Method and kit for detecting sodium cyclamate in white spirit

Abstract

The invention discloses a method and a kit for detecting sodium cyclamate in baijiu, and belongs to the technical field of food safety detection. The hapten is obtained, the sodium cyclamate monoclonal antibody is prepared according to the hapten, the enzyme linked immunosorbent assay kit is further prepared through specific binding of the sodium cyclamate monoclonal antibody and sodium cyclamate, and qualitative and quantitative detection can be conducted on sodium cyclamate in baijiu. The detection method is rapid, efficient and high in sensitivity, the lowest detection limit of sodium cyclamate in various types of baijiu is as low as 2.75-2.82 mu g / L, the detection variable coefficient RSD is smaller than 5%, the detection result is accurate and sensitive, and the problems that an existing detection method is complex in sample pretreatment step, high in instrument and equipment condition requirement and the like are effectively solved.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety detection, and specifically relates to a detection method and a kit for detecting sodium cyclamate in liquor. Background Art

[0002] my country's liquor culture has a long history, and liquor brewing technology is one of my country's precious intangible cultural heritage. Liquor is made from grains as the main raw materials, and is made through cooking, saccharification, fermentation, distillation, storage, and blending with sugar fermentation agents. High-quality liquor has the characteristics of sweet, mellow, soft and sweet, which is related to the flavor substances such as higher alcohols and polyols naturally produced in brewing. Since adding cyclamate to liquor can make the wine taste full, sweet and sweet, some wine workshops or merchants add sweeteners to the liquor without authorization, and pass it off as good. In addition, there are merchants who use edible alcohol or poor quality raw liquor to blend finished wine. In order to cover up the bad taste of the wine such as bitterness, they add cyclamate to the wine without authorization to increase the sweetness. This not only harms the vital interests of liquor consumers, but also has a negative impact on the image of Chinese liquor culture. Therefore, it is necessary and of practical significance to do a good job in cyclamate detection and study a method that can accurately and quickly determine cyclamate in liquor.

[0003] Sodium cyclamate, chemical name is sodium cyclamate, and its structural formula is: , is a commonly used additive in food production. It has a good taste and low price. Its sweetness is 30-40 times that of sucrose, and its price is only one-third of that of sucrose. Whether cyclamate is harmful to the human body has long been controversial since its launch. Some countries such as the United States, the United Kingdom, Japan and Southeast Asia prohibit its use as an additive. In my country, although the use of cyclamate is still allowed, it has strict limit requirements. my country's "GB2760-2014 National Food Safety Standard Food Additives Usage Standard" stipulates the limit value of cyclamate in different foods. The limit value is 0.65-1.6 g / kg (calculated as cyclohexylaminosulfonic acid). The above addition range does not include liquor. In liquor, cyclamate is a sweetener that is strictly prohibited from being added.

[0004] At present, the methods for detecting cyclamate in food at home and abroad mainly include high performance liquid chromatography, gas chromatography, spectrocolorimetry, thin layer chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry and ion chromatography. These methods each have their own scope of application and advantages and disadvantages, and are suitable for different samples and detection requirements. The inventor found that the detection method of cyclamate listed in the current national standard is used to detect cyclamate in white wine, and there are problems such as false positives and the detection limit cannot meet the detection requirements, which affects people's health on the one hand and domestic product export trade on the other hand. The inventor believes that the current equipment and method for detecting cyclamate are not suitable for white wine, and the substances similar to cyclamate produced during the fermentation and storage of white wine are misjudged, resulting in false positive detection results.

[0005] In addition, the inventors found that the existing detection methods for sodium cyclamate in liquors such as white wine also have the problems of complex sample pretreatment steps and high requirements for instrument and equipment conditions. For example, patent document CN 116908317 A discloses a method for detecting food additives in wine using high performance liquid chromatography-triple quadrupole mass spectrometry technology, wherein the sample pretreatment method is: weigh 2.5g of wine sample, place it in a 50mL beaker, place it in a 60℃ water bath for 30min, wash the residue with the initial mobile phase and transfer it all to a 25mL volumetric flask, fix the volume and shake it well, and filter it through a 0.22μm aqueous microporous filter membrane. For another example, the sample pretreatment method disclosed in patent document CN 113295794 A is: use a nitrogen blowing method to blow the volume of the sample to be tested to 10-30% of the original volume. The above sample processing methods all have the defects of many steps and long time consumption. The inventors analyzed that this is related to the low sensitivity of the corresponding detection method. Regarding detection instruments, patent document CN106324150 A discloses the use of high performance liquid chromatography low temperature evaporative light scattering method to determine the sweetener in liquor; CN 108613969 A discloses the detection of cyclamate in liquor by Raman spectrometer; CN108508128 A discloses the use of liquid chromatography-tandem mass spectrometry to detect cyclamate in liquor. In summary, the methods disclosed in the prior art allow the detection work to be carried out only in laboratories with relevant instruments, with many steps, long detection time and high detection cost. At present, there is no report on the immunoaffinity detection method based on artificial cyclamate antigen. Therefore, it is crucial to study a fast, efficient and sensitive detection method for the detection of cyclamate in liquor. Summary of the invention

[0006] Based on the above background, the present invention provides a method for detecting cyclamate in liquor, wherein the detection method realizes qualitative and quantitative detection of cyclamate by specifically binding cyclamate monoclonal antibody to cyclamate, and the detection method is fast, efficient and highly sensitive. In various types of liquor, the minimum detection limit of cyclamate is as low as 0.75-0.82 μg / L, and the detection accuracy is good and the precision is high. And because it is a method of specific binding of antibody and antigen, it effectively avoids the false positive detection results caused by substances similar to cyclamate produced during the fermentation and storage process of liquor under normal circumstances, and the detection accuracy is high. In the second aspect of the present invention, the present invention provides a rapid detection kit for cyclamate in liquor. In the third aspect of the present invention, the present invention provides a hapten for preparing a cyclamate monoclonal antibody and a preparation method thereof. In the fourth aspect of the present invention, the present invention provides a cyclamate monoclonal antibody prepared according to the hapten.

[0007] The present invention includes the following technical solutions: In the first aspect, the present invention provides a method for detecting cyclamate in white wine, characterized in that the method comprises: utilizing an indirect competitive ELISA method, using a cyclamate-coated ELISA plate, adding a sample solution to be tested, and simultaneously adding a cyclamate monoclonal antibody (primary antibody), incubating, washing, and patting dry, adding an enzyme-labeled sheep anti-mouse antibody (secondary antibody), developing color with a substrate, terminating, and determining the absorbance value with an ELISA instrument.

[0008] Preferably, the detection method further comprises preparing an ELISA standard curve, and substituting the percentage absorbance value of the sample to be tested into the ELISA standard curve to obtain the cyclamate concentration of the sample to be tested.

[0009] The preparation method of the ELISA standard curve includes: using cyclamate to coat the original ELISA plate, adding cyclamate standard solution with gradient concentrations, adding cyclamate monoclonal antibody at the same time, incubating, washing and patting dry, adding enzyme-labeled goat anti-mouse antibody, color development and termination by substrate, and measuring the absorbance value with an ELISA reader; drawing the ELISA standard curve with the standard concentration as the horizontal axis and the percentage absorbance value as the vertical axis.

[0010] The percentage absorbance value calculation formula of the present invention is as follows: Percent absorbance = B / B 0 ×100% Where: B-average absorbance value of standard (or sample); B 0 - Average absorbance value of the standard at 0 ppb concentration.

[0011] The cyclamate monoclonal antibody of the present invention highly specifically binds to cyclamate contained in a sample, and the cyclamate monoclonal antibody is obtained by the following preparation method: (a1) coupling the hapten represented by formula I with a carrier protein to prepare an immunogen; (I) (a2) preparing a sodium cyclamate monoclonal antibody according to the immunogen described in step (a1); The carrier protein is selected from any one or a combination of two or more of thyroglobulin (BTG), bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), ovalbumin (OVA), diphtheria toxoid, a non-toxic mutant of diphtheria toxin, tetanus toxoid and bacterially expressed proteins.

[0012] In a specific embodiment of the present invention, the carrier protein used to prepare the immunogen is bovine serum albumin (BSA).

[0013] The preparation of the sodium cyclamate monoclonal antibody according to the immunogen in step (a2) is a routine operation in the art, and the present invention does not limit the specific preparation method of the monoclonal antibody. It should be understood by those skilled in the art that the preparation of the sodium cyclamate monoclonal antibody by the hapten provided by the present invention, only the replacement or modification of the technical features such as the carrier protein and / or the preparation method therein, is within the technical scope of the present invention.

[0014] In a specific embodiment of the present invention, the method for preparing sodium cyclamate monoclonal antibody according to the immunogen comprises the following steps: (1) Immunization of mice: The immunogen was dissolved in physiological saline at 100 μg / mouse and mixed with an equal volume of Freund's adjuvant to immunize BALB / c mice. Complete Freund's adjuvant was used for the first immunization. On the 7th, 14th, and 28th days after the first immunization, the immunogen was mixed with an equal volume of incomplete Freund's adjuvant and boosted once each time. The last boosted immunization was performed without Freund's adjuvant. The serum titer was detected by indirect ELISA. (2) Cell fusion and cloning: The screened mouse spleen cells and mouse myeloma cells were fused and cultured, and the positive cell wells were detected by indirect ELISA. The indirect competitive ELISA method was further used to screen the cell wells with strong positive and high inhibition rate. The positive cell wells with the best inhibition were subcloned multiple times by limiting dilution method, and finally the hybridoma cell line 24D1 was screened and obtained; (3) Preparation of monoclonal antibodies: The hybridoma cells were injected into the peritoneal cavity of BALB / c mice using the in vivo induction method. The ascites was collected and purified to obtain the cyclamate monoclonal antibody.

[0015] The sodium cyclamate coating agent for coating the ELISA plate of the present invention is prepared by coupling the hapten shown in formula I with a carrier protein.

[0016] The carrier protein is selected from any one or a combination of two or more of thyroglobulin (BTG), bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), ovalbumin (OVA), diphtheria toxoid, a non-toxic mutant of diphtheria toxin, tetanus toxoid and bacterially expressed proteins.

[0017] In a specific embodiment of the present invention, the carrier protein used to prepare the coating source is ovalbumin (OVA).

[0018] The enzyme-labeled goat anti-mouse antibody of the present invention refers to a goat anti-mouse antibody labeled with horseradish peroxidase (HRP).

[0019] In a second aspect, the present invention provides a rapid detection kit for cyclamate in liquor, characterized in that the kit comprises an antibody working solution, an ELISA plate, an enzyme marker working solution and a cyclamate standard working solution.

[0020] The antibody working solution is obtained by diluting the sodium cyclamate monoclonal antibody prepared by the present invention by 5000-6000 times.

[0021] The ELISA plate is obtained by coating with sodium cyclamate.

[0022] The enzyme labeling working solution is obtained by diluting goat anti-mouse antibody labeled with horseradish peroxidase (HRP).

[0023] The cyclamate standard working solution is a PBS solution with cyclamate concentrations of 0 ppb, 0.1 ppb, 0.3 ppb, 0.9 ppb, 2.7 ppb, and 8.1 ppb.

[0024] Furthermore, the kit also includes a sample diluent, an antibody diluent, a washing solution, a substrate color developing solution and a stop solution.

[0025] The sample diluent is a PBS buffer solution containing a light stabilizer, bovine serum albumin and a surfactant, with a pH of 7.2, wherein the mass concentrations of the light stabilizer, bovine serum albumin and the surfactant are 0.2 g / L, 0.1 g / L and 0.1 g / L.

[0026] In the kit provided by the present invention, an antibody diluent is used to dilute antibodies, including sodium cyclamate monoclonal antibodies and enzyme-labeled antibodies. The antibody diluent is a PBS buffer containing Proclin 300 and Triton X-100, wherein the mass concentrations of Proclin 300 and Triton X-100 are 200 μL / L and 500 μL / L, respectively.

[0027] The washing solution is a PBS buffer solution containing Tween 20 and Proclin 300, wherein the concentrations of Tween 20 and Proclin 300 are 20 mL / L and 300 μL / L.

[0028] The substrate color developing solution is an aqueous solution of 1.0 g / L urea peroxide, 5.0 g / L sodium acetate, 0.5 g / L light stabilizer, 2.5 mL / L phosphoric acid, and 5.0 g / L tetramethylbenzidine.

[0029] The stop solution is a 0.05 mol / L sulfuric acid aqueous solution.

[0030] In a specific embodiment of the present invention, the light stabilizer is selected from UV328, ie, 2-(2'-hydroxy-3',5'-dipentylphenyl)benzotriazole, and the surfactant is selected from Tween-20.

[0031] The kit provided by the present invention is used for detecting cyclamate according to the following principles: adopting the indirect competitive ELISA method, using a coating source to coat an ELISA plate, using a cyclamate monoclonal antibody as the first antibody, a solid phase antigen and a liquid phase antigen in a sample to compete for binding to the first antibody, using a goat anti-mouse antibody labeled with horseradish peroxidase (HRP) as the second antibody, adding a substrate solution for color development after the reaction is completed, terminating the reaction, reading the absorbance value of the ELISA plate at dual wavelengths of 450 nm and 630 nm, and obtaining the absorbance value of cyclamate in the sample; drawing an ELISA standard curve, and substituting the absorbance value into the standard curve to obtain the concentration of cyclamate in the sample.

[0032] In a third aspect, the present invention provides a hapten for preparing a cyclamate full antigen, characterized in that the hapten is a compound represented by formula I: (I).

[0033] In a fourth aspect, the present invention provides a method for preparing a hapten represented by formula I, characterized in that the preparation method comprises the following steps: (S1) dissolving 4-aminocyclohexanecarboxylic acid in acetonitrile solution, adding chlorosulfonic acid dropwise in a molar mass ratio of 1:1 under an ice bath, reacting for two hours, and after the reaction is completed, adding ice water dropwise and carefully quenching, and then extracting with dichloromethane for multiple times to obtain 4-(sulfonylamino)cyclohexane-1-carboxylic acid; (S2) 4-(sulfonylamino)cyclohexane-1-carboxylic acid was dissolved in DMF solution, NHS and EDC were added for activation, 4-aminobutyric acid methyl ester was added according to the molar mass ratio of 4-(sulfonylamino)cyclohexane-1-carboxylic acid to 4-aminobutyric acid methyl ester of 1:1, and the reaction was carried out overnight to obtain a compound shown in formula II, with the structure (II); (S3) The compound represented by formula II is dissolved in MeOH solution, and sodium hydroxide is added according to formula II and sodium hydroxide aqueous solution at a molar mass ratio of 1:5, and the reaction is carried out overnight. Then, the pH value of the solution is adjusted to 3 with dilute HCl to obtain the compound represented by formula I.

[0034] In a fifth aspect, the present invention provides a cyclamate monoclonal antibody, characterized in that the cyclamate monoclonal antibody is an immunogen obtained by coupling a hapten shown in Formula I with a carrier protein, and is prepared by a monoclonal antibody preparation technology.

[0035] (I) The carrier protein is selected from any one or a combination of two or more of thyroglobulin (BTG), bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), ovalbumin (OVA), diphtheria toxoid, a non-toxic mutant of diphtheria toxin, tetanus toxoid and bacterially expressed proteins.

[0036] In a fifth aspect, the present invention provides a rapid test kit according to the second aspect of the present invention, the hapten according to the third aspect of the present invention, and the use of the sodium cyclamate monoclonal antibody according to the fifth aspect of the present invention in at least one of the following: (b1) Application in the detection of sodium cyclamate in liquor; (b2) Application in the preparation of products for detecting cyclamate in liquor.

[0037] The products include but are not limited to test strips, membrane strips, test kits, chips, models or detection platforms.

[0038] The detection method and kit for sodium cyclamate in liquor provided by the present invention have the following beneficial technical effects: In the GB5009.97-2023 determination standard of cyclohexylaminosulfonate in food, the detection limit in the gas chromatography standard is 0.01 g / kg, the detection limit in the high performance liquid chromatography standard is 0.01 g / kg, and the detection limit of the high performance liquid chromatography is 0.03 mg / kg. The detection limit of cyclamate in liquor determined by the high performance liquid chromatography low temperature evaporative light scattering method disclosed in patent CN 106324150 A is 0.6 mg / kg, and the detection limit of cyclamate in liquor detected by Raman spectrometer disclosed in patent CN 108613969 A is 0.5 mg / L. Patent CN117147512A discloses a rapid detection method for cyclamate by optical probe method, with a detection limit of 0.95 μmol / L. The detection limit standard was not met. The literature (Study on the method of simultaneous determination of 6 trace sweeteners in liquor by ultra-high pressure liquid chromatography-mass spectrometry, Journal of Food and Biotechnology, Wu Shijia, Vol. 29, No. 5, p. 670) uses liquid chromatography-mass spectrometry, and the detection limit of cyclamate can reach 0.01 mg / L. The method recorded in the literature (Study on the method of simultaneous determination of 4 sweeteners in liquor by liquid chromatography-tandem mass spectrometry, Xia Yulin, China Brewing, No. 3, 2011, p. 156) can reach a detection limit of 2 μg / L for cyclamate in liquor. The detection method for cyclamate in liquor provided by the invention has a minimum detection limit of 2.75-2.82 μg / L and a quantitative limit of 5.05-5.29 μg / L. The method recovery rate is between 98-105%, and the coefficient of variation (RSD) of each added concentration is less than 5%.

[0039] The detection method of the present invention does not require the use of large-scale instruments such as chromatography and mass spectrometry, and the detection kit can reach the detection standard of liquid chromatography-mass spectrometry by using a low-cost, small-volume, and portable detection kit. The specificity, sensitivity, and detection speed of the detection of sodium cyclamate in liquor are improved, the detection cost and operation difficulty are reduced, and the detection method of sodium cyclamate in liquor is made faster and more accurate, which is convenient for detection in the food safety detection and supervision process, and can effectively combat such illegal addition behaviors. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Cyclamate ELISA standard curve. DETAILED DESCRIPTION

[0041] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] 1. Preparation of cyclamate hapten and full antigen (1) Preparation and structural identification of cyclamate hapten The reaction route diagram is as follows:

[0043]

[0044] ; S1: 4-aminocyclohexanecarboxylic acid was dissolved in acetonitrile solution, and chlorosulfonic acid was added dropwise in an ice bath at a molar mass ratio of 1:1-2. The reaction was allowed to react for two hours. After the reaction was completed, ice water was added dropwise to quench the reaction, and then the mixture was extracted with dichloromethane for several times to obtain compound 1, i.e. 4-(sulfonyl)cyclohexane-1-carboxylic acid. S2: 4-(sulfonylamino)cyclohexane-1-carboxylic acid (compound 1) was dissolved in DMF solution, NHS and EDC were added for activation, 4-(sulfonylamino)cyclohexane-1-carboxylic acid (compound 1) and 4-aminobutyric acid methyl ester (compound 2) were added in a molar mass ratio of 1:1, and the reaction was carried out overnight to obtain a compound shown in formula II; S3: The compound represented by formula II is dissolved in MeOH solution, sodium hydroxide is added according to formula II and sodium hydroxide at a molar ratio of 1:5, the reaction is allowed to proceed overnight, and then the pH value of the solution is adjusted to 3 with dilute HCl to obtain the compound represented by formula I.

[0045] The hydrogen nuclear magnetic resonance data of the compound represented by formula I are as follows: 1H NMR (500 MHz, Chloroform ) δ4.17 (dt, J = 16.1, 11.8 Hz, 2H), 3.43 (t, J = 15.0 Hz, 2H), 3.29 (t, J = 15.2Hz, 1H), 2.76 (dt, J = 16.1, 11.8 Hz, 2H), 2.55 – 2.37 (m, 1H), 2.31 (t, J =10.8 Hz, 2H), 2.17 – 1.59 (m, 4H), 1.25 (dt, J = 15.2, 11.8 Hz, 2H).

[0046] (2) Preparation of complete cyclamate antigen 2.1. Synthesis of immunogens S1: Dissolve 8.25 mg of sodium cyclamate hapten in 1.5 mL of DMF, stir at 200 rpm for 10 min, add 12.7 mg of EDC to dissolve, then add 7.4 mg of NHS, and activate at room temperature for 2-3 h; S2: Weigh 50 mg BSA and dissolve it in 3.5 mL 0.1 M sodium bicarbonate solution. Stir at 200 rpm for 10 min to fully dissolve it. Cool it to 0-4 °C in an ice bath. Stir at 1000 rpm and add the reaction solution from step (1) dropwise (1 mL / min). Stir at 500 rpm for 24 h. S3: The reaction product was placed in a dialysis bag rinsed with distilled water, and dialyzed with 1 L of 0.01 M PBS (1×, pH 7.2) at 4°C with stirring (100 rpm) for 3 days. The solution was changed 3 times a day. The dialyzed product was centrifuged at 5000 rpm for 6 min, aliquoted, and stored at -20°C for later use.

[0047] 2.2 Synthesis of coating precursor S1: Dissolve 8.25 mg of cyclamate hapten in 1.5 mL of DMF, stir at 200 rpm for 10 min, add 12.7 mg of EDC and then add 7.4 mg of NHS, and activate at room temperature for 2-3 h; S2: Weigh 33.6 mg of OVA and dissolve it in 3.5 mL of 0.1 M sodium bicarbonate solution. Stir at 200 rpm for 10 min to fully dissolve it. Cool it to 0-4 °C in an ice bath. Stir at 1000 rpm and add the reaction solution of step 1 dropwise (1 mL / min). Stir at 500 rpm for 24 h. S3: The reaction product was placed in a dialysis bag rinsed with distilled water, and dialyzed with 1 L of 0.01 M PBS (1×, pH 7.2) at 4°C with stirring (100 rpm) for 3 days. The solution was changed 3 times a day. The dialyzed product was centrifuged at 5000 rpm for 6 min, aliquoted, and stored at -20°C for later use.

[0048] 2. Preparation of monoclonal antibodies by immunizing animals with cyclamate immunogen The sodium cyclamate monoclonal antibody was prepared according to the conventional method, and the specific steps are as follows: 1. Animal immunization The prepared immunogen was dissolved in physiological saline at 100 μg / mouse, and mixed with an equal volume of Freund's complete adjuvant. The mixture was injected subcutaneously on the back of the neck to immunize 6-8 week old Balb / c female mice. On the 7th, 14th and 28th days after the initial immunization, the immunogen was mixed with an equal volume of Freund's incomplete adjuvant for booster immunization each time. Three days before fusion, 100 μg / mouse of immune complex was used for booster immunization without Freund's adjuvant.

[0049] 2. Cell fusion and cloning According to the conventional method, spleen cells of immunized mice were mixed with mouse myeloma cells (SP2 / 0) in the logarithmic growth phase, and then preheated fusion agent (PEG4000) was slowly added within 30 seconds for fusion. The cells were evenly suspended with HAT medium, and then an appropriate amount of feeder cells were added and cultured in a 96-well culture plate at 37°C and 5% CO. 2 Culture in an incubator, change half of the medium with HT medium after 7 days, and change the medium completely after 9 days.

[0050] After cell fusion, when the cells grow to 7-10 days, the hybridoma cells are screened by step-by-step screening. The indirect ELISA method is used for the initial screening, and 834 positive wells are screened; the screened positive wells are then screened by the indirect competitive ELISA method, and the cell culture wells with strong positive and high inhibition rates are selected for subclone screening. After multiple subclone screenings, the positive rate is 100%, and finally multiple hybridoma cell lines that can secrete monoclonal antibodies to cyclamate are obtained. Among them, the cell line 24D1, IC 50 It is 0.56 ng / mL.

[0051] The monoclonal antibody of sodium cyclamate was prepared by inducing ascites in mice. 0.5 mL of liquid paraffin was injected into the peritoneum of 8-10 week-old Balb / c mice. 1-2×10 hybridoma cells were injected into the peritoneum 7-10 days later. 5 / mouse, 7-10 days later, the ascites of the mice were extracted and centrifuged at 4℃, 5000 rpm for 10 min to remove the upper oil and lower precipitate, and then purified by caprylic acid-saturated ammonium sulfate method to obtain cyclamate monoclonal antibody and stored at -20℃.

[0052] 3. Preparation of ELISA kit for cyclamate in liquor The enzyme-linked immunosorbent assay kit for cyclamate in liquor includes: antibody working solution, ELISA plate, enzyme marker working solution, cyclamate standard working solution containing different gradient concentrations, sample diluent, antibody diluent, washing solution, substrate color developing solution, and stop solution.

[0053] (1) Preparation of ELISA plate Cyclamate ELISA plate: dilute the obtained cyclamate coating source to 10.0 μg / mL, add 100 μL of the coating source to each well, incubate at 37°C for 2 h, pour off the liquid, wash twice with 20-fold diluted washing solution, 30 seconds each time, pat dry, then add 150 μL of blocking solution to each well, incubate at 37°C for 1 h, pour off the liquid in the wells, dry to obtain the cyclamate ELISA plate, and store it in a vacuum-sealed aluminum film.

[0054] (2) Preparation of antibody working solution The cyclamate monoclonal antibody was diluted 6000 times to obtain the cyclamate antibody working solution.

[0055] (3) Enzyme marker working solution Horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody.

[0056] (4) Preparation of standard working solution The concentrations of the cyclamate standard working solutions are 0 ppb, 0.1 ppb, 0.3 ppb, 0.9 ppb, 2.7 ppb, and 8.1 ppb, respectively.

[0057] (5) Preparation of other reagents The sample diluent is a PBS buffer solution containing a light stabilizer UV328, bovine serum albumin and a surfactant Tween 20, with a pH of 7.2, wherein the mass concentrations of the light stabilizer, bovine serum albumin and the surfactant are 0.2 g / L, 0.1 g / L and 0.1 g / L.

[0058] The antibody diluent is a PBS buffer containing Proclin 300 and Triton X-100, wherein the mass concentrations of Proclin 300 and Triton X-100 are 200 μL / L and 500 μL / L, respectively.

[0059] The washing solution is a PBS buffer solution containing Tween 20 and Proclin 300, wherein the concentrations of Tween 20 and Proclin 300 are 20 mL / L and 300 μL / L.

[0060] The substrate color developing solution is an aqueous solution of 1.0 g / L urea peroxide, 5.0 g / L sodium acetate, 0.5 g / L light stabilizer UV328, 2.5 mL / L phosphoric acid, and 5.0 g / L tetramethylbenzidine.

[0061] The stop solution is a 0.05 mol / L sulfuric acid aqueous solution.

[0062] 4. How to use the cyclamate ELISA kit 1. Kit detection method S1: Insert the strips into the ELISA plate rack and record the positions of the standards and samples. Make two parallel holes. Seal the unused strips with a ziplock bag and store them immediately at 4°C. S2: Add 50 μL of each standard working solution / sample solution into the corresponding standard / sample wells; S3: Add 50 μL of antibody working solution to each well; S4: Gently shake the ELISA plate for 10 s to mix thoroughly, and react at room temperature (25±2℃) in the dark for 30 min; S5: Pour out the liquid in the wells, add 260 μL of washing solution to each well, wash thoroughly 4 times, soaking for 15-30 s each time; pour out the liquid in the wells and pat the ELISA plate dry; S6: Add 50 μL of enzyme marker working solution to each well and repeat steps S4-S5; S7: Add 100 μL of substrate solution to each well; S8: Gently shake the ELISA plate for 10 s to mix thoroughly, and react at room temperature (25±2℃) in the dark for 15 min; S9: Add 50 μL of stop solution to each well and gently shake the ELISA plate for 10 s to mix thoroughly; S10: Within 5 min after termination, read the absorbance of the ELISA plate at dual wavelengths of 450 nm and 630 nm using an ELISA reader.

[0063] 2. Analysis of test results 2.1. Calculate the percent absorbance value The average absorbance value of each standard (or sample to be tested) is divided by the absorbance value of the zero standard (standard with a concentration of 0 μg / L) and multiplied by 100% to obtain the percentage of absorbance corresponding to each standard (or sample to be tested), that is, the percent absorbance value.

[0064] Absorbance percentage = B / B 0 ×100% Where: B-average absorbance value of standard (or sample); B 0 - Average absorbance value of the standard at 0 ppb concentration.

[0065] 2.2. Make a standard curve The standard curve is drawn with the percentage absorbance of each standard as the ordinate and the concentration of sodium cyclamate (μg / L) in each standard working solution as the abscissa. Figure 1 shown.

[0066] 2.3. Calculate the content of sodium cyclamate in the sample Substituting the percentage absorbance value of the sample to be tested into the standard curve, the corresponding residual concentration of the sample to be tested can be obtained, and then multiplied by the dilution factor of the corresponding sample to obtain the actual content of cyclamate in the original sample to be tested.

[0067] (III) Specificity, accuracy and precision testing of cyclamate ELISA kit 1. Specificity test of the kit: The specificity of the cyclamate ELISA kit is determined by conducting cross-reaction tests with corresponding substances.

[0068] Make serial dilutions of sodium cyclamate, saccharin sodium and acesulfame potassium, respectively, and operate according to the detection method of the kit to make standard curves, and find the 50% inhibition concentration (IC 50 ), the specific method is as follows: get the concentration of sodium cyclamate (μg / L) corresponding to the vertical axis value equal to 50%, that is, IC 50 The cross-reaction rate of the test kit to sodium cyclamate, saccharin sodium and acesulfame potassium was calculated using the following formula: Cross-reaction rate (%) = (concentration of sodium cyclamate causing 50% inhibition / concentration of cross-reactant causing 50% inhibition) × 100%. The results are shown in Table 1.

[0069] Table 1 Specificity of the kit .

[0070] Experiments show that the kit of the present invention has good specificity for cyclamate and can be used for the detection of cyclamate.

[0071] 2. Determination of minimum detection limit Take blank samples of Maotai-flavor, Luzhou-flavor, and Light-flavor liquors and test them according to the kit test method. Calculate the measured value based on the standard curve, calculate the average value, and add 3 times the standard deviation to get the minimum detection limit (LOD). The results are shown in Table 2.

[0072] Table 2 Statistical table of detection limit and quantification limit results (μg / L) .

[0073] The results showed that the minimum detection limit of saccharin in Maotai-flavor liquor was 2.86 μg / L and the limit of quantification was 5.29 μg / L, the minimum detection limit of saccharin in Luzhou-flavor liquor was 2.82 μg / L and the limit of quantification was 5.12 μg / L, and the minimum detection limit of saccharin in light-flavor liquor was 2.75 μg / L and the limit of quantification was 5.05 μg / L.

[0074] 3. Test kit accuracy and precision Blank samples of Maotai-flavor liquor, Luzhou-flavor liquor, and Light-flavor liquor were pretreated and then added with saccharin to obtain the test sample solutions. The concentrations of saccharin in the samples were low concentration group (5 μg / L), medium concentration group (30.0 μg / L), and high concentration group (100.0 μg / L), respectively. Each batch of samples was tested 5 times in parallel and the average value was taken.

[0075] Table 3 Accuracy and precision .

[0076] The results showed that the recovery rates of all samples at various concentrations were between 98% and 105%. The coefficient of variation (RSD) of each concentration was less than 5%. This indicates that the method of the present invention has good accuracy and precision, and can effectively complete the rapid and accurate detection of trace cyclamate in liquor.

Claims

1. A method for detecting sodium cyclamate in liquor, characterized in that: The method comprises: using an indirect competitive ELISA method, using a sodium cyclamate coating source to coat an ELISA plate, adding a sample solution to be tested, adding a sodium cyclamate monoclonal antibody at the same time, incubating, washing and patting dry, adding an enzyme-labeled goat anti-mouse antibody, developing color through a substrate, terminating, and measuring the absorbance value with an ELISA instrument; the sodium cyclamate coating source and the sodium cyclamate monoclonal antibody are both prepared from the hapten shown in formula I: （I）。 2. The detection method according to claim 1, characterized in that: The detection method further comprises preparing an ELISA standard curve, substituting the percentage absorbance value of the sample to be tested into the ELISA standard curve to obtain the sodium cyclamate concentration of the sample to be tested; The preparation method of the ELISA standard curve includes: using cyclamate to coat the original ELISA plate, adding cyclamate standard solution with gradient concentrations, adding cyclamate monoclonal antibody at the same time, incubating, washing and patting dry, adding enzyme-labeled goat anti-mouse antibody, color development and termination by substrate, and measuring the absorbance value with an ELISA reader; drawing the ELISA standard curve with the standard concentration as the horizontal axis and the percentage absorbance value as the vertical axis.

3. The detection method according to claim 1, characterized in that: The cyclamate monoclonal antibody highly specifically binds to the cyclamate contained in the sample, and the cyclamate monoclonal antibody is obtained by the following preparation method: (a1) coupling the hapten represented by formula I with a carrier protein to prepare an immunogen; （I） (a2) preparing a sodium cyclamate monoclonal antibody according to the immunogen described in step (a1); The carrier protein is selected from bovine serum albumin.

4. The detection method according to claim 1, characterized in that: The cyclamate coating is prepared by coupling the hapten shown in formula I with a carrier protein, and the carrier protein is selected from ovalbumin.

5. A rapid test kit for sodium cyclamate in liquor, characterized in that: The kit comprises an antibody working solution, an ELISA plate, an enzyme marker working solution and a cyclamate standard working solution; the antibody working solution is obtained by diluting the cyclamate monoclonal antibody prepared according to claim 3 by 5000-6000 times.

6. The kit according to claim 5, characterized in that The ELISA plate is coated with the cyclamate coating material prepared according to claim 4; the enzyme labeling working solution is obtained by diluting the sheep anti-mouse antibody labeled with horseradish peroxidase; the cyclamate standard working solution is a PBS solution with cyclamate concentrations of 0 ppb, 0.1 ppb, 0.3 ppb, 0.9 ppb, 2.7 ppb, and 8.1 ppb.

7. The kit according to claim 5, characterized in that The kit also includes a sample diluent, an antibody diluent, a washing solution, a substrate color developing solution and a stop solution.

8. A hapten for preparing a cyclamate full antigen, characterized in that: The hapten is a compound represented by formula I: （I）。 9. A sodium cyclamate monoclonal antibody, characterized in that: The sodium cyclamate monoclonal antibody is an immunogen obtained by coupling the hapten shown in Formula I with a carrier protein, and is prepared by a monoclonal antibody preparation technology: （I） The carrier protein is selected from any one or a combination of two or more of thyroglobulin, bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, diphtheria toxoid, a non-toxic mutant of diphtheria toxin, tetanus toxoid and bacterially expressed proteins.

10. Use of the kit according to any one of claims 5 to 7, the hapten according to claim 8, and the sodium cyclamate monoclonal antibody according to claim 9 in at least one of the following: (b1) Application in the detection of sodium cyclamate in liquor; (b2) Application in the preparation of a product for detecting sodium cyclamate in liquor; The products include test strips, membrane strips, test kits, chips, models or detection platforms.

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