CN113045643B - Carbamazepine antigen and preparation method thereof - Google Patents
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Abstract
The invention relates to the field of antigens, and discloses a carbamazepine antigen and a preparation method thereof, wherein the preparation method of the carbamazepine antigen comprises the following steps: (1) performing amidation reaction on 10-methoxy-5H-dibenzo (b, f) azepine by chlorosulfonyl isocyanate to obtain 10-methoxy-5H-dibenzo [ b, f ] azepine-5-formamide; (2) breaking a basic ether bond of 10-methoxy-5H-dibenzo [ b, f ] azepine-5-formamide by using boron tribromide to obtain 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-formamide; (3) reacting 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-formamide with succinic anhydride to obtain carboxyl-containing hapten; (4) the carbamazepine antigen is obtained after coupling the carbamazepine hapten with carrier protein. The carbamazepine antigen of the invention completely reserves the molecular structure of the carbamazepine, thus having higher specificity and sensitivity.
Description
Technical Field
The invention relates to the field of antigens, in particular to a carbamazepine antigen and a preparation method thereof.
Background
Carbamazepine, known by the scientific name "5H-dibenzo [ b, f ] azepine-5-carboxamide," has the following structural formula:
carbamazepine belongs to dibenzoazepine antiepileptic drugs. It is mainly used for treating epilepsy, central nervous uremia, antimanic depression, and arrhythmia. Because carbamazepine is safer and has better curative effect, it becomes the first choice of antiepileptic drugs for most people.
Because carbamazepine is widely applied, adverse reactions of carbamazepine are more and more valued by people. Researches show that the liver and kidney function can be damaged by taking carbamazepine in a large amount for a long time, and the kidney function failure is caused; patients taking carbamazepine often have allergic reactions such as headache, heat in the head, skin plague and the like. In addition, carbamazepine has a significant inhibitory effect on the embryonic reproduction of zebrafish. Therefore, the content detection of carbamazepine plays an important role.
The common detection and analysis method for carbamazepine mainly comprises the following steps: gas Chromatography (GC), gas-mass spectrometry (GC-MS), High Performance Liquid Chromatography (HPLC), liquid-mass spectrometry (LC-MS), and the like. Instrumental methods of analysis have extremely high sensitivity and precision, but require expensive instrumentation, equipment and specially trained technicians, and are not suitable for screening assays of bulk samples and for point-of-care assays. The immunoassay developed in recent years has the advantages of simple and convenient operation, high efficiency, sensitivity, suitability for large-scale detection and the like, and is widely applied to the detection of various contraband products and the like.
To establish an immunoassay for carbamazepine, it is necessary to obtain a carbamazepine whole antigen with antigenic activity.
Disclosure of Invention
In order to solve the technical problems, the invention provides a carbamazepine antigen and a preparation method thereof. The carbamazepine antigen of the invention completely reserves the molecular structure of the carbamazepine, thus having higher specificity and sensitivity.
The specific technical scheme of the invention is as follows:
a carbamazepine antigen having the structural formula:
wherein R is carrier protein.
Preferably, the carrier protein is one of Bovine Serum Albumin (BSA), hemocyanin (KLH), chicken Ovalbumin (OVA), and Bovine Gamma Globulin (BGG).
A preparation method of the carbamazepine antigen comprises the following steps:
(1) carrying out substitution reaction on 10-methoxy-5H-dibenzo (b, f) azepine and chlorosulfonyl isocyanate to obtain 10-methoxy-5H-dibenzo [ b, f ] azepine-5-formamide;
(2) reacting 10-methoxy-5H-dibenzo [ b, f ] azepine-5-formamide with boron tribromide to obtain 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-formamide;
(3) reacting 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-formamide with succinic anhydride to obtain a carbamazepine hapten; (4) the carbamazepine antigen is obtained after coupling the carbamazepine hapten with carrier protein.
The mechanism for preparing the carbamazepine antigen is as follows: amidation of 10-methoxy-5H-dibenzo (b, f) azepine with chlorosulfonyl isocyanate to give 10-methoxy-5H-dibenzo [ b, f ] azepine-5-carboxamide. And (3) breaking the ether bond by using boron tribromide to obtain the 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-formamide. Reacting the hapten with succinic anhydride to obtain a carboxyl-containing hapten, and reacting the carboxyl with amino in carrier protein to realize the coupling of carbamazepine and the carrier protein so as to obtain the immunogenic carbamazepine antigen.
In addition, in the finally obtained carbamazepine antigen, the carbon chain between the carboxyl group for coupling with the carrier protein and the N-containing heterocyclic ring is longer, so that the carboxyl group is not easily interfered by the steric hindrance of the bulky N-containing heterocyclic ring in the process of coupling with the carrier protein, and the effective coupling is easier to carry out.
Preferably, the specific process of step (1) is as follows: under the protection of inert gas, dissolving 10-methoxy-5H-dibenzo (b, f) azepine in dichloromethane, reducing the temperature to 0-5 ℃, adding chlorosulfonyl isocyanate, and raising the temperature to 20-30 ℃ after the addition to react for 12-18H; adding water, adding alkali to adjust the pH value to 8-9, adding dichloromethane, extracting, layering, washing an organic layer with water, drying the organic layer, and concentrating under reduced pressure to obtain 10-methoxy-5H-dibenzo [ b, f ] azepine-5-formamide.
Preferably, in the step (1), the mass ratio of the carbamazepine to the chlorosulfonyl isocyanate is 1: 0.8-1.
Preferably, the specific process of step (2) is as follows: under the protection of inert gas, dissolving 10-methoxy-5H-dibenzo [ b, f ] azepine-5-formamide in dichloromethane, reducing the temperature to-5-0 ℃, adding boron tribromide, and raising the temperature to 20-30 ℃ after the addition to react for 12-18H; adding water, adding alkali to adjust the pH value to 8-9, adding dichloromethane, extracting, layering, washing an organic layer with water, drying the organic layer, and concentrating under reduced pressure to obtain 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-formamide.
Preferably, in the step (2), the mass ratio of the 10-methoxy-5H-dibenzo [ b, f ] azepine-5-formamide to the boron tribromide is 1: 2-3.
Preferably, the specific process of step (3) is as follows: dissolving 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-formamide in pyridine, adding succinic anhydride, reacting at the temperature of 100 ℃ for 12-18H, decompressing and concentrating a dry solvent, and performing column chromatography on a concentrate to obtain the carbamazepine hapten.
Preferably, in the step (3), the mass ratio of the 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-formamide to the succinic anhydride is 1: 0.7-1.1.
Preferably, the specific process of step (4) is as follows: dissolving carbamazepine hapten in N, N-dimethylformamide, adding N-hydroxysuccinimide and dicyclohexylcarbodiimide, reacting at 10-30 ℃ for 18-24h, centrifuging, taking clear liquid, and adding the clear liquid into PBS (phosphate buffer solution) of carrier protein, wherein the mass ratio of the carbamazepine hapten, NHS and DCC is 1: 0.32-0.35: 0.58-0.60; putting the reaction solution into PBS solution with pH of 7.4, dialyzing for 70-80h, and replacing the PBS solution every 20-30 h; and centrifuging the dialyzed reaction solution, and taking supernatant to obtain the carbamazepine antigen.
The invention has the following advantages:
(1) the carbamazepine antigen completely reserves the molecular structure of the carbamazepine, thereby having stronger specificity and higher sensitivity;
(2) the steps of the whole preparation process of the carbamazepine antigen are relatively simple, and the product yield is high.
Detailed Description
The present invention will be further described with reference to the following examples.
General examples
A preparation method of a carbamazepine antigen comprises the following steps:
(1) synthesis of 10-methoxy-5H-dibenzo [ b, f ] azepine-5-carboxamide (II)
Under the protection of nitrogen, dissolving 10-methoxy-5H-dibenzo (b, f) azepine (I) in dichloromethane, reducing the temperature to 0-5 ℃, adding chlorosulfonyl isocyanate, wherein the mass ratio of carbamazepine to chlorosulfonyl isocyanate is 1: 0.8-1, and raising the temperature to 20-30 ℃ after the addition to react for 12-18H. Adding water, adjusting pH to 8-9 with 1N sodium hydroxide solution, adding dichloromethane, extracting, layering, washing organic layer with water, drying organic layer with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain 10-methoxy-5H-dibenzo [ b, f ] azepine-5-formamide (II).
(2) Synthesis of 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-carboxamide (III)
Under the protection of nitrogen, dissolving 10-methoxy-5H-dibenzo [ b, f ] azepine-5-formamide (II) in dichloromethane, reducing the temperature to-5-0 ℃, adding boron tribromide, wherein the mass ratio of the 10-methoxy-5H-dibenzo [ b, f ] azepine-5-formamide to the boron tribromide is 1: 2-3, and raising the temperature to 20-30 ℃ for reaction for 12-18H. Adding water, adjusting pH to 8-9 with 1N sodium hydroxide solution, adding dichloromethane, extracting, layering, washing organic layer with water, drying organic layer with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-formamide (III).
(3) Synthesis of carbamazepine hapten (IV)
Dissolving 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-formamide (III) in pyridine, adding succinic anhydride, reacting at the mass ratio of 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-formamide to succinic anhydride of 1: 0.7-1.1 at the temperature of 110 ℃ for 12-18H, concentrating the dry solvent under reduced pressure, and carrying out column chromatography on the concentrate to obtain the carbamazepine hapten (IV).
(4) Synthesis of carbamazepine antigen (V)
Dissolving carbamazepine hapten (IV) in N, N-Dimethylformamide (DMF), adding N-hydroxysuccinimide (NHS) and Dicyclohexylcarbodiimide (DCC), reacting at 10-30 ℃ for 18-24h, centrifuging, taking clear liquid, adding the clear liquid into a PBS (phosphate buffer solution) solution of carrier protein, wherein the carrier protein is one of bovine serum albumin, hemocyanin, egg albumin and bovine gamma globulin (when the carrier protein is bovine serum albumin, the carbamazepine is 1: 2-3), and reacting at 4-10 ℃ for 18-24 h. Placing the reaction solution in a PBS solution with the pH value of 7.4 for dialysis for 72 hours, and replacing the PBS solution every 24 hours; and centrifuging the dialyzed reaction solution, and taking supernatant to obtain the carbamazepine antigen (V).
Example 1
A preparation method of a carbamazepine antigen comprises the following steps:
(1) synthesis of 10-methoxy-5H-dibenzo [ b, f ] azepine-5-carboxamide (II)
Under nitrogen protection, 45mg of 10-methoxy-5H-dibenzo (b, f) azepine (I) was dissolved in 10mL of dichloromethane, the temperature was reduced to 0-5 ℃, 40mg of chlorosulfonyl isocyanate was added, and the temperature was raised to 25 ℃ to react for 15H. 10mL of water was added, pH8.2 was adjusted with 1N sodium hydroxide solution, 30mL of methylene chloride was added, extraction was performed for separation, the organic layer was washed with 10mL of water, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 58mg of 10-methoxy-5H-dibenzo [ b, f ] azepine-5-carboxamide (II).
(2) Synthesis of 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-carboxamide (III)
Under the protection of nitrogen, 58mg of 10-methoxy-5H-dibenzo [ b, f ] azepine-5-formamide (II) is dissolved in 30mL of dichloromethane, the temperature is reduced to-5-0 ℃, 130mg of boron tribromide is added, and the temperature is raised to 20-30 ℃ after the addition for reaction for 14H. Adding 10mL of water, adjusting pH to 8.5 with 1N sodium hydroxide solution, adding 30mL of dichloromethane, extracting, separating, washing the organic layer with 10mL of water, drying the organic layer with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain 62mg of 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-carboxamide (III).
(3) Synthesis of carbamazepine hapten (IV)
Dissolving 62mg of 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-carboxamide (III) in 5mL of pyridine, adding 57mg of succinic anhydride, reacting at 105 ℃ for 16H, concentrating the dried solvent under reduced pressure, and performing column chromatography on the concentrate to obtain 34mg of carbamazepine hapten (IV).
(4) Synthesis of carbamazepine antigen (V)
34mg of carbamazepine hapten (IV) was dissolved in 2mL of N, N-Dimethylformamide (DMF), 11mg of N-hydroxysuccinimide (NHS) and 19mg of Dicyclohexylcarbodiimide (DCC) were added to the solution, and the mixture was reacted at 20 ℃ for 21 hours, and after centrifugation, the clear solution was added to 10mL of a PBS solution containing 7mg/mL of BSA and reacted at 4 to 10 ℃ for 20 hours. Placing the reaction solution in a PBS solution with the pH value of 7.4 for dialysis for 72 hours, and replacing the PBS solution every 24 hours; the dialyzed reaction solution was centrifuged, and the supernatant was collected to obtain 10.4mL of 4.2mg/mL carbamazepine antigen (V).
Example 2
A preparation method of a carbamazepine antigen comprises the following steps:
(1) synthesis of 10-methoxy-5H-dibenzo [ b, f ] azepine-5-carboxamide (II)
45mg of 10-methoxy-5H-dibenzo (b, f) azepine (I) was dissolved in 12mL of dichloromethane under nitrogen, the temperature was reduced to 0-5 ℃ and 41mg of chlorosulfonyl isocyanate was added, and the temperature was raised to 25 ℃ to react for 16 hours. 10mL of water was added, pH8.7 was adjusted with 1N sodium hydroxide solution, 40mL of methylene chloride was added, extraction was performed for separation, the organic layer was washed with 10mL of water, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 59mg of 10-methoxy-5H-dibenzo [ b, f ] azepine-5-carboxamide (II).
(2) Synthesis of 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-carboxamide (III)
Under the protection of nitrogen, 59mg of 10-methoxy-5H-dibenzo [ b, f ] azepine-5-formamide (II) is dissolved in 30mL of dichloromethane, the temperature is reduced to-5-0 ℃, 134mg of boron tribromide is added, and the temperature is raised to 20-30 ℃ after the addition for reaction for 15H. Adding 10mL of water, adjusting pH to 8.1 with 1N sodium hydroxide solution, adding 30mL of dichloromethane, extracting, separating, washing the organic layer with 10mL of water, drying the organic layer with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain 64mg of 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-carboxamide (III).
(3) Synthesis of carbamazepine hapten (IV)
Dissolving 64mg of 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-carboxamide (III) in 5mL of pyridine, adding 60mg of succinic anhydride, reacting at 105 ℃ for 17H, concentrating the dried solvent under reduced pressure, and carrying out column chromatography on the concentrate to obtain 36mg of carbamazepine hapten (IV).
(4) Synthesis of carbamazepine antigen (V)
36mg of carbamazepine hapten (IV) was dissolved in 2mL of N, N-Dimethylformamide (DMF), 12mg of N-hydroxysuccinimide (NHS) and 21mg of Dicyclohexylcarbodiimide (DCC) were added to the solution, and the mixture was reacted at 20 ℃ for 20 hours, and after centrifugation, the clear solution was added to 11mL of a PBS solution containing 7mg/mL of BSA and reacted at 4 to 10 ℃ for 20 hours. Placing the reaction solution in a PBS solution with the pH value of 7.4 for dialysis for 72 hours, and replacing the PBS solution every 24 hours; the dialyzed reaction solution was centrifuged, and the supernatant was collected to obtain 11.1mL of 4.0mg/mL carbamazepine antigen (V).
Example 3
A preparation method of a carbamazepine antigen comprises the following steps:
(1) synthesis of 10-methoxy-5H-dibenzo [ b, f ] azepine-5-carboxamide (II)
45mg of 10-methoxy-5H-dibenzo (b, f) azepine (I) was dissolved in 12mL of dichloromethane under nitrogen, the temperature was reduced to 0-5 ℃ and 43mg of chlorosulfonyl isocyanate was added, and the temperature was raised to 25 ℃ to react for 16 hours. 10mL of water was added, pH8.8 was adjusted with 1N sodium hydroxide solution, 40mL of methylene chloride was added, extraction was performed for separation, the organic layer was washed with 10mL of water, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 56mg of 10-methoxy-5H-dibenzo [ b, f ] azepine-5-carboxamide (II).
(2) Synthesis of 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-carboxamide (III)
Under the protection of nitrogen, 56mg of 10-methoxy-5H-dibenzo [ b, f ] azepine-5-formamide (II) is dissolved in 30mL of dichloromethane, the temperature is reduced to-5-0 ℃, 138mg of boron tribromide is added, and the temperature is raised to 25 ℃ after the addition to react for 15H. Adding 10mL of water, adjusting pH to 8.7 with 1N sodium hydroxide solution, adding 30mL of dichloromethane, extracting, separating, washing the organic layer with 10mL of water, drying the organic layer with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain 60mg of 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-formamide (III).
(3) Synthesis of carbamazepine hapten (IV)
Dissolving 60mg of 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-carboxamide (III) in 5mL of pyridine, adding 62mg of succinic anhydride, reacting at 105 ℃ for 17H, concentrating the dried solvent under reduced pressure, and performing column chromatography on the concentrate to obtain 35mg of carbamazepine hapten (IV).
(4) Synthesis of carbamazepine antigen (V)
35mg of carbamazepine hapten (IV) was dissolved in 2mL of N, N-Dimethylformamide (DMF), 11mg of N-hydroxysuccinimide (NHS) and 29mg of Dicyclohexylcarbodiimide (DCC) were added to the solution, and the mixture was reacted at 20 ℃ for 20 hours, and after centrifugation, the clear solution was added to 11mL of a 7mg/mL PBS solution of BSA and reacted at 4 to 10 ℃ for 20 hours. Placing the reaction solution in a PBS solution with the pH value of 7.4 for dialysis for 72 hours, and replacing the PBS solution every 24 hours; the dialyzed reaction solution was centrifuged, and the supernatant was collected to obtain 11.8mL of 4.3mg/mL carbamazepine antigen (V).
Comparative example 1
A preparation method of a carbamazepine antigen comprises the following steps (patent CN109678947A method):
5.0g of the compound 5H-dibenzo [ b, f ] azepine-5-carbonyl chloride (VI) was dissolved in 50mL of tetrahydrofuran, and 3.4g of methyl 4-aminobutyrate hydrochloride and 6.75g of triethylamine were added. The reaction was then refluxed overnight, stopped, and extracted with ethyl acetate to give 7.5g of compound (VII).
2.0g of Compound (VII) was dissolved in 5mL of tetrahydrofuran and 5mL of water, and 0.6g of lithium hydroxide was added to the solution to react at room temperature overnight. The pH was adjusted with 1N hydrochloric acid solution to 3, filtered and concentrated under reduced pressure to give 1.2g of Compound (VIII).
8.9mg of Compound (VIII), 8.8mg of N-hydroxysuccinimide (NHS) and 16.2mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were dissolved in 0.6mL of N, N-Dimethylformamide (DMF) (solution A) and reacted at room temperature for 5 hours. 10mg of BSA was weighed and dissolved in 0.6mL of borate buffer (solution B). And dropwise adding the solution A into the solution B at room temperature, and reacting at room temperature overnight. Placing the reaction solution in a PBS solution with the pH value of 7.4 for dialysis for 72 hours, and changing the PBS solution every 24 hours; the dialyzed reaction solution was centrifuged, and the supernatant was collected to obtain 1.1mL of 5.3mg/mL carbamazepine antigen (IX).
The activity of the carbamazepine antigens prepared in the examples 1 to 3 and the comparative example 1 was detected by a colloidal gold immunochromatography under the following conditions: spraying a carbamazepine antigen on an NC membrane with the spraying amount of 1.0 mu g/cm by using a film spraying machine, marking colloidal gold by using a carbamazepine antibody, combining glass fiber paper and absorbent paper to assemble a test strip, and detecting the test strip. The results are shown in Table 1.
TABLE 1
From table 1, it can be seen that: in examples 1-3, the preparation parameters for preparing the hapten are adjusted within the parameter range, the negative detection T-line color development intensity of the obtained antigen reaches G8.5, and the result proves that the synthesized carbamazepine antigen has higher activity and can be identified by the antibody and efficiently combined; positive detection proves that the PBS solution of the carbamazepine has competitive inhibition on the prepared carbamazepine antigen and has higher sensitivity and gradient. Comparative example 1 the potency and sensitivity of the antigen prepared by the method of patent CN109678947A were lower than those of the antigen prepared by the method of the present patent.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (10)
1. A carbamazepine antigen having the structural formula:
wherein R is carrier protein.
2. The carbamazepine antigen of claim 1, wherein said carrier protein is one of bovine serum albumin, hemocyanin, chicken egg albumin, and bovine gamma globulin.
3. A process for the preparation of a carbamazepine antigen according to claim 1 or 2, characterised in that it comprises the following steps:
(1) carrying out substitution reaction on 10-methoxy-5H-dibenzo (b, f) azepine and chlorosulfonyl isocyanate to obtain 10-methoxy-5H-dibenzo [ b, f ] azepine-5-formamide;
(2) reacting 10-methoxy-5H-dibenzo [ b, f ] azepine-5-formamide with boron tribromide to obtain 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-formamide;
(3) reacting 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-formamide with succinic anhydride to obtain a carbamazepine hapten;
(4) the carbamazepine antigen is obtained after coupling the carbamazepine hapten with carrier protein.
4. The method according to claim 3, wherein the specific process of step (1) is as follows: under the protection of inert gas, dissolving 10-methoxy-5H-dibenzo (b, f) azepine in dichloromethane, reducing the temperature to 0-5 ℃, adding chlorosulfonyl isocyanate, and raising the temperature to 20-30 ℃ after the addition to react for 12-18H; adding water, adding alkali to adjust the pH value to 8-9, adding dichloromethane, extracting, layering, washing an organic layer with water, drying the organic layer, and concentrating under reduced pressure to obtain 10-methoxy-5H-dibenzo [ b, f ] azepine-5-formamide.
5. The method according to claim 3 or 4, wherein in the step (1), the mass ratio of carbamazepine to chlorosulfonyl isocyanate is 1: 0.8-1.
6. The method according to claim 3, wherein the specific process of step (2) is as follows: under the protection of inert gas, dissolving 10-methoxy-5H-dibenzo [ b, f ] azepine-5-formamide in dichloromethane, reducing the temperature to-5-0 ℃, adding boron tribromide, and raising the temperature to 20-30 ℃ after the addition to react for 12-18H; adding water, adding alkali to adjust the pH value to 8-9, adding dichloromethane, extracting, layering, washing an organic layer with water, drying the organic layer, and concentrating under reduced pressure to obtain 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-formamide.
7. The method according to claim 3 or 6, wherein in the step (2), the mass ratio of the 10-methoxy-5H-dibenzo [ b, f ] azepine-5-carboxamide to the boron tribromide is 1: 2-3.
8. The method according to claim 3, wherein the specific process of step (3) is as follows: dissolving 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-formamide in pyridine, adding succinic anhydride, reacting at the temperature of 100 ℃ for 12-18H, decompressing and concentrating a dry solvent, and performing column chromatography on a concentrate to obtain the carbamazepine hapten.
9. The method according to claim 3 or 8, wherein in the step (3), the mass ratio of the 10-hydroxy-5H-dibenzo [ b, f ] azepine-5-carboxamide to succinic anhydride is 1: 0.7-1.1.
10. The method according to claim 3, wherein the specific process of step (4) is as follows: dissolving carbamazepine hapten in N, N-dimethylformamide, adding N-hydroxysuccinimide and dicyclohexylcarbodiimide, wherein the mass ratio of the carbamazepine hapten to NHS to DCC is 1: 0.32-0.35: 0.58-0.60, reacting at 10-30 ℃ for 18-24h, centrifuging, taking clear liquid, adding the clear liquid into PBS (phosphate buffer solution) of carrier protein, putting the reaction liquid into the PBS (phosphate buffer solution) with the pH of 7.4, dialyzing for 70-80h, and changing the PBS once every 20-30 h; and centrifuging the dialyzed reaction solution, and taking supernatant to obtain the carbamazepine antigen.
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