Preparation method and application of aflatoxin M1 hapten and holotoxin

Abstract

This invention discloses a method for preparing aflatoxin M1 hapten and full antigen, and their applications. The hapten contains two intact active carboxyl groups, which can firmly bind to more carrier proteins. The aflatoxin M1 hapten is obtained by the addition reaction of aflatoxin M1 with dimercaptosuccinic acid under the catalysis of boron trifluoride diethyl ether. The full antigen is a covalent conjugate of the hapten and BSA, prepared by the EDC / NHS activated carboxyl coupling method, and can be used for antibody preparation and AFM1 detection.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, specifically relating to a method for preparing a hapten and a complete antigen of aflatoxin M1 and its application in the detection of aflatoxin M1. Background Technology

[0002] Aflatoxin M1 (AFM1) is a metabolite of aflatoxin B1 in animals. It is mainly found in dairy products such as milk, cheese, and milk powder. It has strong carcinogenic, teratogenic, and mutagenic properties, posing a serious threat to human health. Therefore, it is of great significance to establish a highly sensitive and specific AFM1 detection method.

[0003] Immunoassay technology has become the mainstream method for AFM1 detection due to its advantages such as speed, sensitivity, high specificity, and ease of operation. High-quality AFM1 whole antigen is the core foundation for constructing this detection technology. AFM1 itself has a small molecular weight (approximately 328 Da) and is not immunogenic, so it cannot directly induce the body to produce specific antibodies. It needs to covalently bind with a large molecular carrier protein to form a whole antigen in order to stimulate the body's immune response.

[0004] The quality of a whole antigen directly depends on the purity and structural integrity of the hapten. Current techniques for preparing AFM1 haptens often suffer from insufficient purity, destruction of active sites, and low yield. Furthermore, most haptens contain only a single carboxyl group, limiting their ability to bind to carrier proteins and preventing them from firmly binding to more carrier proteins. This results in low efficiency of subsequent whole antigen conjugation, poor binding stability, and poor immunogenicity. Simultaneously, current AFM1 whole antigen preparation often employs a dual-carrier approach using bovine serum albumin (BSA) and ovalbumin (OVA) as the immunogen and coating agent, respectively. This approach is cumbersome and costly. In addition, existing whole antigens also suffer from poor product uniformity, a tendency for protein aggregation, and poor stability, severely impacting the stability and reliability of subsequent antibody preparation and detection methods.

[0005] Several patents have been disclosed in the existing technology for AFM1-related immunoassays and antigen preparation. However, these patents have not solved the aforementioned technical problems, such as insufficient purity of the AFM1 hapten, unreasonable preparation process, inability to firmly bind more carrier proteins with only a single carboxyl group, and cumbersome whole antigen preparation process with low coupling efficiency. Therefore, developing a high-purity, high-activity AFM1 hapten containing two carboxyl groups that can firmly bind more carrier proteins, a simple and efficient preparation method thereof, and an efficient whole antigen preparation method based on this hapten have become urgent technical problems to be solved in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, such as insufficient purity of the AFM1 hapten, complex preparation process, low yield, and the inability to firmly bind more carrier proteins due to the presence of only a single carboxyl group, as well as low conjugation efficiency, poor product uniformity, insufficient immunogenicity, poor specificity, and poor stability of the AFM1 whole antigen, which requires separate preparation of the immunogen and coating antigen, resulting in cumbersome procedures and high costs, this invention provides an aflatoxin M1 hapten, its preparation method, a whole antigen based on the hapten, a whole antigen preparation method, and its applications. This hapten has high purity, an intact active site, contains two carboxyl groups that can firmly bind more carrier proteins, and has a stable yield. Its preparation method features mild reaction conditions, simple operation, and strong reproducibility. The whole antigen uses only BSA as the sole carrier protein, simplifying the preparation process, improving conjugation efficiency and product uniformity. The immunogenicity, specificity, and stability are all superior to existing technologies, effectively solving the aforementioned technical problems.

[0007] The core technical solution of this invention revolves around the preparation and application of aflatoxin M1 hapten and whole antigen, highlighting the advantage that the hapten contains two carboxyl groups, which can firmly bind to more carrier proteins. The specific details are summarized below:

[0008] A hapten for the detection of aflatoxin M1, the hapten having the following structural formula: .

[0009] A method for preparing a hapten for the detection of aflatoxin M1 includes the following steps: mixing aflatoxin M1 with dimercaptosuccinic acid under N2 protection at 0°C, adding boron trifluoride diethyl ether to the reaction system, stirring overnight at room temperature, and purifying the mixture.

[0010] The purification steps are as follows: the product solution is depressurized to remove the solvent, ultrapure water is added to the residue, and the product is extracted twice with ethyl acetate solution. The organic layers are then combined. The combined organic layers are washed three times with 1 mol / L hydrochloric acid solution and saturated sodium chloride solution, and then dried with anhydrous sodium sulfate. The solvent is removed under reduced pressure to obtain the crude product.

[0011] The crude product obtained from the above steps was purified by silica gel column chromatography to obtain aflatoxin M1 hapten;

[0012] Aflatoxin M1 was dissolved in anhydrous CH2Cl2 under N2 protection at 0°C, and a DMF solution containing dimercaptosuccinic acid was added. Boron trifluoride diethyl ether was then added to the reaction system. The reaction system was stirred overnight at room temperature. After the reaction was completed by TLC, the solvent was removed under reduced pressure, and H2O was added to the residue. The residue was extracted twice with ethyl acetate. The organic layers were combined and washed three times with 1M HCl solution and saturated sodium chloride solution. The organic layer was then dried with anhydrous Na2SO4, and the solvent was removed under reduced pressure. Finally, the aflatoxin M1 hapten was purified by silica gel column chromatography.

[0013] The molar ratio of aflatoxin M1 to dimercaptosuccinic acid is 1:1.5, and the molar ratio of aflatoxin M1 to boron trifluoride ether is 1:4.2.

[0014] An aflatoxin M1 whole antigen is formed by the covalent binding of the aflatoxin M1 hapten with bovine serum albumin (BSA).

[0015] A method for preparing the aflatoxin M1 whole antigen, employing the EDC / NHS activated carboxyl coupling method, includes the following steps:

[0016] (1) Hapten activation: Take the hapten for the aflatoxin M1 detection, dissolve it in anhydrous ethanol by sonication, add MES buffer to make up the volume to obtain a hapten solution; add EDC·HCl and NHS to the hapten solution, and activate it by magnetic stirring at room temperature for 30 min to obtain an activated hapten solution.

[0017] (2) Coupling reaction: Take BSA, add PBS buffer, and stir magnetically at 4℃ until completely dissolved to obtain BSA solution; slowly add the activated hapten solution obtained in step (1) dropwise to BSA solution, adjust the pH of the system to 7.0-7.2, and stir magnetically at 4℃ for 12h to form hapten BSA covalent conjugate; wherein, the molar ratio of aflatoxin M1 hapten to BSA is 20:1;

[0018] (3) Purification: The hapten BSA covalent conjugate from step (2) was transferred into a dialysis bag with a molecular weight cutoff of 8000-14000 Da. 0.01 mol / L PBS buffer at pH 7.4 was used as the external dialysis solution. Dialysis was performed at 4°C for 72 h, and the external solution was changed every 8 h. After dialysis, the mixture was centrifuged at 4°C and 8000 r / min for 10 min. The supernatant was taken, which is the aflatoxin M1 whole antigen (AFM1-BSA).

[0019] The application of the aforementioned aflatoxin M1 whole antigen is for the detection of anti-aflatoxin M1.

[0020] The aflatoxin M1 hapten contains two intact active carboxyl groups, which can firmly bind to more carrier proteins, significantly improving the binding efficiency and stability of the hapten to the carrier protein, providing a solid guarantee for whole antigen conjugation and antibody linking, and solving the problem of weak binding ability of existing haptens to carriers; its structure has been verified by ¹H NMR and HRMS to ensure the activity of the carboxyl groups and the integrity of the structure.

[0021] The aflatoxin M1 whole antigen can be directly used for the preparation of polyclonal / monoclonal antibodies against aflatoxin M1. The strong binding of the two carboxyl groups of the hapten to BSA can stimulate the body to produce more potent specific antibodies. It can also be used to establish AFM1 immunoassay methods such as indirect enzyme-linked immunosorbent assay (ELISA) and indirect competitive ELISA. The strong binding of the hapten to the carrier makes the whole antigen coating more stable, improving the sensitivity, specificity and repeatability of the detection, which can meet the needs of rapid detection of AFM1 in dairy products.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The hapten has high purity (≥95%) and complete carboxyl activity. Its core advantage lies in the presence of two carboxyl groups, which can firmly bind more carrier proteins. Compared with existing single carboxyl haptens, the binding efficiency with carrier proteins is improved and the binding stability is high, which provides a guarantee for whole antigen conjugation and antibody linkage. The yield is stable and the preparation process is simple and controllable.

[0024] (2) The whole antigen uses a single BSA carrier, which has the functions of both immunogen and coating agent, simplifying the preparation process, reducing costs, and avoiding the problem of poor matching between the two carriers. At the same time, by taking advantage of the two carboxyl groups of the hapten, which can firmly bind more carrier proteins, the whole antigen coupling efficiency and product uniformity are significantly better than the existing technology, the binding stability is stronger, and the protein aggregation phenomenon is less likely to occur.

[0025] (3) The whole antigen has high immunogenicity, strong specificity and excellent stability. This is due to the strong binding of the two carboxyl groups of the hapten to BSA, which makes the whole antigen less likely to dissociate during storage and detection, further improving detection sensitivity, repeatability and recovery rate, meeting the requirements of practical application, and having strong anti-interference ability.

[0026] (4) The overall preparation method has mild reaction conditions, repeatable operation, no need for complicated instruments, and is easy to scale up production. The whole antigen has a wide range of applications and extremely high practical application value. At the same time, the design of the two carboxyl groups of the hapten can achieve a strong binding with the carrier protein without additional modification, which simplifies the preparation process and reduces production costs. Attached Figure Description

[0027] Figure 1 The 1H NMR spectrum is that of the aflatoxin M1 hapten. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can understand it. It should be noted that the following embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0029] The raw materials and instruments used in this embodiment are all commercially available products. The purity of aflatoxin M1 standard is ≥98%; dimercaptosuccinic acid, boron trifluoride ether, anhydrous dichloromethane, DMF, and ethyl acetate are all analytical grade; BSA has a molecular weight of 66kDa and a purity of ≥98%; EDC·HCl and NHS have a purity of ≥98%; the instruments include an electronic analytical balance, a magnetic stirrer, a UV-Vis spectrophotometer, a high-speed refrigerated centrifuge, a pH meter, a constant temperature incubator, an ELISA reader, a silica gel column, and a vacuum freeze dryer.

[0030] Example 1: Preparation of aflatoxin M1 hapten

[0031] Prepare aflatoxin M1 hapten according to the following steps, focusing on ensuring the integrity and activity of the two carboxyl groups so that they can bind firmly to more carrier proteins:

[0032] Aflatoxin M1 (328.06 mg, 1 mmol) was dissolved in 8 mL of anhydrous CH2Cl2 under N2 protection at 0 °C. 8 mL of DMF solution containing dissolved dimercaptosuccinic acid (273.33 mg, 1.5 mmol) was added, followed by the addition of boron trifluoride ether (292 μL, 4.2 mmol). The reaction mixture was stirred overnight at room temperature. After the reaction was complete as monitored by TLC, the solvent was removed under reduced pressure. 10 mL of H2O was added to the residue, and the mixture was extracted twice with ethyl acetate (10 mL each time). The organic layers were combined and washed three times with 1 M HCl solution (5 mL) and saturated sodium chloride solution (10 mL). The organic layers were then dried with anhydrous Na2SO4, and the solvent was removed under reduced pressure. Finally, the mixture was purified by silica gel column chromatography to obtain 284.38 mg of aflatoxin M1 hapten, with a yield of 57.8%. The 1H NMR spectrum of the aflatoxin M1 hapten is shown below. Figure 1 As shown.

[0033] 1HNMR (400MHz, DMSO-d6): δ2.63 (t, J=4.8 Hz, 2H); 3.12 (s, 1H); 3.39 (t, J=5.1Hz, 2H); 3.94 (s, 3H); 4.14 (s, 1H); 4.32 (s, 1H); 4.92 ( s, 1H); 6.25 (s, 1H); 6.40 (s, 1H); 6.55 (s, 1H); 11.45 (s, 1H); 12.15 (s, 1H); HRMS (ESI): m / z 492.0211; Calcd for C 21 H 16 O 10 S2: 492.0185.

[0034] Example 2: Preparation of aflatoxin M1 whole antigen (AFM1-BSA)

[0035] Using the aflatoxin M1 hapten containing two carboxyl groups prepared in Example 1, and taking advantage of its ability to firmly bind to more carrier proteins, the AFM1-BSA full antigen was prepared according to the following steps:

[0036] (1) Hapten activation: Accurately weigh 49.2 mg (0.1 mmol) of aflatoxin M1 hapten prepared in Example 1, place it in a 10 mL brown reaction bottle, add 1 mL of anhydrous ethanol, and sonicate at 200 W for 1 min to obtain hapten ethanol solution; add 0.05 mol / L, pH 6.0 MES buffer to the solution and adjust the volume to 5 mL to prepare a 20 mmol / L hapten solution; add 57.2 mg (0.3 mmol) of EDC·HCl and 34.6 mg (0.3 mmol) of NHS to the hapten solution, and activate it at room temperature (25 °C) with magnetic stirring (200 r / min) for 30 min to obtain activated hapten solution for later use; the activation process specifically activates the two carboxyl groups of the hapten, so that it can fully bind to BSA and exert the advantage of firmly binding more carrier proteins.

[0037] (2) Coupling reaction: Accurately weigh 66.0 mg (1 μmol) of BSA and place it in a 50 mL beaker. Add 10 mL of 0.01 mol / L PBS buffer (containing 0.8% NaCl, 0.02% KCl, 0.144% Na2HPO4, and 0.024% KH2PO4) at pH 7.4. Stir magnetically at 4°C (150 r / min) until completely dissolved and free of visible flocculent material to obtain a BSA solution. Slowly add the above activated hapten solution dropwise to the BSA solution using a pipette at a rate of 1 drop / 2 s. After the addition is complete, adjust the pH of the system to 7.1 using a pH meter. Stir magnetically at 4°C (150 r / min) for 12 h to form a covalently bound hapten BSA. The hapten binds more BSA carrier proteins firmly through two carboxyl groups, improving coupling efficiency and binding stability, and avoiding dissociation during coupling.

[0038] (3) Purification: The hapten BSA covalent conjugate from step (2) was transferred into a dialysis bag with a pretreated molecular weight cutoff of 8000~14000 Da. 0.01 mol / L PBS buffer at pH 7.4 was used as the dialysis fluid. The liquid-liquid ratio of the dialysis fluid to the hapten BSA covalent conjugate was 500:1. Dialysis was performed at 4°C for 72 h. The dialysis fluid was changed every 8 h to remove small molecule impurities such as free hapten, EDC, and NHS. After dialysis, the liquid in the bag was transferred to a centrifuge tube and centrifuged at 4°C and 8000 r / min for 10 min to remove a small amount of aggregated protein precipitate. The supernatant was taken as the AFM1-BSA whole antigen. The purification process protects the two carboxyl groups of the hapten and the binding sites of BSA, maintains the binding strength, and ensures the uniformity of the whole antigen.

[0039] (4) Preservation: The obtained AFM1-BSA whole antigen is aliquoted into sterile cryovials, labeled for immunization and coating, and stored at -20℃ in the dark for later use; low temperature preservation can further maintain the binding stability of the two carboxyl groups of the hapten with BSA, avoid dissociation during storage, and ensure the stability of the whole antigen performance.

[0040] Example 3: Characterization and detection of the complete AFM1-BSA antigen

[0041] 1. Qualitative characterization by ultraviolet-visible spectrophotometry

[0042] Hapten solution (anhydrous ethanol:PBS=1:9), BSA solution, and AFM1-BSA solution with a concentration of 0.5 mg / mL were prepared respectively. Using the corresponding blank buffer as a reference, ultraviolet scanning was performed in the wavelength range of 200~400 nm, and the absorption spectra and characteristic absorption peaks were recorded.

[0043] The results showed that the characteristic absorption peaks of aflatoxin M1 hapten were 365 nm (benzene ring conjugated system) and 280 nm (sulfur-containing group); the characteristic absorption peak of BSA was 280 nm (tryptophan and tyrosine residues); AFM1-BSA showed obvious absorption peaks at both 280 nm and 365 nm, and the absorption peak shape was the superposition of hapten and BSA, with no peak shift or disappearance, and the peak intensity ratio was stable (the absorbance ratio of 365 nm to 280 nm was 0.33), proving that the hapten and BSA were successfully coupled. Furthermore, because the hapten contains two carboxyl groups, it can firmly bind to BSA, the coupling is stable, there is no BSA denaturation, and no hapten dissociation.

[0044] 2. Quantitative characterization of molar ratio and coupling rate

[0045] Based on the absorbance values ​​obtained from ultraviolet scanning, the molar ratio (coupling rate) of the hapten to BSA in the whole antigen was calculated using the Lambert-Beer law. The results showed that the molar ratio of AFM1-BSA whole antigen was 16.8:1, meaning each BSA molecule was coupled to 16-17 hapten molecules. The coupling efficiency was (16.8 / 20) × 100% = 84%, ≥80%. This molar ratio is within the optimal range (10-20:1) for immunoassay antigens, without excessive coupling leading to a decrease in immunogenicity. The high coupling efficiency further confirms the advantage that the two carboxyl groups of the hapten can firmly bind to more carrier proteins, representing a more than 35% improvement in coupling efficiency compared to existing single-carboxyl haptens.

[0046] Example 4: Validation of the efficacy of AFM1-BSA whole antigen

[0047] The AFM1-BSA whole antigen prepared in Example 2 was tested for immunogenicity, specificity, and stability. The results highlight the performance advantages brought by the strong binding of the two carboxyl groups of the hapten to the carrier protein, as detailed below:

[0048] 1. Animal Immunization and Antiserum Preparation

[0049] Ten 6-8 week old SPF-grade Balb / c mice, weighing 18-22g, with half males and half females, were selected for intraperitoneal immunization:

[0050] (1) First immunization: Emulsify AFM1-BSA whole antigen and Freund's complete adjuvant in equal volume (final concentration 1 mg / mL), and inject 0.2 mL into each mouse intraperitoneally;

[0051] (2) Boosting immunization: On days 14 and 28 after the first immunization, the AFM1-BSA whole antigen was emulsified with an equal volume of Freund's incomplete adjuvant and injected intraperitoneally into each mouse.

[0052] (3) Blood collection from tail: On the 7th day after booster immunization, blood was collected from the ocular venous plexus of mice, left to stand at 37°C for 1 hour, refrigerated at 4°C overnight, centrifuged at 3000r / min for 10 min, and the antiserum was separated and stored at -20°C.

[0053] 2. Indirect ELISA detection of immunogenicity

[0054] (1) Coating: Dilute the AFM1-BSA whole antigen with 0.05mol / L carbonate buffer (CBS, pH 9.6) to 1μg / mL, coat 100μL per well of a 96-well microplate, and coat at 4℃ for 12h; because the two carboxyl groups of the hapten are firmly bound to BSA, the whole antigen coating is highly stable and not easy to fall off.

[0055] (2) Blocking: Discard the coating solution, wash 3 times with PBST (PBS containing 0.05% Tween-20) for 3 min each time, add 200 μL of 5% skim milk powder to each well, and block at 37℃ for 1 h;

[0056] (3) Sample addition: Dilute the antiserum with PBST at a ratio of 1:5000, add 100 μL to each well, and set up blank wells (PBST) and negative wells. Incubate at 37°C for 1 h.

[0057] (4) Add secondary antibody: After washing, add 100 μL of HRP-goat anti-mouse IgG diluted 1:5000 to each well and incubate at 37℃ for 40 min;

[0058] (5) Color development and termination: After washing, add 100 μL of TMB substrate color development solution to each well, develop color at 37℃ in the dark for 15 min, and add 50 μL of 2mol / L H2SO4 to terminate the reaction;

[0059] (6) Reading: The OD value of each well was measured at a wavelength of 450nm using an ELISA reader. The positive criterion was P / N≥2.1 (P is the OD value of the sample and N is the OD value of the negative well). The highest dilution factor corresponding to this value was the titer of the antiserum.

[0060] The results showed that the antiserum of all 10 immunized mice was positive, with an average titer of 1:92800, which was much higher than the minimum titer requirement (1:10000) for antiserum used in immunoassay, proving that the whole antigen has strong immunogenicity. The high immunogenicity is due to the strong binding of the two carboxyl groups of the hapten to BSA, which allows the hapten to present the antigen epitope more stably and stimulate the body to produce more potent specific antibodies.

[0061] 3. Validation of the stability of the whole antigen

[0062] The AFM1-BSA whole antigen was stored at -20℃ for 3 months, at 4℃ for 7 days, and at room temperature (25℃) for 24 hours. Its ultraviolet absorption spectrum and immunotiter were detected to observe its stability. The focus was on verifying the strong binding of the two carboxyl groups of the hapten to BSA.

[0063] The results showed that under different storage conditions, the UV characteristic absorption peak of the whole antigen remained unchanged, and the molar ratio fluctuation was ≤5%; after storage at -20℃ for 3 months, the antiserum immunogenicity fluctuation was ≤10%, and the coating binding capacity fluctuation was ≤8%; after storage at 4℃ for 7 days and at room temperature for 24 hours, the immunogenicity fluctuation was ≤15%, and the coating binding capacity fluctuation was ≤12%, proving that the whole antigen has excellent stability and can meet the storage requirements in practical applications; the good stability further confirms the strong binding of the two carboxyl groups of the hapten to BSA, avoiding the dissociation of the hapten from BSA during storage and ensuring the performance stability of the whole antigen.

[0064] Example 5: Application of AFM1-BSA complete antigen

[0065] For the preparation of polyclonal antibodies against aflatoxin M1:

[0066] Using the animal immunization method described in Example 4, Balb / c mice were immunized. Blood was collected on day 7 after booster immunization, and antiserum was separated. The antiserum was purified by protein G affinity chromatography to obtain an anti-aflatoxin M1 polyclonal antibody. This antibody has strong binding specificity to AFM1. An indirect competitive ELISA detection method was established using the AFM1-BSA whole antigen prepared in this invention for the detection of AFM1 in actual milk samples. Specific performance data are as follows: detection range was 0.003~5 ng / mL, linear correlation coefficient R² = 0.992; AFM1 was not detected in any of the 30 blank milk samples (below the detection limit of 0.003 ng / mL). Ten known positive milk samples (AFM1 concentration 0.1~0.4 ng / mL) were tested, and the deviation between the detected value and the actual value was ≤7.3%. A comparison with high-performance liquid chromatography (HPLC) of 20 milk samples showed a correlation coefficient R²=0.987 between the two methods, with no significant difference (P>0.05). This demonstrates that the whole antigen, combined with the antibody prepared from it, provides reliable detection results and can replace traditional instrument detection methods, enabling rapid, sensitive, and accurate detection of AFM1 in dairy products. This excellent application effect is inseparable from the advantage that the two carboxyl groups of the hapten can firmly bind to more carrier proteins, providing a stable foundation for antibody preparation and detection applications.

Claims

1. A hapten for detecting aflatoxin M1, characterized in that: The hapten has a structural formula of .

2. A method for preparing a hapten for aflatoxin M1 detection as described in claim 1, characterized in that, Includes the following steps: Aflatoxin M1 was mixed with dimercaptosuccinic acid under N2 protection at 0°C. Boron trifluoride ether was then added to the reaction system, and the mixture was stirred overnight at room temperature to obtain the final product.

3. A complete aflatoxin M1 antigen, characterized in that, It is formed by the covalent binding of the hapten for the aflatoxin M1 detection described in claim 1 and bovine serum albumin.

4. A method for preparing the aflatoxin M1 whole antigen as described in claim 3, characterized in that, The EDC / NHS activated carboxyl coupling method includes the following steps: (1) Hapten activation: Take the hapten for the detection of aflatoxin M1 as described in claim 1, dissolve it in anhydrous ethanol by sonication, add MES buffer to make up the volume to obtain a hapten solution; add EDC·HCl and NHS to the hapten solution, and activate it by magnetic stirring at room temperature for 30 min to obtain an activated hapten solution. (2) Coupling reaction: Take BSA, add PBS buffer, and stir magnetically at 4℃ until completely dissolved to obtain BSA solution; slowly add the activated hapten solution obtained in step (1) dropwise to BSA solution, adjust the pH of the system to 7.0-7.2, and stir magnetically at 4℃ for 12h to form hapten BSA covalent conjugate; wherein, the molar ratio of aflatoxin M1 hapten to BSA is 20:1; (3) Purification: The hapten BSA covalent conjugate from step (2) was transferred into a dialysis bag with a molecular weight cutoff of 8000-14000 Da. 0.01 mol / L PBS buffer at pH 7.4 was used as the external dialysis solution. Dialysis was performed at 4°C for 72 h, and the external solution was changed every 8 h. After dialysis, the mixture was centrifuged at 4°C and 8000 r / min for 10 min. The supernatant was taken, which is the aflatoxin M1 whole antigen.

5. The application of the aflatoxin M1 whole antigen as described in claim 3, characterized in that, Used for the detection of aflatoxin M1 in dairy products.

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