KR20170134217A - General complete antigen hapten, specific antibody, test strip, and rapid immunochromatographic method for identification of a recovered oil from kitchen - Google Patents

Abstract

The present invention relates to a general specific antibody for an anti-capsaicin material, a strip, and an immunochromatographic method for rapidly identifying kitchen waste oil. The general specific antibody for an anti-capsaicin material is a general specific antibody for an anti-capsaicin material obtained by immunizing an animal with a general artificial immunogenic antigen compound for a capsaicin material according to formula I, separating and refining the same, and then freeze-drying the same. An immunochromatographic strip for kitchen waste oil manufactured by the same is for kitchen waste oil, and is for rapidly detecting the total amount of capsaicin materials in the kitchen waste oil, has high detection sensitivity, has a wide range of pH applications, and is simple and convenient by a one-step operation. In addition, a capsaicin-material standard solution is unnecessary to be used as a positive control group.

Description

[Technical Field] The present invention relates to a method for rapidly distinguishing an antigenic hapten, an anti-capsaicin substance, an artificial hapten, a specific antibody, a strip and a kitchen waste oil by immunochromatography,

  The present invention relates to a method for rapidly distinguishing immunoglobulin-sensitive antibodies, strips and kitchen waste oil for anti-capsaicin substances, which belong to the field of immunochemistry.

Diet milk is a general concept and is a generic term for various low-grade oils. Ordinary oil derived from foodstuffs is the main raw material of the digestive milk. It is one of the questions. In Chinese food culture, peppers containing capsaicin are one of the indispensable seasonings. Capsaicin compounds are the main chemical substances that make them spicy and irritating in pepper fruit, and there are more than 20 structures already known. Capsaicin materials are widely used in the food processing field because of their high nutritional value. Synthetic capsaicin has a structure similar to that of natural capsaicin and has spicy characteristics. It is one of the capsaicin materials and is mainly used in industrial fields. However, due to the low manufacturing cost, some illegal molecules are used as natural capsaicin And is applied to the field of food. Capsaicin, dihydro capsaicin, and synthetic capsaicin are key components in capsaicin materials. Capsaicin materials have high nutritional value and are applied in the field of food processing. Capsaicin materials are characterized by high fat-solubility, good stability, and high boiling point. Currently, digger milk processing methods are difficult to completely remove substances with these characteristics. According to several studies, normal cooking oil is basically hard to cap. Therefore, the capsaicin component can be a characteristic index for distinguishing kitchen waste oil, and it is possible to determine whether the kitchen waste oil is mixed in edible vegetable oil by measuring the content of capsaicin. Currently, there is a method for detecting capsaicin substances in edible vegetable oil mainly by high performance liquid chromatography, GCMS, and the like, which is high in sensitivity and high in accuracy. However, And it is difficult to realize rapid detection because a demand for a professional operator is demanded. The immunological analysis method which has been developed rapidly in recent years overcomes the shortcomings of the above method, has strong specificity, high sensitivity, high analytical capacity, simple and low cost, , And agriculture.

The immunochromatographic technique in which the colloidal gold-labeled antibody specifically binds to the antigen is widely used in the qualitative, on-line, and rapid detection of trace residues such as large pesticide residues because the detection results thereof can be visually observed. However, at present in China and abroad, this method has not yet detected the capsaicin substances in kitchen waste oil. The key elements of the colloidal gold immunochromatography technique are complexity of the specific antibody or kitchen waste oil substrate components, the pH of the sample extract is difficult to be determined, the specific antibody activity is low, and the detection sensitivity is low. Therefore, we developed anti-capsaicin substance-specific antibodies with high affinity, good specificity, resistance to organic solvents, and a wide pH range, and developed a colloidal gold immunochromatographic strip detection method for distinguishing kitchen waste oil using this antibody It is an urgent schedule to do.

The object of the present invention is to provide an anti-capsaicin substance-specific antibody, a kit waste colloidal gold immunochromatographic strip, and a method for rapidly discriminating a kit waste oil immunochromatography. The anti-capsaicin substance-specific antibody affinity provided in the present invention has high antibody affinity, good specificity, resistance to organic solvents, and wide pH range. The immunochromatographic strips prepared using the same are for detecting capsaicin substances in kitchen waste oil, and are characterized by being simple to operate, low in cost, high in sensitivity, and wide in pH range.

In order to solve the above technical problems, the technical solutions used in the present invention are as follows.

와 같다.The present invention provides artificial immunoantigen compounds for capsaicin,

.

The present invention provides a method for producing an artificial immunoantigen compound for capsaicin, which comprises the following specific steps. The hydrochloric acid in vanillylamine hydrochloride is neutralized with sodium hydroxide to obtain a compound of Formula III and then reacted with maleic anhydride to obtain an artificial hapten compound and the capsaicin material artificial hapten is reacted with N, N'-dicyclohexylcarbodiimide Is obtained by activation with DCC and N-hydroxysuccinimide NHS, or by activation with tri-n-butylamine and isobutyl chloroformate followed by coupling with vector protein BSA.

The synthesis route of the artificial hapten compound is as follows.

The method for producing the artificial immunoantigen compound for capsaicin type substance comprises specifically the following steps.

(1) dissolving vanillylamine hydrochloride and sodium hydroxide having a molar ratio of 1 :( 0.7-1.1) in water, stirring at room temperature for 10-30 minutes, filtering to obtain vanillylamine (formula IV compound) as a white precipitate;

Thoroughly dried vanillylamine and maleic anhydride are dissolved in chloroform at a molar ratio of 1: 0.8-1.2, reacted for 1-2 h at room temperature, filtered, and capsaicin material artificial hapten is obtained.

(2) In N, N-dimethylformamide, an artificial hapten for capsaicin material and N-hydroxysuccinimide (NHS) were reacted at room temperature for 30 min to 2h, and then N, N-diisopropylcarbodiimide Dimethylformamide solution was added and the mixture was allowed to react at room temperature for 4 to 6 hours. After standing overnight, the supernatant, that is, activated capsaicin, dihydro capsaicin and synthetic capsaicin artificial hapten solution was taken, The molar ratio of the amide (NHS) to dicyclohexylcarbodiimide is 1: 1-1.2;

Or N, N-dimethylformamide, tri n-butylamine and isobutyl chloroformate are successively added to an artificial hapten solution for capsaicins, and the mixture is allowed to react at room temperature for 0.5-3 hours to obtain a reaction solution, that is, an activated capsaicin material The molar ratio of tri-n-butylamine to isobutyl chloroformate is 1: 1-1.2.

(3) adding the activated captenic acid supernatant obtained in (1) above to a phosphate buffer solution of vector protein having a vector protein concentration of greater than or equal to 2 mg / mL and reacting for 4-8 h at room temperature, The molar ratio of the artificial hapten to the vector protein in the step (1) is greater than 10: 1, and the capsaicin artificial antigen is obtained by dialyzing the conjugate.

According to the above solution, the phosphate buffer solution of the vector protein is obtained by dissolving the vector protein with a phosphate buffer solution of 0.2M pH 8.0.

According to the above solution, the dialysis solution is exchanged with the dialysis solution once every 4-8 h while dialysis with 0.01 M PBS buffer for 72 h.

The specific antibody for the anti-capsaicin substance substance is an antibody specific for the anti-capsaicin substance, which is obtained by immunizing an animal with an artificial immunoantigen compound for capsaicin according to the formula I, separating and purifying the animal, followed by lyophilization;

이다.

to be.

According to the above solution, the immunization is emulsified with a Freund ' s complete adjuvant or a Freund ' s incomplete adjuvant, followed by immunization with the animal at intervals, wherein the first immunization uses Freund's complement adjuvant, Immunization is carried out using Freund's incomplete adjuvant until the antitumor activity reaches 10,000 or more. Carotid artery blood is collected until death, centrifuged, and the antiserum is purified to obtain a capsaicin antibody . The antiserum reactivity obtained by immunizing an animal with the antigen provided in the present invention can reach 320000 and the inhibitory concentration half-value IC ₅₀ of the obtained antibody against capsaicin, dihydro capsaicin and synthetic capsaicin is in the range of pH 5.0 to 8.0, 0.27 - 0.41 μg / mL. This is an antibody for capsaicin, dihydro capsaicin, and synthetic capsaicin, which can specifically react with capsaicin, dihydro capsaicin and synthetic capsaicin, showing good affinity, high sensitivity, and good stability. And high salt ion concentration, and high pH coverage.

According to the above solution, the immunosuppressive capacity of the artificial immunoantigen compound for capsules according to the above formula I is calculated as 0.2-1.0 mg by the OVA protein therein; The number of immune layers is 4-6 times; The first immunization emulsifies the immunogen with the same volume of Freund's adjuvant, administering subcutaneous multi-point injections to the rabbit; After the first immunization, the second immunization is performed at intervals of 2 to 4 weeks, the second and subsequent immunizations are performed at intervals of 2 to 3 weeks, and the immunogen is emulsified with the same volume of Freund's incomplete adjuvant, Subcutaneous multi-point injections are given to the back. From the fourth time, every 6th day after immunization, the rabbit ear vein was collected and the serum titer was measured by the indirect competitive ELISA method. After the test was completed, the carotid artery blood was collected until the potency reached 10000, After centrifuging the blood, the antiserum is purified using octanoic acid-ammonium sulfate to obtain a capsaicin material antibody.

The kit waste oil immunochromatographic strip (as shown in FIG. 3) includes a cardboard, and sequentially adheres the absorption pad, the detection pad, the gold pad, and the sample pad from one side to the other side of the cardboard, And the detection pad is provided with a nitrocellulose membrane as a base pad, a longitudinal quality control line and a detection line are provided in the nitrocellulose membrane from top to bottom, and the quality control line is connected with a rabbit anti mouse The cloned antibody is coated, and the detection line is coated with a coated capsaicin material-coated antigen; The gold pad is sprayed with a nano-gold-specific antibody specific for the capsaicin-like substance.

According to the above solution, the absorption pad has a length of 10 to 15 mm and a width of 3 to 4 mm, a detection pad has a length of 25 to 30 mm and a width of 3 to 4 mm; The gold pad is 6 to 9 mm long and 3 to 4 mm wide; The sample pads are 12 to 15 mm long and 3 to 4 mm wide, and the overlap length of adjacent pads is 1 to 3 mm.

According to the above solution, the absorbent pad is an absorbent paper.

According to the above solution, the distance between the detection line on the detection pad and the nitrocellulose membrane is 8 to 20 mm, and the distance between the detection line and the quality control line is 5 to 10 mm.

이다.The molecular structure of the capsaicin-coated material antigen is represented by Formula II,

to be.

According to the above solution, the coating amount of the coated antigen required for every centimeter on the detection line of the detection pad is 0.1 to 0.6 mu g; The coating amount of the rabbit anti-mouse polyclonal antibody, which is required every centimeter on the quality control line, is 0.1 to 0.6 μg.

According to the above solution, the particle size of the nano gold used for the gold pad is 15 to 20 nm.

The specificity of the antibody specific for the anti-capsaicin substance labeled with nano gold, which is required every centimeter on the gold pad, is 0.40 to 0.98 μg.

The method of producing a colloidal gold immunochromatographic strip for rapidly detecting a capsaicin material as described above includes the following steps.

(1) Fabrication of Absorbent Pads

Cutting the absorbent paper to obtain an absorbent pad;

(2) Manufacture of detection pad

Coating of the detection line: Capsaicin-coated material was prepared as a coating solution having a concentration of 0.17 to 1.0 mg / mL. The coating solution was coated on the nitrocellulose membrane in a transverse direction at a position 8 to 20 mm away from the nitrocellulose membrane by the pre- To obtain a detection line, and the coating amount of the artificial complete antigen-coated antigen for a capsaicin-like material conjugate required by the detection line of every centimeter is 0.1 to 0.6 μg, followed by drying at 37 ° C for 30 to 60 minutes;

Quality control line coating: A rabbit anti-mouse polyclonal antibody was prepared with a coating solution having a concentration of 0.17 to 1.0 mg / mL, and this was coated on the nitrocellulose membrane in a transverse direction at a position 5 to 10 mm away from the detection line, To obtain a quality control line, and the covering amount of the rabbit anti-mouse polyclonal antibody required by the quality control line of every centimeter is 0.1 to 0.6 μg, and then drying at 37 ° C for 30 to 60 minutes;

(3) Preparation of sample pad

The glass fiber membrane is put in a blocking solution, the glass fiber membrane is taken out, the glass fiber membrane is taken out and dried at 37 to 40 ° C for 6 to 12 hours to obtain a sample pad,

(4) Manufacture of gold pads

The glass fiber membrane was put in a blocking solution, and the glass fiber membrane was taken out. The glass fiber membrane was taken out from the glass fiber membrane and dried at 37 to 40 ° C for 6 to 12 hours. On the dried glass fiber membrane, a capsaicin material labeled with nano- The general polyclonal antibody solution was sprayed in a transverse direction and the dose of polyclonal antibody for nano-gold labeled anti-capsaicin substance required by the spray coating length of every centimeter was 0.4 to 0.98 ng. Lyophilized for 2 to 6 hours, placed in a drier, and stored at room temperature;

(5) assembly of strips

A pad, a gold pad, and a sample pad are successively bonded from one side of the cardboard to one side of the cardboard, from top to bottom, and the adjacent pads are connected to each other so as to overlap with each other, and the overlapping length is 1 to 3 mm, And obtaining a waste oil immunochromatographic test paper.

According to the above solution, the capsaicin substance-coated antigen ((2Z) -4 - [(4-hydroxy-3-methoxy) benzylamino] -4-oxo-2-butenoic acid-OVA) The coating buffer solution contained 0.1 g of ovalbumin OVA, 0.002 g of sodium azide, 0.08 g of sodium chloride, 0.029 g of disodium phosphate dodecahydrate, 0.002 g of potassium chloride and 0.002 g of potassium dihydrogenphosphate per 10 mL of the solution; The coating buffer used for the above rabbit anti-mouse polyclonal antibody coating solution contained 0.1 g of bovine serum albumin, 0.002 g of sodium azide, 0.08 g of sodium chloride, 0.029 g of disodium phosphate dodecahydrate, 0.002 g of potassium chloride, 0.002 g of potassium dihydrogenphosphate is contained;

According to the above solution, 1 to 2 g of ovalbumin, 2 to 5 g of sucrose, 0.02 to 0.05 g of sodium azide, 0.8 g of sodium chloride, 100 g of disodium phosphate dodecane, 0.29 g of car- hydrate, 0.02 g of potassium chloride, and 0.02 g of potassium dihydrogenphosphate.

According to the above solution, the polyclonal antibody solution labeled with the nanoparticles of the present invention is prepared by an unsaturated labeling method. 50.0 mL of a nano gold solution having a commercial mass concentration of 0.01% was taken, and the pH value was adjusted with 0.5 mL of 0.1 mol / L potassium carbonate aqueous solution, and 2.0 mL of 0.1 mg / mL of anti-capsaicin substance was slowly used Adding an aqueous clonogenic antibody solution, and continuously stirring for 30 minutes; Add an aqueous ovalbumin (OVA) solution having a mass concentration of 10%, add until the final mass concentration of OVA reaches 1%, and continue stirring for 30 minutes; Left at 4 캜 for 2 hours, centrifuged at 3000 r / min for 15 minutes, take the supernatant, and remove the precipitate; The supernatant is centrifuged at 12000 r / min for 30 min, the supernatant is removed, and 40.0 mL of label wash stock is added; Centrifuged again at 12,000 r / min for 30 minutes, the supernatant liquid was removed, and the saliva label wash liquid was resuspended, and 5.0 mL concentrate was obtained and placed in a refrigerator at 4 ° C.

According to the above solution, the 0.1 mol / L potassium carbonate aqueous solution was obtained by filtering 13.8 g of potassium carbonate into purified water to 1000 mL and filtering through a 0.22 mu m filtration membrane; The label wash preservative solution was obtained by filtering 2.0 g polyethylene glycol-20000, 0.2 g sodium azide and 0.1235 g boric acid to 1000 mL in purified water and filtering through a 0.22 mu m filtration membrane.

The steps of the kitchen waste oil immunochromatography rapid discrimination method are as follows. Measured samples of kitchen waste oil were taken and the capsaicin substances contained therein were extracted. The extract was dried and then re-eluted by adding a solvent. The ratio of the measurement atmospheric sample to the re-leaching solvent was 20 g: 1 ml, (Preferably 80-200 [mu] L) of the measurement solution, and using this measurement atmospheric sample solution as a detection solution, dropwise dropwise to a sample pad of a kit waste oil immunochromatography test strip, which is used as a detection strip, The solution was added dropwise to the sample pads of another kitchen waste oil immunochromatography test paper. Using this as a control strip, after a predetermined time (10-20 minutes), the detection strips And performing a light color contrast on the control strip,

Detection result:

(1) Negative: When the quality control line on the detection strip is colored and the color of the detection line is close to the color of the detection line on the control strip, the content of capsaicin, dihydro capsaicin or synthetic capsaicin, / mL, with the provable method being able to determine whether the sample is a kitchen waste oil or a kitchen waste oil doped oil sample; If the content is less than 3 ng / mL, the measurement atmospheric sample is not likely to be kitchen waste oil and needs to be further confirmed in a provable manner;

(2) Positive: When the quality control line on the detection strip is colored and the color of the detection line is lighter than the color of the detection line on the control strip, the capsaicin, dihydro capsaicin or synthetic capsaicin content in the measurement atmospheric sample solution is more than 3 ng / mL It can be judged that this sample is an oil sample of kitchen waste oil or kitchen waste oil doped;

(3) Invalid: If the quality control line is not colored, even if the detection line of the detection strip is colored, it is judged that the strip is invalid;

When this immunochromatographic strip is used to detect kitchen wastepaper capsaicin, dihydro capsaicin or synthetic capsaicin: When the atmospheric sample solution is added to the sample pad at the bottom of the strip, the atmospheric sample solution is capped with strips The nanoparticle labeled capsaicin, dihydro capsaicin or polyclonal antibody for synthetic capsaicin is dissolved when it moves to the gold pad. When the sample contains capsaicin, dihydro capsaicin or synthetic capsaicin, the capsaicin material binds to the polyclonal antibody conjugated with the nano-gold labeled anti-capsaicin substance on the gold pad and migrates up together with the capsaicin, When the antigen reaches the detection line of the immobilized coated antigen, the antigen is bound to the limited antigen binding site on the polyclonal antibody commonly used with capsaicin, dihydro capsaicin or synthetic capsaicin labeled nanoparticles labeled with anti-capsaicin The higher the content of capsaicin, dihydrocapsacin or synthetic capsaicin in the sample, the more the polyclonal antibody labeled with nanoparticles capable of binding to the antigen on the detection line becomes less and the detection The color of the colored band formed on the line gradually fades. Anti-capsaicin binding to antigen on the detection line When the number of polyclonal antibodies is less than a certain number, no red line appears on the detection line. Whether or not the sample contains a capsaicin substance or not, the anti-capsaicin substance antibody or nano gold-labeled anti-capsaicin substance antibody that is not captured by the antigen on the detection line, and capsaicin, dihydro capsaicin or The conjugate of the synthetic capsaicin migrates to the quality control line and is colored by binding with the rabbit anti-mouse polyclonal antibody on the quality control line and concentrating. Thus, by subjecting the dark-colored control to the detection line of the capsaicin-coated antigen on the detection strip and the corresponding detection line color on the control strip to obtain the capsaicin, dihydro capsaicin or synthetic capsaicin content situation in the sample, It can be judged whether it is waste oil.

According to the above solution, the extraction is carried out by adding an aqueous ethanol solution having a volume concentration of 95% to a sample to be measured, uniformly stirring, refluxing at 60 to 90 ° C for 1 hour and cooling to obtain an extract; The re-eluting solvent is 10% methanol-PBS.

Advantageous Effects of the Invention:

(1) In the present invention, the amide group of vanilylamine in capsaicin is modified to retain the characteristic structure of capsaicin, dihydro capsaicin and synthetic capsaicin (vanillylamine and partial fat chain radical in capsaicin major molecular structure) And one large covalent bond structure is formed in the haptic connection arm region, which can lead to the transfer of electrons in the haptenic structure, which also has a constant stiffness and is advantageous in exposing active portions in the hapten , An antigenic compound obtained by coupling with OVA can be an antigenic determinant and may have an activating group that can be coupled with a vector protein. The resulting antigenic compound is an immunological antigen and has high immunoaffinity, high sensitivity, and high specificity Capsaicin antibody that can tolerate organic solvent and salt ion concentration and has a wide pH range .

(2) The kitchen waste oil immunochromatographic rapid discrimination method provided in the present invention can detect the total amount of capsaicin substances in kitchen waste oil; The detection sensitivity is high and the pH range is wide.

(3) The kitchen waste oil immunochromatographic rapid discrimination method provided in the present invention is simple in operation. The sample solution of the kitchen waste oil is dropped dropwise onto the sample pad of the strip, and it is simple and convenient in a one-step operation. At the time of sample detection, it is not necessary to use a capsaicin material standard solution as a positive control, and a blank sample may be used as a negative control. In addition, the detection result of this method can be immediately read visually.

1 is an artificial complete antigen ultraviolet spectrogram for the anti-capsaicin substance substance synthesized in the present invention.
FIG. 2 is an artificial complete antigen-coated antigen ultraviolet spectrogram for an anti-capsaicin substance substance synthesized in the present invention.
3 is a structural view of a kitchen waste oil immunochromatographic strip provided in the present invention.

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

Materials, reagents and the like used in the following examples are all obtained through commercial routes, unless otherwise specified.

Example 1 Preparation of capsaicin material artificial hapten

(1) 1 g of vanilylamine hydrochloride was dissolved in 11.77 ml of ultrapure water and 2.12 ml of 2M NaOH was dropwise added dropwise with vigorous stirring, followed by reaction at room temperature for 10 minutes. The white precipitate is obtained by filtration and the white precipitate is thoroughly dried before proceeding the next reaction.

(2) 60 mg of the product obtained in the above step is dissolved in 30 ml of chloroform (after dehydration treatment with anhydrous sodium sulfate), stirred at room temperature for 40 minutes, added with 40 mg of maleic anhydride, and reacted at room temperature for 1 hour. The precipitate obtained after filtration was a mixture of capsaicin material artificial hapten (2Z) -4 - [(4-hydroxy-3-methoxy) benzylamino] -4- -hydroxy-3-methoxybenzyl) amino] -4-oxobut-2-enoic acid). The molecular formula is C ₁₂ H ₁₃ NO ₅ .

Example 2 Preparation of capsaicin material artificial total antigen-immunizing antigen

20.8 mg (about 0.08 mmol), 11.6 mg (about 0.1 mmol) of NHS and 20.8 mg (about 0.08 mmol) of the capsaicin material were placed in a reaction cup and dissolved in a reaction cup by addition of 400ul DMF and stirred at room temperature for 30 minutes. 0.1 mmol) DCC is dissolved in 50 ul DMF, and the DCC / DMF solution is added dropwise to the reaction cup dropwise, stirred at room temperature for 4 hours, and allowed to stand overnight at 4 캜. The active ester supernatant is taken under the condition of 8000 rpm / 5 min.

The active ester supernatant is added dropwise dropwise to 6 ml of 7 mg / ml BSA solution, and the reaction buffer is a phosphate buffer solution of 0.2 M pH 8.0. The reaction proceeds at room temperature for 4 h through magnetic stirring. The reaction solution is put into a dialysis bag, dialyzed at 4 ° C with 0.01 M pH 7.4 PBS, and dialyzed for 72 h while exchanging the dialysis solution once every 4 h. This results in a capsaicin artificial antigen-immunizing antigen. The ultraviolet-visible spectral continuous scan spectrum, as shown in Figure 1, indicates that the artificial antigen binding has been successful.

Example 3 Preparation of capsaicin material artificial full antigen-coated antigen

4.52 mg of the capsaicin material artificial hull was dissolved in 200 uL of anhydrous DMF, and 4.27 μL of tri-n-butylamine and 2.34 μL of isobutyl chloroformate were added successively, followed by stirring at room temperature for 1 h while avoiding direct sunlight , Activated capsaicin, dihydro capsaicin and synthetic capsaicin hapten solution.

Activated capsaicin, dihydro capsaicin and synthetic capsaicin hapten solution are added dropwise to 10 ml of 4.5 mg / ml OVA solution, and the reaction buffer is a phosphate buffer solution of 0.2 M pH 8.0. The reaction proceeds at room temperature for 4 h through magnetic stirring. The reaction solution is put into a dialysis bag, dialyzed at 4 ° C with 0.01 M pH 7.4 PBS, and dialyzed for 72 h while exchanging the dialysis solution once every 4 h. This gives capsaicin artificial antigen-coated antigen. The ultraviolet-visible spectral continuous scan spectrum, as shown in Figure 2, indicates that the artificial antigen binding has been successful.

Example 4: Preparation of capsaicin substance-specific antibody

The prepared anti-capsaicin material artificial complete antigen (immunizing antigen) is used to immunize Japanese white rabbits of 3 kg or more. On average, the antigen dose used each time is about 0.84 mg (calculated as the amount of protein). The first immunization emulsifies the immunogen with the same volume of Freund's adjuvant, administering subcutaneous multi-point injections to the rabbit; After the first immunization, the immunogen is emulsified with the same volume of Freund's incomplete adjuvant at intervals of 3 weeks, followed by intervals of 2 weeks, and subcutaneous multipoint injection is administered to the rabbit and the like. From the fourth, every week after the immunization, the rabbit ear vein is collected and the serum titer is measured by an indirect competitive ELISA method. After the fifth immunization, the carotid artery is drawn until the rabbit has died. The collected blood is allowed to stand at 37 DEG C for 1 hour and then centrifuged at 4 DEG C overnight. The supernatant is taken, mixed with a small amount of the same volume of glycerin, and stored at -20 DEG C for a while. The remaining antiserum is purified using octanoic acid-ammonium sulfate, and finally the lyophilized antibody is obtained, which is stored at -20 ° C.

Example 5: Measurement of capsaicin substance-specific antibody

1) Determine capsaicin-specific antibody titer using an indirect non-competitive ELISA method.

Coat: Capsaicin material Artificial complete antigen-coated antigen is diluted to 1 mg / mL with 0.1 mol / L pH 9.6 carbonate buffer, and reacted at 100 ml / well at 4 ° C overnight. The coating solution is poured out, the PBST is filled in the well plate, washed three times, and dried.

Blocking: 1% OVA PBST solution, 200 μL / well, incubate at 37 ° C for 1 h, and PBST is filled in the well plate, washed 3 times and dried.

Antibody antigen specificity reaction: The capsaicin substance-specific antibody was prepared into a 1 mg / mL solution using a 0.01 M PH7 phosphate buffer solution. The antibody solution was diluted to 1: 10000 and diluted to a drainage proportion, The negative group and the control group were set, the serum of the negative group was diluted to 2000-fold, the PBST was added only to the control group, the cells were incubated at 37 占 폚 for 1 hour, and the PBSTs were filled in the well plates Washed 3 times and dried;

Secondary antibody addition: The cells were incubated at 37 ° C for 1 h, and the PBSTs were washed six times in the well plates and washed six times with the PBST solution, diluted 5000 fold with the anti-anti-mouse enzyme labeled secondary antibody and uniformly mixed. do.

Fresh color: Prepare 100 μL / well of 1 liter of urea hydrogen peroxide containing 1 mg / mL tetramethylbenzidine 0.5 mL and 9.5 mL of citric acid buffer, and incubate for 15 min at 37 ° C while avoiding direct sunlight do.

Stop: Use 2 mol / L H2SO4, 50 μL / well, and immediately measure the absorbance at 450 nm, OD450, using an enzyme immunoassay.

Result reading: If the OD450 value is greater than or equal to 1, the highest dilution factor of the corresponding antibody solution is the ELISA titer of the antibody, which is 320000.

2) An indirect competition ELISA method is used to detect half of the inhibition concentration of capsaicin-specific antibody

(1) Determine the working concentration of the antibody using the above-mentioned indirect ELISA method, and set the corresponding antibody concentration to the optimum working concentration when OD ₄₅₀ is one side.

(2) Coating, Washing and Blocking: Method manipulation is the same as indirect non-competitive ELISA method.

(3) Preparation of capsaicin standard solution: Capsaicin, dihydro capsaicin and synthetic capsaicin are prepared in a methanol solution at a concentration of 10 mg / ml, wherein the ratio of the capsaicin material is 1: 1: 1. Before adding the sample, dilute to 5 concentrations starting from 20 μg / mL with 5% methanol / PBS (0.01 mol / L pH 7.4) solution. Capsaicin, dihydro capsaicin and synthetic capsaicin diluted in a proportion of 50 μL per well was added to the wells, and 50 μL of diluted antiserum with a dilution of 1,60000 per well was further added thereto, followed by reaction at 37 ° C. for 1 h.

(4) Addition of secondary antibody, color development, stop and data reading: The method manipulation is the same as the indirect noncontamination ELISA method.

(5) Data processing: A standard curve was prepared by plotting the logarithm of each concentration of capsaicin, dihydro capsaicin and synthetic capsaicin as the abscissa and using the OD value corresponding to each concentration of capsaicin, dihydro capsaicin and synthetic capsaicin as the ordinate, % Inhibition concentration is calculated as 0.3 μg / mL.

Standard curves were prepared by changing the concentration of methanol in the standard dilution solution according to the above method and the 50% inhibitory concentrations of the antibodies were adjusted to 0.31 μg / ml, respectively, under the conditions of methanol concentration of 10%, 20% and 40% mL, 0.36 μg / mL, and 0.34 μg / mL, respectively. Indicating that this antibody has a strong ability to withstand organic solvents.

The standard curves were prepared according to the method described above by changing the salt ion concentration value of the antibody diluted solution and the 50% inhibitory concentration of the antibody was 0.28 < RTI ID = 0.0 > μg / mL, 0.33 μg / mL, and 0.37 μg / mL, respectively. Indicating that this antibody can withstand a relatively high salt ion concentration.

The standard curve was prepared and the 50% inhibitory concentration of this antibody was adjusted to 0.41 μg / mL, pH 5.0 / 6.0 / 7.0 / 7.4 / 8.0, 0.33 μg / mL, 0.29 μg / mL, 0.27 μg / mL, and 0.28 μg / mL, respectively. Indicating that the range of pH values tolerated by this antibody is wide.

Example 6: The preparation method of kitchen waste oil immunochromatography test paper is as follows.

(1) Fabrication of Absorbent Pads

Cutting the absorbent paper into a standard having a length of 15 mm and a width of 4 mm to obtain an absorbent pad;

(2) Manufacture of detection pad

(2Z) -4 - [(4-hydroxy-3-methoxy) benzylamino] -4-oxo-2-butenoic acid-OVA) at a concentration of 0.4 mg / mL , And was coated on the nitrocellulose membrane in the transverse direction at a position 9 mm away from the nitrocellulose membrane by the pre-spraying method to obtain the detection line, and the capsaicin material-coated antigen Was 0.24 쨉 g, and then dried at 37 째 C for 30 minutes;

The coating buffer used for the coating solution of the coating antigen ((2Z) -4 - [(4-hydroxy-3-methoxy) benzylamino] -4-oxo-2-butenoic acid-OVA) 0.1 g of albumin OVA, 0.002 g of sodium azide, 0.08 g of sodium chloride, 0.029 g of disodium phosphate dodecahydrate, 0.002 g of potassium chloride and 0.002 g of potassium dihydrogenphosphate;

Quality control line coating: A rabbit anti-mouse polyclonal antibody was prepared with a coating solution having a concentration of 0.5 mg / mL. The antibody was coated on the nitrocellulose membrane in a transverse direction at a position 10 mm away from the detection line, The amount of coating of rabbit anti-mouse polyclonal antibody required by every centimeter quality control line is 0.3 μg, followed by drying at 37 ° C for 30 minutes;

The coating buffer used for the above rabbit anti-mouse polyclonal antibody coating solution contained 0.1 g of bovine serum albumin, 0.002 g of sodium azide, 0.08 g of sodium chloride, 0.029 g of disodium phosphate dodecahydrate, 0.002 g of potassium chloride, 0.002 g of potassium dihydrogenphosphate is contained;

The nitrocellulose membrane has a length of 28 mm and a width of 4 mm.

(3) Preparation of sample pad

The glass fiber membrane is cut into a standard size of 15 mm in length and 4 mm in width. The glass fiber membrane is then impregnated with a blocking solution, which is then taken out and dried at 37 ° C for 8 hours to obtain a sample pad, which is then placed in a drier and stored at room temperature.

The above blocking solution contains 1 g of ovalbumin, 2 g of sucrose, 0.02 g of sodium azide, 0.8 g of sodium chloride, 0.29 g of disodium phosphate dodecahydrate, 0.02 g of potassium chloride, and 0.02 g of potassium dihydrogenphosphate per 100 mL of the solution.

(4) Manufacture of gold pads

The glass fiber membrane was cut into a specimen having a length of 8 mm and a width of 4 mm and then put in a blocking liquid to be taken out. The specimen was taken out of the glass fiber membrane and dried for 8 hours under a condition of 37 캜. In the dried glass fiber membrane, The polyclonal antibody solution for the labeled anti-capsaicin substance was applied by spraying in the transverse direction, and the dose of the polyclonal antibody used for the anti-capsaicin substance labeled with nano gold required by the spray coating length of every centimeter was 0.5 μg, Then freeze-drying in a vacuum for 6 hours, placing in a drier and preserving at room temperature;

1 g of ovalbumin, 2 g of sucrose, 0.02 g of sodium azide, 0.8 g of sodium chloride, 0.29 g of disodium phosphate dodecahydrate, 0.02 g of potassium chloride and 0.02 g of potassium dihydrogenphosphate are contained per 100 mL of the above blocking solution.

The polyclonal antibody solution for the anti-capsaicin substance labeled with the nano-gold is prepared by an unsaturated labeling method, and the specific method thereof is as follows. 50.0 mL of a nano gold solution having a commercially available mass concentration of 0.01% was taken and the pH value was adjusted with 0.5 mL of a 0.1 mol / L potassium carbonate aqueous solution, and 2.0 mL of 0.1 mg / mL of the anti-capsaicin substance Antibody solution was added, and the mixture was continuously stirred for 30 minutes; Add an aqueous ovalbumin (OVA) solution having a mass concentration of 10%, add until the final mass concentration of OVA reaches 1%, and continue stirring for 30 minutes; Left at 4 캜 for 2 hours, centrifuged at 3000 r / min for 15 minutes, take the supernatant, and remove the precipitate; The supernatant is centrifuged at 12000 r / min for 30 min, the supernatant is removed, and 40.0 mL of label wash stock is added; Centrifuged again at 12,000 r / min for 30 minutes, the supernatant liquid was removed, and the saliva label wash liquid was resuspended, and 5.0 mL concentrate was obtained and placed in a refrigerator at 4 ° C.

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

The aqueous 0.1 mol / L potassium carbonate solution was obtained by filtering 13.8 g of potassium carbonate into purified water to 1000 mL and filtering through a 0.22 μm filtration membrane; The label wash preservative solution was obtained by filtering 2.0 g polyethylene glycol-20000, 0.2 g sodium azide and 0.1235 g boric acid to 1000 mL in purified water and filtering through a 0.22 mu m filtration membrane.

(5) assembly of strips

As shown in FIG. 1, the absorbent pad, the detection pad, the gold pad, and the sample pad are sequentially bonded from one side to the other side of the cardboard, and the adjacent pad portions are connected to overlap each other. 1 mm, thereby obtaining a kitchen waste oil immunochromatographic test paper.

The above-mentioned colloidal gold immunochromatographic strips can be applied rapidly to distinguish kitchen waste oil

Take 20 grams of each of three edible vegetable oil blank samples, with pH values of 5.3, 5.8, and 6.4, respectively, and numbers of 1, 2, and 3, respectively. Ethanol solution having a volume concentration of 50 ml of 95% was added, and the mixture was uniformly mixed and refluxed at 90 ° C for 1 hour. After cooling, the extract was rotary-dried in vacuo and re-eluted by adding 1 ml of 10% methanol- 200 μL of the measurement atmospheric sample solution was taken and added dropwise to the sample pad of the colloidal gold immunochromatography strip of the kit for the determination of waste oil rapidly as a detection solution. This was used as a detection strip, and the same volume of methanol Separately, the methanol-PBS solution was added dropwise to the sample pad of the colloidal gold immunochromatographic strip for rapid discrimination of kitchen waste oil, and this was used as a control strip. Ten minutes later, the detection strip and the control strip And the result is read as follows.

Detection Results: A red band appeared on the quality control line of the three sample detection strips, and no color appeared on the detection line. This was judged as a positive result, and the content of capsaicin was higher than 3 ng / ml, All of which are kitchen waste oil samples. Capsaicin immunoimmune affinity column - high performance liquid chromatography - double mass spectrometry, the results show that the total amount of capsaicin and dihydro capsaicin in samples 1,2,3 is 36, 86, and 1213 ng / mL, respectively It can be determined that the waste oil is kitchen waste oil. These results are consistent with the determination results of the method for rapid classification of waste colloidal gold colloid strips of a kit according to the present invention.

In the present invention, the emotion standard of the kitchen waste oil sample is mainly based on the following causes. The capsaicin content of a large number of actual samples (including samples of many peanut oil, soybean oil, sunflower seed oil, rapeseed oil, and waste oil) was detected by capsaicin immunoaffinity column and HPLC-MS method, 1. The results showed that no capsaicin material was detected in edible vegetable oils other than peanut oil, the capsaicin content in peanut oil was lower than 1.53 ug / kg (1.53 ng / ml), the content of capsaicin substances in waste milk Is higher than 12.27 ug / kg (12.7 ng / ml), indicating that the capsaicin content of the material is greater than or equal to 3 ng / ml, which is the screening standard for oil samples in kitchen or kitchen waste oil.

Table 1 Capsaicin compound content in vegetable oils Sample number Capsaicin (μg / kg) Dihydro
Capsaicin (μg / kg) Total amount (μg / kg) Waste oil -1 4.43 11.38 15.81 Waste oil-2 2.77 9.50 12.27 Waste oil -3 39.92 71.55 111.47 Waste oil -4 28.58 57.50 86.08 Waste oil -5 56.83 92.15 148.98 Waste oil -6 10.93 7.21 18.14 Waste Oil-7 3.99 12.19 16.18 Waste oil -8 9.72 26.69 36.41 Waste oil -9 274.0 343.5 617.5 Waste oil -10 491.3 721.8 1213.1 Waste oil -11 1385.0 1573.4 2958.4 Peanut oil 1 0.67 0.36 1.03 Peanut oil 2 0.61 0.38 0.99 Peanut oil 3 0.59 0.35 0.94 Peanut oil 4 0.72 0.40 1.12 Peanut oil 5 0.92 0.61 1.53 Peanut oil 6 0.58 0.28 0.86 Peanut oil 7 0.37 0.22 0.59 Peanut oil 8 0.39 0.25 0.64 Peanut oil 9 0.42 0.28 0.7 Peanut oil 10 0.48 0.31 0.79 Peanut oil 11 0.36 0.33 0.69 Peanut oil 12 0.49 0.29 0.78 Soybean oil 1 - Soybean oil 11 - - - Sunflower seed oil 1 - Sunflower seed oil 11 - - - Oilseed rape 1 - Oilseed rape 10 - - - Sesame oil 1 - sesame oil 12 - - - Corn germ oil 1-corn germ oil 11 - - - Olive oil 1 - Olive oil 10 - - - Tea seed oil 1 - Tea seed oil 10 - - Walnut Oil 1 - Walnut Oil 10 - - - Rice bran oil 1 - Rice bran oil 11 - - - Palm oil 1-palm oil 10 - - - Grape seed oil 1-grape seed oil 10 - - -

NOTE: '-' not detected.

Example 7: The manufacturing method of kitchen waste oil immunochromatography test paper has the following steps.

(1) Fabrication of Absorbent Pads

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

(2) Manufacture of detection pad

(2Z) -4 - [(4-hydroxy-3-methoxy) benzylamino] -4-oxo-2-butenoic acid-OVA) at a concentration of 0.2 mg / mL And coated with a nitrocellulose membrane at a position 10 mm away from the nitrocellulose membrane by a line spraying method in a transverse direction to the nitrocellulose membrane to obtain a detection line and a capsaicin material coated antigen Lt; RTI ID = 0.0 > 37 C < / RTI > for 30 minutes;

The coating buffer used for the coating solution of the coating antigen ((2Z) -4 - [(4-hydroxy-3-methoxy) benzylamino] -4-oxo-2-butenoic acid-OVA) 0.1 g of albumin OVA, 0.002 g of sodium azide, 0.08 g of sodium chloride, 0.029 g of disodium phosphate dodecahydrate, 0.002 g of potassium chloride and 0.002 g of potassium dihydrogenphosphate;

Quality control line coating: A rabbit anti-mouse polyclonal antibody was prepared with a coating solution having a concentration of 0.2 mg / mL, and was coated laterally on the nitrocellulose membrane by a line spray method at a position 8 mm away from the detection line, And the amount of coating of the rabbit anti-mouse polyclonal antibody required by the quality control line of every centimeter is 0.12 μg, followed by drying at 37 ° C for 30 minutes;

The coating buffer used for the above rabbit anti-mouse polyclonal antibody coating solution contained 0.1 g of bovine serum albumin, 0.002 g of sodium azide, 0.08 g of sodium chloride, 0.029 g of disodium phosphate dodecahydrate, 0.002 g of potassium chloride, 0.002 g of potassium dihydrogenphosphate is contained;

The nitrocellulose membrane has a length of 25 mm and a width of 3.8 mm.

(3) Preparation of sample pad

The glass fiber membrane is cut into a standard size of 13 mm in length and 3.8 mm in width, and is put into a blocking solution to be penetrated. The glass fiber membrane is taken out of the glass fiber membrane and dried for 8 hours under a condition of 37 캜 to obtain a sample pad.

The above blocking solution contains 1 g of ovalbumin, 2 g of sucrose, 0.02 g of sodium azide, 0.8 g of sodium chloride, 0.29 g of disodium phosphate dodecahydrate, 0.02 g of potassium chloride, and 0.02 g of potassium dihydrogenphosphate per 100 mL of the solution.

(4) Manufacture of gold pads

The glass fiber membrane was cut into a size of 9 mm in length and 3.8 mm in width and impregnated with the blocking solution. The glass fiber membrane was taken out from the glass fiber membrane and dried for 8 hours under the condition of 37 캜. In the dried glass fiber membrane, Labeled capsaicin substance was sprayed in the transverse direction and the dose of the polyclonal antibody for anti-capsaicin substance labeled with nano gold required by the spray coating length of every centimeter was 0.6 μg , Lyophilized for 6 hours in a vacuum state, placed in a drier, and stored at room temperature;

1 g of ovalbumin, 2 g of sucrose, 0.02 g of sodium azide, 0.8 g of sodium chloride, 0.29 g of disodium phosphate dodecahydrate, 0.02 g of potassium chloride and 0.02 g of potassium dihydrogenphosphate are contained per 100 mL of the above blocking solution.

The polyclonal antibody solution for the anti-capsaicin substance labeled with the nano-gold is prepared by an unsaturated labeling method, and the specific method thereof is as follows. 50.0 mL of a nano gold solution having a commercially available mass concentration of 0.01% was taken and the pH value was adjusted with 0.5 mL of a 0.1 mol / L potassium carbonate aqueous solution, and 2.0 mL of 0.1 mg / mL of the anti-capsaicin substance Antibody solution was added, and the mixture was continuously stirred for 30 minutes; Add an aqueous ovalbumin (OVA) solution having a mass concentration of 10%, add until the final mass concentration of OVA reaches 1%, and continue stirring for 30 minutes; Left at 4 캜 for 2 hours, centrifuged at 3000 r / min for 15 minutes, take the supernatant, and remove the precipitate; The supernatant is centrifuged at 12000 r / min for 30 min, the supernatant is removed, and 40.0 mL of label wash stock is added; Centrifuged again at 12,000 r / min for 30 minutes, the supernatant liquid was removed, and the saliva label wash liquid was resuspended, and 5.0 mL concentrate was obtained and placed in a refrigerator at 4 ° C.

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

The aqueous 0.1 mol / L potassium carbonate solution was obtained by filtering 13.8 g of potassium carbonate into purified water to 1000 mL and filtering through a 0.22 μm filtration membrane; The label wash preservative solution was obtained by filtering 2.0 g polyethylene glycol-20000, 0.2 g sodium azide and 0.1235 g boric acid to 1000 mL in purified water and filtering through a 0.22 mu m filtration membrane.

 (5) assembly of strips

As shown in FIG. 1, the absorbent pad, the detection pad, the gold pad, and the sample pad are sequentially bonded from one side to the other side of the cardboard, and the adjacent pad portions are connected to overlap each other. 1 mm, thereby obtaining a kitchen waste oil immunochromatographic test paper.

The above-mentioned colloidal gold immunochromatographic strips can be applied rapidly to distinguish kitchen waste oil

The collected kitchen waste oil samples and 20 g of the unused edible vegetable mixed oil samples were taken, and the pH values were 5.4 and 7.2, respectively. An aqueous ethanol solution having a volume concentration of 50 ml of 95% was added thereto, uniformly mixed, After cooling, the extract solution was vacuum-dried, and the residue was re-eluted with 1 ml of a 10% methanol-PBS solution to obtain a measurement atmospheric sample solution. 180 占 퐇 of this atmospheric sample solution was again taken and capsaicins A rapid drop of methanol was added dropwise to the sample pad of the colloidal gold immunochromatographic strip, which was used as a detection strip. A methanol-PBS solution corresponding to the same volume of methanol was separately taken as a negative control solution, Dropwise to the sample pad of the immunochromatographic test strip as a control strip, after 10 minutes, The contrast control is performed on the control strip and the result is read as follows.

A red band appears on the quality control line of the unused edible vegetable blended oil sample detection strip and all of the detection lines are close to the detection limited color of the blank control strip, which is determined as a negative result, and the unused edible vegetable mixed milk sample It is determined that the content of the capsaicin material in the mixed oil is less than 3 ng / ml, indicating that the mixed oil is not doped or a very small amount of doped is the kitchen waste oil sample. A red band appeared on the quality control line of the collected kitchen waste oil sample detection strip and no color appeared on the detection line. This was judged to be a positive result, and the content of capsaicin substances in collected kitchen waste oil samples was higher than 3 ng / ml .

1: cardboard, 2: absorptive pad, 5: detection pad, 6: gold pad, 7: sample pad, 3: quality control line, 4: detection line.

Claims (11)

The artificial immunogenic antigen compound for capsaicin class substance, according to the formula I,

Wherein the antibody is an antibody specific for an anti-capsaicin substance, which is obtained by immunizing an animal with an artificial immunoantigen compound for capsaicin according to Formula I, separating and purifying the animal, followed by lyophilization.

The method according to claim 1,
Wherein said immunization is emulsified using a Freund ' s complement or Freund ' s incomplete adjuvant and then immunized with the animal in duplicate at intervals wherein the first immunization uses a Freund ' s complete adjuvant, Using an incomplete adjuvant until the antitumor activity reaches 10,000 or more, carotid artery blood collection until death, centrifugation of the collected blood, and purification of the antiserum to obtain a capsaicin material antibody Antibodies specific for anti-capsaicin substances.
The method according to claim 1,
The immunosuppressive capacity of the artificial immunoantigen compound for capsaicin according to the above formula I is calculated to be 0.2-1.0 mg by the OVA protein therein; The number of immune layers is 4-6 times; The first immunization emulsifies the immunogen with the same volume of Freund's adjuvant, administering subcutaneous multi-point injections to the rabbit; After the first immunization, the second immunization is performed at intervals of 2 to 4 weeks, the second and subsequent immunizations are performed at intervals of 2 to 3 weeks, and the immunogen is emulsified with the same volume of Freund's incomplete adjuvant, Administer subcutaneous multi-point injections to the back; From the fourth time, every 6th day after immunization, the rabbit ear vein was collected and the serum titer was measured by the indirect competitive ELISA method. After the test was completed, the carotid artery blood was collected until the potency reached 10000, Wherein the blood is centrifuged and the antisera is purified using ammonium octanoate-ammonium sulfate to obtain a capsaicin material antibody.
A detection pad, a gold pad, and a sample pad are successively attached to one surface of a cardboard from top to bottom in such a manner that adjacent pad-to-pad portions overlap each other, and the detection pad is connected to a nitrocellulose membrane A longitudinal quality control line and a detection line are provided on the nitrocellulose membrane from top to bottom as a base pad, the quality control line is coated with a rabbit anti-mouse polyclonal antibody, and the detection line is coated with a capped- Coated antigen is coated; Wherein the gold pad is sprayed in a longitudinal direction with a specific antibody for capsaicin-type material according to claim 1 labeled with nano gold.
6. The method of claim 5,
The absorbent pad has a length of 10 to 15 mm and a width of 3 to 4 mm; the detection pad has a length of 25 to 30 mm and a width of 3 to 4 mm; The gold pad is 6 to 9 mm long and 3 to 4 mm wide; The sample pad has a length of 12 to 15 mm and a width of 3 to 4 mm, and the overlapping length of neighboring pads is 1 to 3 mm;
Wherein the absorbent pad is an absorbent paper.
6. The method of claim 5,
The gap between the detection line on the detection pad and the nitrocellulose membrane is 8 to 20 mm, the gap between the detection line and the quality control line is 5 to 10 mm; Wherein the nano gold used in the gold pad has a particle diameter of 15 to 20 nm.
6. The method of claim 5,
The molecular structure of the capsaicin-coated antigens is shown in Formula II,

;
The coating amount of the coated antigen required for each centimeter on the detection line of the detection pad is 0.1 to 0.6 mu g; The coverage of rabbit anti-mouse polyclonal antibody required per centimeter on the quality control line is 0.1-0.6 μg;
Wherein the amount of the specific antibody for the anti-capsaicin substance labeled with nano gold required for each centimeter on the gold pad is 0.40-0.98 micrograms.
(1) Fabrication of Absorbent Pads
Cutting the absorbent paper to obtain an absorbent pad;
(2) Manufacture of detection pad
Coating of the detection line: Capsaicin-coated material was prepared as a coating solution having a concentration of 0.17 to 1.0 mg / mL. The coating solution was coated on the nitrocellulose membrane in a transverse direction at a position 8 to 20 mm away from the nitrocellulose membrane by the pre- To obtain a detection line, and the coating amount of the artificial complete antigen-coated antigen for a capsaicin-like material conjugate required by the detection line of every centimeter is 0.1 to 0.6 μg, followed by drying at 37 ° C for 30 to 60 minutes;
Quality control line coating: A rabbit anti-mouse polyclonal antibody was prepared with a coating solution having a concentration of 0.17 to 1.0 mg / mL, and this was coated on the nitrocellulose membrane in a transverse direction at a position 5 to 10 mm away from the detection line, To obtain a quality control line, and the covering amount of the rabbit anti-mouse polyclonal antibody required by the quality control line of every centimeter is 0.1 to 0.6 μg, and then drying at 37 ° C for 30 to 60 minutes;
(3) Preparation of sample pad
The glass fiber membrane is put in a blocking solution, the glass fiber membrane is taken out, the glass fiber membrane is taken out and dried at 37 to 40 ° C for 6 to 12 hours to obtain a sample pad, followed by putting it in a drier and preserving it at room temperature;
(4) Manufacture of gold pads
The glass fiber membrane was put in a blocking solution, and the glass fiber membrane was taken out. The glass fiber membrane was taken out from the glass fiber membrane and dried at 37 to 40 ° C for 6 to 12 hours. On the dried glass fiber membrane, a capsaicin material labeled with nano- The general polyclonal antibody solution was spray coated in the transverse direction and the dose of the polyclonal antibody for nanoparticle-labeled anti-capsaicin substance, which requires spray coating length of several centimeters, was 0.4 to 0.98 ng. Lyophilized for 2 to 6 hours, placed in a drier and preserved at room temperature;
(5) assembly of strips
A pad, a gold pad, and a sample pad are successively bonded from one side of the cardboard to one side of the cardboard, from top to bottom, and the adjacent pads are connected to each other so as to overlap with each other, and the overlapping length is 1 to 3 mm, A method for rapid classification of kitchen waste oil immunochromatography according to claim 5, comprising the step of obtaining a waste oil immunochromatographic test paper.
Measured samples of kitchen waste oil were taken and the capsaicin substances contained therein were extracted. The extract was dried and then re-eluted by adding a solvent. The ratio of the measurement atmospheric sample to the re-leaching solvent was 20 g: 1 ml, , And the measurement was performed while dropwise dropwise adding to the sample pad of the waste oil immunochromatography test paper as the detection solution, using this measurement atmospheric sample solution as a detection strip, taking an aqueous methanol solution corresponding to the same volume of methanol concentration separately As a negative control solution, dropwise dropwise to a sample pad of another kitchen waste oil immunochromatography test strip, using the same as a control strip, and performing a light control on the detection strip and the control strip after a predetermined time,
Detection result:
(1) Negative: When the quality control line on the detection strip is colored and the color of the detection line is close to the color of the detection line on the control strip, the content of capsaicin, dihydro capsaicin or synthetic capsaicin, / mL, with the provable method being able to determine whether the sample is a kitchen waste oil or a kitchen waste oil doped oil sample; If the content is less than 3 ng / mL, the measurement atmospheric sample is not likely to be kitchen waste oil and needs to be further confirmed in a provable manner;
(2) Positive: When the quality control line on the detection strip is colored and the color of the detection line is lighter than the color of the detection line on the control strip, the content of capsaicin, dihydro capsaicin or synthetic capsaicin in the atmospheric sample solution is less than 3 ng / mL Or higher, it can be determined that the sample is an oil sample of kitchen waste oil or kitchen waste oil doped;
(3) Invalid: When the quality control line is not colored, even if the detection line of the detection strip is colored, the strip is determined to be invalid.
11. The method of claim 10,
The extraction method includes the step of mixing an aqueous alcohol solution having a volume concentration of 95% in a measurement atmospheric sample, uniformly mixing, refluxing at 60 to 90 ° C for 1 hour, and cooling to obtain an extract; Wherein the re-eluting solvent is 10% methanol-PBS.

Images