Method for detecting residual wheat-specific proteins in rice products

Abstract

The application belongs to the technical field of immunochromatographic detection, and particularly relates to a detection method of residual wheat-specific protein in rice products. The detection method comprises the following steps: step S1: preparing an antibody; step S2: preparing an immunochromatographic test paper; step S3: preparing a developing solution; and step S4: wheat-specific protein detection. The application significantly improves the detection sensitivity and specificity of wheat prolamin by optimizing the formula of the developing solution to screen inhibitory proteins, synergizing with surfactants, and combining with the preparation process of multiple batches of antibodies.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of immunochromatographic detection, and particularly relates to a detection method for residual wheat specific protein in rice products. BACKGROUND

[0002] Allergic diseases are clinical diseases caused by the body's incorrect immune response to external stimuli. Allergens exist in all aspects of life. Currently known allergens that can cause food allergies include grains, eggs, beans, fish, milk, seafood, mangoes and the like.

[0003] Immunoassay detects whether a certain type of antibody that causes an allergic reaction exists in the patient's body or whether an antigen that can cause an allergic reaction exists in the food through the specific reaction of antibodies and antigens in food.

[0004] However, the existing method for detecting allergens in food by using immunization has the following defects:

[0005] 1. Due to the variety and diversity of allergens in food, there are often cases where such allergens produce incorrect responses to antibodies of other types, interfering with the detection results.

[0006] 2. Even for the same allergen in the same food, since the allergen in the food is usually a protein, and the same protein can have different substructures, such substructure antigens usually do not cause a response of antibodies of other substructures.

[0007] In summary, due to the complexity and diversity of the microstructure of antigens and antibodies, and the diversity of the sources of food allergens, the use of immunization for detection often results in false positives and false negatives. SUMMARY

[0008] The purpose of the present application is to provide a detection method for residual wheat specific protein in rice products, which solves the problems of false positives and false negatives in existing immunization detection methods.

[0009] In order to achieve the above purpose, the present application provides the following technical solutions:

[0010] A detection method for residual wheat specific protein in rice products, comprising the following steps:

[0011] Step S1: preparing antibodies, using standard wheat specific proteins of different molecular weights to immunize mice, and preparing antibodies corresponding to the wheat specific proteins;

[0012] Step S2: preparing an immunochromatographic test paper, using the standard wheat specific proteins of different molecular weights in step S1 and the prepared antibodies to prepare an immunochromatographic test paper, the immunochromatographic test paper comprising sample chromatography paper, a developing liquid carrier and an absorber connected in sequence;

[0013] Step S3: preparing a developing solution, extracting wheat-specific proteins from the rice product, dissolving the extracted wheat-specific proteins in the developing solution to obtain an analysis solution;

[0014] Step S4: detecting wheat-specific proteins, adding the analysis solution dropwise to the sample chromatography paper, detecting the wheat-specific proteins in the rice product by observing the color development of the analysis solution on the developing solution carrier;

[0015] The developing solution comprises a buffer, a non-specific reaction inhibitor, a non-ionic surfactant, a chelating agent, and a preservative; the non-specific reaction inhibitor comprises a non-specific reaction inhibiting protein and an ionic surfactant.

[0016] In the step S1, the mice are immunized with standard wheat-specific proteins of different molecular weights to prepare antibodies corresponding to the wheat-specific proteins, including the following steps:

[0017] Step S11: taking 5-6-week-old healthy mice for three times of immunization: the first time of basic immunization, 0.08-0.12 g of standard wheat-specific proteins is mixed with water-soluble adjuvant containing the same amount of effective components, and then intraperitoneal injection is performed; two weeks after the immunization, the same method is used for the second time of immunization, and four weeks after the immunization, the same method is used for the third time of immunization;

[0018] Step S12: cell fusion: the mice are killed by the cervical dislocation method, and the bone marrow cells and the spleen cells after the immunization are taken; at the same time, the same age mice without immunization are treated by the cervical dislocation method, distilled water is injected into the abdominal cavity, and the supernatant is obtained after blowing and sucking after the centrifugation to obtain a feeder cell solution;

[0019] The bone marrow cells and the spleen cells are washed once with I MDM (I scove's Modified Dulbecco's Medium), centrifuged at 1200-1600 r / min for 5 minutes, the supernatant is discarded, the feeder cell solution is added to the precipitate to obtain a cell fusion suspension;

[0020] Step S13: the cell fusion suspension is inoculated into a fusion plate, and the culture solution is replaced every 6-7 days at room temperature; 14 days later, the I MDM complete culture solution is replaced according to the proliferation;

[0021] Step S14: the supernatant of the plate hole of the fusion plate is taken regularly for antibody detection, and after the cells are completely cloned, the mouse abdominal cavity is inoculated, the ascites is taken after the ascites is sufficient, and the antibody corresponding to the wheat-specific proteins is obtained by immunization affinity chromatography purification.

[0022] In the step S12, the cell number ratio of the bone marrow cells to the spleen cells is 1.5:1 to 1:1.5.

[0023] In the step S2, the detection area and the control area are arranged in sequence along the sample moving direction in the development liquid carrier.

[0024] The detection area is formed by uniformly applying the marker solution to the area where the detection area is located; and the control area is formed by uniformly applying the control liquid to the area where the control area is located.

[0025] The marker solution is prepared by the following method:

[0026] In step S21, the prepared antibody is diluted to 2-4 mg / mL with 0.008-0.012 mol / L PBS buffer to obtain an antibody liquid.

[0027] In step S22, the antibody liquid prepared in step S21 is added to the colloidal gold suspension in a ratio of 5:1 to 4:1 by volume, and is left to stand at room temperature for ten minutes.

[0028] In step S23, 0.08-0.12 times the volume of potassium carbonate solution and 0.008-0.012 times the volume of polyethylene glycol are further added to the colloidal gold suspension, which is fully stirred and then centrifuged at 10000-12000 r / min for 5 minutes. The precipitate is resuspended with 100-105 times the volume of 0.8-1.2% bovine serum albumin solution to obtain the marker solution.

[0029] The control liquid is prepared by the following method:

[0030] In step S2a, the wheat-specific protein is diluted to 2-4 mg / mL with 0.008-0.012 mol / L PBS buffer to obtain a wheat-specific protein liquid.

[0031] In step S2b, the wheat-specific protein liquid prepared in step S2a is added to the colloidal gold suspension in a ratio of 5:1 to 4:1 by volume, and is left to stand at room temperature for ten minutes.

[0032] In step S2c, 0.08-0.12 times the volume of potassium carbonate solution and 0.008-0.012 times the volume of polyethylene glycol are further added to the colloidal gold suspension, which is fully stirred and then centrifuged at 10000-12000 r / min for 5 minutes. The precipitate is resuspended with 100-105 times the volume of 0.8-1.2% bovine serum albumin solution to obtain the control liquid.

[0033] In the step S3,

[0034] The buffer of the developing solution is selected from acetic acid buffer, boric acid buffer, Tris (Tris (hydroxymethy l) aminomethane) buffer or sodium citrate buffer;

[0035] The non-ionic surfactant of the developing solution is selected from amine oxide or oxirane;

[0036] The chelating agent of the developing solution is selected from ethylenediamine;

[0037] The preservative of the developing solution is selected from benzoic acid or sorbic acid;

[0038] The non-specific reaction inhibiting protein is selected from any one of fetal bovine serum, casein or gelatin;

[0039] The ionic surfactant is selected from lecithin or sodium dodecylbenzenesulfonate.

[0040] The mass concentration of the non-ionic surfactant, the chelating agent, the preservative, the non-specific reaction inhibiting protein and the ionic surfactant in the developing solution is 0.18-0.22%, 0.08-0.12%, 0.08-0.12%, 5-10% and 3-8% respectively.

[0041] In the step S3, the wheat specific protein is extracted by the following method:

[0042] In step S31, 500g of rice products are ultrasonically pulverized, 0.9-1.1L of 0.1-0.2M sodium chloride aqueous solution is added, 10000-12000r / min centrifugation is carried out for 5 minutes, the supernatant is taken, sodium sulfate is slowly added until the precipitation appears, and the precipitation is placed at 4 degrees Celsius overnight.

[0043] In step S32, 10000-12000r / min centrifugation is carried out for 5 minutes, the supernatant is collected, and sodium sulfate is slowly added until the precipitation appears.

[0044] In step S33, step S32 is repeated for three to four times, and the precipitation of the last time is collected.

[0045] In step S34, the precipitation is vacuum frozen to obtain the wheat specific protein.

[0046] The detected wheat specific protein is wheat prolamin.

[0047] Compared with the prior art, the advantages and beneficial effects of the present application are:

[0048] 1. In response to the diverse types, complex structures, and interfering detection of food allergens, the inventors attempted to improve the formulation of the developing solution to enhance the specificity of the reaction. Different antigens require different inhibitors to suppress the specific reaction of their corresponding interfering antigens (relative molecular mass, determinant structure, etc., all affect the binding of antigen and antibody). The inventors first conducted a preliminary screening of non-specific reaction inhibitors using a simplified procedure: various non-specific reaction inhibitors were mixed with various food allergen standards, and then mixed with the labeled solution prepared in this invention. The color development of the solution was observed. Preliminary judgment indicated that protein-based non-specific reaction inhibitors had a more ideal inhibitory effect on non-specific reactions in plant allergens. Therefore, fetal bovine serum, casein, gelatin, β-lactoglobulin, and lactalbumin were further screened as non-specific reaction inhibitors. Rigorous antigen extraction, test strip preparation, and immunochromatographic experiments were then conducted, ultimately selecting a non-specific reaction inhibitor protein specifically suitable for the detection of wheat gliadin that met both sensitivity and specificity standards.

[0049] 2. Nonspecific reaction inhibitory proteins are expensive, and adding too little is insufficient to inhibit nonspecific reactions, while adding too much saturates the inhibitory effect, resulting in wasted costs. Therefore, it is necessary to explore the optimal addition range for various nonspecific reaction inhibitory proteins. Based on this, in order to further reduce the amount of nonspecific reaction inhibitory proteins used, the inventors attempted to verify whether the surfactants required for the preparation of the developing solution have a synergistic effect on the inhibition of nonspecific reactions. Surfactants broadly include nonionic surfactants and ionic surfactants. Experiments showed that adding only the latter could not achieve interfacial homogenization of wheat gliadin, while adding only the former could not synergistically promote the inhibition of nonspecific reactions. Therefore, the inventors hypothesized that nonionic surfactants are necessary for the uniform distribution of wheat gliadin in the developing solution. Based on this, the inventors further selected small-molecule ionic surfactants to find those that could synergistically inhibit nonspecific reactions. It was verified that lecithin or sodium dodecylbenzenesulfonate can indeed improve the inhibitory effect of nonspecific reaction inhibitory proteins on nonspecific reactions.

[0050] 3. Considering the relatively large molecular weight range of wheat gliadin, the inventors purchased multiple batches of wheat gliadin standards during antibody preparation to cover as many types of proteins as possible found in natural foods. Furthermore, by combining multiple booster immunizations and cell co-cultures during antibody preparation with multiple centrifugation processes during antigen extraction, the protein extracted from natural wheat achieved the same detection intensity as the standards during antigen testing. This further demonstrates that the present invention significantly improves detection accuracy through the combination of antibody preparation, antigen extraction, and other processes. Attached Figure Description

[0051] Figure 1 A flow chart of the steps of the method for detecting residual wheat specific proteins in rice products of the present application. DETAILED DESCRIPTION

[0052] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0053] I. Screening of non-specific reaction inhibitor components

[0054] Embodiment 1:

[0055] Four wheat gliadin standard samples with different molecular weights were purchased from the Merck website: standard sample 1 to standard sample 4, with the following relative molecular weights: standard sample 1: 25000-35000, standard sample 2: 32000-38000, standard sample 3: 35000-40000, and standard sample 4: 38000-45000. The mixed standard sample was prepared by mixing equal amounts of the above standard samples.

[0056] Step S1: Preparation of antibodies;

[0057] Step S11: 5-6-week-old healthy mice were immunized three times: the first time was basic immunization, 0.1 g of the mixed standard sample was mixed with 0.1 g of complete Freund's adjuvant dissolved in water (1 mL) to obtain a mixture, and the mixture was injected into the abdominal cavity; two weeks after immunization, the same method was used for booster immunization, and four weeks after immunization, the same method was used for booster immunization again;

[0058] Step S12: The mice were sacrificed by cervical dislocation, and the bone marrow cells and spleen cells after immunization were taken; at the same time, the same age mice without immunization were treated by cervical dislocation, and distilled water was injected into the abdominal cavity, and after blowing and sucking, the feeder cell solution was obtained; the bone marrow cells and spleen cells (cell ratio 1:1) were washed once with IMDM incomplete culture solution, centrifuged at 1500 r / min for 5 minutes, the supernatant was discarded, and the feeder cell solution was added to the precipitate to obtain a cell fusion suspension;

[0059] Step S13: The cell fusion suspension was inoculated into a fusion plate and cultured at room temperature, the culture solution was replaced every seven days, and after 14 days, the IMDM complete culture solution was replaced according to the proliferation situation;

[0060] Step S14: periodically take the supernatant of the fusion plate hole for antibody detection, after the cell is completely cloned, the mouse abdominal cavity is inoculated, after the ascites is sufficient, the ascites is taken, and the antibody corresponding to the wheat specific protein is obtained through the immunization affinity chromatography purification.

[0061] Step S2: prepare an immunochromatography test paper;

[0062] Steps S21 to S23: prepare a label solution;

[0063] The prepared antibody is diluted to 3 mg / mL with 0.01 mol / L PBS buffer solution to obtain an antibody solution; the antibody solution is added to the colloidal gold suspension (purchased from Xingboseng Biological) at a volume ratio of 5:1, and after standing at room temperature for ten minutes, 0.1 times the volume of a potassium carbonate solution (concentration 0.2 mol / L) and 0.01 times the volume of polyethylene glycol (purchased from the Merck website, product number 202398, relative molecular mass 380-420, density 1.128 g / mL) are continuously added; after fully stirring, centrifugation is performed at 12000 r / min for 5 minutes, and the precipitate is resuspended with 100 times the volume of a bovine serum albumin solution with a mass concentration of 1% to obtain a label solution;

[0064] Steps S2a to S2b: prepare a control solution;

[0065] The mixed standard is diluted to 3 mg / mL with 0.01 mol / L PBS buffer solution to obtain a wheat gliadin solution; the wheat gliadin solution is added to the colloidal gold suspension (purchased from Xingboseng Biological) at a volume ratio of 5:1, and after standing at room temperature for ten minutes; 0.1 times the volume of a potassium carbonate solution (concentration 0.2 mol / L) and 0.01 times the volume of polyethylene glycol (purchased from the Merck website, product number 202398, relative molecular mass 380-420, density 1.128 g / mL) are continuously added; after fully stirring, centrifugation is performed at 12000 r / min for 5 minutes, and the precipitate is resuspended with 100 times the volume of a bovine serum albumin solution with a mass concentration of 1% to obtain a control solution;

[0066] The label solution and the control solution are coated on the detection area and the control area of the development liquid carrier respectively, and dried, and then the sample chromatography paper, the development liquid carrier and the absorber are connected from left to right in sequence to obtain an immunochromatography test paper; wherein the materials of the sample chromatography paper, the development liquid carrier and the absorber are known in the art, and will not be described here in detail.

[0067] Step S3: prepare a development liquid;

[0068] An acetic acid buffer solution is used as a solvent to dissolve 0.2% of an amine oxide, 0.1% of ethylenediamine, 0.1% of benzoic acid and 8% of fetal bovine serum to prepare a development liquid.

[0069] Step S31 to Step S34: allergen extraction;

[0070] Take 500g of peanuts, milk, millet, and soybeans respectively, and ultrasonically pulverize them. Add 1L of a 0.15M sodium chloride aqueous solution, and centrifuge at 12000r / min for 5 minutes. Take the supernatant, slowly add sodium sulfate until a precipitate appears, and let it stand overnight at 4 degrees Celsius. Repeat the centrifugation 5 times, each time at 10000-12000r / min for 5 minutes. Collect the precipitate from the last time, and vacuum freeze the precipitate to obtain the allergen.

[0071] Step S4: wheat-specific protein detection;

[0072] Dissolve the obtained allergen and mixed standard into the developing solution prepared in Step S3 above to obtain allergen solutions of different concentrations, and perform immunochromatographic detection. Observe the color development results, as shown in Tables 1-3.

[0073] Example 2

[0074] The difference from Example 1 is that in Step S3, the non-specific inhibitory protein selected for the prepared developing solution is casein.

[0075] Example 3:

[0076] The difference from Example 1 is that in Step S3, the non-specific inhibitory protein selected for the prepared developing solution is gelatin.

[0077] Example 4:

[0078] The difference from Example 1 is that in Step S3, the non-specific inhibitory protein selected for the prepared developing solution is β-lactoglobulin.

[0079] Example 5:

[0080] The difference from Example 1 is that in Step S3, the non-specific inhibitory protein selected for the prepared developing solution is lactalbumin.

[0081] Table 1: Color development results of each allergen at a concentration of 2ppm

[0082]

[0083] Table 2: Color development results of each allergen at a concentration of 5ppm

[0084]

[0085] Table 3: Color development results of each allergen at a concentration of 10ppm

[0086]

[0087]

[0088] As can be seen from Tables 1 to 3, when any one of fetal bovine serum, casein or gelatin is selected as the non-specific reaction inhibitor, accurate and sensitive specific reaction can be achieved, and when the concentration of the detection substance is 2 ppm, wheat gliadin can be specifically detected, and the allergens in other foods will not interfere with the detection results. In addition, through the detection substance antigen extraction step and the antibody preparation step provided by the system, the same sensitivity as the standard product can be achieved for the detection substance.

[0089] When β-lactoglobulin is selected as the non-specific reaction inhibitor, specific detection can also be achieved, and the allergens in other foods will not interfere, but the detection sensitivity is low, and the test paper will produce color development when the concentration of the detection substance reaches 10 ppm.

[0090] When lactoglobulin is selected as the non-specific reaction inhibitor, high sensitivity detection can be achieved for wheat gliadin, but color development also occurs for other foods that do not contain wheat gliadin, such as peanuts and milk, so the specificity cannot meet the requirements.

[0091] In summary, fetal bovine serum, casein and gelatin are relatively ideal non-specific reaction inhibitors for wheat gliadin detection.

[0092] II. Investigation of the concentration of the non-specific reaction inhibitor

[0093] 1. Preparation of each component and each concentration of the developing solution

[0094] The developing solutions with different components and different concentrations (first group of developing solutions) were prepared in the same manner as in Example 1, and the components and concentrations of the non-specific inhibitors of the different developing solutions are shown in Table 4. Meanwhile, on this basis, a second group of developing solutions was additionally prepared, which had more ionic surfactants (when fetal bovine serum and casein were used as non-specific inhibitors, the ionic surfactant was lecithin (specifically, soy lecithin); when gelatin was used as the non-specific inhibitor, the ionic surfactant was sodium dodecyl benzene sulfonate) compared to the first group of developing solutions.

[0095] The immunochromatographic test paper was obtained in the same manner as in Example 1, and then the first part of the allergen prepared from wheat and the standard product were respectively dissolved in the first group of developing solutions and the second group of developing solutions to obtain allergen solutions with different concentrations (the concentration was 2 ppm), and immunochromatographic detection was performed, and the color development results were observed as follows:

[0096] Table 4: Evaluation of the non-specific inhibition effect of fetal bovine serum with different concentrations

[0097]

[0098] Table 5: Evaluation of non-specific inhibition effect of different concentrations of casein

[0099]

[0100]

[0101] Table 6: Evaluation of non-specific inhibition effect of different concentrations of gelatin

[0102]

[0103]

[0104] From Tables 4 to 6, it can be seen that the amount of the added non-specific reaction inhibitor and the detection intensity of the immunochromatographic test paper are positively correlated within a certain range. If the amount of the added non-specific reaction inhibitor is too small, the immunochromatographic test paper cannot reach the color development threshold, and the non-specific reaction cannot be promoted. If the amount of the added non-specific reaction inhibitor reaches a certain degree, the promotion effect on the color development intensity is saturated, and further increasing the content will only increase the preparation cost of the developing solution, because the manufacturing cost of the immunochromatographic test paper is indirectly increased. In addition, it can be seen from Tables 4 to 6 that no matter which non-specific reaction inhibitor is selected, the addition of the ionic surfactant reduces the minimum amount of the non-specific reaction inhibitor corresponding to the start of color development of the immunochromatographic test paper. Therefore, the addition of the ionic surfactant can synergistically promote the non-specific reaction inhibitor to promote the non-specific reaction, and can reduce the amount of the added non-specific reaction inhibitor.

[0105] In the specific examples corresponding to Tables 4 to 6, the amount of the added ionic surfactant is 5% (mass concentration after being added to the developing solution). Further verification by the present application shows that when the amount of the added ionic surfactant is between 3% and 8%, the effect is the same as that of Tables 4 to 6, and therefore, it is not necessary to repeat the details here.

[0106] For fetal bovine serum, casein and gelatin, in the case of adding the ionic surfactant, in order to achieve sensitive color development, the respective amounts of the added non-specific reaction inhibitors are as follows: 5% to 10%, 5% to 10% and 8% to 12%.

[0107] The application significantly improves the detection performance of wheat prolamin by innovatively optimizing the detection system. First, specific inhibitory proteins such as fetal bovine serum and casein are screened, and the inhibitory effect on cross reaction is verified through step-by-step experiments, which greatly improves the detection specificity. Secondly, the synergistic system of non-ionic surfactant (guaranteeing protein homogenization) and small molecule ionic surfactant (lecithin / sodium dodecyl benzene sulfonate) is innovatively adopted, which reduces the use amount of expensive inhibitory proteins while ensuring the detection sensitivity, and reduces the cost by more than 30%. In addition, through the process combination of multiple batches of standard immunization and multiple stages of centrifugal purification, the antibody covers the prolamin in different molecular weight intervals, and the natural sample detection sensitivity reaches 0.1 ppm, and the false positive rate is less than 5%. The overall scheme has high specificity, economy and detection accuracy, and is suitable for on-site rapid screening of complex food matrix.

[0108] Although embodiments of the application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the application, and the scope of the application is defined by the appended claims and their equivalents.

Claims

1. A method for detecting wheat-specific protein residues in rice products, characterized in that, Includes the following steps: Step S1: Prepare antibodies by immunizing mice with standard wheat-specific proteins of different molecular weights to prepare antibodies corresponding to wheat-specific proteins; Step S2: Prepare immunochromatographic test strips. Using standard wheat-specific proteins of different molecular weights from step S1 and the prepared antibodies, prepare immunochromatographic test strips. The immunochromatographic test strips include sample chromatography paper, developing liquid carrier, and absorber connected in sequence. Step S3: Prepare the developing solution. Extract wheat-specific protein from rice products and dissolve the extracted wheat-specific protein in the developing solution to obtain the analytical solution. Step S4: Detection of wheat-specific proteins. The analytical solution is dropped onto the sample chromatography paper, and the wheat-specific proteins in rice products are detected by observing the color development of the analytical solution on the developing liquid carrier. The developing solution includes a buffer, a non-specific reaction inhibitor, a nonionic surfactant, a chelating agent, and a preservative; the non-specific reaction inhibitor includes a non-specific reaction inhibitory protein and an ionic surfactant. In step S3, the buffer for the developing solution is selected from acetate buffer, borate buffer, Tris buffer or sodium citrate buffer. The nonionic surfactant in the developing solution is selected from amine oxide or ethylene oxide; The chelating agent for the developing solution is selected from ethylenediamine; The preservative for the developing solution is selected from benzoic acid or sorbic acid; The nonspecific reaction inhibitory protein is selected from any one of fetal bovine serum, casein, or gelatin; The ionic surfactant is selected from lecithin or sodium dodecylbenzenesulfonate; In the developing solution, the mass concentrations of nonionic surfactant, chelating agent, preservative, and ionic surfactant are 0.18–0.22%, 0.08–0.12%, 0.08–0.12%, and 3–8%, respectively. The nonspecific reaction inhibitory protein is selected from fetal bovine serum, casein, or gelatin at mass concentrations of 5–10%, 5–10%, and 8–12%, respectively.

2. The method for detecting residual wheat-specific proteins in rice products according to claim 1, characterized in that: In step S1, mice are immunized using standard wheat-specific proteins of different molecular weights to prepare antibodies corresponding to the wheat-specific proteins, including the following steps: Step S11: Take 5-6 week old healthy mice and perform three immunizations: The first basic immunization is to mix 0.08-0.12g of standard wheat-specific protein with a water-soluble adjuvant containing an equal mass of active ingredient and inject it into the peritoneum; two weeks after immunization, perform a booster immunization in the same way; four weeks after immunization, perform a booster immunization in the same way. Step S12: Cell fusion: Mice were euthanized by cervical dislocation, and immunized bone marrow cells and spleen cells were collected; at the same time, unimmunized mice of the same age were also treated by cervical dislocation, and distilled water was injected into the peritoneal cavity, blown out and aspirated to obtain feeder cell fluid. Bone marrow cells and spleen cells were washed once with IMDM incomplete culture medium, centrifuged at 1200-1600 r / min for 5 minutes, the supernatant was discarded, and feeder cell solution was added to the precipitate to obtain a cell fusion suspension. Step S13: Seed the cell fusion suspension into a fusion plate and culture at room temperature. Change the culture medium every 6-7 days. After 14 days, change to IMDM complete culture medium according to the proliferation status. Step S14: Periodically collect the supernatant from the wells of the fusion plate for antibody detection. After the cells are completely cloned, inoculate mice into the peritoneum. Once the ascites is sufficient, collect the ascites and purify it by immunoaffinity chromatography to obtain the antibody corresponding to the wheat-specific protein.

3. The method for detecting residual wheat-specific proteins in rice products according to claim 2, characterized in that: In step S12, the ratio of bone marrow cells to spleen cells is 1.5:1 to 1:1.

5.

4. The method for detecting residual wheat-specific proteins in rice products according to claim 1, characterized in that: In step S2, the developing liquid carrier is provided with a detection area and a control area in sequence along the sample movement direction; The detection zone is formed by uniformly applying the label solution to the area where the detection zone is located; the control zone is formed by uniformly applying the control solution to the area where the control zone is located.

5. The method for detecting residual wheat-specific proteins in rice products according to claim 4, characterized in that, The labeled solution was prepared by the following method: Step S21: Dilute the prepared antibody with 0.008-0.012 mol / L PBS buffer to 2-4 mg / mL to obtain antibody solution; Step S22: Add the antibody solution prepared in step S21 to the colloidal gold suspension at a volume ratio of 5:1 to 4:1, and let it stand at room temperature for ten minutes. Step S23: Continue to add 0.08 to 0.12 times the volume of colloidal gold suspension in potassium carbonate solution and 0.008 to 0.012 times the volume of polyethylene glycol. After stirring thoroughly, centrifuge at 10,000 to 12,000 r / min for 5 minutes, and reconstitute the precipitate with 100 to 105 times the volume of bovine serum albumin solution with a mass concentration of 0.8 to 1.2% to obtain the labeled solution.

6. The method for detecting residual wheat-specific protein in rice products according to claim 4, characterized in that, The control solution was prepared by the following method: Step S2a: Dilute the wheat-specific protein with 0.008–0.012 mol / L PBS buffer to 2–4 mg / mL to obtain a wheat-specific protein solution; Step S2b: Add the wheat-specific protein solution prepared in step S2a to the colloidal gold suspension at a volume ratio of 5:1 to 4:1, and let it stand at room temperature for ten minutes. Step S2c: Continue to add 0.08 to 0.12 times the volume of colloidal gold suspension in potassium carbonate solution and 0.008 to 0.012 times the volume of polyethylene glycol. After stirring thoroughly, centrifuge at 10,000 to 12,000 r / min for 5 minutes, and reconstitute the precipitate with 100 to 105 times the volume of bovine serum albumin solution with a mass concentration of 0.8 to 1.2% to obtain the control solution.

7. The method for detecting residual wheat-specific protein in rice products according to claim 1, characterized in that, In step S3, wheat-specific proteins are extracted using the following method: Step S31: Take 500g of rice products, ultrasonically pulverize them, add 0.9-1.1L of sodium chloride aqueous solution with a concentration of 0.1-0.2M, centrifuge at 10000-12000r / min for 5 minutes, take the supernatant, slowly add sodium sulfate until a precipitate appears, and let stand overnight at 4 degrees Celsius. Step S32: Centrifuge at 10000-12000 r / min for 5 minutes, collect the supernatant, and slowly add sodium sulfate until a precipitate appears; Step S33: Repeat step S32 three to four times, and collect the precipitate from the last time; Step S34: Vacuum freeze the precipitate to obtain wheat-specific protein.

8. The method for detecting residual wheat-specific protein in rice products according to any one of claims 1 to 7, characterized in that, The wheat-specific protein detected was wheat gliadin.

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