CN109438424B - Ribavirin hapten and artificial antigen as well as preparation method and application thereof - Google Patents
Ribavirin hapten and artificial antigen as well as preparation method and application thereof Download PDFInfo
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- CN109438424B CN109438424B CN201811367056.5A CN201811367056A CN109438424B CN 109438424 B CN109438424 B CN 109438424B CN 201811367056 A CN201811367056 A CN 201811367056A CN 109438424 B CN109438424 B CN 109438424B
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- ribavirin
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- 229960000329 ribavirin Drugs 0.000 title claims abstract description 117
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- HZCAHMRRMINHDJ-DBRKOABJSA-N ribavirin Natural products O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1N=CN=C1 HZCAHMRRMINHDJ-DBRKOABJSA-N 0.000 title claims abstract description 113
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
- C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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Abstract
The invention relates to ribavirin hapten and artificial antigen as well as a preparation method and application thereof. The structure of the ribavirin hapten is shown as a formula (I) or a formula (II):
Description
Technical Field
The invention belongs to the technical field of biochemical engineering, and particularly relates to ribavirin haptens and artificial antigens as well as preparation methods and applications thereof.
Background
Ribavirin (RBV), also known as Ribavirin or Ribavirin, is a synthetic purine nucleoside analog. The product was researched and developed by ICN company in USA in 1972, first marketed in Mexico in 1974, and officially approved for production in China in 1980. Ribavirin is a broad-spectrum antiviral drug, has an inhibiting effect on various RNA viruses and DNA viruses, is most sensitive to influenza A and B viruses, has an action mechanism that the ribavirin is metabolized into ribavirin groups (1,2, 4-triazole-3-carboxamide) in vivo, and can inhibit viral polymerase, interfere the synthesis of viral RNA and block the replication of viruses. Additionally, ribavirin can induce T cell phenotypic transformation, thereby enhancing T cell-mediated humoral immune responses.
The ribavirin has been widely noticed to exert the antiviral effect, and the toxic and side effects of the ribavirin are increasingly noticed, so that the ribavirin is widely applied to the livestock and poultry breeding process for preventing the livestock and poultry influenza for a long time. In order to ensure the safety of human medication and animal epidemic prevention, the 2005 ministry of agriculture issues a publication No. 560, which prohibits the use of antiviral drugs such as ribavirin, amantadine, moroxydine, etc. in animal farming. However, the phenomenon that ribavirin is illegally used in the livestock and poultry breeding process still exists generally, and the quality safety of livestock and poultry products is threatened to a certain extent.
At present, the detection means of ribavirin is mainly liquid chromatography (L C) and liquid chromatography-mass spectrometry (L C-MS/MS), but large instruments are expensive, complex to operate and poor in portability, so that the first-line detection is difficult to realize, and the effective supervision on the use of ribavirin is severely restricted.
Disclosure of Invention
The invention aims to provide ribavirin hapten and artificial antigen as well as preparation methods and application thereof.
In order to achieve the object of the present invention, in a first aspect, the present invention provides ribavirin haptens having the structure represented by formula (I) or formula (II):
in a second aspect, the present invention provides a preparation method of the hapten, wherein when the ribavirin hapten is a compound represented by the formula (I), the preparation method comprises the following steps:
1) dissolving 48.0g of ribavirin, 6.17g of p-toluenesulfonic acid and 39.2m of L2, 2-dimethoxypropane in 500m of L acetone, reacting the mixed solution at 60 ℃ for 3h, adding 1m of L5% of sodium bicarbonate to terminate the reaction, removing the precipitate, collecting and concentrating the filtrate, and purifying by column chromatography to obtain the compound shown in the formula (III);
2) dissolving 35.0g of p-fluorobenzoic acid and 15.0g of dimethylaminopyridine in 500m of L tert-butyl alcohol, cooling to 0 ℃, adding 107.50g of di-tert-butyl dicarbonate to the mixed solution, stirring at room temperature for 16h to obtain a colorless oily product, dissolving the colorless oily product, 15.0g of the compound of formula (III) and 60.0g of cesium carbonate in 300m of L dimethyl sulfoxide, continuously heating at 110 ℃ for 16h, removing the dimethyl sulfoxide by reduced pressure distillation, adding 200m of L water to the residue, extracting with dichloromethane, drying with sodium sulfate, concentrating, purifying the residue by column chromatography, dissolving the obtained 6.0g of a white solid product in 100m L of a mixed solution of hydrochloric acid and tetrahydrofuran at room temperature, stirring for 16h, adjusting the pH to 7-9 with sodium bicarbonate, filtering the mixture to obtain a crude product, and further crystallizing the crude product with methanol.
Wherein, the mobile phase used for carrying out column chromatography in the step 2) is formed by mixing methanol and dichloromethane according to the volume ratio of 50:1, and the used stationary phase is 200-mesh 300-mesh silica gel.
The mixed liquid of the hydrochloric acid and the tetrahydrofuran is formed by mixing the tetrahydrofuran and the hydrochloric acid with the concentration of 12 mol/L according to the volume ratio of 9: 1.
When the ribavirin hapten is a compound shown as a formula (II), the preparation method is as follows:
putting 1.0g of ribavirin into a 100m L two-necked bottle, adding acetone 20m L, stirring at room temperature for 10min, adding 50mg of p-toluenesulfonic acid, reacting at 60 ℃ for 4h, spin-drying a reaction system, purifying residues by column chromatography to obtain a white solid product, adding the white solid product into a microwave tube, adding 3m L pyridine, stirring at room temperature for 10min, adding 426mg of succinic anhydride, reacting at 120 ℃ for 20min, transferring the product obtained after the reaction system is spin-dried and purified by column chromatography to a 100m L flask, adding 15m L pyridine, stirring at room temperature for 10min, adding 5m L trifluoroacetic acid, reacting at 50 ℃ for 12h, and spin-drying the reaction system to obtain the ribavirin-free ribavirin injection.
Wherein, the mobile phase used for carrying out column chromatography is formed by mixing methanol and dichloromethane according to the volume ratio of 1:20, and the stationary phase is 200-mesh 300-mesh silica gel.
In a third aspect, the invention provides a ribavirin artificial antigen, which is obtained by coupling the ribavirin hapten with a carrier protein. The ribavirin artificial antigen can be used as immunogen or coating antigen.
Wherein the carrier protein is selected from bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroid protein, human serum albumin; bovine serum albumin and keyhole limpet hemocyanin are preferred.
In a fourth aspect, the present invention provides a method for preparing the artificial antigen, wherein an activated ester method is used to couple a carrier protein to the carboxyl carbon of the hapten as defined in claim 1.
Preferably, the compound of formula (I) is coupled to the carrier protein in a molar ratio of 9.6: 1. The coupling molar ratio of the compound shown in the formula (II) to the carrier protein is 7.0: 1.
In a fifth aspect, the invention provides specific antibodies, including polyclonal antibodies and monoclonal antibodies, preferably polyclonal antibodies, prepared from the ribavirin artificial antigen. The polyclonal antibody can be obtained by immunizing experimental animals (such as New Zealand white rabbits) with ribavirin artificial antigen, collecting serum, and purifying.
In a sixth aspect, the present invention provides any one of the following uses of the ribavirin hapten or the ribavirin artificial antigen:
① application in preparing anti-ribavirin specific antibody;
② for use in detecting anti-ribavirin specific antibodies.
In a seventh aspect, the invention provides a ribavirin detection reagent or kit prepared from said specific antibody.
In an eighth aspect, the invention provides any one of the following uses of the specific antibody:
(1) the application of the ribavirin detecting reagent in the detection of ribavirin;
(2) the application in the preparation of an immunochromatographic test strip of ribavirin;
(3) the application in preparing the colloidal gold test strip of ribavirin.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention discloses two novel ribavirin haptens, artificial antigens and preparation methods thereof for the first time, and a specific antibody with high titer and high sensitivity can be obtained by immunizing animals with the ribavirin artificial antigens. The ribavirin hapten and the antibody prepared by the ribavirin hapten provide a new means for establishing a rapid, simple, cheap, sensitive and specific ribavirin detection method.
The conjugate of the hapten and the carrier protein provided by the invention is used for preparing the ribavirin antibody (polyclonal antibody), the preparation process is simple and economic, the detection sensitivity of the antibody can reach 0.31ng/m L, and the practical value is high.
Drawings
FIG. 1 is a flow chart of the preparation of ribavirin haptens represented by formula (I) in example 1 of the present invention.
FIG. 2 is a flow chart of the preparation of ribavirin haptens represented by formula (II) in example 1 of the present invention.
FIG. 3 is a mass spectrum of ribavirin hapten shown in formula (I) in example 1 of the present invention.
FIG. 4 is a mass spectrum of ribavirin hapten shown in formula (II) in example 1 of the present invention.
FIG. 5 is the nuclear magnetic spectrum of the ribavirin hapten shown in the formula (I) in the example 1 of the invention.
FIG. 6 is a nuclear magnetic spectrum of ribavirin hapten shown in formula (II) in example 1 of the present invention.
FIG. 7 is a structural diagram of an artificial antigen prepared from ribavirin hapten shown in formula (I) in example 2 of the invention.
FIG. 8 is a structural diagram of an artificial antigen prepared from ribavirin hapten shown in formula (II) in example 2 of the invention.
FIG. 9 is a MA L DI-TOF-MS map of RBV ① -BSA in example 2 of the present invention.
FIG. 10 is a MA L DI-TOF-MS map of RBV ② -BSA in example 2 of the present invention.
FIG. 11 is a standard graph of the detection of ribavirin using polyclonal antibodies in example 4 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the 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 commercially available products.
In the quantitative tests in the following examples, three replicates were set up and the results averaged, the PBS buffer used in the examples was pH7.4, 0.01M PBS buffer, and the carbonate buffer used in the examples was 0.05 mol/L of sodium carbonate buffer, pH 9.6.
NHS for N-hydroxy succinimide abbreviation EDC for 1- (3-two amino propyl) -3-ethyl carbon two imine hydrochloride abbreviation DMF for N, N-two methyl formamide abbreviation NHS, EDC, bovine serum albumin (Albumin serum, BSA), Keyhole limpet Hemocyanin (Keyhole L Impet Hemocyanin, K L H) and Freund complete adjuvant, Freund incomplete adjuvant all from Sigma company, column chromatography used stationary phase is 200-.
Example 1 preparation and characterization of ribavirin haptens
Preparation of ribavirin hapten
1. Preparation of ribavirin hapten shown as formula (I)
48.0g of ribavirin, 6.17g of p-toluenesulfonic acid and 39.2m of L2, 2-dimethoxypropane are dissolved in 500m of L acetone, the mixed solution reacts for 3h at 60 ℃, 1m of L5% of sodium bicarbonate is added to stop the reaction, the precipitate is removed, the filtrate is collected and concentrated, and the intermediate product shown in the formula (III) is obtained after column chromatography purification.
35.0g of p-fluorobenzoic acid and 15.0g of dimethylaminopyridine are dissolved in 500m of L tert-butanol and cooled to 0 ℃, 107.50g of di-tert-butyl dicarbonate is added to the mixture, then the mixture is stirred at room temperature for 16h and purified by column chromatography to obtain a colorless oily product, the oily product is dissolved in 300m of L of dimethyl sulfoxide together with 15.0g of the intermediate product of the formula (III) and 60.0g of cesium carbonate, the mixture is heated continuously at 110 ℃ for 16h, the dimethyl sulfoxide is removed by distillation under reduced pressure, 200m of L water is added to the residue, the residue is extracted with dichloromethane and dried with sodium sulfate and concentrated, the residue is purified by column chromatography with methanol/dichloromethane (volume ratio 50:1) as a mobile phase, 6.0g of a white solid product is stirred at room temperature for 16h with sodium bicarbonate to adjust the pH to 9, the mixture is filtered to obtain a crude product, the crude product is further crystallized with methanol 3 times to obtain 2.6g of a white solid product, namely a pure ribavirin of the formula I (see fig. 1).
2. Preparation of ribavirin hapten shown as formula (II)
Weighing 1.0g of ribavirin, putting the ribavirin into a 100ml two-necked bottle, adding acetone 20m L, stirring at room temperature for 10min, adding 50mg of p-toluenesulfonic acid, reacting at 60 ℃ for 4h, spin-drying a reaction system, purifying a residue by column chromatography with a mobile phase of methanol/dichloromethane (volume ratio of 1:20) to obtain a white solid product, adding the white solid product into a microwave tube, adding 3m L pyridine, stirring at room temperature for 10min, adding 426mg of succinic anhydride, reacting at 120 ℃ for 20min, spin-drying the reaction system, purifying by column chromatography to obtain a product, adding the product into a 100m L flask, adding 15m L pyridine, stirring at room temperature for 10min, adding 5m L trifluoroacetic acid, reacting at 50 ℃ for 12h, and spin-drying the reaction system to obtain a target product, namely the ribavirin hapten shown in formula (II), wherein a specific synthetic route is shown in figure 2.
Characterization of the Lonicerin hapten
1. Identification by mass spectrometry
Step one ribavirin hapten (molecular formula is C) shown as formula (I)15H16N4O7) The mass spectrum identification result of (1) MS M/z [ M + H]+364.3 as a theoretical value; observed value is 365.0, which coincides with the molecular weight of the target product, and the mass spectrum is shown in FIG. 3.
Step one ribavirin hapten (molecular formula is C) shown as formula (II)12H16N4O8) The mass spectrum identification result of (1) is MSm/z [ M + H ]]+344.3 as a theoretical value; observed value 343.1, which coincides with the molecular weight of the target product, is shown in FIG. 4 by mass spectrum.
2. Nuclear magnetic resonance identification
The nuclear magnetic identification result of the ribavirin hapten shown as the formula (I) in the step one is as follows:1H NMR(400MHz,CDCl3) Nuclear magnetic data of 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) indicates that the compound synthesized by the above method is the target product, and the magnetic resonance identification result is shown in fig. 5.
The nuclear magnetic identification result of the ribavirin hapten shown in the formula (II) in the step one is as follows:1H NMR(400MHz,CD3OD), 8.68(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), nuclear magnetic data indicate that the compound synthesized by the above method is the target product, and the result of magnetic resonance identification is shown in fig. 6.
Example 2 preparation and characterization of ribavirin Artificial antigens
The immunogen and the coating antigen are prepared by a method which is different from the method for preparing the coating antigen in the using type of the carrier protein, the immunogen carrier protein mainly adopts K L H, the coating antigen carrier protein mainly adopts BSA, and the coupling method is an active ester method, the structure of the artificial antigen prepared by the ribavirin haptens shown in the formula (I) and the formula (II) is shown in figures 7 and 8.
Synthesis and identification of ribavirin envelope antigen
1. Preparation of ribavirin coated antigen
(1) 20mg of the compound represented by the formula (I) prepared in example 1 was dissolved in 2m L DMF, and 10mg of NHS and 10mg of DCC were added thereto, followed by stirring overnight at room temperature to obtain solution I.
(2) 7mg of BSA was added to 7m L PBS buffer solution, and the mixture was dissolved sufficiently to obtain solution II.
(3) Slowly dripping the solution I into the solution II, slowly stirring for 24h at 4 ℃, then putting into a dialysis bag, dialyzing for 72h in physiological saline at 4 ℃ (the water is changed for 6 times) to obtain a ribavirin envelope original solution, and storing at-20 ℃.
(4) The ribavirin coating antigen synthesized by the compound shown in the formula (II), RBV ② -BSA for short, is prepared by the same method of the three steps.
2. Identification of ribavirin capsulogen
The binding ratio of BSA to hapten in RBV ① -BSA solution and RBV ② -BSA solution was determined by Matrix-Assisted laser Desorption/Ionization Time of Flight Mass Spectrometry (Matrix-Assisted L ASER Desorption/Ionization Time of Flight Mass Spectrometry, MA L DI-TOF-MS) method, respectively, and the results are shown in FIGS. 9 and 10.
Binding ratio { M (conjugate) -M (protein) }/M (hapten)
The molecular weight of BSA was 65700.3, the molecular weight of hapten of formula (I) was 364.3, and the molecular weight of the conjugate was 69212.9, calculated as the binding ratio of BSA to hapten was 9.6, i.e., an average of 9.6 haptens were conjugated to one BSA molecule in RBV ① -BSA.
The hapten of formula (II) has a molecular weight of 344.3, the molecular weight of the conjugate is 68119.3 from the highest peak mass spectrometry, and the binding ratio of BSA to hapten is calculated to be 7.0, namely 7.0 haptens are averagely coupled on one BSA molecule in RBV ② -BSA.
Synthesis of di-and ribavirin immunogens
1. Preparation of ribavirin immunogens
K L H was used instead of BSA, and the same procedure as step two was followed by 1.
The ribavirin immunogens synthesized by the compounds shown in the formula (I) and the formula (II) are respectively abbreviated as RBV ① -K L H and RBV ② -K L H.
EXAMPLE 3 preparation of ribavirin antiserum
The RBV ① -K L H and RBV ② -K L H solutions prepared in example 2 are respectively used for immunizing 2 groups of female New Zealand white rabbits with the age of 3-4 months and the weight of 1.5-2.0kg, each group comprises 2 immunogens, the immunogens are diluted to 1mg/m L by physiological saline and emulsified with equivalent Freund's adjuvant, Freund's complete adjuvant is adopted for first immunization, multipoint injection is carried out in the neck and back, the immunization dose is 1 mg/4 weeks later, the immunization is enhanced, 1 immunization is carried out every 4 weeks, 3 times of immunization are carried out in total, the adjuvant is changed into Freund's incomplete adjuvant, the immunization dose is not changed, multipoint injection is carried out in the neck and back, after 1 week of 4 immunization, massive blood sampling is carried out by a heart blood sampling method, the blood is kept for 2 hours at 37 ℃, then kept overnight at 4 ℃, then centrifuged at 3000rpm for 20 minutes, and the supernatant is collected, namely antiserum is subpackaged and preserved at-20 ℃.
Example 4 measurement of ribavirin antisera
Firstly, an indirect E L ISA method is adopted to detect the titer of antiserum, and the specific operation steps are as follows:
1) coating the antigen of example 2 was diluted in 0.05M carbonate buffer pH9.6 at 10. mu.g/M L in two-fold ratio at 100. mu. L/well and reacted at 37 ℃ for 2 h.
2) Washing: the plate was decanted, spun-dried and washed 3 times with washing solution for 3min each time.
3) Sealing, namely after patting to dry, adding 200 mu L/hole sealing solution, reacting for 2h at 37 ℃, washing and drying for later use.
4) Adding the antiserum, diluting the antiserum by a multiple ratio from 1:1000, adding the antiserum into coated wells with various dilutions, reacting for 1h at 37 ℃ at 100 mu L/well, fully washing, adding HRP-goat anti-rabbit IgG diluted by 1:3000 at 100 mu L/well, and reacting for 1h at 37 ℃.
5) And (3) color development, namely taking the enzyme label plate out, fully washing, adding 100 mu L of TMB color development liquid into each hole, and carrying out light-shielding reaction at 37 ℃ for 15 min.
6) Stop and assay the reaction was stopped by adding 100. mu. L stop solution per well and thenOD of each well was measured by microplate reader450The value is obtained.
7) And (4) interpretation of results: by OD450The highest dilution factor of the serum with the value more than or equal to 2.1 times of that of the negative control hole (namely P/N more than or equal to 2.1) is the E L ISA titer of the serum.
Second, minimum detection limit, half inhibition and detection of specificity
The specific operation steps are as follows:
1) the indirect E L ISA method is used to determine whether RBV ① -BSA is used as a coating antigen, serum obtained from RBV ① -K L H immunized rabbit is used as an antibody, and OD is used450The corresponding antigen and antibody concentrations are the optimal working concentrations at values around 1.5.
2) Coating, diluting the coating source with coating buffer solution 20000 times, 100 mu L per well, and reacting at 37 ℃ for 2 h.
3) Washing and blocking the process was carried out as described above for the indirect E L ISA process.
4) Preparing a ribavirin standard solution, namely preparing a ribavirin standard solution into a mother solution of 5mg/m L by using a PBS solution with the concentration of 0.01 mol/L and the pH value of 7.4, and then diluting the mother solution into required concentrations by using a PBS solution with the concentration of 0.01 mol/L and the pH value of 7.4 in a multiple ratio (the RBV concentration is respectively 0.005ng/m L, 0.05ng/m L, 0.25ng/m L, 0.5ng/m L, 5ng/m L and 50ng/m L) before loading.
5) Adding samples, namely adding 50 mu L times diluted ribavirin concentration standard substance into each well, then adding 50 mu L/well antiserum with the optimal dilution factor, reacting for 1h at 37 ℃, fully washing, adding 1:3000 diluted HRP-goat anti-rabbit IgG, 100 mu L/well, and reacting for 1h at 37 ℃.
6) And (3) color reaction, namely taking out the enzyme label plate, fully washing, adding 100 mu L of TMB color developing solution into each hole, and carrying out light-shielding reaction at 37 ℃ for 15 min.
7) Stop and assay 100. mu. L stop buffer was added to each well to stop the reaction, and the OD of each well was measured using a microplate reader450The value is obtained.
8) Data processing: taking the logarithm of each concentration of ribavirin as the abscissa, taking the OD value corresponding to each concentration of ribavirin as the ordinate, using Origin 8.5 software, and fitting the four-parameter logarithm to draw a standard curve, as shown in FIG. 11, by calculating IC50Value (median)Manufactured concentration) to determine whether the antiserum is specific for ribavirin.
The result shows that after the quadruplicate immunization, the rabbit antiserum titer can reach 50000, and the detection limit is 0.02ng/m L50The value was 0.31ng/m L, and the linear detection range was 0.06-1.67ng/m L.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (11)
1. The ribavirin hapten is characterized by having a structure shown as a formula (I):
2. the method for producing the hapten according to claim 1, comprising the steps of:
1) dissolving 48.0g of ribavirin, 6.17g of p-toluenesulfonic acid and 39.2m of L2, 2-dimethoxypropane in 500m of L acetone, reacting the mixed solution at 60 ℃ for 3h, adding 1m of L5% of sodium bicarbonate to terminate the reaction, removing the precipitate, collecting and concentrating the filtrate, and purifying by column chromatography to obtain the compound shown in the formula (III);
2) dissolving 35.0g of p-fluorobenzoic acid and 15.0g of dimethylaminopyridine in 500m L t-butanol, cooling to 0 ℃, adding 107.50g of di-tert-butyl dicarbonate to the mixed solution, stirring at room temperature for 16h to obtain a colorless oily product, dissolving the colorless oily product, 15.0g of the compound shown in the formula (III) and 60.0g of cesium carbonate in 300m L dimethyl sulfoxide, continuously heating at 110 ℃ for 16h, removing the dimethyl sulfoxide through reduced pressure distillation, adding 200m L water to the residue, extracting with dichloromethane, drying with sodium sulfate, concentrating, purifying the residue through column chromatography, dissolving the obtained 6.0g of white solid product in 100m L of the mixed solution of hydrochloric acid and tetrahydrofuran at room temperature, stirring for 16h, adjusting the pH to 7-9 with sodium bicarbonate, filtering the mixture to obtain a crude product, and further crystallizing the crude product with methanol to obtain the product;
wherein, the mobile phase used for carrying out column chromatography in the step 2) is formed by mixing methanol and dichloromethane according to the volume ratio of 50:1, the used stationary phase is 200-mesh 300-mesh silica gel, and the mixed liquid of the hydrochloric acid and the tetrahydrofuran is formed by mixing the tetrahydrofuran and hydrochloric acid with the concentration of 12 mol/L according to the volume ratio of 9: 1.
3. A ribavirin artificial antigen obtained by coupling the ribavirin hapten as described in claim 1 with a 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 of claim 3, wherein the carrier protein is bovine serum albumin or keyhole limpet hemocyanin.
5. The method for producing 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 an activated ester method.
6. The method of claim 5, wherein the compound of formula (I) is coupled to the carrier protein at a molar ratio of 9.6: 1.
7. Use of any one of the haptens of claim 1 or artificial antigens of claims 3 or 4:
① application in preparing anti-ribavirin specific antibody;
② for use in detecting anti-ribavirin specific antibodies.
8. A specific antibody prepared from the artificial antigen of claim 3 or 4, wherein 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. Use of a specific antibody according to claim 8 or 9, for any one of the following applications:
(1) the application of the ribavirin detecting reagent in the detection of ribavirin;
(2) the application in the preparation of an immunochromatographic test strip of ribavirin;
(3) the application in preparing the colloidal gold test strip of ribavirin.
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