CN111879937A - Immunoassay kit for detecting aflatoxin B1 in grain and oil - Google Patents

Abstract

The invention discloses a time fluorescence resolution fluorescence immunoassay kit for detecting aflatoxin B1 in grain and oil. The time-resolved fluorescence immunoassay detection kit for detecting the aflatoxin B1 is composed of a porous coated plate, a buffer solution, an aflatoxin B1 standard substance, an antibody freeze-dried product of the aflatoxin B1, a europium-labeled goat anti-mouse antibody, a washing solution and an enhancement solution. The detection method of the time fluorescence immunoassay kit for detecting the aflatoxin B1 comprises the following steps: (1) preparing an immunogen; (2) preparing a coating source; (3) preparing a monoclonal antibody; (4) and (4) pretreating and detecting a sample. The invention has the advantages of short detection time, high average recovery rate, simple sample pretreatment, capability of field operation detection, wide application, low detection cost, strong detection specificity, small difference between batches, high sensitivity, simple and quick operation, particular suitability for the detection of mass samples and the like.

Description

Immunoassay kit for detecting aflatoxin B1 in grain and oil

Technical Field

The invention belongs to the field of biological detection, and particularly relates to a time-resolved fluorescence immunoassay kit for detecting aflatoxin B1 in grain and oil.

Background

Aflatoxins (AFT) are secondary metabolites of fungi, mainly produced by a number of strains of aspergillus flavus (a. flavus), aspergillus parasiticus (a. parasiticus) and aspergillus wasabis (a. nonius); the toxin production capacity of aspergillus flavus is greatly different due to different strains; all strains of aspergillus parasiticus are capable of producing aflatoxins. Aflatoxin is a hepatotoxin with strong toxicity, can cause acute or chronic damage to the liver, and is considered as the two main causes of liver cancer together with hepatitis B virus; besides damaging the liver of an organism, aflatoxin can also cause serious damage to other various tissues and organs such as the kidney and the like, and more seriously, aflatoxin is proved to have carcinogenic, teratogenic and cytogenic effects. Aflatoxins are widely found in cereal agricultural products such as peanuts, corns, wheat and rice, and are often found in food such as macaroni, seasonings, grain and oil, edible oil and the like, and seriously harm the health of people, livestock and poultry. Aflatoxin B1 is recognized as the most carcinogenic natural substance at present, and aflatoxin was classified as a class i carcinogen by the cancer research institute of the World Health Organization (WHO) in 1993. The species of aflatoxins have increased since their discovery in 1961. Twelve aflatoxins, B1, B2, G1, G2, B1, M2, B2a, G2a, GB1, R0 and toxol, have been isolated and identified so far; from the chemical structure, the structures of various aflatoxins are very similar, and all contain a bifuran ring which is a basic toxic structure and an o-naphthone (coumarin) which may be related to carcinogenesis. Aflatoxins are many times more toxic than cyanide and arsenic. It has been ascertained that aflatoxins exhibit severe toxicity to humans and a wide variety of animals, and have significant carcinogenic potential; aflatoxin B1 was the most toxic and most carcinogenic, G1, B1, and B2, G2 and M2 were the second to be weaker. Aflatoxin B1, in addition to being carcinogenic, can cause mutations and malformations. The LD 50 of the aflatoxin B1 is 0.249 mg/kg and less than 1 mg/kg, belongs to a very toxic substance, and has the toxicity 10 times that of potassium cyanide and 68 times that of arsenic. Aflatoxin B1 was found to be the most carcinogenic of all known carcinogens at present.

Many chemical detection methods have appeared on aflatoxin detection methods, such as Gas Chromatography (GC), High Performance Liquid Chromatography (HPLC), thin layer chromatography, spectrophotometry, liquid mass spectrometer (LC-MS), gas mass spectrometer (GC-MS), and the like. The chemical detection method has the defects of high test cost, long analysis time, large sample amount (generally more than 500 mL) required by detection, complicated pretreatment steps and the like, and can not systematically, comprehensively and quickly analyze the content of the aflatoxin in a larger range.

The time-resolved fluoroimmunoassay (TR-FIA) is increasingly regarded and adopted by people due to its advantages of strong specificity, high sensitivity, simple operation, low cost, and being particularly suitable for the detection of large-scale samples. At present, no patent and literature report of a time fluorescence immunoassay method aiming at the detection of aflatoxin B1 exists.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a detection time-resolved fluorescence immunoassay kit for aflatoxin B1 residue in grain and oil.

The second purpose of the invention is to provide a detection method of a time-resolved fluoroimmunoassay kit for quickly and simply detecting aflatoxin B1 in grain and oil, which is used for quantitatively or qualitatively detecting the residual amount of aflatoxin B1 in crops.

One of the objects of the invention is achieved by: the time-resolved fluoroimmunoassay kit for detecting aflatoxin B1 is characterized by consisting of a porous coated plate, a buffer solution, an aflatoxin B1 standard solution, an anti-aflatoxin B1 antibody freeze-dried product, a europium-labeled rabbit anti-mouse antibody, a washing solution and an enhanced liquid.

The second purpose of the invention is realized by the following steps: the detection method of the time-resolved fluoroimmunoassay kit for detecting the aflatoxin B1 comprises the preparation of immunogen, coating antigen and monoclonal antibody, the pretreatment and detection of a sample, and is characterized in that:

(1) preparation of immunogen: coupling hapten aflatoxin B1 with Bovine Serum Albumin (BSA) to obtain immunogen (aflatoxin B1-BSA);

(2) preparation of coating antigen: coupling hapten aflatoxin B1 with egg serum albumin (OVA) to obtain a coating antigen (aflatoxin B1-OVA);

(3) preparation of monoclonal antibody:

a. immunizing a mouse by using the immunogen (aflatoxin B1-BSA) obtained in the step (1), and obtaining a hybridoma cell strain secreting a monoclonal antibody against aflatoxin B1 by using a hybridoma technology;

b. preparing a large amount of antibodies by an in vivo induced ascites method, and purifying by using a Protein G column to obtain monoclonal antibody IgG of anti-aflatoxin B1;

c. coating the 96-well coated plate with the coating original coating in the step (2);

(4) pretreatment and detection of a sample:

taking a porous coating plate coated with a coating source (aflatoxin B1-OVA), adding 50 muL of aflatoxin B1 into respective micropores, adding 50 muL of anti-aflatoxin B1 antibody diluted by buffer solution, oscillating for 0.5-1 h at 25-37 ℃, washing for 3 times by using washing solution, and adding 100 muL of Eu for dilution by using buffer solution³⁺And (3) oscillating the rabbit anti-mouse antibody at 25-37 ℃ for 0.5-1 hour, washing the rabbit anti-mouse antibody for 6 times by using washing liquid, adding 200 mu L of enhancing liquid, oscillating the rabbit anti-mouse antibody for 5 minutes, measuring the fluorescence intensity cps, and calculating the content of aflatoxin B1 in the sample according to a standard curve.

The solid phase carrier is a multi-hole coated plate, and a 96-hole multi-hole coated plate is used as the solid phase carrier.

The invention mainly adopts a time-resolved fluorescence immunoassay method to detect aflatoxin B1. The technique of time-resolved fluoroimmunoassay has two main aspects: firstly, preparing a specific monoclonal antibody, immunizing a mouse by using a coupled immunogen, and obtaining a hybridoma cell strain secreting the monoclonal antibody against aflatoxin B1 by using a hybridoma technology; preparing a large amount of antibodies by an in vivo induced ascites method, and purifying by using a Protein G column to obtain the monoclonal antibody IgG of the anti-aflatoxin B1. Second, Eu³⁺And (3) preparing a labeled antibody.

The determination method of the invention comprises the following steps: the basis of the assay is a labeled immune response. Adding a test sample into each micropore of a porous coated plate coated with aflatoxin B1-OVA, adding an anti-aflatoxin B1 antibody, carrying out oscillation reaction, enabling free aflatoxin B1 to compete with aflatoxin B1-OVA on the microporous plate for resisting aflatoxin B1 antibody, washing with a washing solution, and removing the unconnected aflatoxin B1 antibody in the washing step. Adding Eu³⁺Rabbit anti-mouse antibodies, carrying out a labeled immune reaction,washing with a washing solution, and washing the Eu which is not connected after reaction³⁺Rabbit anti-mouse antibodies are removed in a washing step. After the enhancement liquid is added and oscillated, strong fluorescence is emitted under the excitation of an ultraviolet lamp, the fluorescence intensity cps is measured by a time-resolved fluorescence instrument, the fluorescence intensity is in inverse proportion to the concentration in the sample, and the amount of the aflatoxin B1 in the sample can be determined by contrasting with a standard curve.

The detection method provided by the invention does not need expensive instruments, is simple in sample pretreatment, can be used for field operation detection, is wide in application, is sensitive, accurate and rapid, is simple and convenient to operate, has strong specificity, and is suitable for rapid detection of a large number of samples.

Detailed Description

Examples

1. Preparation of immunogen and coatingen

Synthesis of the immunogen (aflatoxin B1-BSA) of the invention: accurately weighing aflatoxin B1324mg, dissolving in 2mLN, N-dimethylformamide, dropwise adding gamma-aminobutyric acid solution while stirring, stirring for reaction for 3 hours, and adjusting the pH of the reaction solution to about 10. The precipitate was removed by centrifugation. Adding the above reaction dropwise into BSA solution (320 mg BSA dissolved in 5mL physiological saline), adding 23mg N-hydroxysuccinimide (NHS), 45.4mg N, N-Dicyclohexylcarbodiimide (DCC), reacting overnight at 4 deg.C, centrifuging to remove precipitate, collecting supernatant, dialyzing with Phosphate Buffer Solution (PBS) for 3 days, changing dialysate every 6 hr, lyophilizing the obtained product, and storing at-20 deg.C;

synthesis of coating antigen (aflatoxin B1-OVA): in the above reaction, after the OVA was replaced with BSA, the aflatoxin B1-OVA was obtained as a reaction conjugate, which was used as a coating antigen in the TR-FIA assay.

2. Preparation of monoclonal antibodies

2.1 animal immunization

And (3) immunizing female Balb/c mice with the immunogen prepared in the step 1 for 6 weeks, wherein the immunization dose of each mouse is 100 mug/0.2 mL. The first immunization, the immunogen (aflatoxin B1-BSA) is dissolved by sterile 0.01mol/L PBS (pH7.4), then the mixture is mixed with an equal amount of Freund's complete adjuvant, the complete emulsification is carried out, and the injection is carried out by 2-3 points under the skin of the back; the immunization is strengthened, the immunogen is dissolved by 0.01mol/L PBS (pH7.4PBS) and is mixed with equivalent Freund's complete adjuvant, the mixture is fully emulsified, and the mouse is injected into the abdominal cavity. The interval of each time is 14-21 days, blood is collected from the tail vein of the immunized mouse 7-10 days after 3 rd immunization, and the serum titer of the mouse is detected by ELISA. After last immunization, the interval is more than 4 weeks, aflatoxin B1-BSA antigen is intraperitoneally injected for 3-4 days before cell fusion, 100 microgram/0.2 mL/mouse is observed every day after injection, and the good state of the mouse before fusion is ensured.

2.2 monoclonal antibody preparation

Splenocytes from immunized mice were isolated and homogenized to prepare immunized splenocytes. 1 immunized Balb/c mouse was taken, serum was isolated from orbital bleeds as negative serum and sacrificed. Mice were soaked in 75% alcohol for 5min for total disinfection. Fixing the four limbs of the mouse, clamping the skin of the lower abdomen of the mouse by using forceps, cutting a small opening, tearing the skin by using the forceps to expose the peritoneum, replacing a set of forceps and scissors, and cutting a small opening on the central peritoneum of the abdomen by using the scissors. The peritoneum is cut off by the scissors to expose the spleen, the spleen is clamped by the forceps by the scissors, the spleen adventitia is cut off by the scissors, and then the spleen is placed into a homogenizer which is sterilized in advance. Adding appropriate amount of basal medium (RPMI-1640) into homogenizer, grinding, extruding splenocytes, taking out homogenizing rod of homogenizer, adding appropriate amount of basal medium (RPMI-1640), standing for 2min, sucking upper layer cell liquid, placing into abdominal cavity macrophage centrifuge tube, and repeating the above operation for 1 time. Centrifuging at 1200r/min for 10min, and removing supernatant. Will 10⁸Immune spleen cells and 1-2 x 10⁷SP2/0 myeloma cells were added to the centrifuge tube at a ratio of 1:10 or 1:5, mixed well, and then centrifuged at 1500 rpm for 10min, and the supernatant was discarded. The centrifuge tube was inverted over sterilized absorbent paper and the liquid in the tube was blotted. Gently knocking the bottom of the tube with fingers or a table to loosen the settled cells, and placing the centrifuge tube in a 37 ℃ water bath. 0.8mL of 50% PEG was slowly dropped into the centrifuge tube over 1min while gently stirring with the pipette tip to pellet the cells. Further stirring for 30s, standing for 1min, and slowly adding 40mL of basal medium pre-warmed at 37 deg.C(RPMI-1640). The method for adding the basic culture medium comprises the following steps: dropwise adding 1mL of the culture medium in 1min, dropwise adding 2mL of the culture medium in 2min, dropwise adding 3mL of the culture medium in 3min, dropwise adding 4mL of the culture medium in 4min, slowly adding the culture medium in each time, slightly stirring the culture medium, and finally slowly adding the rest of the RPMI-1640 culture medium. Centrifuging at 1000r/min for 5min, and removing supernatant. The mixed cells were then suspended in HAT medium and the feeder spleen cells were added. And adding a proper amount of HAT culture medium according to needs, uniformly mixing, and dropwise adding the cell fusion liquid containing the feeder cells onto a 96-hole cell culture plate, wherein the dropwise adding amount is about 150 muL/hole. The plates were incubated at 37 ℃ with 5% CO₂And (5) culturing in a saturated humidity incubator. Positive cell clones were screened by established indirect ELISA. Wells in which strong positive colonies grew were selected and cloned by limiting dilution. And performing 24-hole expanded culture on other positive holes, detecting the supernatant of the expanded culture holes by using indirect ELISA and indirect competition ELISA, and performing liquid nitrogen cryopreservation on the cells of the positive holes of both the indirect ELISA and the indirect competition ELISA. And (3) carrying out fusion detection, and carrying out subcloning for 3 times to obtain the hybridoma cell strain. The hybridoma cell strain is subjected to multiple passages, cryopreservation and resuscitation, and the hybridoma cell secretes stable antibody. Counting chromosomes of the hybridoma cells, randomly selecting 20 cells from each hybridoma cell, counting the number of chromosomes of the cells, and calculating the average value of the number of chromosomes of the cells. The number of chromosomes of mouse spleen cells is 40, the average number of chromosomes of SP2/0 cells is 62-68, and the number of chromosomes of 20 hybridoma cells obtained in the experiment is 92-103, and the average number of chromosomes is 96.8. The number of chromosomes of the hybridoma cell is higher than that of the chromosome of the amphiphilic parent cell, which indicates that the hybridoma cell is a hybridization product of the two cells. The culture supernatant secreted by the cell line cells is diluted by 1:10, and then the antibody subtype secreted by the cell line is IgG1 by a sandwich ELISA method. The mouse ascites is purified by the octanoic acid-ammonium sulfate method. The monoclonal antibody can be used for preparing a time-resolved fluorescence detection kit.

2.3 purification of monoclonal antibodies

The mouse ascites is purified by adopting an octanoic acid-ammonium sulfate method: taking 10mL of mouse ascites, adding equal volume of barbital buffer solution and proper amount of silicon dioxide, mixing, and oscillating for 30min at room temperature. Standing at room temperature for 15min, taking the supernatant, centrifuging at 4 deg.C and 1800r/min for 20 min; taking 18mL of supernatant, adding 36mL of 0.06mol/L sodium acetate buffer solution, adjusting the pH value to 4.5 by using HCl, and slowly adding 297 muL of caprylic acid within 30min under full stirring; continuously stirring for 10min, then transferring to a refrigerator at 4 ℃ for standing for 2h, centrifuging at 4 ℃ at 15000r/min for 30min, and filtering the supernatant with a filter membrane of 0.45 mu m to obtain a supernatant with a volume of 50 mL; adding 5mL of 0.1mol/L phosphate buffer solution, adjusting the pH value to 7.6 by using NaOH, and slowly adding ammonium sulfate to a final concentration of 0.277g/mL under stirring; standing in a refrigerator at 4 ℃ for 2h, centrifuging at 12000r/min at 4 ℃ for 30min, and removing the supernatant; the precipitate is resuspended with 5mL of 0.1mol/L phosphate buffer, put into a dialysis bag, dialyzed fully with 5000mL of 0.01mol/L PBS buffer with pH7.2, dialyzed with 2000 mL of distilled water, and finally dialyzed with 3000mL of triple-distilled deionized water; then centrifuging at 12000r/min for 30min at 4 ℃, discarding the precipitate, collecting the supernatant, and measuring the protein concentration. SDS-PAGE electrophoresis is carried out to identify the purity of the monoclonal antibody.

2.4 preparation of Rabbit anti-mouse IgG antibodies

A health New Zealand white rabbit is immunized by Balb/C mouse IgG to prepare high-titer rabbit anti-mouse IgG hyperimmune serum, the serum is subjected to crude extraction by adopting a saturated ammonium sulfate precipitation method, and the high-purity rabbit anti-mouse IgG is obtained after G-200 column chromatography.

3.1 preparation kit and detection sample

Taking 1-2 mL of 5g/L rabbit anti-mouse antibody dissolved in 50mmol/L PBS (pH7.0), converting buffer conditions by a PD-10 column, and eluting with 50mmol/L Na containing 0.155mmol/L NaCl₂CO₃-NaHCO₃pH8.5 buffer solution. The protein peak was collected, quantified by UV absorbance analysis (1.46A 280-0.74A 260), and the rabbit anti-mouse antibody was diluted to 2g/L with the above eluent. Adding 0.2-0.4 mg Eu into 500-1000 μ L diluted rabbit anti-mouse antibody³⁺-N₂- [ p-isocyanato-benzyl radical]-diethylenetriamine tetraacetic acid (Eu)³⁺-DTTA), the reaction was magnetically stirred for 20 hours at 30 ℃. The reaction solution was chromatographed on a Sepharose CL-6B column (1X 40 cm) equilibrated with 80mmol/L Tris-HCl pH7.8 buffer, A₂₈₀Monitoring and collecting eggAnd (5) white peak, diluting and subpackaging for later use.

3.2 preparation of coated plate solid phase antigen

Aflatoxin B1-OVA was used at 50mmol/LNa₂CO₃-NaHCO₃The buffer solution with pH9.6 is diluted to 1mg/L coating solution, 100 mu.L of the solution is added to each well of a 96-well coating plate, and the plate is placed at 4 ℃ overnight. The coating solution was discarded, washed three times, blocked with 150. mu.L of the above buffer containing 3g/L of OVA, and left overnight at 4 ℃. Discarding the sealing liquid, vacuum-pumping, sealing the lath, and freezing and storing at-20 deg.C.

3.3 preparation of reagents

(1) Preparing an aflatoxin B1 standard solution: diluting aflatoxin B1 standard substance into 0ng/mL, 0.01ng/mL, 0.025ng/mL, 0.1ng/mL, 0.25ng/mL, 1ng/mL, 2.5ng/mL, 10ng/mL, 25ng/mL, 100ng/mL series of concentrations, and diluting with 0.1mol/L pH7.5 phosphate buffer solution;

(2) buffer solution: 8mmol/L NaCl, 0.2% OVA, 50. mu. mol/L diethylenetriaminepentaacetic acid (DTPA), 0.1mL/LTweeen-80 and 0.1% NaN₃50mmol/L Tris-HCl pH 7.8;

(3) the washing liquid is: 14.5mmol/L NaCl, 0.2mL/L Tween-80 and 0.2% NaN₃50mmol/L Tris-HCl pH 7.8;

(4) preparing an enhancement solution: is prepared by adding 15 mu mol of beta-naphthoyl trifluoroacetone, 50 mu mol of tri-n-octylphosphine oxide and 1mL of triton X-100 into a pH3.2 potassium hydrogen phthalate buffer solution and then fixing the volume to 1L.

3.4 reagents provided by the kit

Based on the prepared reagent, the time-resolved fluorescence immunoassay kit for detecting aflatoxin B1 comprises the following materials:

(1) 96-hole enzyme label plate is multiplied by 1 block;

(2) aflatoxin B1 standard substance 1 mg/mL/bottle;

(3) the aflatoxin B1-resistant antibody freeze-dried product is dissolved in 0.5 mL of distilled water when in use;

(4)Eu³⁺-a lyophilized rabbit anti-mouse antibody product, which is dissolved in 0.5 mL of distilled water at the time of use;

(5) enhancing liquid: 15 mL;

(6)10 × washing solution: 30 mL;

(7) buffer solution: 30 mL.

3.5 considerations before the assay:

A. all reagents were brought back to room temperature (18-30 ℃) prior to use;

B. immediately after use, all reagents were returned to 2-8 ℃;

C. use of a multichannel pipettor if the sample size is large;

D. in all constant temperature incubation processes, light irradiation is avoided, and the micropores are covered by covers;

E. the required number of microplates and frames were removed, the unused microplates were placed in the original tinfoil bag and resealed with the provided desiccant and stored at 2-8 ℃.

3.6 the concrete detection steps are as follows:

taking aflatoxin B1-OVA laths, adding 50 mu L of aflatoxin B1 into respective micropores, adding 50 mu L of anti-aflatoxin B1 antibody diluted by buffer solution, oscillating for 0.5-1 h at 25-37 ℃, washing with washing solution for 3 times, and adding 100 mu L of Eu diluted by buffer solution³⁺And (3) oscillating the rabbit anti-mouse antibody at 25-37 ℃ for 0.5-1 hour, washing the rabbit anti-mouse antibody for 6 times by using washing liquid, adding 200 mu L of enhancing liquid, oscillating the rabbit anti-mouse antibody for 5 minutes, measuring the fluorescence intensity cps, and calculating the content of aflatoxin B1 in the sample from the standard curve.

3.7 preparing the kit and detecting the grain and oil samples according to the following steps:

(1) the kit is prepared as in the example;

(2) the specific detection steps are as follows:

taking aflatoxin B1-OVA laths, adding 50 mu L of aflatoxin B1 into respective micropores, adding 50 mu L of anti-aflatoxin B1 antibody diluted by buffer solution, oscillating for 0.5-1 h at 25-37 ℃, washing with washing solution for 3 times, and adding 100 mu L of Eu diluted by buffer solution³⁺And (3) oscillating the rabbit anti-mouse antibody at 25-37 ℃ for 0.5-1 hour, washing the rabbit anti-mouse antibody for 6 times by using washing liquid, adding 200 mu L of enhancing liquid, oscillating the rabbit anti-mouse antibody for 5 minutes, measuring the fluorescence intensity cps, and calculating the content of aflatoxin B1 in the sample according to a standard curve.

Claims (6)

1. A time-resolved fluorescence immunoassay kit for detecting aflatoxin B1 in grain and oil is characterized in that: the kit consists of a porous coating plate, a buffer solution, an aflatoxin B1 standard product, an antibody freeze-dried product of aflatoxin B1, a europium-labeled goat anti-mouse antibody, a washing solution and an enhancing solution.

2. The time-resolved fluoroimmunoassay kit for detecting aflatoxin B1 according to claim 1, which comprises the preparation of immunogen, coating antigen and monoclonal antibody and the pretreatment of sample, and is characterized in that:

(1) coupling aflatoxin B1 with bovine serum albumin to obtain an immunogen;

(2) coupling aflatoxin B1 with egg serum protein to obtain a coating antigen;

(3) immunizing a mouse by using the immunogen in the step (1), and obtaining a hybridoma cell strain secreting the monoclonal antibody against aflatoxin B1 by using a hybridoma technology;

(4) preparing a large amount of antibody by in vivo induced ascites method, purifying by Protein G column to obtain monoclonal antibody of anti-aflatoxin B1

(5) Coating the solid phase carrier with the coating antigen obtained in the step (2);

(6) firstly, acidolysis extracting animal tissues, purifying the animal tissues by an MAX column, and finally adding a derivative reagent and a catalyst for treatment to obtain a product to be detected;

(7) and (4) measuring the fluorescence intensity cps of the object to be detected in the step (6), and calculating the content of aflatoxin B1 and aflatoxin B1 in the sample by contrasting with the standard curve.

3. The time fluorescence immunoassay kit for detecting aflatoxin B1 of claim 1, which is characterized in that: the solid phase carrier is a porous coated plate, and a 96-hole microporous coated plate is used as the solid phase carrier.

4. The time-resolved fluorescence immunoassay kit for detecting aflatoxin B1 of claim 1, which is characterized in that: the derivatizing agent is butylamine.

5. The time-resolved fluoroimmunoassay kit for detecting aflatoxin B1 of claim 1, wherein: the catalyst is nitrile diethyl phosphate.

6. The time fluorescence immunoassay kit for detecting aflatoxin B1 of claim 1, which is characterized in that: the steps (6) and (7) are specifically to take a micropore coated plate coated with aflatoxin B1-OVA, add 50 muL of treated samples to respective micropores, add 50 muL of aflatoxin B1 antibody diluted by buffer solution, oscillate for 0.5-1 hour at 25-37 ℃, wash for three times by using washing solution, and add 100 muL of Eu diluted by buffer solution³⁺Oscillating the goat anti-mouse antibody at 25-37 ℃ for 0.5-1 hour, washing the goat anti-mouse antibody for six times by using washing liquid, adding 200 mu L of enhancing liquid, oscillating for 5 minutes, measuring fluorescence intensity cps, and calculating the content of aflatoxin B1 in the sample from the standard curve.