CN114044782A

CN114044782A - Aflatoxin B1 hapten, artificial antigen, and preparation method and application thereof

Info

Publication number: CN114044782A
Application number: CN202111230122.6A
Authority: CN
Inventors: 李斌; 梁巧雯; 梁科; 郑婉洋; 刘芸; 石松; 任季玉
Original assignee: Guangdong C&d Testing Technology Co ltd; Guangdong Dayuan Oasis Food Safety Technology Co ltd
Current assignee: Guangdong C&d Testing Technology Co ltd; Guangdong Dayuan Oasis Food Safety Technology Co ltd
Priority date: 2021-09-18
Filing date: 2021-10-21
Publication date: 2022-02-15
Anticipated expiration: 2041-10-21
Also published as: CN114044782B

Abstract

The invention discloses an aflatoxin B1 hapten, an artificial antigen, a preparation method and application thereof, wherein the structure of the aflatoxin B1 hapten is shown as a formula I, and the structural formula of the aflatoxin B1 artificial antigen is shown as a formula IV. The preparation method of the aflatoxin B1 hapten is simple, the aflatoxin B1 hapten has an active group, and the structural characteristics of the aflatoxin B1 are kept, so that the electron cloud density of the hapten and the target object to be detected is consistent. Moreover, the artificial antigen and the antibody prepared based on the method have good specificity and can accurately identify the target substance. The test strip prepared based on the test paper has good specificity and high sensitivity, the test sensitivity of the test strip to aflatoxin B1 can reach 0.02 mug/L, the test strip is in a linear relation at 0.02-0.32 mug/L, and the test strip has a very high detection effect on aflatoxin B1.

Description

Aflatoxin B1 hapten, artificial antigen, and preparation method and application thereof

Technical Field

The invention relates to the field of biochemical engineering, and in particular relates to ochratoxin hapten, an artificial antigen, and preparation methods and applications thereof.

Background

Aflatoxins (AF) are secondary metabolites of aspergillus flavus (aspergillus flavus) and aspergillus parasiticus (aspergillus parasiticus), and are widely varied, more than 12 types of aflatoxins have been isolated and identified at present, and the aflatoxins are commonly identified as B1, B2, G1, G2, M1, M2, B2a, G2a, BM2a, GM2a and the like. Wherein, aflatoxin B1 has the advantages of widest distribution, strongest toxicity and greatest harm. Aflatoxins are harmful to the life safety of animals and humans mainly by means of contaminating feed or food. After the mammal ingests the feed or food polluted by the aflatoxin B1, a part of toxins can be accumulated at the edible parts of the animal, and the other part of toxins can be subjected to hydroxylation after being metabolized in vivo and are converted into the aflatoxin M1, so that the aflatoxin M1 exists in milk and urine secreted by the animal and further pollutes other animals. Most animals, especially ruminants, have weak sensitivity to aflatoxin B1, and intake of a large amount of aflatoxin B1 at one time or a small dose for a long time causes a plurality of problems of malnutrition, slow production and immunity reduction.

In the related art, the detection methods of aflatoxin B1 mainly include thin layer chromatography, high performance liquid chromatography, immunological methods and the like. However, the thin layer chromatography has low detection sensitivity and low specificity to aflatoxin B1, is easy to be interfered by other fluorescent substances, and has long analysis time. Although the high performance liquid chromatography is excellent in sensitivity, the detection instrument degree is high, the equipment is expensive, the operation technical requirement is high, the pretreatment purification requirement is high, the whole detection difficulty is not easy to operate by ordinary people, the field detection and the rapid preliminary inspection cannot be really realized, and great inconvenience is brought to daily detection work.

Therefore, the development of a novel detection method which is rapid, high in sensitivity, high in specificity, low in cost and convenient to carry and operate has great significance in the fields of environment and food detection and the like, and the development is focused on obtaining the aflatoxin B1 hapten or artificial antigen with higher quality.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the hapten of the aflatoxin B1, the artificial antigen, the preparation method and the application thereof are provided, the hapten not only has an active group, but also completely retains the structural characteristics of the aflatoxin B1, so that the electron cloud density of the hapten and the aflatoxin B1 is consistent. The artificial antigen and the specific antibody prepared based on the hapten have the advantages of strong specificity, high sensitivity and extremely high application value.

The first aspect of the invention provides an aflatoxin B1 hapten, wherein the structure of the aflatoxin B1 hapten is shown as a formula I:

the arm introduced by the hapten of the aflatoxin B1 designed by the invention not only has an active group, but also completely retains the structural characteristics of the aflatoxin B1, so that the density of the electron cloud of the hapten and the density of the electron cloud of the aflatoxin B1 are consistent. Based on the structure, the artificial antigen and the specific antibody prepared by the artificial antigen and the specific antibody have extremely strong specificity.

In a second aspect of the present invention, there is provided a method for preparing aflatoxin B1 hapten as described in the first aspect of the present invention, comprising the steps of:

reacting a compound shown in a formula II and a compound shown in a formula III at room temperature to obtain the compound;

wherein the structural formula of the compound shown in the formula II is as follows:

the structural formula of the compound shown in the formula III is as follows:

according to a second aspect of the invention, in some embodiments of the invention, the reaction further comprises a catalyst, a solvent, and an acid scavenger.

In some preferred embodiments of the present invention, the acid scavenger comprises at least one of sodium bicarbonate, sodium carbonate, and potassium carbonate.

In some more preferred embodiments of the invention, the acid scavenger is potassium carbonate.

In some preferred embodiments of the present invention, the solvent comprises at least one of acetonitrile, acetone, tetrahydrofuran.

In some more preferred embodiments of the invention, the solvent is acetonitrile.

In some preferred embodiments of the present invention, the catalyst comprises at least one of benzyltriethylammonium chloride, tetrabutylammonium bromide, tetrabutylammonium chloride, tetrabutylammonium hydrogen sulfate, trioctylmethylammonium chloride and tetrabutylammonium iodide.

In some more preferred embodiments of the invention, the catalyst is tetrabutylammonium bromide.

In some more preferred embodiments of the present invention, the preparation method comprises the following specific steps:

mixing a compound with a structural formula shown as a formula II, acetonitrile, pure water, a compound with a structural formula shown as a formula III, potassium carbonate and tetrabutylammonium bromide, and reacting for more than 12 hours at room temperature (23-27 ℃). And removing the solvent (acetonitrile), adding a sodium chloride aqueous solution, adjusting the pH value to 4-5, filtering, and collecting filter residues to obtain the aflatoxin B1 hapten.

In a third aspect of the invention, an artificial antigen of aflatoxin B1 is provided, and the artificial antigen of aflatoxin B1 is obtained by coupling an aflatoxin B1 hapten and a carrier protein.

According to the third aspect of the invention, in some embodiments of the invention, the aflatoxin B1 artificial antigen is specifically prepared by:

(1) aflatoxin B1 hapten (3mg) was taken and dissolved well in Dimethylformamide (DMF). And then adding carbodiimide (EDC) and N-hydroxysuccinimide (NHS) for activation to obtain the aflatoxin B1 hapten activated ester.

(2) And adding the obtained sodium pentachlorophenate hapten activated ester into a bovine serum albumin solution to obtain the aflatoxin B1 artificial antigen.

In some preferred embodiments of the present invention, the activation reaction conditions in step (1) are: stirred at room temperature for 4h in the dark.

In some preferred embodiments of the present invention, in step (2), the reaction conditions of the sodium pentachlorophenol hapten-activated ester and the bovine serum albumin are as follows: stirring for more than 16h at room temperature.

According to a third aspect of the invention, in some embodiments of the invention, the carrier protein comprises at least one of bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.

Of course, other carrier proteins can be coupled by those skilled in the art according to the actual use requirement.

According to the third aspect of the invention, in some embodiments of the invention, the structural formula of the aflatoxin B1 artificial antigen is shown in formula IV,

wherein protein in formula IV represents a carrier protein.

In a fourth aspect of the present invention, there is provided a use of the aflatoxin B1 hapten of the first aspect of the present invention or the aflatoxin B1 hapten prepared by the preparation method of the second aspect of the present invention in any one of the following (1) to (2);

(1) preparing an aflatoxin B1 specific antibody;

(2) detecting an aflatoxin B1-specific antibody.

According to a fourth aspect of the invention, in some embodiments of the invention, the aflatoxin B1-specific antibodies include monoclonal and polyclonal antibodies.

In some preferred embodiments of the invention, the aflatoxin B1-specific antibody is a monoclonal antibody.

In the fifth aspect of the invention, an aflatoxin B1 specific antibody is provided, and the aflatoxin B1 specific antibody is obtained by immunizing an animal with the artificial antigen of aflatoxin B1 of the third aspect of the invention as an immunogen.

According to a fifth aspect of the invention, in some embodiments of the invention, the animal comprises at least one of a rabbit, a sheep, a guinea pig, a mouse, a chicken, a goat and a horse.

According to a fifth aspect of the invention, in some embodiments of the invention, the aflatoxin B1-specific antibodies include monoclonal and polyclonal antibodies.

In a sixth aspect of the present invention, there is provided use of an aflatoxin B1-specific antibody according to the fifth aspect of the present invention in any one of (1) to (2) below;

(1) detecting aflatoxin B1;

(2) preparing aflatoxin B1 detection product.

According to a sixth aspect of the invention, in some embodiments of the invention, the aflatoxin B1-specific antibodies include monoclonal and polyclonal antibodies.

According to a sixth aspect of the invention, in some embodiments of the invention, the product comprises: at least one of a detection reagent, a detection kit, a detection test paper or a detection card.

In a seventh aspect of the present invention, a detection reagent or a detection test paper is provided, wherein the detection reagent or the detection test paper contains the aflatoxin B1 specific antibody according to the fifth aspect of the present invention.

According to a seventh aspect of the invention, in some embodiments of the invention, the aflatoxin B1-specific antibodies include monoclonal and polyclonal antibodies.

In an eighth aspect of the present invention, there is provided a test kit, wherein the test kit contains the detection reagent or test strip according to the seventh aspect of the present invention.

The invention has the beneficial effects that:

1. the preparation method of the aflatoxin B1 hapten is simple and unique in design, has active groups, and completely retains the structural characteristics of aflatoxin B1, so that the electron cloud density of the hapten and the aflatoxin B1 is consistent. Based on the structure, the artificial antigen and the specific antibody prepared by the artificial antigen and the specific antibody have extremely strong specificity. .

2. The artificial antigen and the antibody prepared based on the aflatoxin B1 hapten have good specificity, can accurately identify a target substance, can be used for preparing a rapid detection test strip, a kit and other related detection equipment aiming at the aflatoxin B1, and have extremely high application prospect.

3. The detection test strip prepared based on the artificial antigen and the specific antibody has high specificity and strong sensitivity, the detection sensitivity to the aflatoxin B1 can reach 0.02 mu g/L, the linear relation is formed between 0.02 and 0.32 mu g/L, and the detection test strip has extremely high detection effect on the aflatoxin B1.

Drawings

FIG. 1 is a synthetic route to aflatoxin B1 hapten in an example of the invention;

FIG. 2 is a mass spectrum of aflatoxin B1 hapten in an example of the invention;

FIG. 3 is a synthetic roadmap of an aflatoxin B1 artificial antigen in an example of the invention;

FIG. 4 is a standard curve of aflatoxin B1 standards at various concentrations in an example of the invention;

fig. 5 is a quantitative standard curve of the fluorescent quantitative immunochromatographic test strip in the embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.

Preparation of aflatoxin B1 hapten

Taking 0.020g (0.067mmol) of the compound shown in the structural formula II into a 50mL round-bottom flask, sequentially adding 0.5mL of acetonitrile, 0.5mL of pure water, 0.047g (0.335mmol) of the compound shown in the structural formula III, 0.019g (0.134mmol) of potassium carbonate and 0.005mg of n-tetrabutylammonium bromide, and reacting for more than 12 hours at room temperature (23-27 ℃). After the reaction was completed, the solvent (acetonitrile) was removed under reduced pressure. And adding 2mL of sodium chloride aqueous solution, adjusting the pH value to 4-5 by using 1M dilute hydrochloric acid, filtering, and collecting filter residues to obtain the aflatoxin B1 hapten.

the structural formula of the compound shown in the formula III is as follows:

in this example, 0.011g of aflatoxin B1 hapten was obtained altogether.

The aflatoxin B1 hapten ESI-MS obtained in this example was: 355.0[ M-1 ].

The synthetic route of aflatoxin B1 hapten in this example is shown in figure 1.

The mass spectrum is shown in FIG. 2.

Preparation of aflatoxin B1 artificial antigen

(1) Bovine serum albumin-aflatoxin B1 hapten coupling to obtain an artificial antigen:

the aflatoxin B1 hapten (3mg) prepared in the above example was taken and dissolved thoroughly in 0.2mL of Dimethylformamide (DMF). Then 5mg of carbodiimide (EDC) and 5mg of N-hydroxysuccinimide (NHS) are added, and the mixture is stirred for 4 hours at room temperature in the dark to fully react to obtain the aflatoxin B1 hapten activated ester.

The sodium pentachlorophenol hapten activated ester obtained is then added dropwise to a bovine serum albumin solution (BSA, 10mg BSA +1mL 0.01mol/L PBS buffer) with stirring and stirred at room temperature for more than 16 h. Dialyzing with 0.01mol/L PBS at room temperature for 3 days, and changing the dialyzate for 3 times every day to remove unreacted micromolecule substances in the solution, thus obtaining the aflatoxin B1 artificial antigen.

The obtained aflatoxin B1 artificial antigen can be stored at 4 deg.C for use.

(2) Artificial antigen obtained by coupling hemocyanin-aflatoxin B1 hapten:

the method is the same as (1) and is characterized in that:

in this example, a hemocyanin (KLH) solution was prepared by mixing 18mg KLH +1.8mL of 0.01mol/L PBS buffer.

The synthetic route of the aflatoxin B1 artificial antigen is shown in figure 3.

Application of aflatoxin B1 artificial antigen

(1) The application of the artificial antigen of aflatoxin B1 in preparing the monoclonal antibody of aflatoxin B1 comprises the following steps:

the preparation method comprises the following steps:

the artificial antigen obtained by coupling the hemocyanin-aflatoxin B1 hapten in the embodiment is taken as immunogen, and is added with the Freund's adjuvant with the same volume for full emulsification, and then the BALB/C mouse is immunized. Each BALB/C mouse was inoculated with 80. mu.g of immunogen at 2 week intervals for each immunization. After 3 times of immunization, tail venous blood of the mice is collected, and the antibody titer in serum is detected. If the antibody titer does not meet the requirement, the immunization needs to be strengthened.

After the antibody titer is not increased any more, the whole antigen (artificial antigen obtained by coupling hemocyanin-aflatoxin B1 hapten) with an inoculation amount of 100 mu g is used for subcutaneous boosting immunization.

5 days after the booster immunization, the test mouse spleen cells were taken and subjected to cell fusion with SP20 cells (mouse myeloma cells). The fused cells are incubated and screened in HAT medium, and cultured in complete medium after 5 days of incubation.

And (3) detecting the obtained fusion cell supernatant by using an ELISA method, carrying out clonal culture on the fusion cells with strong positive detection results by using a limiting dilution method, and detecting through 3 times of clonal culture to obtain the fusion cells with positive results, namely the hybridoma of the aflatoxin B1 monoclonal antibody. After the hybridoma cells are subjected to amplification culture, the hybridoma cells are inoculated to the abdominal cavity of a mouse to generate ascites containing the aflatoxin B1 monoclonal antibody. Purifying the ascites by an octanoic acid-ammonium sulfate precipitation method, and freeze-drying to obtain the aflatoxin B1 monoclonal antibody.

(2) The application of the aflatoxin B1 artificial antigen in immunoassay:

in this embodiment, the application of the aflatoxin B1 artificial antigen in immunoassay is demonstrated by taking ELISA as an example, and the specific steps are as follows:

the artificial antigen obtained by coupling the bovine serum albumin-aflatoxin B1 hapten in the above example was diluted to 0.08. mu.g/mL with carbonate buffer (pH 9.6) as a coating diluent. Then added to a polystyrene microplate in an amount of 100. mu.L/well and coated overnight at 4 ℃. The wells were drained, 1% BSA (phosphate buffer as a diluent) was added at 250. mu.L/well, blocked at 37 ℃ for 1h, and dried to obtain coated assay plates. And sequentially adding an aflatoxin B1 standard solution (100 mu L/hole) and an aflatoxin B1 monoclonal antibody (20 mu L/hole) into the coated detection plate, and incubating at 37 ℃ for 0.5 h. And (4) drying the liquid in the holes, adding 300 mu L/hole of washing liquid, washing for 3 times, and patting dry. Add 100 u L/hole enzyme labeled second antibody, 37 degrees C reaction for 0.5 h. Washing for 3 times again, drying, adding 100 μ L/well color development solution, and reacting at 37 deg.C in dark for 15 min. The reaction was stopped by adding 50. mu.L/well of 2M sulfuric acid, and the OD value of each well was measured at a wavelength of 450 nm. The results are shown in Table 1.

TABLE 1 OD values of standard solutions of aflatoxin B1 series of metabolites at different concentrations

The data in table 1 were fitted to a four parameter Logistic curve using ELISA Calc software to generate a standard curve (figure 4).

It can be found that the linear equation of the obtained aflatoxin B1 standard solution is as follows:

y＝(A-D)/[1+(x/C)^B]+D；

wherein r is²＝0.996，A＝2.42421，B＝1.41044，C＝0.04033，D＝0.16792。

x represents the concentration of the substance in the sample;

y represents an OD value.

Found by calculation, the obtained IC_s0The value is 0.05. mu.g/L, and is in a linear relationship of 0.02 to 0.32. mu.g/L.

(3) The application of the aflatoxin B1 artificial antigen in preparing the fluorescence quantitative immunochromatographic test strip for detecting aflatoxin B1 is as follows:

the preparation method comprises the following steps:

the method comprises the steps of taking a nitrocellulose membrane (NC membrane) as a reaction membrane, regulating the concentration of an artificial antigen obtained by coupling bovine serum albumin-aflatoxin B1 hapten in the embodiment to 0.1-0.5 mg/mL by using a coating buffer solution, and regulating the concentration of rabbit IgG to 0.1-1 mg/mL by using the coating buffer solution. And respectively spraying the artificial antigen and the rabbit IgG on the surfaces of a detection area (T line) and a control area (C line) corresponding to the reaction membrane according to the membrane liquid amount of 0.8-1.2 mu L/cm. The interval between the detection area and the control area is 5mm, and the detection area and the control area are placed in an oven for processing for 3-35 h at the temperature of 40-45 ℃.

Wherein, the coating buffer solution is 0.01M PBS buffer solution containing 1% of sucrose, 0.5% of BSA and 0.05% of sodium azide by mass percentage.

The reaction membrane is superposed in the middle of a PVC plate back lining, and a sample pad (obtained by soaking the sample pad in a sample pad treatment solution for 5min and drying at 37 ℃ for 16-24 h, wherein the sample pad treatment solution is 0.01M PBS buffer solution containing 1% of sucrose, 0.5% of BSA, 0.5% of Triton-100 and 0.05% of sodium azide by mass percent) and a water absorption pad are respectively superposed at two ends of the PVC plate back lining. The reaction membrane is respectively connected with the water absorption pad and the sample pad, the detection area is close to the sample pad, the control area is close to the water absorption pad, the fluorescence quantitative immunochromatography test paper board for detecting the aflatoxin B1 is obtained, and the test paper board is cut to obtain the test paper.

The fluorescent quantitative immunochromatographic test strip obtained in this example was used for detection, and the detection sensitivity was evaluated.

The fluorescent microspheres are used as markers, and an immunofluorescence method is adopted for detection, and the method comprises the following specific steps:

(a) taking 500 mu L of fluorescent microsphere solution (the solid content of the fluorescent microsphere is 1 percent, namely 5mg of the fluorescent microsphere is contained), respectively adding 5mg of N-hydroxysulfosuccinimide (NHS) and 4mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (CEDC or DEC) as coupling agents under the stirring condition, controlling the temperature of the fluorescent microsphere solution to be 4-10 ℃, and activating for 40min at the temperature. After activation is completed, adding 0.1M potassium carbonate solution, adjusting the pH to 8-9, and keeping the temperature of the fluorescent microsphere solution at 4-10 ℃ during the period. And adding 0.25mg of the aflatoxin B1 monoclonal antibody prepared in the embodiment, uniformly stirring, carrying out ice bath, naturally heating to room temperature, and stirring at room temperature for 4-6 h for coupling. After the coupling was completed, the reaction solution was centrifuged at 12000rpm for 10min, the supernatant was removed, 1mL of a fluorescent buffer was added, and after ultrasonic resuspension, centrifugation was performed, the supernatant was removed, and the procedure was repeated 3 times. Adding 0.5mL of fluorescent buffer solution, performing ultrasonic resuspension again, and storing in a refrigerator at 2-8 ℃ for later use.

The fluorescence buffer in this example is 0.01M PBS buffer containing 0.5% PEG20000, 2% sucrose, 0.1% tween 20, 0.5% BSA, and 0.05% sodium azide.

(b) Coupling the fluorescent microspheres with goat anti-rabbit IgG by the method in (a).

(c) And (B) mixing the aflatoxin B1 monoclonal antibody marked by the fluorescent microspheres in the step (a) with goat anti-rabbit IgG marked by the fluorescent microspheres, and diluting by 100-1000 times with a fluorescent buffer solution to obtain the aflatoxin B1 monoclonal antibody detection solution.

(d) Preparing a series of aflatoxin B1 standard solutions (each concentration is 100. mu.L) with 0.1M PBS buffer, adding aflatoxin B1 monoclonal antibody detection solution (20. mu.L) obtained in (c), and reacting for 1 min. And respectively dripping 80 mu L of mixed solution into the sample area of the prepared fluorescent quantitative immunochromatographic test strip. Standing for 15min, reading the ratio of the fluorescence signal values of T/C by a fluorescence quantitative detector, and repeating the 3 groups.

The results are shown in Table 2.

TABLE 2 test results of aflatoxin B1 standard substance detected by fluorescent quantitative immunochromatographic test strip

Aflatoxin B1 Standard concentration (. mu.g/L)	0	0.02	0.04	0.08	0.016	0.032
							T/C value	6.503	5.104	3.985	2.257	0.980	0.362
B/B0	100％	78.49％	61.28％	34.71％	15.07％	5.57％

Wherein B represents the T/C value of aflatoxin B1 standard substances with different concentrations, and B₀The T/C value of the aflatoxin B1 standard at a concentration of 0 is shown.

As can be seen from the above results, when the concentration of the standard aflatoxin B1 was 0.02. mu.g/L, its B/B ratio was determined to be₀The value was 78.49%, indicating that the T/C value measured at this concentration was comparable to that of the aflatoxin B1 standard at a concentration of 0. mu.g/L. Therefore, the detection sensitivity of the prepared fluorescence quantitative immunochromatographic test strip to the aflatoxin B1 is 0.02 mu g/L.

From the data in the table above, a quantitative standard curve of the fluorescence quantitative immunochromatographic test strip was drawn by performing four-parameter Logistic curve fitting using ELISA Calc software (the standard curve is shown in fig. 5).

The corresponding standard curve linear equation is as follows:

y＝(A-D)/[1+(x/C)^B]+D；

wherein r is²＝0.99924，A＝6.48145，B＝1.40739，C＝0.05503，D＝-0.18972；

x represents the concentration of the substance to be detected (aflatoxin B1) in the sample to be detected;

y represents a T/C value.

A four-parameter Logistic curve is established through T/C values corresponding to the concentrations of the standard substances with different concentrations, and four parameter values obtained by the curve are recorded into calibration software of the fluorescence quantitative detection instrument, so that the fluorescence quantitative immunochromatographic test strip can be rapidly and quantitatively detected on the fluorescence immunochromatographic analyzer.

Meanwhile, the fluorescent quantitative immunochromatographic test strip obtained in the above embodiment is used for stability detection.

The normal storage condition of the fluorescent quantitative immunochromatographic test strip obtained in the above example is room temperature storage. However, in this example, in order to more truly express the stability of the test strip, an accelerated destructive test was performed on the test strip, and the quantitative fluorescence immunochromatographic test strip obtained in the above example was continuously left for 60 days at room temperature and 45 ℃ (high temperature accelerated product aging), and the fluorescence signal values were measured on days 1, 3, 6, 15, 30, and 60 using the same sample (repeat 3 groups).

The results are shown in Table 3.

TABLE 3 relationship between storage conditions and T/C values of the fluorescence quantitative immunochromatographic test strip

As can be seen from the above table, after the fluorescence quantitative immunochromatographic test strip obtained in the above example is stored for 60 days under the sealed storage condition at room temperature and 45 ℃, the T/C values of the two test strips have no significant change, which indicates that the fluorescence quantitative immunochromatographic test strip obtained in the above example can be stably stored for at least 60 days under the extreme condition (45 ℃) and the detection effect has no significant change compared with the normal test strip. The fluorescent quantitative immunochromatographic test strip stored at normal temperature is stably stored at room temperature for more than one year, has no significant influence on the detection effect, and can completely meet the requirements of the market in the storage and transportation processes.

And (3) carrying out precision detection on the fluorescent quantitative immunochromatographic test strip obtained in the embodiment.

In this embodiment, the precision of the quantitative fluorescence immunochromatographic strip prepared by the above preparation method is measured by the in-plate error (the same plate of the strip, which can be understood as the same batch) and the inter-plate error (different plate of the strip, which can be understood as different batches) of the T/C value of the strip.

The in-plate variation coefficient is measured by the T/C value data of 10 fluorescence quantitative immunochromatographic test strips of the same plate, the inter-plate variation coefficient is measured by the T/C value data of 10 fluorescence quantitative immunochromatographic test strips of different plates, and both the in-plate variation coefficient and the inter-plate variation coefficient need to be analyzed in discrete degree.

The coefficient of variation formula is:

CV% -standard deviation SD value determination mean value X100%

The results are shown in Table 4.

TABLE 4 in the above examples, the in-plate variation coefficient and the inter-plate variation coefficient of the fluorescence quantitative immunochromatographic test strip

Serial number	In-plane T/C value	T/C value between plates
			1	6.533	6.297
2	6.48	6.581
			3	6.377	6.495
4	6.669	6.483
			5	6.731	6.521
6	6.566	6.388
			7	6.539	6.664
8	6.831	6.759
			9	6.493	6.530
10	6.757	6.689
			CV value	2.17％	2.13％

As can be seen from Table 4, the above-mentioned fluorescent quantitative immunochromatographic test strip shows a small change in T/C values in both in-plate and in-plate tests. The in-plate and inter-plate variation coefficients obtained by further calculation through a variation coefficient calculation formula are respectively 2.17% and 2.13%, which shows that the in-plate and inter-plate variation coefficients of the fluorescence quantitative immunochromatographic test strip are small, and the test strip can be considered to have high precision in a statistical sense and can meet the quantitative requirements of the conventional test strip at present.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. The aflatoxin B1 hapten is characterized in that the structure of the aflatoxin B1 hapten is shown as a formula I:

2. a process for the preparation of aflatoxin B1 hapten as claimed in claim 1 which comprises the steps of:

reacting the compound shown in the formula II and the compound shown in the formula III at room temperature to obtain the compound;

the structural formula of the compound shown in the formula III is as follows:

wherein, the reaction preferably also contains a catalyst, a solvent and an acid-binding agent;

the acid-binding agent preferably comprises at least one of sodium bicarbonate, sodium carbonate and potassium carbonate;

the solvent preferably comprises at least one of acetonitrile, acetone and tetrahydrofuran;

the catalyst preferably comprises at least one of benzyltriethylammonium chloride, tetrabutylammonium bromide, tetrabutylammonium chloride, tetrabutylammonium hydrogen sulfate, trioctylmethylammonium chloride and tetrabutylammonium iodide.

3. The aflatoxin B1 artificial antigen, which is characterized in that the aflatoxin B1 artificial antigen is obtained by coupling the aflatoxin B1 hapten as claimed in claim 1 with a carrier protein.

4. The aflatoxin B1 artificial antigen of claim 3 wherein the carrier protein comprises at least one of bovine serum albumin, ovalbumin, human serum albumin and hemocyanin.

5. The aflatoxin B1 artificial antigen of claim 3, which is characterized in that the structural formula of the aflatoxin B1 artificial antigen is shown in a formula IV,

wherein protein in formula IV represents a carrier protein.

6. The use of the aflatoxin B1 hapten as claimed in claim 1 or the aflatoxin B1 hapten as prepared by the preparation method as claimed in claim 2 in any one of the following (1) to (2);

(1) preparing an aflatoxin B1 specific antibody;

(2) detecting an aflatoxin B1-specific antibody.

7. The aflatoxin B1 specific antibody, which is characterized in that the aflatoxin B1 specific antibody is obtained by immunizing an animal with the artificial antigen of aflatoxin B1 of any one of claims 3-5 as immunogen.

8. The use of an aflatoxin B1-specific antibody of claim 7 in any one of (1) to (2) below;

(1) detecting aflatoxin B1;

(2) preparing an aflatoxin B1 detection product;

wherein the product preferably comprises: at least one of a detection reagent, a detection kit, a detection test paper and a detection card.

9. A detection reagent or test strip comprising an aflatoxin B1-specific antibody of claim 7.

10. A test kit comprising the detection reagent or test strip of claim 9.