CN112142756A

CN112142756A - Preparation of aflatoxin M1 hapten and holoantigen as well as preparation and application of rapid transfer quantitative kit

Info

Publication number: CN112142756A
Application number: CN202011116334.7A
Authority: CN
Inventors: 吴小平
Original assignee: Beijing Ceer Kang Bio Tech Co ltd
Current assignee: Beijing Ceer Kang Bio Tech Co ltd
Priority date: 2020-10-19
Filing date: 2020-10-19
Publication date: 2020-12-29

Abstract

The invention discloses preparation of aflatoxin M1 hapten and holoantigen and preparation and application of a rapid transfer quantitative kit thereof. The aflatoxin M1 hapten provided by the invention is connected with a carrier protein to obtain the aflatoxin M1 antigen. The aflatoxin M1 antigen can be applied to preparation of an aflatoxin M1 specific antibody. The preparation method is simple, convenient and feasible, and has lower cost and higher hapten yield. The aflatoxin M1 holoantigen and the aflatoxin M1 specific antibody can be used for preparing a chemiluminescence enzyme-linked immunoassay kit for detecting aflatoxin M1 residues, and have the advantages of simplicity, rapidness, large sample treatment amount, high sensitivity, strong specificity and the like.

Description

Preparation of aflatoxin M1 hapten and holoantigen as well as preparation and application of rapid transfer quantitative kit

Technical Field

The invention relates to a hapten, an antigen and a preparation method thereof, in particular to preparation of aflatoxin M1 hapten and holoantigen and preparation and application of a rapid transfer quantitative kit thereof.

Background

Aflatoxin M1(AflatoxinM1, abbreviated AFM1) whose basic structure is an oxanaphthalenone with a difuran ring. Molecular formula C₁₇H₁₂O₇The shape of the crystal is rectangular flake and colorless crystal. The physical and chemical properties are quite stable and not destroyed by pasteurization. AFM1 belongs to one of Aflatoxins (Aflatoxins) which are one of compounds with similar structures, the Aflatoxins are metabolites produced by common aspergillus flavus (Aspergillus flavus) and aspergillus parasiticus (Aspergillus parasiticus), wherein aflatoxin B1 is the most important toxin, and after a mammal ingests feed or food polluted by AFB1, under the catalysis of in vivo liver microsome monooxygenase system, AFM1 is generated by hydroxylation of furan ring C-10 at the end of AFB1 through the regulation of cytochrome P-448. AFM1 can also be produced directly from some aspergillus flavus and aspergillus parasiticus, and is second only toxic to aflatoxin B1. Research shows that aflatoxin M1 is a strong carcinogen, and is classified as a class I carcinogen by WHO, and is a key monitoring object in all countries in the world. After aflatoxin M1 enters human or animal bodies, the aflatoxin M1 inhibits the synthesis of DNA and RNA, and also inhibits the synthesis of liver protein, thereby causing human poisoning. The harm is mainly shown in 3 aspects: damage to tissue and organs; carcinogenesis, teratogenesis and mutagenesis, and can induce liver cancer, renal cancer, gastric cancer, etc. of animals and human beings; inhibiting immune function. The toxicity of the arsenic trioxide powder is 10 times that of potassium cyanide and 68 times that of arsenic trioxide, and the main harmful parts are in the liver. The world health organization establishes that the maximum allowable concentration of aflatoxin in the food is 15 mug/kg, the content of milk consumed by human beings cannot exceed 0.5 mug/kg, and the content of other animal feeds cannot exceed 300 mug/kg according to related laws of the federal government in the United states. The limit of aflatoxin M1 in food in milk and dairy products is specified in national standard GB2761-2011 limit of mycotoxins in food published this year in ChinaThe amount was 0.5. mu.g/kg. While the eu national regulations are more stringent, the M1 limit in raw milk, heat-treated milk and processed milk products is 0.05 μ g/kg, and the M1 limit in infant food (including baby milk) is 0.025 μ g/kg. Therefore, it is urgent to establish a specific and sensitive detection method for aflatoxin M1.

In the aspect of the detection method of aflatoxin M1, the most commonly used methods at present mainly include methods such as High Performance Liquid Chromatography (HPLC), Thin Layer Chromatography (TLC), enzyme-linked immunosorbent assay (ELISA), immunoaffinity column method, and the like. The HPLC method has the advantages of high detection accuracy and low false positive rate, and has the main defects of high instrument price, more complicated operation, long time consumption, high detection cost and high operation technical requirement; and the selection of the extraction conditions of the early samples also has great influence on the detection sensitivity and accuracy. The TLC method has poor specificity and sensitivity, and the required standard substance has high concentration and high potential pollution. The immunoaffinity column has good specificity, but can be quantitatively detected only by combining an HPLC (high performance liquid chromatography) instrument and a fluorescence photometer, and has high detection technical requirements and cost. The enzyme-linked immunosorbent assay is one of the most widely applied and developed immunoenzyme technologies at present, and has the main advantages of rapid detection, simple sample pretreatment, simple detection system operation, less pollution, higher sensitivity, low cost, convenience for large-scale detection and accordance with the development direction of the current international detection field.

Disclosure of Invention

The invention aims to provide preparation of aflatoxin M1 hapten and holoantigen and preparation and application of a rapid transfer quantitative kit.

In order to solve the technical problems, the technical scheme of the invention is as follows:

the aflatoxin M1 hapten provided by the invention is a compound shown in a formula 1.

Formula 1

The invention also discloses a preparation method of the compound shown in the formula 1, which comprises the following steps:

weighing 4.6mg of aflatoxin M1 and 8mg of carboxyl methoxy amine hydrochloride by a ten-thousandth electronic balance, dissolving in 8mL of pyridine, adding into a reflux bottle, and controlling the temperature to be 80-90 ℃ for refluxing for 16 hours;

evaporating the obtained solution to dryness by a rotary evaporator, adjusting the pH value to about 9.0 by using 0.1mol/L potassium hydroxide, immediately adding 10mL ethyl acetate, uniformly mixing, standing and layering;

and thirdly, removing the lower aqueous phase, taking the upper organic phase ethyl acetate, and drying the upper organic phase ethyl acetate by using a rotary evaporator to obtain a light yellow solid oily substance which is identified as aflatoxin M1 hapten.

The aflatoxin M1 antigen provided by the invention is a conjugate obtained by coupling a compound shown in formula 1 and a carrier protein.

As the carrier protein, there can be used, for example, Bovine Serum Albumin (BSA), Ovalbumin (OVA), Human Serum Albumin (HSA), Murine Serum Albumin (MSA), thyroid protein (TG) or hemocyanin (KLH), etc.

The structural schematic diagram of AFM1 antigen obtained by coupling the compound shown in the formula 1 with BSA is shown in figure 1.

The invention also discloses a preparation method of the AFM1 holoantigen, which comprises the following steps:

weighing 30mgBSA by a precision electronic balance, dissolving in 10ml carbonate buffer solution with the concentration of 0.1MOL/L to obtain solution A;

dissolving the hapten with 2ml of N, N-dimethylformamide, and adding 20mg of DCC and 20mg of NHS to obtain solution B;

③ after 30 minutes of activation, the solution B is slowly dripped into the solution A, after stirring for 16 hours at room temperature, 0.01MOL/LPBS buffer solution is selected for dialysis for 7 working days, the solution is replaced for 3 times every day in a refrigerator at 2-8 ℃, after dialysis, the lower layer sediment is removed by centrifugation, and the supernatant is taken and stored separately.

The aflatoxin M1 antigen can be used as an immunogen to prepare an aflatoxin M1 specific antibody, and can also be used as a coating antigen to prepare a microporous plate.

The specific antibody prepared by using the aflatoxin M1 antigen can be a monoclonal antibody or a polyclonal antibody.

The invention also discloses a preparation method of the AFM1 polyclonal antibody, which comprises the following steps:

taking aflatoxin M1 whole antigen as an immunogen solution, diluting the aflatoxin M1 whole antigen with PBS (phosphate buffer solution) with pH of 7.2 and 0.02M to obtain an immunogen diluent for preparing an aflatoxin M1 polyclonal antibody, and taking a black goat bred in Jiangkou county Vanjinna mountain in southwest as an aflatoxin M1 polyclonal antibody immune animal, wherein the immune steps are as follows:

first immunization: mixing aflatoxin M1 immunogen diluent with a complete adjuvant prepared by Beijing Keerkang biotechnology limited company with the same volume to prepare an emulsifier, and performing subcutaneous multipoint injection on the neck and back, wherein the immunization dose is 5000 ug/mouse at one time;

② strengthening immunity: after 30 days, 60 days and 90 days of primary immunization, respectively carrying out primary boosting immunization, mixing and emulsifying aflatoxin M1 immunogen diluent and an incomplete adjuvant prepared by Beijing Keerkang biotechnology limited with the same volume, carrying out subcutaneous multi-point injection on different parts and the neck and back, wherein the single immunization dose is 5000 ug/mouse;

(iii) last immunization: performing last immunization 120 days after the first immunization, and directly injecting aflatoxin M1 immunogen diluent subcutaneously at multiple points on the neck and back, wherein the immunization dose is 5000 ug/mouse; after 14 days of the last immunization, blood is collected and serum is separated, namely the aflatoxin M1 polyclonal antibody corresponding to the immunogen can be prepared by using the kit.

The aflatoxin M1 antigen and the specific antibody can be applied to detection of aflatoxin M1 drug residues.

The invention discloses a rapid transfer quantitative kit prepared by using an aflatoxin M1 antigen and an aflatoxin M1 specific antibody.

The rapid transfer quantification kit comprises: the kit comprises aflatoxin M1 standard substance working solution, aflatoxin M1 high-concentration standard substance, aflatoxin M1 enzyme-labeled plate, aflatoxin M1 enzyme-labeled concentrate, concentrated washing solution, substrate solution and stop solution.

The invention designs and synthesizes small molecule target analyte hapten by depending on immunology, immunochemistry basic principle and residue analysis technical means, and couples with carrier protein to prepare effective artificial antigen, and immunizes animals to prepare specific antibody aiming at small molecule analyte. And quantitatively detecting trace micromolecular target analytes in the sample by utilizing the specific immunological reaction of the antigen and the antibody. The preparation method is simple, convenient and feasible, and has lower cost and higher hapten yield. The invention overcomes the defects that the pretreatment of the aflatoxin M1 sample is complex and time-consuming, a large amount of organic solvent is required for extraction, and a precise and expensive detection instrument is required in the detection process, so that the method is not suitable for popularization and use in the prior detection technology. The aflatoxin M1 antigen can generate a specific antibody aiming at aflatoxin M1 by immunizing animals, is used for quickly detecting aflatoxin M1 residues in food, and has the advantages of simple and quick operation, large sample treatment amount, high sensitivity, strong specificity and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a schematic diagram of the structure of aflatoxin M1 antigen in the present invention.

FIG. 2 is a schematic structural diagram of a reagent rack and a placement groove in the aflatoxin M1 rapid transfer quantification kit.

FIG. 3 is a schematic structural diagram of a micro-scale test well plate in the aflatoxin M1 rapid transfer quantification kit in the invention.

In the figure, 2 is a standard solution bottle tank, 2 is a high-concentration standard product bottle tank, 3 is a substrate solution A solution bottle tank, and 4 is a substrate solution

The kit comprises a solution bottle tank B, a stop solution bottle tank 5, an enzyme marker bottle tank 6, an antibody working solution bottle tank 7, a concentrated washing solution bottle tank 8, a placement tank 9, a reagent rack 10, a pore plate rack 11 and a pore plate strip 12.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 preparation of aflatoxin M1 hapten

The preparation method of the aflatoxin M1 hapten comprises the following specific operation steps:

The structural formula of the hapten is shown in formula 1:

formula 1

Example 2 preparation of aflatoxin M1 whole antigen

The preparation method of the aflatoxin M1 holoantigen comprises the following specific operation steps:

weighing 30mgBSA by a precision electronic balance, dissolving in 10ml of carbonate buffer solution with the concentration of 0.1MOL/L to obtain solution A;

② dissolving the hapten by 2ml of N, N-dimethylformamide, adding 20mg of DCC and 20mg of NHS as B liquid;

The structure of the whole antigen is shown in formula 2:

formula 2

Example 3 preparation of Aflatoxin M1 polyclonal antibody

The preparation method of the aflatoxin M1 polyclonal antibody comprises the following specific operation steps:

taking the immunogen solution obtained in the embodiment 2 as an immunogen solution, diluting the immunogen solution by using PBS (phosphate buffer solution) with the pH value of 7.2 and the concentration of 0.02M to obtain an immunogen diluent for preparing the aflatoxin M1 polyclonal antibody, and taking a black goat bred in Jiangkou county of south West as the aflatoxin M1 polyclonal antibody to immunize animals, wherein the immunization step is as follows:

Example 4 preparation of an Aflatoxin M1 enzyme marker

Weighing 12mg of horseradish peroxidase by a precision electronic balance, and dissolving the horseradish peroxidase in 4.8 ml of carbonate buffer solution with the concentration of 0.1MOL/L to obtain solution A;

② taking 1.2 ml of the liquid from the hapten, adding 10mgDCC and 10mgNHS as B liquid, and activating for 30 minutes;

thirdly, slowly dripping the solution B into the solution A, and stirring for 16 hours at room temperature;

and fourthly, selecting 0.01MOL/LPBS buffer solution for dialysis for 7 working days, placing the solution in a refrigerator at the temperature of 2-8 ℃, changing the solution for 3 times every day, centrifuging the solution after dialysis to remove the lower-layer precipitate, and taking the supernatant for split charging and storage.

The structure of the enzyme label is shown as formula 3:

formula 3

Determination of aflatoxin M1 antibody titre standard aflatoxin M1 was purchased from Sigma.

And determining the working concentration of the aflatoxin M1-coated antibody and the prepared aflatoxin M1 enzyme-labeled antigen by using a matrix titration method, wherein the working concentration of the aflatoxin M1-coated antibody is 2 mug/L, and the working concentration of the enzyme-labeled antigen is 1: 5000.

Aflatoxin M1 determination of antigenic potency aflatoxin M1 standard was purchased from Sigma.

And determining the working concentration of the aflatoxin M1-coated antibody and the prepared aflatoxin M1 enzyme-labeled antigen by using a matrix titration method, wherein the working concentration of the aflatoxin M1-coated antigen is 2 mug/L, and the working concentration of the enzyme-labeled antigen is 1: 5000.

Example 5 Rapid transfer quantification kit for detecting aflatoxin M1 and preparation method thereof

Working solution of aflatoxin M1 standard: aflatoxin M1 was dissolved in a phosphate buffer solution of ph7.4, 0.05M to give standard working solutions having concentrations of 0.025ppb, 0.05ppb, 0.1ppb, 0.4ppb and 1.6ppb, respectively. Phosphate buffer solution of pH7.4 and 0.05M was used as a negative control solution of the standard solution, and was referred to as 0 solution.

Aflatoxin M1 elisa plate: the aflatoxin M1 antibody solution prepared in example 3 was diluted with coating buffer to give a coated antibody dilution with a protein concentration of 2. mu.g/mL. Coating 96-well microporous plates with 100. mu.L of each well, incubating at 37 ℃ for 2 hours, pouring out the coating solution, washing 3 times with a washing solution, 10s each time, drying, adding 150. mu.L of a confining solution into each well, incubating at 37 ℃ for 2 hours, pouring out the liquid in the wells, drying, and preserving in a vacuum seal manner by using an aluminum film.

③ aflatoxin M1 high-concentration standard substance: aflatoxin M1 was dissolved in 0.05M phosphate buffer at ph7.4 to give a high-concentration standard of 100 ppb.

Concentrate of aflatoxin M1 enzyme label: diluting an aflatoxin M1 enzyme marker by 100 times with an enzyme marker diluent to obtain an aflatoxin M1 enzyme marker concentrated solution, wherein the working concentration is 1: 5000; enzyme label diluent: 5mg of BSA was taken, dissolved in 0.02M PBS buffer solution (pH7.4), and the volume was adjusted to 1000mL to obtain the enzyme-labeled diluent.

Fifth, concentrating the washing liquid: mixing 10mL of tween-20, 5g of sodium azide and 990mL of phosphate buffer solution to obtain the washing solution; the phosphate buffer had a concentration of 0.01MpH and a value of 7.4.

Substrate solution: the luminous liquid consists of a liquid A and a liquid B, wherein the liquid A and the liquid B are respectively in one bottle;

the preparation method of the solution A comprises the following steps: taking 0.2g luminol monosodium salt, 0.1g p-iodophenol, 0.16g sodium chloride and 0.18g EDTA-Na2, dissolving with 0.1M Tris-HCl buffer solution with pH of 8.4, and fixing the volume to 1000mL,

and B, liquid B: Tris-HCl buffer (0.1M) with pH8.4 containing 0.3mMH2O2 and 5mM EDTA-Na 2.

Seventh, stop solution: 50mL of sulfuric acid was added to 1L of water to obtain the stop solution.

Example 6 Rapid transfer quantitation kit Using method of Aflatoxin M1

Firstly, preparing a working solution:

sample diluent: deionized water was added in a ratio of 1: 10 (1 part concentrated sample dilution +9 parts deionized water) dilution.

Enzyme-labeled working solution: diluting with sample (see: 1): and 5 (1 part of enzyme marker concentrated solution +4 parts of sample diluent) for dilution.

③ washing the working solution: deionized water was added in a ratio of 1: 20 (1 part concentrated wash +19 parts deionized water).

Second, pretreatment of raw milk sample

Accurately measuring 2mL of raw milk sample in a 4mL centrifuge tube;

fully whirling for 30s, and taking 1mL of clear liquid in a new centrifugal tube;

and taking 70 mu L for detection.

Note that: if the content of the crude milk oil is high, centrifuging for 5min at 4000g after whirling to remove the upper layer oil, and then detecting; if the oil is carelessly transferred into a new centrifugal tube after the vortex motion, the oil is removed and then the detection is carried out so as to avoid influencing the detection result;

dilution factor of raw milk: 1.

third, fast transfer quantitative reagent kit detection

Inserting an enzyme label plate coated by the required antibody into the enzyme label plate frame, recording the positions of each standard substance and each sample, suggesting that parallel experiments are carried out, sealing unused enzyme label plates by a self-sealing bag, and immediately storing in an environment of 2-8 ℃; respectively adding 70 mu L of each standard substance working solution/sample solution into the corresponding standard substance/sample hole; adding 70 mu L of enzyme marker working solution into each hole; slightly oscillating the ELISA plate for 10s, fully and uniformly mixing, immediately transferring the liquid in the uniformly mixed micropores to corresponding micropores of the ELISA plate strips coated with the required antibody, and covering a cover plate film; reacting at room temperature (25 +/-2 ℃) in a dark place for 30 min; uncovering the cover plate film; pouring out liquid in the plate holes, adding 260 mu L of washing working solution into each hole, and fully washing for 4 times, wherein each time of soaking is 15-30 times; pouring out liquid in the plate hole, pouring the enzyme label plate on absorbent paper, and patting dry; immediately adding 100 mu of LA and B mixed solution into each hole; note: the substrate A liquid and the substrate B liquid are mixed according to the volume of 1:1, and must be fully mixed, the mixed liquid is used within 5min, the use of metal containing and stirring reagents is avoided! Covering a cover plate film, slightly oscillating the ELISA plate for 10s, fully and uniformly mixing, and reacting for 15-20min at room temperature (25 +/-2 ℃) in a dark place; uncovering the cover plate membrane, adding 50 mu L of stop solution into each hole, slightly oscillating the ELISA plate for 10s, and fully and uniformly mixing; and reading the absorbance value of the ELISA plate by using an ELISA reader at the dual wavelength of 450nm and 630nm within 5min after termination.

Making of standard curve

The average luminescence intensity (RLU) of the standard solutions at each concentration was divided by the average luminescence intensity (RLU0) of the 0 solution, and multiplied by 100%, i.e., the inhibition ratio. Calculating the formula: inhibition (%) = RLU/RLU0 × 100%. And drawing a standard curve chart by taking the semilogarithmic value of the aflatoxin M1 concentration (mu g/L) in the standard solution as an X axis and the inhibition rate as a Y axis.

And (4) according to a regression equation of the standard curve, the concentration of the aflatoxin M1 in the sample solution to be detected can be obtained. The analysis of the detection result can utilize professional software, can realize the rapid analysis of a large number of samples, and can be completed in 30 minutes in the whole detection process. In order to facilitate the calculation of the result by the detection personnel, after the standard curve is drawn, the professional software replaces the semilogarithmic value of the concentration (mu g/L) of the aflatoxin M1 in the standard solution by the concentration (mu g/L) of the aflatoxin M1 in the standard solution. The resulting calibration curve is shown in FIG. 2. The corresponding aflatoxin M1 concentration in the standard solution when the inhibition rate (RLU/RLU 0) is 50% is the IC50 value. According to a standard curve chart, the aflatoxin M1 rapid transfer quantification kit IC50=0.05 μ g/L.

Fifthly, measuring the concentration of aflatoxin M1 in the sample

The inhibition was obtained by dividing the mean value of the luminescence intensity (RLU) of each test sample solution by the mean value of the luminescence intensity of 0 solution (RLU0), and multiplying by 100%. And (3) corresponding to the inhibition rate of each detection sample solution, reading the semilogarithmic value of the detection sample solution from the standard curve, converting the residual quantity of the aflatoxin M1 in the sample solution according to the semilogarithmic value of the sample solution, and multiplying the residual quantity by the dilution multiple of the pretreatment process of each sample to calculate the concentration of the aflatoxin M1 in the sample.

Example 7 evaluation of the detection Effect of the Aflatoxin M1 Rapid transfer quantification kit

First, sensitivity of kit

The lowest detection limit is taken as the sensitivity index of the kit. 20 parts of blank samples are taken and detected according to the using method of the embodiment 6, the average value of the luminous intensity values (RLU) of the blank samples is calculated and is substituted into a standard curve to obtain the corresponding sample concentration, the Standard Deviation (SD) of each corresponding concentration value is calculated, the lowest detection Limit (LOD) of the sample is obtained by adding three times of the standard deviation to the average value, and the result is shown in the table 1.

TABLE 1 lowest detection limit of the kit in raw milk, reconstituted milk, yogurt

Second, test of accuracy and precision

Adding aflatoxin M1 standard substance into raw milk without aflatoxin M1 to make the final concentrations of aflatoxin M1 standard substance in the raw milk sample respectively 0.125, 0.25 and 0.5 μ g/kg; the added samples were pretreated as described in example 6, respectively, to obtain test sample solutions.

3 kits were extracted from each of the 3 different batches of kits for detection, the detection method was as described in example 6, each sample was repeated 5 times, and the intra-batch inter-batch coefficient of variation was calculated separately. The results are shown in tables 2 to 5, respectively.

The results show that: the average adding recovery rate of the raw milk sample is 90.0% +/-30%, which indicates that the accuracy of the kit is good; the intra-batch variation coefficient is 4.2-10.0%, and the inter-batch variation coefficient is 4.5-8.1%. The inter-batch variation coefficient is less than 10%, which indicates that the precision of the kit is good.

TABLE 2 test results of the detection accuracy and precision of aflatoxin M1 in raw milk

Third, the shelf life of the kit

The storage condition of the kit is 2-8 ℃, and the luminous intensity value of 0 solution, the 50% inhibition concentration and the sample addition recovery rate of the kit are within the normal range after 15 months of determination. Considering that abnormal storage conditions occur in the transportation and use processes, the kit is placed for 9 days under the condition of being stored at 37 ℃ for accelerated aging experiments, and the results show that all indexes of the kit completely meet the requirements. In consideration of the occurrence of the freezing condition of the kit, the kit is placed into a refrigerator at the temperature of-20 ℃ for freezing for 9 days, and the determination result also shows that all indexes of the kit are completely normal. From the above results, it can be found that the kit can be stored at 2 to 8 ℃ for at least 12 months.

Fourth, Cross reactivity test

Other drugs with similar structures or functions to aflatoxin M1 are selected to prepare standard solutions according to the method described in example 5, standard curves are drawn according to the method described in example 6, and the 50% inhibition concentration of each drug is calculated according to the standard curves of the drugs. The cross-reactivity of the kit to other analogues was calculated using the following formula. The smaller the cross reaction rate with other medicines, the better the detection specificity of the aflatoxin M1 chemiluminescence enzyme-linked immunoassay kit to aflatoxin M1. The results are shown in Table 3.

The cross reaction rate (%) = (IC 50 value of aflatoxin M1/IC 50 value of drug to be detected) × 100% test result shows that the cross reaction rate of the kit of the invention to aflatoxin M1 is 100%; the cross reaction rate to the aspergillus flavus M2 is less than 1%, the cross reaction rate to the aspergillus flavus B1 and G1 is more than 100%, so the specificity of the kit to the aflatoxin M1 is good, namely the kit can detect the metabolite of the aflatoxin M1.

TABLE 3 Aflatoxin M1 kit Cross-reactivity Rate

Claims

1. An aflatoxin M1 hapten which is a compound shown as a formula 1:

formula 1.

2. A method for preparing a compound represented by formula 1, comprising the steps of:

3. An aspergillus flavus M1 holoantigen is shown in a formula 2, and is a conjugate obtained by coupling a compound shown in a formula 1 and a carrier protein.

4.

And (3) formula 2.

The A aflatoxin M1 whole antigen of claim 3, wherein the Bovine Serum Albumin (BSA), Ovalbumin (OVA), Human Serum Albumin (HSA), Murine Serum Albumin (MSA), thyroid protein (TG) or hemocyanin (KLH).

5. The process for preparing the whole antigen of A. flavus M1 as claimed in claim 3 or 4, which comprises the steps of:

③ after 30 minutes of activation, slowly dripping the solution B into the solution A, stirring for 16 hours at room temperature, dialyzing with 0.01MOL/LPBS buffer solution for 7 working days, placing in a refrigerator at 2-8 ℃, changing the solution 3 times per day, centrifuging to remove the lower layer precipitate after dialysis, and collecting the supernatant for separate storage;

6. Use of the aflatoxin M1 antigen of claim 3 or 4 in the preparation of antibodies specific for aflatoxin M1.

7. Specific antibodies and polyclonal antibodies prepared by using the aflatoxin M1 antigen of claim 3 or 4.

8. The method for producing a polyclonal antibody according to claim 7, comprising the steps of:

9. The aflatoxin M1 antigen of claim 3 or 4, the use of the specific antibody of claim 7 for detecting aflatoxin M1.

10. A kit for rapid transfer quantification prepared by using the aflatoxin M1 antigen of claim 3 or 4 and the specific antibody of claim 7.

11. The rapid transfer quantification kit of claim 10, comprising: the kit comprises aflatoxin M1 standard substance working solution, aflatoxin M1 high-concentration standard substance, aflatoxin M1 enzyme-labeled plate, aflatoxin M1 enzyme-labeled concentrate, concentrated washing solution, substrate solution and stop solution.