A highly stable vomitoxin kit and its preparation method and detection method

Abstract

The present application relates to a highly stable vomitoxin test kit and its preparation method and detection method; a highly stable vomitoxin test kit, comprising a gold-labeled microwell and a test strip, wherein the gold-labeled microwell comprises mouse anti-hazelnut monoclonal antibodies labeled with vomitoxin and mouse anti-vomitoxin monoclonal antibodies labeled with colloidal gold; the test strip comprises a bottom plate, on which a sample pad, a reaction membrane and a water-absorbing pad are sequentially arranged along the chromatography direction; on the reaction membrane, a detection line coated with hazelnut antigen and a quality control line coated with sheep anti-mouse IgG are sequentially arranged along the chromatography direction. The test kit of the present application can improve the disadvantage of traditional vomitoxin quantitative test strips being sensitive to temperature and humidity, freeing it from the limitation of temperature control machines, thereby greatly saving costs and improving detection efficiency.

Description

Technical Field

[0001] The present application relates to the field of immunochromatographic test strips, and more specifically, to a highly stable vomitoxin test kit and a preparation method and a detection method thereof. Background Art

[0002] Vomitoxin, also known as deoxynivalenol (DON), has a chemical name of 3α,7α,15-trihydroxyfusarium-9-ene-8-one, and is mainly derived from the genus Fusarium. Vomitoxin is named because it can cause vomiting in pigs. When humans and animals ingest food contaminated with vomitoxin, they will experience acute poisoning symptoms such as anorexia, vomiting, diarrhea, fever, unsteady standing, and slow reaction. In severe cases, it can damage the hematopoietic system and cause death. Therefore, it is of great significance to quickly and accurately detect vomitoxin in food.

[0003] At present, there are two main methods for detecting vomitoxin: instrumental analysis and immunological detection; instrumental analysis mainly includes thin layer chromatography (TLC), gas chromatography (GC), high pressure liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (HPLC-MS), etc.; immunological detection mainly includes immunoaffinity column-fluorescence detection (IAC-FLD), enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography, etc. The main advantage of instrumental analysis is accurate results, but because it requires the use of large-scale instruments and equipment, the site requirements are relatively high, and the operation process is complicated and time-consuming, so it is not suitable for large-scale sample screening and daily internal control testing. The ELISA method of enzyme-linked immunosorbent assay and the colloidal gold rapid test card are commonly used rapid detection methods by enterprises. Among them, the colloidal gold rapid quantitative test card plays an important role. However, since the DON antigen-antibody reaction is seriously affected by temperature and humidity, the vomitoxin quantitative test strips produced by most manufacturers on the market currently need to be incubated in a machine that can provide a constant temperature and humidity environment for a certain period of time before accurate quantification can be achieved. Not only is the detection cost high, but it also limits the number of samples tested at one time to some extent, reducing the detection rate. Summary of the invention

[0004] In order to solve the above technical problems, the present application provides a highly stable vomitoxin test strip and a preparation method and a detection method thereof. The kit of the present application can not only improve the shortcomings of traditional vomitoxin quantitative test strips that are sensitive to temperature and humidity, but also free them from the limitations of temperature control machines, thereby greatly saving costs.

[0005] In a first aspect, the present application provides a highly stable vomitoxin kit, which adopts the following technical solution:

[0006] A high-stability vomitoxin test kit, comprising a gold-labeled microwell and a test strip;

[0007] The gold-labeled microwells include mouse anti-hazelnut monoclonal antibodies labeled with vomitoxin and mouse anti-vomitoxin monoclonal antibodies labeled with colloidal gold;

[0008] The test strip comprises a bottom plate, on which a sample pad, a reaction membrane and a water absorbent pad are arranged in sequence along the chromatography direction;

[0009] The reaction membrane is provided with a detection line coated with hazelnut antigen and a quality control line coated with goat anti-mouse IgG in sequence along the chromatography direction.

[0010] The present application uses a gold-labeled microwell and a test strip to detect vomitoxin in a sample. After extraction, the sample is added to the gold-labeled microwell to complete the antigen-antibody reaction, and then the test strip is used for detection. The detection principle of the present application is: when the sample extract contains vomitoxin, the vomitoxin (DON) in the sample competes with the mouse anti-hazelnut monoclonal antibody (DON-HZmAb) labeled with vomitoxin for binding to the mouse anti-vomitoxin monoclonal antibody (DONmAb-Gdd) labeled with colloidal gold. Only the conjugate of DON-HZmAb and DONmAb-Gdd can bind to the hazelnut antigen coated with the detection line (T line) on the reaction membrane. The higher the concentration of vomitoxin in the sample, the lower the amount of the conjugate formed, and the lighter the color of the T line. Conversely, the darker the color. Since the hazelnut antigen-antibody reaction in the present application is not sensitive to temperature and humidity, and the vomitoxin antigen-antibody reaction is completed in the aqueous solution of the gold-labeled microwell, after a certain incubation time, the sensitivity of the test strip to temperature and humidity will be greatly reduced. By adopting the above technical solution, the kit of the present application can not only improve the disadvantage that the vomitoxin quantitative test strip is sensitive to temperature and humidity, but also free it from the limitation of the temperature control machine, thereby greatly saving costs and improving detection efficiency.

[0011] Optionally, the gold-labeled microwell is prepared by the following method:

[0012] Mouse anti-hazelnut monoclonal antibody labeled with vomitoxin was taken, mixed evenly with the lyophilized diluent, and then dispensed into a low-adsorption ELISA plate, and freeze-dried to obtain lyophilized powder cake I;

[0013] The colloidal gold-labeled mouse anti-vomiting toxin monoclonal antibody is mixed evenly with the lyophilized diluent, and then divided into low-adsorption ELISA plates, and freeze-dried to obtain lyophilized powder II;

[0014] The freeze-dried powder cake II is transferred to the microwell where the freeze-dried powder cake I is located to obtain the gold-labeled microwell.

[0015] Optionally, the lyophilized diluent comprises components in the following concentrations: 3-7% by mass volume fraction of trehalose and 0.5-2% by mass volume fraction of casein.

[0016] By adopting the above technical solution, the freeze-dried diluent has a certain influence on the stability of the gold-labeled micropores after freeze-drying; when the gold-labeled micropores prepared by the above formula are used in combination with test strips, when detecting vomitoxin, they are less affected by the temperature and humidity of the environment, which is conducive to simplifying the detection operation steps and improving the detection efficiency.

[0017] Optionally, the volume ratio of the deoxynivalenol-labeled mouse anti-hazelnut monoclonal antibody to the lyophilized diluent is 0.01-1:60;

[0018] The volume ratio of the colloidal gold-labeled mouse anti-vomitoxin monoclonal antibody to the lyophilized diluent is 1-10:56.

[0019] Optionally, the colloidal gold-labeled mouse anti-vomitoxin monoclonal antibody is prepared by the following method: add carbonate solution to colloidal gold, mix evenly, add mouse anti-vomitoxin monoclonal antibody while stirring, react at room temperature for 1-3 hours, then add carrier protein solution dropwise, react at room temperature for 1-2 hours, collect the precipitate by centrifugation and resuspend it with a resuspended solution to obtain colloidal gold-labeled mouse anti-vomitoxin monoclonal antibody.

[0020] Optionally, the carrier protein is one of bovine serum albumin, human serum albumin, keyhole limpet hemocyanin, and chicken ovalbumin.

[0021] Preferably, the carrier protein is bovine serum albumin.

[0022] Optionally, the carbonate is one of sodium carbonate and potassium carbonate.

[0023] Preferably, the carbonate is potassium carbonate.

[0024] Optionally, the reconstituted solution comprises components in the following concentrations: bovine serum albumin with a mass volume concentration of 3-7%, trehalose with a mass volume concentration of 1-5%, and Tween-20 with a mass volume concentration of 0.5-2%.

[0025] Optionally, the deoxynivalenol-labeled mouse anti-hazelnut monoclonal antibody is prepared by the following method:

[0026] Dissolve vomitoxin in anhydrous tetrahydrofuran, add N,N-carbonyldiimidazole, and react at room temperature with stirring for 1-3 hours; then dry the tetrahydrofuran, and dissolve the residue in dimethyl sulfoxide to obtain a mixed solution;

[0027] Take mouse anti-hazelnut monoclonal antibody and dissolve it in PBS solution to obtain solution A;

[0028] The mixed solution was slowly added dropwise to solution A; the mixture was reacted at room temperature overnight; the reaction solution was then collected, dialyzed, and frozen to obtain mouse anti-hazelnut monoclonal antibodies labeled with vomitoxin.

[0029] Optionally, the concentration of the hazelnut antigen coated on the detection line is 0.1-10 mg / mL, and the concentration of the sheep anti-mouse IgG on the quality control line is 0.1-10 mg / mL.

[0030] By adopting the above technical solution, the kit has higher sensitivity within this concentration range.

[0031] In a second aspect, the present application provides a method for preparing a highly stable vomitoxin kit, using the following technical solution:

[0032] A method for preparing a highly stable vomitoxin kit comprises the following steps:

[0033] Preparation of gold-labeled microwells:

[0034] Mouse anti-hazelnut monoclonal antibody labeled with vomitoxin was taken, mixed evenly with the lyophilized diluent, divided into ELISA plates, and freeze-dried to obtain lyophilized powder cake I;

[0035] The colloidal gold-labeled mouse anti-vomiting toxin monoclonal antibody is mixed evenly with the lyophilized diluent, and then divided into ELISA plates, and freeze-dried to obtain lyophilized powder II;

[0036] Transfer the freeze-dried powder cake II to the microwell where the freeze-dried powder cake I is located to obtain the gold-labeled microwell;

[0037] Assembly of the test strips:

[0038] The cut reaction membrane is pasted on a PVC plate, goat anti-mouse IgG is diluted and sprayed on the reaction membrane to obtain a quality control line; hazelnut antigen is diluted and sprayed on the reaction membrane to obtain a test line; the reaction membrane after the marking is dried and set aside;

[0039] A water-absorbing pad is attached to the PVC sheet near one end of the reaction membrane quality control line, a sample pad is attached to the PVC sheet near one end of the reaction membrane detection line, and a MAX line is attached to the PVC sheet near one end of the sample pad to obtain a test strip.

[0040] In a third aspect, the present application provides a method for detecting vomitoxin, which adopts the following technical solution:

[0041] A method for detecting vomitoxin, using the kit of the present application for detection, the detection method comprising the following steps:

[0042] ① Take the sample to be tested, add solvent and shake and extract for 5-10 minutes; take the supernatant, mix it with the sample diluent in a volume ratio of 1:8-10 to obtain the test solution;

[0043] ② Take 100-200 μL of the test solution, add it to the gold-labeled microwell, pipette 2-3 times, and incubate at room temperature for 3-10 minutes;

[0044] ③Insert the test strip into the gold-labeled microwell after incubation in step ② and keep it for 3-8 minutes, then place the test strip in the quantitative instrument to read the test data.

[0045] Preferably, the solvent is water; and the sample diluent is PBS buffer solution.

[0046] In summary, this application has the following beneficial effects:

[0047] The present application adopts a method of combining hazelnut antigen-antibody and vomitoxin antigen-antibody to prepare a test strip. Since the hazelnut antigen-antibody reaction in the present application is not sensitive to temperature and humidity, and the vomitoxin antigen-antibody reaction is completed in an aqueous solution of a gold-labeled micropore, after a certain incubation time, the sensitivity to temperature and humidity will be greatly reduced. The resulting kit can not only improve the disadvantage of the vomitoxin quantitative test strip being sensitive to temperature and humidity, but also free it from the limitations of the temperature control machine, thereby greatly saving costs and improving detection efficiency. DETAILED DESCRIPTION

[0048] The present application is further described below in conjunction with embodiments.

[0049] Example

[0050] Unless otherwise specified, the raw materials in the examples can be obtained commercially. Among them, vomitoxin, hazelnut antigen, mouse anti-vomitoxin monoclonal antibody, and mouse anti-hazelnut monoclonal antibody were purchased from Wuxi Guanyi Biotechnology Co., Ltd. The performance index requirements of the above raw materials are: the titer of vomitoxin antigen is not less than 5000; the titer of mouse anti-vomitoxin monoclonal antibody is not less than 5000, and the sensitivity IC50 is 50-100ppb; the titer of mouse anti-hazelnut monoclonal antibody is not less than 5000, and the sensitivity is 10-100ppb. Sheep anti-mouse IgG and bovine serum albumin were purchased from Beijing Solebow Technology Co., Ltd.; the PBS buffer used in the examples is a phosphate buffer with a pH of 7.4 and a concentration of 10mM; the bovine serum albumin solution used in the examples is prepared with PBS buffer as the solvent.

[0051] The reconstituted solution and lyophilized diluent used in the examples were prepared by the following method:

[0052] The reconstitution solution was prepared by the following method: 2 g of trehalose, 1 g of Tween-20 and 5 g of bovine serum albumin were taken and the volume was made up to 100 mL with PBS buffer solution.

[0053] The lyophilized diluent was prepared by the following method: 5 g of trehalose and 1 g of casein were taken and the volume was fixed to 100 mL with PBS buffer solution.

[0054] Example 1: A highly stable vomitoxin kit is prepared by the following steps:

[0055] 1. Preparation of Colloidal Gold

[0056] Colloidal gold was prepared by the trisodium citrate reduction method. The main process was as follows: 800 mL of ultrapure water (UP grade water) was accurately measured and added to a 1000 mL conical flask, and then 8 mL of 1% chloroauric acid solution was added. The solution was heated to boiling while stirring, and then 1 mL of a 10.4% trisodium citrate aqueous solution was quickly added. The solution changed from gray to black within 2 minutes and finally to red. The heating and stirring were continued for 10 minutes, cooled to room temperature, and ultrapure water was added to make up to 100 mL. The solution was stored away from light for later use.

[0057] 2. Preparation of colloidal gold-labeled mouse anti-DON monoclonal antibody (DONmAb-Gdd)

[0058] Take 100mL of the prepared colloidal gold, add 1mL of 0.1M potassium carbonate aqueous solution, mix well, add 600μg of mouse anti-vomiting toxin monoclonal antibody while stirring, react at room temperature for 2h, then add 1000μL of 10wt% bovine serum albumin solution dropwise, react at room temperature for 1h, then centrifuge at 13000r / min for 15min, discard the supernatant, take the precipitate and resuspend it with 10mL of re-solution to obtain DONmAb-Gdd for use.

[0059] 3. Preparation of DON-labeled mouse anti-hazelnut monoclonal antibody (DON-HZmAb)

[0060] ① Dissolve 5 mg of DON in 1 mL of anhydrous tetrahydrofuran (THF), add 40 mg of N,N-carbonyldiimidazole (CDI), and stir at room temperature for 2 h;

[0061] ② Dry THF with argon gas, and dissolve the residue in 500 μL dimethyl sulfoxide (DMSO) to obtain a mixed solution;

[0062] ③ Weigh 10 mg of mouse anti-hazelnut monoclonal antibody and dissolve it in 1.5 mL of 0.1 M pH 7.2 PBS solution to obtain solution A;

[0063] ④ Take the mixed solution in step ② and slowly add it dropwise to solution A in step ③; react at room temperature overnight; collect the reaction solution and put it into a dialysis bag, and dialyze it with 1L, 0.01M, pH 7.4 PBS solution while stirring, changing the solution every 2 hours. After changing the solution 3 times, the obtained DON-HZmAb was packaged and frozen at -80°C for future use.

[0064] 4. Preparation of Gold-labeled Microwells

[0065] ① After DON-HZmAb was restored to room temperature, 0.1 mL of DON-HzmAb was taken and mixed evenly with 60 mL of lyophilized diluent; 60 μL of each well was dispensed into a low-adsorption ELISA plate and precooled in a -80°C refrigerator for 12 h; then placed in a freeze dryer and frozen for 20 h at an absolute vacuum of 0.001 MPa and a temperature of -60°C, and then the ELISA plate cap was closed to obtain lyophilized powder I;

[0066] ② Take 4 mL of DONmAb-Gdd, mix it evenly with 56 mL of lyophilized diluent, and dispense it into a low-adsorption ELISA plate at 60 μL per well, and precool it in a -80°C refrigerator for 12 hours; then put it into a freeze dryer, freeze it at an absolute vacuum of 0.001 MPa and a temperature of -60°C for 20 hours, and then buckle the ELISA plate cap to obtain lyophilized powder II;

[0067] ③ Transfer the freeze-dried powder cake II to the microwell where the freeze-dried powder cake I is located, buckle the enzyme labeling plate cap, store it in a desiccant, and obtain the gold-labeled microwell for use.

[0068] 5. Test strip assembly

[0069] (1) Cut the nitrocellulose membrane (reaction membrane) into 30 cm strips, stick them on a PVC board, and equilibrate them at 25°C and 50% humidity for 30 min.

[0070] (2) Film cutting

[0071] ① Line C: Dilute goat anti-mouse IgG with PBS buffer solution and spray it on the reaction membrane with a film scribing instrument to obtain a quality control line (line C) with a coating concentration of 1 mg / mL;

[0072] ②T line: dilute the hazelnut antigen with PBS buffer solution and apply it on the nitrocellulose membrane with a film streaker to obtain a test line (T line) with a coating concentration of 1 mg / mL;

[0073] ③ Drying: Dry the cut film at 37°C for 24 hours.

[0074] (3) Assembly

[0075] The absorbent pad, reaction membrane, sample pad and MAX line were fixed on the PVC board respectively, wherein the overlapped parts of the absorbent pad and sample pad with the reaction membrane were 2 mm each; and then they were cut by a strip cutter to obtain a test strip with a width of 3.5 mm.

[0076] Example 2: A method for detecting vomitoxin, comprising the following steps:

[0077] ① Take 5g of the sample to be tested, add 20mL of water to it, and vigorously shake and extract at a speed of 20000r / min for 5min; take 100μL of the supernatant and mix it with 900μL of sample diluent (PBS buffer) to obtain the test solution;

[0078] ② Take 100 μL of the test solution, add it to the gold-labeled microwell of Example 1, blow it 3 times, and incubate it at 25° C. for 5 min;

[0079] ③ Insert the test strip of Example 1 into the gold-labeled microwell of step ② and keep it for 5 minutes, and place the test strip in a quantitative instrument to read the test data.

[0080] Comparative Example

[0081] Comparative Example 1: The difference between this comparative example and Example 1 is that: the gold-labeled micropores are prepared by freeze-drying colloidal gold-labeled mouse anti-deoxyribonucleic acid toxin monoclonal antibodies; the detection line of the test strip is coated with a deoxyribonucleic acid toxin-bovine serum albumin conjugate, and the deoxyribonucleic acid toxin-bovine serum albumin conjugate is purchased from Wuxi Guanyi Biotechnology Co., Ltd.

[0082] A vomitoxin test strip of Comparative Example 1 was prepared by the following method:

[0083] 1. Preparation of colloidal gold: same as in Example 1.

[0084] 2. Preparation of colloidal gold-labeled mouse anti-deoxytoxin monoclonal antibody (DONmAb-Gdd): Same as Example 1.

[0085] 3. Preparation of gold-labeled microwells: Take 4 mL of DONmAb-Gdd, mix it evenly with 56 mL of freeze-dried diluent, and dispense it into a low-adsorption ELISA plate at a volume of 60 μL per well, and precool it in a -80°C refrigerator for 12 hours; then put it into a freeze dryer and freeze it for 20 hours under the conditions of absolute vacuum of 0.001 MPa and temperature of -60°C, and then buckle the ELISA plate cap to obtain gold-labeled microwells.

[0086] 4. Test strip assembly

[0087] (1) Cut the nitrocellulose membrane (reaction membrane) into 30 cm strips, stick them on a PVC board, and equilibrate them at 25°C and 50% humidity for 30 min.

[0088] (2) Film cutting

[0089] ① Line C: Dilute goat anti-mouse IgG with PBS buffer solution and spray it on the reaction membrane with a film scribing instrument to obtain a quality control line (line C) with a coating concentration of 1 mg / mL;

[0090] ②T line: dilute the DON-BSA conjugate with PBS buffer solution and apply it on the nitrocellulose membrane with a film stripper to obtain a test line (T line) with a coating concentration of 1 mg / mL;

[0091] ③ Drying: Dry the cut film at 37°C for 24 hours.

[0092] (3) Assembly: Same as Example 1.

[0093] The method of using the vomitoxin test strip of Comparative Example 1 is the same as that of Example 2.

[0094] Performance Testing

[0095] The performance of the test kit of Example 1 and the test strip of Comparative Example 1 were tested respectively according to the following methods.

[0096] 1. Sensitivity experiment

[0097] A series of concentrations of vomitoxin standard products were prepared, and curves were established using vomitoxin antigen-antibody and hazelnut antigen-antibody combined with vomitoxin antigen-antibody, respectively. The test strips of comparative example 1 and example 1 were respectively read with a quantitative detector (LFR-TC multifunctional quantitative detector produced by Prebond) to read the T / C values, and the sensitivity of the test strips was evaluated by calculating the inhibition rate. The inhibition rate was calculated as follows: inhibition rate = [T / C(0)-T / C(X)] / T / C(0)*100; wherein, T / C(0): T / C value of standard product 0; T / C(X): T / C value of standard product X.

[0098] The greater the inhibition rate of the standard product with the same concentration, the higher the sensitivity. The data are recorded in Table 1 and Table 2.

[0099] Table 1: Data table of curves established using only DON antigen and antibody

[0100] Standard concentration, ppb 0 10 30 90 270 T / C 3.12 2.56 1.77 1.18 0.61 Inhibition rate % / 17.9 43.2 62.2 80.4

[0101] Table 2: Data table of curves established using hazelnut antigen-antibody and vomitoxin antigen-antibody combination

[0102] Standard concentration, ppb 0 10 30 90 270 T / C 3.31 2.31 1.71 1.06 0.52 Inhibition rate % / 30.2 48.3 68 84.3

[0103] From the comparison between Table 1 and Table 2, it can be seen that the method using the combination of hazelnut antigen-antibody and vomitoxin antigen-antibody is slightly more sensitive than the method using only vomitoxin antigen-antibody.

[0104] 2. Temperature sensitivity experiment

[0105] Test strips prepared using vomitoxin antigen-antibody (Comparative Example 1) and test strips prepared using a combination of hazelnut antigen-antibody and vomitoxin antigen-antibody (Example 1) were tested under different temperature conditions, and the T / C values ​​of the test strips were read by a quantitative detector, and the coefficient of variation (CV) was calculated. The data were recorded in Tables 3 and 4.

[0106] Table 3: Coefficient of variation of the test strips of comparative example 1 at different temperature conditions

[0107]

[0108] Table 4: Coefficient of variation of the test strips of Example 1 under different temperature conditions

[0109]

[0110] From the comparison of Table 3 and Table 4, it can be seen that the coefficient of variation of the test strips of Comparative Example 1 under different temperature conditions is greater than 10%, while the coefficient of variation of the test strips of Example 1 under different temperature conditions is less than 5%, indicating that the test strips prepared by the method of the present application are not sensitive to the reaction temperature.

[0111] 3. Humidity sensitivity experiment

[0112] Test strips prepared using vomitoxin antigen-antibody (Comparative Example 1) and test strips prepared using a combination of hazelnut antigen-antibody and vomitoxin antigen-antibody (Example 1) were tested under different humidity conditions, and the T / C values ​​of the test strips were read by a quantitative detector, and the coefficient of variation (CV) was calculated. The data were recorded in Tables 5 and 6.

[0113] Table 5: Coefficient of variation of the test strips of Comparative Example 1 under different humidity conditions

[0114]

[0115] Table 6: Coefficient of variation of the test strips of Example 1 under different humidity conditions

[0116]

[0117] From the comparison between Table 5 and Table 6, it can be seen that the coefficient of variation of the test strips of Comparative Example 1 under different humidity conditions is greater than 15%, while the coefficient of variation of the test strips of Example 1 under different humidity conditions is less than 5%, indicating that the test strips prepared by the method of the present application are not sensitive to the reaction humidity.

[0118] In summary, when the kit of the present application is used for the detection of vomitoxin, it has low sensitivity to the temperature and humidity of the reaction, and can get rid of the limitation of the temperature control machine, thereby greatly saving costs and operation time. In addition, the kit of the present application also has a high sensitivity. When used with a dedicated quantitative detector, it can read the T line and C line data and calculate the T / C value, and then the vomitoxin content can be calculated and the positive and negative can be judged through the built-in standard curve of the instrument, which can achieve more accurate quantitative detection.

[0119] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed. However, as long as it is within the scope of the claims of the present application, it shall be protected by the patent law.

Claims

1. A highly stable vomitoxin kit, characterized in that: Includes gold-labeled microwells and test strips; The gold-labeled microwells include mouse anti-hazelnut monoclonal antibodies labeled with vomitoxin and mouse anti-vomitoxin monoclonal antibodies labeled with colloidal gold; The test strip comprises a bottom plate, on which a sample pad, a reaction membrane and a water absorbent pad are arranged in sequence along the chromatography direction; The reaction membrane is provided with a detection line coated with hazelnut antigen and a quality control line coated with goat anti-mouse IgG in sequence along the chromatography direction.

2. A highly stable vomitoxin kit according to claim 1, characterized in that: The gold-labeled micropores are prepared by the following method: Mouse anti-hazelnut monoclonal antibody labeled with vomitoxin was taken, mixed evenly with the lyophilized diluent, and then dispensed into a low-adsorption ELISA plate, and freeze-dried to obtain lyophilized powder cake I; The colloidal gold-labeled mouse anti-vomiting toxin monoclonal antibody is mixed evenly with the lyophilized diluent, and then divided into low-adsorption ELISA plates, and freeze-dried to obtain lyophilized powder II; The freeze-dried powder cake II is transferred to the microwell where the freeze-dried powder cake I is located to obtain the gold-labeled microwell.

3. A highly stable vomitoxin kit according to claim 2, characterized in that: The freeze-dried diluent comprises components with the following concentrations: trehalose with a mass volume fraction of 3-7% and casein with a mass volume fraction of 0.5-2%.

4. A highly stable vomitoxin kit according to claim 2, characterized in that: The volume ratio of the mouse anti-hazelnut monoclonal antibody labeled with vomitoxin to the lyophilized diluent is 0.01-1:60; The volume ratio of the colloidal gold-labeled mouse anti-vomitoxin monoclonal antibody to the lyophilized diluent is 1-10:

56.

5. The highly stable vomitoxin kit according to claim 1, characterized in that: The colloidal gold-labeled mouse anti-vomitoxin monoclonal antibody is prepared by the following method: adding carbonate solution to colloidal gold, mixing evenly, adding mouse anti-vomitoxin monoclonal antibody while stirring, reacting at room temperature for 1-3 hours, then adding carrier protein solution dropwise, reacting at room temperature for 1-2 hours, collecting the precipitate by centrifugation and resuspending it with a resuspending solution to obtain the colloidal gold-labeled mouse anti-vomitoxin monoclonal antibody.

6. A highly stable vomitoxin kit according to claim 5, characterized in that: The reconstituted solution comprises components with the following concentrations: bovine serum albumin with a mass volume concentration of 3-7%, trehalose with a mass volume concentration of 1-5%, and Tween-20 with a mass volume concentration of 0.5-2%.

7. A highly stable vomitoxin kit according to claim 1, characterized in that: The deoxynivalenol-labeled mouse anti-hazelnut monoclonal antibody is prepared by the following method: Dissolve vomitoxin in anhydrous tetrahydrofuran, add N,N-carbonyldiimidazole, and react at room temperature with stirring for 1-3 hours; then dry the tetrahydrofuran, and dissolve the residue in dimethyl sulfoxide to obtain a mixed solution; Take mouse anti-hazelnut monoclonal antibody and dissolve it in PBS solution to obtain solution A; The mixed solution was slowly added dropwise to solution A; the mixture was reacted at room temperature overnight; the reaction solution was then collected, dialyzed, and frozen to obtain mouse anti-hazelnut monoclonal antibodies labeled with vomitoxin.

8. The highly stable vomitoxin kit according to claim 1, characterized in that: The concentration of the hazelnut antigen coated on the detection line is 0.1-10 mg / mL, and the concentration of the goat anti-mouse IgG on the quality control line is 0.1-10 mg / mL.

9. The method for preparing a highly stable vomitoxin kit according to any one of claims 1 to 8, characterized in that: The steps include: Preparation of gold-labeled microwells: Mouse anti-hazelnut monoclonal antibody labeled with vomitoxin was taken, mixed evenly with the lyophilized diluent, divided into ELISA plates, and freeze-dried to obtain lyophilized powder cake I; The colloidal gold-labeled mouse anti-vomiting toxin monoclonal antibody is mixed evenly with the lyophilized diluent, and then divided into ELISA plates, and freeze-dried to obtain lyophilized powder II; Transfer the freeze-dried powder cake II to the microwell where the freeze-dried powder cake I is located to obtain a gold-labeled microwell; Assembly of the test strips: The cut reaction membrane is pasted on a PVC plate, goat anti-mouse IgG is diluted and sprayed on the reaction membrane to obtain a quality control line; hazelnut antigen is diluted and sprayed on the reaction membrane to obtain a test line; the reaction membrane after the marking is dried and set aside; A water-absorbing pad is attached to the PVC sheet near one end of the reaction membrane quality control line, a sample pad is attached to the PVC sheet near one end of the reaction membrane detection line, and a MAX line is attached to the PVC sheet near one end of the sample pad to obtain a test strip.

10. A method for detecting vomitoxin, characterized in that: The detection is performed using the kit as claimed in claim 1, wherein the detection method comprises the following steps: ① Take the sample to be tested, add solvent and shake and extract for 5-10 minutes; take the supernatant, mix it with the sample diluent in a volume ratio of 1:8-10 to obtain the test solution; ② Take 100-200 μL of the test solution, add it to the gold-labeled microwell, pipette 2-3 times, and incubate at room temperature for 3-10 minutes; ③Insert the test strip into the gold-labeled microwell after incubation in step ② and keep it for 3-8 minutes, then place the test strip in the quantitative instrument to read the test data.

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