Nifuroxol metabolite enzyme-linked immunosorbent assay kit and application

Abstract

The invention provides a nifuralol metabolite enzyme-linked immunosorbent assay kit. The nifuralol metabolite enzyme-linked immunosorbent assay kit comprises a microporous elisa plate, a nifuralol antibody working solution, a nifuralol standard substance, a horse radish peroxidase-labeled anti-antibody working solution, a 20 * concentrated washing solution, a substrate solution A, a substrate solution B and a stop solution, the microporous ELISA plate is coated with a nifurasol hapten-carrier protein conjugate, and a nifurasol hapten-carrier protein conjugate is coated on the microporous ELISA plate; the nifurasol hapten is prepared by the following steps: weighing 1.0 g of nifurasol, adding 20 mL of acetone and 20 mL of pyridine, and uniformly stirring; and weighing 0.5 g of succinic anhydride, adding the succinic anhydride into the solution obtained in the previous step, stirring and reacting at 80 DEG C for 6 hours, drying by distillation at 100 DEG C, loading on a silica gel column, eluting by using an eluent of petroleum ether and ethyl acetate in a ratio of 1: 1, and separating and purifying to obtain the nifurasol hapten product. The nifurasol enzyme-linked immunosorbent assay kit disclosed by the invention is relatively good in technical parameters such as detection sensitivity and the like, and can be stored for a long time.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology detection, and specifically relates to an enzyme-linked immunosorbent assay kit for nitrofuroxol metabolites and an application thereof. Background Art

[0002] Nifursol, also known as nifuroxil, is a nitrofuran antibacterial drug. The structure and properties of nifuroxil are similar to those of other nitrofuran drugs such as nifuroxil metabolites. The original drug is rapidly metabolized in animal tissues, but its labeled metabolites (DNSH / DNSAH) can bind tightly to proteins in the animal body to form stable binding residues, which can exist in the body for a considerable period of time and have certain toxicity.

[0003] Nitrofuran drugs have potential teratogenic, carcinogenic, and mutagenic effects. Their abuse will cause veterinary drug residues in animals, ultimately endangering the health of consumers. In 2020, my country's Ministry of Agriculture and Rural Affairs Announcement No. 250 listed nitrofuran drugs in the "List of Drugs and Other Compounds Prohibited for Use in Food Animals." The announcements in Annex IV of EU Directives 1756 / 2002 / EC and 90 / 2377 / EC prohibit the use of nitrofuran as a veterinary drug additive. In addition, Regulation (EU) 2019 / 1971 also requires that from November 28, 2022, the determination limit of nitrofuran metabolites (nitrofurosol metabolites, a total of 5 types) in animal-derived foods shall be 0.5 μg / kg.

[0004] At present, the main detection methods for nitrofuran drugs include liquid chromatography-tandem mass spectrometry, gas chromatography, etc. Instrumental analysis has the advantages of high detection accuracy and good repeatability, but it has disadvantages such as cumbersome pre-treatment procedures, long measurement time, high instrument cost, and high professional requirements for detection personnel. It is not conducive to the screening of large quantities of samples, nor is it conducive to effective supervision of the use of nitrofuran drugs. Enzyme-linked immunosorbent assay is a rapid screening method based on the specific binding reaction between antigens and antibodies. It uses enzymes as reaction media. It has the characteristics of simple and rapid detection, large sample volume, and low requirements for detection personnel. Summary of the invention

[0005] The purpose of the present invention is to provide an enzyme-linked immunosorbent assay kit for nitrofuroxol metabolites, which has strong detection stability, high detection sensitivity, strong specificity and short detection time.

[0006] Technical solution of the present invention:

[0007] A nitrofurosol metabolite enzyme-linked immunosorbent assay kit comprises a microporous ELISA plate coated with a nitrofurosol hapten-OVA conjugate, a nitrofurosol antibody working solution, a nitrofurosol standard, a horseradish peroxidase-labeled anti-antibody working solution, a 20× concentrated washing solution, a substrate solution A, a substrate solution B, and a stop solution. The microporous ELISA plate is coated with a nitrofurosol hapten-carrier protein conjugate. The nitrofurosol hapten is prepared by the following steps: weighing 1.0 g of nitrofurosol, adding 20 mL of acetone and 20 mL of pyridine, and stirring evenly; weighing 0.5 g of succinic anhydride, adding it to the solution in the above step, stirring and reacting at 80° C. for 6 h, evaporating to dryness at 100° C., applying to a silica gel column, eluting with an eluent of petroleum ether:ethyl acetate=1:1, separating and purifying, and finally obtaining 0.8-1 g of a nitrofurosol hapten product.

[0008] Further, the nitrofurosol hapten-carrier protein conjugate is prepared by the following steps: 15 mg of nitrofurosol hapten is weighed and dissolved in 0.5 mL of DMF to obtain a hapten dilution, and then 10 μL of isobutyl chloroformate is added, and stirred at room temperature for 0.5 h to obtain an activated hapten dilution. Weigh 50 mg of the carrier protein BSA or OVA and fully dissolve it in 5 mL, 0.05 mol / L pH 9.0 sodium carbonate solution. Add the activated hapten dilution dropwise to the dissolved carrier protein solution, and stir the reaction at room temperature for 24 h. Dialyze with 0.01 mol / L PBS, dialyze at 4 ° C for three days, change the solution three times a day, and retain the molecular weight of 8000-14000D. After preparation, it is packaged and stored at -20 ° C.

[0009] Furthermore, 5% glycerol, 2.5% trehalose, 2.5% L-histidine, 0.05% benzamidine hydrochloride, and 0.05mol / LpH6.0 2-morpholineethanesulfonic acid buffer were added to the nitrofuroxol antibody working solution as antibody stabilizers, and 50mmol / LpH8.0 barbituron-hydrochloric acid buffer containing 1% PEG-2000, 0.2% magnesium chloride, 0.05% fatty alcohol polyoxyethylene ether 23, and 1.5% bovine serum albumin was added to the horseradish peroxidase labeled anti-antibody working solution as an enzyme conjugate stabilizer. The addition of the stabilizer protection solution effectively improves the stability of the reagent and extends the shelf life of the reagent.

[0010] Furthermore, the nifuroxol antibody is prepared by immunizing mice or rabbits with the nifuroxol hapten-carrier protein conjugate as an immunogen.

[0011] Furthermore, the concentrated washing solution contains 1% Tween-20 and 0.1 mol / L PBS buffer.

[0012] Furthermore, the substrate A liquid is hydrogen peroxide, and the substrate B liquid is TMB, which are mixed within 10 minutes before use.

[0013] Furthermore, the stop solution is 1 mol / L H2SO4.

[0014] The nitrofuroxol metabolite enzyme-linked immunosorbent assay kit also includes a sample reconstitution solution when detecting a specific sample. The sample reconstitution solution is a PBS solution containing 50% methanol and 0.05 mol / L pH 8.0, and the percentage is a volume ratio.

[0015] Another object of the present invention is to provide the use of the above-mentioned nitrofurosol metabolite enzyme-linked immunosorbent assay kit in detecting nitrofurosol metabolites in fish samples.

[0016] Beneficial effects of the present invention:

[0017] The nitrofuroxol metabolite enzyme-linked immunosorbent assay kit of the present invention coats the coupled antigen on a microplate, uses an antibody working solution to compete with the sample reaction for the coating, then adds an enzyme-labeled secondary antibody for detection and develops color through a substrate, analyzes and determines the residual amount of the nitrofuroxol metabolite, and establishes an indirect competitive ELISA method. When preparing the nitrofuroxol metabolite enzyme-linked immunosorbent assay kit of the present invention, the PBS buffer containing 2.5% sucrose, 1% gelatin, 0.5% casein, and 0.1 mol / L pH 8.0 is used for blocking in the process of preparing the ELISA plate, an antibody stabilizer is added to the antibody working solution, and a horseradish peroxidase conjugate stabilizer is added to the anti-antibody labeled with horseradish peroxidase, which effectively improves the stability of the detection kit of the present invention, greatly prolongs the shelf life of the kit, and avoids the phenomenon of inaccurate detection results due to reagent instability, resulting in waste of reagents. The nitrofuroxol metabolite enzyme-linked immunosorbent assay kit of the present invention has a detection sensitivity of up to 0.1 μg / kg for nitrofuroxol, a determination recovery rate between 88% and 104%, and a coefficient of variation of less than 8%. The kit of the present invention has a simple structure, high detection sensitivity, high stability, good accuracy and precision detection data, and low detection cost, and is suitable for rapid screening of nitrofuroxol metabolites in large batches of samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Synthetic route of nitrofuroxol hapten-carrier protein;

[0019] Figure 2 Kit standard curve graph. DETAILED DESCRIPTION

[0020] In order to further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention. Unless otherwise specified, the reagents of the present invention are all conventional reagents and can be purchased from the market.

[0021] Embodiment 1

[0022] 1. The nitrofuroxol metabolite enzyme-linked immunosorbent assay kit contains the following components:

[0023] (1) A 96-well ELISA plate coated with nitrofuroxol hapten-OVA conjugate antigen.

[0024] (2) Nitrofuroxol antibody working solution, specifically Nitrofuroxol monoclonal antibody, 7 mL / bottle. The antibody working solution is added with 5% glycerol, 2.5% trehalose, 2.5% L-histidine, 0.05% benzamidine hydrochloride, 0.05 mol / L pH 6.0 2-morpholineethanesulfonic acid buffer as an antibody stabilizer, and the percentages are weight volume percentages.

[0025] (3) Nifurosol standard: 0 μg / L, 0.1 μg / L, 0.3 μg / L, 0.9 μg / L, 2.7 μg / L, 8.1 μg / L, 1 mL / bottle.

[0026] (4) Horseradish peroxidase labeled goat anti-mouse anti-antibody working solution, 12 mL / bottle, to which 1% PEG-2000, 0.2% magnesium chloride, 0.05% fatty alcohol polyoxyethylene ether 23, and 1.5% bovine serum albumin in 50 mmol / L pH 8.0 barbiturate-hydrochloric acid buffer were added as an enzyme conjugate stabilizer, and the percentages are weight volume percentages.

[0027] (5) 20× concentrated washing solution, containing 1% Tween-20, 0.1 mol / L pH 7.2 PBS buffer, 30 mL / bottle, diluted with deionized water at a ratio of 1:19 when used.

[0028] (6) Sample reconstitution solution, composed of 50% methanol, 0.05 mol / L pH 8.0 PBS solution, 8 mL / bottle, percentages are by volume.

[0029] (7) Substrate A solution is hydrogen peroxide, and substrate B solution is TMB, both at 7 mL / bottle.

[0030] (8) The stop solution is 1 mol / L H2SO4, 7 mL / bottle.

[0031] 2. Preparation of main components:

[0032] 2.1. Synthesis of hapten, immunogen and coating

[0033] 2.1.1 Preparation of nitrofuroxol hapten

[0034] Weigh 1.0 g of nitrofurosol, add 20 mL of acetone and 20 mL of pyridine, and stir evenly; weigh 0.5 g of succinic anhydride and add it to the solution in the above step, stir and react at 80 ° C for 6 h, evaporate to dryness at 100 ° C, apply to a silica gel column, use petroleum ether: ethyl acetate = 1:1 eluent for elution, separation and purification, and finally obtain 0.8-1 g of nitrofurosol hapten product, see Figure 1 .

[0035] 2.1.2 The immunogen is nitrofuroxol hapten-BSA, and the coating is nitrofuroxol hapten-OVA. The preparation process is as follows:

[0036] Weigh 15 mg of nitrofurosol hapten and dissolve it in 0.5 mL of DMF to obtain a hapten dilution, then add 10 μL of isobutyl chloroformate and stir at room temperature for 0.5 h to obtain an activated hapten dilution. Weigh 50 mg of carrier protein BSA or OVA and fully dissolve it in 5 mL, 0.05 mol / L pH 9.0 sodium carbonate solution. Add the activated hapten dilution dropwise to the dissolved carrier protein solution and stir the reaction at room temperature for 24 h. Dialyze with 0.01 mol / L PBS at 4 ° C for three days, change the liquid three times a day, and retain the molecular weight of 8000-14000D. After preparation, aliquot and store at -20 ° C.

[0037] 2.2 Preparation of monoclonal antibodies against nitrofuroxol

[0038] 2.2.1 Animal immunization

[0039] Healthy female Balb / c mice aged 6 weeks were selected and subcutaneously injected with nitrofuroxol hapten-BSA conjugate containing Freund's complete adjuvant. The immunization dose was 150 μg / mouse. Immunizations were performed at 2, 4 and 8 weeks respectively, and the last immunization was at 10 weeks. Splenocytes were collected three days after the last booster immunization.

[0040] 2.2.2 Preparation of myeloma cells and spleen cells

[0041] SP2 / 0 myeloma cells were cultured in RPMI-1640 culture medium to make the cells in logarithmic growth phase to prepare SP2 / 0 myeloma cell suspension; spleen of immunized mice was taken, broken, filtered through 200-mesh stainless steel gauze to prepare spleen cell suspension, counted and set aside.

[0042] 2.2.3 Cell fusion and monoclonalization

[0043] SP2 / 0 myeloma cells and spleen cells were fused and positive wells were screened by indirect competitive ELISA. Positive wells were monocloned by limiting dilution method until a hybridoma cell line that stably secreted nitrofuroxol monoclonal antibody was obtained.

[0044] 2.2.4 Cell cryopreservation and thawing

[0045] The nitrofuroxol monoclonal hybridoma cell line was made into a cell suspension and frozen in liquid nitrogen. When thawing, the cryopreserved tube was taken out and immediately placed in a 37°C water bath for rapid thawing, transferred to a conical flask, added with complete culture medium, and cultured in a CO2 incubator.

[0046] 2.2.5 Mass production and purification of monoclonal antibodies

[0047] Healthy Balb / c female mice were intraperitoneally injected with paraffin oil (0.5 mL / mouse). Ten days later, nitrofuroxol monoclonal hybridoma cell line (10 μg / mL) was intraperitoneally injected. 7 The ascites was collected after 7 days of observation and centrifugation at 4000 rpm for 10 min. The supernatant was collected and the purified antibodies were stored at -20°C.

[0048] 2.2.6 Specificity of nitrofuroxol monoclonal antibody

[0049] Antibody specificity refers to the ability of an antibody to bind to antigenic determinants of different structures, which is expressed by the cross-reaction rate. A small cross-reaction rate can prove that the antibody has high specificity. The 50% inhibition concentration (IC 50 ). Use the following formula to calculate the cross-reaction rate of the kit to other drugs:

[0050] Cross-reaction rate (%) = (nitrofuroxol IC 50 / Nitrofuroxol analog IC 50 )×100%

[0051] It is measured that the cross-reaction rate of the nifuroxol metabolite enzyme-linked immunosorbent assay kit of the present invention to nifuroxol and nifuroxol metabolites is 100%, and the cross-reaction rate to furazolidone metabolites, furazolidone, nitrofurantoin metabolites, nitrofurantoin, furaltadone metabolites, furaltadone, nitrofurazone metabolites, and nitrofurazone is less than 1%, and the content of nifuroxol and nitrofurazone metabolites in samples can be specifically detected.

[0052] 2.3. Preparation of goat anti-mouse antibody labeled with horseradish peroxidase

[0053] The goat anti-mouse anti-antibody labeled with horseradish peroxidase is obtained by coupling horseradish peroxidase with goat anti-mouse anti-antibody by using sodium periodate method.

[0054] 2.4. ELISA plate

[0055] The nitrofuroxol hapten-OVA conjugate was diluted to 2 μg / mL with 0.05 mol / L, pH 9.6 carbonate buffer, 100 μL was added to each well, incubated at 37°C for 2 hours, washed twice, and dried; then 250 μL of coating blocking solution was added to each well for blocking, incubated at 37°C for 2 hours, the liquid in the well was poured out, patted dry with absorbent paper, and then vacuum sealed with aluminum film for storage. The coating blocking solution is a PBS buffer containing 2.5% sucrose, 1% gelatin, 0.5% casein, and 0.1 mol / L pH 8.0. The coating blocking solution can play a blocking role, reduce the background effect of the reagent wells, and improve the detection accuracy of the kit.

[0056] The coated ELISA plate and other reagents of the present invention were placed in a 37°C constant temperature box for aging test. On the 45th day, the absorbance value was measured and the A value decreased by less than 10%.

[0057] The stability of the ELISA plate and reagents is a key indicator for measuring product quality and an important guarantee for the safety and effectiveness of the kit. In order to significantly improve the stability of the kit of the present invention. In the process of developing the kit of the present invention, 5% glycerol, 2.5% trehalose, 2.5% L-histidine, 0.05% benzamidine hydrochloride, 0.05mol / LpH6.0 2-morpholineethanesulfonic acid buffer was added to the nitrofuroxol antibody working solution as an antibody stabilizer, and 1% PEG-2000, 0.2% magnesium chloride, 0.05% fatty alcohol polyoxyethylene ether 23, 1.5% bovine serum albumin 50mmol / LpH8.0 barbituron-hydrochloric acid buffer was added to the goat anti-mouse anti-antibody labeled with horseradish peroxidase as an enzyme conjugate stabilizer, which significantly improved the stability of the kit of the present invention and extended the shelf life of the reagent.

[0058] The assembled test kit may also include instructions, a cover film, a ziplock bag, a certificate of conformity, etc. The shelf life of the test kit can reach 18 months under conditions of 2 to 8°C.

[0059] 3. Detection principle of nitrofuroxol metabolite enzyme-linked immunosorbent assay kit:

[0060] Taking advantage of the specific binding property of nitrofuroxol antibody and nitrofuroxol antigen, the antigen is first pre-coated on the ELISA plate, and then the standard or sample and nitrofuroxol antibody are added. Nitrofuroxol competes with the antigen fixed on the ELISA plate for the nitrofuroxol antibody, and then goat anti-mouse antibody labeled with horseradish peroxidase is added for signal amplification and detection. Finally, the substrate is added to catalyze the color development. At this time, the color development depth is inversely proportional to the nitrofuroxol content in the standard or sample.

[0061] 4. Specific detection steps of the kit

[0062] (1) Take out the required number of ELISA strips and insert them into the ELISA plate rack, and record the positions of each standard and sample. To reduce the fluctuation of the test value, perform a double-well parallel experiment.

[0063] (2) Add 50 μL of the standard / sample to the corresponding microwells, then add 50 μL / well of the nitrofuroxol antibody working solution, gently shake for 5 seconds to mix, cover the plate and place in a dark environment at 25°C for 30 minutes.

[0064] (3) Remove the cover film, shake off the liquid in the wells, add 250 μL / well of washing solution, wash twice, pour out the washing solution, and pat dry with absorbent paper.

[0065] (4) Add 100 μL of enzyme-labeled goat anti-mouse antibody to each microwell, shake gently for 5 seconds to mix, cover the plate and place in a dark environment at 25°C for 30 minutes.

[0066] (5) Remove the cover film, shake off the liquid in the wells, add 250 μL / well of washing solution, wash twice, pour out the washing solution, and pat dry with absorbent paper.

[0067] (6) Add 100 μL / well of substrate colorimetric solution, mix well, cover the plate and place in a dark environment at 25°C for 15 min. The substrate colorimetric solution is a mixture of substrate A solution and substrate B solution in a 1:1 volume ratio. The mixture should be used within 10 min.

[0068] (7) Add 50 μL / well of stop solution, mix well, set the microplate reader at dual wavelengths of 450 nm and 630 nm, and measure the OD value.

[0069] 5. Analysis of test results

[0070] Assume that the average absorbance of each standard (or sample) is B, the zero standard is a standard with a concentration of 0 μg / L, and the average absorbance B0 is (B / B0)×100% as the percentage of the absorbance corresponding to each standard (or sample): use a semi-logarithmic system to substitute the percentage of the absorbance corresponding to the standard, and fit the standard curve with the concentration of the standard. Substitute the percentage of the absorbance of the sample to be tested into the fitted standard curve equation to obtain the concentration corresponding to the sample, and finally multiply it by the corresponding dilution factor of the sample to obtain the content of the test substance in the sample.

[0071] Experimental Example: Detection of Nitrofuroxol in Fish Samples

[0072] 1. Sample pretreatment method

[0073] Weigh 1±0.01g of the homogenized fish sample and place it in a 50mL centrifuge tube; add 4mL of ethyl acetate; vortex at room temperature for 1min, centrifuge at 4000rpm for 5min; take 1mL of the supernatant to a centrifuge tube, blow dry with nitrogen in a 50-60℃ water bath; add 0.5mL of n-hexane, then add 1mL of the sample reconstitution solution, vortex for 1min, centrifuge at 4000rpm for 5min; remove the n-hexane layer with a pipette or organic phase aspirator, and take the lower layer solution for analysis. The sample reconstitution solution is a PBS solution containing 50% methanol, 0.05mol / LpH8.0.

[0074] 2. Standard curve fitting

[0075] The standard curve consists of 6 concentrations (0 μg / L, 0.1 μg / L, 0.3 μg / L, 0.9 μg / L, 2.7 μg / L, 8.1 μg / L). Each of the 6 standard points was measured twice to determine the working concentration range and IC 50 The standard curve was prepared with the logarithmic value of the concentration of nitrofuroxol standard as the horizontal axis and the inhibition rate (B / B0%) of each corresponding concentration as the vertical axis. 50 0.336 μg / L, see Figure 2 .

[0076] 3. Sample precision and accuracy test

[0077] Accuracy is expressed as recovery rate, and precision is expressed as coefficient of variation. Nitrofuroxol was added to the fish sample for recovery, so that the final concentration was 0.5 μg / kg. Five parallels were drawn for each concentration, and different kits were used for determination. The same sample was determined 3 times, and the sample recovery rate and coefficient of variation were calculated respectively. The results are shown in Table 1.

[0078] Table 1 Recovery rate and coefficient of variation determination

[0079]

[0080] The results showed that the recovery rate of fish meat samples added with 0.5 μg / kg nitrofuroxol was between 88-104%, and the coefficient of variation was less than 8%.

[0081] Embodiment 2

[0082] (1) A 96-well ELISA plate coated with a nitrofuroxol hapten-OVA conjugate.

[0083] (2) Nitrofuroxol polyclonal antibody working solution, 7 mL / bottle.

[0084] (3) Nifurosol standard: 0 μg / L, 0.1 μg / L, 0.3 μg / L, 0.9 μg / L, 2.7 μg / L, 8.1 μg / L, 1 mL / bottle.

[0085] (4) Horseradish peroxidase-labeled goat anti-rabbit antibody, 12 mL / bottle.

[0086] (5) 20× concentrated washing solution, containing 1% Tween-20, 0.1 mol / L pH 7.2 PBS buffer, 30 mL / bottle, diluted with deionized water at a ratio of 1:19 when used.

[0087] (6) Sample reconstitution solution, consisting of 50% methanol, 0.05 mol / L PBS solution, pH 8.0, 8 mL / bottle.

[0088] (7) Substrate A solution is hydrogen peroxide, and substrate B solution is TMB, both at 7 mL / bottle.

[0089] (8) The stop solution is 1 mol / L H2SO4, 7 mL / bottle.

[0090] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A nitrofuroxol metabolite enzyme-linked immunosorbent assay kit, comprising a microporous ELISA plate, a nitrofuroxol antibody working solution, a nitrofuroxol standard, a horseradish peroxidase-labeled anti-antibody working solution, a 20× concentrated washing solution, a substrate solution A, a substrate solution B, and a stop solution; the microporous ELISA plate is coated with a nitrofuroxol hapten-carrier protein conjugate; the nitrofuroxol hapten is prepared by the following steps: weighing 1.0 g of nitrofuroxol, adding 20 mL of acetone and 20 mL of pyridine, and stirring evenly; weighing 0.5 g of succinic anhydride, adding it to the solution in the above step, stirring and reacting at 80° C. for 6 h, evaporating at 100° C. and applying it to a silica gel column, eluting with an eluent of petroleum ether:ethyl acetate=1:1, separating and purifying, and obtaining a nitrofuroxol hapten product.

2. The nitrofuroxol metabolite enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: The nitrofurosol hapten-carrier protein conjugate is obtained by the following steps: 1) Weigh 15 mg of nitrofurosol hapten and dissolve it in 0.5 mL of DMF to obtain a hapten dilution solution, then add 10 μL of isobutyl chloroformate and stir at room temperature for 0.5 h to obtain an activated hapten dilution solution; 2) Weigh 50 mg of carrier protein BSA or OVA and fully dissolve it in 5 mL of 0.05 mol / L pH 9.0 sodium carbonate solution; 3) adding the activated hapten dilution in step 1) dropwise into the carrier protein solution in step 2) and stirring the mixture at room temperature for 24 hours; 4) The mixture was dialyzed against 0.01 mol / L PBS at 4° C. to prepare a nitrofurosol hapten-carrier protein conjugate.

3. The nitrofuroxol metabolite enzyme-linked immunosorbent assay kit according to claim 1 or 2, characterized in that: The nitrofurosol antibody working solution is added with 5% glycerol, 2.5% trehalose, 2.5% L-histidine, 0.05% benzamidine hydrochloride, and 0.05mol / LpH6.0 2-morpholineethanesulfonic acid buffer as an antibody stabilizer; the horseradish peroxidase labeled anti-antibody working solution is added with 50mmol / LpH8.0 sodium barbiturate-hydrochloric acid buffer containing 1% PEG-2000, 0.2% magnesium chloride, 0.05% fatty alcohol polyoxyethylene ether 23, and 1.5% bovine serum albumin as an enzyme conjugate stabilizer.

4. The nitrofuroxol metabolite enzyme-linked immunosorbent assay kit according to claim 1 or 2, characterized in that: The substrate A liquid A is hydrogen peroxide, and the substrate B liquid is TMB.

5. The nitrofuroxol metabolite enzyme-linked immunosorbent assay kit according to claim 1 or 2, characterized in that: The concentrated washing solution contains 1% Tween-20, 0.1 mol / L PBS buffer, and has a pH of 7.

2.

6. The nitrofuroxol metabolite enzyme-linked immunosorbent assay kit according to claim 1 or 2, characterized in that: The stop solution is 1 mol / L H2SO4.

7. The nitrofuroxol metabolite enzyme-linked immunosorbent assay kit according to claim 1 or 2, characterized in that: The kit also includes a sample reconstitution solution, which contains 50% methanol. 0.05mol / L PBS solution, pH 8.

0.

8. The nitrofuroxol metabolite enzyme-linked immunosorbent assay kit according to claim 1 or 2, characterized in that: The nitrofuroxol antibody is prepared by using the nitrofuroxol hapten-carrier protein conjugate as an immunogen.

9. Use of the nifuroxol metabolite enzyme-linked immunosorbent assay kit according to any one of claims 1 to 8 in detecting nifuroxol metabolite drug residues in animal tissues.

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