CN109438424B - Ribavirin hapten and artificial antigen as well as preparation method and application thereof - Google Patents

Abstract

所述利巴韦林人工抗原是由式(I)或式(II)所示半抗原与载体蛋白偶联得到。利用所述利巴韦林人工抗原免疫动物，可得到效价高，灵敏度高的特异性抗体。本发明提供的利巴韦林半抗原及其制备的抗体，为建立快速、简便、价廉、灵敏、特异的利巴韦林检测方法提供了新手段。The present invention relates to ribavirin hapten and artificial antigen as well as preparation method and application thereof. The structure of the ribavirin hapten is shown in formula (I) or formula (II):

The ribavirin artificial antigen is obtained by coupling the hapten represented by formula (I) or formula (II) with a carrier protein. Using the ribavirin artificial antigen to immunize animals, specific antibodies with high titer and high sensitivity can be obtained. The ribavirin hapten and the prepared antibody provided by the invention provide a new means for establishing a rapid, simple, inexpensive, sensitive and specific ribavirin detection method.

Description

Ribavirin hapten and artificial antigen and preparation method and application thereof

technical field

The invention belongs to the technical field of biochemical industry, in particular to a ribavirin hapten and an artificial antigen and a preparation method and application thereof.

Background technique

Ribavirin (RBV), also known as ribavirin, ribavirin, is a synthetic purine nucleoside analog. In 1972, it was researched and developed by the American ICN company, and it was first listed in Mexico in 1974. Ribavirin is a broad-spectrum antiviral drug that has inhibitory effects on a variety of RNA viruses and DNA viruses. Influenza A and B viruses are the most sensitive. The mechanism of action is that it is metabolized into ribavirin groups in the body (1, 2,4-triazole-3-carboxamide), which can inhibit viral polymerase and interfere with viral RNA synthesis, thereby blocking viral replication. In addition, ribavirin can also induce phenotypic transformation of T cells, thereby enhancing T cell-mediated humoral immune responses.

Ribavirin has a wide range of antiviral effects, and its toxic and side effects have also attracted increasing attention. For a long time, in order to prevent livestock and avian influenza, ribavirin has also been widely used in livestock and poultry breeding. In order to ensure the safety of human drug use and animal epidemic prevention, the Ministry of Agriculture issued Announcement No. 560 in 2005, prohibiting the use of ribavirin, amantadine, morpholineguanidine and other antiviral drugs in animal breeding. However, the illegal use of ribavirin in the livestock and poultry breeding process is still widespread, and the quality and safety of livestock and poultry products are threatened to some extent.

At present, the detection methods of ribavirin are mainly liquid chromatography (LC) and liquid chromatography-mass spectrometry (LC-MS/MS). , severely restricting the effective regulation of ribavirin use. The immunoassay technology based on antigen-antibody specific reaction has the advantages of fast detection speed and low cost. Therefore, it is particularly important to design an immune hapten molecule and develop a simple and fast antibody for the detection of ribavirin.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide ribavirin hapten and artificial antigen and preparation method and application thereof.

In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a ribavirin hapten, the structure of which is shown in formula (I) or formula (II):

In the second aspect, the present invention provides a preparation method of the hapten, when the ribavirin hapten is a compound represented by formula (I), the preparation method comprises the following steps:

1) Dissolve 48.0 g of ribavirin, 6.17 g of p-toluenesulfonic acid and 39.2 mL of 2,2-dimethoxypropane in 500 mL of acetone, and react the mixture at 60°C for 3 h; add 1 mL of 5% sodium bicarbonate Terminate the reaction; after removing the precipitate, collect and concentrate the filtrate, and obtain the compound of formula (III) after purification by column chromatography;

2) Dissolve 35.0 g of p-fluorobenzoic acid and 15.0 g of dimethylaminopyridine in 500 mL of tert-butanol and cool to 0°C; add 107.50 g of di-tert-butyl dicarbonate to the mixture, and stir at room temperature for 16 h to obtain The colorless oily product was dissolved in 300 mL of dimethyl sulfoxide with 15.0 g of the compound of formula (III) and 60.0 g of cesium carbonate, and heated at 110 °C for 16 h; the dimethyl sulfoxide was removed by distillation under reduced pressure 200 mL of water was added to the residue, extracted with dichloromethane, dried over sodium sulfate and concentrated, the residue was purified by column chromatography, and the obtained 6.0 g of white solid product was dissolved in a mixture of hydrochloric acid and tetrahydrofuran at room temperature In 100 mL of liquid, stir for 16 h, adjust the pH to 7-9 with sodium bicarbonate, and filter the mixture to obtain a crude product; the crude product is further crystallized with methanol to obtain.

Wherein, the mobile phase used in the column chromatography in step 2) is formed by mixing methanol and dichloromethane in a volume ratio of 50:1, and the used stationary phase is 200-300 mesh silica gel.

The mixed solution of the hydrochloric acid and tetrahydrofuran is formed by mixing tetrahydrofuran and hydrochloric acid with a concentration of 12 mol/L in a volume ratio of 9:1.

When the ribavirin hapten is a compound represented by formula (II), the preparation method is as follows:

Put 1.0g of ribavirin into a 100mL two-necked flask, add 20mL of acetone, stir at room temperature for 10min, put in 50mg of p-toluenesulfonic acid, react at 60°C for 4h, spin the reaction system to dryness, and purify the residue by column chromatography to obtain a white solid product and put it into a microwave tube, add 3 mL of pyridine, stir at room temperature for 10 min, put in 426 mg of succinic anhydride, and react at 120 ° C for 20 min. Add 15 mL of pyridine, stir at room temperature for 10 min, put in 5 mL of trifluoroacetic acid, react at 50°C for 12 h, and spin the reaction system to dryness.

Wherein, the mobile phase used in the column chromatography is formed by mixing methanol and dichloromethane in a volume ratio of 1:20, and the used stationary phase is 200-300 mesh silica gel.

In a third aspect, the present invention provides an artificial ribavirin antigen obtained by coupling the ribavirin hapten with a carrier protein. The ribavirin artificial antigen can be used as an immunogen or a coating original.

Wherein, the carrier protein is selected from bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroid protein, human serum albumin; preferably bovine serum albumin and keyhole limpet hemocyanin.

In a fourth aspect, the present invention provides a method for preparing the artificial antigen, wherein the carrier protein is coupled to the carboxyl carbon of the hapten according to claim 1 by using an activated ester method.

Preferably, the coupling molar ratio of the compound represented by formula (I) to the carrier protein is 9.6:1. The coupling molar ratio of the compound represented by formula (II) and the carrier protein is 7.0:1.

In a fifth aspect, the present invention provides specific antibodies prepared from the ribavirin artificial antigen, including polyclonal antibodies and monoclonal antibodies, preferably polyclonal antibodies. The polyclonal antibody can be obtained by immunizing experimental animals (such as New Zealand white rabbits) with ribavirin artificial antigen, and collecting serum and purifying.

In the sixth aspect, the present invention provides any following application of the ribavirin hapten or the ribavirin artificial antigen:

① Application in the preparation of anti-ribavirin-specific antibodies;

② Application in the detection of anti-ribavirin-specific antibodies.

In a seventh aspect, the present invention provides a ribavirin detection reagent or kit prepared from the specific antibody.

In an eighth aspect, the present invention provides any of the following applications of the specific antibody:

(1) Application in the detection of ribavirin;

(2) the application in the preparation of the immunochromatographic test strip of ribavirin;

(3) Application in the preparation of ribavirin colloidal gold test strips.

By the above-mentioned technical scheme, the present invention at least has the following advantages and beneficial effects:

The present invention discloses for the first time two new ribavirin haptens, artificial antigens and preparation methods thereof. Using the ribavirin artificial antigen to immunize animals, specific antibodies with high titer and high sensitivity can be obtained. The ribavirin hapten and the prepared antibody provided by the invention provide a new means for establishing a rapid, simple, inexpensive, sensitive and specific ribavirin detection method.

Using the conjugate of hapten and carrier protein provided by the invention to prepare ribavirin antibody (polyclonal antibody), the preparation process is simple and economical, the detection sensitivity of the antibody can reach 0.31ng/mL, and the practical value is high. The invention has a good application prospect in the detection of veterinary drug residues.

Description of drawings

Fig. 1 is a flow chart of the preparation of the ribavirin hapten represented by formula (I) in Example 1 of the present invention.

2 is a flow chart of the preparation of the ribavirin hapten represented by formula (II) in Example 1 of the present invention.

3 is a mass spectrum of the ribavirin hapten represented by formula (I) in Example 1 of the present invention.

4 is a mass spectrum of the ribavirin hapten represented by formula (II) in Example 1 of the present invention.

Fig. 5 is the nuclear magnetic spectrum of the ribavirin hapten represented by formula (I) in Example 1 of the present invention.

Fig. 6 is the nuclear magnetic spectrum of the ribavirin hapten represented by formula (II) in Example 1 of the present invention.

7 is a structural diagram of an artificial antigen prepared from the ribavirin hapten represented by formula (I) in Example 2 of the present invention.

8 is a structural diagram of an artificial antigen prepared from the ribavirin hapten represented by formula (II) in Example 2 of the present invention.

FIG. 9 is a MALDI-TOF-MS diagram of RBV①-BSA in Example 2 of the present invention.

10 is a MALDI-TOF-MS diagram of RBV②-BSA in Example 2 of the present invention.

FIG. 11 is a graph of the standard curve for detecting ribavirin using polyclonal antibodies in Example 4 of the present invention.

Detailed ways

The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available commodities.

The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged. The PBS buffers used in the examples are all PBS buffers with pH 7.4 and 0.01M. The carbonate buffers used in the examples are all sodium carbonate buffers with pH 9.6 and 0.05mol/L.

NHS is an abbreviation for N-hydroxysuccinimide. EDC is an abbreviation for 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. DMF is an abbreviation for N,N-dimethylformamide. NHS, EDC, bovine serum albumin (Albuminfrombovine serum, BSA), Keyhole Limpet Hemocyanin (KLH), complete Freund's adjuvant, incomplete Freund's adjuvant were purchased from Sigma company. The stationary phase used in column chromatography is 200-300 mesh silica gel.

Example 1 Preparation and characterization of ribavirin hapten

1. Preparation of ribavirin hapten

1, the preparation of ribavirin hapten represented by formula (I)

48.0 g of ribavirin, 6.17 g of p-toluenesulfonic acid and 39.2 mL of 2,2-dimethoxypropane were dissolved in 500 mL of acetone, and the mixture was reacted at 60° C. for 3 h. The reaction was quenched by the addition of 1 mL of 5% sodium bicarbonate. After removal of the precipitate, the filtrate was collected and concentrated. The intermediate product represented by formula (III) is obtained after purification by column chromatography.

35.0 g of p-fluorobenzoic acid and 15.0 g of dimethylaminopyridine were dissolved in 500 mL of tert-butanol and cooled to 0°C. 107.50 g of di-tert-butyl dicarbonate was added to the mixed solution, followed by stirring at room temperature for 16 h. Purify by column chromatography to obtain a colorless oily product. The oily product, 15.0 g of the intermediate product represented by formula (III) and 60.0 g of cesium carbonate were dissolved in 300 mL of dimethyl sulfoxide, and heated at 110 °C for 16 h. Dimethyl sulfoxide was removed by distillation under reduced pressure. 200 mL of water was added to the residue, extracted with dichloromethane, dried over sodium sulfate, and concentrated. The residue was purified by column chromatography with a mobile phase of methanol/dichloromethane (volume ratio 50:1) to obtain 6.0 g The white solid product was stirred with a mixture of hydrochloric acid/tetrahydrofuran (12 mol/L concentrated hydrochloric acid 10 ml and tetrahydrofuran 90 ml) at room temperature for 16 h. The pH was adjusted to 9 with sodium bicarbonate and the crude product was obtained after filtration of the mixture. The crude product was further crystallized with methanol three times to obtain 2.6 g of pure product in the form of a white solid, which is the ribavirin hapten represented by formula (I), with a yield of 55%. The specific synthetic route is shown in Figure 1.

2. Preparation of ribavirin hapten represented by formula (II)

Weigh 1.0g of ribavirin into a 100ml two-necked flask, add 20mL of acetone, stir at room temperature for 10min, put in 50mg of p-toluenesulfonic acid, react at 60°C for 4h, spin the reaction system to dryness, and the residue passes through the mobile phase to methanol Purify by column chromatography of /dichloromethane (volume ratio 1:20) to obtain a white solid product and add it to a microwave tube, add 3 mL of pyridine, stir at room temperature for 10 min, put in 426 mg of succinic anhydride, and react at 120 ° C for 20 min. After spin-drying, the product obtained after purification by column chromatography was added to a 100 mL flask, 15 mL of pyridine was added, stirred at room temperature for 10 min, put into 5 mL of trifluoroacetic acid, and reacted at 50 °C for 12 h, the reaction system was spin-dried to obtain the target product, which is formula (II) Ribavirin hapten shown. The specific synthetic route is shown in Figure 2.

2. Characterization of ribavirin hapten

1. Mass spectrometry identification

The mass spectrometry identification result of the ribavirin hapten (molecular formula is C ₁₅ H ₁₆ N ₄ O ₇ ) shown in the formula (I) of step 1: MS m/z [M+H] ⁺ theoretical value: 364.3; measured value: 365.0, which is consistent with the molecular weight of the target product, and the mass spectrum is shown in Figure 3.

Mass spectrometry identification result of ribavirin hapten (molecular formula C ₁₂ H ₁₆ N ₄ O ₈ ) represented by formula (II) in step 1: MSm/z[M+H] ⁺ theoretical value: 344.3; measured value: 343.1 , which is consistent with the molecular weight of the target product, and the mass spectrum is shown in Figure 4.

2. NMR identification

The nuclear magnetic identification results of the ribavirin hapten represented by the formula (I) in the first step: ¹ H NMR (400MHz, CDCl ₃ ), 1.35(s, 3H), 1.52(s, 9H), 1.53(s, 3H) .4.16-4.27(m, 2H), 4.60-4.63(m, 1H), 5.08-5.10(m, 1H), 5.30(d, J=2H, 1H), 6.33(s, 1H), 6.91(d, J=7.2Hz, 2H), 7.70(s, br, 1H), 7.79(d, J=8.8Hz, 2H), 8.01(s, br, 1H), 8.79(s, 1H). NMR data show that the above method The synthesized compound is the target product, and the magnetic resonance identification results are shown in Figure 5.

NMR identification results of the ribavirin hapten represented by formula (II) in step 1: ¹ H NMR (400MHz, CD ₃ OD), δ8.68(s, 1H), 5.93(s, 4H), 4.55-4.54 (m,1H),4.46-4.39(m,2H),4.26-4.25(m,2H),2.59(brs,4H). NMR data show that the compound synthesized by the above method is the target product, and the magnetic resonance identification results are shown in Fig. 6.

Example 2 Preparation and Characterization of Ribavirin Artificial Antigen

In the preparation method of the immunogen and the coating original, the difference lies in the type of carrier protein used. The immunogen carrier protein mainly adopts KLH, the coating original carrier protein mainly adopts BSA, and the coupling method used is active ester. Law. The structures of artificial antigens prepared from ribavirin haptens represented by formula (I) and formula (II) are shown in Figures 7 and 8 .

1. Synthesis and identification of ribavirin coating

1. Preparation of ribavirin coating

(1) Dissolve 20 mg of compound (I) prepared in Example 1 in 2 mL of DMF, add 10 mg of NHS and 10 mg of DCC, and stir overnight at room temperature to obtain solution I.

(2) 7mg BSA was added into 7mL PBS buffer, and it was fully dissolved, which was solution II.

(3) Slowly add solution I dropwise to solution II, slowly stir at 4°C for 24h, put it into a dialysis bag, and dialyze it in physiological saline at 4°C for 72h (with 6 water changes in between) to obtain the original ribavirin-coated solution , -20 ℃ storage. The ribavirin coating synthesized by the compound represented by formula (I) was originally abbreviated as RBV①-BSA.

(4) The ribavirin-coated source synthesized by the compound represented by formula (II) is prepared by the same method in the above three steps, abbreviated as RBV②-BSA.

2. Identification of the original ribavirin coating

Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) method was used to determine the binding ratio of BSA to hapten in RBV①-BSA solution and RBV②-BSA solution, respectively. The results are shown in Figure 9 and Figure 10.

Binding ratio = {M(conjugate)-M(protein)}/M(hapten)

The molecular weight of BSA is 65700.3, the molecular weight of the hapten of formula (I) is 364.3, the molecular weight of the conjugate analyzed by the highest peak of mass spectrometry is 69212.9, and the binding ratio of BSA to hapten is calculated to be 9.6, which is one of RBV①-BSA An average of 9.6 haptens were coupled to the BSA molecule.

The molecular weight of the hapten of formula (II) is 344.3, and the molecular weight of the conjugate analyzed by the highest peak of mass spectrometry is 68119.3. It is calculated that the binding ratio of BSA and hapten is 7.0, that is, the average coupling on a BSA molecule in RBV②-BSA 7.0 haptens.

2. Synthesis of Ribavirin Immunogen

1. Preparation of ribavirin immunogen

Substitute KLH for BSA, otherwise the same as 1 in step 2.

The compounds represented by formula (I) and formula (II) are abbreviated as RBV①-KLH and RBV②-KLH respectively for the synthesis of ribavirin immunogens.

Example 3 Preparation of ribavirin antiserum

The RBV①-KLH and RBV②-KLH solutions prepared in Example 2 were immunized respectively with 2 groups of 3-4 month old female New Zealand white rabbits weighing 1.5-2.0 kg, 2 in each group. Each immunogen was diluted with physiological saline to 1 mg/mL and emulsified with an equal amount of Freund's adjuvant. Freund's complete adjuvant was used for the first immunization, with multiple intradermal injections on the back of the neck, and the immunization dose was 1 mg per animal. After 4 weeks, booster immunization was carried out, and the booster immunization was performed once every 4 weeks, for a total of 3 times. The adjuvant was changed to Freund's incomplete adjuvant. One week after the fourth immunization, a large amount of blood was collected by the method of cardiac blood collection. After blood collection, the blood was allowed to stand at 37°C for 2 hours, then at 4°C overnight, and then centrifuged at 3000 rpm for 20 min. The supernatant was collected, which was antiserum, and was stored in aliquots at -20°C.

Example 4 Determination of ribavirin antiserum

1. Use indirect ELISA method to detect antiserum titer, the specific operation steps are as follows:

1) Coating: The antigen in Example 2 was double-diluted with 0.05M, pH9.6 carbonate buffer starting from 10 μg/mL, 100 μL/well, and reacted at 37° C. for 2 h.

2) Washing: Pour off the solution in the plate, spin dry, and wash three times with washing solution for 3 minutes each time.

3) Blocking: After patting dry, add 200 μL/well of blocking solution, and react at 37°C for 2 hours. Dry after washing.

4) Sample loading: The antiserum was doubling dilution from 1:1000, and added to the coated wells of various dilutions, 100 μL/well, and reacted at 37°C for 1 h; after thorough washing, adding 1:3000 diluted HRP-goat anti-rabbit IgG, 100 μL/well, reacted at 37°C for 1 h.

5) Color development: Take the ELISA plate out, wash it thoroughly, add 100 μL of TMB color developing solution to each well, and react at 37°C for 15 minutes in the dark.

6) Stop and measure: Add 100 μL of stop solution to each well to stop the reaction, and then measure the OD ₄₅₀ value of each well with a microplate reader.

7) Interpretation of results: the highest dilution ratio of serum corresponding to OD ₄₅₀ value ≥ 2.1 times of the negative control well (ie P/N ≥ 2.1) is the ELISA titer of serum.

2. Detection of minimum detection limit, half inhibition and specificity

The specific operation steps are as follows:

1) Use the above-mentioned indirect ELISA method to determine that RBV①-BSA is used as the coating source, the serum obtained from immunized rabbits with RBV①-KLH is used as the antibody, and the corresponding antigen and antibody concentration when the OD ₄₅₀ value is about 1.5 is the optimal working concentration. .

2) Coating: Dilute the original coating 20,000 times with coating buffer, 100 μL/well, and react at 37°C for 2 hours.

3) Washing and blocking: the method operation is the same as the above-mentioned indirect ELISA method.

4) Prepare ribavirin standard solution: prepare ribavirin standard solution with 0.01mol/L, pH7.4 PBS solution to prepare 5mg/mL stock solution, then, before adding samples, add 0.01mol/L , the pH7.4 PBS solution was diluted to the required concentration (the concentrations of RBV were 0.005ng/mL, 0.05ng/mL, 0.25ng/mL, 0.5ng/mL, 5ng/mL, 50ng/mL, respectively).

5) Sample loading: Add 50 μL of ribavirin standard at each concentration dilution to each well, then add 50 μL/well of antiserum at the optimal dilution, and react at 37°C for 1 h. After thorough washing, 1:3000 diluted HRP-goat anti-rabbit IgG was added, 100 μL/well, and reacted at 37°C for 1 h.

6) Color development reaction: Take out the ELISA plate, and after sufficient washing, add 100 μL of TMB color development solution to each well, and react at 37°C for 15 minutes in the dark.

7) Stop and measure: Add 100 μL of stop solution to each well to stop the reaction, and then measure the OD ₄₅₀ value of each well with a microplate reader.

8) Data processing: take the logarithm of each concentration of ribavirin as the abscissa, and take the OD value corresponding to each concentration of ribavirin as the ordinate, use Origin 8.5 software, and draw a standard curve according to four-parameter logarithmic fitting, As shown in FIG. 11 , whether the antiserum was specific for ribavirin was determined by calculating the _IC50 value (the median inhibitory concentration).

The results showed that after four immunizations, the titer of rabbit antiserum could reach 50000, the detection limit was 0.02ng/mL, the IC ₅₀ value was 0.31ng/mL, and the linear detection range was 0.06-1.67ng/mL.

Although the present invention has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

Claims (11)

1. Ribavirin hapten, is characterized in that, its structure is as shown in formula (I):

2. the preparation method of the described hapten of claim 1, is characterized in that, comprises the steps: 1) Dissolve 48.0 g of ribavirin, 6.17 g of p-toluenesulfonic acid and 39.2 mL of 2,2-dimethoxypropane in 500 mL of acetone, and react the mixture at 60°C for 3 h; add 1 mL of 5% sodium bicarbonate Terminate the reaction; after removing the precipitate, collect and concentrate the filtrate, and obtain the compound of formula (III) after purification by column chromatography;

2) Dissolve 35.0 g of p-fluorobenzoic acid and 15.0 g of dimethylaminopyridine in 500 mL of tert-butanol and cool to 0°C; add 107.50 g of di-tert-butyl dicarbonate to the mixture, and stir at room temperature for 16 h to obtain The colorless oily product was dissolved in 300 mL of dimethyl sulfoxide with 15.0 g of the compound of formula (III) and 60.0 g of cesium carbonate, and heated at 110 °C for 16 h; the dimethyl sulfoxide was removed by distillation under reduced pressure 200 mL of water was added to the residue, extracted with dichloromethane, dried over sodium sulfate and concentrated, the residue was purified by column chromatography, and the obtained 6.0 g of white solid product was dissolved in a mixture of hydrochloric acid and tetrahydrofuran at room temperature In 100 mL of liquid, stir for 16 h, adjust the pH to 7-9 with sodium bicarbonate, and filter the mixture to obtain a crude product; the crude product is further crystallized with methanol to obtain; Wherein, the mobile phase used in the column chromatography in step 2) is formed by mixing methanol and dichloromethane in a volume ratio of 50:1, and the stationary phase used is 200-300 mesh silica gel; the mixed solution of hydrochloric acid and tetrahydrofuran is composed of tetrahydrofuran and tetrahydrofuran. Hydrochloric acid with a concentration of 12mol/L is mixed in a volume ratio of 9:1. 3. ribavirin artificial antigen, is characterized in that, is obtained after the ribavirin hapten described in claim 1 is coupled with carrier protein; Wherein, the carrier protein is selected from bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroid protein, and human serum albumin. 4. The ribavirin artificial antigen according to claim 3, wherein the carrier protein is bovine serum albumin or keyhole limpet hemocyanin. 5 . The method for preparing the artificial antigen according to claim 3 or 4 , wherein the carrier protein is coupled to the carboxyl carbon of the hapten according to claim 1 by using an activated ester method. 6 . 6. The method of claim 5, wherein the coupling molar ratio of the compound represented by formula (I) to the carrier protein is 9.6:1. 7. the following any application of the described hapten of claim 1 or the described artificial antigen of claim 3 or 4: ① Application in the preparation of anti-ribavirin-specific antibodies; ② Application in the detection of anti-ribavirin-specific antibodies. 8. The specific antibody prepared from the artificial antigen of claim 3 or 4, the specific antibody is a polyclonal antibody or a monoclonal antibody. 9. The specific antibody according to claim 8, wherein the specific antibody is a polyclonal antibody. 10. A ribavirin detection reagent or kit prepared from the specific antibody of claim 8 or 9. 11. the following any application of the described specific antibody of claim 8 or 9: (1) Application in the detection of ribavirin; (2) the application in preparing the immunochromatographic test strip of ribavirin; (3) Application in the preparation of ribavirin colloidal gold test strips.

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