CN112462047A

CN112462047A - Nicarbazin detection kit and application thereof

Info

Publication number: CN112462047A
Application number: CN202011270800.7A
Authority: CN
Inventors: 王世恩; 郝士元; 杨昌松
Original assignee: Beijing Yuanen Biotechnology Co ltd
Current assignee: Beijing Yuanen Biotechnology Co ltd
Priority date: 2020-11-13
Filing date: 2020-11-13
Publication date: 2021-03-09
Anticipated expiration: 2040-11-13
Also published as: CN112462047B

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 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 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 disclosed by the invention is used for detecting the nicarbazin marker by adopting a one-step method, is high in detection sensitivity, and good in accuracy and precision of a detection result, has a simple structure, is convenient to use, is good in portability, has low requirements on detection personnel, and is suitable for rapid detection of large-batch samples on site.

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 complex 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.

The nicarbazin is widely applied at present, but can cause residues in the muscles and other tissues of poultry to different degrees after being fed, and is harmful to the life health of human beings. The residual marker of nicarbazin in chicken tissues is DNC and the residual limit of nicarbazin in chicken muscles, skins, fat, livers and kidneys is 200 mug/kg.

At present, the detection method of the nicarbazin residual marker mainly comprises an instrumental analysis method and an immunological analysis method. The instrument analysis method has the disadvantages of complicated pretreatment procedures, long measurement time, high instrument cost and high requirement on detection personnel, and cannot meet the requirement on real-time rapid detection of nicarbazin. The immunoassay method is a rapid screening method mainly using enzyme as a reaction medium, and 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, wherein the enzyme linked immunosorbent assay kit is provided with an ELISA plate coated with a nicarbazin coupling antigen, a nicarbazin monoclonal antibody, an enzyme marker anti-antibody, a nicarbazin standard solution, a substrate color development 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 solution, a substrate B solution 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 pH 6.0.

Wherein the 20 Xconcentrated complex solution contains 50% DMF, 0.01mol/L carbonate buffer solution, and 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 H₂SO₄。

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 the mixture in an oil bath at 65 ℃ for 4 hours, dotting the mixture, adding 80mL of deionized water into the reaction solution, extracting the mixture by using dichloromethane, combining organic phases, washing the organic phases by using the deionized water for 1 time, washing the organic phases by using saturated common salt for 1 time, and then using anhydrous Na₂SO₄Drying and removal of the solvent under reduced pressure gave an oil which was purified on column with DCM: MeOH ═ 100:1 to give 8.4g of product i as a colourless 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 H₂The reaction is carried out for 8h in an atmosphere of (1), the reaction is carried out on a point plate, DCM: MeOH is 10:1, Rf is 0.1, and the reaction is carried outPd/C was removed by filtration, and then methanol was removed under reduced pressure to obtain 4.0g of a product II.

3) Synthesis of product III

Dissolving 2.2g of the 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-on-plate reaction, precipitating a large amount of white solid by DCM (methyl alcohol) ═ 10:1 and Rf ═ 0.1, and carrying out suction filtration 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 phase with deionized water for 2 times, washing the organic phase with saturated salt water for 1 time, and adding anhydrous Na, wherein the solution is stirred for 5h at room temperature, carrying out spotting reaction, adding DCM (DCM): MeOH ═ 10:1 and Rf ═ 0.2, removing the methanol under reduced pressure, adjusting the pH of the obtained solution to 2-3 with 1mol/L of HCl₂SO₄Drying, removing solvent under reduced pressure to obtain oil, purifying with column, and collecting white solid product (IV) (1.2 g) as nicarbazin hapten, wherein DCM is MeOH (100: 1).

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 8000-14000 u, 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. 8 percent of glycerol, 0.1 percent of EDTA, 5 percent of trehalose and 0.01 percent of Proclin300 which is taken as an antibody stabilizer are added into the enzyme-labeled antibody working solution, and the percentages are mass percentages.

(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, diluted with deionized water at a ratio of 1:19 at the time of use.

(5)20 times the concentration of the complex solution, the components of which are 50% DMF, 0.01mol/L carbonate buffer solution, pH9.0, 10 mL/bottle, and when in use, the complex solution is diluted by methanol according to the proportion of 1: 19.

(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 H₂SO₄7 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-bromobutyric acid tert-butyl ester (10g, 44mmoL) and potassium carbonate (6g, 48mmoL) in 80mL DMF, stirring for 4h in an oil bath at 65 ℃, completing the spotting reaction (DCM: MeOH 10:1, Rf 0.7), adding 80mL of water to the reaction solution, extracting with dichloromethane (800mLX3), combining the organic phases, washing the organic phase with water for 1 time, washing with saturated salt water for 1 time, and washing with anhydrous Na₂SO₄Drying, removing the solvent under reduced pressure to obtain an oil, and purifying the oil by a column (DCM: MeOH: 100:1) to obtain a colorless liquid product.

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 H₂Was reacted for 8h under an atmosphere of (1), (DCM: MeOH ═ 10:1, Rf ═ 0.1), and then Pd/C was removed by filtration, followed by removal of methanol under reduced pressure to obtain a product.

3) Dissolving 2.2g of the product obtained in the step 2) in30 mL of toluene, dropwise adding 1.64g of 4-nitrophenylisocyanate under the cooling of ice water, stirring for 2h, carrying out dot-on-plate reaction (DCM: MeOH: 10:1, Rf: 0.1), precipitating a large amount of white solid, and carrying out suction filtration under reduced pressure to obtain a solid product.

4) 1.92g of the product of step 3) was weighed into 10mL of methanol, 10mL of a 2mol/L NaOH solution was added, the mixture was stirred at room temperature for 5 hours, the reaction was spotted (DCM: MeOH: 10:1, Rf: 0.2), and the formazan was removed under reduced pressureAdjusting pH of the obtained solution to 2-3 with 1mol/L HCl, adding 20mL deionized water into the reaction solution, extracting with dichloromethane (50mLX3), combining organic phases, washing the organic phase with deionized water for 2 times, washing with saturated salt water for 1 time, and adding anhydrous Na₂SO₄Drying, removal of the solvent under reduced pressure and column purification of the resulting oil (DCM: MeOH ═ 100:1) afforded the nicarbazin hapten as a white solid.

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 carrier protein KLH or OVA50mg, and dissolving in 3.8ml 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 mass of 8000-14000 u, dialyzing at 4 ℃ for 3 days, changing the dialysate 3 times per day, subpackaging after the preparation and storing at-20 ℃.

2.3 preparation of nicarbazin monoclonal antibody

2.3.1 animal immunization

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

2.3.2 preparation of myeloma and splenocytes

Culturing SP2/0 myeloma cells in RPMI-1640 culture medium to make the cells in logarithmic growth phase to obtain 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 × 10⁸SP2/0 myeloma cellsSum of cells 4X 10⁸Splenocytes 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 10⁶one/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 CO₂The 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 days⁶Every other day, and ascites were collected 7-10 days later. Ascites was centrifuged at 4000rpm for 10min and the supernatant was collected. The purified antibody was stored at-20 ℃.

2.3.6 specificity of nicarbazin monoclonal antibody

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 drug₅₀). The cross-reactivity of the kit to other drugs was calculated using the following formula:

cross reaction rate (%) (nicarbazin marker IC)₅₀Nicarbazin marker analog IC₅₀)×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 confining liquid is PBS containing 1% of peptone, 0.1% of EDTA, 0.1% of PEG4000, 5% of trehalose and 0.02mol/L, the pH value is 7.4, and the percentages are mass fractions. 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 further comprise an instruction book, a cover plate film, a self-sealing bag and a certificate, and the storage 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 15 min.

(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 μ L of stop solution per well, mixing, setting enzyme-labeling instrument at 450nm, and measuring 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 B₀With (B/B)₀) X 100% is the percentage of absorbance corresponding to each standard (or sample): and (5) substituting a semi-logarithmic system into the percentage of absorbance corresponding to the standard substance, and fitting a standard curve with the concentration of the standard substance. 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. Chicken sample pretreatment method

Weighing 1 plus or minus 0.05g of sample into a 10mL centrifuge tube, adding 3mL of absolute ethyl alcohol, and whirling for 5 min; centrifuging at 5000rpm for 5 min; taking 25 mu L of upper clear liquid, adding 975 mu L of redissolving working solution, and shaking and mixing for 30 s; 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 concentration₀%) as ordinate, making the markA quasi-curve. 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.

(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)20 times the concentration of the complex solution, the composition is 50% DMF, 0.01mol/L carbonate buffer, pH9.0, 10 mL/bottle.

(7) The stop solution is 2mol/L H₂SO₄7 mL/bottle.

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection 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

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, and the enzyme label is a nicarbazin specific antibody marked by horseradish peroxidase.

2. The nicarbazin assay kit of claim 1, wherein: the structural formula of nicarbazin hapten is

3. The nicarbazin assay kit of claim 1 or 2, 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 the mixture in an oil bath at 65 ℃ for 4 hours, dotting the mixture, adding 80mL of deionized water into the reaction solution, extracting the mixture by using dichloromethane, combining organic phases, washing the organic phases by using the deionized water for 1 time, washing the organic phases by using saturated common salt for 1 time, and then using anhydrous Na₂SO₄Drying, removing solvent under reduced pressure to obtain oil, purifying by column, and obtaining colorless liquid product I8.4 g by DCM: MeOH ═ 100: 1;

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 H₂The reaction is carried out for 8h in the atmosphere of (1), the reaction is carried out on a point plate, DCM is used for MeOH 10:1, Rf is 0.1, Pd/C is removed by filtration, and then the methanol is removed under reduced pressure to obtain a product II4.0g；

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 2 hours, carrying out dot-on-plate reaction, precipitating a large amount of white solid by DCM (methyl alcohol) ═ 10:1 and Rf ═ 0.1, and carrying out vacuum filtration to obtain 3.1g of a solid product III;

4) synthesis of product IV, nicarbazin hapten

4. The nicarbazin assay kit of claim 1 or 2, 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.

5. The nicarbazin assay kit of claim 1 or 2, wherein: the substrate A solution is hydrogen peroxide, the substrate B solution is TM, and the stop solution is 2mol/L H₂SO₄。

6. The nicarbazin assay kit of claim 1 or 2, wherein: the concentrated washing solution contained 1% polyethylene glycol 400 monooleate, 0.1mol/L MES buffer, pH 6.0.

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

8. The nicarbazin assay kit of claim 1 or 2, wherein: the nicarbazin specific antibody is prepared by taking a nicarbazin hapten-KLH conjugate as an immunogen.

9. The nicarbazin assay kit of claim 8, 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;

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

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