Amanita peptide hapten, monoclonal antibody, hybridoma cell strain, test strip and application

Abstract

The invention relates to an amanita peptide hapten, a monoclonal antibody, a hybridoma cell strain, a test strip and application, and belongs to the technical field of amanita peptide detection. The hapten is prepared by the following steps: reacting amatoxin with pyridine dichromate to obtain an intermediate product, and condensing the intermediate product with 4-maleimide butyryl hydrazine. The test strip provided by the invention has the characteristics of simplicity in operation, high sensitivity, high detection speed, low cost and the like.

Description

Technical Field

[0001] The present invention relates to an amatoxin hapten, a monoclonal antibody, a hybridoma cell line, a test strip and applications thereof, and belongs to the technical field of amatoxin detection. Background Art

[0002] Food poisoning caused by mushrooms has always been one of the key food safety issues that many countries in the world focus on. In recent years, poisoning incidents caused by accidentally eating poisonous mushrooms in China have occurred frequently, and the mortality rate remains high. China is rich in mushroom resources, and there are more than 480 recorded poisonous mushrooms. Among them, mushrooms of the genus Amanita contain a toxic cyclic peptide, namely amatoxin. Amanitoxin can form a stable quaternary complex with eukaryotic cell RNA polymerase II, DNA template and newly synthesized RNA, thereby hindering protein synthesis and ultimately leading to necrosis of tissue cells such as the liver. Amanitoxin mainly includes α-amanitoxin, β-amanitoxin and γ-amanitoxin. Among them, α-amanitoxin has the strongest toxicity, and β-amanitoxin follows. Once poisoning symptoms appear after accidentally eating poisonous mushrooms containing amatoxin, the rescue effect is often not good and the mortality rate is relatively high. Therefore, early diagnosis is particularly important for the treatment of amatoxin poisoning.

[0003] At present, the methods for detecting amatoxin mainly include instrumental methods such as high performance liquid chromatography and liquid chromatography-mass spectrometry, which have the advantages of high sensitivity and good accuracy. However, the pretreatment of test samples is cumbersome and time-consuming, requires expensive large-scale instruments and equipment, and is operated and managed by professional detection technicians. It cannot be used for large-scale on-site detection, has poor timeliness and is difficult to popularize. Immunological detection and analysis technology has been widely used in the field of drug residue detection due to its advantages of high sensitivity, high specificity, rapidity and simple operation, and has many advantages compared with instrumental detection methods.

[0004] When establishing an immunological detection method and applying this detection method to detect amatoxin, the key technology lies in being able to obtain antibodies with strong specificity and high sensitivity. To achieve this goal, the prerequisite is to synthesize and prepare a suitable amatoxin hapten. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an amatoxin hapten, a monoclonal antibody, a hybridoma cell line, a test strip and applications thereof.

[0006] To achieve the above purpose, the technical solution of the present invention is as follows.

[0007] An amatoxin hapten, the structural formula of the amatoxin hapten is:

[0008]

[0009] A method for preparing amanitin hapten according to the present invention, the method steps include:

[0010] (1) Amanitin reacts with pyridinium dichromate. After purification of the reaction product, an intermediate product is obtained; the structural formula of the intermediate product is:

[0011] (2) The intermediate product undergoes a condensation reaction with 4-maleimidobutyric hydrazide. After purification of the condensation reaction product, an amanitin hapten is obtained.

[0012] Preferably, in step (1), amanitin is dissolved in pyridine, then pyridinium dichromate is added. After stirring and mixing evenly, glacial acetic acid is added. At 20-25 °C, the reaction is carried out for 2-3 h. After purification of the reaction product, an intermediate product is obtained; more preferably, the dosage ratio of amanitin, pyridine, and pyridinium dichromate is 10 mg: 8-12 ml: 6-7 mg, and the addition amount of glacial acetic acid is 2-4% of the volume of pyridine.

[0013] Preferably, in step (2), the intermediate product is dissolved in methanol, then 4-maleimidobutyric hydrazide is added. The condensation reaction is carried out at 20-25 °C for 3-4 h. After purification of the condensation reaction product, an amanitin hapten is obtained; more preferably, the dosage ratio of the intermediate product, methanol, and 4-maleimidobutyric hydrazide is 7-8 mg: 10-20 ml: 2.5-3.5 mg.

[0014] Application of an amanitin hapten according to the present invention in the preparation of a reagent for detecting amanitin.

[0015] A hybridoma cell line secreting amanitin monoclonal antibody, the preservation unit is the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, the preservation number is CGMCC No. 46334, and the preservation name is Amanitin Monoclonal Antibody Hybridoma Cell Line AMA-1-1.

[0016] Application of a hybridoma cell line secreting amanitin monoclonal antibody according to the present invention in the preparation of a reagent for detecting amanitin.

[0017] An amanitin monoclonal antibody, secreted by the hybridoma cell line according to the present invention.

[0018] Application of an amanitin monoclonal antibody according to the present invention in the preparation of a reagent for detecting amanitin.

[0019] Application of an amanitin antigen according to the present invention in the preparation of a reagent for detecting amanitin.

[0020] A test strip for detecting amanitoxins, comprising a sample absorption pad, a conjugate release pad, a reaction membrane, an absorbent pad and a base plate. An amanitoxin antigen of the present invention is coated on the test line of the reaction membrane; an anti-mouse antibody is coated on the quality control line of the reaction membrane; a marker is sprayed on the conjugate release pad; the conjugate release pad, the reaction membrane are assembled with the sample absorption pad, the absorbent pad and the base plate to form a test strip;

[0021] Wherein, the marker is prepared from an amanitoxin monoclonal antibody of the present invention and colloidal gold; the amanitoxin antigen is obtained by conjugating an amanitoxin hapten of the present invention with a carrier protein; preferably, the carrier protein is keyhole limpet hemocyanin or ovalbumin.

[0022] Use of a test strip for detecting amanitoxins of the present invention in the preparation of a reagent for detecting amanitoxins.

[0023] Beneficial effects

[0024] The amanitoxin hapten provided by the present invention is obtained by first reacting amanitoxin with pyridinium dichromate to obtain an intermediate product, and then condensing the intermediate product with 4-maleimidobutyric acid hydrazide. Compared with traditional coupling methods, this preparation method has the advantages of high yield and few impurities, and the monoclonal antibody generated by the immunogen prepared based on this method has excellent affinity.

[0025] The test strip for detecting amanitoxins provided by the present invention adopts a highly specific antibody-antigen reaction and competitive inhibition immunochromatographic analysis technology. The monoclonal antibody-gold conjugate of amanitoxins is fixed on the conjugate release pad. During the flow of the sample, the amanitoxins in the sample bind to the monoclonal antibody-gold conjugate on the conjugate release pad, forming amanitoxin-antibody-gold conjugate. The amanitoxins in the sample compete with the amanitoxin antigen on the test line of the reaction membrane for binding to the monoclonal antibody-gold conjugate, and it is determined whether the test sample solution contains amanitoxins according to the presence or absence or the color depth of the red strip on the test line. During the detection, the sample is processed and then dropped into the hole of the test strip. When the concentration of amanitoxins in the sample is lower than the detection limit or zero, the monoclonal antibody-gold conjugate will bind to the amanitoxin antigen fixed on the reaction membrane during the chromatography process, and a red strip will appear in each of the test line (T) and the control line (C), and the color of the T line is darker than or the same as that of the C line; if the concentration of amanitoxins in the sample is equal to or higher than the detection limit, the monoclonal antibody-gold conjugate will bind to all the amanitoxins, so that due to the competitive reaction, it will not bind to the amanitoxin antigen at the T line, and no red strip will appear or the color is lighter than that of the C line. The test strip of the present invention has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, suitable for use in various units, simple storage, and long shelf life. The method for detecting amanitoxins with the test strip of the present invention is simple, rapid, intuitive, accurate, wide in application range, low in cost, and easy to promote and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a flowchart for preparing the amanitoxin hapten of the present invention.

[0027] Figure 2 It is a 1H NMR spectrum of the amanitoxin hapten.

[0028] Figure 3 It is a schematic cross-sectional structure diagram of the test strip. 1 is the sample absorption pad, 2 is the conjugate release pad, 3 is the reaction membrane, 4 is the water absorption pad, 5 is the test line (T), 6 is the control line (C), and 7 is the bottom plate.

[0029] Figure 4 It is a diagram for judging the test result of the test strip. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] The present invention will be further described in detail below with reference to specific embodiments.

[0031] Example 1 Preparation of Amanitoxin Hapten

[0032] The preparation process of the amanitoxin hapten is as Figure 1As shown, 10 mg of amanitin was dissolved in 10 mL of pyridine, 6.4 mg of pyridinium dichromate (PDC) was added, and the mixture was stirred thoroughly. Then, 0.3 mL of glacial acetic acid was added, and the reaction was stirred at 20 - 25 °C for 2 h. The reaction was stopped, and the solvent was removed by rotary evaporation. 50 mL of ethyl acetate was added, and the mixture was shaken well. The residue was removed by filtration. The mixture was loaded onto a silica gel column and eluted and separated with a dichloromethane - methanol mixed solution with a volume ratio of 10:1 to obtain 7.2 mg of the intermediate product.

[0033] 7.2 mg of the intermediate product was dissolved in 10 mL of methanol, 3 mg of 4 - maleimidobutyric acid hydrazide was added, and the mixture was stirred at 20 - 25 °C for 3 h. The reaction was stopped, and methanol was removed by rotary evaporation. The mixture was loaded onto a silica gel column and eluted and separated with a dichloromethane - methanol mixed solution with a volume ratio of 10:1 to obtain 6.8 mg of the amanitin hapten with a yield of 78%.

[0034] The hapten was measured by hydrogen nuclear magnetic resonance ( 1 H - NMR), and the results are as Figure 2 shown: 11H NMR (500 MHz, Chloroform-d): δ (ppm) 9.55 (s, 1H), 8.95 (s, 1H), 8.08 (d, J = 8.8 Hz, 1H), 7.73 (dd, J = 8.8, 1.1 Hz, 2H), 7.50 (d, J = 9.7 Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 7.13–7.08 (m, 2H), 7.07–7.01 (m, 2H), 6.99 (dd, J = 8.3, 1.7 Hz, 1H), 6.71 (s, 2H), 6.63 (dd, J = 8.9, 1.9 Hz, 1H), 6.44 (t, J = 6.2 Hz, 1H), 6.15 (t, J = 5.9 Hz, 1H), 4.87 (dt, J = 9.7, 7.1 Hz, 1H), 4.76 (dt, J = 8.8, 6.6 Hz, 1H), 4.68 (dt, J = 9.1, 6.0 Hz, 1H), 4.44–4.32 (m, 3H), 4.32–4.20 (m, 2H), 4.08 (td, J = 6.0, 1.2 Hz, 2H), 4.00 (d, J = 5.1 Hz, 1H), 3.92 (dd, J = 16.8, 6.2 Hz, 2H), 3.85–3.80 (m, 1H), 3.84–3.77 (m, 1H), 3.54 (dd, J = 12.4, 2.6 Hz, 1H), 3.48 (d, J = 5.7 Hz, 1H), 3.33 (dd, J = 12.4, 4.5 Hz, 1H), 3.21–3.14 (m, 3H), 3.16–3.09 (m, 1H), 3.02 (dd, J = 16.7, 6.0 Hz, 1H), 2.90 (dd, J = 17.6, 7.1 Hz, 1H), 2.51–2.39 (m, 3H), 2.07–1.96 (m, 4H), 1.86 (ddd, J = 12.5, 6.1, 5.2 Hz, 1H), 1.41 (dqd, J = 13.2, 7.5, 5.7 Hz, 1H), 1.27 (dqd, J = 13.3, 7.6, 5.7 Hz, 1H), 1.03 (dd, J = 6.9, 1.5 Hz, 3H), 0.94 (dd, J = 6.9, 1.5 Hz, 3H), 0.86 (t, J = 7.6 Hz, 3H). In the 1H NMR spectrum, the absorption peaks with chemical shifts δ of 2.51–2.39 (m, 3H), 2.07–1.96 (m, 4H), 4.08 (td, J = 6.0, 1.2 Hz, 2H), 4.00 (d, J = 5.1 Hz, 1H), and 6.71 (s, 2H) are the absorption peaks of the hydrogens on the spacer arm. The presence of these peaks proves that the spacer arm is successfully coupled, and the hapten structure is:

[0035]

[0036] Example 2 Preparation of Amanitin Antigen

[0037] Take 10 mg of keyhole limpet hemocyanin (KLH), dissolve it in 1 mL of PB buffer solution with pH 8.0, add 1 mg of N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), react at room temperature for 4 h, then add 0.2 mL of an aqueous solution containing 0.8 mg of tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl), and continue to react for 20 min; add 0.2 mL of a DMF solution containing 3.6 mg of the amanitin hapten prepared in Example 1, continue to react for 4 h, and dialyze and purify with 0.02 mol / L PB buffer solution for 3 days, changing the solution 3 times a day, to obtain an amanitin antigen conjugated with keyhole limpet hemocyanin. Aliquot and store at -20 °C for later use.

[0038] Replace KLH with ovalbumin (OVA), and repeat the above reaction with 3 mg of amanitin hapten and 30 mg of OVA to obtain an amanitin antigen conjugated with ovalbumin.

[0039] Example 3 Preparation of Amanitin Detection Test Strip

[0040] 1. Preparation of Amanitin Monoclonal Antibody

[0041] (1) Animal Immunization

[0042] Inject the amanitin antigen conjugated with keyhole limpet hemocyanin prepared in Example 2 into Balb / c mice at an immunization dose of 150 μg per mouse to produce antiserum.

[0043] (2) Cell Fusion and Cloning

[0044] Take the spleen cells of immunized Balb / c mice and fuse them with SP2 / 0 myeloma cells at a ratio of 8:1 (quantity ratio). Use indirect competitive ELISA to measure the cell supernatant and screen positive wells. Clone the positive wells using the limiting dilution method until a hybridoma cell line that stably secretes monoclonal antibody is obtained. The preservation unit of this monoclonal antibody hybridoma cell line is the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, the preservation number is CGMCC No. 46334, and the preservation name is Amanitin Monoclonal Antibody Hybridoma Cell Line AMA-1-1. Preservation date: March 20, 2025.

[0045] (3) Cell Cryopreservation and Resuscitation

[0046] Prepare the hybridoma cells into a cell suspension of 1×10 6 cells / mL with cryopreservation solution and store it in liquid nitrogen for a long time. When resuscitating, take out the cryopreservation tube, immediately place it in a 37 °C water bath for rapid thawing, centrifuge to remove the cryopreservation solution, and then transfer it to a culture flask for cultivation.

[0047] (4) Preparation and purification of monoclonal antibodies

[0048] Incremental culture method: The hybridoma cells are placed in a cell culture medium and cultured at 37 °C. The obtained culture solution is purified by the caprylic acid-ammonium sulfate saturation method to obtain monoclonal antibodies, which are stored at -20 °C.

[0049] The cell culture medium is prepared by adding calf serum and sodium bicarbonate to RPMI1640 medium, so that the final concentration of calf serum in the cell culture medium is 20% (mass fraction), and the final concentration of sodium bicarbonate in the cell culture medium is 0.2% (mass fraction); the pH of the cell culture medium is 7.4.

[0050] 2. Preparation of goat anti-mouse anti-antibody

[0051] Using goats as immunized animals, immunize pathogen-free goats with mouse-derived antibodies as immunogens to obtain goat anti-mouse anti-antibodies.

[0052] 3. Preparation of amanitin monoclonal antibody-gold colloid conjugate

[0053] (1) Preparation of colloidal gold

[0054] Dilute 1% chloroauric acid with double-distilled deionized water to 0.01% (mass fraction), take 100 mL and place it in a conical flask, heat it to boiling with a constant-temperature electromagnetic stirrer, add 1.5 mL of 1% trisodium citrate under continuous high temperature and continuous stirring, continue to stir and heat evenly until the solution turns bright red, then stop, cool to room temperature and restore the volume to the original volume with deionized water, and store at 4 °C. The prepared colloidal gold has a pure, transparent appearance, no precipitation and floating substances, and the color is wine red when observed under sunlight.

[0055] (2) Preparation of amanitin monoclonal antibody-gold colloid conjugate

[0056] Under magnetic stirring, adjust the pH value of the colloidal gold to 7.2 with 0.2 mol / L potassium carbonate solution, add the above-prepared amanitin monoclonal antibody to the colloidal gold solution according to the standard of adding 5 - 50 μg of antibody per milliliter of colloidal gold solution, and continue to stir and mix evenly for 30 min; after standing for 10 min, add 10 μL of 5% casein blocking solution and stand for 10 min. Centrifuge at 11000 r / min at 4 °C for 5 min, discard the supernatant, wash the precipitate twice with reconstitution buffer, resuspend the precipitate with reconstitution buffer with a volume of 1 / 10 of the initial volume of the colloidal gold, and place it at 4 °C for standby.

[0057] Reconstitution buffer: 0.02 mol / L phosphate buffer containing 0.1% - 0.5% (mass fraction) of BSA, 2% - 4% (mass fraction) of sucrose, and pH 7.2.

[0058] 4. Preparation of the conjugate release pad

[0059] Soak the conjugate release pad in 0.02 mol / L phosphate buffer containing 0.5% BSA, 5% sucrose, and pH 7.4 for 2 h to uniformly wet it, and then dry it at 37°C for later use. Use a Bio dot membrane applicator to uniformly spray the prepared anti-amanitin monoclonal antibody-gold conjugate on the conjugate release pad. After spraying 0.7 μL of the anti-amanitin monoclonal antibody-gold conjugate per 1 cm of the conjugate release pad, place it in an environment at 37°C (humidity < 20%) and dry it for 1 h, then take it out, seal it, and store it for later use.

[0060] 5. Preparation of the sample absorption pad

[0061] Soak the sample absorption pad in 0.02 mol / L phosphate buffer containing 1% BSA and pH 7.2 for 2 h, and dry it at 37°C for 2 h for later use.

[0062] 6. Preparation of the reaction membrane

[0063] Coat the reaction membrane with the anti-amanitin antigen conjugated to ovalbumin to form the test line, and coat the reaction membrane with goat anti-mouse antibody to form the control line.

[0064] Coating process: Dilute the anti-amanitin antigen conjugated to ovalbumin to 1 mg / mL with 0.1 mol / L phosphate buffer at pH 7.2, and use a Bio dot membrane applicator to coat it on the test line (T line) of the nitrocellulose membrane, with a coating amount of 1.0 μL / cm; dilute the goat anti-mouse antibody to 200 μg / mL with 0.1 mol / L phosphate buffer at pH 7.2, and use a Bio dot membrane applicator to coat it on the control line (C line) of the nitrocellulose membrane, with a coating amount of 1.0 μL / cm. Place the coated reaction membrane at 37°C and dry it for 16 h for later use.

[0065] 7. Assembly of the test strip

[0066] As Figure 3The cross-sectional structure of the test strip is shown as follows. The reaction membrane (3) is pasted on the PVC bottom plate (7), and then the conjugate release pad (2) and the sample absorption pad (1) are sequentially pasted at one end, and the water absorption pad (4) is pasted at the other end. The conjugate release pad and the sample absorption pad are connected to each other, and both the conjugate release pad and the water absorption pad press the reaction membrane by 1-2 mm. There are a test line (5) and a quality control line (6) on the reaction membrane. Both the test line (T line) and the quality control line (C line) are strip-shaped bands perpendicular to the length of the test strip. The test line is located on one side near the end of the conjugate release pad; the quality control line is located on one side far from the end of the conjugate release pad. The test strip is cut into small strips with a width of 3.96 mm by a machine, packed in a special plastic cartridge, sealed with an aluminum foil bag, and stored in an environment of 4-30 °C, with a shelf life of 12 months.

[0067] Detection of Amanitoxins in Samples of Example 4

[0068] 1. Detection with the test strip

[0069] Use a micropipette to vertically pipette 100 μL of the sample solution to be tested into the sample addition hole; start timing when the liquid flows, react for 5 min, and judge the result.

[0070] 2. Analysis of the test results

[0071] Negative (-): The color development of the T line is stronger than that of the C line or there is no obvious difference in color development from the C line, indicating that the sample does not contain Amanitoxins or its concentration is below the detection limit, such as Figure 4 a and b in it.

[0072] Positive (+): The color development of the T line is significantly weaker than that of the C line or the T line does not develop color, indicating that the concentration of Amanitoxins in the sample is equal to or higher than the detection limit, such as Figure 4 c and d in it.

[0073] Invalid: The C line does not appear, indicating an incorrect operation process or the test strip has deteriorated and become invalid, such as Figure 4 e and f in it.

[0074] Sample Detection of Example 5

[0075] 1. Detection limit test

[0076] Take blank mushroom samples, add α-Amanitoxin to them to a final concentration of 2.5 μg / kg, 5 μg / kg, and 10 μg / kg respectively, take the test strip for detection, and repeat the determination three times for each sample.

[0077] When detecting mushroom samples with a test strip, when there is no α-amanitin in it and its added concentration is 2.5 μg / kg, the test strip shows that the color development of the T line is stronger than that of the C line or there is no obvious difference in color development from the C line, showing a negative result; when the added concentration of α-amanitin is 5 μg / kg and 10 μg / kg, the test strip shows that the color development of the T line is significantly weaker than that of the C line or the T line does not develop color, showing a positive result, indicating that the detection limit of this test strip for α-amanitin in mushrooms is 5 μg / kg.

[0078] 2. False positive rate and false negative rate tests

[0079] Take 20 blank mushroom samples and 20 positive mushroom samples with α-amanitin added to a final concentration of 5 μg / kg, and use test strips produced in 3 batches to detect them respectively, and calculate their positive and negative rates.

[0080] The results show that: when using test strips produced in 3 batches to detect positive samples, the results are all positive, so the positive coincidence rate is 100%, and the false negative rate is 0; when detecting negative samples, the results are all negative, so the negative coincidence rate is 100%, and the false positive rate is 0. It shows that the test strip for detecting amanitin of the present invention can quickly detect amanitin in mushroom samples.

[0081] 3. Specificity test

[0082] Dilute β-amanitin and γ-amanitin with a phosphate buffer solution of pH 7.2 and 0.2 mol / L to 4, 5, 6, 7, 8, 9, 10 μg / L in gradients, and use an amanitin test strip to detect. The results show that when using this test strip to detect β-amanitin and γ-amanitin, the minimum concentrations for positive detection results are 5 μg / L for β-amanitin and 6 μg / L for γ-amanitin respectively. According to the cross-reaction rate calculation formula in the "Opinions of the State Administration for Market Regulation on Regulating the Use of Food Rapid Detection" (Guo Shi Jian Shi Jian Gui 〔2023〕 No. 1): Cross-reaction rate (%) = Detection limit of the target substance × 100% / Minimum concentration when the interfering substance is detected positively, calculate the cross-reaction rates of the test strip for β-amanitin and γ-amanitin to be 100% and 80% respectively, indicating that this test strip can detect β-amanitin and γ-amanitin simultaneously.

[0083] In summary, the invention includes but is not limited to the above embodiments. Any equivalent replacement or partial improvement made under the spirit and principle of the present invention will be regarded as within the protection scope of the present invention.

Claims

1. An amatoxin hapten, characterized in that: The structural formula of the amatoxin hapten is as follows:

2. A preparation method of the amatoxin hapten as described in claim 1, characterized in that: The method steps include: (1) Amanitoxins react with pyridinium dichromate, and after purification of the reaction product, an intermediate product is obtained; the structural formula of the intermediate product is: (2) The intermediate product is subjected to a condensation reaction with 4-maleimidobutyric hydrazide. After purification of the condensation reaction product, an amatoxin hapten is obtained.

3. The preparation method of an amatoxin hapten according to claim 2, characterized in that: In step (1), the amatoxin is dissolved in pyridine, then pyridinium dichromate is added. After stirring and mixing evenly, glacial acetic acid is added. The reaction is carried out at 20-25 °C for 2-3 h. After purification of the reaction product, an intermediate product is obtained. More preferably, the dosage ratio of amatoxin, pyridine, and pyridinium dichromate is 10 mg: 8-12 ml: 6-7 mg, and the addition amount of glacial acetic acid is 2-4% of the volume of pyridine. Preferably, in step (2), the intermediate product is dissolved in methanol, then 4-maleimidobutyric hydrazide is added. The condensation reaction is carried out at 20-25 °C for 3-4 h. After purification of the condensation reaction product, an amatoxin hapten is obtained. More preferably, the dosage ratio of the intermediate product, methanol, and 4-maleimidobutyric hydrazide is 7-8 mg: 10-20 ml: 2.5-3.5 mg.

4. Application of an amatoxin hapten as described in claim 1 in the preparation of a reagent for detecting amatoxin.

5. A hybridoma cell line secreting anti-amanitin monoclonal antibody, characterized in that: The preservation unit is the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, the preservation number is CGMCC No. 46334, and the preservation name is the hybridoma cell line AMA-1-1 of amatoxin monoclonal antibody.

6. Application of a hybridoma cell line secreting an amatoxin monoclonal antibody as described in claim 5 in the preparation of a reagent for detecting amatoxin.

7. An amatoxin monoclonal antibody, characterized in that: It is secreted and produced by the hybridoma cell line described in claim 5.

8. Application of an amatoxin monoclonal antibody as described in claim 7 in the preparation of a reagent for detecting amatoxin.

9. A test strip for detecting amanitin, characterized in that: It includes a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad, and a bottom plate. The detection line of the reaction membrane is coated with an amatoxin antigen of the present invention; the quality control line of the reaction membrane is coated with a goat anti-mouse antibody; the conjugate release pad is sprayed with a marker; the conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad, and the bottom plate are assembled into a test strip; Among them, the marker is prepared from an amatoxin monoclonal antibody described in claim 7 and colloidal gold; the amatoxin antigen is obtained by conjugating an amatoxin hapten described in claim 1 with a carrier protein. Preferably, the carrier protein is keyhole limpet hemocyanin or ovalbumin.

10. Application of a test strip for detecting amatoxin as described in claim 9 in the preparation of a reagent for detecting amatoxin.

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