CN112462047B - Nicarbazin detection kit and application thereof - Google Patents

Abstract

The invention provides a nicarbazin detection kit, which comprises a 96-hole enzyme label plate, an enzyme label, a nicarbazin label standard substance, 20 multiplied by concentrated washing liquid, 20 multiplied by concentrated complex solution, a substrate A liquid, a substrate B liquid and stop solution, wherein the 96-hole enzyme label plate is coated with nicarbazin hapten-carrier protein, and the enzyme label is a horseradish peroxidase-labeled nicarbazin specific antibody. The invention also provides application of the nicarbazin detection kit in detection of chicken samples. The nicarbazin detection kit adopts a one-step method to detect the nicarbazin marker, has high detection sensitivity, and has better accuracy and precision of detection results.

Description

Nicarbazin detection kit and application thereof

Technical Field

The invention belongs to the field of biotechnology detection, and particularly relates to a nicarbazin enzyme-linked immunosorbent assay rapid detection kit and application thereof.

Background

Nicarbazin, also known as coccidiostatin, is a 1:1 compound composed of dinitro-sym-Diphenylurea (DNC) and hydroxy-dimethyl-pyrimidine (HDP), and is a broad-spectrum, high-efficiency and stable-performance feed additive. Nicarbazin is used to prevent coccidiosis in chickens and turkeys. Although nicarbazin is a compound consisting of two components, namely DNC and HDP, because the HDP component in the compound can be rapidly excreted out of the body through urine in an animal body, and the coccidiostat active component DNC is slowly excreted through feces, dinitro-sym-Diphenylurea (DNC) is internationally specified as a nicarbazin residue marker.

Nicarbazin is widely used at present, but is remained in the muscle and other tissues of poultry to different degrees after being fed, and is harmful to human life and health. The residual marker of nicarbazin in chicken tissue is DNC and the residual limit of nicarbazin in chicken muscle, skin, fat, liver and kidney is 200 microgram/kg.

At present, the detection method of the nicarbazin residual marker mainly comprises an instrumental analysis method and an immunological analysis method. The method has the disadvantages of complicated pretreatment procedure, long measuring time, high instrument cost, high requirement on the detection personnel and incapability of meeting the real-time rapid detection of nicarbazin. The immunoassay method mainly uses enzyme as a reaction medium for rapid screening, and uses an antigen antibody as a core detection reagent. The immunoassay method has the advantages of simple and rapid detection and is suitable for the detection of a large number of samples. The invention discloses an enzyme linked immunosorbent assay kit for detecting nicarbazin and application thereof, which is disclosed by the invention patent with the application publication number of CN106771137, and discloses the enzyme linked immunosorbent assay kit, which is provided with an ELISA plate coated with nicarbazin coupling antigen, a nicarbazin monoclonal antibody, an enzyme marker anti-antibody, a nicarbazin standard solution, a substrate color developing solution and a stop solution.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide the nicarbazin detection kit which has the advantages of high detection sensitivity, short on-site detection reaction time and simple and convenient detection, and has higher precision and accuracy.

The technical scheme of the invention is as follows:

the nicarbazin detection kit comprises a 96-hole enzyme label plate, an enzyme label, a nicarbazin label standard substance, 20 multiplied concentrated washing liquid, 20 multiplied concentrated complex solution, a substrate A liquid, a substrate B liquid and stop solution, wherein the 96-hole enzyme label plate is coated with a nicarbazin hapten-carrier protein conjugate, and the enzyme label is a nicarbazin specific antibody marked by horseradish peroxidase.

Wherein, the standard substance of the nicarbazin marker is 0 mug/L, 0.025 mug/L, 0.075 mug/L, 0.225 mug/L and 0.675 mug/L.

Wherein the 20 × concentrated washing solution contains 1% polyethylene glycol 400 monooleate, 0.1mol/L MES buffer, and pH6.0.

The 20 Xconcentrated double solution contained 50% of DMF,0.01mol/L carbonate buffer, pH 9.0.

Wherein, the substrate A liquid is hydrogen peroxide, and the substrate B liquid is TMB.

Wherein the stop solution is 2mol/L H ₂ SO ₄ 。

Further, the structural formula of the nicarbazin hapten is shown in the specification

Prepared by the following steps, and the preparation scheme is shown in figure 1.

1) Synthesis of product I

Weighing 5.52g of 4-nitroaniline, 10g of tert-butyl 4-bromobutyrate and 6g of potassium carbonate, placing the mixture in 80mL of DMF, stirring for 4h in an oil bath at 65 ℃, spotting and reacting, adding 80mL of deionized water into a reaction solution, extracting with dichloromethane, combining organic phases, washing the organic phases with deionized water for 1 time, washing with saturated salt for 1 time, and then washing with anhydrous Na ₂ SO ₄ Drying and removal of the solvent under reduced pressure gave an oil which was purified on column with DCM: meOH =100 to give 8.4g of product i as a colorless liquid.

2) Synthesis of product II

5.04g of product I are weighed out into 40mL of methanol, 0.2g of 10% Pd/C is added and the mixture is washed with water and washed with H ₂ Reacted for 8h under an atmosphere of DCM: meOH =10, rf =0.1, filtered to remove Pd/C, and then methanol was removed under reduced pressure to obtain 4.0g of product ii.

3) Synthesis of product III

2.2g of the product II is dissolved in30 mL of toluene, 1.64g of 4-nitrophenyl isocyanate is added dropwise under the cooling of ice water, the mixture is stirred for 2 hours, the reaction is performed by a spot plate, DCM: meOH =10, rf =0.1, a large amount of white solid is separated out, and the mixture is filtered by suction under reduced pressure to obtain 3.1g of a solid product III.

4) Synthesis of product IV, nicarbazin hapten

Weighing 1.92g of the product III, placing the product III in 10mL of methanol, adding 10mL of 2mol/L NaOH solution, stirring at room temperature for 5h, carrying out spotting reaction, adding 20mL of deionized water into the reaction solution, extracting with dichloromethane, combining organic phases, washing the organic phases with deionized water for 2 times, washing the organic phases with saturated common salt water for 1 time, and washing the organic phases with anhydrous Na, wherein the mixture is subjected to methanol removal under reduced pressure, the pH of the obtained solution is adjusted to 2-3 by 1mol/L of HCl, the reaction solution is added with 20mL of deionized water, the mixture is extracted with dichloromethane, the organic phases are combined, and the organic phases are washed with the deionized water for 2 times, saturated common salt water for 1 time, and anhydrous Na ₂ SO ₄ Drying, removing the solvent under reduced pressure to obtain oil, purifying by a column, and obtaining 1.2g of a white solid product (IV) which is the nicarbazin hapten by DCM: meOH = 100.

The nicarbazin antibody is prepared by taking a nicarbazin hapten-KLH conjugate as an immunogen. The immunogen preparation steps are as follows:

1) Weighing 12mg of nicarbazin hapten and dissolving in 1mL of DMF;

2) Weighing 15mgEDC and 10mgNHS and dissolving in 0.2mL deionized water;

3) Adding the solution obtained in the step 2) into the solution obtained in the step 1), and stirring and reacting for 30min at room temperature;

4) Weighing 50mg of KLH, dissolving the KLH in 3.8mL of PBS, adding the solution obtained in the step 3), and stirring at room temperature for reacting for 16 hours;

5) Dialyzing with 0.01mol/L PBS, intercepting the molecular weight of 8000-14000u, dialyzing for 3 days at 4 ℃, and changing the dialyzate every day to obtain the immunogen.

The invention also provides an application of the nicarbazin detection kit in detecting the nicarbazin residue in chicken, which specifically comprises the following steps: processing a chicken sample; detecting by using the nicarbazin detection kit; and analyzing the detection result.

The invention has the beneficial effects that:

the nicarbazin detection kit disclosed by the invention coats the antigen on an enzyme-labeled microporous plate, uses an enzyme-labeled antibody to react with a sample to compete for the coating, performs analysis and determination on the residual quantity of the nicarbazin marker through substrate color development, and establishes a direct competition ELISA method. The detection sensitivity of the nicarbazin detection kit on the nicarbazin marker is up to 0.025 mu g/L, the variation coefficient in the plate is between 2.6 and 4.6 percent, the variation coefficient between plates is between 4.1 and 6.4 percent, and the recovery rate is between 90 and 110.5 percent. The nicarbazin detection kit disclosed by the invention is simple in structure, high in detection sensitivity, better in accuracy and precision detection data, low in detection cost, low in requirements on detection personnel, good in portability of detection products, suitable for field rapid detection of large-batch samples, and ideal in nicarbazin residue detection products.

Drawings

FIG. 1 scheme for synthesis of nicarbazin hapten

FIG. 2 standard curve diagram of kit

Detailed Description

For a further understanding of the present invention, reference will now be made to the preferred embodiments of the present invention by way of examples, but it is to be understood that these descriptions are intended only to further illustrate the features and advantages of the present invention, and not to limit the scope of the claims, and that the reagents of the present invention, unless otherwise specified, are conventional and commercially available.

Example one

1. The nicarbazin detection kit comprises the following components:

(1) A 96-hole enzyme label plate, wherein the enzyme label plate is coated with nicarbazin hapten-OVA conjugate;

(2) An enzyme-labeled antibody working solution, specifically a horseradish peroxidase-labeled nicarbazin monoclonal antibody, 7 mL/bottle. 0.01% of proclin300 as an antibody stabilizer, which was obtained by adding 8% of glycerol, 0.1% of EDTA and 5% of trehalose to the enzyme-labeled antibody working solution, and the percentages were mass percentages.

(3) Nicarbazin marker standard 0. Mu.g/L, 0.025. Mu.g/L, 0.075. Mu.g/L, 0.225. Mu.g/L, 0.675. Mu.g/L, 2.025. Mu.g/L, 1 mL/bottle.

(4) The washing solution was concentrated at 20X, and the ingredients contained 1% polyethylene glycol 400 monooleate, 0.1mol/L MES buffer, pH6.0, 30 mL/bottle, diluted with deionized water at the ratio of 1.

(5) 20X the double solution was concentrated to a concentration of 50% DMF,0.01mol/L carbonate buffer, pH9.0, 10 mL/bottle, and diluted with methanol at a ratio of 1.

(6) The substrate A solution is hydrogen peroxide, and the substrate B solution is TMB, which are all 7 mL/bottle.

(7) The stop solution is 2mol/L H ₂ SO ₄ 7 mL/bottle.

The percentage is mass to volume ratio.

2. Preparation of main components:

2.1 Synthesis of nicarbazin hapten

1) Weighing 4-nitroaniline (5.52g, 44mmoL), 4-tert-butyl bromobutyrate (10g, 44mmoL) and potassium carbonate (6g, 48mmoL) in 80mLDMF, stirring for 4h in an oil bath at 65 ℃,the dot plate reaction was complete (DCM: meOH =10, rf = 0.7), water 80mL was added to the reaction solution and extracted with dichloromethane (800 mLX), the organic phases were combined, washed with water 1 time, saturated brine 1 time, anhydrous Na ₂ SO ₄ Drying, removal of solvent under reduced pressure gave an oil which was purified by column chromatography (DCM: meOH = 100) to give the product as a colorless liquid.

2) 5.04g of the product of step 1) are weighed into 40mL of methanol, 0.2g of 10% Pd/C is added, and the mixture is stirred in H ₂ Was reacted for 8h in an atmosphere of (DCM: meOH =10, rf = 0.1), the Pd/C was removed by filtration, and then methanol was removed under reduced pressure to obtain the product.

3) Dissolving 2.2g of the product obtained in the step 2) in30 mL of toluene, dropwise adding 1.64g of 4-nitrophenyl isocyanate under the cooling of ice water, stirring for 2h, carrying out dot-on-plate reaction (DCM: meOH =10, rf = 0.1), precipitating a large amount of white solid, and carrying out suction filtration under reduced pressure to obtain a solid product.

4) Weighing 1.92g of the product of step 3) into 10mL of methanol, adding 10mL of 2mol/L NaOH solution, stirring at room temperature for 5h, performing spot plate reaction (DCM: meOH =10, 1, rf = 0.2), removing methanol under reduced pressure, adjusting the pH of the obtained solution to 2-3 with 1mol/L HCl, adding 20mL of deionized water into the reaction solution, extracting with dichloromethane (50 mLX), combining organic phases, washing the organic phase with deionized water for 2 times, washing with saturated brine for 1 time, and then washing with anhydrous Na ₂ SO ₄ The solvent was removed under reduced pressure and the resulting oil was purified on a column (DCM: meOH = 100).

2.2 Synthesis of immunogen and coatingen

The immunogen is nicarbazin hapten-KLH, the coating antigen is nicarbazin hapten-OVA, and the preparation process is as follows:

weighing 12mg of nicarbazin hapten and dissolving the nicarbazin hapten in 1mL of DMF to obtain hapten diluent; weighing 15mgEDC and 10mgNHS, dissolving in 0.2mL deionized water, adding into hapten diluent, and stirring at room temperature for 30min to obtain activated hapten diluent; weighing 50mg of carrier protein KLH or OVA, and fully dissolving the carrier protein KLH or OVA in 3.8ml of PBS; dropwise and slowly adding the activated hapten diluent into the prepared carrier protein solution, stirring at room temperature for reaction for 10 hours, dialyzing with 0.01mol/L PBS (phosphate buffer solution), intercepting the molecular weight of 8000-14000u, dialyzing at 4 ℃ for 3 days, changing the dialysate 3 times per day, subpackaging and storing at-20 ℃ after the preparation is finished.

2.3 preparation of nicarbazin monoclonal antibody

2.3.1 animal immunization

8-week-old Balb/c mice were selected, injected subcutaneously with nicarbazin hapten-KLH conjugate containing Freund's complete adjuvant at an immunization dose of 150 μ g/mouse, boosted at 2,4 and 8 weeks, and finally immunized at week 12 to generate polyclonal antibody serum.

2.3.2 preparation of myeloma and splenocytes

Culturing SP2/0 myeloma cells by using RPMI-1640 culture solution to enable the cells to be in a logarithmic growth phase to prepare SP2/0 myeloma cell suspension; taking the spleen of the immunized mouse, crushing, filtering by a 200-mesh stainless steel gauze to prepare spleen cell suspension, and counting for later use.

2.3.3 cell fusion and Mono-cloning

Will be 1 × 10 ⁸ SP2/0 myeloma cells and 4X 10 ⁸ Splenocytes were fused under PEG4000 and positive wells were screened using an indirect competitive ELISA assay for secreted antibody culture wells. And (3) adopting a limiting dilution method to perform monoclonality on the positive hole until obtaining the hybridoma cell strain which stably secretes the nicarbazin monoclonal antibody.

2.3.4 cell cryopreservation and Resuscitation

The nicarbazin monoclonal hybridoma cell strain is changed into 3 multiplied by 10 ⁶ one/mL cell suspension, 500. Mu.L each in cryotubes, was stored in liquid nitrogen. Taking out the frozen tube during recovery, immediately putting into 37 ℃ water bath for fast melting, transferring into a conical flask, adding into 15mL of complete culture solution, and using CO ₂ The incubator is used for culture.

2.3.5 Mass production and purification of monoclonal antibodies

Taking a healthy Balb/c female mouse, injecting 0.5mL of paraffin oil into the abdominal cavity, and injecting the nicarbazin monoclonal hybridoma cell strain 10 into the abdominal cavity after 10 days ⁶ Every other day, ascites was collected 7-10 days later. The ascites fluid was centrifuged at 4000rpm for 10min and the supernatant was collected. Purified antibody-20 deg.CAnd (5) storing.

2.3.6 nicarbazin monoclonal antibody specificity

Antibody specificity refers to the ability of an antibody to bind to an epitope of a different structure, expressed as the rate of cross-reactivity. The cross reaction rate is low, and the specificity of the antibody can be proved to be high. The 50% inhibitory concentrations (i.e., IC) were obtained from the standard curves for each drug ₅₀ ). The cross-reactivity of the kit to other drugs was calculated using the following formula:

cross reaction rate (%) = (nicarbazin marker IC) ₅₀ Nicarbazin marker analog IC ₅₀ )×100％

The detection kit for nicarbazin has the cross reaction rate of 100 percent on the nicarbazin marker, has no cross reaction on the commonly detected medicines in chicken, such as neomycin, gentamicin, nitroimidazoles, diclazuril, salinomycin, sulfonamides and the like, and can specifically detect the residual nicarbazin marker in chicken tissues.

2.4 preparation of enzyme-labeled antibodies

The enzyme-labeled antibody is a nicarbazin monoclonal antibody labeled by horseradish peroxidase. The horseradish peroxidase and nicarbazin monoclonal antibody are reacted by a sodium periodate method to obtain the nicarbazin monoclonal antibody.

2.5 Elisa plate

Diluting nicarbazin hapten-OVA to 0.10-0.15 mu g/mL by using 0.05mol/L phosphate coating solution with pH7.2, adding 100 mu L of nicarbazin hapten-OVA into each hole, incubating for 2h at 37 ℃, washing for 2 times by using washing liquid, patting to dry, adding 0.05mol/L phosphate buffer solution with 1% gelatin and pH7.2 into each hole for sealing, incubating for 2h at 37 ℃, pouring out liquid in the hole, drying, and storing by using an aluminum film in a vacuum sealing way. The blocking solution contained 1% peptone, 0.1% EDTA, 0.1% PEG4000, 5% trehalose, and 0.02mol/L PBS (pH 7.4), the percentages being by mass. The confining liquid not only plays a role in reducing the background interference of the reagent holes, but also can ensure the activity of macromolecular protein, prevent degradation and improve the stability of the ELISA plate.

The enzyme label plate after being coated and other reagents of the invention are put in a constant temperature box at 37 ℃ for aging test, and the test is carried out until the 28 th day, and the absorbance value is measured and still in the normal value range.

The assembled kit can also comprise an instruction book, a cover plate film, a self-sealing bag and a certificate, and the shelf life of the kit is more than 3 years at the temperature of 2-8 ℃.

3. The detection principle of the nicarbazin detection kit is as follows:

the method comprises the steps of pre-coating an antigen on an enzyme label plate by utilizing the property that a nicarbazin antibody and a nicarbazin marker can generate specific binding, then adding a standard substance or a sample and the nicarbazin antibody, enabling the nicarbazin marker and the antigen fixed on the enzyme label plate to compete for the nicarbazin antibody, and finally adding a substrate to catalyze and develop color, wherein the color development depth is in inverse proportion to the content of nicarbazin in the standard substance or the sample.

4. Specific detection steps of kit

(1) And taking out the enzyme label plates with required quantity, inserting the enzyme label plates into the enzyme label plate frame, recording the positions of each standard product and each sample, and performing a double-hole parallel experiment for reducing the fluctuation of detection values.

(2) Adding 50 μ L of standard substance/sample into corresponding micropores, adding 50 μ L/pore of enzyme-labeled antibody working solution, gently shaking for 5s, mixing, and placing the cover plate at the rear part in a dark environment at 25 deg.C for reaction for 15min.

(3) Uncovering the cover plate film, drying liquid in the holes, adding 250 mu L of washing liquid into the holes, washing for 2 times, pouring the washing liquid, and patting the washing liquid dry by absorbent paper.

(4) Adding 50 mu L/hole of substrate color development A liquid hydrogen peroxide, adding 50 mu L/hole of substrate color development B liquid tetramethyl benzidine, mixing uniformly, covering the plate, and reacting for 10min in a dark environment at 25 ℃.

(5) Adding 50 mu L of stop solution per hole, mixing uniformly, setting an enzyme-labeling instrument at 450nm, and determining the OD value.

5. Analysis of detection results

Let the average absorbance of each standard (or sample) be B, zero standard, i.e. the standard with a concentration of 0. Mu.g/L, and the average absorbance B ₀ With (B/B) ₀ ) X 100% is the percentage of absorbance corresponding to each standard (or sample): substituting the semi-logarithmic system into the standard for the percentage of absorbance corresponding to the standard, andand fitting a standard curve according to the product concentration. And substituting the percentage of the absorbance of the sample to be detected into the fitted standard curve equation to obtain the corresponding concentration of the sample, and finally multiplying the corresponding dilution times of the sample by the corresponding concentration times to obtain the content of the detected substance in the sample.

Example 2 detection of nicarbazin marker in Chicken samples

1. Pretreatment method of chicken sample

Weighing 1 plus or minus 0.05g of sample into a 10mL centrifuge tube, adding 3mL of absolute ethyl alcohol, and whirling for 5min; centrifuging at 5000rpm for 5min; taking 25 mu L of upper clear liquid, adding 975 mu L of redissolving working solution, and shaking and mixing for 30s; 50 μ L was taken for analysis.

2. Fitting of standard curve

The standard curve consisted of 6 concentrations (0. Mu.g/L, 0.025. Mu.g/L, 0.075. Mu.g/L, 0.225. Mu.g/L, 0.675. Mu.g/L, 2.025. Mu.g/L) and was measured 2 times for each of the 6 standard points to determine the working concentration range. Taking the concentration logarithm value of the standard substance of nicarbazin marker as the abscissa, the inhibition rate (B/B) of each corresponding concentration ₀ %) was plotted on the ordinate, and a calibration curve was prepared. The results are shown in FIG. 2.

3. Sample precision and accuracy testing

Accuracy is expressed as recovery and precision as coefficient of variation. The chicken samples were recovered by addition with nicarbazin marker to a final concentration of 20 μ g/kg. And extracting three batches of kits with five parallel concentrations, measuring the same sample for 3 times by each batch of kits, and respectively calculating the recovery rate and the coefficient of variation of the measured sample. The results are shown in the following table.

The results show that the adding recovery rate of the chicken sample is between 90 and 110.5 percent, the intra-plate coefficient of variation is between 2.6 and 4.6 percent, and the inter-plate coefficient of variation is between 4.1 and 6.4 percent by adding the nicarbazin marker of 20 mu g/kg.

EXAMPLE III

(1) A 96-hole enzyme label plate, wherein the enzyme label plate is coated with nicarbazin hapten-HSA conjugate;

(2) An enzyme-labeled antibody working solution, specifically a horseradish peroxidase-labeled nicarbazin polyclonal antibody, 7 mL/bottle.

(3) Nicarbazin marker standard 0 μ g/L,0.025 μ g/L,0.075 μ g/L,0.225 μ g/L,0.675 μ g/L,2.025 μ g/L,1 mL/bottle.

(4) The washing solution was concentrated at 20X, and the ingredients contained 1% polyethylene glycol 400 monooleate, 0.1mol/L MES buffer, pH6.0, 30 mL/bottle.

(5) 20X the double solution was concentrated to a concentration of 50% DMF,0.01mol/L carbonate buffer, pH9.0, 10 mL/bottle.

(6) The substrate A solution is hydrogen peroxide, and the substrate B solution is TMB, which are all 7 mL/bottle.

(7) The stop solution is 2mol/L H ₂ SO ₄ 7 mL/bottle.

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited to the above embodiments, and all technical solutions that belong to the idea of the present invention belong to the scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.

Claims (9)

1. A nicarbazin detection kit comprises a 96-hole enzyme label plate, an enzyme label, a nicarbazin label standard substance, 20 multiplied concentrated washing liquid, 20 multiplied concentrated complex solution, a substrate A solution, a substrate B solution and stop solution, wherein nicarbazin hapten-carrier protein is coated on the 96-hole enzyme label plate, the enzyme label is a horseradish peroxidase-labeled nicarbazin specific antibody, and the structural formula of the nicarbazin hapten is shown in the specification

2. The nicarbazin assay kit of claim 1, wherein: the nicarbazin hapten is obtained by the following steps,

1) Synthesis of product I

Weighing 5.52g of 4-nitroaniline, 10g of tert-butyl 4-bromobutyrate and 6g of potassium carbonate, placing the mixture in 80mL of DMF, stirring for 4h in an oil bath at 65 ℃, spotting and reacting, adding 80mL of deionized water into a reaction solution, extracting with dichloromethane, combining organic phases, washing the organic phases with deionized water for 1 time, washing with saturated salt for 1 time, and then washing with anhydrous Na ₂ SO ₄ Drying and removing the solvent under reduced pressure to obtain an oil which is purified by a column, and DCM: meOH = 100;

2) Synthesis of product II

5.04g of product I are weighed out into 40mL of methanol, 10% Pd/C0.2 g are added and the mixture is washed with water and dried in H ₂ The reaction was carried out for 8h under an atmosphere of DCM: meOH =10, rf =0.1, pd/C was removed by filtration, and then methanol was removed under reduced pressure to obtain 4.0g of product ii;

3) Synthesis of product III

Dissolving 2.2g of a product II in30 mL of toluene, dropwise adding 1.64g of 4-nitrophenyl isocyanate under the cooling of ice water, stirring for 2h, carrying out dot-plate reaction, precipitating a large amount of white solid when DCM is used, meOH =10, rf =0.1, and carrying out vacuum filtration to obtain 3.1g of a solid product III;

4) Synthesis of product IV, nicarbazin hapten

Weighing 1.92g of the product III, placing the product III in 10mL of methanol, adding 10mL of 2mol/L NaOH solution, stirring at room temperature for 5h, carrying out spotting reaction, adding 20mL of deionized water into the reaction solution, extracting with dichloromethane, combining organic phases, washing the organic phases with deionized water for 2 times, washing the organic phases with saturated common salt water for 1 time, and washing the organic phases with anhydrous Na, wherein the mixture is subjected to methanol removal under reduced pressure, the pH of the obtained solution is adjusted to 2-3 by 1mol/L of HCl, the reaction solution is added with 20mL of deionized water, the mixture is extracted with dichloromethane, the organic phases are combined, and the organic phases are washed with the deionized water for 2 times, saturated common salt water for 1 time, and anhydrous Na ₂ SO ₄ Drying and removing the solvent under reduced pressure to obtain an oil, purifying the oil by a column, and obtaining 1.2g of a white solid product (IV) which is the nicarbazin hapten by DCM: meOH = 100.

3. The nicarbazin assay kit of claim 1, wherein: the concentrations of the nicarbazin marker standard substance are respectively 0 mug/L, 0.025 mug/L, 0.075 mug/L, 0.225 mug/L, 0.675 mug/L and 2.025 mug/L.

4. The nicarbazin assay kit of claim 1, wherein: the substrate A solution is hydrogen peroxide, the substrate B solution is TMB, and the stop solution is 2mol/L H ₂ SO ₄ 。

5. The nicarbazin assay kit of claim 1, wherein: the concentrated washing solution contained 1% polyethylene glycol 400 monooleate, 0.1mol/L MES buffer, pH6.0.

6. The nicarbazin assay kit of claim 1, wherein: the concentrated double solution contained 50% of DMF,0.01mol/L carbonate buffer, pH 9.0.

7. The nicarbazin assay kit of claim 1, wherein: the nicarbazin specific antibody is prepared by taking a nicarbazin hapten-KLH conjugate as an immunogen.

8. The nicarbazin assay kit of claim 7, wherein: the nicarbazin hapten-KLH conjugate is prepared by the following steps:

1) Weighing 12mg nicarbazin hapten and dissolving in 1 mLDMF;

2) Weighing 15mgEDC and 10mgNHS and dissolving in 0.2mL deionized water;

3) Adding the solution obtained in the step 2) into the solution obtained in the step 1), and stirring and reacting for 30min at room temperature;

4) Weighing 50mg of KLH, dissolving the KLH in 3.8mL of PBS, adding the solution obtained in the step 3), and stirring at room temperature for reacting for 16 hours;

5) Dialyzing with 0.01mol/L PBS, intercepting the molecular weight of 8000-14000u, dialyzing for 3 days at 4 ℃, and changing the dialyzate every day to obtain the nicarbazin hapten-KLH conjugate.

9. Use of the nicarbazin assay kit of any one of claims 1-8 to detect nicarbazin residues in chicken.

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