CN110724190A

CN110724190A - Naringenin antigen and preparation method and application thereof

Info

Publication number: CN110724190A
Application number: CN201911067958.1A
Authority: CN
Inventors: 崔永亮; 焦必宁; 赵静; 赵其阳; 张耀海; 陈爱华; 王成秋; 何悦
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2019-11-04
Filing date: 2019-11-04
Publication date: 2020-01-24
Anticipated expiration: 2039-11-04
Also published as: CN110724190B

Abstract

The invention discloses a naringenin antigen and a preparation method and application thereof, wherein naringenin reacts with a compound B containing amino and carboxyl at the same time to obtain a compound C; carrying out coupling reaction on the compound C and N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide or 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to obtain a compound D; coupling reaction is carried out on the compound D and carrier protein to obtain naringenin antigen; the naringenin antigen is applied to the preparation of an enzyme linked immunosorbent kit, a luminous immunoassay kit or an immunoaffinity chromatographic column for detecting naringenin in a sample. The method for preparing the naringenin antigen can conveniently and quickly obtain the naringenin antigen, and has the advantages of concise and clear synthesis steps, low synthesis cost and good effect. The antibody obtained by immunizing the naringenin antigen prepared by the method has good specificity and low minimum detection limit. Has wide prospect in the rapid immunodetection application of naringenin.

Description

Naringenin antigen and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a preparation method of a naringenin antigen, the naringenin antigen prepared by the preparation method and application thereof.

Background

Naringenin is aglycone of naringin, widely exists in rutaceae plants and is commonly known as Naringenin; the chemical name is as follows: 4', 5, 7-trihydroxyflavone; the trade name is: naringin, naringenin; CAS registry number 480-41-1; the molecular formula is: c₁₅H₁₂O₅(ii) a The relative molecular weight is: 272.25. the chemical structural formula is as follows:

naringenin (Naringin) has estrogen-like activity, and thus has the effect of promoting endothelial cells to secrete NO; it can reduce oxidative damage of cells; it can effectively reduce the damage of retina function and tissue structure, improve visual function; naringenin also has obvious effects of relieving cough, eliminating phlegm and relieving asthma; naringenin can directly inhibit activation of T lymphocyte, thereby preventing secretion of various inflammatory factors and further having protective effect on immunological acute liver injury.

At present, naringenin extraction methods mainly comprise a reflux method, an organic solvent method, a solid phase extraction method and an ultrasonic extraction method. The naringenin content analysis method comprises a spectrophotometric method, a High Performance Liquid Chromatography (HPLC), an Ultra Performance Liquid Chromatography (UPLC), an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), a reversed phase high performance liquid chromatography-diode array detection method (HPLC-DAD), a capillary zone electrophoresis method, an RP-HPLC method and the like, and has high sensitivity and good accuracy. Compared with an instrumental analysis method, the immunoassay method has the advantages of rapidness, simplicity, convenience, real-time performance, easiness in field detection, simplicity in sample pretreatment, high sensitivity, strong selectivity, suitability for high-throughput analysis and the like, and can also greatly reduce the detection cost.

Based on the analysis, a naringenin antigen which can rapidly, simply and conveniently carry out on-site detection on naringenin in real time, has high sensitivity and strong selectivity and is suitable for high-throughput analysis is urgently needed in the industry at present.

Disclosure of Invention

In view of the defects, the invention provides a method for preparing a naringenin antigen which has high sensitivity and strong selectivity and is suitable for high-throughput analysis, the naringenin antigen prepared by the method and application thereof.

The invention is realized by the following means:

the naringenin antigen provided by the invention has a structural general formula shown in formula A,

in the formula A, X is O, S, CH2 or NH, and n is an integer of 0-6; protein represents a carrier Protein selected from at least one of bovine serum albumin, ovalbumin and hemocyanin.

The invention provides a method for preparing naringenin antigen (formula A), which comprises the following steps:

(1) reacting naringenin with a compound which is shown in a formula B and contains amino and carboxyl to obtain a compound shown in a formula C;

H₂N-X-(CH₂)n-COOH

structural formula B

In the formulas B and C, X is O, S, CH2 or NH, and n is an integer of 0-6;

(2) carrying out coupling reaction on the compound shown in the formula C and N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide or 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to obtain a compound shown in a formula D;

in the formula D, X is O, S, CH2 or NH, and n is an integer of 0-6;

(3) coupling reaction is carried out on the compound shown in the formula D and carrier protein to obtain the naringenin antigen shown in the formula A; the carrier protein is at least one selected from bovine serum albumin, ovalbumin and hemocyanin.

Further, in the step (1), the compound shown in the formula B is at least one selected from carboxymethoxyamine, hydrazinoacetic acid, aminoacetic acid, aminopropionic acid, aminobutyric acid, aminopentanoic acid and aminocaproic acid.

Further, in the step (1), the feeding molar ratio of the compound shown in the formula B to naringenin is (0.1-10): 1

The reaction temperature is 0-100 ℃, and the reaction time is 6-48 hours;

the solvent for the reaction is at least one selected from pyridine, N-dimethylformamide, dimethyl sulfoxide and tetrahydrofuran.

Further, in the step (2), the molar part ratio of the compound shown in the formula C, the N-hydroxysuccinimide and the dicyclohexylcarbodiimide is 1: (1-5): (1-5);

the molar part ratio of the compound shown as the formula C to the N-hydroxysuccinimide to the 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride is 1: (1-5): (1-5);

the temperature of the coupling reaction is 0-50 ℃, and the time is 4-24 hours.

Further, in the step (3), the molar part ratio of the compound shown in the formula D to the carrier protein is (5-100): 1;

the temperature of the coupling reaction is 0-50 ℃, and the time is 8-36 hours; the coupling reaction is carried out under the condition that the pH value is 4-11;

carrying out coupling reaction on the compound shown in the formula D in a carrier protein solution, wherein the carrier protein solution is obtained by adding the carrier protein into a buffer solution, the buffer solution is selected from at least one of carbonate buffer solution, phosphate buffer solution, borate buffer solution and 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution, and the pH value of the buffer solution is 7.4;

further, the method further comprises the step of dialyzing the reaction system of the coupling reaction; in the dialysis step, the used dialysate is phosphate buffer solution with the pH value of 4-10 and the concentration of 0.01-0.2 mol/L.

The naringenin antigen prepared by the method also belongs to the protection scope of the invention. The naringenin antigen is a conjugate formed by connecting naringenin and carrier protein through amido bond; the amido bond is formed by carboxyl on the formula C and amino on the carrier protein through active ester.

The antibody obtained from the naringenin antigen provided by the invention, the application of the antigen and/or the antibody in detecting naringenin in a sample, the application in preparing an enzyme linked immunosorbent kit for detecting naringenin in the sample, a chemiluminescence immunoassay kit for naringenin or an immunoaffinity chromatographic column belong to the protection scope of the invention. Wherein the detection sample is water, medicine, food or soil.

The invention has the beneficial effects that:

the method for preparing the naringenin antigen can conveniently and quickly obtain the naringenin antigen, and has the advantages of concise and clear synthesis steps, low synthesis cost and good effect. The antibody obtained by immunizing the naringenin antigen prepared by the method has good specificity and low minimum detection limit. The method for preparing the naringenin antigen and the naringenin antigen obtained by the method have wide prospects in the application of rapid immunodetection of naringenin.

Drawings

FIG. 1 is a scheme of the synthesis of naringenin antigen;

FIG. 2 is a standard curve of the established naringenin indirect ELISA method.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Dicyclohexylcarbodiimide (DCC), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) Freund's complete adjuvant, Freund's incomplete adjuvant, bovine serum albumin and ovalbumin were purchased from Sigma, goat anti-mouse IgG-HRP from Jackson, o-phenylenediamine (OPD), carboxymethoxylamine hemihydrochloride, naringenin and other conventional reagents were purchased from Shanghai Arlatin Biotechnology Ltd.

Example 1

Preparation of naringenin-ovalbumin (naringenin-OVA) antigen (synthetic route is shown in figure 1)

1) Synthesis of naringenin hapten compounds of formula I:

in a 25mL three-necked flask, 0.2g of naringenin and 0.3g of carboxymethoxyamine hemihydrochloride were added, then 5mL of pyridine was added, heated to 100 ℃, reacted for 24 hours and cooled to room temperature, the reaction solution was poured into 20mL of water, the pH was adjusted to 3 with concentrated hydrochloric acid, extracted with ethyl acetate (3X 25mL), dried over anhydrous sodium sulfate, rotary desolventized, and column chromatography gave 0.13g of the product in 51.4% yield.

¹H NMR(600MHz，dmso)δ10.44(s，1H)，7.30(d，J＝8.2Hz，2H)，6.78(d，J＝8.3Hz，2H)，5.91(s，1H)，5.88(s，1H)5.06(d，J＝11.5Hz，1H)，4.68(s，2H)，3.30(d，J＝17.1Hz，1H)，2.91(dd，J＝17.0，11.8Hz，1H).

¹³C NMR(151MHz，dmso)δ171.06，161.64，159.43，158.88，157.97，155.69，129.84，128.58，115.57，96.94，96.56，95.96，76.03，70.98，40.34.

ESI-MS m/z 346.16[M+H]⁺，368.14[M+Na]⁺。

Product warp¹H-NMR、¹³And C-NMR and mass spectrum prove that the compound is the compound shown in the formula I.

2) Coupling:

method (1): weighing naringenin hapten (0.036mmol), NHS (0.047mmol) and DCC (0.040mmol) shown in formula I obtained in the step 1), dissolving the naringenin hapten and NHS (0.047mmol) and DCC (0.040mmol) in 1mL of anhydrous DMF, stirring at room temperature and 25 ℃ for reacting for 6 hours, centrifuging the reaction solution for 5 minutes at 8000 turns, and taking a supernatant to obtain a compound shown in formula B;

method (2): weighing naringenin hapten (0.036mmol), NHS (0.047mmol) and EDC (0.040mmol) which are obtained in the step 1) and shown in the formula I, dissolving the naringenin hapten and NHS and EDC in 1mL of water, and stirring the mixture at room temperature and 25 ℃ for reacting for 6 hours to obtain a compound shown in a formula II;

3) slowly dropwise adding the compound shown in the formula B obtained in the step 2) into an OVA solution (which is prepared by dissolving 105mg of OVA in 10mL Phosphate (PBS) buffer solution with the pH value of 7.5 and uniformly mixing), wherein the feeding molar ratio of the compound shown in the formula B to the carrier protein is 15:1, and stirring at 4 ℃ overnight.

4) And (3) dialysis: dialyzing the reaction solution obtained in the step 3) with PBS solution with the pH value of 7.5 and the concentration of 0.1mol/L for three days, diluting the completely dialyzed reaction product solution (naringenin-OVA) into 1mg/mL solution, and freezing and storing at-40 ℃ for later use. The dialysis is used for removing unreacted naringenin hapten or other small molecules to obtain a conjugate of naringenin shown in a formula III 1 and OVA, namely naringenin antigen shown in a formula C.

Wherein the PBS solution is prepared according to the following method: mixing NaCl and KH₂PO₄And Na₂HPO₄·12H₂O is mixed according to a mass ratio of 8.0: 0.2: the solution is dissolved in water in the proportion of 2.96, and the volume is adjusted to 1L by water.

Example 2

Preparation of naringenin-bovine serum albumin (naringenin-BSA) antigen

1) The synthesis and activation of naringenin hapten as shown in formula I is not different from that in example 1, and will not be described herein.

2) The compound of formula II obtained in step 2) of example 1 was slowly added dropwise to a carrier protein solution of 157.5mg BSA in 10mL Phosphate Buffered Saline (PBS) at pH 7.4, at a 15:1, stirred at 4 ℃ overnight.

3) And (3) dialysis: dialyzing the reaction solution obtained in the step 2) with PBS solution with the pH value of 7.4 and the concentration of 0.1mol/L for three days, diluting the completely dialyzed reaction product solution (naringenin-BSA) into 1mg/mL solution, and freezing and storing at-40 ℃ for later use. The dialysis is used for removing unreacted naringenin or other unreacted small molecules to obtain naringenin conjugate of formula III 2 and OVA, i.e. naringenin antigen of formula C.

Example 3

Application of naringenin-bovine serum albumin (naringenin-BSA) antigen

Preparation of antibody by naringenin-bovine serum albumin (naringenin-BSA) antigen

(1) 6-8 week-old Balb/c mice were used as experimental animals.

(2) Basic immunity: the diluted naringenin-BSA antigen solution (concentration: 1mg/mL) obtained in example 2 was filtered through a sterile filter, added with an equal volume of Freund's complete adjuvant, and emulsified by stirring with a magnetic stirrer until it did not diffuse when dropped into water. Injecting Balb/c mice with emulsified complete antigen via abdominal cavity and back subcutaneous multipoint injection at the dose of 0.1mg emulsified antigen per mouse.

(3) And (3) boosting immunity: after 2 weeks of basic immunization, 1mL of the diluted naringenin-BSA antigen solution was taken, 1mL of Freund's incomplete adjuvant was added, and the mixture was stirred and emulsified with a magnetic stirrer until the solution was not dispersed when dropped into water. The emulsified antigen was injected into Balb/C mice at a dose of 0.1mg of the emulsified diluted antigen per mouse by intraperitoneal and dorsal subcutaneous multiple injections (8-week-old Balb/C mice weigh about 23-25 g).

The boosting immunization is performed once every 15 days, from the beginning of the third boosting immunization, blood is collected from the eye socket of the mouse 3-5 days after each immunization, the titer of the antibody is determined, the coating is diluted 500 times by 1mg/mL naringenin-OVA, after the titer is greater than 1:8000 (the titer is defined as the color value of a control hole as 1, the dilution times of serum), the eyeball is removed for blood collection, the blood is kept still for 1 hour at room temperature, then kept still for 2 hours in a refrigerator at 4 ℃, and then centrifuged for 5 minutes at 8000r/min in a centrifuge, and antiserum is separated, so that the naringenin-BSA antibody is obtained. Was used in each of the following experiments.

Second, antibody effect detection

The various buffers used in the following experiments were as follows:

(1) coating buffer solution: 0.05M, pH 9.6.6 carbonate buffer;

(2) phosphate buffered saline PBS (pH 7.5): weigh 4.0g NaCl, 0.1g KH₂PO₄、1.48g Na₂HPO₄·12H₂O is diluted to 500mL by distilled water, and the concentration of O is 0.1M, pH which is 7.5 phosphate buffer solution;

(3) sample dilution PBSTG: is prepared by mixing 0.5mL of Tween 20, 0.5g of gelatin and 500mL of PBS buffer solution with the concentration of 0.1M, pH being 9.6;

(4) citrate-phosphate buffer: is prepared from trisodium citrate and Na₂HPO₄And water; the concentration of trisodium citrate in citrate-phosphate buffer is 0.01M, Na₂HPO₄Concentration in citrate-phosphate buffer 0.03M; the pH value of the citrate-phosphate buffer solution is 5.5;

(5) substrate buffer: 20.0mg of o-phenylenediamine (OPD) was dissolved in 10.0mL of citrate-phosphate buffer, followed by addition of 4. mu.L of 30% H by volume₂O₂The solution obtained by the aqueous solution, wherein the citrate-phosphate buffer solution is the solution obtained in the step (4);

(6) stop buffer: 2.0M aqueous sulfuric acid;

(7) washing liquid: prepared from NaCl and KH₂PO₄、Na₂HPO₄·12H₂O, Tween-20 and water; the concentration of NaCl in the washing solution was 8.0g/L, KH₂PO₄The concentration in the washing solution was 0.2g/L, Na₂HPO₄·12H₂The concentration of O in the washing solution is 2.96g/L, and the volume percentage content of Tween-20 in the washing solution is 1: 1000.

test example 1

Antibody inhibition assay

1. Preparation of naringenin-OVA coating antigen solution

After completely thawing the diluted naringenin-OVA antigen of 1mg/mL prepared in example 1, the naringenin-OVA antigen was diluted in a gradient of 1:1000, 1:2000, 1:4000, and 1:8000 with a coating buffer solution to obtain a coating antigen solution of naringenin-OVA with different concentrations.

2. Preparation of naringenin standard solution

(1) Weighing 1mg naringenin standard sample, and fully dissolving in 10mL of anhydrous methanol to obtain 1mg/mL naringenin standard solution;

(2) and (3) preparing the naringenin standard solution with the concentration of 2000ng/mL from the 1mg/mL naringenin standard solution obtained in the step (1) by using a sample diluent.

3. Preparation of naringenin-BSA antiserum diluent

And (3) carrying out gradient dilution on the naringenin-BSA antibody prepared in the first step by using a sample diluent according to the ratio of 1:1000, 1:2000, 1:4000 and 1:8000 to obtain naringenin-BSA antiserum diluent.

4. Checkerboard experiment of antigen and antibody

Coating: and (3) adding 100 mu L of naringenin-OVA coating antigen solution prepared in the step (1) into each well of a 96-well enzyme label plate, coating for 3 hours at 37 ℃, and washing for 4 times by using a washing solution.

And (3) sealing: sealing with 3% skimmed milk powder sealing solution 180 μ L/well in 37 deg.C wet box for 1 hr, discarding sealing solution, and washing for 3 times.

Competition: and (3) adding 50 mu l of sample diluent into each control hole, and adding 50 mu l of naringenin standard solution prepared in the step (2) into each inhibition hole. Diluting naringenin-BSA antiserum obtained in the step 3 (from 2.5X 10)³Multiple to 40 × 10³Fold) was added to the ELISA plate (50. mu.l/well), placed in a wet box at 37 ℃ for 30min, and the plate was washed 4 times.

Adding an enzyme-labeled secondary antibody: a goat anti-mouse enzyme-labeled secondary antibody (IgG-HRP, Jackson, catalog No. 79556) (0.1mg/mL) was diluted 1000-fold in 0.1M PBSTG (pH 9.6) at 100. mu.L per well, placed in a wet box at 37 ℃ for 30min, and the plate was washed 4 times.

Color development: substrate buffer was added to the microplate in 100. mu.l per well. And (4) shading and developing for 15 min.

And (4) terminating: add 50. mu.L of stop buffer to each well and measure the OD of each well at 492nm using a microplate reader.

Titers were defined as the fold of serum dilution at zero well OD value of 1.

The results are shown in Table 1.

TABLE 1 serum titer test for anti-naringenin mice (OPD Room temperature development 15min, 2000ng standard inhibition)

Note: i denotes the inhibition well in the microplate, C denotes the control well in the microplate.

According to the results in table 1, when the dilution of the coating antigen is 1:8000, at an antiserum dilution of 1:4000, the inhibition rate was best at this timeAnd 93.6% [ inhibition ratio (a) ]₀-A₂₀₀₀)/A₀×100％]That is, the suppression effect is the best at this time. It is demonstrated that naringenin-BSA prepared in example 2 above can be used as an immunogen to prepare an antibody for detecting naringenin.

A₀OD values for control wells; a. the₂₀₀₀To inhibit well OD values;

the calculation formula of the inhibition rate is as follows: inhibition rate ═ A₀-A₂₀₀₀)/A₀×100％。

Establishment of naringenin standard curve

Diluting the prepared naringenin standard solution with sample diluent to different concentrations as follows: 2000ng/mL, 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.6ng/mL, 7.8 ng/mL.

(1) Coating the original coating: diluting the prepared naringenin-OVA antigen according to the ratio of 1:8000, adding the diluted naringenin-OVA antigen into an enzyme label plate, incubating for 3 hours at 37 ℃ with 100 mu L of each hole; pouring out the solution in the ELISA plate, washing the plate for 4 times by using a washing solution, and drying;

(2) respectively adding the naringenin standard substance solutions (experimental wells) with different concentrations into the ELISA plate in the step (1), wherein each well is 50 mu L, and adding 50 mu L of sample diluent without adding the naringenin standard substance solution into a control well;

(3) adding naringenin-BSA antiserum diluent with the dilution ratio of 1:8000 into the experimental wells and the control wells respectively, wherein each well contains 50 mu L of naringenin-BSA antiserum diluent; incubation at 37 ℃ for 30 min; pouring off the solution in the ELISA plate, washing the plate for 4 times by using a washing solution, and spin-drying;

(4) mu.L of IgG-HRP (Jackson, catalog No. 79556) (0.1mg/mL) diluted 1:1000 was added to each of the experimental and control wells and incubated at 37 ℃ for 30 minutes; washing the plate for 4 times by using a washing solution, pouring off the solution in the ELISA plate, and spin-drying;

(5) adding 100 mu L of substrate buffer solution into the experimental wells and the control wells respectively, incubating for 15 minutes at room temperature, and adding 50 mu L of 2.0M sulfuric acid solution into each well to terminate the reaction;

(6) measuring absorbance at 492 nm;

(7) drawing a standard curve: taking naringenin standard solutions with different concentrations (ng/mL) as X axis, and taking ratio of absorbance values (B/B)₀ X 100%, wherein B is the average absorbance value of naringenin standard solution, B₀Average absorbance values for control wells) as the Y-axis, a standard graph is plotted.

The experiment was repeated 3 times, and the results of the three experiments were averaged to obtain a standard curve as shown in FIG. 2. The results show that it has sensitivity (IC)₅₀) 65.5ng/mL, and the detection range is 22.0ng/mL-220.8 ng/mL. It is demonstrated that naringenin-BSA prepared in example 1 has a very good effect as an antibody obtained by immunizing a mouse with an antigen.

(III) detection of antibody specificity

1. Preparation of naringenin analogue standard solution

Preparation of naringenin analogue standard substance

And (3) preparing a standard sample of the naringenin analogue according to the preparation method of the naringenin standard in the step (I).

Diluting the naringenin with a sample diluent to the following concentrations respectively: 20000ng/mL, 10000ng/mL, 5000ng/mL, 2500ng/mL, 1250ng/mL, 625ng/mL, 312.5 ng/mL.

2. Establishing standard curve, and determining the concentration IC in the inhibition₅₀(the concentration of the standard sample at which the inhibition rate reached 50%).

The method for establishing the standard curve is the same as the method for establishing the naringenin standard curve.

Rate of cross reaction (%) (naringenin IC)₅₀) /(naringenin analogue IC₅₀)×100％。

The experiment was repeated 3 times, and the results of the three experiments were averaged and shown in Table 2.

TABLE 2 specific detection of antibodies prepared from naringenin-BSA

Analyte	IC₅₀(ng/mL)	Cross reaction Rate (%)
			Naringenin	65.5	100
Naringenin	>20000	-
			Naringin	>20000	-
Hesperidin	>20000	-
			Hesperetin (Hesperetin)	>20000	-

The results in Table 2 show that the cross-reactivity of the antibody prepared from naringenin-BSA and its analogues is very low, indicating that the antibody prepared from naringenin-BSA has very good specificity to naringenin.

It will be appreciated by those of ordinary skill in the art that the examples described herein are intended to assist the reader in understanding the manner in which the invention is practiced, and it is to be understood that the scope of the invention is not limited to such specifically recited statements and examples. Those skilled in the art can make various other specific changes and combinations based on the teachings of the present invention without departing from the spirit of the invention, and these changes and combinations are within the scope of the invention.

Claims

1. A naringenin antigen characterized by: the structural general formula is shown as a formula A,

2. The method for preparing naringenin antigen of claim 1, comprising the steps of:

H₂N-X-(CH₂)n-COOH

structural formula B

In the formulas B and C, X is O, S, CH2 or NH, and n is an integer of 0-6;

in the formula D, X is O, S, CH2 or NH, and n is an integer of 0-6;

3. The method of claim 2, wherein: in the step (1), the compound shown in the formula B is at least one selected from carboxymethoxylamine, hydrazinoacetic acid, aminoacetic acid, aminopropionic acid, aminobutyric acid, aminopentanoic acid and aminocaproic acid.

4. The method of claim 2, wherein: in the step (1), the feeding molar ratio of the compound shown in the formula B to naringenin is (0.1-10): 1;

the reaction temperature is 0-100 ℃, and the reaction time is 6-48 hours;

5. The method of claim 2, wherein: in the step (2), the molar part ratio of the compound shown in the formula C, N-hydroxysuccinimide and dicyclohexylcarbodiimide is 1 (1-5) to 1-5;

the molar ratio of the compound shown in the formula C to the N-hydroxysuccinimide to the 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride is 1 (1-5) to 1-5;

6. The method of claim 2, wherein: in the step (3), the molar part ratio of the compound shown in the formula D to the carrier protein is (5-100): 1;

and (2) carrying out coupling reaction on the compound shown in the formula D in a carrier protein solution, wherein the carrier protein solution is obtained by adding the carrier protein into a buffer solution, the buffer solution is at least one selected from carbonate buffer, phosphate buffer, borate buffer and 4-hydroxyethyl piperazine ethanesulfonic acid buffer, and the pH value of the buffer solution is 7.4.

7. The method of claim 2, wherein: the method further comprises a step of dialyzing the reaction system of the coupling reaction; in the dialysis step, the used dialysate is phosphate buffer solution with the pH value of 4-10 and the concentration of 0.01-0.2 mol/L.

8. The method of claim 2, wherein: the naringenin antigen is a conjugate formed by connecting naringenin and carrier protein through an amido bond; the amido bond is formed by carboxyl on the formula C and amino on the carrier protein through active ester.

9. Use of the naringenin antigen of claims 1-8 wherein the use comprises: the application of the naringenin in detecting samples, the application of the naringenin in preparing enzyme linked immunosorbent assay kits for detecting the naringenin in the samples, and the application of the naringenin in preparing luminescent immunoassay kits or immunoaffinity chromatographic columns for detecting the naringenin in the samples.

10. The method of application according to claim 9, characterized in that: the detection sample is water, medicine, food or soil.