CN106084059B - Anti-capsaicinoids general-specific antibodies, test strips, and rapid immunochromatographic identification method for kitchen waste oils and fats - Google Patents

Abstract

The invention relates to a general specific antibody against capsaicinoids, a test strip and a rapid identification method for the immunochromatographic identification of kitchen waste oils and fats. The general-specific anti-capsaicinoid antibody is characterized in that it is an anti-capsaicinoid general-specificity antibody obtained by immunizing animals with the general-purpose artificial immune antigen compound of capsaicinoids described in formula I, and is separated and purified by freeze-drying. Antibody. The kitchen waste oil and fat immunochromatographic immune test strip prepared by it can be used in the kitchen waste oil and can be used to quickly detect the total amount of capsaicinoids in the kitchen waste oil; the detection sensitivity is high, and the pH adaptability range is wide; one-step method Easy to operate. A capsaicinoid standard solution is not required as a positive control.

Description

Anti-capsaicinoids general-specific antibodies, test strips and kitchen waste oil free Immunochromatographic rapid identification method

technical field

The invention relates to a general-specific specific antibody against capsaicinoids, a test strip and a rapid immunochromatographic identification method for kitchen waste oil, belonging to the technical field of immunochemistry.

Background technique

Waste oil is a general concept, which is a general term for all kinds of low-quality oil, generally including swill oil, frying waste oil, food and waste oil produced by related enterprises, and swill oil from the catering industry is the main raw material source of waste oil one. In Chinese food culture, pepper containing capsaicin is one of the essential condiments. Capsaicinoids are the main chemical substances present in capsicum fruits that make them pungent and stimulating. There are more than 20 kinds of compounds whose structures have been proven so far. Capsaicinoids have high nutritional value and are widely used in the field of food processing. Synthetic capsaicin also has a structure similar to natural capsaicinoids, and also has spicy characteristics. It is a member of capsaicinoids and is mainly used in the industrial field. However, because of its low preparation cost, some criminals use it to replace natural peppers. element, used in the food field. Capsaicin, dihydrocapsaicin and synthetic capsaicin are the main members of capsaicinoids. Capsaicinoids have high nutritional value and are widely used in the field of food processing. Capsaicinoids have the characteristics of strong fat solubility, good stability, and high boiling point. The current waste oil processing technology is difficult to completely remove substances with such characteristics. A number of studies have shown that normal cooking oil basically does not contain capsaicin, while kitchen waste oils that have been exposed to chili inevitably contain such ingredients. Therefore, capsaicinoids can be used as characteristic indicators to identify kitchen waste oils. By detecting the content of capsaicinoids, it is possible to determine whether edible vegetable oils are mixed with kitchen waste oils. At present, the methods for detecting capsaicinoids in edible vegetable oil mainly include modern instrument detection methods such as high performance liquid chromatography and chromatography-mass spectrometry, which have high sensitivity and good accuracy, but the equipment is expensive and requires high experimental environment. And require professional operators, it is difficult to achieve rapid detection. The immunoassay method developed rapidly in recent years has been widely used in medicine, various fields of agriculture.

The immunochromatographic technique based on the specific binding reaction between colloidal gold-labeled antibody and antigen is visible to the naked eye, does not require large-scale equipment, has low detection cost, and short analysis time. It has been widely used in qualitative, online and rapid detection. However, at present, this method has not been applied to the detection of capsaicinoids in kitchen waste oils and fats at home and abroad. The core component of colloidal gold immunochromatography technology is specific antibody, but the matrix composition of kitchen waste oil is complex, and the pH of the sample extract is difficult to determine, which usually reduces the activity of specific antibody and the detection sensitivity. Therefore, there is an urgent need to develop an anti-capsaicinoid-specific antibody with high affinity, good specificity, resistance to organic solvents, and a wide range of pH adaptation, and use this antibody to establish a colloidal gold immunochromatographic test strip detection method for identification. Kitchen waste grease.

Contents of the invention

The problem to be solved by the present invention is to provide an anti-capsaicin-like substance-specific antibody, a colloidal gold immunochromatographic test strip of kitchen waste oil, and a rapid identification method for kitchen waste oil immunochromatography. The anti-capsaicinoid-specific antibody provided by the invention has high affinity and good specificity, is resistant to organic solvents, and has a wide range of pH adaptation. The immunochromatographic test strip made with it is used for the detection of capsaicin-like substances in kitchen waste oils and fats, and has the characteristics of simple operation, low cost, high sensitivity and wide adaptable pH range.

In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is:

Provide capsaicin-like substance universal artificial immune antigen compound, its structural formula is as described in formula I:

Provide the preparation method of the general artificial immune antigen compound of capsaicinoid substances, including the following specific steps: neutralize the hydrochloric acid in vanillin amine hydrochloride with sodium hydroxide to obtain the compound of formula III, and then react with maleic anhydride, To obtain artificial hapten compounds, artificial haptens of capsaicinoids are activated by N,N'-dicyclohexylcarbodiimide DCC and N-hydroxysuccinimide NHS, or by tri-n-butylamine and isobutyl chloroformate Ester activation, and then coupled with the carrier protein BSA.

The synthetic route of artificial hapten compound is as follows:

The preparation method of the above-mentioned capsaicinoid general artificial immune antigen compound specifically comprises the following steps:

(1) Dissolve vanillinamine hydrochloride and sodium hydroxide with a molar ratio of 1: (0.7-1.1) in water, stir at room temperature for 10-30min, and filter to obtain white precipitated vanillinamine (compound of formula IV) ;

Dissolve thoroughly dried vanillin amine and maleic anhydride in chloroform at a molar ratio of 1:0.8-1.2, react at room temperature for 1-2 hours, and filter to obtain capsaicin-like artificial haptens.

(2) React capsaicinoids with N-hydroxysuccinimide (NHS) at room temperature for 30min-2h in N,N-dimethylformamide, and then add dicyclohexylcarbodiimide N,N-dimethylformamide solution, react at room temperature for 4-6 hours, let it stand overnight, take the supernatant, which is activated capsaicin, dihydrocapsaicin and synthetic capsaicin artificial hapten solution, the N-hydroxy The molar ratio of succinimide (NHS) and dicyclohexylcarbodiimide is 1:1-1.2;

Or dissolve the general artificial hapten of capsaicin-like substances in N,N-dimethylformamide, add tri-n-butylamine and isobutyl chloroformate sequentially, react at room temperature for 0.5-3 hours, and the resulting reaction solution is activated capsaicin Substance artificial hapten solution, the molar ratio of tri-n-butylamine and isobutyl chloroformate is 1:1-1.2.

(3) adding the activated capsaicin-like substance general artificial hapten supernatant obtained in the above (1) dropwise to the phosphate buffer solution of the carrier protein with a carrier protein concentration greater than or equal to 2mg/mL, and reacting at room temperature for 4-8h, Wherein: the molar ratio of the general artificial hapten of capsaicin-like substances to the carrier protein in the step (1) is greater than 10:1, and then dialyzed to obtain the capsaicin artificial antigen.

According to the above scheme, the phosphate buffer of the carrier protein is obtained by dissolving the carrier protein with 0.2M phosphate buffer of pH 8.0.

According to the above-mentioned scheme, the dialysis is dialysis with 0.01M PBS buffer solution for 72 hours, during which, the dialysis solution is changed every 4-8 hours.

Anti-capsaicinoid general-specific antibody, which is an anti-capsaicinoid general-specific antibody obtained by immunizing animals with the general artificial immune antigen compound of capsaicinoid described in formula I, separating and purifying, and freeze-drying;

According to the above scheme, the immunization is repeated immunization of animals at intervals after emulsification with Freund's complete adjuvant or Freund's incomplete adjuvant, wherein: Freund's complete adjuvant is used for the initial immunization, and Freund's incomplete adjuvant is used for subsequent immunization, When the antiserum titer reaches above 10,000, blood is collected from the carotid artery until death. After the collected blood is centrifuged, the antiserum is purified to obtain antibodies to capsaicinoids. The titer of antiserum obtained by immunizing animals with the antigen provided by the present invention can reach 320,000, and the half-inhibitory concentration IC ₅₀ of the obtained antibody to capsaicin, dihydrocapsaicin and synthetic capsaicin is under the condition of pH 5.0-8.0, It is 0.27-0.41μg/mL. It shows that the antibody can specifically react with capsaicin, dihydrocapsaicin and synthetic capsaicin, and can be used as a general antibody for capsaicin, dihydrocapsaicin and synthetic capsaicin, with good affinity, high sensitivity, good stability, including tolerance Strong capacity for organic solvent and salt ion concentration, wide range of pH application.

According to the above-mentioned scheme, the immunization dose of the capsaicin-like substance general artificial immunization antigen compound described in the formula I is 0.2-1.0 mg based on the OVA protein; the number of immune layers is 4-6 times; the first immunization adopts an equal volume of Freund's complete adjuvant emulsified immunogen, injected subcutaneously in rabbits at multiple points; the second immunization was carried out at an interval of 2-4 weeks after the first immunization, and the second and subsequent immunization intervals were 2-3 weeks. The immunogen was emulsified with complete adjuvant and injected subcutaneously at multiple points on the back of rabbits. Beginning from the fourth time, within 6-10 days after each immunization, the rabbit ear vein blood collection non-competitive indirect ELISA method was used to determine the serum titer. After centrifugation, the antiserum was purified by caprylic acid-ammonium sulfate method to obtain capsaicinoid antibody.

Kitchen waste oil and fat immunochromatographic test strips (see Figure 3), including cardboard, one side of the cardboard is pasted with water-absorbing pads, detection pads, gold standard pads and sample pads in sequence from top to bottom, and adjacent pads overlap at the joints The detection pad is based on a nitrocellulose membrane, and a horizontal quality control line and a detection line are arranged on the nitrocellulose membrane from top to bottom, and the quality control line is coated with a rabbit anti-mouse polyclonal antibody. The above detection line is coated with capsaicin-like substance-coated antigen; the gold-labeled pad is laterally sprayed with the above-mentioned anti-capsaicin-like substance general-specific antibody labeled with nano-gold.

According to the above scheme, the absorbent pad is 10-15mm long and 3-4mm wide; the detection pad is 25-30mm long and 3-4mm wide; the gold standard pad is 6-9mm long and 3-4mm wide; the sample pad is 12-15mm long , 3-4mm wide, and the overlapping length of adjacent pads is 1-3mm.

According to the above scheme, the absorbent pad is absorbent paper.

According to the above scheme, the distance between the detection line on the detection pad and the upper edge of the nitrocellulose membrane is 8-20 mm, and the distance between the detection line and the quality control line is 5-10 mm.

The molecular structural formula of the capsaicinoid-coated antigen is shown in Formula II:

According to the above scheme, the coating amount of coated antigen required per centimeter on the detection pad detection line is 0.1-0.6 μg; the coating amount of rabbit anti-mouse polyclonal antibody required per centimeter on the quality control line is 0.1 ~0.6 μg.

According to the above scheme, the particle size of the nano gold used in the gold standard pad is 15-20 nm.

According to the above-mentioned scheme, the amount of nano-gold-labeled anti-capsaicinoid general specific antibody required per centimeter of spraying length on the gold-marked pad is 0.40-0.98 μg.

The preparation method of the colloidal gold immunochromatographic test strip for rapid detection of capsaicinoids as described above comprises the following steps:

(1) Preparation of absorbent pad

Cut the absorbent paper to get the absorbent pad;

(2) Preparation of detection pad

Coating of detection line: Prepare capsaicin-like substance-coated antigen to prepare a coating solution with a concentration of 0.17-1.0 mg/mL, and place it at a position 8-20 mm away from the edge of the nitrocellulose membrane, spray it horizontally Coated on a nitrocellulose membrane to obtain a detection line, the amount of capsaicin-like substance universal artificial complete antigen-coated antigen required for each centimeter detection line is 0.1-0.6 μg, and then dried at 37°C for 30-30 μg 60 minutes;

Coating of quality control line: make rabbit anti-mouse polyclonal antibody into a coating solution with a concentration of 0.17-1.0 mg/mL, and coat it horizontally with nitric acid at a position 5-10 mm away from the test line. On the cellulose membrane, get the quality control line, the coating amount of rabbit anti-mouse polyclonal antibody required for each centimeter quality control line is 0.1-0.6 μg, and then dry at 37°C for 30-60 minutes;

(3) Preparation of sample pad

Soak the glass fiber membrane in the blocking solution, take it out, and dry it at 37-40°C for 6-12 hours to obtain a sample pad, and then store it in a desiccator at room temperature;

(4) Preparation of gold standard pad

Soak the glass fiber membrane in the sealing solution, take it out, and dry it at 37-40°C for 6-12 hours. On the dried glass fiber membrane, spray it horizontally on the dried glass fiber membrane by point spraying. Nanogold-labeled universal polyclonal antibody solution against capsaicin-like substances, the amount of nano-gold-labeled universal polyclonal antibody against capsaicinoid-like substances required per centimeter of spraying length is 0.4-0.98ng, and then vacuum freeze-dried for 2-6 Hours, stored in a desiccator at room temperature;

(5) Assembly of test strips

Paste absorbent pads, test pads, gold standard pads, and sample pads on one side of the cardboard in sequence, and overlap and connect adjacent pads at the joints, with an overlapping length of 1-3 mm to obtain an immune layer of kitchen waste grease Analysis test paper.

According to the above scheme, capsaicinoids are coated with antigen ((2Z)-4-[(4-hydroxy-3-methoxy)benzylamino]-4-carbonyl-2-enoic acid-OVA) in the coating solution The coating buffer used is: 0.1g of ovalbumin OVA, 0.002g of sodium azide, 0.08g of sodium chloride, 0.029g of disodium hydrogen phosphate dodecahydrate, 0.002g of potassium chloride, and 0.002g of potassium chloride per 10mL. Potassium hydrogen 0.002g; The coating buffer used in the described rabbit anti-mouse polyclonal antibody coating liquid is: every 10mL contains bovine serum albumin 0.1g, sodium azide 0.002g, sodium chloride 0.08g , 0.029g disodium hydrogen phosphate dodecahydrate, 0.002g potassium chloride, 0.002g potassium dihydrogen phosphate;

According to the above scheme, each 100 mL of the blocking solution in the step (3) and step (4) contains: 1-2 g of ovalbumin, 2-5 g of sucrose, 0.02-0.05 g of sodium azide, and 0.8 g of sodium chloride , 0.29 g of disodium hydrogen phosphate dodecahydrate, 0.02 g of potassium chloride, and 0.02 g of potassium dihydrogen phosphate.

According to the above scheme, the universal polyclonal antibody solution against capsaicinoids labeled with nano-gold is prepared by an unsaturated labeling method. 0.5mL of 0.1mol/L potassium carbonate aqueous solution to adjust the pH value, slowly add 2.0mL of 0.1mg/mL anti-capsaicinoid universal polyclonal antibody aqueous solution under stirring, and continue stirring for 30min; the mass concentration of adding is 10% Ovalbumin (OVA) aqueous solution to OVA final mass concentration of 1%, continue to stir for 30min; after 2h at 4°C, centrifuge at 3000r/min for 15min, take the supernatant, discard the precipitate; centrifuge the supernatant at 12000r/min for 30min , discard the supernatant, add 40.0mL labeled washing and storage solution; then centrifuge at 12000r/min for 30min, discard the supernatant, resuspend the precipitate with the labeling washing and storage solution to obtain 5.0mL concentrate, put it in the refrigerator at 4℃ for later use .

According to the above scheme, the 0.1mol/L potassium carbonate aqueous solution is: 13.8g potassium carbonate dissolved in pure water to 1000mL, obtained by filtering through a 0.22 μm filter membrane; the described label washing preservation solution is: 2.0g polyethylene glycol Alcohol-20000, 0.2g sodium azide, 0.1235g boric acid, dilute pure water to 1000mL, and filter through a 0.22μm filter membrane.

The method for rapid identification of kitchen waste oil by immunochromatography, the steps are as follows: take the test sample of kitchen waste oil, extract the capsaicin-like substances therein, dry the extract, add a solvent and redissolve it, and use the test sample and reconstitution The ratio of the solvent is 20g: 1ml to obtain the sample solution to be tested, and then the sample solution to be tested (preferably 80-200 μL) is added dropwise as a detection solution to the sample pad of the kitchen waste oil and fat immunochromatography test paper for detection. For the test strip, take another methanol aqueous solution with the same volume of methanol as the negative control solution, add it dropwise to the sample pad of another kitchen waste oil immunochromatography test paper, and use it as the control test strip for a period of time (10- After 20 minutes), the detection test strip and the contrast test strip are carried out for color contrast:

Test results:

(1) Negative: When the quality control line on the test strip develops color and the color of the test line is close to the color of the test line on the control test strip, it indicates that the capsaicinoids capsaicin and dihydrocapsicum in the sample solution to be tested are If the content of capsaicin or synthetic capsaicin is less than 3ng/mL, combined with the confirmatory detection method to determine whether the sample is kitchen waste oil or a grease sample mixed with kitchen waste oil; if the content is less than 3ng/mL, it can be judged that the sample to be tested is The possibility of kitchen waste oils and fats is unlikely, and further confirmatory testing methods are needed to confirm;

(2) Positive: When the quality control line on the test strip develops color and the color of the test line is lighter than that on the control test strip, it indicates that capsaicin, dihydrocapsaicin or synthetic capsicum in the sample solution to be tested If the element content is equal to or higher than 3ng/mL, it can be determined that the sample is kitchen waste oil or a fat sample mixed with kitchen waste oil;

(3) Invalid: When the quality control line does not develop color, no matter whether the detection line of the test strip develops color, the test strip is judged as invalid;

The working principle of the immunochromatographic test strip in the detection of kitchen waste oil capsaicin, dihydrocapsaicin or synthetic capsaicin: when the sample solution to be tested is added to the sample pad at the lower end of the test strip, the sample solution to be tested passes through Capillary action moves along the test strip to the absorbent pad, and when it moves to the gold-labeled pad, the nano-gold-labeled universal polyclonal antibody against capsaicin, dihydrocapsaicin or synthetic capsaicin is dissolved. When the sample contains capsaicin, dihydrocapsaicin or synthetic capsaicin, the capsaicinoids will bind to the nano-gold-labeled anti-capsaicinoids universal polyclonal antibody on the gold-labeled pad and swim upward together, and reach the immobilized When the capsaicinoid-coated antigen detection line is applied, the antigen will compete with capsaicin, dihydrocapsaicin or synthetic capsaicin for the limited antigen-binding sites on the nano-gold-labeled anti-capsaicinoid universal polyclonal antibody, The higher the content of capsaicin, dihydrocapsaicin or synthetic capsaicin in the sample, the less nano-gold-labeled anti-capsaicin, dihydrocapsaicin or synthetic capsaicin universal polyclonal antibody will be able to bind the antigen on the detection line. The lighter the color band formed on the detection line. When the anti-capsaicinoid universal polyclonal antibody combined with the antigen on the detection line is less than a certain amount, no red line will appear on the detection line. Regardless of whether the sample contains capsaicinoids, the nano-gold-labeled anti-capsaicinoid antibody that is not intercepted by the antigen on the detection line or the nano-gold-labeled anti-capsaicinoid antibody combined with capsaicin, dihydrocapsaicin or synthetic The capsaicin conjugate will continue to move to the quality control line and combine with the rabbit anti-mouse polyclonal antibody on the quality control line and be enriched for color development. Accordingly, the detection line of the capsaicin-like substance-coated antigen on the detection test strip is compared with the color of the corresponding detection line on the control test strip, and the concentration of capsaicin, dihydrocapsaicin or synthetic capsaicin in the sample can be obtained. content, so as to judge whether the oil sample is kitchen waste oil or not.

According to the above scheme, the extraction is to add an aqueous ethanol solution with a volume concentration of 95% to the sample to be tested, mix well, reflux at 60-90° C. for 1 hour, and cool to obtain the extract; the solvent for reconstitution is 10% methanol-PBS.

Beneficial effects of the present invention:

(1) The artificial hapten provided by the present invention by modifying the amide group of vanillylamine in capsaicin has not only retained the characteristic structure of capsaicin, dihydrocapsaicin and synthetic capsaicin to the greatest extent (in the main molecular structure of capsaicin) vanillylamine and part of the aliphatic chain group), and a large conjugated structure is formed at the linking arm of the hapten, which can promote the flow of electrons in the hapten structure. In addition, it has a certain rigidity, so there may be It is beneficial to the exposure of the active site in the hapten, and then the antigenic compound obtained by coupling with OVA can be used as an antigenic determinant, and has an active group that can be coupled with the carrier protein. The obtained antigenic compound is used as an immune antigen Capsaicin antibodies with high affinity, high sensitivity, strong specificity, resistance to organic solvents and salt ion concentrations, and a wide range of pH adaptation can be obtained by immunization.

(2) The rapid immunochromatographic identification method for kitchen waste oils and fats provided by the invention can detect the total amount of capsaicinoids in kitchen waste oils and fats; the detection sensitivity is high, and the pH adaptability range is wide.

(3) The rapid identification method of kitchen waste oil and fat immunochromatography provided by the present invention is simple to operate: only need to add the detection solution after the treatment of the kitchen waste oil sample to the sample pad of the test strip drop by drop, one-step operation, easy and convenient. When testing samples, it is not necessary to use capsaicinoid standard solution as a positive control, and only need to use a blank sample as a negative control. And the detection result of this method can be judged by naked eyes.

Description of drawings

Fig. 1 is the ultraviolet spectrogram of the universal artificial complete antigen of anti-capsaicinoid substances synthesized by the present invention.

Fig. 2 is the ultraviolet spectrogram of the general artificial complete antigen-coated antigen synthesized by the present invention.

Fig. 3 is a structural schematic diagram of the immunochromatographic test strip of kitchen waste oil provided by the present invention. 1 cardboard; 2 absorbent pad; 5 test pad; 6 gold label pad; 7 sample pad; 3 quality control line; 4 test line.

Detailed ways

The test methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The preparation of embodiment 1 capsaicinoid artificial hapten

(1) Weigh 1 g of vanillin amine hydrochloride and dissolve it in 11.77 ml of ultrapure water, add 2.12 ml of 2M NaOH drop by drop under vigorous stirring, and react at room temperature for 10 min. The white precipitate was obtained by filtration, and the next reaction was carried out after the white precipitate was thoroughly dried.

(2) Weigh and weigh 60 mg of the product from the previous step, dissolve it in 30 ml of chloroform (after dehydration with anhydrous sodium sulfate), stir at room temperature for 40 min, add 40 mg of maleic anhydride, and react at room temperature for 1 h. The resulting precipitate after filtration is capsaicinoid artificial hapten (2Z)-4-[(4-hydroxy-3-methoxy)benzylamino]-4-carbonyl-2-enoic acid ((2Z)-4- [(4-hydroxy-3-methoxybenzyl)amino]-4-oxobut-2-enoic acid). The molecular formula is C ₁₂ H ₁₃ NO ₅ .

Example 2 Preparation of capsaicinoids artificial complete antigen-immune antigen

Weigh 20.8mg (about 0.08mmol) of the artificial hapten of the above-mentioned capsaicinoids and 11.6mg (about 0.1mmol) of NHS in the reaction flask, add 400ulDMF to dissolve in the reaction flask, stir at room temperature for 30min, weigh 20.6mg (about 0.1mmol) mmol) DCC was dissolved in 50 ul of DMF, and the DCC/DMF solution was added dropwise to the above-mentioned reaction flask, stirred at room temperature for 4 h, and left standing at 4° C. overnight. 8000rpm/5min, take the supernatant active ester solution.

The supernatant active ester solution was added dropwise to 6ml of 7mg/ml BSA solution for reaction, and the reaction buffer was 0.2M phosphate buffer at pH 8.0. The reaction was carried out at room temperature for 4 h under magnetic stirring. The reaction solution was placed in a dialysis bag, and dialyzed with 0.01M PBS, pH 7.4 at 4°C with stirring, and the dialysate was changed every 4 hours for a total of 72 hours. That is, capsaicin artificial antigen-immune antigen is obtained. The continuous scanning pattern of ultraviolet-visible spectrum is shown in Fig. 1, and the identification result shows that the coupling of the artificial antigen is successful.

Example 3 Preparation of capsaicinoids artificial complete antigen-coated antigen

Dissolve 4.52 mg of the above artificial hapten of capsaicinoids in 200 μL of anhydrous DMF, then add 4.27 μL of tri-n-butylamine and 2.34 μL of isobutyl chloroformate in sequence, and stir at room temperature for 1 hour in the dark to obtain activated capsicum. Capsaicin, dihydrocapsaicin and synthetic capsaicin hapten solutions.

The activated capsaicin, dihydrocapsaicin and synthetic capsaicin hapten solutions were added dropwise to 10ml of 4.5mg/ml OVA solution for reaction, and the reaction buffer was 0.2M phosphate buffer at pH 8.0. The reaction was carried out at room temperature for 4 h under magnetic stirring. The reaction solution was placed in a dialysis bag and dialyzed with 0.01M PBS with pH 7.4 at 4°C with stirring, and the dialysate was changed every 4 hours for a total of 72 hours. That is, capsaicin artificial antigen-coated antigen is obtained. The ultraviolet-visible spectrum continuous scanning spectrum is shown in Fig. 2, and the identification result shows that the artificial antigen is successfully coupled.

Embodiment 4: Preparation of capsaicinoid-specific antibody

The prepared anti-capsaicinoid artificial complete antigen (immune antigen) was used to immunize Japanese big-eared rabbits with more than 3 kg. The average dosage of each antigen is about 0.84mg (based on the amount of protein). For the first immunization, the immunogen emulsified with an equal volume of Freund's complete adjuvant was used, and the rabbit was injected subcutaneously at multiple points; after the first immunization, the immunogen was emulsified with an equal volume of Freund's incomplete adjuvant every 2 weeks. Originally, it was injected subcutaneously at multiple points on the back of rabbits. Beginning from the fourth time, one week after each immunization, blood was collected from the vein of the rabbit's ear margin to determine the serum titer by non-competitive indirect ELISA. After five immunizations, blood was collected from the carotid artery of the rabbit until death. The collected blood was placed at 37°C for about 1 hour, then overnight at 4°C, centrifuged, the supernatant was taken, and a small amount of glycerol was mixed with an equal volume and temporarily stored at -20°C for later use. The rest of the antiserum was purified by caprylic acid-ammonium sulfate method, and finally freeze-dried antibodies were obtained, which were stored at -20°C for future use.

Embodiment 5: Determination of capsaicinoid-specific antibody

1) Use the indirect non-competitive ELISA method to detect the specific antibody titer of capsaicinoids

Coating: Capsaicin-like substance artificial complete antigen-coating antigen diluted to 1 μg/mL with 0.1 mol/L pH9.6 carbonate buffer, 100 μl/well, react overnight at 4°C. Pour off the coating solution, wash the wells with PBST three times, and tap dry.

Blocking: 1% OVA PBST solution, 200 μL/well, incubate at 37°C for 1 hour, wash the wells with PBST three times, and blot dry.

Antibody antigen-specific reaction: Prepare the capsaicinoid-specific antibody with 0.01M pH7.4 phosphate buffer to make a 1mg/mL solution, then dilute the antibody solution from 1:10000, and add it to each dilution Add 100 μL/well to the coated wells with a concentration of 100 μL/well, set up negative and blank control wells, the negative control serum was diluted 2 000 times, only PBST was added to the blank, incubated at 37°C for 1 hour, washed with PBST three times in the wells, and then dried;

Add secondary antibody: Dilute goat anti-mouse enzyme-labeled secondary antibody 5000 times with PBST solution, mix well, add 100 μL/well, incubate at 37°C for 1 hour, wash all wells with PBST 6 times, and blot dry.

Color development: color development solution (containing 0.5 mL of 1 mg/mL tetramethylbenzidine, 9.5 mL of citric acid buffer, and 32 μL of 1% urea hydrogen peroxide) is prepared and used immediately, 100 μL/well, and incubated at 37°C in the dark 15min.

Termination: 2 mol/L H2SO4, 50 μL/well, immediately use a microplate reader to measure the absorbance at 450 nm, that is, the OD450 value.

Interpretation of the results: The ELISA titer of the antibody is 320,000 when the OD450 value is greater than or equal to 1 corresponding to the highest dilution of the antibody solution.

2) Indirect competitive ELISA method was used to detect the half-inhibitory concentration of capsaicinoid-specific antibody

(1) Use the above-mentioned indirect ELISA method to determine the working concentration of the antibody, and the antibody concentration corresponding to OD ₄₅₀ of about 1 is the optimal working concentration.

(2) Coating, washing and blocking: the operation of the method is the same as that of the indirect non-competitive ELISA method.

(3) Prepare capsaicin standard solution: prepare capsaicin, dihydrocapsaicin and synthetic capsaicin with methanol solution to prepare 10 mg/ml mother liquor (the ratio of capsaicinoids is 1:1:1). Before adding the sample, use 10% methanol/PBS (0.01mol/L pH7.4) solution to start 5-fold dilution from 20 μg/mL, and dilute 5 concentrations in total. Add 50 μL of diluted capsaicin, dihydrocapsaicin and synthetic capsaicin standard solution to each well, and then add 50 μL of antiserum with a dilution factor of 160,000 to each well, and react at 37°C for 1 h.

(4) Adding secondary antibody, color development, termination and reading: the operation of the method is the same as that of the indirect non-competitive ELISA method.

(5) Data processing: take the logarithm of each concentration of capsaicin, dihydrocapsaicin and synthetic capsaicin as the abscissa, and the OD value corresponding to each concentration of capsaicin, dihydrocapsaicin and synthetic capsaicin as the ordinate, draw the standard curve, the calculated 50% inhibitory concentration was 0.3 μg/mL.

Change the concentration of methanol in the diluent of the standard substance, and after detection according to the above method, draw a standard curve, and calculate the 50% inhibitory concentration of the antibody under the conditions of 10%, 20%, and 40% methanol concentration to be 0.31 μg/mL and 0.36 μg/mL, respectively. mL, 0.34 μg/mL. It shows that the antibody has a strong ability to tolerate organic solvents.

Change the salt ion concentration value of the antibody diluent, after detection according to the above method, draw a standard curve, and calculate the 50% inhibitory concentration of the antibody at 0.01M, 0.16M, and 0.32M under the conditions of 0.28μg/mL respectively , 0.33 μg/mL, 0.37 μg/mL. It shows that the antibody can tolerate higher salt ion concentration.

Change the pH value of the standard diluent, and after detection according to the above method, draw a standard curve and calculate the 50% inhibitory concentration of the antibody at pH 5.0/6.0/7.0/7.4/8.0 to be 0.41 μg/mL, 0.33 μg/mL mL, 0.29μg/mL, 0.27μg/mL, 0.28μg/mL. It shows that the antibody tolerates a wide range of pH values.

Embodiment 6: the preparation method of kitchen waste oil and fat immunochromatography test paper, the steps are as follows:

(1) Preparation of absorbent pad

Cut the absorbent paper to a length of 15mm and a width of 4mm to obtain an absorbent pad;

(2) Preparation of detection pad

Coating of detection line: Coating antigen ((2Z)-4-[(4-hydroxy-3-methoxy)benzylamino]-4-carbonyl-2-enoic acid-OVA) with capsaicinoids Coating solution with a concentration of 0.4 mg/mL is coated on the nitrocellulose membrane in a horizontal direction at a position 9 mm away from the upper edge of the nitrocellulose membrane by a line spray method to obtain a detection line, and the pepper required for each centimeter of the detection line The coating amount of the antigen-coated antigen is 0.24 μg, and then dried at 37°C for 30 minutes;

Coating buffer used in the coating antigen ((2Z)-4-[(4-hydroxy-3-methoxy)benzylamino]-4-carbonyl-2-enoic acid-OVA) coating solution The solution is: every 10mL contains 0.1g of ovalbumin OVA, 0.002g of sodium azide, 0.08g of sodium chloride, 0.029g of disodium hydrogen phosphate dodecahydrate, 0.002g of potassium chloride, and 0.002g of potassium dihydrogen phosphate;

Coating of quality control line: make rabbit anti-mouse polyclonal antibody into a coating solution with a concentration of 0.5 mg/mL, and coat it horizontally on the nitrocellulose membrane at a position 10 mm away from the test line by line spraying , to get the quality control line, the coating amount of rabbit anti-mouse polyclonal antibody required for each centimeter quality control line is 0.3 μg, and then dry at 37°C for 30 minutes;

The coating buffer used in the rabbit anti-mouse polyclonal antibody coating solution is: every 10mL contains bovine serum albumin 0.1g, sodium azide 0.002g, sodium chloride 0.08g, dodecahydrate phosphoric acid Disodium Hydrogen 0.029g, Potassium Chloride 0.002g, Potassium Dihydrogen Phosphate 0.002g;

The nitrocellulose membrane is 28mm long and 4mm wide

(3) Preparation of sample pad

Cut the glass fiber membrane to a size of 15mm in length and 4mm in width, soak it in the blocking solution, take it out, and dry it at 37°C for 8 hours to obtain a sample pad, and then store it in a desiccator at room temperature.

Each 100mL of the blocking solution contains: ovalbumin 1g, sucrose 2g, sodium azide 0.02g, sodium chloride 0.8g, disodium hydrogen phosphate dodecahydrate 0.29g, potassium chloride 0.02g, dihydrogen phosphate Potassium 0.02g.

(4) Preparation of gold standard pad

Cut the glass fiber membrane to a size of 8mm in length and 4mm in width, soak it in the blocking solution, take it out, and dry it at 37°C for 8 hours. Horizontally spray the anti-capsaicinoid universal polyclonal antibody solution labeled with nano-gold on the membrane, and the amount of universal polyclonal antibody labeled with nano-gold for each centimeter of spraying length is 0.5 μg, and then vacuum freeze-dry for 6 Hours, stored in a desiccator at room temperature;

Each 100mL of the blocking solution contains: ovalbumin 1g, sucrose 2g, sodium azide 0.02g, sodium chloride 0.8g, disodium hydrogen phosphate dodecahydrate 0.29g, potassium chloride 0.02g, dihydrogen phosphate Potassium 0.02g.

The universal polyclonal antibody solution against capsaicinoids labeled with nano-gold is prepared by an unsaturated labeling method. The specific method is: take 50.0 mL of commercially available nano-gold solution with a mass concentration of 0.01%, and use 0.5 mL of 0.1 mol /L potassium carbonate aqueous solution to adjust the pH value, slowly add 2.0mL0.1mg/mL anti-capsaicinoids universal polyclonal antibody aqueous solution under stirring, continue to stir for 30min; add mass concentration and be 10% ovalbumin (OVA) Aqueous solution until the final mass concentration of OVA is 1%, continue to stir for 30min; after standing at 4°C for 2h, centrifuge at 3000r/min for 15min, take the supernatant, discard the precipitate; centrifuge the supernatant at 12000r/min for 30min, discard the supernatant , add 40.0mL marked washing and preservation solution; then centrifuge at 12000r/min for 30min, discard the supernatant, resuspend the precipitate with the marking washing and preservation solution to obtain 5.0mL concentrate, put it in a 4°C refrigerator for later use.

The particle size of nano gold in the described nano gold solution is 15nm;

The 0.1mol/L potassium carbonate aqueous solution is: 13.8g potassium carbonate dissolved in pure water to 1000mL, filtered through a 0.22 μm filter membrane; the label washing preservation solution is: 2.0g polyethylene glycol-20000, 0.2g sodium azide, 0.1235g boric acid, dilute pure water to 1000mL, and filter through a 0.22μm filter membrane.

(5) Assembly of test strips

Paste absorbent pads, test pads, gold standard pads, and sample pads on one side of the cardboard in order, and overlap and connect adjacent pads at the joints, with an overlapping length of 1 mm, to obtain the kitchen waste oil and fat immunochromatographic test paper. . see picture 1.

Application of the above-mentioned colloidal gold immunochromatographic test strips in the rapid identification of kitchen waste oils and fats

Weigh 20g of 3 blind samples of edible vegetable oil, the pH values are 5.3, 5.8, 6.4 respectively, and the numbers are 1, 2, 3. Add 50ml of ethanol aqueous solution with a volume concentration of 95%, mix well, and reflux at 90°C for 1 hour. After cooling, the extract is vacuum-rotated and dried, and 1ml of 10% methanol-PBS solution is added to redissolve to obtain the sample solution to be tested. Take 200 μL of the sample solution to be tested as the detection solution and add it dropwise to the sample pad of the colloidal gold immunochromatography test strip for rapid identification of kitchen waste oil for detection. Methanol-PBS solution was used as a negative control solution, and was added dropwise to the sample pad of another colloidal gold immunochromatographic test strip for rapid identification of kitchen waste oil, which was used as a control test strip. After 10 minutes, the test strip and the control strip were mixed Test strips for color control, read the results:

Test results: The quality control lines of the test strips of the three samples showed red bands, but the test lines showed no color, which can be judged as positive results, indicating that the contents of capsaicinoids were all higher than 3ng/ml, and the three samples All are kitchen waste oil samples. After detection by capsaicin immunoaffinity column-high performance liquid chromatography-mass spectrometry, the results showed that the total amount of capsaicin and dihydrocapsaicin in samples 1, 2, and 3 were 36, 86, and 1213 ng/mL, respectively, which can be determined For kitchen waste grease. This result is consistent with the judgment result of the colloidal gold immunochromatographic test strip rapid identification method for kitchen waste oil provided by the present invention.

The identification standard of the waste oil and fat sample in the Chinese kitchen of the present invention is mainly based on the following reasons: the capsaicin content of a large number of actual samples (including a plurality of peanut oil, soybean oil, sunflower oil, rapeseed oil and waste oil and fat samples) Detection, detection method: capsaicin immunoaffinity column liquid mass method, the results are shown in Table 1. The results showed that no capsaicinoids were detected in edible vegetable oils except peanut oil, the capsaicinoids in peanut oil were lower than 1.53ug/kg (1.53ng/ml), while the capsaicinoids in waste The content of the substance is higher than 12.27ug/kg (12.7ng/ml), so the content of capsaicinoids is equal to or greater than 3ng/ml as a sieve for identifying the sample as kitchen waste oil or oil mixed with kitchen waste oil Check the standard.

Table 1 Content of capsaicinoids in vegetable oil

Note: '-' was not detected.

Embodiment 7: The preparation method of kitchen waste oil and fat immunochromatography test paper, the steps are as follows:

(1) Preparation of absorbent pad

Cut the absorbent paper to a length of 16mm and a width of 3.8mm to obtain an absorbent pad;

(2) Preparation of detection pad

Coating of detection line: Coating antigen ((2Z)-4-[(4-hydroxy-3-methoxy)benzylamino]-4-carbonyl-2-enoic acid-OVA) with capsaicinoids Coating solution with a concentration of 0.2 mg/mL is coated on the nitrocellulose membrane in a horizontal direction at a position 10 mm away from the upper edge of the nitrocellulose membrane by a line spray method to obtain a detection line, and the pepper required for each centimeter of the detection line The coating amount of the antigen-coated antigen is 0.12 μg, and then dried at 37°C for 30 minutes;

Coating buffer used in the coating antigen ((2Z)-4-[(4-hydroxy-3-methoxy)benzylamino]-4-carbonyl-2-enoic acid-OVA) coating solution The solution is: every 10mL contains 0.1g of ovalbumin OVA, 0.002g of sodium azide, 0.08g of sodium chloride, 0.029g of disodium hydrogen phosphate dodecahydrate, 0.002g of potassium chloride, and 0.002g of potassium dihydrogen phosphate;

Coating of quality control line: make rabbit anti-mouse polyclonal antibody into a coating solution with a concentration of 0.2 mg/mL, and coat it horizontally on the nitrocellulose membrane at a position 8mm away from the test line by line spraying , to obtain the quality control line, the coating amount of rabbit anti-mouse polyclonal antibody required for each centimeter of the quality control line is 0.12 μg, and then dried at 37°C for 30 minutes;

The coating buffer used in the rabbit anti-mouse polyclonal antibody coating solution is: every 10mL contains bovine serum albumin 0.1g, sodium azide 0.002g, sodium chloride 0.08g, dodecahydrate phosphoric acid Disodium Hydrogen 0.029g, Potassium Chloride 0.002g, Potassium Dihydrogen Phosphate 0.002g;

The nitrocellulose membrane is 25mm long and 3.8mm wide

(3) Preparation of sample pad

Cut the glass fiber membrane to a size of 13mm in length and 3.8mm in width, soak it in the blocking solution, take it out, and dry it at 37°C for 8 hours to obtain a sample pad, and then store it in a desiccator at room temperature.

Each 100mL of the blocking solution contains: ovalbumin 1g, sucrose 2g, sodium azide 0.02g, sodium chloride 0.8g, disodium hydrogen phosphate dodecahydrate 0.29g, potassium chloride 0.02g, dihydrogen phosphate Potassium 0.02g.

(4) Preparation of gold standard pad

Cut the glass fiber membrane to a size of 9mm in length and 3.8mm in width, soak it in the blocking solution, take it out, and dry it at 37°C for 8 hours. The anti-capsaicinoid universal polyclonal antibody solution labeled with gold nanometers is sprayed horizontally on the fiber membrane, and the amount of universal polyclonal antibody against capsaicinoids labeled with gold nanometers required for each centimeter of spraying length is 0.6 μg, and then vacuum freeze-dried 6 hours, store in a desiccator at room temperature;

Each 100mL of the blocking solution contains: ovalbumin 1g, sucrose 2g, sodium azide 0.02g, sodium chloride 0.8g, disodium hydrogen phosphate dodecahydrate 0.29g, potassium chloride 0.02g, dihydrogen phosphate Potassium 0.02g.

The universal polyclonal antibody solution against capsaicinoids labeled with nano-gold is prepared by an unsaturated labeling method. The specific method is: take 50.0 mL of commercially available nano-gold solution with a mass concentration of 0.01%, and use 0.5 mL of 0.1 mol /L potassium carbonate aqueous solution to adjust the pH value, slowly add 2.0mL0.1mg/mL anti-capsaicinoids universal polyclonal antibody aqueous solution under stirring, continue to stir for 30min; add mass concentration and be 10% ovalbumin (OVA) Aqueous solution until the final mass concentration of OVA is 1%, continue to stir for 30min; after standing at 4°C for 2h, centrifuge at 3000r/min for 15min, take the supernatant, discard the precipitate; centrifuge the supernatant at 12000r/min for 30min, discard the supernatant , add 40.0mL marked washing and preservation solution; then centrifuge at 12000r/min for 30min, discard the supernatant, resuspend the precipitate with the marking washing and preservation solution to obtain 5.0mL concentrate, put it in a 4°C refrigerator for later use.

The particle size of nano gold in the described nano gold solution is 15nm;

The 0.1mol/L potassium carbonate aqueous solution is: 13.8g potassium carbonate dissolved in pure water to 1000mL, filtered through a 0.22 μm filter membrane; the label washing preservation solution is: 2.0g polyethylene glycol-20000, 0.2g sodium azide, 0.1235g boric acid, dilute pure water to 1000mL, and filter through a 0.22μm filter membrane.

(5) Assembly of test strips

Paste absorbent pads, test pads, gold standard pads, and sample pads on one side of the cardboard in sequence, and overlap and connect adjacent pads at the joints. The overlapping length is 1mm, and the kitchen waste oil and fat immunochromatographic test paper is obtained. . see picture 1.

Application of the above-mentioned colloidal gold immunochromatographic test strips in the rapid identification of kitchen waste oils and fats

Weigh 20g of the collected kitchen waste oil sample and unused edible vegetable blend oil sample, the pH values are 5.4 and 7.2 respectively, add 50ml of ethanol aqueous solution with a volume concentration of 95%, mix well, and reflux at 90°C After cooling for 1 hour, spin the extract to dry in vacuum, add 1ml of 10% methanol-PBS solution to redissolve to obtain the sample solution to be tested, and then take 180 μL of the sample solution to be tested as the test solution and add it dropwise to the rapid detection of capsaicinoids The sample pad of the colloidal gold immunochromatography test strip of the substance is used as the detection test strip, and an equal volume of methanol-PBS solution with the same methanol concentration is taken as the negative control solution, and added dropwise to another kitchen waste On the sample pad of the oil immunochromatography test paper, it is used as a control test strip, and after 10 minutes, the detection test strip and the control test strip are carried out for color comparison, and the result is read:

Test results: the quality control line of the unused edible vegetable blend oil sample test strip shows a red band, and the detection line is similar to the detection limit color of the blank control test strip, then it is judged as a negative result, and thus judged : The content of capsaicinoids in the unused edible vegetable blend oil sample is lower than 3ng/mL, indicating that the blend oil is not mixed with or mixed with a very small amount of kitchen waste oil sample. The quality control line of the collected kitchen waste oil sample detection test strip shows a red band, but the detection line does not develop color, it is judged as a positive result, indicating that the content of capsaicinoids in the collected kitchen waste oil sample exceeds 3ng/mL.

Claims (10)

1. the anti-general specific antibody of Capsaicinoids, it is characterised in that: it is that the Capsaicinoids as described in formula I are general Animal is immunized in artificial immunity antigen compound, and the general specificity of anti-Capsaicinoids of acquisition is freeze-dried after isolating and purifying Antibody；

2. the general specific antibody of anti-Capsaicinoids according to claim 1, it is characterised in that: described being immunized be Immune animal is repeated using interval after Freund's complete adjuvant or incomplete Freund's adjuvant emulsification, in which: initial immunity Freund is complete Full adjuvant, follow-up immunization incomplete Freund's adjuvant, until antiserum titre reaches 10000 or more, arteria carotis is taken a blood sample to its death, After the centrifugal blood adopted, antiserum is purified to obtain Capsaicinoids antibody.

3. the general specific antibody of anti-Capsaicinoids according to claim 1, it is characterised in that: described in the formula I The immune dosage of the general artificial immunity antigen compound of Capsaicinoids 0.2-1.0mg is calculated as with BSA albumen therein；Exempt from Epidemic disease number is 4-6 times；First immunisation uses isometric Freund's complete adjuvant emulsified immunogen, the multi-point injection under rabbit；It is first It is secondary it is immune after be spaced carry out within 2-4 weeks second it is immune, for the second time and later immunization interval 2-3 weeks, not by isometric Freund Freund's complete adjuvant emulsified immunogen, in the subcutaneous multi-point injection of rabbit back, since the 4th time, after being immunized every time in 6-10 days, rabbit ear The non-competing indirect ELISA method of edge venous blood collection measures serum titer, and after potency reaches 10000, i.e. rabbit arteria carotis is taken a blood sample to it Death after the centrifugal blood adopted, antiserum is purified using caprylic acid-ammonium, obtains Capsaicinoids antibody.

4. kitchen waste grease immuno-chromatographic test paper strip, it is characterised in that: including cardboard, the one side of cardboard is successively glued from top to bottom Water absorption pad, detecting pad, gold-labelled pad and sample pad are pasted, adjacent each pad is in the overlapping connection in junction, and the detecting pad is with cellulose nitrate Plain film is base wad, and lateral nature controlling line is arranged from top to bottom on nitrocellulose filter and detection line, the nature controlling line are coated with rabbit Anti- mouse polyclonal antibody is coated with Capsaicinoids envelope antigen in the detection line；The gold-labelled pad, which is laterally coated with, to be received The anti-general specific antibody of Capsaicinoids described in claim 1 of rice gold label.

5. kitchen waste grease immuno-chromatographic test paper strip according to claim 4, it is characterised in that: the water absorption pad is long 10~15mm, wide 3~4mm, detecting pad long 25~30mm, wide 3~4mm；Gold-labelled pad grows 6~9mm, wide 3~4mm；Sample pad is long 12~15mm, wide 3~4mm, the adjacent overlapping length respectively padded are 1~3mm；

The water absorption pad is blotting paper.

6. kitchen waste grease immuno-chromatographic test paper strip according to claim 4, it is characterised in that: on the detecting pad The spacing on edge is 8~20mm on detection line and nitrocellulose filter, and the spacing of the detection line and nature controlling line is 5~10mm；Institute The partial size for stating nanogold used in gold-labelled pad is 15~20nm.

7. kitchen waste grease immuno-chromatographic test paper strip according to claim 4, it is characterised in that: the capsaicine class The molecular structural formula of substance envelope antigen is as shown in formula II:

In the detecting pad detection line it is per cm required for envelope antigen package amount be 0.1~0.6 μ g；Every li on nature controlling line The package amount of rabbit-anti mouse polyclonal antibody required for rice is 0.1~0.6 μ g；

In the gold-labelled pad it is per cm spraying length needed for nano gold mark the general specific antibody of anti-Capsaicinoids Dosage be 0.40~0.98 μ g.

8. the preparation method of kitchen waste grease immuno-chromatographic test paper strip as claimed in claim 4, it is characterised in that: including following Step:

(1) preparation of water absorption pad

Blotting paper is cut out up to water absorption pad；

(2) preparation of detecting pad

The coating of detection line: being configured to the coating buffer that concentration is 0.17~1.0mg/mL for Capsaicinoids envelope antigen, in Away from the position on nitrocellulose filter along 8~20mm, its transverse direction is coated on nitrocellulose filter with line spray mode, is examined Survey line, the package amount of the general artificial complete antigen-envelope antigen of Capsaicinoids needed for detection line per cm are 0.1~0.6 μ G is then 30~60 minutes dry under the conditions of 37 DEG C；

The coating of nature controlling line: rabbit-anti mouse polyclonal antibody is made into the coating buffer that concentration is 0.17~1.0mg/mL, in away from detection Its transverse direction is coated on nitrocellulose filter with line spray mode, obtains nature controlling line, nature controlling line per cm by the position of 5~10mm of line The package amount of required rabbit-anti mouse polyclonal antibody is 0.1~0.6 μ g, then 30~60 minutes dry under the conditions of 37 DEG C；

(3) preparation of sample pad

Glass fibre membrane is put into confining liquid and is soaked, is taken out, it is 6~12 hours dry under the conditions of 37~40 DEG C, sample pad is obtained, Then room temperature preservation in drier is set；

(4) preparation of gold-labelled pad

Glass fibre membrane is put into confining liquid and is soaked, is taken out, dry 6~12 hours under the conditions of 37~40 DEG C, in having dried Glass fibre membrane on, the anti-capsaicine class of nano gold mark is laterally sprayed on the glass fibre membrane dried with spray mode The general Anti-TNF-α liquid solution of substance, general more grams of anti-Capsaicinoids of nano gold mark needed for spraying length per cm The dosage of grand antibody is 0.4~0.98 μ g, and then vacuum freeze drying 2~6 hours, set room temperature preservation in drier；

(5) assembling of test strips

Water absorption pad, detecting pad, gold-labelled pad and sample pad are successively pasted from top to bottom in the one side of cardboard, and adjacent each pad is in junction Overlapping connection, overlapping length are 1~3mm to get kitchen waste grease immune chromatography test paper.

9. kitchen waste grease immunochromatography discrimination method, it is characterised in that: steps are as follows: pick-up kitchen waste grease waits for test sample Product extract Capsaicinoids therein, by extracting solution it is dry after solubilizer methanol-PBS solution redissolve, the sample to be tested and The ratio of redissolution solvent is 20g:1ml, obtains testing sample solution, then be added dropwise using testing sample solution as detection liquid It is detected on to the sample pad of kitchen waste grease immuno-chromatographic test paper strip as claimed in claim 4, is used as Test paper Item separately takes the consistent methanol-PBS solution of isometric methanol concentration as negative controls, another claim 4 is added dropwise In the sample pad of the kitchen waste grease immuno-chromatographic test paper strip, it is used as control stripes item, will test after a period of time Test strips and control stripes item carry out colour developing control:

Testing result:

(1) negative: when nature controlling line colour developing in test strip, and to detect the color of detection line on line color and control stripes item When close, show that Capsaicinoids capsaicine in testing sample solution, Dihydrocapsaicin or synthetic capsaicin content are lower than 3ng/mL determines whether the sample is kitchen waste grease or mixed with the grease of kitchen waste grease in conjunction with authenticity detection method Sample；Content is lower than 3ng/mL, can determine whether that a possibility that sample to be tested is kitchen waste grease is little, need to further use authenticity Detection method is determined；

(2) positive: when nature controlling line colour developing in test strip, and to detect color of the line color than detection line in control test strips When shallow, show that capsaicine in testing sample solution, Dihydrocapsaicin or synthetic capsaicin content are equal to or higher than 3ng/mL, then may be used Determine the sample for kitchen waste grease or mixed with the oil sample of kitchen waste grease；

(3) invalid: when nature controlling line does not develop the color, no matter whether the detection line of test strip develops the color, which is judged in vain.

10. kitchen waste grease immunochromatography discrimination method according to claim 9, it is characterised in that:

The extracting method is that the ethanol water that volumetric concentration is 95% is added into sample to be tested, is mixed, 60~90 It flows back 1 hour at DEG C, cooling obtains extracting solution；The redissolution solvent is 10% methanol-PBS.