Enzyme-linked immunosorbent assay kit and method for detecting capsaicin in animal and vegetable oils

Abstract

The invention discloses an enzyme-linked immunosorbent assay kit and method for detecting capsaicin in animal and vegetable oils, and belongs to the technical field of immunoassay. The enzyme-linked immunosorbent assay kit comprises a standard diluent, a sample diluent, an antibody / enzyme conjugate diluent, a capsaicin standard, an enzyme-labeled plate coated with a capsaicin antigen, a capsaicin antibody, an enzyme conjugate, a washing solution, a color developing solution, and a stop solution. The enzyme-linked immunosorbent assay kit provided by the invention has wide applicability, can stably, rapidly, and efficiently perform quantitative detection of capsaicinoids in a variety of animal and vegetable oils, and has the advantages of high sensitivity, low detection limit, etc. The sensitivity of the kit is 0.1 μg / kg, the detection limit is 0.3 μg / kg, and the quantitative range is 0.4-10 μg / kg. Therefore, it has good application prospects in the rapid detection of vegetable crude oil and kitchen recycling oil.

Description

Technical Field

[0001] The invention belongs to the technical field of immune detection, and in particular relates to an enzyme-linked immunosorbent assay kit and a method for detecting capsaicin in animal and vegetable oils. Background Art

[0002] After extensive research on waste cooking oil by the Chinese government and scholars from relevant research institutions, it was found that capsaicinoids can be used as exogenous markers in the identification and detection of waste cooking oil, with an accuracy rate of over 80%. Capsaicinoids as exogenous markers have a wide range of content in waste cooking oil, so the detection method is required to be not only fast and simple, but also meet the requirements of high sensitivity and accuracy. The existing waste oil on-site rapid identification technology in my country cannot meet the urgent need for rapid detection in the current quality and safety supervision of edible animal and vegetable oils.

[0003] At present, the detection of adulteration markers in edible oils and fats mainly relies on various instrumental detection methods, including classic chromatography-mass spectrometry, new spectroscopy, nuclear magnetic resonance, etc. Although instrumental detection methods have the advantages of high sensitivity and good stability, their equipment is expensive, the pre-treatment process is cumbersome, and professional operators are required. Therefore, the above detection methods cannot meet the requirements of rapid, low-cost and efficient detection.

[0004] In view of this, it is necessary to develop an enzyme-linked immunosorbent assay kit that can detect capsaicin in a variety of animal and vegetable oils to solve the shortcomings of the existing technology. Summary of the invention

[0005] The purpose of the present invention is to provide an enzyme-linked immunosorbent assay kit and method for detecting capsaicin in animal and vegetable oils. The enzyme-linked immunosorbent assay kit of the present invention can stably, quickly and efficiently perform quantitative detection of capsaicin in animal and vegetable oils, and has the advantages of high sensitivity and low detection limit, and therefore has a good application prospect in the detection of capsaicin in animal and vegetable oils.

[0006] In a first aspect, the present invention provides an enzyme-linked immunosorbent assay kit for detecting capsaicin in animal and vegetable oils, comprising a standard diluent, a sample diluent and an antibody / enzyme conjugate diluent; wherein the standard diluent comprises: a NaCl aqueous solution with a mass concentration of 0.1-5%, methanol with a volume percentage of 1-10%, 0.01-0.5M BB buffer, and a pH value of 7.5-9.0; the sample diluent comprises: a NaCl aqueous solution with a mass concentration of 0.1-10%, methanol with a volume percentage of 1-10%, 0.01-0.5M BB buffer, and a pH value of 7.5-9.0; the antibody / enzyme conjugate diluent comprises: 0.01-0.05M PB buffer, trehalose with a mass concentration of 0.1~10%, β-cyclodextrin with a mass concentration of 0.01~1%, sorbitol with a mass concentration of 1~10%, BSA with a mass concentration of 0.01~1%, skimmed milk powder with a mass concentration of 0.01~1%, thimerosal with a mass concentration of 0.005~0.1%, proclin 950 with a mass concentration of 0.05~0.5%, polylysine with a mass concentration of 0.1~5%, β-mercaptoethanol with a mass concentration of 0.001~0.2%, PEG4000 with a mass concentration of 0.5~5%, and Tween20 with a mass concentration of 0.01~2%.

[0007] In the standard diluent provided by the present invention, the mass concentration of the NaCl aqueous solution can be, for example, 0.1%, 1%, 5% or other values ​​within the range; the volume percentage of methanol can be, for example, 1%, 5%, 10% or other values ​​within the range; the concentration of the BB buffer can be, for example, 0.01M, 0.1M, 0.5M or other values ​​within the range; the pH value can be, for example, 7.5, 8.5, 9.0 or other values ​​within the range; in the sample diluent provided by the present invention, the mass concentration of the NaCl aqueous solution can be, for example, 0.1%, 2%, 10% or other values ​​within the range; the volume percentage of methanol can be, for example, 1%, 5%, 10% or other values ​​within the range; the concentration of the BB buffer can be, for example, 0.01M, 0.1M, 0.5M or other values ​​within the range; the pH value can be, for example, 7.5, 8.5, 9.0 or other values ​​within the range the mass concentration of thimerosal can be, for example, 0.005%, 0.01%, 0.01%, 0.01%, 0.01%, 0.006%, 0.010%, 0.011%, 0.012%, 0.013%, 0.014%, 0.015%, 0.016%, 0.017%, 0.018%, 0.019%, 0.101%, 0.011%, 0.012%, 0.013%, 0.014%, 0.016%, 0.017%, 0.018%, 0.019%, 0.011%, 0.013%, 0.014%, 0.015%, 0.016%, 0.018%, 0.019%, 0.011%, 0.012%, 0.013%, 0.014%, 0.015%, 0.016%, 0.018%, 0.019%, 0.01 ... The mass concentration of 950 can be, for example, 0.05%, 0.2%, 0.5% or other values ​​within this range; the mass concentration of polylysine can be, for example, 0.1%, 1%, 5% or other values ​​within this range; the mass concentration of β-mercaptoethanol can be, for example, 0.001%, 0.01%, 0.2% or other values ​​within this range; the mass concentration of PEG4000 can be, for example, 0.5%, 2%, 5% or other values ​​within this range; the mass concentration of Tween20 can be, for example, 0.01%, 0.5%, 2% or other values ​​within this range.

[0008] In some embodiments, the standard diluent includes: a 1% NaCl aqueous solution, a 5% methanol by volume, a 0.1 M BB buffer, and a pH value of 8.5.

[0009] In the present invention, the inventors have found that increasing the salt ion concentration in the standard diluent can improve the sensitivity of the product, but at the same time it will increase the amount of antibody or enzyme conjugate used and fail to achieve the desired effect. Therefore, it is important to screen a suitable salt ion concentration. At the same time, since capsaicin is easily soluble in alcohol solvents such as ethanol and methanol, but difficult to dissolve in water, a certain concentration of methanol or ethanol and other water-miscible alcohol solvents need to be added to the standard diluent as a cosolvent to improve the solubility of capsaicinoids in aqueous solution. However, alcohol solvents such as methanol or ethanol can cause denaturation of protein components in antibodies, enzymes, antigens, etc., thereby increasing costs and decreasing performance, such as decreasing sensitivity. Therefore, it is necessary to control the concentration of alcohol substances within a suitable range to increase the solubility of capsaicinoids while ensuring the sensitivity of the product. In addition, introducing a pH buffer component into the standard diluent can maintain the pH stability of the reaction system and increase the stability of the product. Further, the present invention obtains a NaCl aqueous solution containing a mass concentration of 1%, a methanol solution with a volume percentage of 5%, and a 0.1M BB buffer, pH 8.5 standard diluent can not only meet the requirements of improving product sensitivity, but also keep the dosage of antibody and enzyme conjugate at appropriate concentrations.

[0010] In some embodiments, the sample diluent includes: a 2% NaCl aqueous solution, a 4% methanol by volume, a 0.1 M BB buffer, and a pH value of 8.5.

[0011] In the present invention, the inventors further discovered that a suitable salt ion concentration in the sample diluent can improve the sample recovery rate of the product, but at the same time it will affect the background value of the sample, resulting in a false negative or false positive phenomenon in the test result. At the same time, since capsaicin is easily soluble in alcohol solvents such as ethanol and methanol, but difficult to dissolve in water, it is necessary to add a certain concentration of methanol or ethanol or other water-miscible alcohol solvents as co-solvents in the sample diluent to improve the solubility of capsaicinoids in aqueous solution. However, alcohol solvents such as methanol or ethanol can cause denaturation of protein components in antibodies, enzymes, antigens, etc., thereby increasing costs and decreasing performance, such as low or high sample recovery rates; therefore, it is necessary to control the concentration of alcohol substances within a suitable range to improve the solubility of capsaicinoids while ensuring the sample recovery rate; in addition, introducing a pH buffer component into the sample diluent can maintain the pH stability of the reaction system and increase the stability of the product. Taking into account the influence of the sample matrix, the mass concentration of the NaCl aqueous solution in the sample diluent is controlled to 2%, the volume percentage of methanol is controlled to 4%, the concentration of the BB buffer is controlled to 0.1M, and the pH value is controlled to 8.5. While improving the product sample recovery rate, the probability of false negative or false positive results is greatly reduced.

[0012] In some embodiments, the antibody / enzyme conjugate diluent comprises: 0.01M PB buffer, 10% trehalose by mass, 0.1% β-cyclodextrin by mass, 10% sorbitol by mass, 0.1% BSA by mass, 0.1% skim milk powder by mass, 0.1% thimerosal by mass, 0.2% proclin 950 by mass, 5% polylysine by mass, 0.01% β-mercaptoethanol by mass, 5% PEG4000 by mass, 0.5% Tween20 by mass, and the pH value is 7.4.

[0013] In the present invention, the inventors further studied and found that some components in the diluent commonly used to dilute antibodies or secondary antibodies in the prior art would greatly reduce the performance of the product. However, in the present invention, by selecting an antibody / enzyme conjugate diluent with specific components and contents, it can maintain its protective effect on the antibody and enzyme conjugate, allowing the antibody / enzyme conjugate to maintain its activity for a long time, and will not affect the sensitivity of the product.

[0014] In some embodiments, the animal and vegetable oils include crude oil and / or recycled kitchen oil; and the capsaicin includes at least one of natural capsaicin, dihydrocapsaicin and synthetic capsaicin.

[0015] The enzyme-linked immunosorbent assay kit provided by the present invention can quantitatively detect capsaicin in animal and vegetable oils, wherein the animal and vegetable oils can include crude oil and / or kitchen recycled oil, and can also include other common animal and vegetable oils in the prior art. Capsaicin can include only natural capsaicin, or only dihydrocapsaicin, or only synthetic capsaicin, or both natural capsaicin and dihydrocapsaicin, or both natural capsaicin and synthetic capsaicin, or both dihydrocapsaicin and synthetic capsaicin, or both natural capsaicin, dihydrocapsaicin and synthetic capsaicin. The enzyme-linked immunosorbent assay kit of the present invention can detect the total content of capsaicinoid substances.

[0016] In some embodiments, the ELISA kit further comprises at least one of a capsaicin standard, an enzyme-labeled well plate coated with a capsaicin antigen, a capsaicin antibody, an enzyme conjugate, a washing solution, a color developing solution, and a stop solution.

[0017] In some embodiments, the enzyme conjugate includes horseradish peroxidase-coupled goat anti-mouse IgG; the washing solution includes: 0.005-0.05M PBS buffer and Tween20 with a mass concentration of 0.01-0.2%.

[0018] In the washing solution provided by the present invention, the concentration of PBS buffer can be, for example, 0.005M, 0.02M, 0.05M or other values ​​within this range; the mass concentration of Tween20 can be, for example, 0.01%, 0.05%, 0.2% or other values ​​within this range.

[0019] In a second aspect, the present invention provides a method for detecting capsaicin in animal and vegetable oils using any of the above-mentioned enzyme-linked immunosorbent assay kits, comprising the following steps: using a first organic solvent to extract a sample to be tested, extracting and drying the supernatant with a second organic solvent, and then re-dissolving it with a sample diluent to obtain a solution to be tested; diluting a capsaicin standard with a standard diluent to different concentrations, and then adding the standard solutions and the solution to be tested of different concentrations to the microwells of an enzyme-labeled well plate coated with a capsaicin antigen, respectively, adding an enzyme conjugate working solution and a capsaicin antibody working solution to the microwells, mixing them, and standing at room temperature for reaction; pouring out the unreacted liquid in the microwells, washing with a washing solution, adding a color developing solution for color development, and finally adding a stop solution, and measuring the absorbance of each well at 450nm; drawing a standard curve according to the concentration and absorbance value of the capsaicin standard solution, thereby determining the concentration of capsaicinoids in the solution to be tested.

[0020] In the detection method provided by the present invention, antigens are pre-coated in the microwells of the ELISA plate, and the test substance in the standard product / sample competes with the antigen in the microwell to bind to the capsaicin antibody. After the enzyme conjugate reaction, unnecessary substances are removed by washing, and then a substrate is added for color development, and finally a stop solution is added. A log-logit standard curve of concentration and absorbance is established by a series of known concentrations of capsaicin standards and their absorbances, and the concentration of capsaicinoid substances in the sample to be tested can be obtained by substituting the absorbance of the sample to be tested. The content of capsaicinoid substances in the sample is negatively correlated with its absorbance.

[0021] In some embodiments, during the preparation of the test solution, the first organic solvent includes 50-90% (V / V) methanol aqueous solution, and the second organic solvent includes n-hexane.

[0022] The volume percentage of the methanol aqueous solution provided by the present invention can be, for example, 50%, 70%, 90% or other values ​​within this range.

[0023] In some embodiments, during the determination of the absorbance of each well, the enzyme conjugate working solution and the capsaicin antibody working solution are both diluted from the antibody / enzyme conjugate diluent; the room temperature standing reaction step specifically includes: the reaction time is 10 to 30 minutes; the color development step specifically includes: the color development time is 5 to 15 minutes.

[0024] In the room temperature standing reaction step provided by the present invention, the reaction time can be, for example, 10 min, 20 min, 30 min or other values ​​within the range; in the color development step provided by the present invention, the color development time can be, for example, 5 min, 10 min, 15 min or other values ​​within the range.

[0025] In some preferred embodiments, the enzyme conjugate working solution is obtained by diluting horseradish peroxidase-coupled goat anti-mouse IgG with antibody / enzyme conjugate diluent at a ratio of 1:500; the capsaicin antibody working solution is obtained by diluting capsaicin antibody with antibody / enzyme conjugate diluent at a ratio of 1:10000.

[0026] It should be noted that the dilution multiples of the enzyme conjugate working solution and the capsaicin antibody working solution can be routinely adjusted according to actual use needs.

[0027] The beneficial effects of the present invention are as follows: different from the prior art, the enzyme-linked immunosorbent assay kit provided by the present invention has wide applicability, can stably, rapidly and efficiently perform quantitative detection of capsaicinoids in a variety of animal and vegetable oils, and has the advantages of high sensitivity and low detection limit. The sensitivity of the kit is 0.1 μg / kg, the detection limit is 0.3 μg / kg, and the quantitative range is 0.4-10 μg / kg. Therefore, the kit has good application prospects in the rapid detection of crude vegetable oil and recycled kitchen oil. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a standard curve diagram after dilution with antibody / enzyme conjugate diluent in the present invention;

[0029] Figure 2 This is a standard curve diagram after dilution with antibody / enzyme conjugate diluent 1 in the present invention;

[0030] Figure 3 This is a standard curve diagram after dilution with antibody / enzyme conjugate diluent 2 in the present invention;

[0031] Figure 4 The results are the consistency between the results of using the kit of the present invention to detect capsaicin in actual samples and the results of LC-MS / MS detection. DETAILED DESCRIPTION

[0032] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0033] The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions and conditions described in the manual, or according to conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all available from commercial channels unless otherwise specified.

[0034] The invention provides an enzyme-linked immunosorbent assay kit and a method for detecting capsaicin in animal and vegetable oils.

[0035] Specifically, the enzyme-linked immunosorbent assay kit includes a standard diluent, a sample diluent, an antibody / enzyme conjugate diluent, a capsaicin standard, an enzyme-labeled well plate coated with a capsaicin antigen, a capsaicin antibody, an enzyme conjugate, a washing solution, a color developing solution and a stop solution.

[0036] The standard diluent includes: a NaCl aqueous solution with a mass concentration of 0.1-5%, methanol with a volume percentage of 1-10%, 0.01-0.5M BB buffer, and a pH value of 7.5-9.0; the sample diluent includes: a NaCl aqueous solution with a mass concentration of 0.1-10%, methanol with a volume percentage of 1-10%, 0.01-0.5M BB buffer, and a pH value of 7.5-9.0; the antibody / enzyme conjugate diluent includes: 0.01-0.05M PB buffer, trehalose with a mass concentration of 0.1-10%, β-cyclodextrin with a mass concentration of 0.01-1%, sorbitol with a mass concentration of 1-10%, BSA with a mass concentration of 0.01-1%, skimmed milk powder with a mass concentration of 0.01-1%, thimerosal with a mass concentration of 0.005-0.1%, and proclin with a mass concentration of 0.05-0.5%. 950, polylysine with a mass concentration of 0.1-5%, β-mercaptoethanol with a mass concentration of 0.001-0.2%, PEG4000 with a mass concentration of 0.5-5%, Tween20 with a mass concentration of 0.01-2%, and a pH value of 7.4.

[0037] The method for detecting capsaicin in animal and vegetable oils using the enzyme-linked immunosorbent assay kit comprises the following steps:

[0038] S1. Add 1 mL of 70% methanol aqueous solution to 0.5 g of vegetable oil and extract for 2 min, then centrifuge for 5 min to obtain the supernatant. Take 0.5 mL of the supernatant and add it to 2 mL of deionized water to mix well. Then add 2 mL of n-hexane and extract for 1 min. After standing, take 1 mL of the upper n-hexane layer and blow dry it with nitrogen in a water bath. Add 0.1 mL of sample diluent and vortex for 1 min to obtain the test solution.

[0039] S2, diluting the capsaicin standard with a standard diluent to different concentrations, then adding the standard solutions of different concentrations and the test solution to the microwells of the enzyme-labeled well plate coated with capsaicin antigen, adding the enzyme conjugate working solution and the capsaicin antibody working solution to the microwells, mixing, and standing at room temperature for 20 minutes; pouring out the unreacted liquid in the microwells, washing with a washing solution, adding a color developing solution for 10 minutes, and finally adding a stop solution, and measuring the absorbance of each well at 450nm;

[0040] S3. Draw a standard curve according to the concentration and absorbance value of the capsaicin standard solution to determine the concentration of capsaicin in the test solution.

[0041] Example 1 Optimization of the formula of standard dilution solution

[0042] Screening of buffer components: 0.02M citric acid buffer (pH 4.0), 0.02M citric acid buffer (pH 5.0), 0.02M citric acid buffer (pH 6.0), 0.02M PB buffer (pH 6.0), 0.02M PB buffer (pH 7.0), 0.02M PB buffer (pH 8.0), 0.02M BB buffer (pH 7.5), 0.02MBB buffer (pH 8.0), 0.02M BB buffer (pH 9.0), a total of 9 aqueous solutions and double distilled water for the control group were prepared. The above 10 reagents were used as standard diluents to dilute the capsaicin standard (100ppm, dissolved in methanol) to 0μg / L and 0.5μg / L, and then ELISA detection was performed. The results are shown in Table 1.

[0043] Table 1 Results of buffer component screening experiment

[0044]

[0045] Among them, 1) the relative change of the absorbance of the 0 standard was calculated by subtracting the absorbance of the control double distilled water from the absorbance of each standard dilution and then dividing it by the absorbance of double distilled water; 2) the inhibition rate of 0.5 μg / L capsaicin was calculated by (1-0.5 μg / L absorbance / corresponding standard dilution absorbance) × 100%.

[0046] From the data analysis in Table 1, it can be seen that when 0.02M citrate buffer (pH 4.0-6.0) is used as the diluent of the capsaicin standard, the absorbance of the 0 standard is reduced by more than 40% compared with the control group; when the pH value of the 0.02M PB buffer is 6.0, the absorbance of the 0 standard is reduced by about 40%, and when the pH is 7.0-8.0, the absorbance of the 0 standard is reduced by only about 10%; and when 0.02M BB buffer is used as the diluent of the standard, when the pH is increased from 7.5 to 8.0, the absorbance of the 0 standard increases by about 25%, and when the pH of the buffer is further increased to 9.0, the absorbance of the 0 standard decreases by about 5%. The increase in the absorbance of the 0 standard indicates an increase in potency, which means a decrease in the cost of raw materials; further, compared with the control group, the acidic reaction system can increase the inhibition rate of the capsaicin standard. When the pH of the standard diluent is 7.5-9.0, the inhibition rate is consistent with the control group, and the increase in the inhibition rate means an increase in sensitivity.

[0047] Based on the above analysis, BB buffer was selected as the buffer component of the standard dilution solution.

[0048] In the screening experiment of the co-solvent component, methanol and ethanol aqueous solutions with volume percentages of 5%, 10%, and 20% were prepared as standard diluents, and double distilled water was used as a control. The capsaicin standard (100 ppm, dissolved in methanol) was diluted to 0 μg / L and 0.5 μg / L, and then ELISA detection was performed. The results are shown in Table 2 below.

[0049] Table 2 Screening results of co-solvent components

[0050]

[0051] Among them, 1) the relative change of the absorbance of the 0 standard was calculated by subtracting the absorbance of the control double distilled water from the absorbance of each standard dilution and then dividing it by the absorbance of double distilled water; 2) the inhibition rate of 0.5 μg / L capsaicin was calculated by (1-0.5 μg / L absorbance / corresponding standard dilution absorbance) × 100%.

[0052] From the data analysis in Table 2, it can be seen that compared with the double distilled water control, when the methanol content in the standard diluent is between 0-20%, the absorbance change of the 0 standard is less than 10%, so the change in the raw material potency caused can be ignored, and thus the raw material cost will not increase. However, when the ethanol content in the standard diluent exceeds 10%, the absorbance change of the 0 standard is greater than 10%, which will lead to a decrease in the raw material potency and an increase in the cost of the raw materials. Furthermore, when the methanol or ethanol content in the standard diluent increases, the inhibition rate of 0.5μg / L capsaicin gradually decreases, and thus the sensitivity decreases. When the methanol or ethanol content in the standard diluent is ≤10%, the inhibition rate change caused is less than 10%, and the slight decrease in sensitivity can be compensated by optimization.

[0053] Based on the above analysis, methanol was selected as the co-solvent component of the standard dilution solution.

[0054] 3) Experiment on optimal standard dilution

[0055] Based on the principle of cheap and easy availability of raw materials, the molar concentration of BB buffer (0.01-0.5M) and pH (7.0-9.0), methanol volume percentage (1-10%), and mass concentration of NaCl (0.1-5%) were used as four factors. A 4-factor 3-level orthogonal experimental design was performed based on the concentration range obtained by the single-factor experiment. The following 9 standard dilution formulas were obtained and prepared into corresponding standard dilution solutions. Then, these standard dilutions were used to prepare the capsaicin standard into standard solutions of 0μg / L, 0.1μg / L, 0.3μg / L, 0.9μg / L, 2.7μg / L, and 8.1μg / L; these 9 standard dilutions were numbered 1-9, as follows:

[0056] 1: 0.01M BB pH 7.0, containing 0.1% NaCl (m / v), 1% methanol (v / v) in water;

[0057] 2: 0.01M BB pH 8.0, containing 1% NaCl (m / v), 5% methanol (v / v) in water;

[0058] 3: 0.01M BB pH 8.5, containing 5% NaCl (m / v), 10% methanol (v / v) in water;

[0059] 4: 0.1 M BB pH 8.5, containing 1% NaCl (m / v), 5% methanol (v / v) in water;

[0060] 5: 0.1 M BB pH 7.0, containing 5% NaCl (m / v), 10% methanol (v / v) in water;

[0061] 6: 0.1M BB pH 8.0, containing 0.1% NaCl (m / v), 1% methanol (v / v) in water;

[0062] 7: 0.5 M BB pH 8.0, containing 5% NaCl (m / v), 10% methanol (v / v) in water;

[0063] 8: 0.5 M BB pH 8.5, containing 0.1% NaCl (m / v), 1% methanol (v / v) in water;

[0064] 9: 0.5M BB pH 7.0, containing 1% NaCl (m / v), 5% methanol (v / v) in water;

[0065] Horseradish peroxidase-coupled goat anti-mouse IgG was diluted with antibody / enzyme conjugate diluent at a ratio of 1:500 to obtain enzyme conjugate working solution;

[0066] Dilute the capsaicin antibody and the antibody / enzyme conjugate diluent at a ratio of 1:10000 to obtain an antibody working solution;

[0067] The standard solutions of different concentrations diluted by the above 9 kinds of standard diluents were added to the microwells of the enzyme-labeled plate coated with capsaicin antigen, and the enzyme conjugate working solution and capsaicin antibody working solution were added to the microwells, mixed well, and allowed to react at room temperature for 20 minutes; the liquid in the microwells was poured out, washed with washing solution, and then the color developing solution was added to develop the color for 10 minutes, and finally the stop solution was added, and the absorbance of each well was measured at 450nm;

[0068] The inhibition rate of different concentrations of capsaicin can be calculated by the absorbance value (OD value). The results are shown in Table 3 below.

[0069] Table 3 Optimization results of the standard dilution formula in the present invention

[0070]

[0071] From the data in Table 3, it can be seen that considering the sensitivity of the product and the utilization rate of raw materials, 1% NaCl, 0.1M BB buffer with a pH value of 8.5 and a volume percentage of 5% methanol (v / v) aqueous solution were selected as the standard diluent.

[0072] Example 2 Optimization of sample diluent formula

[0073] Weigh 100 g of negative edible oil sample, add capsaicin standard to it so that the final capsaicin content in the sample is 5 μg / kg, mix thoroughly, and obtain a positive sample;

[0074] Taking into account the interference and solubility of the sample matrix, the concentrations of NaCl and methanol were adjusted on the basis of the standard dilution solution to obtain the following 9 sample dilution solutions;

[0075] Sample dilution 1: 0.1M BB pH 8.5, containing 0.1% NaCl (m / v), 3% methanol (v / v) in water;

[0076] Sample dilution 2: 0.1M BB pH 8.5, containing 0.1% NaCl (m / v), 4% methanol (v / v) in water;

[0077] Sample dilution 3: 0.1M BB pH 8.5, containing 0.1% NaCl (m / v), 5% methanol (v / v) in water;

[0078] Sample dilution 4: 0.1 M BB pH 8.5, containing 1% NaCl (m / v), 3% methanol (v / v) in water;

[0079] Sample dilution 5: 0.1 M BB pH 8.5, containing 1% NaCl (m / v), 4% methanol (v / v) in water;

[0080] Sample dilution 6: 0.1M BB pH 8.5, containing 1% NaCl (m / v), 5% methanol (v / v) in water;

[0081] Sample dilution 7: 0.1 M BB pH 8.5, containing 2% NaCl (m / v), 3% methanol (v / v) in water;

[0082] Sample dilution 8: 0.1 M BB pH 8.5, containing 2% NaCl (m / v), 4% methanol (v / v) in water;

[0083] Sample dilution 9: 0.1 M BB pH 8.5, containing 2% NaCl (m / v), 5% methanol (v / v) in water;

[0084] Weigh 9 groups of samples, each group contains one tube of negative sample and one tube of positive sample, 0.5g per tube, add 1mL 70% methanol aqueous solution, extract for 2min, then centrifuge for 5min to obtain the supernatant, take 0.5mL of the supernatant and add it to 2mL deionized water to mix, then add 2mL of n-hexane and extract for 1min, after standing, take 1mL of the upper n-hexane layer and blow dry it with nitrogen in a water bath, add 0.1mL of sample diluent, vortex and oscillate for 1min to obtain the test solution;

[0085] Prepare a solution containing 1% NaCl aqueous solution, 5% methanol by volume, 0.1M BB buffer, and pH 8.5 as a standard diluent, and dilute the capsaicin standard into standard solutions of 0 μg / L, 0.1 μg / L, 0.3 μg / L, 0.9 μg / L, 2.7 μg / L, and 8.1 μg / L;

[0086] Horseradish peroxidase-coupled goat anti-mouse IgG was diluted with antibody / enzyme conjugate diluent at a ratio of 1:500 to obtain enzyme conjugate working solution;

[0087] Dilute the capsaicin antibody and the antibody / enzyme conjugate diluent at a ratio of 1:10000 to obtain an antibody working solution;

[0088] The standard solution / test solution is added to the microwells of the enzyme-labeled plate coated with capsaicin antigen, and the enzyme conjugate working solution and the capsaicin antibody working solution are added to the microwells, mixed, and allowed to react at room temperature for 20 minutes; the liquid in the microwells is poured out, washed with a washing solution, and a color developing solution is added for 10 minutes, and finally a stop solution is added, and the absorbance of each well is measured at 450nm;

[0089] A standard curve was drawn based on the concentration and absorbance of the capsaicin standard solution to calculate the concentration of capsaicin in the test solution. The results are shown in Table 4 below.

[0090] Table 4 Optimization results of sample dilution formula in the present invention

[0091]

[0092] From the data in Table 4, it can be seen that considering the sample background value and sample recovery rate, 0.1M BB pH8.5, containing 2% NaCl (m / v), 4% methanol (v / v) aqueous solution was selected as the sample diluent.

[0093] Example 3 Optimization of Antibody / Enzyme Conjugate Diluent Formula

[0094] An antibody / enzyme conjugate diluent was prepared so that the concentration of PB buffer was 0.01 M, the mass concentration of trehalose was 10%, the mass concentration of β-cyclodextrin was 0.1%, the mass concentration of sorbitol was 10%, the mass concentration of BSA was 0.1%, the mass concentration of skimmed milk powder was 0.1%, the mass concentration of thimerosal was 0.1%, the mass concentration of proclin 950 was 0.2%, the mass concentration of polylysine was 5%, the mass concentration of β-mercaptoethanol was 0.01%, the mass concentration of PEG4000 was 5%, the mass concentration of Tween20 was 0.5%, and the pH value was adjusted to 7.4.

[0095] Dilute horseradish peroxidase-coupled goat anti-mouse IgG and antibody / enzyme conjugate diluent at a ratio of 1:500 to obtain an enzyme conjugate working solution; divide the solution into two portions, one portion is placed in a refrigerator at 4°C, and the other portion is placed in a 37°C incubator;

[0096] Dilute the capsaicin antibody and antibody / enzyme conjugate diluent at a ratio of 1:10000 to obtain the antibody working solution, and divide it into two portions, one portion is placed in a refrigerator at 4°C, and the other portion is placed in a 37°C incubator;

[0097] For comparison, two other commonly used antibody / enzyme conjugate diluents (hereinafter referred to as antibody / enzyme conjugate diluent 1 and antibody / enzyme conjugate diluent 2) can be selected to dilute the capsaicin antibody and enzyme conjugate in appropriate proportions;

[0098] Prepare a solution containing 1% NaCl aqueous solution, 5% methanol by volume, 0.1M BB buffer, and pH 8.5 as a standard diluent, and dilute the capsaicin standard into standard solutions of 0 μg / , 0.1 μg / L, 0.3 μg / L, 0.9 μg / L, 2.7 μg / L, and 8.1 μg / L;

[0099] The standard solution was added to the microwells of the enzyme-labeled plate coated with capsaicin antigen, and the enzyme conjugate working solution and capsaicin antibody working solution (treated with the antibody / enzyme conjugate diluent, antibody / enzyme conjugate diluent 1 and antibody / enzyme conjugate diluent 2 of the present invention, respectively) were added to the microwells, mixed, and allowed to react at room temperature for 20 minutes; the liquid in the microwells was poured out, washed with a washing solution, and a color developing solution was added for 10 minutes, and finally a stop solution was added, and the absorbance of each well was measured at 450nm, and a standard curve was drawn according to the concentration and absorbance value of the capsaicin standard solution. The results are as follows: Figure 1-3 shown.

[0100] from Figure 1-3 It can be seen that after dilution with the antibody / enzyme conjugate diluent of the present invention and the antibody / enzyme conjugate diluent 1, the standard curve obtained is better.

[0101] Further, comparative experiments of refrigeration and acceleration of antibody working solution and enzyme conjugate working solution were performed on the antibody / enzyme conjugate diluent of the present invention and antibody / enzyme conjugate diluent 1 on the 1st day, the 2nd day, the 3rd day, the 7th day and the 15th day; the results are shown in Tables 5 and 6.

[0102] Table 5 Effect of antibody / enzyme conjugate diluent in the present invention

[0103]

[0104] Table 6 Effect of antibody / enzyme conjugate dilution 1

[0105]

[0106] It can be seen from the data in Tables 5 and 6 that, taking all factors into consideration, the best effect is achieved by using the antibody / enzyme conjugate diluent of the present invention for dilution.

[0107] Application test case

[0108] The capsaicin antigen is coated into the wells of the ELISA plate in a suitable ratio, and the desired ELISA plate is obtained after treatment;

[0109] Prepare a solution containing 1% NaCl aqueous solution, 5% methanol by volume, 0.1M BB buffer, and pH 8.5 as a standard diluent, and dilute the capsaicin standard into standard solutions of 0 μg / L, 0.1 μg / L, 0.3 μg / L, 0.9 μg / L, 2.7 μg / L, and 8.1 μg / L;

[0110] A solution containing 2% NaCl aqueous solution, 4% methanol by volume, 0.1M BB buffer, and pH 8.5 was prepared as a sample diluent;

[0111] An antibody / enzyme conjugate diluent was prepared in which the concentration of PB buffer was 0.01 M, the mass concentration of trehalose was 10%, the mass concentration of β-cyclodextrin was 0.1%, the mass concentration of sorbitol was 10%, the mass concentration of BSA was 0.1%, the mass concentration of skimmed milk powder was 0.1%, the mass concentration of thimerosal was 0.1%, the mass concentration of proclin 950 was 0.2%, the mass concentration of polylysine was 5%, the mass concentration of β-mercaptoethanol was 0.01%, the mass concentration of PEG4000 was 5%, the mass concentration of Tween20 was 0.5%, and the pH value was adjusted to 7.4.

[0112] Horseradish peroxidase-coupled goat anti-mouse IgG was diluted with antibody / enzyme conjugate diluent at a ratio of 1:500 to obtain enzyme conjugate working solution;

[0113] Dilute the capsaicin antibody and the antibody / enzyme conjugate diluent at a ratio of 1:10000 to obtain an antibody working solution;

[0114] Weigh 0.5 g of sample in a centrifuge tube, add 1 mL of 70% methanol aqueous solution, extract for 2 min, then centrifuge for 5 min to obtain the supernatant, take 0.5 mL of the supernatant and add it to 2 mL of deionized water to mix well, then add 2 mL of n-hexane and extract for 1 min. After standing, take 1 mL of the upper n-hexane layer and blow dry it with nitrogen in a water bath, add 0.1 mL of sample diluent, and vortex for 1 min to obtain the test solution;

[0115] Take out the appropriate amount of balanced strips, put the rest back into the aluminum film bag, add 50 μL of standard / sample to the corresponding microwells, add 50 μL of enzyme conjugate and 50 μL of antibody to all microwells, cover the plate sticker, incubate at 20-28°C for 20 minutes, pour out the liquid in the microwells, fill the microwells with washing solution, soak for 15 seconds and pour out, repeat 4 times, a total of 5 times, add 100 μL of substrate to the microwells, incubate at 20-28°C for 10 minutes, add 100 μL of stop solution to the microwells, and read at 450 / 630 nm wavelength;

[0116] A standard curve was drawn based on the concentration and absorbance of the capsaicin standard solution to calculate the concentration of capsaicin in the solution to be tested. The results are shown in Table 7 below.

[0117] Table 7 Actual sample test results

[0118]

[0119] As can be seen from Table 7, the kit of the present invention can accurately quantify the capsaicin content in 20 samples (capsaicin content of 0-50 μg / kg, including 10 parts of refined oil and 10 parts of other oils), and the detection results of the kit of the present invention are highly consistent with the LC-MS / MS determination results (see Figure 4 ), the results show that the kit of the present invention can stably, quickly and efficiently perform quantitative detection of capsaicinoids in animal and vegetable oils, and has the advantages of high sensitivity and low detection limit.

[0120] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0121] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An enzyme-linked immunosorbent assay kit for detecting capsaicin in animal and vegetable oils, characterized in that: It consists of standard diluent, sample diluent and antibody / enzyme conjugate diluent; The standard diluent comprises: a NaCl aqueous solution with a mass concentration of 1-5%, a methanol with a volume percentage of 5-10%, a 0.01-0.5M BB buffer solution, and a pH value of 7.5-8.5; The sample diluent includes: a NaCl aqueous solution with a mass concentration of 1-2%, a methanol with a volume percentage of 3-5%, a 0.01-0.5M BB buffer solution, and a pH value of 7.5-8.5; The antibody / enzyme conjugate diluent comprises: 0.01M PB buffer, 10% trehalose, 0.1% β-cyclodextrin, 10% sorbitol, 0.1% BSA, 0.1% skim milk powder, 0.1% thimerosal, 0.2% proclin 950, 5% polylysine, 0.01% β-mercaptoethanol, 5% PEG4000, 0.5% Tween20, and pH value is 7.4; Horseradish peroxidase-coupled goat anti-mouse IgG was diluted with antibody / enzyme conjugate diluent at a ratio of 1:500 to obtain enzyme conjugate working solution; The capsaicin antibody was diluted with the antibody / enzyme conjugate diluent at a ratio of 1:10000 to obtain the antibody working solution.

2. The enzyme-linked immunosorbent assay kit according to claim 1, characterized in that The standard diluent includes: a NaCl aqueous solution with a mass concentration of 1%, methanol with a volume percentage of 5%, and a 0.1M BB buffer solution with a pH value of 8.

5.

3. The enzyme-linked immunosorbent assay kit according to claim 1, characterized in that The sample diluent includes: a 2% mass concentration NaCl aqueous solution, a 4% volume percentage methanol, a 0.1M BB buffer solution, and a pH value of 8.

5.

4. The enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: The animal and vegetable oils include crude oil and / or recycled cooking oil; The capsaicin includes at least one of natural capsaicin, dihydrocapsaicin and synthetic capsaicin.

5. The enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: The enzyme-linked immunosorbent assay kit also includes at least one of a capsaicin standard, an enzyme-labeled well plate coated with a capsaicin antigen, a capsaicin antibody, an enzyme conjugate, a washing solution, a color developing solution and a stop solution.

6. The enzyme-linked immunosorbent assay kit according to claim 5, characterized in that: The enzyme conjugate includes horseradish peroxidase-coupled goat anti-mouse IgG; The washing solution comprises: 0.005-0.05M PBS buffer and Tween20 with a mass concentration of 0.01-0.2%.

7. A method for detecting capsaicin in animal and vegetable oils using the enzyme-linked immunosorbent assay kit according to any one of claims 1 to 6, characterized in that: The steps include: The sample to be tested is extracted with a first organic solvent, the supernatant is extracted with a second organic solvent, blown dry, and then redissolved with a sample diluent to obtain a solution to be tested; Dilute the capsaicin standard with a standard diluent to different concentrations, then add the standard solutions of different concentrations and the test solution to the microwells of the enzyme-labeled well plate coated with capsaicin antigen, add the enzyme conjugate working solution and the capsaicin antibody working solution to the microwells, mix well, and stand at room temperature for reaction; pour out the unreacted liquid in the microwells, wash with a washing solution, add a color developing solution for color development, finally add a stop solution, and measure the absorbance of each well at 450nm; A standard curve is drawn according to the concentration and absorbance value of the capsaicin standard solution to determine the concentration of capsaicinoids in the test solution.

8. The method according to claim 7, characterized in that In the preparation process of the test solution, the first organic solvent includes 50-90% methanol aqueous solution, and the second organic solvent includes n-hexane.

9. The method according to claim 7, characterized in that: In the process of measuring the absorbance of each well, the enzyme conjugate working solution and the capsaicin antibody working solution are both diluted by antibody / enzyme conjugate diluent; the specific steps of the room temperature static reaction include: the reaction time is 10 to 30 minutes; the specific color development step includes: the color development time is 5 to 15 minutes.

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