Enzyme linked immunosorbent assay kit for detecting capsaicin in edible oil and application of enzyme linked immunosorbent assay kit

Abstract

The invention relates to an enzyme linked immunosorbent assay kit for detecting capsaicin in edible oil and application of the enzyme linked immunosorbent assay kit. The kit disclosed by the invention adopts a competitive enzyme-linked immunosorbent assay and is matched with a matched calculation template, so that the content of capsaicin in a grease sample can be detected only by simultaneously detecting one standard substance during sample detection. The kit and the detection method thereof have the characteristics of simple sample treatment steps, short time consumption, high specificity, high sensitivity, high accuracy, high stability and the like, the arrangement of a single standard curve point is suitable for high-throughput sample detection, the cost can be saved under the condition of small sample size, and the detection method is suitable for large-scale popularization and application. The curve difference caused by personnel operation and the like is avoided, and the detection efficiency is greatly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of immune detection, and particularly relates to an enzyme-linked immunosorbent assay kit for detecting capsaicin in edible oil and an application thereof. Background Art

[0002] Fats and oils are an important part of people's diet. "Edible oil safety" is an important part of "food safety". Its product quality will directly affect the health and safety of consumers. The quality and safety testing of edible oils and oils has always been a difficult and hot issue in the field of food analysis, including the issue of edible oils mixed with waste cooking oils.

[0003] According to Chinese cuisine and dining habits, chili peppers are commonly used as cooking seasonings, and capsaicin can be widely used in the field of food processing as a condiment. Capsaicin is the active ingredient of chili peppers, and usually exists in chili peppers together with other capsaicinoids, of which capsaicin and dihydrocapsaicin account for about 90% of the total amount of capsaicinoids. Normal edible oils basically do not contain capsaicin, while waste cooking oils that have been in contact with capsaicin products inevitably contain capsaicinoids. Capsaicinoids have the characteristics of strong fat solubility and good stability, and are not easily destroyed in oils and fats. Therefore, capsaicin can be used as an exogenous marker for edible oils. By detecting the content of capsaicinoids in oil samples, it can be determined whether the edible oil is mixed with waste cooking oils and fats, and the accuracy rate is more than 80%.

[0004] At present, the detection methods of capsaicin in edible oil are mainly based on instrumental detection methods, including classic chromatography-mass spectrometry, high performance liquid chromatography, high performance liquid chromatography-mass spectrometry, new spectroscopic methods, nuclear magnetic resonance methods, etc. Although the instrumental detection method has the advantages of high sensitivity and good stability, its equipment is expensive, the pretreatment process is cumbersome, time-consuming, and requires a large amount of organic reagents. Therefore, it needs to be carried out in a laboratory with relatively complete facilities, and it must be equipped with professional operators. In addition, there are some electrochemical method equipment and reagents on the market, but the sensitivity is relatively low, and it is easily interfered by the sample matrix, and the detection sample range is limited. Therefore, none of the above detection methods can meet the requirements of rapid, low-cost and efficient detection. In view of many factors, it is urgent to develop a detection method that has fast detection speed, strong specificity, high sensitivity and low cost compared with the instrumental method, which can detect capsaicin in a variety of vegetable oils. And the immunoassay technology based on antigen-antibody specific reaction is just right. The common immunoassay techniques currently used to detect capsaicin include: colloidal gold immunochromatography, fluorescent immunoassay, enzyme-linked immunosorbent assay, etc. Colloidal gold is low-cost, easy to use and fast, but it can only detect qualitatively, not quantitatively; although fluorescent immunoassay can detect quantitatively, the problem of non-specific staining has not been solved, the objectivity of the result judgment is insufficient, and the procedure is relatively complicated. Although the traditional enzyme-linked immunosorbent assay kit solves the shortcomings of the existing technology, it is more suitable for high-throughput sample testing because this detection method requires at least 5 standard points for each detection. For detection experiments with a small sample volume, the finished product is slightly higher, and the detection personnel need to have certain professional skills, so it is easy to lead to low test precision and poor repeatability. Therefore, the development of a more convenient and stable enzyme-linked immunosorbent assay kit has important economic and social benefits. Summary of the invention

[0005] The present invention aims to overcome the defects of the existing enzyme-linked immunosorbent assay technology and provide an enzyme-linked immunosorbent assay kit for detecting capsaicin in edible oil. The difference between the present kit and the general enzyme-linked immunosorbent assay kit is that the present kit no longer contains a set of standard products, but only a zero standard. The present kit has high sensitivity, simple composition, short sample processing time, convenient operation, good stability, and the kit without a standard curve can also reduce the detection cost.

[0006] The present invention provides a capsaicin enzyme-linked immunosorbent assay kit, comprising a box body, an ELISA plate arranged in the box body, reagents and a calculation template, wherein the reagents include a standard substance, an enzyme conjugate working solution and a sample diluent.

[0007] The wells of the ELISA plate are coated with antigens that can bind to capsaicin-specific antibodies, and the coating solution is preferably 0.1M Tris-HCl buffer (pH 8.5);

[0008] The enzyme conjugate working solution includes: horseradish peroxidase-labeled capsaicin-specific antibody (the enzyme-labeled antibody is from the Oil Crops Research Institute of Hubei Academy of Agricultural Sciences), the enzyme-labeled antibody is diluted and stored with enzyme conjugate diluent, and the ratio of the two is 1:1000-1:100000;

[0009] The enzyme conjugate diluent comprises: 0.01-0.1M Tris-HCl buffer, casein at a mass concentration of 0.01%-0.1%, BSA at a mass concentration of 0.2-0.3%, skimmed milk powder at a mass concentration of 0.2-0.3%, sucrose at a mass concentration of 1-3%, enzyme stabilizer SG09 at a mass concentration of 0.1-0.3%, proclin 950 at a mass concentration of 0.05-0.15%, β-mercaptoethanol at a mass concentration of 0.01-0.1%, Tween20 at a mass concentration of 0.01-0.1%, and the pH value is adjusted to 8-9;

[0010] The standard is a zero capsaicin content standard, that is, a standard diluent, including: 1-10% methanol by volume, 1-5% NaCl aqueous solution by mass, less than 5% glycerol by mass, less than 1% PVP-40 by mass, no more than 1% EDTA by mass, less than 10% trehalose by mass, 0.05-0.15M BB buffer, and a pH value of 8-9;

[0011] The sample diluent includes: a 1-2% by mass NaCl aqueous solution, less than 5% by mass glycerol, less than 1% by mass PVP-40, no more than 1% by mass EDTA, less than 10% by mass trehalose, 0.05-0.15M BB buffer, and a pH value of 8-9;

[0012] The calculation template is a standard curve template obtained by linear fitting.

[0013] In the kit of the present invention, the ELISA plate is composed of a plastic support and separate microporous strips.

[0014] Furthermore, the standard diluent includes: 5% methanol by volume, 1% NaCl aqueous solution by mass, no more than 1% glycerol by mass, no more than 0.3% PVP-40 by mass, no more than 1% EDTA by mass, no more than 5% trehalose by mass, 0.1M BB buffer, and a pH value of 8.5.

[0015] Furthermore, the sample diluent includes: 2% by mass NaCl aqueous solution, no more than 1% by mass glycerol, no more than 0.3% by mass PVP-40, no more than 1% by mass EDTA, no more than 5% by mass trehalose, 0.1M BB buffer, and a pH value of 8.5.

[0016] Furthermore, the enzyme conjugate diluent comprises: 0.01M Tris-HCl buffer, casein with a mass concentration of 0.05%, BSA with a mass concentration of 0.24%, skimmed milk powder with a mass concentration of 0.24%, sucrose with a mass concentration of 2%, enzyme stabilizer SG09 with a mass concentration of 0.2%, proclin 950 with a mass concentration of 0.1%, β-mercaptoethanol with a mass concentration of 0.01%, Tween20 with a mass concentration of 0.05%, and the pH value is adjusted to 8.5.

[0017] Furthermore, the calculation template is obtained by linear fitting with the logarithm of the standard concentration as the x-axis and the logit value of the corresponding percentage absorbance value as the y-axis. The calculation template of the present invention is specially made for the kit and used for the analysis of the test results. When the sample is tested, the curve required for the test result analysis of the kit can be directly determined by a single standard point in combination with the reagent of the present invention, which reduces the use of the ELISA plate wells and can achieve "testing as soon as there is a sample" even when the sample amount is relatively small.

[0018] Furthermore, the enzyme-linked immunosorbent assay kit also includes a substrate solution, a stop solution, and a 10× concentrated washing solution. The substrate solution is composed of a color developing solution A and a color developing solution B. The color developing solution A is hydrogen peroxide or urea peroxide, and the color developing solution B is o-phenylenediamine or tetramethylbenzidine. Before use, the color developing solution A and the color developing solution B are mixed in a 1:1 ratio and used immediately. They are prepared on demand. The stop solution is a 1-2 mol / L hydrochloric acid buffer solution, and the 10× concentrated washing solution is a phosphate buffer solution containing 0.5% Tween 20.

[0019] The present invention also provides a method for detecting capsaicin in a sample using any of the above-mentioned kits, comprising the following steps:

[0020] (1) Sample pretreatment;

[0021] (2) testing with a kit;

[0022] (3) Use the matching calculation template to calculate the test results.

[0023] Furthermore, the pretreatment of the sample is mainly to extract capsaicin from the edible oil for subsequent detection; the detection steps include: adding capsaicin zero content standard solution or sample solution to the microwells of the ELISA plate, adding enzyme conjugate working solution, shaking incubation and then washing, shaking off the liquid in the wells, and absorbing residual moisture with absorbent paper, color development, termination, and measuring the absorbance value with an ELISA instrument; calculation of the test results includes: entering the absorbance values ​​of the capsaicin zero content standard and the sample in the corresponding positions in the calculation template, and if necessary, entering one to two other parameters to calculate the capsaicin content in the sample to be tested.

[0024] Furthermore, the sample pretreatment specifically includes: weighing 1 g of the edible oil sample, adding 1 mL of 70% methanol aqueous solution, extracting for 2 minutes, and then centrifuging for 5 minutes to obtain a supernatant, taking 0.1 mL of the supernatant, adding it to 0.9 mL of the sample diluent, and vortexing to mix to obtain a test solution.

[0025] Further, add 50 μL of zero capsaicin content standard solution or test solution to the microwells of the ELISA plate coated with capsaicin-coupled antigen, then add 50 μL of enzyme conjugate working solution, place on an oscillator and incubate at room temperature for 20-30 minutes, wash and pat dry, add 100-200 μL of substrate solution to each well, incubate at room temperature in the dark for 10-20 minutes, add 100 μL of stop solution, measure the absorbance value at a wavelength of 450 nm / 630 nm, enter the absorbance value of the capsaicin zero content standard and the sample in the corresponding position in the calculation template, and enter one to two other parameters if necessary to calculate the capsaicin content in the sample to be tested.

[0026] The present invention also provides the use of any of the above-mentioned kits in detecting the content of capsaicin in a sample.

[0027] Beneficial effects:

[0028] The kit of the present invention adopts competitive enzyme-linked immunosorbent assay, and cooperates with a matching calculation template. When testing samples, only one standard product needs to be tested at the same time to detect the content of capsaicin in the oil sample. In addition, the sample processing steps are simple, time-consuming, and have the characteristics of high specificity, high sensitivity, and high accuracy. The setting of a single calibration point is not only suitable for high-throughput sample detection, but also can save costs when the sample amount is small.

[0029] The reagent of the present invention reduces the number of standard solutions, uses a matching calculation template to analyze and calculate the content of capsaicin in a sample, saves costs, and avoids the influence of curve differences caused by human operation and other reasons on the final result of the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0031] Figure 1 : Schematic diagram of the composition of the capsaicin detection kit.

[0032] Figure 2 : Calculate the template graph;

[0033] Figure 3 :Calculation software interface diagram corresponding to the calculation template. DETAILED DESCRIPTION

[0034] The following examples are only used to more clearly illustrate the technical scheme of the present invention, and are therefore only used as examples, and cannot limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise stated, the reagents and materials used in the following examples are commercially available.

[0035] Example 1 Preparation of the kit

[0036] 1. Preparation of ELISA Plate

[0037] 1) Coating solution 1: prepare 0.1 mol / L carbonate buffer with a pH of 9.6.

[0038] 2) Coating solution 2: prepare 0.1 mol / L Tris-HCl buffer with a pH of 8.5.

[0039] 3) Coating solution 3: Prepare 0.1 mol / L PB buffer with a pH of 7.4.

[0040] 4) Dilute the capsaicin-coupled antigen to 0.1 μg / mL with coating buffer, add 100 μL to each well, incubate at 4°C overnight, pour off the coating solution, wash three times with 1× washing solution, 15 seconds each time, shake off the liquid in the wells, absorb the residual water with absorbent paper, add 200 μL of blocking solution to each well, incubate at 37°C for 2 hours, pour off the liquid in the wells, dry and vacuum seal with aluminum film, and store in a dry place at 4°C for later use.

[0041] 5) The blocking solution is a phosphate buffer solution containing 3% skimmed milk powder, pH 7.4, and 0.02 mol / L, and the percentages are weight-to-volume ratios.

[0042] 6) Determined by square array titration test, with absorbance value of 0.8-1.2 as reference standard, the enzyme conjugate was mixed with 1× washing solution at a ratio of 1:1000-1:100000 to obtain enzyme conjugate working solution, 50 μL of deionized water and 50 μL of enzyme conjugate working solution were added to the above different ELISA plate wells, mixed and incubated at room temperature for 20 minutes, washed 5 times, patted dry, 100 μL of substrate solution was added to each well, incubated at room temperature in the dark for 10 minutes, 100 μL of stop solution was added, and the absorbance value was measured at a wavelength of 450nm / 630nm, and the coefficient of variation of the plate was calculated. The results are shown in Table 1.

[0043] Table 1 Coefficient of variation of ELISA plate wells

[0044]

[0045]

[0046] The results showed that using 0.1 mol / L Tris-HCl buffer (pH 8.5) as the coating solution and coating according to the process can make the difference between the plate wells smaller, thereby ensuring the stability of subsequent test results.

[0047] 2. Preparation of enzyme conjugate diluent

[0048] An enzyme conjugate diluent was prepared so that the concentration of Tris-HCl buffer was 0.01 M, the mass concentration of casein was 0.05%, the mass concentration of BSA was 0.24%, the mass concentration of skimmed milk powder was 0.24%, the mass concentration of sucrose was 2%, the mass concentration of enzyme stabilizer SG09 was 0.2%, the mass concentration of proclin 950 was 0.1%, the mass concentration of β-mercaptoethanol was 0.01%, the mass concentration of Tween20 was 0.05%, and the pH value was adjusted to 8.5.

[0049] It was determined by a square array titration test that the absorbance value was 1.8-2.2 as a reference standard, the enzyme-labeled antibody was mixed with the enzyme conjugate diluent at a ratio of 1:1000-1:100000 to obtain an enzyme conjugate working solution, 50 μL of the standard diluent and 50 μL of the enzyme conjugate working solution were added to the wells of the above-determined ELISA plate, mixed and incubated at room temperature for 20 minutes, washed 5 times, patted dry, 100 μL of substrate solution was added to each well, incubated at room temperature in the dark for 10 minutes, 100 μL of stop solution was added, and the absorbance value was measured at a wavelength of 450nm / 630nm to determine that the dilution multiple of the enzyme-labeled antibody was 4000 times, and it was divided into two parts, one was placed in a refrigerator at 4°C, and the other was placed in a 37°C constant temperature box. Accelerated comparison experiments were performed at different intervals of different days to detect the stability of the enzyme conjugate working solution. The results are shown in Table 2.

[0050] Table 2 Accelerated comparison experiment of enzyme conjugate working solution

[0051]

[0052]

[0053] The results showed that the enzyme conjugate diluent could be used to dilute and preserve enzyme-labeled antibodies.

[0054] 3. Preparation of standard products

[0055] The main components of the standard diluent are 1% NaCl, 0.1M BB buffer solution with a pH value of 8.5 and a 5% methanol (v / v) aqueous solution. Adding other stabilizing components thereto can make the standard curve more stable without affecting the OD value and inhibition.

[0056] 1) Determination of the composition of the standard dilution solution

[0057] The specific operations are:

[0058] Add glycerol to the main components of the standard diluent to make its mass fraction 0.1%, 1%, and 5%;

[0059] PVP-40 was added to the main components of the standard dilution solution to make the mass fractions 0.1%, 0.3%, and 1%;

[0060] Add EDTA to the main components of the standard diluent to make the mass fractions 0.1%, 0.3%, and 1%;

[0061] Trehalose was added to the main components of the standard dilution solution to make the mass fractions 1%, 5% and 10%.

[0062] A total of 12 solutions and the main components of the control group standard dilution solution, the above 13 reagents were used as standard dilution solutions, capsaicin standard (100ppm, dissolved in methanol) was diluted to 0μg / kg and 0.5μg / kg, the enzyme-labeled antibody was mixed with the enzyme conjugate dilution solution at a ratio of 1:4000 to obtain an enzyme conjugate working solution, 50μL of standard and 50μL of enzyme conjugate working solution were added to the above-determined ELISA plate wells, mixed and incubated at room temperature for 20 minutes, washed 5 times, patted dry, 100μL of substrate solution was added to each well, incubated at room temperature in the dark for 10 minutes, 100μL of stop solution was added, and the absorbance value was measured at a wavelength of 450nm / 630nm. The results are shown in Table 3.

[0063] Table 3 Experiment on the influence of the components of standard dilution solution

[0064]

[0065]

[0066] The data show that preparing 0.1M BB buffer with a pH value of 8.5, a volume percentage of 5% methanol (v / v), a mass fraction of 1% NaCl aqueous solution, adding glycerol to it so that its mass fraction is within 1%, adding PVP-40 to it so that its mass fraction is within 0.3%, adding EDTA to it so that its mass fraction is within 1%, and adding trehalose to it so that its mass fraction is within 5%, neither affects the inhibition of the standard nor the OD value of the zero standard.

[0067] 2) Optimization of reaction conditions

[0068] The present invention finds that the oscillation operation is used in the process of enzyme-linked immunosorbent assay to improve the utilization rate of the enzyme-labeled antibody and improve its sensitivity.

[0069] The specific operation is as follows: using the standard diluent of the present invention, the capsaicin standard (100ppm, dissolved in methanol) is diluted to 0μg / kg, 0.03μg / kg and 0.1μg / kg, the enzyme-labeled antibody is mixed with the enzyme conjugate diluent at a ratio of 1:8000 (which can be adjusted according to actual conditions) to obtain an enzyme conjugate working solution, 50μL of the standard and 50μL of the enzyme conjugate working solution are added to the wells of the ELISA plate determined above, the plate is placed on an oscillator at 400r / min and incubated at room temperature for 20 minutes, washed 5 times, patted dry, 100μL of substrate solution is added to each well, the plate is incubated at room temperature in the dark for 10 minutes, 100μL of stop solution is added, and the absorbance value is measured at a wavelength of 450nm / 630nm. The control group is subjected to static reaction under the same conditions. The results are shown in Table 4.

[0070] Table 4 Comparison results of reaction conditions

[0071]

[0072] The results showed that the absorbance value and raw material sensitivity could be improved by 400r / min shaking operation during the enzyme-linked immunosorbent assay.

[0073] 3) Curve experiment

[0074] The specific operations are:

[0075] Prepare 0.1M BB buffer with a pH value of 8.5, a volume percentage of 5% methanol (v / v), a mass fraction of 1% NaCl aqueous solution, add glycerol thereto to make its mass fraction 1%, add PVP-40 thereto to make its mass fraction 0.3%, add EDTA thereto to make its mass fraction less than 1%, add trehalose thereto to make its mass fraction 5%, use this reagent as a standard diluent, prepare the capsaicin standard into standard solutions of 0μg / kg, 0.03μg / kg, 0.1μg / kg, 0.3μg / kg, 1μg / kg, and 3μg / kg, use the main component of the standard diluent as a control group, prepare the capsaicin standard into standard solutions of 0μg / kg, 0.03μg / kg, 0.1μg / kg, 0.3μg / kg, 1μg / kg, and 3μg / kg, and carry out ELISA detection at 35°C, 25°C, and 15°C, respectively. The results are shown in Table 5.

[0076] Table 5 Optimization results of standard dilution formula

[0077]

[0078]

[0079] The results show that, by preparing a 0.1M BB buffer solution with a pH value of 8.5, 5% methanol (v / v) by volume, a 1% NaCl aqueous solution by mass, and adding a certain amount of glycerol, PVP-40, EDTA and trehalose, using the reagents in the kit of the present invention, and assisting with a vibration plate operation during the reaction process, the standard curve can maintain a basically consistent inhibition rate at high, medium and low values.

[0080] 4. Determination of sample dilution

[0081] To keep consistent with the standard, the sample diluent can be adjusted based on the standard diluent to offset matrix interference. The specific operation is to remove the methanol component in the standard diluent (since the sample extract is a certain concentration of methanol, the final sample test solution still contains a certain proportion of methanol after dilution, so there is no need to add methanol to the sample diluent again), and then add NaCl to it so that its final mass fraction is 1%-5%, and prepare the following sample diluent.

[0082] Sample dilution 1: prepare 0.1M BB buffer solution with a pH value of 8.5, add glycerol to make its mass fraction 1%, add PVP-40 to make its mass fraction 0.3%, add EDTA to the main component of the standard dilution solution to make its mass fraction less than 1%, add trehalose to the main component of the standard dilution solution to make its mass fraction 5%, and add NaCl to make its final mass fraction 1%;

[0083] Sample dilution 2: Add NaCl to standard dilution 1 to make the final mass fraction 2%;

[0084] Sample dilution 3: Add NaCl to standard dilution 1 to make the final mass fraction 3%;

[0085] Sample dilution 4: Add NaCl to standard dilution 1 to make the final mass fraction 4%;

[0086] Sample dilution 5: Add NaCl to standard dilution 1 to make the final mass fraction 5%.

[0087] Weigh 100g of capsaicin-negative edible oil sample, add capsaicin standard to it, theoretically make the final capsaicin content in the sample 5μg / kg, mix thoroughly and send to a third party for inspection, and obtain a capsaicin-positive sample with a total capsaicin content of 4.86μg / kg;

[0088] Weigh 5 groups of samples, each group contains one tube of negative sample and one tube of positive sample, 1 g per tube, add 1 mL of 70% methanol aqueous solution, extract for 2 min, and then centrifuge for 5 min to obtain the supernatant, take 0.1 mL of the supernatant and add it to 0.9 mL of the above sample dilution and mix well to obtain the sample solution to be tested.

[0089] The capsaicin standard (100ppm, dissolved in methanol) was diluted to 0μg / kg, 0.03μg / kg, 0.1μg / kg, 0.3μg / kg, 1μg / kg, and 3μg / kg using the standard diluent. The enzyme-labeled antibody was mixed with the enzyme conjugate diluent at a ratio of 1:8000 to obtain an enzyme conjugate working solution. 50μL of the standard / test sample solution and 50μL of the enzyme conjugate working solution were added to the wells of the ELISA plate determined above, and the plate was placed on an oscillator at 400r / min and incubated at room temperature for 20 minutes. The plate was washed 5 times and patted dry. 100μL of substrate solution was added to each well, and the plate was incubated at room temperature for 10 minutes in a dark place. 100μL of stop solution was added, and the absorbance value was measured at a wavelength of 450nm / 630nm. The control group was allowed to react under the same conditions. The results are shown in Table 6.

[0090] Table 6 Sample dilution formula optimization results

[0091]

[0092]

[0093] Taking into account the sample background value and sample recovery rate, 0.1M BB buffer solution with a pH value of 8.5 was prepared, glycerol was added to make its mass fraction 1%, PVP-40 was added thereto to make its mass fraction 0.3%, EDTA was added to the main component of the standard diluent to make its mass fraction within 1%, trehalose was added to the main component of the standard diluent to make its mass fraction 5%, and NaCl was added to make its final mass fraction 2%.

[0094] 5. Calculation template determination

[0095] The standard diluent is prepared according to the present invention, the capsaicin standard is diluted into 0 μg / kg, 0.03 μg / kg, 0.1 μg / kg, 0.3 μg / kg, 1 μg / kg, 3 μg / kg standard solutions, the enzyme-labeled antibody is mixed with the enzyme conjugate diluent at a ratio of 1:8000 to obtain the enzyme conjugate working solution, and 50 μL of the capsaicin standard solution is added to the plate wells with a coating concentration of 0.1 μg / mL. 0μg / kg, 0.03μg / kg, 0.1μg / kg, 0.3μg / kg, 1μg / kg, 3μg / kg of these six concentrations of standard, and then add 50μL enzyme conjugate working solution, incubate at room temperature for 20 minutes on an oscillator at 400r / min, wash 5 times, pat dry, add 100μL substrate solution to each well, incubate at room temperature in the dark for 10 minutes, add 100μL stop solution, measure the absorbance at a wavelength of 450nm / 630nm, and calculate the percentage absorbance of the standard, that is, the percentage absorbance of the standard is equal to the absorbance of the standard divided by the absorbance of the first standard (0μg / kg), multiplied by 100%, that is

[0096]

[0097] B—average absorbance value of the standard

[0098] B0—average absorbance value of 0μg / kg standard

[0099] Repeat the test several times and record the percent absorbance value of the standard, take the average value, and the results are shown in Table 7.

[0100] Table 7 Percent absorbance values ​​of standard products

[0101]

[0102] Based on this data, a linear fit is performed with the logarithm of the standard concentration as the x-axis and the logit value of the corresponding percentage absorbance value as the y-axis to produce a special calculation template suitable for this batch of kits, such as Figure 2 The calculation template shown in FIG. 1 is used to design the corresponding calculation software based on the calculation template. Figure 3As shown, the absorbance values ​​of the capsaicin zero content standard and the sample can be input in the corresponding positions in the calculation software (one to two other parameters can be input if necessary), and the content of capsaicin in the sample can be finally calculated. Since the reagents of the present invention can ensure the stability of the kit of the present invention, a complete real-time curve can be fitted through the calculation template and the absorbance values ​​of a single standard point, and then the content of capsaicin in the sample can be calculated through the sample absorbance value. The calculation template is based on the use of all reagents of the present invention.

[0103] 6. Sample data comparison

[0104] Weigh 10 groups of samples, each group containing one tube of negative sample (ND) and one tube of positive sample (4.86μg / kg), 1g in each tube, add 1mL 70% methanol aqueous solution, extract for 2min, and then centrifuge for 5min to obtain the supernatant, take 0.1mL of the supernatant and add it to 0.9mL of the above sample dilution and mix well to obtain the sample solution to be tested. These 10 groups of sample solutions to be tested are divided into 10 different people for testing.

[0105] The capsaicin standard (100 ppm, dissolved in methanol) was diluted to 0 μg / kg, 0.03 μg / kg, 0.1 μg / kg, 0.3 μg / kg, 1 μg / kg, and 3 μg / kg using the standard diluent, and the enzyme-labeled antibody was mixed with the enzyme conjugate diluent at a ratio of 1:8000 to obtain an enzyme conjugate working solution. 50 μL of standard / test sample solution and 50 μL of enzyme conjugate working solution were added to the wells of the ELISA plate determined above, and the plates were placed on an oscillator at 400 r / min and incubated at room temperature for 20 minutes. The plates were washed 5 times and patted dry. 100 μL of substrate solution was added to each well, and the plates were incubated at room temperature in the dark for 10 minutes. 100 μL of stop solution was added, and the absorbance value was measured at a wavelength of 450 nm / 630 nm using an automatic ELISA reader. A standard curve was drawn according to the concentration and absorbance value of the capsaicin standard solution to determine the capsaicin content in the test solution (i.e., the full standard curve measurement value). At the same time, the capsaicin content (i.e., the single standard measurement value) was calculated using the standard curve attached to the kit of the present invention through the absorbance values ​​of the 0 standard and the test sample solution. The two were compared. The results are shown in Table 8.

[0106] Table 8 Comparison results between full-calibration curve measurement and single-calibration measurement

[0107]

[0108] The data show that when the kit of the present invention is used, the single-label measurement results are more stable, which reduces the detection errors caused by differences in human operation, environmental differences, etc. to a certain extent.

[0109] Example 2 Construction of an ELISA kit for detecting capsaicin in edible oil

[0110] like Figure 1 As shown, an enzyme-linked immunosorbent assay kit for detecting capsaicin in edible oil is constructed to include the following components:

[0111] (1) ELISA plate coated with capsaicin-conjugated antigen;

[0112] (2) Capsaicin-specific antibody was labeled with horseradish peroxidase (the enzyme-labeled antibody was obtained from the Oil Crops Research Institute of Hubei Academy of Agricultural Sciences);

[0113] (3) 1 bottle of capsaicin standard solution, i.e., the standard dilution solution of the present invention;

[0114] (4) Substrate liquid: substrate liquid A liquid and substrate liquid B liquid;

[0115] (5) The stop solution is 1 mol / L hydrochloric acid;

[0116] (6) 10× concentrated washing solution is a 0.2 mol / L phosphate buffer solution with a pH value of 7.4 and containing 0.5% Tween-20. The percentages are weight-to-volume ratios.

[0117] (7) The sample diluent is the sample diluent described in the present invention.

[0118] (8) Calculation template for the same batch.

[0119] Example 3 Detection of capsaicin in actual edible oil

[0120] 1. Sample pretreatment

[0121] Weigh 1 g of edible oil sample, add 1 mL of 70% methanol aqueous solution, extract for 2 min, and then centrifuge for 5 min to obtain the supernatant. Take 0.1 mL of the supernatant and add it to 0.9 mL of sample diluent and mix well to obtain the test solution.

[0122] 2. Detection with a kit

[0123] Add 50 μL of zero capsaicin content standard solution to the microwells of the ELISA plate coated with capsaicin-coupled antigen, then add 50 μL of enzyme conjugate working solution, place on an oscillator at 400 r / min and incubate at room temperature for 20 minutes, wash 5 times, pat dry, add 100 μL of substrate solution to each well, incubate at room temperature in the dark for 10 minutes, add 100 μL of stop solution, measure the absorbance value at a wavelength of 450 nm / 630 nm, enter the absorbance value of the capsaicin zero content standard and the sample at the corresponding position in the standard curve (if necessary, enter one to two other parameters) to calculate the capsaicin content in the sample, and based on the test results, it can be determined whether the edible oil is mixed with kitchen waste oil (i.e., gutter oil). The results are shown in Table 9.

[0124] Table 9 Actual sample test results

[0125]

[0126]

[0127] The results show that the values ​​measured by the kit of the present invention are very close to those measured by the instrument.

[0128] Example 4 Determination of Kit Quality

[0129] 1. Sample precision test

[0130] The edible oil was tested at a concentration of 0.40 μg / kg capsaicin. Three different batches of test kits were used and the test was repeated 5 times at each concentration. The coefficient of variation was calculated. The results are shown in Table 10.

[0131] Table 10 Repeatability test of edible oil samples

[0132]

[0133] The results showed that the coefficient of variation of the edible oil samples was within 15%, which was in line with the reference evaluation criteria of the test kit.

[0134] 2-sample accuracy test

[0135] Two edible oil samples with capsaicin contents of 1.04 μg / kg and 2.43 μg / kg were taken and tested according to the kit method of the present invention. Four parallels were made for each sample, and the accuracy was calculated. The results are shown in Table 11.

[0136] Table 11 Accuracy of the kit

[0137]

[0138] The results showed that the accuracy of edible oil samples was 80%-115%, and the coefficient of variation was within 15%, which met the reference evaluation criteria of the test kit.

[0139] 3. Kit Storage Experiment

[0140] The storage condition of the test kit is 2-8°C. After 12 months of measurement, the maximum absorbance value (zero standard), 50% inhibition concentration, and actual measured value of capsaicin addition of the test kit are all within the normal range. Considering that abnormal storage conditions may occur during transportation and use, the test kit is placed at 37°C for 12 days and an accelerated aging experiment is performed. The results show that the various indicators of the test kit fully meet the requirements. From the above results, it can be concluded that the test kit of the present invention can be stored at 2-8°C for more than 12 months.

[0141] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A capsaicin enzyme-linked immunosorbent assay kit, characterized in that: It includes a box body, an ELISA plate arranged in the box body, reagents and a calculation template, wherein the reagents include a standard substance, an enzyme conjugate working solution, and a sample diluent. The wells of the ELISA plate are coated with antigens that can bind to capsaicin-specific antibodies, and the coating solution is preferably 0.1 M Tris-HCl buffer; The enzyme conjugate working solution includes: a capsaicin-specific antibody labeled with horseradish peroxidase, the enzyme-labeled antibody is diluted and stored with an enzyme conjugate diluent, and the ratio of the two is 1:1000-1:100000; The enzyme conjugate diluent comprises: 0.01-0.1M Tris-HCl buffer, casein at a mass concentration of 0.01%-0.1%, BSA at a mass concentration of 0.2-0.3%, skimmed milk powder at a mass concentration of 0.2-0.3%, sucrose at a mass concentration of 1-3%, enzyme stabilizer SG09 at a mass concentration of 0.1-0.3%, proclin 950 at a mass concentration of 0.05-0.15%, β-mercaptoethanol at a mass concentration of 0.01-0.1%, Tween20 at a mass concentration of 0.01-0.1%, and the pH value is adjusted to 8-9; The standard is a zero capsaicin content standard, that is, a standard diluent, including: 1-10% methanol by volume, 1-5% NaCl aqueous solution by mass, less than 5% glycerol by mass, less than 1% PVP-40 by mass, no more than 1% EDTA by mass, less than 10% trehalose by mass, 0.05-0.15M BB buffer, and a pH value of 8-9; The sample diluent includes: a 1-2% mass fraction NaCl aqueous solution, less than 5% mass fraction glycerol, less than 1% mass fraction PVP-40, no more than 1% mass fraction EDTA, less than 10% mass fraction trehalose, 0.05-0.15MBB buffer, and a pH value of 8-9; The calculation template is a standard curve template obtained by linear fitting.

2. The enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: The standard diluent includes: 5% methanol by volume, 1% NaCl aqueous solution by mass, no more than 1% glycerol by mass, no more than 0.3% PVP-40 by mass, no more than 1% EDTA by mass, no more than 5% trehalose by mass, 0.1MBB buffer, and a pH value of 8.

5.

3. The enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: The sample diluent comprises: 2% NaCl aqueous solution by mass, no more than 1% glycerol by mass, no more than 0.3% PVP-40 by mass, no more than 1% EDTA by mass, no more than 5% trehalose by mass, 0.1M BB buffer, and a pH value of 8.

5.

4. The enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: The enzyme conjugate diluent comprises: 0.01M Tris-HCl buffer, casein with a mass concentration of 0.05%, BSA with a mass concentration of 0.24%, skimmed milk powder with a mass concentration of 0.24%, sucrose with a mass concentration of 2%, enzyme stabilizer SG09 with a mass concentration of 0.2%, proclin 950 with a mass concentration of 0.1%, beta-mercaptoethanol with a mass concentration of 0.01%, Tween20 with a mass concentration of 0.05%, and the pH value is adjusted to 8.

5.

5. The enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: The calculation template is obtained by performing linear fitting with the logarithm of the standard concentration as the x-axis and the logit value of the corresponding percentage absorbance value as the y-axis.

6. A method for detecting capsaicin in a sample using the kit according to any one of claims 1 to 5, characterized in that: The steps include: (1) Sample pretreatment; (2) testing with a kit; (3) Use the matching calculation template to calculate the test results.

7. The method according to claim 6, characterized in that The sample pretreatment includes: extracting capsaicin from edible oil; the detection step includes: adding a capsaicin zero-content standard solution or a sample solution into the microwells of the ELISA plate, adding an enzyme conjugate working solution, washing after shaking and incubation, shaking off the liquid in the wells, and absorbing residual moisture with absorbent paper, coloring, terminating, and measuring the absorbance value with an ELISA instrument; the calculation of the detection result includes: inputting the absorbance value of the capsaicin zero-content standard and the sample into a calculation template, so as to calculate the content of capsaicin in the sample to be tested.

8. The method according to any one of claims 6-7, characterized in that: The sample pretreatment specifically includes: weighing 1 g of edible oil sample, adding 1 mL of 70% methanol aqueous solution, extracting for 2 min, and then centrifuging for 5 min to obtain the supernatant, taking 0.1 mL of the supernatant, adding it to 0.9 mL of sample diluent, and vortexing to mix to obtain the test solution.

9. The method according to any one of claims 6-7, characterized in that: Add 50 μL of zero capsaicin content standard solution or test solution to the microwells of the ELISA plate coated with capsaicin-coupled antigen, then add 50 μL of enzyme conjugate working solution, incubate on an oscillator at room temperature for 20-30 minutes, wash and pat dry, add 100-200 μL of substrate solution to each well, incubate at room temperature in the dark for 10-20 minutes, add 100 μL of stop solution, measure the absorbance value at a wavelength of 450 nm / 630 nm, enter the absorbance value of the capsaicin zero content standard and the sample in the calculation template, and calculate the capsaicin content in the sample to be tested.

10. Use of the kit according to any one of claims 1 to 5 in detecting the content of capsaicin in a sample.

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