Preparation method and application of S-adenosyl homocysteine artificial complete antigen

Abstract

The invention provides a preparation method of an S-adenosylhomocysteine artificial complete antigen, which comprises the following steps: mixing S-adenosylhomocysteine and an amino protective agent, and carrying out condensation reaction to obtain a first intermediate; mixing the first intermediate and a resin carrier, and carrying out esterification reaction to obtain a second intermediate; removing an amino protecting group on the second intermediate to obtain a third intermediate; mixing the first intermediate and the third intermediate, and carrying out polymerization reaction to obtain a fourth intermediate; removing an amino protecting group on the fourth intermediate to obtain a fifth intermediate; stripping the resin carrier on the fifth intermediate to obtain a sixth intermediate; and mixing the sixth intermediate with a carrier protein, and coupling the sixth intermediate with the carrier protein to obtain the immunogenicity enhanced S-adenosine homocysteine artificial complete antigen. The immunogenicity enhanced S-adenosine homocysteine artificial complete antigen prepared by the invention has the advantages of strong immunogenicity and good specificity.

Description

Technical Field

[0001] The present invention relates to the technical field of artificial complete antigen preparation, and in particular to a preparation method and application of S-adenosylhomocysteine ​​artificial complete antigen. Background Art

[0002] S-adenosylhomocysteine ​​is a hapten and has no immunogenicity by itself. It needs to be coupled with a suitable carrier protein to prepare an S-adenosylhomocysteine ​​artificial complete antigen. However, the S-adenosylhomocysteine ​​prepared by the existing method for preparing the S-adenosylhomocysteine ​​artificial complete antigen has the disadvantage of unstable antigenicity, which results in the prepared S-adenosylhomocysteine ​​artificial complete antigen having the disadvantages of weak immunogenicity and poor specificity, limiting its wide application and downstream products, such as the preparation of monoclonal antibodies. Summary of the invention

[0003] In view of this, the present invention provides a method for preparing an immunogenicity-enhanced S-adenosylhomocysteine ​​artificial complete antigen to solve the shortcomings of weak immunogenicity and poor specificity of the S-adenosylhomocysteine ​​artificial complete antigen prepared by the existing method for preparing the S-adenosylhomocysteine ​​artificial complete antigen.

[0004] The above technical objectives of the present invention are achieved through the following technical solutions:

[0005] A method for preparing an artificial complete S-adenosylhomocysteine ​​antigen comprises the following steps:

[0006] S1, providing S-adenosylhomocysteine, an amino protecting agent, a resin carrier, and a carrier protein;

[0007] S2, mixing the S-adenosylhomocysteine ​​and the amino protecting agent, and the S-adenosylhomocysteine ​​and the amino protecting agent undergo a condensation reaction to obtain a first intermediate;

[0008] S3, mixing the first intermediate and a resin carrier, and causing an esterification reaction between the first intermediate and the resin carrier to obtain a second intermediate;

[0009] S4, removing the amino protecting group on the second intermediate to obtain a third intermediate;

[0010] S5, mixing the first intermediate and the third intermediate, and subjecting the first intermediate and the third intermediate to a polymerization reaction to obtain a fourth intermediate;

[0011] S6, removing the amino protecting group on the fourth intermediate to obtain a fifth intermediate;

[0012] S7, stripping the resin carrier on the fifth intermediate to obtain a sixth intermediate;

[0013] S8, mixing the sixth intermediate and a carrier protein, coupling the sixth intermediate and the carrier protein to obtain the S-adenosylhomocysteine ​​artificial complete antigen.

[0014] Further preferably, in step S6, the first intermediate and the fourth intermediate from which the amino protecting group is removed are mixed, and the first intermediate and the fourth intermediate from which the amino protecting group is removed undergo a polymerization reaction to remove the amino protecting group, thereby obtaining a fifth intermediate.

[0015] Further preferably, in step S6, the first intermediate and the fourth intermediate from which the amino protecting group is removed are mixed, the first intermediate and the fourth intermediate from which the amino protecting group is removed undergo a polymerization reaction, the amino protecting group is removed, and the polymerization reaction and the steps of removing the amino protecting group on the product are repeated several times to obtain a fifth intermediate.

[0016] Further preferably, in step S8, a coupling agent is added to the mixture of the sixth intermediate and the carrier protein.

[0017] More preferably, the coupling agent is at least one of isobutyl chloroformate, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide / N-hydroxysuccinimide, hexafluorophosphate or tetramethyluronium tetrafluoroborate.

[0018] More preferably, in step S1, the amino protecting agent is at least one of 9-fluorenylmethoxycarbonyl chloride and 9-fluorenylmethyl succinimidyl carbonate.

[0019] More preferably, the amino protecting groups on the second intermediate and the fourth intermediate are removed by using a deamino protecting agent.

[0020] More preferably, a stripping agent is used to strip the resin carrier from the fifth intermediate.

[0021] The present invention also provides an S-adenosylhomocysteine ​​artificial complete antigen, which is prepared by the above-mentioned method for preparing the S-adenosylhomocysteine ​​artificial complete antigen.

[0022] The present invention also provides an application of an S-adenosylhomocysteine ​​artificial complete antigen, and the application of the S-adenosylhomocysteine ​​artificial complete antigen in antibody preparation.

[0023] In summary, the present invention has the following beneficial effects: In the method for preparing the artificial complete S-adenosylhomocysteine ​​antigen provided by the present invention, the S-adenosylhomocysteine ​​and the amino protecting agent undergo a nucleophilic reaction to protect the α-amino antigen epitope and the primary amino group at the purine end in the S-adenosylhomocysteine ​​to prevent the amino group from being destroyed in the subsequent steps. The first intermediate and the resin carrier undergo an esterification reaction to obtain a second intermediate. The amino protecting group on the second intermediate is removed to obtain a third intermediate so that the third intermediate can polymerize with the first intermediate through the amino group. The first intermediate and the third intermediate are mixed, and the first intermediate and the third intermediate undergo a polymerization reaction to obtain a fourth intermediate, thereby increasing the molecular weight of the subsequently prepared S-adenosylhomocysteine ​​artificial complete antigen and increasing the number of key groups of the subsequently prepared S-adenosylhomocysteine ​​artificial complete antigen. The amino protecting group on the fourth intermediate is removed to obtain a fifth intermediate to expose the amino group on the fifth intermediate for the subsequent coupling reaction. The resin carrier on the fifth intermediate is stripped to obtain the sixth intermediate. The sixth intermediate and the carrier protein are mixed, and the sixth intermediate and the carrier protein are coupled to obtain the S-adenosylhomocysteine ​​artificial complete antigen. Since the amino group on the S-adenosylhomocysteine ​​is not damaged by the amino protective agent, and the molecular weight of the S-adenosylhomocysteine ​​increases after polymerization, the key antigen epitope is repeated, so that the S-adenosylhomocysteine ​​artificial complete antigen has strong specificity and immunogenicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a reaction principle diagram of the method for preparing the immunogenicity-enhanced S-adenosylhomocysteine ​​artificial complete antigen provided in an embodiment of the present invention.

[0025] Figure 2 1 is a graph showing the relationship between the crystallization yield and pH of the first intermediate provided in an embodiment of the present invention.

[0026] Figure 3 This is the Fourier infrared identification spectrum of the immunogenicity-enhanced S-adenosylhomocysteine ​​provided in the embodiment of the present invention.

[0027] Figure 4 It is the Fourier infrared identification spectrum of the first intermediate provided in the embodiment of the present invention.

[0028] Figure 5 It is the reverse phase high performance liquid chromatography identification spectrum of the first intermediate, the second intermediate and the fifth intermediate provided in the embodiment of the present invention.

[0029] Figure 6 This is a laser confocal Raman identification spectrum of the first intermediate provided in an embodiment of the present invention.

[0030] Figure 7 This is a laser confocal Raman identification spectrum of the fifth intermediate provided in an embodiment of the present invention.

[0031] Figure 8 This is a detection diagram of the antiserum induced by the immunogenicity-enhanced S-adenosylhomocysteine ​​artificial antigen of Comparative Example 1 in ELISA competition with S-adenosylhomocysteine ​​of different concentrations provided by an embodiment of the present invention.

[0032] Fig. 9 This is a detection diagram of the antiserum induced by the immunogenicity-enhanced S-adenosylhomocysteine ​​artificial complete antigen of Example 1 in ELISA competition with S-adenosylhomocysteine ​​of different concentrations provided by the embodiments of the present invention.

[0033] Fig.10 This is a surface plasmon resonance identification diagram of the affinity of the antiserum induced by the immunogenicity-enhanced S-adenosylhomocysteine ​​artificial complete antigen of Example 1 to S-adenosylhomocysteine ​​provided in an embodiment of the present invention.

[0034] Fig.11 This is a surface plasmon resonance identification diagram of the affinity of the antiserum induced by the S-adenosylhomocysteine ​​artificial antigen of Comparative Example 1 and S-adenosylhomocysteine ​​provided in an embodiment of the present invention.

[0035] Fig.12 It is a comparison chart of the detection results after the antiserum induced by the S-adenosylhomocysteine ​​artificial complete antigen of Example 1 and the antiserum induced by the S-adenosylhomocysteine ​​artificial antigen of Comparative Example 1 were cross-reacted with the S-adenosylmethionine standard substance. DETAILED DESCRIPTION

[0036] In order to make those skilled in the art better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with embodiments. It will be understood by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0037] See also Figure 1 The present invention provides a method for preparing an artificial complete S-adenosylhomocysteine ​​antigen, comprising the following steps:

[0038] Step S1: providing S-adenosylhomocysteine ​​(SAH), an amino protecting agent (Fmoc), a resin carrier, and a carrier protein;

[0039] Step S2: mixing the S-adenosylhomocysteine ​​and the amino protecting agent, and the S-adenosylhomocysteine ​​and the amino protecting agent undergo a nucleophilic reaction to obtain a first intermediate (abbreviated as Fmoc-SAH);

[0040] Step S3: mixing the first intermediate and the resin carrier, and the first intermediate and the resin carrier undergo an esterification reaction to obtain a second intermediate;

[0041] Step S4: removing the amino protecting group on the second intermediate to obtain a third intermediate;

[0042] Step S5: mixing the first intermediate and the third intermediate, and polymerizing the first intermediate and the third intermediate to obtain a fourth intermediate;

[0043] Step S6: removing the amino protecting group on the fourth intermediate to obtain a fifth intermediate;

[0044] Step S7: peeling off the resin carrier on the fifth intermediate to obtain a sixth intermediate; and

[0045] Step S8: mixing the sixth intermediate and the carrier protein, coupling the sixth intermediate and the carrier protein to obtain the S-adenosylhomocysteine ​​artificial complete antigen.

[0046] It can be understood that the sixth intermediate is an S-adenosylhomocysteine ​​polymer.

[0047] In one embodiment, the amino protecting agent is at least one of 9-fluorenylmethoxycarbonyl chloride and 9-fluorenylmethyl succinimidyl carbonate. The amino protecting agent is used to protect the key groups of S-adenosylhomocysteine, such as α-amino antigen epitope, primary amino group at the purine end, and carboxyl group. Thus, unnecessary cross-linking of S-adenosylhomocysteine ​​can be avoided, achieving the purpose of controlling the cross-linking degree and the number of key groups of the sixth intermediate.

[0048] In one embodiment, the resin carrier is at least one of Wang resin, Sasrin resin, PMA resin, and HMPB resin.

[0049] In one embodiment, the resin carrier can be added to the swelling agent for 20 to 40 minutes before being mixed with the first intermediate. The swelling agent can be dichloromethane.

[0050] In one embodiment, the carrier protein comprises at least one of bovine serum albumin, keyhole limpet hemocyanin, ovalbumin and human serum albumin. The surface of the carrier protein has amino groups.

[0051] In one embodiment, the amino protecting groups on the second intermediate and the fourth intermediate are removed using a dimethylformamide solution or a diethylamine solution containing 20 wt % piperidine to expose the amino groups.

[0052] In one embodiment, a stripping agent is used to strip the resin carrier from the fifth intermediate. The stripping agent contains trifluoroacetic acid, water, and triisopropyl borate to strip the resin carrier from the fifth intermediate. Wherein, the mass ratio of trifluoroacetic acid, water, and triisopropyl borate is 80-100:1-5:1-5, for example, 80:1:1, 90:1:1, 100:1:1, 80:1:2, 90:1:3, 100:1:4, 80:2:1, 90:2:1, 100:2:1, 80:3:1, 90:4:1, 100:5:1, 80:5:1, 90:5:1, or 100:5:1.

[0053] In another embodiment, the stripping agent contains 0.1% trifluoroacetic acid in 50% acetonitrile aqueous solution.

[0054] In one embodiment, the resin carrier and the fifth intermediate can be separated by acid solution to obtain the sixth intermediate, i.e., S-adenosylhomocysteine ​​enhanced polymer. Specifically, the acid solution can be an acetonitrile aqueous solution containing trifluoroacetic acid, wherein the volume percentage of acetonitrile is 50% and the volume percentage of trifluoroacetic acid is 0.1%.

[0055] In one embodiment, the obtained fourth intermediate can be washed with dichloromethane several times.

[0056] In one embodiment, the S-adenosylhomocysteine ​​can be dissolved in a solution and then mixed with an amino protective agent. Specifically, the S-adenosylhomocysteine ​​can be added to an alkaline solution, and then an amphiphilic solubilizing agent (such as tetrahydrofuran and dioxane) is added dropwise to the alkaline solution in an ice bath environment, and magnetic stirring is performed at 0° C. for 15 minutes to dissolve the S-adenosylhomocysteine.

[0057] In one embodiment, the amount of the amino protecting agent is 1.5 to 4 molar equivalents of S-adenosylhomocysteine, for example, 1.5 molar equivalents, 2 molar equivalents, 2.5 molar equivalents, 3 molar equivalents, 3.5 molar equivalents, or 4 molar equivalents.

[0058] In one embodiment, the mass ratio of the first intermediate to the resin carrier is 8-10:1-3, for example, 8:1, 8:2, 8:3, 9:1, 9:2, 9:3, 10:1, 11:2, or 12:3.

[0059] In one embodiment, the mass ratio of the sixth intermediate to the carrier protein is 1-3:2-4, for example, 1:2, 1:3, 1:4, 2:2, 2:3, 2:4, 3:2, 3:3, or 3:4.

[0060] In one embodiment, the nucleophilic reaction is carried out at a temperature of -2 to 2°C for 20 to 40 minutes, and then the temperature is raised to 20 to 30°C for 10 to 14 hours in a light-proof environment.

[0061] In one embodiment, the first intermediate can be stored at 4° C. until use.

[0062] In the preparation method of the immunogenicity-enhanced S-adenosylhomocysteine ​​artificial complete antigen provided by the present invention, the S-adenosylhomocysteine ​​and the amino protecting agent undergo a nucleophilic reaction to protect the α-amino antigen epitope and the primary amino group at the purine end in the S-adenosylhomocysteine ​​to prevent the amino group from being destroyed in the subsequent steps. The first intermediate and the resin carrier undergo an esterification reaction to obtain a second intermediate to support the subsequent intermediate. The amino protecting group on the second intermediate is removed to obtain a third intermediate so that the third intermediate can polymerize with the first intermediate through the amino group. The first intermediate and the third intermediate are mixed, and the first intermediate and the third intermediate undergo a polymerization reaction to obtain a fourth intermediate, thereby increasing the molecular weight of the S-adenosylhomocysteine ​​artificial complete antigen obtained subsequently and increasing the number of key groups of the S-adenosylhomocysteine ​​artificial complete antigen obtained subsequently. The amino protecting group on the fourth intermediate is removed to obtain a fifth intermediate to expose the amino group on the fifth intermediate for the subsequent coupling reaction. The resin carrier on the fifth intermediate is stripped to obtain the sixth intermediate. The sixth intermediate and the carrier protein are mixed, and the sixth intermediate and the carrier protein are coupled to obtain the S-adenosylhomocysteine ​​artificial complete antigen. Since the amino groups on the S-adenosylhomocysteine ​​are protected by the amino protecting agent and are not damaged, and the number of amino groups on the S-adenosylhomocysteine ​​artificial complete antigen is large, the S-adenosylhomocysteine ​​artificial complete antigen has strong specificity and immunogenicity, which lays a solid foundation for the preparation and development of biological methylation rapid detection reagents, etc.

[0063] After removing the amino protecting group on the fourth intermediate, the method for preparing the S-adenosylhomocysteine ​​artificial complete antigen further comprises:

[0064] The first intermediate and the fourth intermediate from which the amino protecting group is removed are mixed, and the first intermediate and the fourth intermediate from which the amino protecting group is removed undergo a polymerization reaction to remove the amino protecting group, thereby obtaining a fifth intermediate.

[0065] In one embodiment, the polymerization reaction and the steps of removing the amino protecting group on the product are repeated several times to obtain a fifth intermediate.

[0066] In one embodiment, during the process of removing the amino protecting group, the substitution rate of S-adenosylhomocysteine ​​loaded on the carrier resin can be calculated to determine the number of times the polymerization reaction and the steps of removing the amino protecting group on the product are repeated. Specifically, 5 mg of the second intermediate / fourth intermediate is added to 2 ml of N, N-dimethylformamide solution, reacted for 30 minutes, and the supernatant is taken and the absorbance is measured at 301 nm in a UV-visible spectrophotometer with N, N-dimethylformamide background zeroed, and then calculated by the following formula: Where abs is the absorbance and m is the mass of the carrier resin.

[0067] In the technical scheme of the present invention, the first intermediate and the fourth intermediate from which the amino protecting group is removed are mixed, and a polymerization reaction occurs between the first intermediate and the fourth intermediate from which the amino protecting group is removed, and the amino protecting group is removed to obtain a fifth intermediate, which can increase the key groups (such as amino group, carboxyl group, etc.) of the S-adenosylhomocysteine ​​artificial complete antigen to enhance the specificity and immunogenicity of the S-adenosylhomocysteine ​​artificial complete antigen.

[0068] The step S2 comprises the following steps:

[0069] Step S21: In an ice bath environment, adding an amphiphilic solubilizing agent to the mixture of S-adenosylhomocysteine ​​and the amino protecting agent.

[0070] In one embodiment, the amphiphilic solubilizing agent may be tetrahydrofuran and dioxane in a volume ratio of 1:1.

[0071] In one embodiment, a base (such as 10% by mass sodium carbonate or 10% by mass potassium carbonate) can be added to the mixture of S-adenosylhomocysteine ​​and the amino-protecting agent to provide an alkaline environment for the nucleophilic reaction to promote the condensation of S-adenosylhomocysteine ​​and the amino-protecting agent.

[0072] In one embodiment, a mixture of S-adenosylhomocysteine, a first condensing agent, a catalyst, and an amphiphilic solubilizing agent can be stirred at 0° C. for 10 to 20 minutes, and after S-adenosylhomocysteine ​​is completely dissolved, the amino protecting agent is added to the mixture, and the mixture is reacted at 0° C. for 20 to 40 minutes, and then heated to 20 to 40° C. and reacted in a light-proof environment for 10 to 15 hours.

[0073] In one embodiment, after the S-adenosylhomocysteine ​​and the amino protecting agent react completely, ether extraction can be used at least once, and after the aqueous phase is separated, the pH is adjusted to 2.5-3, and after standing for 2-4 minutes, the precipitated crystals are filtered and recovered to obtain the first intermediate.

[0074] See also Figure 2 The most crystals precipitated in the pH range of 2.5 to 3.

[0075] See also Figure 3 , the S-adenosylhomocysteine ​​at 3324 cm -1 (νNH2 s), 3132cm -1 There is a strong amino stretching vibration characteristic peak at 1602cm -1 There is a strong amino bending vibration characteristic peak at (δNH2 s), 1675cm -1 (νC-N s), 1205cm -1 (νasC-OC vs), 1015cm -1 (νC-O s), 613cm -1 (νC-S s) are characteristic group stretching vibration peaks on the molecular skeleton of S-adenosylhomocysteine.

[0076] See also Figure 4 , the characteristic peaks of the amino stretching vibration and bending vibration of the first intermediate disappeared, 1142cm -1 (νasC-OC vs), 684cm -1 (νC-S s), and 598cm -1 The characteristic peak of S-adenosylhomocysteine ​​skeleton at (δN-C=O s) still exists. In addition, at 1748 cm -1 The characteristic peak of α-ketoester produced by the introduction of amino protecting group appeared at (νC=O vs).

[0077] In the technical solution of the present invention, an amphiphilic solubilizer can be added to the mixture of S-adenosylhomocysteine ​​and the amino protective agent in an ice bath environment to increase the solubility of the S-adenosylhomocysteine.

[0078] The step S3 comprises the following steps:

[0079] Step S31: dispersing 15-25 mg of the resin carrier in a solvent such as dichloromethane and swelling for 20-40 minutes;

[0080] Step S32: Add 50 to 55 μmol of the first intermediate solution to a solvent containing a resin carrier, then add 4 to 6 times the molar equivalent of the first condensing agent, stir at a temperature of -2 to 2°C for 10 to 20 minutes, then add 1 to 2 times the molar equivalent of the catalyst, react for 50 to 70 minutes, then add 4 to 6 times the molar equivalent of the acetylation reagent and continue to react for 20 to 40 minutes. After the reaction is completed, filter out the particles, rinse three times with N, N dimethylformamide solution, and rinse three times with dichloromethane to obtain a second intermediate.

[0081] In one embodiment, the first condensing agent is at least one of N,N-diisopropylcarbodiimide, N,N-dicyclohexylcarbodiimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

[0082] In one embodiment, the catalyst is at least one of dimethylaminopyridine, 4-(dimethylamino)pyridine-N-oxide, and polypyrrole.

[0083] In one embodiment, the acetylating agent is at least one of acetic anhydride and acetyl chloride.

[0084] The method for preparing the S-adenosylhomocysteine ​​artificial complete antigen also includes:

[0085] A coupling agent is added to the mixture of the sixth intermediate and the carrier protein.

[0086] In one embodiment, the coupling agent is at least one of isobutyl chloroformate, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide / N-hydroxysuccinimide, hexafluorophosphate or tetramethyluronium tetrafluoroborate.

[0087] In one embodiment, 5 to 15 mg of the sixth intermediate can be dissolved in 2 to 4 mL of N,N-dimethylformamide, cooled to -2 to 2°C, equilibrated for 10 to 20 min, and then 10 to 20 μL of isobutyl chloroformate and 15 to 25 μL of triethylamine are added. After reacting in the dark for 50 to 70 min, the mixture is added dropwise to a borate buffer solution containing 20 to 25 mg of a carrier protein, and the mixture is heated to room temperature and reacted for 14 to 18 h, and then concentrated, centrifuged and dialyzed to obtain an S-adenosylhomocysteine ​​artificial complete antigen. The volume of the borate buffer solution is 2 to 6 mL, and the concentration is 0.1 to 0.2 M.

[0088] In the technical solution of the present invention, a coupling agent is added to the mixture of the sixth intermediate and the carrier protein to promote the coupling reaction between the sixth intermediate and the carrier protein.

[0089] See also Figure 5, using 50% acetonitrile aqueous solution as mobile phase, the first intermediate, the second intermediate, and the fifth intermediate were analyzed by reverse phase high performance liquid chromatography. The analysis results showed that the retention time of the fifth intermediate was 5.40 min, the retention time of the first intermediate was 4.83 min, and the second intermediate had the characteristic retention time corresponding to the first intermediate and the fifth intermediate.

[0090] See also Figure 6 , the first intermediate at 684cm -1 and 705cm -1 The peak at is the characteristic peak of carbon-sulfur bond.

[0091] See also Figure 7 The fifth intermediate is at 684 cm -1 and 705cm -1 The characteristic peak of carbon-sulfur bond at 1290cm -1 The characteristic peak of secondary amine after polymerization appeared at , indicating that the fifth intermediate is S-adenosylhomocysteine ​​polymer.

[0092] The step S5 comprises:

[0093] A second condensing agent and an acid binding agent are added to the mixture of the first intermediate and the third intermediate.

[0094] In one embodiment, the second condensing agent is at least one of hexafluorophosphate, benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate, and tripyrrolidinyl phosphorus bromide hexafluorophosphate.

[0095] In one embodiment, the acid binding agent is at least one of diisopropylethylamine, triethylamine, a mixture of triethylamine and magnesium chloride, and a mixture of triethylamine and lithium bromide.

[0096] During the polymerization reaction, Kaiser reagent can be used to detect the content of free amino acids to monitor whether the polymerization reaction is thorough. If the polymerization reaction is not thorough, the polymerization reaction needs to be repeated. In addition, the molecular weight of the sixth intermediate can be purposefully increased by controlling the number of cycles of the polymerization reaction.

[0097] In the technical solution of the present invention, a second condensing agent and an acid binding agent are added to a mixture of the first intermediate and the third intermediate to achieve directional polymerization of the third intermediate and the first intermediate.

[0098] The embodiment of the present invention also provides an immunogenicity-enhanced S-adenosylhomocysteine ​​artificial complete antigen, which is prepared by the above-mentioned immunogenicity-enhanced S-adenosylhomocysteine ​​artificial complete antigen preparation method.

[0099] Since the immunogenicity-enhanced S-adenosylhomocysteine ​​artificial complete antigen is prepared by the above-mentioned method for preparing the immunogenicity-enhanced S-adenosylhomocysteine ​​artificial complete antigen, it has at least all the beneficial effects brought about by the technical solutions of the above-mentioned embodiments, which will not be described one by one here.

[0100] The embodiment of the present invention also provides a use of the immunogenicity-enhanced S-adenosylhomocysteine ​​artificial complete antigen in antibody preparation.

[0101] The present invention is described in detail below through specific embodiments.

[0102] Embodiment 1

[0103] Dissolve 1 mg of sodium carbonate in 10 mL of deionized water. After the sodium carbonate is completely dissolved, add 0.26 mmol of S-adenosyl homocysteine. Slowly drop 2.5 mL of tetrahydrofuran and 2.5 mL of dioxane in an ice bath. After the dropwise addition is complete, stir magnetically at 0°C for 15 min. After the S-adenosyl homocysteine ​​is completely dissolved, add 2.5 times the molar equivalent of 9-fluorenylmethoxycarbonyl chloride. Continue to react at 0°C for 30 minutes, then heat to 25°C and react for 12 hours in a light-proof environment. After the reaction is completed, add 10 mL of ether to extract three times, separate the aqueous phase, adjust the pH to 3, let stand for 3 minutes, filter the precipitated crystals, and freeze-dry and recover the crystals in a freeze dryer to obtain the first intermediate.

[0104] 20 mg of Wang resin was added to 10 mL of dichloromethane and soaked for 30 minutes, then filtered, dried and recovered for later use;

[0105] 54.5 μmol of the first intermediate was dissolved in dichloromethane dispersed with Wang resin, 5 times the molar equivalent of N,N-diisopropylcarbodiimide was added, and the mixture was stirred at 0°C for 15 minutes, and then 1 times the molar equivalent of dimethylaminopyridine was added to react for 1 hour, and then 5 times the molar equivalent of acetic anhydride was added to continue the reaction for 30 minutes. After the reaction, the particles were filtered out, and washed three times with N,N-dimethylformamide solution and three times with dichloromethane to obtain the second intermediate;

[0106] The second intermediate was added to 5 mL of N,N-dimethylformamide solution containing 20% ​​piperidine and reacted for 30 minutes to obtain a third intermediate;

[0107] Add the first intermediate, 4 times the molar equivalent of diisopropylethylenediamine, and 3 times the molar equivalent of hexafluorophosphate to the third intermediate, and react for 30 minutes. After the reaction, rinse with N,N-dimethylformamide three times and dichloromethane three times in sequence to obtain a fourth intermediate.

[0108] The fourth intermediate is added to 5 mL of N,N-dimethylformamide solution containing 20% ​​piperidine and reacted for 30 minutes to obtain a fifth intermediate;

[0109] The fifth intermediate is added with 5 mL of 50% acetonitrile aqueous solution containing 0.1% trifluoroacetic acid, and the carrier resin is removed to obtain a sixth intermediate;

[0110] 10 mg of the sixth intermediate was mixed and dissolved in 3 mL of N,N-dimethylformamide, cooled to 0°C, and equilibrated for 15 min. Then, 16 μL of isobutyl chloroformate and 20 μL of triethylamine were added. After reacting in the dark for 1 hour, the mixture was added dropwise to 4 mL of 0.1 M boric acid buffer containing 25 mg of ovalbumin. The mixture was heated to room temperature and the reaction was continued for 16 hours. The product was concentrated, centrifuged and dialyzed to obtain the immunogenicity-enhanced S-adenosylhomocysteine ​​artificial complete antigen of Example 1, which was stored at 4°C.

[0111] Comparative Example 1

[0112] The difference between Comparative Example 1 and Example 1 is that the first intermediate, 4 times the molar equivalent of diisopropylethylenediamine, and 3 times the molar equivalent of hexafluorophosphate are not added to the third intermediate and reacted for 30 minutes. Instead, the third intermediate is directly treated to remove the amino protecting group, remove the carrier resin, and couple with ovalbumin to obtain the immunogenicity enhanced S-adenosylhomocysteine ​​artificial antigen of Comparative Example 1.

[0113] The other steps are the same as those in the first embodiment and will not be described in detail here.

[0114] The immunogenicity-enhanced S-adenosylhomocysteine ​​artificial complete antigen of Example 1 was diluted with 0.9% saline until the concentration of the immunogenicity-enhanced S-adenosylhomocysteine ​​artificial complete antigen of Example 1 reached 1 mg / mL to obtain the test solution of Example 1. 50 μL of the test solution of Example 1 was mixed with 50 μL of adjuvant and injected into the thigh muscle of 8-week-old BALB / C mice. The second immunization was performed after two weeks. After the third week, the tail blood was collected for the first small molecule enzyme-linked immunosorbent assay (ELISA) competition experiment.

[0115] The S-adenosylhomocysteine ​​artificial antigen of Comparative Example 1 was diluted with 0.9% saline until the concentration of the S-adenosylhomocysteine ​​artificial complete antigen of Comparative Example 1 reached 1 mg / mL to obtain the test solution of Comparative Example 1. 50 μL of the test solution of Comparative Example 1 was mixed with 50 μL of adjuvant and injected into the thigh muscle of 8-week-old BALB / C mice. A second immunization was performed after two weeks. After the third week, the tail blood was collected for a second small molecule enzyme-linked immunosorbent assay (ELISA) competition experiment.

[0116] The first small molecule enzyme-linked immunosorbent assay (ELISA) competition experiment comprises the following steps:

[0117] A 0.05 M carbonate buffer solution with a pH of 9.6 was added to the immunogenicity-enhanced S-adenosylhomocysteine ​​artificial complete antigen of Example 1 to obtain a solution with a concentration of 1 μg / mL. The solution was used to coat the ELISA The 96-well plate was incubated at 4°C overnight, after which the coating solution was discarded, and the plate was washed three times with 0.02M phosphate Tween buffer containing 0.05% Tween20, and then patted dry on absorbent paper. A phosphate Tween buffer containing 1% bovine serum albumin was added, and the plate was blocked at 37°C for 2 hours. S-adenosylhomocysteine ​​was prepared into a series of concentrations (10 mg / mL, 1 mg / mL, 100 μg / mL, 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL) and mixed with the rat tail blood of Example 1 diluted 1000 times at a ratio of 1:1, and then added to the 96-well plate according to the concentration gradient, and incubated at 37°C for one hour, and a blank control well was set. After that, the plate was washed three times with PBST, and then a 1:4000 diluted enzyme-labeled secondary antibody was added, and incubated at 37°C for 1 hour. Then, the plate was washed three times with phosphate-tween, and a color developing solution containing o-phenylenediamine (OPD) was added. After color development for 15 minutes, the reaction was terminated with sulfuric acid stop solution, and the OD value was immediately detected with a microplate reader.

[0118] The second small molecule enzyme-linked immunosorbent assay (ELISA) competition experiment is similar to the first small molecule enzyme-linked immunosorbent assay (ELISA) competition experiment, except that the second small molecule enzyme-linked immunosorbent assay (ELISA) competition experiment uses the S-adenosylhomocysteine ​​artificial antigen of comparative example 1 and the rat tail blood of comparative example 1.

[0119] Ginseng Figure 8 and Fig. 9 The S-adenosylhomocysteine ​​molecules and the coated S-adenosylhomocysteine ​​antigens competitively bound to the antibodies in the rat tail blood of Example 1 and the antibodies in the rat tail blood of Comparative Example 1, and their OD values ​​showed an obvious negative correlation according to the concentration gradient of S-adenosylhomocysteine. Fig. 9The R of the fitted curve in 2 Value compared to Figure 8 R of the fitted curve 2 The value is larger, that is, 0.97152>0.92569. This shows that the S-adenosylhomocysteine ​​artificial complete antigen of Example 1 effectively stimulates the mouse immune B lymphocytes to produce polyclonal antibodies. Compared with the S-adenosylhomocysteine ​​artificial antigen of Comparative Example 1, the S-adenosylhomocysteine ​​artificial complete antigen of Example 1 can stimulate the mouse immune B lymphocytes to produce more polyclonal antibodies. This shows that the S-adenosylhomocysteine ​​artificial complete antigen of Example 1 has better immunogenicity and immune specificity.

[0120] The antiserum obtained by immunization with the immunogenicity-enhanced S-adenosylhomocysteine ​​artificial complete antigen of Example 1 was diluted 1000 times and then fixed to the second lane of the CM5 chip by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide / N-hydroxysuccinimide (EDC-NHS). At the same time, the antiserum obtained by immunization with the S-adenosylhomocysteine ​​artificial antigen of Comparative Example 1 was diluted 1000 times and then fixed to the fourth lane of the CM5 chip by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide / N-hydroxysuccinimide (EDC-NHS). S-adenosylhomocysteine ​​was prepared into solutions with a series of concentrations (1 μg / mL, 200 ng / mL, 40 ng / mL, 8 ng / mL, 1.6 ng / mL, 0.32 ng / mL) as the mobile phase, and the flow rate was set to 10 μL / min. The contact time and elution time were both 120 seconds. The mobile phase flowed through the second and fourth lanes, respectively, and surface plasmon resonance was performed to detect the affinity constants of antibodies obtained by immunization with different antigens for S-adenosylhomocysteine ​​small molecules.

[0121] Ginseng Fig.10 The affinity constant of the antiserum obtained by immunization with the S-adenosylhomocysteine ​​artificial antigen in comparative example 1 is KD=1.72×10 -6 . Fig.11 The affinity constant of the antiserum obtained by immunization with the S-adenosylhomocysteine ​​artificial complete antigen in Example 1 is KD=2.99×10 -8 The smaller the affinity constant, the stronger the affinity. This indicates that the immunogenicity-enhanced S-adenosylhomocysteine ​​artificial complete antigen of Example 1 has better immunogenicity and immune specificity, and can induce the production of antibodies with stronger affinity for S-adenosylhomocysteine ​​small molecules.

[0122] A carbonate buffer solution with a concentration of 0.05 M and a pH of 9.6 was added to the immunogenicity-enhanced S-adenosylhomocysteine ​​artificial complete antigen of Example 1 to obtain a solution with a concentration of 1 μg / mL. The solution was used to coat an ELISA 96-well plate and incubated overnight at 4° C. After that, the coating solution was discarded, and the plate was washed three times with a 0.02 M phosphate Tween buffer solution containing 0.05% Tween 20, and then patted dry on absorbent paper. Then, a phosphate Tween buffer solution containing 1% bovine serum albumin was added to block the plate at 37° C. for 2 hours. After S-adenosylhomocysteine ​​was prepared into a series of concentrations (10 mg / mL, 1 mg / mL, 100 μg / mL, 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL), it was mixed with the antiserum diluted 1000 times obtained by immunization with the immunogenicity enhanced S-adenosylhomocysteine ​​artificial complete antigen of Example 1 as an antigen at a ratio of 1:1, and then added to a 96-well plate according to a concentration gradient, incubated at 37°C for 1 hour, and a blank control well was set. Similarly, S-adenosylhomocysteine ​​was prepared into a series of concentrations (10 mg / mL, 1 mg / mL, 100 μg / mL, 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL), and then mixed with the antiserum diluted 1000 times obtained by immunization with the S-adenosylhomocysteine ​​artificial antigen of Comparative Example 1 as an antigen at a ratio of 1:1, and then added to a 96-well plate according to a concentration gradient, incubated at 37°C for 1 hour, and a blank control well was set. After that, the plate was washed 3 times with phosphate Tween buffer, and a 1:4000 diluted enzyme-labeled secondary antibody was added, incubated at 37°C for 1 hour, and then washed 3 times with phosphate Tween buffer, and a color developing solution containing o-phenylenediamine (OPD) was added. After color development for 15 minutes, the reaction was terminated with sulfuric acid stop solution, and the OD value was immediately detected with an enzyme marker.

[0123] Ginseng Fig.12 The polyclonal antibody obtained after immunization with the immunogenicity-enhanced S-adenosylhomocysteine ​​artificial complete antigen of Example 1 has a smaller degree of cross-reaction with the S-adenosylmethionine standard. This shows that the immunogenicity-enhanced S-adenosylhomocysteine ​​artificial complete antigen of Example 1 has better specificity.

[0124] In one embodiment, the preparation method of phosphate-buffered saline (PBS) buffer comprises:

[0125] 2.3 g Na2HPO4, 0.524 g NaH2PO4·H2O, and 8.77 g NaCl were dissolved in pure water, the volume was adjusted to 1 L with deionized water, and the pH was adjusted to 7.4 to obtain a phosphate buffer solution with a concentration of 0.02 M.

[0126] In one embodiment, a method for preparing a dialysate for dialysis comprises:

[0127] Dilute the phosphate buffer solution 2-fold.

[0128] In one embodiment, the preparation method of phosphate-saline-Tween (PBST) buffer comprises:

[0129] Tween 20 is added to the phosphate buffer having a concentration of 0.02 M until it is completely dissolved. The volume of the Tween 20 is 0.05% of the volume of the phosphate buffer.

[0130] The Tween 20 is a mixture of polyoxyethylene sorbitan monolaurate and a portion of polyoxyethylene bis-dehydrated sorbitan monolaurate.

[0131] In one embodiment, the method for preparing the blocking solution comprises:

[0132] Add bovine serum albumin to the phosphate-Tween buffer and dissolve it completely. The volume of the BSA is 1% of the volume of the phosphate-Tween buffer.

[0133] In one embodiment, the preparation method of carbonate (CBS) buffer comprises:

[0134] Dissolve 1.59 g Na2CO3 and 2.93 g NaHCO3 in deionized water, make up to 100 ml, adjust the pH to 9.6, and obtain a carbonate buffer solution with a concentration of 0.5 M.

[0135] The above embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for preparing an artificial complete S-adenosylhomocysteine ​​antigen, characterized in that: The following steps are involved: S1, providing S-adenosylhomocysteine, an amino protecting agent, a resin carrier, and a carrier protein; S2, mixing the S-adenosylhomocysteine ​​and the amino protecting agent, and the S-adenosylhomocysteine ​​and the amino protecting agent undergo a condensation reaction to obtain a first intermediate; S3, mixing the first intermediate and a resin carrier, and causing an esterification reaction between the first intermediate and the resin carrier to obtain a second intermediate; S4, removing the amino protecting group on the second intermediate to obtain a third intermediate; S5, mixing the first intermediate and the third intermediate, and subjecting the first intermediate and the third intermediate to a polymerization reaction to obtain a fourth intermediate; S6, removing the amino protecting group on the fourth intermediate to obtain a fifth intermediate; S7, stripping the resin carrier on the fifth intermediate to obtain a sixth intermediate; S8, mixing the sixth intermediate and a carrier protein, coupling the sixth intermediate and the carrier protein to obtain the S-adenosylhomocysteine ​​artificial complete antigen.

2. The method for preparing a S-adenosylhomocysteine ​​artificial complete antigen according to claim 1, characterized in that: In step S6, the first intermediate and the fourth intermediate from which the amino protecting group is removed are mixed, and the first intermediate and the fourth intermediate from which the amino protecting group is removed undergo a polymerization reaction to remove the amino protecting group, thereby obtaining a fifth intermediate.

3. The method for preparing a S-adenosylhomocysteine ​​artificial complete antigen according to claim 1, characterized in that: In step S6, the first intermediate and the fourth intermediate from which the amino protecting group is removed are mixed, the first intermediate and the fourth intermediate from which the amino protecting group is removed undergo a polymerization reaction to remove the amino protecting group, and the polymerization reaction and the steps of removing the amino protecting group on the product are repeated several times to obtain a fifth intermediate.

4. The method for preparing a S-adenosylhomocysteine ​​artificial complete antigen according to claim 1, characterized in that: In step S8, a coupling agent is added to the mixture of the sixth intermediate and the carrier protein.

5. The method for preparing a S-adenosylhomocysteine ​​artificial complete antigen according to claim 4, characterized in that: The coupling agent is at least one of isobutyl chloroformate, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide / N-hydroxysuccinimide, hexafluorophosphate or tetramethyluronium tetrafluoroborate.

6. The method for preparing a S-adenosylhomocysteine ​​artificial complete antigen according to claim 1, characterized in that: In step S1, the amino protecting agent is at least one of 9-fluorenylmethoxycarbonyl chloride and 9-fluorenylmethyl succinimidyl carbonate.

7. The method for preparing a S-adenosylhomocysteine ​​artificial complete antigen according to claim 1, characterized in that: The amino protecting groups on the second intermediate and the fourth intermediate are removed by using a deamino protecting agent.

8. The method for preparing a S-adenosylhomocysteine ​​artificial complete antigen according to claim 1, characterized in that: The resin carrier is peeled off from the fifth intermediate using a peeling agent.

9. An artificial complete S-adenosylhomocysteine ​​antigen, characterized in that: It is prepared by the method for preparing an artificial complete S-adenosylhomocysteine ​​antigen according to any one of claims 1 to 8.

10. The use of an artificial complete S-adenosylhomocysteine ​​antigen according to claim 9, characterized in that: The S-adenosylhomocysteine ​​artificial complete antigen is used in antibody preparation.

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