阅读说明：本技术 安乃近人工抗原的合成方法 (Synthetic method of analgin artificial antigen ) 是由 胥传来 李月 匡华 徐丽广 马伟 刘丽强 吴晓玲 宋珊珊 胡拥明 于 2019-11-21 设计创作，主要内容包括：安乃近人工抗原的合成方法,属于生物化工技术领域。本发明采用戊二醛法将4-氨基安替比林H1与载体蛋白偶联,得到安乃近的人工抗原；或先由4-氨基安替比林H1和丁二酸酐反应得到安乃近半抗原H1-HS,再用碳二亚胺法将安乃近半抗原H1-HS与载体蛋白偶联,即得到安乃近人工抗原。本发明成功合成了安乃近人工抗原,合成步骤简单、安全、有效,完全可用于免疫分析中,为以后的研究提供了必需的人工抗原。(A synthetic method of analgin artificial antigen belongs to the technical field of biochemical engineering. The invention adopts a glutaraldehyde method to couple 4-aminoantipyrine H1 with carrier protein to obtain an artificial antigen of analgin; or 4-amino antipyrine H1 and succinic anhydride react to obtain analgin hapten H1-HS, and then the analgin hapten H1-HS is coupled with carrier protein by a carbodiimide method to obtain the analgin artificial antigen. The invention successfully synthesizes the analgin artificial antigen, has simple, safe and effective synthesis steps, can be completely used in immunoassay, and provides the necessary artificial antigen for the later research.)

1. The synthetic method of the analgin artificial antigen is characterized by comprising the following steps: coupling 4-aminoantipyrine H1 with carrier protein by glutaraldehyde method to obtain artificial antigen of analgin.

2. The method for synthesizing analgin artificial antigen as claimed in claim 1, wherein the steps are as follows:

(1) preparation of solution A: weighing 4-aminoantipyrine H12.03 mg, dissolving in 500 μ L of ultrapure water, adding 60 μ L of 2.5% glutaraldehyde solution, stirring in ice bath for 15 min to obtain reaction solution A;

(2) and (3) preparation of a liquid B: weighing 5mg BSA, and dissolving with 1mL phosphate buffer PBS (0.01M, pH = 7.4) to obtain solution B;

(3) coupling: dropwise adding the solution A into the solution B, stirring while dropwise adding, and stirring at room temperature for reaction for 2h to obtain a coupling mixed solution;

(4) and (3) dialysis, namely taking a dialysis bag with 10cm of protein intercepted as 3kDa, boiling for 5 min, cooling to room temperature, adding the coupling mixed solution into the dialysis bag, dialyzing for 3 days by using 0.01M PBS with pH =7.4, changing the dialyzate for times every 6H, removing redundant small molecular compounds to obtain the artificial antigen H1-BSA of analgin, and identifying by adopting an ultraviolet absorption scanning method.

3. The synthetic method of the analgin artificial antigen is characterized by comprising the following steps: the analgin hapten H1-HS is obtained by reacting 4-amino-antipyrine H1 with succinic anhydride, and then the analgin hapten H1-HS is coupled with carrier protein by a carbodiimide method, so that the analgin artificial antigen is obtained.

4. The method for synthesizing analgin artificial antigen as claimed in claim 3, wherein the steps are as follows:

(1) and (3) synthesis of analgin hapten: weighing 4-aminoantipyrine H150 mg, 175 mg of 4-dimethylaminopyridine and 300mg of succinic anhydride, dissolving in 20mL of anhydrous pyridine, and refluxing at 60 ℃ for 12H; evaporating and concentrating the obtained reaction liquid, dissolving the residue in 10mL of water, extracting with 5mL of ethyl acetate for three times, and collecting an organic phase; drying the organic phase by nitrogen to obtain a white solid, namely analgin hapten H1-HS;

(2) preparation of complete antigen: weighing 1.1 mg of analgin hapten H1-HS and 1.3 mg of N-hydroxysuccinimide NHS prepared in the step (1), dissolving in 300 mu L N, N-dimethylformamide DMF, and stirring at room temperature for reaction for 10 min; weighing 2.2 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC, fully dissolving the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC in 50 mu L of 0.01M 2- (n-morpholine) ethylsulfonic acid buffer solution MES with the pH value of 4.6, adding the dissolved solution into the H1-HS solution, and stirring and reacting for 6-8H at room temperature to obtain solution A; dissolving 5mg of KLH with 0.01M PBS to obtain solution B; slowly adding the solution A into the solution B drop by drop, stirring at room temperature for reaction overnight to obtain a conjugate mixed solution; dialyzing the mixed solution in 0.01M PBS solution for three days to finally obtain analgin artificial antigen H1-HS-KLH, and identifying by adopting an ultraviolet absorption scanning method.

Technical Field

The invention relates to a method for synthesizing two kinds of analgin artificial antigens, and belongs to the technical field of biochemical engineering.

Background

Analgin (dipyrone) is which is a clinical antipyretic analgesic and belongs to pyrazolone antipyretic analgesic, and has been used for hundreds of years since the clinical application, analgin can be rapidly hydrolyzed into an active product 4-Methylaminoantipyrine (MAA) in vivo and then enters the systemic circulation, MAA is metabolized into 4-formamidoantipyrine (4-formamidoantipyrine, FA) and 4-aminoantipyrine (4-aminoantipyrine, AA) in vivo, and AA can be continuously metabolized into 4-acetylaminoantipyrine (4-acetylaminoantipyrine, AAA).

The drug has strong antipyretic and analgesic effects, and is currently widely used by in many countries, but the drug can cause severe reactions such as granulocytopenia, aplastic anemia, severe allergy, shock collapse and the like, because of toxic and side effects and an uncertain pharmacodynamic action mechanism, the developed countries in the world have made the regulations for stopping or limiting the use of the drug to food-borne animals, such as the drug is prohibited to be used in food-borne animals in the United states and Sweden, and the positive list of Japan also lists the drug to be in law standard, while the European Union has a limit requirement of 50 mug/kg (calculated by MAA content) for the drug to be used in milk.

The currently reported methods for detecting analgin or analogues thereof include spectrophotometry, flow injection chemiluminescence photometry, thin-layer chromatography (TLC), High Performance Liquid Chromatography (HPLC), liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), and the like, however, these instrumental analysis methods all require complex pretreatment, are time-consuming and labor-consuming, and are not suitable for rapid detection of a large number of samples.

Disclosure of Invention

The invention aims to provide two methods for synthesizing analgin artificial antigens, and the prepared product is used for analgin immunoassay method research and provides necessary artificial antigens for future research.

The technical scheme of the invention comprises two synthetic methods of analgin artificial antigen, , 4-aminoantipyrine (H1) is coupled with carrier protein by adopting a glutaraldehyde method to obtain artificial antigens of analgin, and the hapten is obtained by reacting H1 with succinic anhydride, which is hereinafter referred to as H1-HS, and then the hapten H1-HS is coupled with the carrier protein by adopting a carbodiimide method to obtain another artificial antigens of analgin.

The synthetic method comprises the following steps:

(1) preparation of solution A: weighing 4-aminoantipyrine H12.03 mg, dissolving in 500 μ L of ultrapure water, adding 60 μ L of 2.5% glutaraldehyde solution, stirring in ice bath for 15 min to obtain reaction solution A;

(2) and (3) preparation of a liquid B: weighing 5mg BSA, and dissolving with 1mL phosphate buffer PBS (0.01M, pH = 7.4) to obtain solution B;

(3) coupling: dropwise adding the solution A into the solution B while stirring, and stirring at room temperature for reaction for 2h to obtain a coupling mixed solution;

(4) and (3) dialysis, namely taking a dialysis bag with 10cm of protein intercepted as 3kDa, boiling for 5 min, cooling to room temperature, adding the coupling mixed solution into the dialysis bag, dialyzing for 3 days by using 0.01M PBS with pH =7.4, changing the dialyzate for times every 6H, removing redundant small molecular compounds to obtain the artificial antigen H1-BSA of analgin, and identifying by adopting an ultraviolet absorption scanning method.

The second synthesis method is as follows:

(1) and (3) synthesis of analgin hapten H1-HS:

the synthetic route is as follows:

weighing 4-aminoantipyrine H150 mg, 4-dimethylaminopyridine 175 mg and succinic anhydride 300mg, dissolving in 20mL of anhydrous pyridine, and refluxing at 60 ℃ for 12H. The reaction solution was concentrated by evaporation, the residue was dissolved in 10mL of water and extracted three times with 5mL of ethyl acetate, and the organic phase was collected. And drying the organic phase by nitrogen to obtain a white solid, namely analgin hapten H1-HS, and identifying the structure by nuclear magnetism and mass spectrometry.

(2) Preparation of complete antigen: weighing 1.1 mg of analgin hapten H1-HS and 1.3 mg of N-hydroxysuccinimide (NHS) obtained in the step (1), dissolving in 300 mu L N of N-Dimethylformamide (DMF), and stirring at room temperature for reaction for 10 min; weighing 2.2 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), fully dissolving with 50 μ L of 2- (n-morpholine) ethyl sulfonic acid buffer solution (MES) with pH = 4.6 and 0.01M, adding into the H1-HS solution, and stirring at room temperature for 6-8H to obtain solution A; dissolving 5mg of KLH in 0.01M PBS to obtain solution B; and slowly adding the solution A into the solution B dropwise, and stirring at room temperature for reaction overnight to obtain a conjugate mixed solution. Dialyzing the mixed solution in 0.01M PBS solution for three days to finally obtain analgin artificial antigen H1-HS-KLH, and identifying by adopting an ultraviolet absorption scanning method.

Identification of the obtained analgin artificial antigen:

(1) the hapten is identified by nuclear magnetic resonance and mass spectrometry.

(2) And (3) identifying the coupling effect of the artificial antigen by adopting an ultraviolet method, and calculating the coupling ratio of the conjugate by utilizing the concentrations of small molecules and protein in the conjugate.

And (3) coupling ratio determination: the method for estimating the ratio of two molecules to be conjugated (conjugation ratio) in a conjugate is based on the principle of detecting the amount (or relative amount) of the two molecules to be conjugated in the conjugate, although the determination method is various. The ultraviolet method determines the coupling ratio according to the ratio of the molar concentration of molecules to the molar concentration of proteins in the synthesized artificial antigen.

Coupling ratio = molar concentration of small molecule in the conjugate/molar concentration of protein in the conjugate.

The invention has the beneficial effects that: the invention successfully synthesizes the artificial antigen of analgin, has simple, safe and effective synthesis steps, can be completely used in immunoassay, and provides necessary artificial antigen for the subsequent research.

Drawings

FIG. 1 shows the ultraviolet identification of the analgin artificial antigen H1-BSA.

FIG. 2 is a mass spectrometric identification of the analgin hapten H1-HS.

FIG. 3 shows the UV identification of the artificial antigen H1-HS-KLH of Analgin.

Detailed Description

The coupling result of the artificial antigen is identified by adopting an ultraviolet method in the following examples, and the coupling ratio of the artificial antigen is calculated by utilizing the concentrations of small molecules and proteins in the conjugate.

And (3) coupling ratio determination: the method for estimating the ratio of two molecules to be conjugated (conjugation ratio) in a conjugate is based on the principle of detecting the amount (or relative amount) of the two molecules to be conjugated in the conjugate, although the determination method is various. The ultraviolet method determines the coupling ratio according to the ratio of the concentration of small molecules to the concentration of protein in the synthesized artificial antigen.