CN106565737B - A kind of aflatoxin B1The preparation method of haptens, artificial antigen and its Yolk antibody - Google Patents

Abstract

The invention discloses _a preparation method of aflatoxin B1 hapten, artificial antigen and egg yolk antibody with high sensitivity and high specificity. In the present invention, firstly, the carbon-carbon double bond in the difuran ring on the aflatoxin B1 molecule is subjected to _an addition reaction to introduce _a free carboxyl group to form the aflatoxin B1 hapten, and its structural formula is shown in formula (I): formula (I); then the hapten is coupled with a carrier protein to obtain the aflatoxin B ₁ artificial antigen; the artificial antigen is emulsified and then immunized chickens to obtain the aflatoxin B ₁ egg yolk antibody. The half-inhibitory concentration (IC ₅₀ ) of the antibody against aflatoxin B ₁ is 4.23ng/mL, the quantitative detection range is 0.29~61.83ng/mL, and the detection limit is 0.06ng/mL, and the antibody is effective against AFB ₂ , AFG ₁ and AFG ₂ had a cross-reactivity rate of less than 1%. The antibody of the present invention has the characteristics of high sensitivity and specificity at the same time, and has broad application prospects.

Description

A kind of preparation method of aflatoxin B1 hapten, artificial antigen and egg yolk antibody

technical field

The invention relates to the technical field of food safety detection, in particular to a novel aflatoxin B ₁ hapten, an artificial antigen and a method for preparing the egg yolk antibody with high sensitivity and specificity.

Background technique

Aflatoxin is an important mycotoxin, mainly produced by toxigenic Aspergillus flavus and Aspergillus parasiticus and other molds. It is widely distributed in agricultural products and foods, especially peanuts, corn and their products. Aflatoxin is a highly toxic substance and the strongest carcinogen found so far. In 1993, aflatoxin was classified as a class I carcinogen by the World Health Organization (WTO) Cancer Research Agency, mainly inducing liver cancer. . Aflatoxins mainly include aflatoxin B ₁ , B ₂ , G ₁ and G ₂ , of which aflatoxin B ₁ accounts for more than 70%. Aflatoxin B ₁ (AFB ₁ ) has no immune activity due to its small molecular weight and must be linked Only protein molecules can cause the body's immune response, so it is very necessary to obtain a reliable method for the preparation of aflatoxin artificial antigens.

Currently, aflatoxin detection methods mainly include thin layer chromatography (Thin Layer chromatography, TLC), high performance liquid chromatography (High performance Liquid chromatography, HPLC), gas chromatography (Gaschromatography, GC), mass spectrometry (Mass Spectrum, MS) and High performance liquid chromatography-mass spectrometry (Highperformance Liquid chromatography-Mass spectrum, HPLC-MS) and other methods. Although these methods have high accuracy, the instruments and equipment are expensive, the sample pretreatment is complex and cumbersome, the detection cost is high, and professional personnel are required to operate. The immunoassay method has the characteristics of fast, sensitive, accurate, easy operation, etc., and does not require high sample purity, and is especially suitable for the detection of large batches of samples.

Yolk antibody refers to the antibody against a specific antigen extracted from immunized poultry eggs, also known as egg yolk immunoglobulins (EggYolk Immunoglobulins), referred to as IgY. The obtained antibody can be used to specifically detect trace small molecule toxic substances. The advantage over mammalian antibody IgG lies in: In terms of yield, IgG and IgY are 15 g/monkey and 150mg/monkey, respectively. It can be seen that the yield of IgY antibody is significantly higher than that of IgG; IgY is obviously easier to separate and extract than IgG in egg yolk, and It is not necessary to take blood from animals to purify antibodies; IgY antibodies are more stable and have been used to detect a variety of bacteria and proteins; IgY has the advantages of non-toxicity, no residue, and no drug resistance, and is a potential alternative to antibiotics products; IgY does not activate complement, does not bind to Fc receptors and human rheumatism factors, and can react with more epitopes on mammalian antigens, thus improving the specificity and sensitivity of detection; IgY preparation costs are low and the output rate is high , Purification is simple and fast, and has the advantages of heat resistance, good stability, high titer and long stable period after immunization.

At present, the IgY technology has been reported in the immunological detection of small molecular substances, and there are very few reports. Li Peiwu and others prepared hybridoma cells secreting aflatoxin B ₁ monoclonal antibody, the cross-reactivity rates of AFB ₁ , AFB ₂ , AFG ₁ and AFG ₂ were all between 65.2% and 100%; Chen Fusheng and others prepared aflatoxin B 1 monoclonal antibody Aspergillus toxin B ₁ -IgY antibody, using the oximation method to synthesize aflatoxin B ₁ hapten and then immunize chickens to obtain IgY antibody, but its detection limit is 6 ng/mL, and it is different from other structural analogues of AFB ₁ , AFB ₂ , AFG ₁ and AFG ₂ both had high cross-reactivity rates. The aflatoxin B ₁ antibodies prepared by these existing methods have poor specificity, and generally have strong cross-reactions to other aflatoxins, which will cause false positives when specifically detecting aflatoxin B ₁ , and the aflatoxin B ₁ -IgY antibodies have a higher limit of detection. Therefore, it is urgent to establish a highly sensitive and specific method for the detection of aflatoxin B1.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the technical defects and deficiencies of the lack _of IgY antibodies capable of detecting aflatoxin B1 with high sensitivity and high specificity in the prior art, and to provide _a novel aflatoxin B1 hapten and artificial antigen , the egg yolk antibody (AFB ₁ -IgY antibody) prepared by using this new type of artificial antigen has high sensitivity and specificity at the same time, which provides the core raw material for the establishment of an immunological detection method for specific aflatoxin B ₁ , and has broad development prospect.

The object of the present invention is to provide a novel aflatoxin B ₁ hapten and a preparation method thereof.

Another object of the present invention is to provide an artificial antigen of aflatoxin B ₁ and a preparation method thereof.

Another object of the present invention is to provide an egg yolk antibody to aflatoxin B ₁ and a preparation method thereof.

Another object of the present invention is to provide _a kit for detecting aflatoxin B1.

Above-mentioned purpose of the present invention is given to realize by following technical scheme.

A kind of aflatoxin B ₁ hapten, its structural formula is as formula ( ) as shown in:

Mode( ).

The aflatoxin B ₁ hapten is named after systematic nomenclature: 2-((4-methoxy-1,11-dioxy-2,3,6a,8,9,9a,10,11-cis Cyclopentadien[5,6]methylnaphthol[2,1-b]furo[3,2-d]furan-8-yl)oxy)acetic acid (ie: 2-((4-methoxy- 1,11-dioxo-2,3,6a,8,9,9a,10,11-octahydro-1H-cyclopenta[5,6]naphtho[2,1-b]furo[3,2-d]furan- 8-yl)oxy)acetic acid).

Further, the preparation method of the aflatoxin B ₁ hapten comprises the following steps: after dissolving the aflatoxin B ₁ in acetonitrile, reacting with glycolic acid and trifluoroacetic acid, and reacting the reaction product with ethyl acetate and saturated salt After water extraction, drying with anhydrous sodium sulfate, and finally liquid phase purification to obtain aflatoxin B ₁ hapten.

Wherein, preferably, the mobile phase of the liquid phase purification is acetonitrile and water, or the mobile phase is 0.1% acetic acid, the column temperature is 35°C, and the flow rate is 1 mL/min. More specifically, as a preferred embodiment, the specific preparation method of the aflatoxin B ₁ hapten comprises the following steps:

(1) Aflatoxin B ₁ (AFB ₁ ) was dissolved in acetonitrile and then reacted with glycolic acid and trifluoroacetic acid. After the reaction product was extracted twice with ethyl acetate and saturated sodium chloride solution, it was added to the ethyl acetate phase. Add anhydrous sodium sulfate to dry;

(2) After the liquid was evaporated to dryness in vacuum, the product was purified by liquid phase, the mobile phase was acetonitrile (0.1% acetic acid) and water (0.1% acetic acid), the column temperature was 35°C, the flow rate was 1 mL/min, the target peak was collected and frozen After drying, carry out ¹ H-NMR and mass spectrometric identification to determine the final product.

Wherein, preferably, the mass volume ratio of the aflatoxin B ₁ and 4 mL of acetonitrile is: 2-5 mg: 1 mL.

More preferably, the mass volume ratio of the aflatoxin B ₁ and 4 mL of acetonitrile is: 3 mg: 1 mL.

Preferably, the mass volume ratio of glycolic acid and trifluoroacetic acid is: 1g:3~6mL.

More preferably, the mass volume ratio of glycolic acid and trifluoroacetic acid is: 1g:5mL.

Preferably, the mass ratio of glycolic acid and aflatoxin B1 is ₁ :10-15.

More preferably, the mass ratio of glycolic acid and aflatoxin B1 is _1:12 .

In addition, the artificial antigen of aflatoxin B ₁ obtained by coupling the above-mentioned aflatoxin B ₁ hapten with a carrier protein by the active ester method is also within the protection scope of the present invention.

Preferably, the carrier protein is bovine serum albumin or keyhole limpet hemocyanin or the like.

Preferably, the preparation method of the aflatoxin B ₁ artificial antigen comprises the following steps: formula ( ) The aflatoxin B ₁ hapten shown in ) is coupled to the carrier protein by the active ester method, and the aflatoxin B ₁ artificial antigen is obtained after dialysis.

_A method for preparing aflatoxin B1 egg yolk antibody, comprising emulsifying the above _- mentioned aflatoxin B1 artificial antigen to immunize chickens, collecting eggs, separating and purifying the egg yolk antibody by _a salting-out method to obtain aflatoxin B1 egg yolk antibody.

Preferably, the salting-out method is an ammonium sulfate precipitation method.

As a preferred embodiment, the specific preparation method of the novel aflatoxin B ₁ yolk antibody comprises the following steps:

(1) First use 300 µL of aflatoxin B ₁ artificial antigen (1mg/mL), emulsify with Freund's complete adjuvant at a volume ratio of 1:1, and immunize laying hens, and then add an equal volume of AFB ₁ -GA-BSA Emulsify with Freund's incomplete adjuvant, boost immunization once every three weeks, and boost immunization three times in total;

(2) Collect eggs on the day after the first immunization, and store them at 4°C after labeling;

(3) After washing the eggs, wipe the eggshells with 75% alcohol cotton, remove the eggshells and egg whites, gently break the yolk membrane with a needle, and dilute the yolk liquid with 0.06 mol/L NaAc at a volume of 1:9 , add octanoic acid with a final concentration of 8% while stirring, place at room temperature for 20 minutes, centrifuge at 8000 r/min for 20 minutes, filter to remove precipitates, and obtain fat-free egg yolk water-soluble extract (WSF);

(4) Add 40% saturated ammonium sulfate solution, place at room temperature for 20 minutes, centrifuge at 12000 r/min for 20 minutes, separate the supernatant and precipitate, and the obtained precipitate is the aflatoxin B ₁ -IgY antibody.

The aflatoxin B ₁ egg yolk antibody prepared by the above method is also within the protection scope of the present invention.

A test kit for detecting aflatoxin B ₁ , the test kit includes the above-mentioned aflatoxin B ₁ egg yolk antibody.

In addition, the application of the above-mentioned aflatoxin B ₁ egg yolk antibody or kit in immunodetection of aflatoxin is also within the protection scope of the present invention.

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

(1) The present invention designs a novel hapten structure, and the preparation method of the hapten is simple;

(2) The half-inhibitory concentration (IC ₅₀ ) of the aflatoxin B ₁ -IgY antibody drug obtained in the present invention is 4.23 ng/mL, the quantitative detection range is 0.29-61.83 ng/mL, and the detection limit is 0.06 ng/mL. The cross-reactivity rates of AFB ₂ , AFG ₁ and AFG ₂ are all less than 1%;

(3) The aflatoxin B ₁ -IgY antibody prepared by the present invention has high sensitivity and good specificity, and provides core raw materials for the establishment of an immunological detection method for specific aflatoxin B ₁ , and has broad development prospects.

Description of drawings

Fig. 1 is a flowchart for the preparation of aflatoxin B ₁ -IgY antibody.

Fig. 2 is a schematic diagram of the synthesis process of the aflatoxin B ₁ hapten.

Fig. 3 is the ultraviolet scanning identification curve of the artificial antigen of aflatoxin B ₁ .

Fig. 4 is the indirect competition ELISA standard curve of aflatoxin B ₁ -IgY antibody.

Detailed ways

The present invention will be further elaborated below in combination with the accompanying drawings and specific embodiments. The embodiments are only used to explain the present invention, and are not intended to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

The following examples include the synthesis of the aflatoxin B ₁ hapten, the aflatoxin B ₁ artificial antigen and the preparation of the aflatoxin B ₁ -IgY antibody, as shown in Figure 1 .

Example 1 Synthesis and Identification of Aflatoxin B ₁ Hapten

1. Synthesis of Aflatoxin B ₁ Hapten

(1) Add 1 g of glycolic acid to the reaction round bottom bottle, fill it with nitrogen gas protection, add 5 mL of trifluoroacetic acid, absorb 4 mL of acetonitrile, weigh 12 mg of aflatoxin B ₁ (AFB ₁ ) and completely dissolve it in acetonitrile , added into a round bottom flask, the reaction formula is shown in Figure 2, monitored by TLC until the reaction was complete, extracted twice with ethyl acetate and saturated sodium chloride, and then added anhydrous sodium sulfate to the ethyl acetate phase to dry ;

(2) After the liquid was evaporated to dryness in vacuum, the product was purified by liquid phase, the mobile phase was acetonitrile (0.1% acetic acid) and water (0.1% acetic acid), the column temperature was 35°C, the flow rate was 1 mL/min, the target peak was collected and frozen After drying, ¹ H-NMR and mass spectrometry were performed to determine the final product, which was recorded as aflatoxin B ₁ hapten AFB ₁ -GA.

2. Identification:

The standard product of aflatoxin B ₁ was taken for NMR identification spectrum, and the aflatoxin B ₁ hapten AFB ₁ -GA was taken for hydrogen spectrum identification and mass spectrometry identification spectrum.

The results of hydrogen spectrum are as follows: ¹ H-NMR (600 MHz , CD ₃ OD)

Δ _ppm 6.54 (d, J =6.0 Hz , 1H), 6.51 (s, 1H), 5.47 (d, J =4.8 Hz , 1H), 4.16 (dd, J =9.0, 6.0 Hz , 1H), 3.97 (s , 3H), 3.93 (d, J =16.2 Hz , 1H), 3.77 (d, J =16.2 Hz , 1H), 3.42-3.46(m, 2H), 2.59-2.63 (m, 2H), 2.52 (d, J =13.2 Hz , 1H), 2.34-2.44 (m, 1H).

The mass spectrometry results are as follows: MS: C ₁₉ H ₁₆ O ₉ : 388, ESI ^- [MH] ^- : 387.

Both results indicated that the derivation site was correct and successful. Aflatoxin B ₁ hapten (AFB ₁ -GA) was successfully synthesized.

Example 2 Synthesis and Identification of Aflatoxin B ₁ Artificial Antigen

1. Synthesis of Aflatoxin B ₁ Artificial Antigen

(1) Dissolve 3 mg of AFB ₁ -GA hapten, 2 mg of NHS and 3 mg of EDC in 100uL DMF, and stir overnight;

(2) Weigh 9.4 mg of bovine serum albumin (BSA) and add it to 1 mL of PBS buffer;

(3) Slowly add the solution obtained in step (1) to the solution obtained in step (2) dropwise, and stir for 8 h;

(4) Dialyze with PBS buffer for two days, 4 times a day, and obtain aflatoxin B ₁ -GA -BSA artificial antigen (AFB ₁ -GA-BSA) after dialysis.

Among them, the formula of the phosphate buffer solution: Na ₂ HPO ₄ ·12H ₂ O 2.90 g, NaCl 8.50 g, KCl 0.20 g, KH ₂ PO ₄ 0.20 g, add distilled water to 1000 mL.

Similarly, keyhole limpet hemocyanin (KLH) was used to replace BSA as the carrier protein to obtain the target product aflatoxin B ₁ -GA -KLH, and the process was the same as that for preparing the artificial antigen of aflatoxin B ₁ -GA -BSA.

2. Identification:

The artificial antigen of aflatoxin B ₁ was taken, and the ultraviolet full-wavelength scanning was performed, and the results are shown in Fig. 3 .

BSA, KLH, and artificial antigens _were identified by ultraviolet (200-400 nm) scanning, and the highest absorbance values of each substance before and after coupling were compared. The characteristic absorption peak of aflatoxin B ₁ appears at , so the curve of aflatoxin B ₁ artificial antigen is the cumulative absorption feature of the two, indicating that the aflatoxin B ₁ hapten is successfully coupled with BSA to obtain an artificial antigen.

Example 3 Preparation of Aflatoxin B ₁ -IgY Antibody

The preparation method of aflatoxin B ₁ -IgY antibody is as follows:

(1) Three 20-week-old laying hens were used for immunization, the artificial antigen was AFB ₁ -GA-BSA (1 mg/mL) 300 µL, and the volume ratio of AFB ₁ -GA-BSA was 1:1 for the first immunization Emulsify with Freund's complete adjuvant and immunize chickens. Afterwards, AFB ₁ -GA-BSA was emulsified with Freund's incomplete adjuvant at a volume ratio of 1:1, and a booster immunization was performed every three weeks, for a total of three booster immunizations;

(2) Collect the eggs on the day after the first immunization, and store them at 4°C after labeling;

(3) Wash the preserved eggs and wipe the egg shells with 75% alcohol cotton. After removing the egg shells, use a yolk separator to remove the egg whites as much as possible. Pour the egg yolks into a graduated cylinder, measure and record the volume, and pour the egg yolks into the filter paper. Roll it on and remove the egg white that adheres to the surface of the egg yolk. Use a needle to gently break the egg yolk membrane, the egg yolk flows out into the measuring cylinder, and the egg yolk membrane sticks to the surface of the filter paper, put it into a stirrer, stir on a magnetic stirrer, and use egg yolk diluent 0.06 Sodium acetate of M (pH=5.0) was diluted 1:9 by volume. Pour the egg yolk into a graduated cylinder, measure the volume and record.

(4) Add octanoic acid with a final concentration of 8% while stirring, place at room temperature for 20 minutes, then centrifuge at 8000 r/min for 20 minutes, filter with qualitative filter paper to remove precipitates, and obtain fat-free egg yolk water-soluble extract (WSF);

(5) Then add a saturated ammonium sulfate solution with a final concentration of 40%, place at room temperature for 20 minutes, centrifuge at 12000 r/min for 20 minutes, separate the supernatant and precipitate, and the obtained precipitate is aflatoxin B ₁ -IgY antibody.

Example 4 ELISA Detection of Aflatoxin B ₁ -IgY Antibody

1. ELISA detection

(1) Dilute egg yolk extract with PBST to 1:6400, and set up blank control wells (without adding egg yolk extract Ig Y, replace with PBST);

(2) Dilute aflatoxin B ₁ artificial antigen with coating solution to a concentration of 1 µg/mL, coat a 96-well microtiter plate, add 100 µL to each well, incubate overnight at 37°C, discard the coating solution, wash 2 times;

(3) Add 120 µL of blocking solution (5% skimmed milk powder) to each well, block at 37°C for 30 min, discard the blocking solution, clap the plate, and dry at 37°C for later use;

(4) Dilute egg yolk extract 500 times with PBST, and dilute aflatoxin B ₁ to 1000, 200, 40, 8, 1.6, 0.32, 0.064 ng/mL;

(5) Add 50 µL aflatoxin B ₁ dilution to each row (three parallel groups), add 50 µL egg yolk extract dilution/well, incubate at 37°C for 40 min, and wash 5 times;

(6) Add goat anti-chicken secondary antibody IgY-HRP (5000-fold dilution), incubate at 37°C for 30 min, wash 5 times, and seal;

(7) Add color developing solution to develop color for 10 min;

(8) Add 50 µL of 10% H ₂ SO ₄ to terminate the reaction, and read the OD value at 450 nm;

(9) Replace the above-mentioned drug AFB ₁ with AFB ₂ , AFG ₁ and AFG ₂ , and carry out the above test with the same dilution factor to determine the cross-reactivity rate of the antibody to other structural analogues of aflatoxin.

2. Results, the indirect competition ELISA standard curve of the aflatoxin B ₁ -IgY antibody is shown in FIG. 4 .

The half inhibitory concentration (IC ₅₀ ) of the aflatoxin B ₁ -IgY antibody to AFB ₁ was 4.23 ng/mL, the linear range of quantitative detection (IC ₂₀ ~IC ₈₀ ) was 0.29~61.83 ng/mL, and the minimum detection limit was 0.06 ng/mL, and the cross-reactivity rates to AFB ₂ , AFG ₁ and AFG ₂ were all lower than 1%.

The aflatoxin B ₁ -IgY antibody obtained by the method can meet the detection requirements, and has high sensitivity and high specificity recognition ability for the aflatoxin B ₁ at the same time.

Claims (5)

1. a kind of preparation method of aflatoxin B ₁ yolk antibody, is characterized in that, comprises the steps: (1) Dissolve aflatoxin B ₁ in acetonitrile, react with glycolic acid and trifluoroacetic acid, extract the reaction product with ethyl acetate and saturated saline, dry it with anhydrous sodium sulfate, and finally obtain aflatoxin by liquid phase purification Aspergillus toxin B ₁ hapten; (2) Coupling the aflatoxin B ₁ hapten described in step (1) with BSA by the active ester method to obtain the aflatoxin B ₁ artificial antigen; (3) Use 300 µL of aflatoxin B ₁ artificial antigen (1 mg/mL), emulsify with Freund's complete adjuvant at a volume ratio of 1:1, and immunize laying hens, and then mix an equal volume of AFB ₁ -GA-BSA with Freund's incomplete adjuvant emulsification, booster immunization once every three weeks, a total of three booster immunizations; (4) Collect eggs on the day after the first immunization, and store them at 4°C after labeling; (5) After washing the eggs, wipe the eggshells with 75% alcohol cotton, remove the eggshells and egg whites, gently break the egg yolk membrane with a needle, and dilute the outflowing egg yolk liquid with 0.06 mol/L NaAc at a volume of 1:9 , add octanoic acid with a final concentration of 8% while stirring, place at room temperature for 20 minutes, centrifuge at 8000 r/min for 20 minutes, filter to remove precipitates, and obtain fat-free egg yolk water-soluble extract (WSF); (6) Add 40% saturated ammonium sulfate solution, place at room temperature for 20 minutes, centrifuge at 12000 r/min for 20 minutes, separate supernatant and precipitate, and the obtained precipitate is aflatoxin B ₁ -IgY antibody; The molecular structure of the aflatoxin B ₁ hapten described in step (1) is as follows: ) as shown in: Formula (I). 2. the preparation method of aflatoxin B1 egg yolk antibody according to claim ₁ , is characterized in that, the mobile phase of described liquid phase purification is acetonitrile and water, or mobile phase is 0.1% acetic acid, 35 ℃ of column temperatures, Flow rate 1 mL/min. 3. the aflatoxin B ₁ egg yolk antibody prepared by the method described in claim 1 or 2. 4. _A test kit for detecting aflatoxin B1, characterized in that, said test kit comprises the egg yolk antibody of aflatoxin B1 according to claim ₃ . 5. the aflatoxin B ₁ yolk antibody described in claim 3 or the application of the kit described in claim 4 in the immunodetection of aflatoxin B ₁ .