An ofendaazole hapten, artificial antigen and preparation method and application thereof

Abstract

The application discloses an oxfendazole hapten, an artificial antigen, and a preparation method and application thereof. The oxfendazole artificial antigen provided by the application is an antigen obtained by coupling an oxfendazole hapten shown in formula I with a carrier protein. The oxfendazole artificial antigen provided by the application has the advantages of simple synthesis method, high purity, and high yield, and has great value for preparation of an oxfendazole antibody and detection of oxfendazole drug residues.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing technology, specifically relating to an oxifenedazol hapten, an artificial antigen, and their preparation methods and applications. Background Technology

[0002] Olfendazole is a novel, highly effective, broad-spectrum, and low-toxicity benzimidazole carbamate anti-helmintic drug that has been widely used in veterinary production, such as for the prevention and treatment of gastrointestinal nematodes, lungworms, and liver flukes. However, due to its teratogenic and mutagenic effects in animal safety evaluation studies, most countries and regions, including China, the United States, and the European Union, have designated it as a key food safety monitoring target and established maximum residue limits.

[0003] Currently, the main methods for determining ophenazine residues include gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). HPLC offers advantages such as accurate quantification, but its pretreatment is relatively complex, resulting in low sensitivity in practical applications. GC-MS provides confirmatory detection with high sensitivity, but its pretreatment process is complex and requires derivatization. LC-MS / MS offers advantages such as high sensitivity, good selectivity, accurate qualitative and quantitative analysis, and strong anti-interference capabilities, but it also suffers from complex pretreatment, long processing time, and high cost.

[0004] Immunochemical analysis has unique advantages in the qualitative and quantitative identification of antigens and antibodies. It is simple, rapid, low-cost, highly sensitive, and can analyze large sample volumes, thus compensating for the shortcomings of physicochemical analysis. Therefore, there is an urgent need to develop a rapid and accurate detection method for screening large quantities of animal-derived foods to improve the current situation, fill the gaps in domestic testing methods, enhance and standardize the technical level of the entire testing industry, and strengthen the competitiveness of my country's animal-derived foods in the international market. Summary of the Invention

[0005] The purpose of this invention is to provide oxifenedazole hapten, artificial antigen, preparation method and application thereof.

[0006] The oxendazole artificial antigen provided by this invention is an antigen constructed based on the oxendazole hapten.

[0007] The oxifenedazol hapten is within the scope of protection of this invention, and its structure is shown in Formula I.

[0008]

[0009] The method for preparing the ophenazine hapten provided by this invention may specifically include the following steps:

[0010] 2-Methoxycarbonylamino-3H-benzimidazole-5-carboxylic acid was added to dimethylformamide (DMF) and stirred, followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-bromosuccinimide (NBS) and stirring. 4-(aminomethyl)benzoic acid was then added and reacted. The reaction mixture was extracted, evaporated to dryness, chromatographically analyzed, and then evaporated again to dryness to obtain solid 1. The ratio of 2-methoxycarbonylamino-3H-benzimidazole-5-carboxylic acid, dimethylformamide (DMF), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-bromosuccinimide (NBS), and 4-(aminomethyl)benzoic acid was 500 mg: 5 ml: 448 mg: 269 mg: 446 mg.

[0011] The oxendazole antigen constructed based on the oxendazole hapten is also within the scope of protection of this invention.

[0012] The oxendazole antigen is an antigen obtained by conjugating the oxendazole hapten (Formula I) with a carrier protein. In one embodiment of the present invention, the carrier protein is specifically bovine serum albumin (BSA) or ovalbumin (OVA).

[0013] The method for preparing the ofendaazole antigen is also within the scope of protection of this invention.

[0014] The preparation method of the ofendazol antigen may specifically include the following steps: coupling the ofendazol hapten (Formula I) to a carrier protein via an amide bond to obtain the ofendazol antigen.

[0015] In this invention, the ophenazine antigen is specifically prepared by a method comprising the following steps:

[0016] (1) Dissolve the ofendazol hapten (Formula I) in dimethylformamide (DMF), then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and stir magnetically at 20-25°C for 2-3 hours to obtain solution I;

[0017] The ratio of the oxifenezole hapten (Formula I), the dimethylformamide (DMF), the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and the N-hydroxysuccinimide (NHS) is 13.73 mg: 1.5 ml: 21.5 mg: 13 mg.

[0018] (2) The carrier protein was placed in 0.1M sodium bicarbonate buffer and stirred at 200 rpm for 10 min to dissolve it completely, to obtain solution II; the ratio of the carrier protein to the 0.1M sodium bicarbonate buffer was 33.6-50 mg: 3.5 ml;

[0019] Wherein, if the carrier protein is bovine serum albumin (BSA), the ratio of bovine serum albumin (BSA) to the 0.1M sodium bicarbonate buffer is 50 mg: 3.5 ml; if the carrier protein is ovalbumin (OVA), the ratio of ovalbumin (OVA) to the 0.1M carbonate buffer is 33.6 mg: 3.5 ml;

[0020] (3) Mix the solution I and the solution II, specifically by adding the solution I dropwise to the solution II under 0-4℃ conditions and stirring at 1000 rpm, and stirring at 500 rpm for 24 h to obtain the solution III;

[0021] (4) The solution III was dialyzed at 4°C for 3 days with phosphate buffer (0.01M PBS, pH 7.2) to obtain the ofendazol antigen.

[0022] The application of the oxendazole hapten (Formula I) or the oxendazole antigen in the qualitative or quantitative detection of oxendazole is also within the scope of protection of this invention.

[0023] Antibodies prepared using the ophenazine antigen are also within the scope of protection of this invention. The antibody may be a polyclonal antibody, a monoclonal antibody, or an antiserum.

[0024] The oxendazole hapten and the oxendazole antigen provided by this invention have simple synthesis methods, high purity, and high yield, and are of great value for the preparation of oxendazole antibodies and the detection of oxendazole residues. Attached Figure Description

[0025] Figure 1 This is the mass spectrum of the oxifenedazol hapten.

[0026] Figure 2 This is the standard curve for oxifenedazol. Detailed Implementation

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0029] Example 1: Preparation and identification of oxifenedazol hapten

[0030] 1. Preparation of Ophenazine Hapten

[0031] Rinse the 50ml round-bottom flask thoroughly, dry it with ethanol, fix it on a stirrer, add a stir bar, weigh 500mg of 2-methoxycarbonylamino-3H-benzimidazole-5-carboxylic acid, dissolve it in 5ml DMF, stir for 15min, add 448mg EDC and 269mg NBS, stir magnetically for 16h, then add 446mg of 4-(aminomethyl)benzoic acid and stir. Monitor the reaction by TLC. After the reaction is complete, extract in an ice bath, evaporate the solvent, add silica gel for column chromatography, collect the eluent, evaporate to dryness to obtain the hapten.

[0032] The reaction equation is as follows:

[0033]

[0034] 2. Structural identification of oxifenedazol hapten

[0035] The obtained hapten was detected by mass spectrometry. Figure 1 The results showed that its chemical structure was as shown in Formula I (MW = 368.11), which is the oxifenezole hapten.

[0036]

[0037] Example 2: Preparation of Oxyphenidazole Artificial Antigen

[0038] 1. Synthesis of immunogens

[0039] (1) Dissolve 13.73 mg of ofendazole hapten in 1.5 ml of DMF, stir at 200 rpm for 10 min, add 21.5 mg of EDC to dissolve, then add 13 mg of NHS, stir at room temperature (500 rpm) and activate for 2-3 h.

[0040] (2) Weigh 50 mg BSA and dissolve it in 3.5 ml of 0.1 M sodium bicarbonate solution. Stir at 200 rpm for 10 min to fully dissolve it. Cool it in an ice bath at 0-4 °C. Add the reaction solution from step 1 dropwise (1 ml / min) while stirring at 1000 rpm. Stir at 500 rpm for 24 h.

[0041] (3) Place the reaction product into a dialysis bag (10cm) that has been rinsed with distilled water, add 1L of 0.01M PBS (1×, pH 7.2) and stir at 4℃ (100rpm) for dialysis for 3 days, changing the medium 3 times a day (once in the morning, once in the afternoon, and once in the evening), for a total of 9 changes. Centrifuge the dialysis product at 5000rpm for 6min, aliquot 1.5ml / tube, label the antigen, and store at -20℃ for later use.

[0042] 2. Synthesis of coating antigens

[0043] (1) Dissolve 13.73 mg of ofendazole hapten in 1.5 ml of DMF, stir at 200 rpm for 10 min, add 21.5 mg of EDC to dissolve, then add 13 mg of NHS, stir at room temperature (500 rpm) and activate for 2-3 h.

[0044] (2) Weigh 33.6 mg OVA and dissolve it in 3.5 ml of 0.1 M sodium bicarbonate solution. Stir at 200 rpm for 10 min to fully dissolve it. Cool it in an ice bath at 0-4℃. Add the reaction solution from step 1 dropwise (1 ml / min) while stirring at 1000 rpm. Stir at 500 rpm for 24 h.

[0045] (3) Place the reaction product into a dialysis bag (10cm) that has been rinsed with distilled water, add 1L of 0.01M PBS (1×, pH 7.2) and stir at 4℃ (100rpm) for dialysis for 3 days, changing the medium 3 times a day (once in the morning, once in the afternoon, and once in the evening), for a total of 9 changes. Centrifuge the dialysis product at 5000rpm for 6min, aliquot 1.5ml / tube, label the antigen, and store at -20℃ for later use.

[0046] Example 3: Preparation of monoclonal antibodies by immunizing animals with oxifenedazol artificial antigen

[0047] I. Animal Immunization

[0048] The immunogen (oxendazole-BSA) prepared in Example 2 was dissolved in physiological saline at 100 μg / mouse and mixed with an equal volume of Freund's complete adjuvant. The mixture was then subcutaneously injected into the neck and back of 6-8 week old Balb / c female mice. On days 7, 14, and 28 after the initial immunization, an additional immunization was performed by mixing an equal volume of the immunogen with Freund's incomplete adjuvant. Three days before fusion, an additional immunization was performed by administering 100 μg / mouse of the immune complex without Freund's adjuvant.

[0049] II. Cell Fusion and Cloning

[0050] Following standard procedures, spleen cells from immunized mice were mixed with myeloma cells (SP2 / 0) in the logarithmic growth phase. Preheated fusion agent (PEG4000) was then slowly added over 45 seconds for fusion. The cells were then suspended in HAT medium and homogenized. An appropriate amount of feeder cells was added, and the mixture was cultured in 96-well plates at 37°C in a 5% CO2 incubator. After 5 days, the medium was partially replaced with HT medium, and after 9 days, the medium was completely replaced.

[0051] 3. After cell fusion, when the cells reach 1 / 4 of the culture well area, hybridoma cells are screened using a stepwise screening method. Initial selection uses an indirect ELISA method. The ELISA plate is coated with the coating antigen (pre-titrated using a checkerboard method to determine the optimal coating concentration and positive serum dilution). Culture supernatant from the test wells is added, incubated, washed, and then goat anti-mouse IgG-HRP and IgM-HRP are added for OPD colorimetric reaction. Positive wells are then screened using an indirect competitive ELISA method. Cell supernatant is mixed with an equal volume of 100 μg / ml olfendazole, incubated at 37°C for 30 min, and then added to the coated ELISA plate. PBS is used as a control instead of olfendazole, and the remaining steps are the same. If the OD of the cells blocked by olfendazole is... 450nm If the value drops to below 50% of that of the control well, it is considered positive. Wells that are positive after 2 to 3 tests should be immediately subcloned using the limiting dilution method.

[0052] III. Preparation and Purification of Monoclonal Antibodies

[0053] Hybridoma cells obtained after 2-3 subclonings were cultured extensively, and the supernatant was collected for titer determination by indirect ELISA and then frozen. Eight- to ten-week-old Balb / c mice were intraperitoneally injected with 0.5 ml of liquid paraffin per mouse, and 7-10 days later, 1-2 × 10⁶ hybridoma cells were injected intraperitoneally. 5 Ascites fluid was collected from each mouse 7–10 days later. Cell supernatant or ascites fluid was collected, and its titer was determined using an indirect ELISA method (the titer was expressed as the maximum dilution of cell supernatant or ascites fluid with a P / N > 2.1). The results showed that the titer of the cell supernatant was 1:10000, and the titer of the ascites fluid was 1:50000. Subsequently, the fluid was purified using the octanoic acid-saturated ammonium sulfate method and stored at -20°C.

[0054] Upon testing, the amino acid sequence of the variable region of the heavy chain of the oxendazole monoclonal antibody is shown in Sequence 1 of the sequence listing, and the amino acid sequence of the variable region of the light chain of the oxendazole monoclonal antibody is shown in Sequence 2 of the sequence listing.

[0055] Example 4: Detection of Oxyphenidazole using an Enzyme-Linked Immunosorbent Assay Kit

[0056] I. Assembly of the Ofendazol ELISA Kit

[0057] 1. The components of the Oxyphenidazole ELISA kit are as follows:

[0058] (1) Omphidazole 10× concentrated standard: 6 bottles, 1ml / bottle, with concentrations of 0μg / l, 1μg / l, 3μg / l, 9μg / l, 27μg / l, and 81μg / l;

[0059] (2) Oxyphenidazole ELISA plate: 1 plate (8 wells × 12 strips), which is an ELISA plate coated with “Oxyphenidazole-OVA” prepared in Example 2;

[0060] (3) Oxyphenidazole antibody working solution: 1 bottle (7ml), which is an antibody dilution solution to dilute the antibody 1:40000. The antibody dilution solution is 0.2M PBS containing 6% (volume fraction) goat serum. The oxyphenidazole antibody is the antibody obtained by purification of the antiserum prepared in Example 3.

[0061] (4) Enzyme-conjugated working solution: 1 bottle (12ml), the enzyme conjugate is a goat anti-mouse antibody conjugated with horseradish peroxidase;

[0062] (5) Sample dilution solution A: 1 bottle (150ml), which is 0.1M pH 6.2 disodium hydrogen phosphate-citric acid buffer;

[0063] (6) Sample dilution solution B: 1 bottle (100ml), which is 0.01M pH6.0 disodium hydrogen phosphate-citric acid buffer;

[0064] (7) Concentrated washing solution: 1 bottle (20×, 25ml), which is 0.01M pH7.4 PBST solution;

[0065] (8) One bottle (7 ml) each of substrate A solution and substrate B solution. Substrate A is a 2% urea peroxide aqueous solution. Substrate B is a 1% tetramethylbenzidine aqueous solution.

[0066] (9) Termination solution: 1 bottle (7ml), which is 2M H2SO4 solution;

[0067] (10) Cover film;

[0068] (11) Self-sealing bags.

[0069] 2. Equipment and materials that were required but not provided

[0070] (1) Equipment

[0071] ELISA reader (detection wavelength 450nm, reference wavelength 630nm), homogenizer, balance (accuracy: 0.01g), vortex mixer, centrifuge (4000g), micropipette, timer.

[0072] (2) Reagents

[0073] Tissue extraction solution: Accurately weigh 1.59g sodium carbonate and 2.93g sodium bicarbonate, add 1000ml deionized water to dissolve, mix well and set aside.

[0074] Ethyl acetate, n-hexane.

[0075] 3. Storage

[0076] If you are not using the kit, store the 10× concentrated standard at -20°C to prevent degradation.

[0077] Other components of the kit should be stored at 2-8℃. Do not freeze. Shelf life is 1 year.

[0078] Unused ELISA strips should be sealed and stored at 2-8℃.

[0079] 4. Detection principle of the reagent kit

[0080] Oxyphenidazole in the sample competes with the antigen-specific antibody immobilized on the ELISA plate. An enzyme label is added, catalyzing the substrate to develop a color. The intensity of the color development indicates the amount of oxyphenidazole in the sample. A deeper color indicates a lower concentration, and a lighter color indicates a higher concentration.

[0081] II. Instructions for use of the Ofendazol ELISA kit

[0082] 1. Sample pretreatment

[0083] (1) Beef, mutton, pork, and chicken (dilution factor: 10)

[0084] a) Weigh 3 ± 0.05 g of homogenized sample into a 50 ml centrifuge tube; b) Add 3 ml of tissue extract and 9 ml of ethyl acetate, and vortex thoroughly for 3 min; c) Centrifuge at 4000 g or higher for 5 min; d) Transfer 3 ml of supernatant to a new centrifuge tube; e) Dry the sample in a 50-60℃ water bath under nitrogen; f) Add 1 ml of n-hexane and vortex thoroughly for 30 s; g) Add 750 μl of sample diluent B, cap the tube, and gently invert it 20 times; h) Centrifuge at 4000 g or higher for 5 min; i) Completely discard the upper n-hexane layer and the middle layer impurities; j) Transfer 100 μl of the clear lower layer liquid to a new centrifuge tube, add 900 μl of sample diluent A, and vortex thoroughly for 1 min; k) Immediately take 50 μl for analysis.

[0085] (2) Cow's milk and goat's milk (dilution factor: 10)

[0086] a) Measure 3 ml of the mixed sample into a 50 ml centrifuge tube; b) Add 9 ml of ethyl acetate and vortex thoroughly for 3 min; c) Centrifuge at 4000 g or higher for 5 min; d) Take 3 ml of the supernatant into a new centrifuge tube; dry it in a 50-60℃ water bath under nitrogen; e) Add 1 ml of n-hexane and vortex thoroughly for 30 s; f) Add 1 ml of sample diluent B, cap the tube, and gently invert it 20 times; g) Centrifuge at 4000 g or higher for 5 min; h) Completely discard the upper n-hexane layer and the middle layer impurities; i) Take 100 μl of the clear lower layer liquid and add it into a new centrifuge tube, then add 900 μl of sample diluent A and vortex thoroughly for 1 min; j) Immediately take 50 μl for analysis.

[0087] 2. Detection steps (1) Insert the strips into the microplate rack and record the positions of each standard and sample. It is recommended to perform parallel double wells. Unused strips should be sealed in a self-sealing bag and stored immediately at 2-8℃.

[0088] (2) Add 50 μl of each concentration of oxifenedazol standard working solution (or sample solution to be tested) to the corresponding standard (or sample well);

[0089] (3) Add 50 μl of sample solution to the corresponding sample well;

[0090] (4) Add 50 μl of antibody working solution to each well;

[0091] (5) Cover the plate with the cover film, gently shake the plate for 10 seconds to mix thoroughly, and react at room temperature (25±2℃) in the dark for 30 minutes.

[0092] (6) Remove the cover film;

[0093] (7) Pour out the liquid in the plate wells, add 260 μl of washing working solution to each well, and wash thoroughly 4 times, soaking for 15-30 seconds each time;

[0094] (8) Pour out the liquid from the wells, invert the ELISA plate onto absorbent paper, and pat it dry;

[0095] (9) Add 100 μl of enzyme-conjugated working solution to each well;

[0096] (10) Cover the plate with the cover film, gently shake the plate for 10 seconds to mix thoroughly, and react at room temperature (25±2℃) in the dark for 30 minutes.

[0097] (11) Repeat steps (6)-(8);

[0098] (12) Immediately add 100 μl of the A and B mixture to each well;

[0099] (13) Cover the plate with the cover film, gently shake the plate for 10 seconds to mix thoroughly, and react at room temperature (25±2℃) in the dark for 15-20 minutes.

[0100] (14) Uncover the cover film, add 50 μl of stop solution to each well, gently shake the microplate for 10 seconds to mix thoroughly;

[0101] (15) Within 5 minutes after termination, use an ELISA reader to read the absorbance value of the ELISA plate at dual wavelengths of 450nm and 630nm.

[0102] 3. Result calculation or judgment

[0103] (1) The average absorbance value of each standard (or the sample to be tested) is divided by the absorbance value of the zero standard (the standard with a concentration of 0 μg / l) and multiplied by 100 to obtain the percentage of absorbance corresponding to each standard, i.e., the percentage absorbance value.

[0104] (2) Plot a standard curve with the percentage absorbance of each standard as the ordinate and the corresponding oxifenezole concentration as the abscissa.

[0105] (3) Substitute the percentage absorbance value of the sample to be tested into the standard curve equation to obtain the concentration of the sample to be tested. Then multiply it by the dilution factor of the corresponding sample to obtain the actual content of oxifenezole in the original sample to be tested.

[0106] III. Detection of Ophenazol Using an Enzyme-Linked Immunosorbent Assay Kit

[0107] Following the method of using the kit in Example 2, the sensitivity (IC50) of the kit developed in this invention was determined. 50 ), detection limit, 1, kit sensitivity (IC50) 50 ) Measurement

[0108] Oxyphenidazole standards with concentrations of 0.0, 0.1, 0.3, 0.9, 2.7, and 8.1 μg / L were tested. A standard curve was plotted with the logarithm of the standard concentration on the x-axis and the absorbance B / B0 on ​​the y-axis (B is the OD value of each standard concentration, and B0 is the OD value of 0 μg / L).

[0109] The standard curve for oxifenedazol is shown below. Figure 2 .

[0110] The equation for the oxifenedazole standard curve is: y = 0.0105 + 1.042 / (1 + (x / 0.1941)^1.1262)

[0111] Correlation coefficient (R) 2 The value is 0.9985, IC 50 The concentration was 0.1998 μg / L, and the sensitivity of the ofendazol ELISA kit was 0.20 μg / L.

[0112] 2. Determination of the lowest detection limit

[0113] Twenty blank samples were tested. The measured values ​​were obtained according to the standard curve, and the average value was calculated. The value was then multiplied by three times the standard deviation to obtain the limit of detection. The results are shown in Table 1. The limit of detection for oxifenesole in milk, pork and chicken was 5 μg / kg.

[0114] Table 1. Statistical table of blank sample determination results (μg / kg)

[0115]

[0116] 3. Determination of method accuracy and precision

[0117] Twenty blank samples were measured separately. Pretreatment was performed according to the method provided in this invention. The measured values ​​were obtained from the standard curve, and the average value was calculated. Adding three times the standard deviation gave the limit of detection. The results are shown in Tables 2-4. The results indicate that the recoveries of the various additive concentrations in milk, pork, and chicken samples ranged from 87.69% to 106.09%; the intra-batch coefficient of variation ranged from 4.38% to 9.83%; and the intra-batch coefficient of variation ranged from 7.38% to 10.71%.

[0118] Table 2. Reagent kit accuracy and precision (milk)

[0119]

[0120] Table 3. Reagent kit accuracy and precision (pork)

[0121]

[0122] Table 4. Reagent kit accuracy and precision (chicken meat)

[0123]

[0124] 4. Specific detection

[0125] The specificity of the oxifenezole enzyme-linked immunosorbent assay kit is determined by cross-reactivity testing with the corresponding substance. The smaller the cross-reactivity, the better the specificity.

[0126] Perform serial dilutions of oxendazole and other analogues (carbendazole, albendazole, oxendazole, oxendazole, mebendazole, flubendazole, fenbendazole, thiabendazole, trichlorobendazole) as described in step 2 above, replacing the "oxendazole standard working solution" with the serial dilutions of oxendazole and other analogues. Construct standard curves and determine the 50% inhibitory concentration (IC50) of each on the curves. 50The specific method is as follows: Obtain the concentration of oxifenedazol (μg / L) corresponding to a vertical axis value of 50%, i.e., IC50. 50 Value. Calculate the cross-reactivity of the kit with oxendazole and its analogues using the following formula.

[0127] Cross-reactivity rate (%) = (Concentration of oxendazole causing 50% inhibition / Concentration of oxendazole analog causing 50% inhibition) × 100%

[0128] The results are shown in Table 5. As can be seen from Table 5, the oxendazole enzyme-linked immunosorbent assay kit has a high cross-reactivity rate with various analogs, indicating that the oxendazole enzyme-linked immunosorbent assay kit can be used for the detection of benzimidazole drugs.

[0129] Table 5. Specificity of the Ofendazol ELISA Kit

[0130]

[0131] 5. Comparison of detection results between the oxifenedazol ELISA kit and the instrument method

[0132] Three types of samples (milk, pork, and chicken, a total of 60 samples) were tested using the ELISA method. The results were then compared with those obtained by HPLC-MS / MS for confirmation. The ND result of the reference method indicated no detection. For the ELISA method, a "-" sign indicated results below the limit of detection (LOD), and a "+" sign indicated results above the ELISA LOD but below the LOD of the reference method. The results are shown in Table 6. Table 6 shows that the ELISA method is completely consistent with the liquid chromatography-tandem mass spectrometry method described in GB / T 21324-2007 and GB / T 22972-2008 in terms of result interpretation.

[0133] Table 6 Comparison results of reagent kits and instrument methods

[0134]

[0135] Example 5: Detection of Oxyphenidazole with Colloidal Gold Test Strip

[0136] I. Composition of Ophenazine Colloidal Gold Test Strip

[0137] The oxifenedazol colloidal gold test strip consists of a sample absorption pad, a colloidal gold pad, a reaction membrane, and an absorbent pad.

[0138] Along the axial direction of the test strip, the sample absorption pad, colloidal gold pad, reaction membrane, and absorbent pad are connected in sequence. The end of the sample absorption pad is connected to the beginning of the colloidal gold pad, the end of the colloidal gold pad is connected to the beginning of the reaction membrane, and the end of the reaction membrane is connected to the beginning of the absorbent pad.

[0139] The colloidal gold pad is coated with the ofendazol monoclonal antibody obtained in Example 3, which is labeled with colloidal gold.

[0140] The reaction membrane has a detection area and a control area. Both the detection area (T line) and the control area (C line) are strips perpendicular to the axis of the test strip. The detection area is located on the side near the end of the colloidal gold pad, and the control area is located on the side away from the end of the colloidal gold pad. The detection area is coated with oxifenezole-OVA prepared in Example 2, and the control area is coated with goat anti-mouse secondary antibody.

[0141] The sample well is located on the sample absorbent pad at the end furthest from the colloidal gold pad.

[0142] II. Preparation of test strips

[0143] 1. Colloidal gold-labeled antibodies

[0144] (1) Preparation of colloidal gold solution

[0145] Take 100 ml of 0.01% chloroauric acid aqueous solution and heat it to boiling using a constant temperature electromagnetic stirrer. While stirring continuously, add 2.5 ml of 1% trisodium citrate aqueous solution and continue stirring and heating for 20 minutes. The solution will turn a clear red color. Cool to room temperature, restore to the original volume with deionized water, and store at 2-8℃.

[0146] (2) Preparation of gold-labeled antibody solution

[0147] Adjust the pH of the colloidal gold solution to 8.2 with 0.1 mol / L K2CO3 aqueous solution, then take 10 ml and add it to a 50 ml beaker. Stir with a magnetic stirrer at 250 r / min, add the monoclonal antibody solution dropwise, and add 3 ml of 5 g / 100 ml BSA aqueous solution dropwise. Continue stirring for 10 min.

[0148] (3) Centrifuge the gold-labeled antibody solution at 20-24℃ at low speed (1500r / min) for 20min, discard the precipitate formed by the aggregated gold particles, and take the red supernatant solution.

[0149] (4) Centrifuge the solution from step (3) at 4°C and 11000 r / min for 40 min. The solution will separate into three layers (a clear supernatant, a flowable dark red precipitate at the bottom of the tube, and a layer of dense black gold particles on the bottom wall of the tube). Transfer the flowable dark red precipitate to another centrifuge tube and resuspend it in 0.01 mol / L phosphate buffer containing 1 g / 100 ml BSA to the original volume of the gold-labeled antibody solution. Incubate overnight and centrifuge at 4°C and 11000 r / min for 40 min. Collect the precipitate.

[0150] (5) Resuspend the precipitate from step (4) in 0.01 mol / L phosphate buffer containing 1 g / 100 ml BSA and 0.02 g / 100 ml NaN3 to 1 / 40 of the original gold-labeled antibody solution and store at 2-8 °C.

[0151] 2. Spraying with gold: Spray the suspension obtained in step (1) onto the glass fiber membrane to make a colloidal gold pad.

[0152] 3. Spraying the membrane: Spray the ofendazol-OVA prepared in Example 2 onto the T-line position on the reaction membrane, and spray the goat anti-mouse antibody onto the C-line position.

[0153] 4. Assembly: Assemble the sample absorption pad (cellulose filter membrane), colloidal gold pad, nitrocellulose membrane, and absorbent pad using standard methods, then cut into strips for testing. Alternatively, the test strips can be inserted into plastic cards to form test cards for testing.

[0154] III. Testing with test strips

[0155] 1. Sample pretreatment

[0156] Milk: After the milk sample has been brought to room temperature, shaken well and tested directly.

[0157] 2. Testing Steps

[0158] Remove the test strip, open it, and place it flat on the table. Draw 200 μl of the sample solution to be tested into the gold-labeled microwell, and carefully blow until the purple-red particles at the bottom of the well are completely dissolved. Place the gold-labeled microwell in a 50°C incubator and incubate for 3 minutes. Insert the test strip into the gold-labeled microwell, ensuring that the sample pad is fully immersed in the sample solution to be tested, and react for 5 minutes. Remove the test strip from the gold-labeled microwell, discard the sample pad at the bottom of the test strip, and determine the result.

[0159] 3. Result Determination

[0160] Negative: The C line is visible, and the T line is darker or the same color as the C line, indicating a negative result. Positive: The C line is visible, and the T line is lighter or not visible, indicating a positive result. Invalid: The C line is not visible, and regardless of whether the T line is visible, the test strip is considered invalid.

Claims

1. A compound having the structure shown in Formula I: Equation I.

2. A method for preparing the compound of claim 1, comprising the steps of: 2-Methoxycarbonylamino-3H-benzimidazole-5-carboxylic acid was added to N,N-dimethylformamide and stirred. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-bromosuccinimide were added and stirred. 4-(aminomethyl)benzoic acid was added and reacted. The reaction solution was extracted, evaporated to dryness, chromatographically analyzed, and evaporated to dryness again to obtain solid 1. The ratio of 2-methoxycarbonylamino-3H-benzimidazole-5-carboxylic acid, N,N-dimethylformamide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-bromosuccinimide, and 4-(aminomethyl)benzoic acid was 500 mg: 5 ml: 448 mg: 269 mg: 446 mg.

3. Olfendazol antigen, which is an antigen obtained by conjugating the compound of claim 1 to a carrier protein.

4. The oxifenedazol antigen according to claim 3, characterized in that: The carrier protein is bovine serum albumin, ovalbumin, human serum albumin, hemocyanin, mouse serum albumin, thyroprotein, or rabbit serum albumin.

5. The method for preparing the ofendazol antigen according to claim 3 or 4, comprising the following steps: coupling the compound of claim 1 with a carrier protein via an amide bond to obtain the ofendazol antigen.

6. The method for preparing oxifenedazol antigen according to claim 5, characterized in that: The method includes the following steps: (1) Dissolve the compound in N,N-dimethylformamide, then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and stir magnetically at 20-25°C for 2-3 h to obtain solution I; The ratio of the compound, the N,N-dimethylformamide, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and the N-hydroxysuccinimide is 13.73 mg: 1.5 ml: 21.5 mg: 13 mg. (2) The carrier protein was placed in 0.1M sodium bicarbonate buffer and stirred at 200 rpm for 10 min to dissolve it completely, to obtain solution II; the ratio of the carrier protein to the 0.1M sodium bicarbonate buffer was 33.6-50 mg: 3.5 ml; Wherein, if the carrier protein is bovine serum albumin, the ratio of bovine serum albumin to the 0.1M sodium bicarbonate buffer is 50mg:3.5ml; if the carrier protein is ovalbumin, the ratio of ovalbumin to the 0.1M carbonate buffer is 33.6mg:3.5ml. (3) Mix the solution I and the solution II, specifically by adding the solution I dropwise to the solution II under 0-4℃ conditions and stirring at 1000 rpm, and stirring at 500 rpm for 24 h to obtain the solution III; (4) The solution III was dialyzed at 4°C for 3 days with 0.01M pH7.2 phosphate buffer to obtain the ofendazol antigen.

7. The application of the compound of claim 1 or the oxifenezole antigen of claim 3, characterized in that... Prepare reagents for qualitative or quantitative detection of oxifendazole; prepare oxifendazole antibodies.

8. The antibody prepared using the oxifenedazol antigen according to claim 3, characterized in that... The antibody is an anti-oxifendazole monoclonal antibody; the amino acid sequence of the heavy chain variable region of the anti-oxifendazole monoclonal antibody is shown in Sequence 1 of the sequence listing; the amino acid sequence of the light chain variable region of the anti-oxifendazole monoclonal antibody is shown in Sequence 2 of the sequence listing.

9. The use of the oxifenezole antigen according to claim 3 in the preparation of enzyme-linked immunosorbent assay kits and colloidal gold test card reagents.

10. The use of the oxifenedazol antigen according to claim 9, characterized in that... The enzyme-linked immunosorbent assay kit and colloidal gold test card were used to detect dairy products and tissues. The detection limit for oxifenesole in the above samples was 5 μg / kg, and the sensitivity was 0.20 μg / L.

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