phenazine-1-carboxamide, N-(naphthalene-1-yl)phenazine-1-carboxamide hapten, and N-(naphthalene-1-yl)phenazine-1-carboxamide artificial antigen.

7. Use of the N-(naphthalene-1-yl)phenazine-1-carboxamide polyclonal antibody according to claim 6 in the preparation of a preparation for detecting N-(naphthalene-1-yl)phenazine-1-carboxamide.

8. The use according to claim 7, characterized in that The test strip is a colloidal gold test strip.

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