EC hapten and preparation method thereof, artificial antigen, enzyme-linked immunosorbent assay kit and detection method

Abstract

The present invention provides an EC hapten and a preparation method thereof, an artificial antigen, an enzyme-linked immunosorbent assay kit and a detection method, and belongs to the technical field of immunoassay. The ethyl carbamate hapten of the present invention can retain the structural characteristics of the ethyl carbamate functional group to the greatest extent, does not affect its own antigenic determinant structure, and can also enhance its ability to couple with a macromolecular carrier protein. The unique structure of the ethyl carbamate hapten of the present invention can avoid the shielding effect of the macromolecular carrier protein on it, and after coupling with the carrier protein, the antigenic determinant can be completely exposed, thereby generating good immunogenicity. The ethyl carbamate kit prepared using the ethyl carbamate hapten of the present invention has the characteristics of convenience, rapidity, sensitivity, etc., and is suitable for on-site large-scale sample detection.

Description

Technical Field

[0001] The invention belongs to the technical field of immune detection, and in particular relates to an EC hapten and a preparation method thereof, an artificial antigen, an enzyme-linked immunosorbent assay kit and a detection method. Background Art

[0002] Fermented grains and distiller's grains are mainly a mixture of sorghum, corn, rice, wheat, etc., and are by-products produced during the fermentation and production of alcoholic products. In recent years, with the vigorous development of the alcoholic beverage industry, the output of such by-products has also increased accordingly. Due to its high nutritional value, large output and relatively low price, it is widely used in animal feed, industrial and agricultural production and other fields. However, due to the diversity of raw materials and production process characteristics in the production process of alcoholic beverages, ethyl formate pollution is easily generated, causing certain health hazards and economic losses.

[0003] Ethyl carbamate (EC), also known as urethane, has a chemical formula of C3H7NO2 (CAS No. 51-79-6), a relative molecular mass of about 89.09, is colorless and odorless, and is widely found in fermented foods (such as soy sauce, fermented bean curd, kimchi, etc.) and alcoholic drinks. EC is easily soluble in water and alcohol solvents, with a boiling point of 182℃~184℃, a melting point of 48℃~50℃, and is relatively stable at room temperature and pressure. EC has a variety of production pathways and is easily produced during food fermentation and storage. It is mainly formed by the reaction of carbamyl compounds (urea, citrulline, carbamyl phosphate, diethyl pyrocarbonate, etc.) and ethanol. In addition, cyanide is also an important precursor for EC synthesis. Historically, EC has been used as an anesthetic, a co-solvent for tumor treatment, analgesics and sedatives, etc. However, since its potential carcinogenicity was discovered in 1943, studies on its hazards have been carried out one after another. Many studies have shown that EC has carcinogenic, teratogenic and mutagenic effects. In 2007, EC was upgraded from Group 2B to Group 2A by the International Agency for Research on Cancer (IARC), which means it is "probably carcinogenic to humans". Therefore, it is necessary to strengthen the monitoring of ethyl carbamate content to effectively prevent products contaminated by it from directly or indirectly entering the human food chain.

[0004] At present, the traditional methods for detecting ethyl carbamate in food and feed mainly include gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). Although the above instrument detection methods have good repeatability and accuracy, the operation process is complicated, and relatively expensive detection equipment is required. In addition, there are certain requirements for the professional operation skills of the detection personnel, and they are not suitable for on-site rapid detection of large sample quantities. Summary of the invention

[0005] In view of this, the present invention provides a urethane hapten, an artificial antigen and applications thereof, which can detect urethane with high sensitivity, quickly and simply.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A urethane hapten, the structure of which is shown in formula (1):

[0008]

[0009] Another object of the present invention is to provide a method for preparing the ethyl carbamate hapten, comprising the following steps:

[0010] Ethyl bromoformate, 2-(2-naphthyl)-2-aminoacetic acid and sodium hydroxide are mixed in an acidic solution to carry out a substitution reaction.

[0011] Preferably, the molar ratio of ethyl bromoformate, sodium hydroxide and 2-(2-naphthyl)-2-aminoacetic acid is (1.5-2.5):1:(1.5-2.5).

[0012] Another object of the present invention is to provide an ethyl carbamate artificial antigen, which is prepared by coupling the ethyl carbamate hapten with a carrier protein via carboxyl coupling.

[0013] Preferably, the carrier protein is selected from any one of bovine serum albumin, human serum albumin, rabbit serum albumin, ovalbumin, keyhole limpet hemocyanin, glutathione S-transferase or a multivalent antigen peptide.

[0014] Another object of the present invention is to provide a method for preparing urethane-specific antibodies, wherein the urethane artificial antigen is used to immunize an animal so that the body produces antibodies specific to urethane.

[0015] Another object of the present invention is to provide the use of the antibody prepared by the preparation method of the hapten or the artificial antigen or the urethane-specific antibody in detecting urethane.

[0016] Another object of the present invention is to provide a urethane detection kit, comprising the urethane-specific antibody prepared by the urethane-specific antibody preparation method and an enzyme label plate coated with the artificial antigen.

[0017] Preferably, the artificial antigen-coupled carrier protein for preparing urethane-specific antibodies is the first carrier protein, the artificial antigen-coupled carrier protein coated with the ELISA plate is the second carrier protein, and the first carrier protein and the second carrier protein are of different types.

[0018] Preferably, the urethane detection kit further comprises an enzyme marker antibody working solution, a urethane standard working solution, a sample diluent, and a substrate color developing solution.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides a urethane hapten. The urethane hapten of the present invention retains the structural characteristics of the urethane functional group to the greatest extent, does not affect the antigenic determinant structure of the urethane itself, and can also enhance its ability to couple with a macromolecular carrier protein. The unique structure of the urethane hapten of the present invention can avoid the shielding effect of the macromolecular carrier protein on it, and after coupling with the carrier protein, the antigenic determinant can be completely exposed, thereby generating good immunogenicity. The urethane kit prepared using the urethane hapten of the present invention has the characteristics of convenience, rapidity, sensitivity, etc., and is suitable for on-site large-scale sample detection. DETAILED DESCRIPTION

[0021] The present invention provides an ethyl carbamate hapten, the structure of which is shown in formula (1):

[0022]

[0023] The ethyl carbamate hapten provided by the present invention can retain the structural characteristics of the ethyl carbamate functional group to the greatest extent and not affect the antigenic determinant structure of the ethyl carbamate itself, while also being able to enhance its ability to couple with a macromolecular carrier protein, thereby preventing the macromolecular carrier protein from producing a shielding effect on the hapten, i.e., preventing the antigenic determinant from being embedded in the curled and concave macromolecular structure when the hapten is combined with the carrier. The complete exposure of the antigenic determinant increases the specificity of the generated corresponding antibody, thereby reducing cross-reactions, thereby making the conjugate immunogenic, and further, being able to stimulate the body to produce an immune response and produce antibodies.

[0024] The present invention also provides a method for preparing the ethyl carbamate hapten, comprising the following steps: mixing ethyl bromoformate, 2-(2-naphthyl)-2-aminoacetic acid and sodium hydroxide in an acidic solution to carry out a substitution reaction.

[0025] The ethyl bromoformate, 2-(2-naphthyl)-2-aminoacetic acid and sodium hydroxide of the present invention are reacted in an organic solvent, preferably N,N-dimethylformamide. The reaction system of the present invention also includes a catalyst, preferably triethylamine.

[0026] The molar ratio of ethyl bromoformate, sodium hydroxide and 2-(2-naphthyl)-2-aminoacetic acid in the present invention is (1.5-2.5):1:(1.5-2.5), preferably 2:1:2.

[0027] In the present invention, the ethyl bromoformate and 2-(2-naphthyl)-2-aminoacetic acid can prevent the shielding effect of the macromolecular carrier protein on the hapten while acting as a cross-linking agent, so that the ethyl bromoformate and 2-(2-naphthyl)-2-aminoacetic acid introduce a carboxyl functional group into the ethyl formate molecule without affecting the structure of the antigenic determinant of the ethyl bromoformate molecule.

[0028] The present invention also provides an ethyl carbamate artificial antigen, which is prepared by coupling the ethyl carbamate hapten with a carrier protein via carboxyl groups.

[0029] The carboxyl group in the carboxyl coupling of the present invention refers to the ester group where the 4th carbon atom is located. The present invention preferably uses the activated ester method to esterify the carboxyl group of the ethyl carbamate hapten. As an operative method, the ethyl carbamate hapten of the present invention is dissolved in N,N-dimethylformamide (DMF) for reaction with N-hydroxysuccinimide (NHS) and N,N-dicyclohexylcarbodiimide (DCC). Preferably, the ethyl carbamate hapten and N,N-dimethylformamide are added in a ratio of 10-25:10-15 mg / mL, and the reaction process is carried out at room temperature in the dark with continuous stirring, and the stirring time is 12-14h; preferably, the reaction process is carried out under continuous stirring at room temperature in the dark, and the stirring time is 10-12h. The present invention reacts more completely in a dark environment. As another operative method, the ethyl carbamate hapten of the present invention is reacted with tetrahydrofuran (THF) and N,N'-carbonyldiimidazole (CDI).

[0030] The reaction liquid after esterification of the present invention is added to a solution containing a carrier protein, and the amino groups in the carrier protein and the esterified urethane hapten are coupled through chemical bonds to form a conjugate, that is, the urethane artificial antigen is obtained.

[0031] The carrier protein of the present invention is selected from any one of bovine serum albumin (BSA), human serum albumin (HSA), rabbit serum albumin (RSA), ovalbumin (OVA), keyhole limpet hemocyanin (KLH), glutathione S-transferase (GST), and multivalent antigen peptide (MAP, mainly referring to poly-Lys); preferably, the carrier protein is selected from any one of human serum albumin, ovalbumin or bovine serum albumin, and more preferably human serum albumin.

[0032] The ethyl carbamate hapten provided by the present invention is a small molecule substance, which only has reactogenicity but not immunogenicity in immune response. After being coupled with a macromolecular carrier protein, it will produce immunogenicity, thereby stimulating the body to produce an immune response and then produce antibodies.

[0033] The present invention also provides a method for preparing urethane-specific antibodies, wherein an animal is immunized with urethane artificial antigen to make the body produce antibodies specific to urethane.

[0034] The present invention injects the above urethane artificial antigen into an animal for immunization to prepare antiserum. The antiserum is purified to obtain urethane polyclonal antibodies, and the urethane polyclonal antibodies are diluted in a certain proportion to form a diluent as urethane specific antibody working solution.

[0035] The invention injects urethane artificial antigen into mammals for immunization, and the mammals are selected from any one of rabbits, sheep, mice, guinea pigs or horses.

[0036] The present invention also provides the use of the ethyl carbamate hapten or the ethyl carbamate artificial antigen or the antibody prepared by the ethyl carbamate-specific antibody preparation method in detecting ethyl carbamate.

[0037] The present invention also provides a kit for detecting ethyl formate. Preferably, the kit provided by the present invention is an enzyme-linked immunosorbent assay kit, which comprises an antibody prepared by the method for preparing ethyl formate-specific antibodies and an enzyme-labeled plate coated with the artificial antigen.

[0038] In the present invention, the carrier protein coupled with the artificial antigen for preparing the urethane-specific antibody is the first carrier protein, the carrier protein coupled with the artificial antigen coated with the enzyme label plate is the second carrier protein, and the first carrier protein and the second carrier protein are of different types. The different types of the first carrier protein and the second carrier protein of the present invention help to further improve the specificity of the urethane enzyme-linked immunosorbent assay kit reaction.

[0039] The urethane detection kit of the present invention also includes an enzyme marker working solution, which refers to a phosphate buffer for diluting the enzyme-labeled secondary antibody. As an practicable method, the secondary antibody of the present invention is a goat anti-mouse secondary antibody.

[0040] Urethane detection kit of the present invention also includes substrate color developing solution, and the substrate color developing solution is combined with enzyme-labeled secondary antibody to show the reagent of visible color change. As an implementable mode, the component of substrate color developing solution of the present invention includes p-nitrophenol phosphate, and nitrophenol phosphate is under the action of alkaline phosphatase, and the p-nitrophenol (4-NPP) released is molecularly reshot in alkaline solution to form yellow water-soluble quinone products (maximum light absorption value 405nm-415nm). As another implementable mode, substrate color developing solution of the present invention includes substrate A liquid and substrate B liquid, and the substrate A liquid is 0.21g urea peroxide, 27.2g sodium acetate, and deionized water is settled to 1L, and substrate B liquid is 0.4g sodium ethylenediaminetetraacetate, 1.9g citric acid, 0.3gTMB hydrochloride, and deionized water is settled to 1L. When using, mix substrate A liquid and substrate B liquid in a volume ratio of 1:1, mix thoroughly, and use the mixed solution within 5 minutes. Do not use metal containers or stir the reagents. If substrate A liquid and substrate B liquid have turned blue before use, or turn blue immediately after mixing, it means that the reagents have deteriorated.

[0041] The ethyl carbamate detection kit of the present invention further comprises a concentrated washing solution, which is a 0.02 mol / L phosphate buffer solution, used in the washing step. In a specific embodiment of the present invention, the concentrated washing solution is diluted with deionized water at a ratio of 1:19 and used as the washing solution.

[0042] The ethyl formate detection kit of the present invention also includes a sample diluent.

[0043] The ethyl carbamate detection kit of the present invention further comprises a stop solution, which is 53 mL of concentrated sulfuric acid, which is diluted to 1 L with deionized water.

[0044] The ethyl carbamate detection kit of the present invention is used for detecting ethyl carbamate in fermented grains or lees.

[0045] The present invention also provides a method for using the urethane kit, comprising the following steps: pre-treating a sample to be tested; using the urethane enzyme-linked immunosorbent assay kit to perform enzyme-linked immunosorbent assay on the pre-treated sample to be tested; and analyzing the test result.

[0046] The pretreatment of the sample to be tested according to the present invention includes adding acetonitrile to the test object and mixing, and diluting the mixed solution with a phosphate buffer for standby use. As an implementable method, after the test object is mixed with acetonitrile, it is oscillated on an oscillator for 8-10 minutes at a speed of 150-200×g, or centrifuged at 8000×g for 5 minutes to mix. After mixing, take the upper organic phase at 60°C, slowly blow it to near dryness with nitrogen, add 500μL deionized water, vortex for 30-60s to fully dissolve the residue, and obtain a mixed solution of the test object, which is diluted with 0.2mol / L PB diluent.

[0047] The detection principle of the urethane kit of the present invention is as follows: after the coating source is pre-coated on the ELISA plate, a sample solution to be tested is added, and then an antibody working solution and an enzyme marker working solution are added, the urethane remaining in the sample solution to be tested competes with the coating source coated on the ELISA plate for the antibody, and the antigen-antibody conjugate formed by the coating source combined with the antibody further reacts with the enzyme marker working solution, and then a substrate color developing solution is added, and the alkaline phosphatase on the enzyme marker reacts chemically with the substrate color developing solution to emit yellow light, and the luminous intensity is negatively correlated with the urethane content in the sample solution to be tested, and then the urethane content in the sample solution to be tested can be obtained by comparing with a standard curve.

[0048] The ethyl carbamate in the sample competes with the antigen-specific antibody fixed on the ELISA plate, and the enzyme marker is added to catalyze the color development. The content of ethyl carbamate in the sample is determined according to the depth of the color development. The darker the color development, the less the content; the lighter the color development, the more the content.

[0049] The raw materials used in the present invention, unless otherwise specified, are all commercially available products.

[0050] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0051] Example 1

[0052] This embodiment provides an ethyl carbamate hapten and a preparation method thereof, which are as follows:

[0053] At 4°C, take 20mmoL of ethyl bromoformate, dissolve it in 10mL DMF, add 2mL concentrated sulfuric acid, react at 120°C for 10 hours, cool it and slowly add it dropwise to 2-(2-naphthyl)-2-aminoacetic acid (20mmol) and 10mL NaOH (1mol / L), stir it at an appropriate rate after mixing, react at room temperature for 2h, monitor the reaction by thin layer chromatography, and reflux at 90°C for 25min. Add 2.0mol of acidic solution H3PO4 to obtain precipitate. Wash the precipitate twice with saturated sodium bicarbonate, wash it twice with saturated sodium chloride, wash it once with pure water, and dry it in a vacuum dryer for 1 day. After that, mix 1.0mmoL of the product with 2.0mmoL of triethylamine and react at room temperature for 3h. Then add 3moL of palladium acetate (10%), 1.5moL of sodium bicarbonate and 4.0mL of water, and continue to react at 60°C for 11h. Finally, the ethyl carbamate hapten was separated by gradient elution of dichloromethane and methanol.

[0054] Example 2

[0055] This embodiment provides an ethyl carbamate artificial antigen and a preparation method thereof, which are specifically as follows:

[0056] Weigh 2.8 mg of the ethyl carbamate hapten prepared in Example 1, dissolve it in 2 mL of dimethylformamide (DMF) with 270 mg of NHS and 206 mg of DDC, and stir at room temperature in the dark for 10 h; add the above reaction solution dropwise to 16 mL of 0.05 mol / L carbonate buffer (5% DMF v / v) with pH 9.6 containing 100 mg of bovine serum albumin (BSA) within about 1 h, and stir at the same time. After 1 h, transfer to 4 ° C and stir for 48 h; add 4 mL of 2.5 mol / L NaOH solution dropwise, stir for 0.5 hours in an environment of 80 ° C, cool to 4 ° C and stir, and adjust the pH to 7.1. After reacting for 30 min, transfer to 0.02 mol / L PBS, and dialyze for about 24 h at 4 ° C with magnetic stirring, and change the dialysate every 6 h during this period. When the dialysate has no small molecule absorption peak by ultraviolet scanning, it can be centrifuged to obtain the supernatant, which is the ethyl carbamate artificial immunogen. The product is packaged in centrifuge tubes and stored at -20°C.

[0057] Example 3

[0058] This embodiment provides a urethane polyclonal antibody and a preparation method thereof, which are as follows:

[0059] The urethane artificial antigen prepared in Example 2 was diluted with a PBS buffer solution of pH 7.4 and 0.01 mol / L to obtain an immunogen dilution.

[0060] Balb / c mice were used as immunized animals in the experiment, and the immunization process was as follows (immunization dose is protein amount):

[0061] The first immunization of experimental Balb / c mice: the immunogen diluent was mixed with an equal volume of Freund's complete adjuvant to form an emulsion, which was injected subcutaneously at multiple points in the back of the neck, with an immunization dose of 1 mg / kg·bw. After the first immunization of experimental Balb / c mice, booster immunization was performed every 2 weeks. The immunogen diluent was mixed with Freund's incomplete adjuvant in a ratio of 1:1 to form an emulsified solution, which was injected subcutaneously at multiple points in the back of the neck, with a single immunization dose of 1 mg / kg·bw.

[0062] The final immunization was carried out on the Balb / c mice used in the experiment: the final immunization was carried out 10 weeks after the first immunization, and the immunogen dilution was directly injected subcutaneously at multiple points on the back of the neck. The immunization dose was 1 mg / kg·bw.

[0063] One week after the last immunization, the serum titer was 3.0×10 4 In the above experiments, Balb / c mice were killed, serum was separated and purified by saturated ammonium sulfate method to obtain the polyclonal antibody corresponding to the immunogen (referred to as polyclonal antibody B), which can be packaged and frozen after preparation.

[0064] Example 4

[0065] This embodiment provides an artificial antigen for coating an ELISA plate

[0066] Take 2.5 mg of the ethyl carbamate hapten prepared in Example 1, dissolve it in 500 μL tetrahydrofuran (THF), add 5 mg N,N'-carbonyldiimidazole (CDI), react at room temperature for 6 hours, drain and dissolve it in 500 μL N,N-dimethylformamide (DMF), drop it into a PBS solution containing 20 mg of ovalbumin (OVA), react at 37°C for 4 hours, dialyze with PBS for 1 day, and obtain the coated antigen EC-OVA.

[0067] Example 5

[0068] This embodiment provides a urethane enzyme-linked immunosorbent assay kit.

[0069] The kit comprises an ELISA plate, an enzyme marker antibody working solution, an ethyl carbamate standard working solution, a sample diluent, an antibody working solution, a substrate color developing solution, a stop solution and a washing solution.

[0070] ELISA plate: Dilute the original coating solution prepared in Example 4 with a coating buffer to obtain an original coating dilution solution, wherein the coating buffer solution is a carbonate solution with a pH of 9.6 and a value of 0.02 mol / L. Add 100 μL of the original coating dilution solution to each well of the ELISA plate and incubate at 37°C for 16 h. Pour off the liquid in the well, wash, and pat dry. Add 200 μL of blocking solution to each well, wherein the blocking solution is a mixture of 2.0 g of bovine serum albumin and 1000 mL of a phosphate buffer with a pH of 7.4 and a value of 0.01 mol / L. Incubate at 37°C for 2 h, pour off the liquid in the well, dry, and vacuum seal with aluminum film for storage.

[0071] Enzyme marker working solution: Dissolve 10 mg of alkaline phosphatase in 2 mL of acetic acid-sodium acetate buffer solution with a pH of 5.0, and then add 0.1 mL of 1.0% glutaraldehyde aqueous solution. Add goat anti-mouse secondary antibody at a mass ratio of 1:2 of alkaline phosphatase (ALP) enzyme: antibody, and stir at room temperature for 4 hours. Add NaBH4 at a mass ratio of 1:10 of NaBH4 to alkaline phosphatase (ALP), stir at room temperature for 4 hours to obtain alkaline phosphatase (ALP) labeled secondary antibody solution, dialyze and purify with 0.9% saline solution, and store at 4°C.

[0072] Ethyl carbamate standard working solution: dissolve ethyl carbamate in pH7.4, 0.05mol / L phosphate buffer to obtain standard solutions with concentrations of 0ppb, 1ppb, 3ppb, 9ppb, 27ppb, and 81ppb, respectively. Use pH7.4, 0.05mol / L phosphate buffer as the negative control solution of the standard solution, called the zero standard solution.

[0073] Sample diluent: 0.2 mol / L PB solution.

[0074] Antibody working solution: The polyclonal antibody B prepared in Example 3 was diluted with 1 mol / L phosphate buffer solution at a ratio of 1:50 to form a diluent that is an ethyl carbamate-specific antibody working solution.

[0075] Substrate color developing solution: 0.15 mol / L p-nitrophenol phosphate.

[0076] Stop solution: 53 mL of concentrated sulfuric acid, dilute to 1 L with deionized water.

[0077] The ELISA kit was stored at 2-8°C.

[0078] Example 6

[0079] This embodiment provides a method for using a urethane ELISA kit.

[0080] Pretreatment of the test object: After the sample is crushed, 1.0g of a representative sample is weighed and placed in a 15mL centrifuge tube, and 1.0mL of 70% acetonitrile-PB solution is added to mix it. Place the centrifuge tube on an oscillator and oscillate for 9 minutes at a speed of 180×g. Take the upper organic phase at 60°C, slowly blow it with nitrogen until it is almost dry, add 500μL of deionized water, vortex for 40s to fully dissolve the residue, and obtain a mixed solution of the test object. Take 100μL of the mixed solution, add 400μL of the sample diluent in Example 5, and use it for subsequent detection using the enzyme-linked immunosorbent assay kit in Example 5.

[0081] Take out the ELISA kit in Example 5 and place it at room temperature for equilibration for more than 30 minutes. Each liquid reagent must be shaken before use.

[0082] Take out the required number of ELISA plates, put the unused ELISA plates into the original tin foil bag and reseal them together with the provided desiccant for storage.

[0083] Numbering: Number the corresponding microwells of the samples and standards in sequence, make 2 parallel wells for each sample and standard, and record the positions of the standard wells and sample wells.

[0084] Add standard / sample diluent: Add 50 μL of standard / sample diluent to the corresponding microwells, then add 50 μL / well of antibody working solution and enzyme marker working solution, gently shake to mix, cover the plate with a cover film, and react at 25°C in a dark environment for 15 minutes.

[0085] Washing the plate: Carefully peel off the cover film, shake off the liquid in the wells, wash thoroughly 4 times with 260μL / well of washing working solution, each time with an interval of 10s, and pat dry with absorbent paper (the bubbles that are not removed after patting dry can be punctured with an unused pipette tip).

[0086] Color development: Add 100 μL / well of substrate solution and color development solution, shake gently to mix, cover the plate with cover film, and react at room temperature in a dark environment for 15 minutes.

[0087] Determination: Set the microplate reader at 450nm (it is recommended to use dual wavelength 450 / 630nm detection and read the data within 5 minutes) and determine the OD value of each well.

[0088] Quantitative analysis:

[0089] 1. Calculation of percent absorbance: The percent absorbance of a standard or sample is equal to the average of the percent absorbance values ​​of the standard or sample (dual wells) divided by the absorbance value of the first standard (zero standard solution), multiplied by 100%.

[0090] 2. Drawing and calculation of standard curve: Draw the standard curve with the percentage absorbance of the standard as the ordinate and the logarithm of the concentration of ethyl carbamate standard (μg / kg) as the abscissa. Substitute the percentage absorbance of the sample into the standard curve, read the corresponding concentration of the sample from the standard curve, and multiply it by the corresponding dilution factor to get the actual content of ethyl carbamate in the sample.

[0091] Example 7

[0092] This example tests the sensitivity and quantitative limit of the ethyl carbamate enzyme-linked immunosorbent assay kit.

[0093] The ELISA kit provided in Example 5 was tested using the method for using the ELISA kit provided in Example 6.

[0094] Limit of quantification: Take 20 batches of fermented grains and 20 batches of lees, which are blank samples verified by HPLC method as not containing ethyl formate. The samples to be tested are detected by enzyme-linked immunosorbent assay kit after pretreatment, and the test results are calculated according to the formula for the limit of quantification of the samples. LOQ = MN + 10SD, where SD is the standard deviation and MN is the mean value.

[0095] Sensitivity: IC 50 Value evaluation sensitivity, IC 50 That is, the standard concentration corresponding to when the inhibition rate reaches 50% according to the standard curve.

[0096] Linear range: Prepare standard products with different concentrations, with R 2 The standard working solution of the concentration in the enzyme-linked immunosorbent assay kit of the present invention is arranged as a standard curve. The standard curve is drawn and the linear range of the kit is calculated to be 3.15-62.10 μg / kg, IC 50 It is 13.26μg / kg, and the linear correlation coefficient is 0.9915.

[0097] Recovery rate: The spiked recovery rate measured after adding the standard to the blank sample is used to express the target object to be tested. Two concentrations, low (5 μg / kg) and high (50 μg / kg), are set. The experimental steps of Example 6 are used to perform 6 analyses for each concentration level. Recovery rate = (detection concentration - background value) / spiked concentration * 100%, which is the recovery rate result.

[0098] Precision: The intra-day coefficient of variation was calculated by analyzing the spiked recoveries six times, and the inter-day coefficient of variation was calculated by testing the recoveries for three consecutive days to express precision.

[0099] The present invention uses an enzyme marker for detection, and the detection instrument is equipped with software that can directly display the detection results. After detection, the sensitivity of the enzyme-linked immunosorbent assay kit provided by the present invention is: 13.26μg / kg, the quantitative limit of fermented grains is: 3.15μg / kg, and the quantitative limit of lees is: 3.15μg / kg. In addition, the detection results of this embodiment show that the recovery rate of the kit of the present invention is: 100±20%, and the precision: the intra-day and inter-day coefficients of variation of the kit are both less than 10%, indicating that the detection results of the kit of the present invention are highly accurate.

[0100] Example 8

[0101] This example provides a stability test of the urethane ELISA kit

[0102] The ELISA kit provided in Example 5 was assembled into three batches of kits, with 10 kits in each batch, and stability experiments were performed.

[0103] 1. Stability test (4°C)

[0104] The kit was stored at 4 degrees Celsius in the dark, and one kit was randomly selected from each batch at the 1st week, 2nd week, 4th week, 8th week, 16th week, and 24th week, and its IC was determined according to the operation and treatment method in Example 8. 50 And the spiked recovery rate of EC standard with a concentration of 20 μg / kg. The results are shown in Table 1. IC 50 There was no significant difference in the mean value (M) (t test, p value>0.05). The mean value (M) of the recovery results was between 85% and 110%, and the coefficient of variation (CV) was less than 10%, which was acceptable, indicating that the kit can be stored at 4°C for 24 weeks.

[0105] Table 1 Stability (4°C)

[0106]

[0107] 2. Thermal acceleration test (37°C)

[0108] The kit was sealed and kept away from light in a 37°C oven. One kit was randomly selected from each batch at the 1st, 2nd, 4th, 8th, 16th and 24th week of storage, and its IC was determined according to the operation and treatment method in the example. 50 And the spiked recovery rate of EC standard with a concentration of 20 μg / kg. The results are shown in Table 2. IC 50 There was no significant difference in the mean values ​​(M) (t test, p value>0.05). The mean values ​​(M) of the recovery results were between 85% and 110%, and the coefficient of variation was less than 10%, which was acceptable, indicating that the kit could be stored at 37°C for 24 weeks.

[0109] Table 2 Stability (37°C)

[0110]

[0111] Example 9

[0112] This example tests the specificity of the urethane ELISA kit

[0113] The cross-reaction test was performed using the ELISA kit usage method provided in Example 7 and the ELISA kit provided in Example 5.

[0114] Detecting IC of different samples 50 , calculated according to the formula: Cross-reaction rate (CR) = ethyl carbamate IC 50 / Analog IC 50 × 100%. The cross-reaction rate results are shown in Table 3, which show that the kit provided by the present invention has high specificity in detecting ethyl formate.

[0115] Table 3 Cross-reaction results

[0116] Competitor CR% Ethyl carbamate 100.0 Methyl carbamate 0.005 Butyl carbamate 0.00008 Acrylamide 0.00008

[0117] Comparative Example 1

[0118] This comparative example provides a urethane hapten, an artificial antigen and a preparation method thereof, which are as follows:

[0119] 4-Hydroxybenzylamine (2 g, 16.2 mmol) and NaHCO3 (1.6 g, 190 mmol) were added to 10 mL of dichloromethane, and the mixture was cooled to 0°C. Ethyl bromoformate (1.9 mL, 20 mmol) was added dropwise to the cooled mixture for 1 h. The sample was stirred at 25°C for 2 h. The resulting precipitate was filtered and washed three times with dichloromethane. An excess of 5% HCl was added to the filtrate and mixed evenly. NaCl was added to saturation. The organic layer was extracted and evaporated to dryness to obtain a light red solid. The obtained solid was purified by silica gel column, and the eluent was CHCl3 / MeOH / CH3COOH solution (25:8:1) to obtain an ethyl carbamate hapten control product (0.7 g, 3.6 mmol), as shown in formula (2).

[0120]

[0121] The urethane hapten control provided in this comparative example was used to prepare the corresponding urethane artificial antigen by reaction with carbonyldiimidazole. The reaction process is as follows:

[0122]

[0123] The preparation methods and experimental methods of other components are the same as those of Examples 3-9.

[0124] The results of the effect verification test are as follows:

[0125] Sensitivity of the kit: 29.2 μg / kg;

[0126] The limits of quantification were: fermented grains: 8.24 μg / kg, lees: 8.24 μg / kg;

[0127] Recovery rate: 100±20%;

[0128] Precision: The intra-day and inter-day coefficients of variation of the kit were both greater than 10%.

[0129] The results of the cross-reaction test are shown in Table 4. The kit prepared using the hapten, artificial antigen and antibody provided in Comparative Example 1 has a strong cross-reaction with methyl carbamate, butyl carbamate and acrylamide, and has a low specificity.

[0130] Table 4 Cross-reaction results

[0131] Competitor CR% Ethyl carbamate 100.0 Methyl carbamate 0.08 Butyl carbamate 0.001 Acrylamide 0.001

[0132] According to the above results, the ethyl carbamate kit prepared from the ethyl carbamate hapten provided by the present invention has the characteristics of convenience, rapidity, sensitivity, etc., and is suitable for on-site large-volume sample detection.

[0133] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An ethyl carbamate hapten, characterized in that: The structure of the ethyl carbamate hapten is shown in formula (1):

2. The method for preparing the ethyl carbamate hapten according to claim 1, characterized in that: The steps include: Ethyl bromoformate, 2-(2-naphthyl)-2-aminoacetic acid and sodium hydroxide are mixed in an acidic solution to carry out a substitution reaction.

3. The method for preparing the ethyl carbamate hapten according to claim 2, characterized in that: The molar ratio of ethyl bromoformate, sodium hydroxide and 2-(2-naphthyl)-2-aminoacetic acid is (1.5-2.5):1:(1.5-2.5).

4. An ethyl carbamate artificial antigen, characterized in that: The ethyl carbamate hapten according to claim 1 or the ethyl carbamate hapten obtained by the preparation method according to claim 2 or 3 is prepared by coupling with a carrier protein via carboxyl group.

5. The urethane artificial antigen according to claim 4, characterized in that: The carrier protein is selected from any one of bovine serum albumin, human serum albumin, rabbit serum albumin, ovalbumin, keyhole limpet hemocyanin, glutathione S-transferase or a multivalent antigen peptide.

6. A method for preparing a urethane-specific antibody, characterized in that: The artificial urethane antigen of claim 4 or 5 is used to immunize an animal, so that the body produces antibodies specific to urethane.

7. Use of the urethane hapten according to claim 1, the urethane hapten prepared by the method according to claim 2 or 3, the urethane artificial antigen according to claim 4 or 5, or the urethane specific antibody prepared by the method according to claim 6 in detecting urethane.

8. A urethane detection kit, characterized in that: The invention comprises an ethyl carbamate-specific antibody prepared by the method of claim 6 and an ELISA plate coated with the ethyl carbamate artificial antigen of claim 4 or 5.

9. The kit according to claim 8, characterized in that: The urethane artificial antigen-coupled carrier protein for preparing the urethane-specific antibody is the first carrier protein, and the urethane artificial antigen-coupled carrier protein coated with the ELISA plate is the second carrier protein. The first carrier protein and the second carrier protein are of different types.

10. The kit according to claim 8, characterized in that: It also includes enzyme marker antibody working solution, ethyl carbamate standard working solution, sample diluent, and substrate color developing solution.

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