CN114685527A - Olanzapine derivative, immunogen, anti-olanzapine specific antibody, preparation method and application thereof - Google Patents

Olanzapine derivative, immunogen, anti-olanzapine specific antibody, preparation method and application thereof Download PDF

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CN114685527A
CN114685527A CN202011558915.6A CN202011558915A CN114685527A CN 114685527 A CN114685527 A CN 114685527A CN 202011558915 A CN202011558915 A CN 202011558915A CN 114685527 A CN114685527 A CN 114685527A
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olanzapine
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serum albumin
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CN114685527B (en
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张小可
王永霞
封飒飒
胡振阳
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Changsha Boyuan Medical Technology Co ltd
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Suzhou Evermed Medical Technology Co ltd
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Abstract

The invention discloses an olanzapine derivative, immunogen, an anti-olanzapine specific antibody, a preparation method and application thereof. Firstly, coupling a novel olanzapine derivative with recombinant sheep serum albumin obtained through genetic engineering modification to prepare an olanzapine artificial antigen, immunizing an experimental animal with the olanzapine artificial antigen to obtain an anti-olanzapine specific antibody, wherein ELISA detection shows that the specific antibody has strong specificity and high sensitivity, and interference experiments show that the specific antibody does not have any cross reaction with 100 common drugs; the anti-olanzapine specific antibody is applied to the preparation of an olanzapine detection reagent, the olanzapine detection reagent comprises an olanzapine homogeneous enzyme immunoassay reagent and an olanzapine latex enhanced immunoturbidimetry detection reagent, and the detection reagent can realize high-throughput and rapid detection of olanzapine on a full-automatic biochemical analyzer.

Description

Olanzapine derivative, immunogen, anti-olanzapine specific antibody, preparation method and application thereof
Technical Field
The invention relates to an olanzapine derivative, immunogen, an anti-olanzapine specific antibody, a preparation method and application thereof, and belongs to the technical field of biomedical detection.
Background
Olanzapine (Olanzapine) is a novel atypical neuroleptic drug which can bind to dopamine receptors, 5-hydroxytryptamine (5-HT) receptors and cholinergic receptors and exert antagonistic action. Olanzapine is used clinically mainly for the acute phase and maintenance treatment of schizophrenia and other psychoses with severe positive and/or negative symptoms; can also play a certain role in relieving the common secondary emotional symptoms of schizophrenia and related diseases. Olanzapine is chemically named as 2-methyl-4- (4-methyl-1-piperazinyl) -10H-thieno [2, 3-b ] [1,5] benzodiazepine, sulfur, propionaldehyde and malononitrile are used as raw materials to close a ring, the ring is reacted with o-nitrofluorobenzene to obtain 2- (2-nitroanilino) -3-cyano-5-methylthiophene, and an olanzapine product is obtained through reduction and reaction with N-methylpiperazine in one step. The clinical applications of olanzapine are: (ii) treatment of schizophrenia and other psychotic disorders. Olanzapine can obviously reduce positive symptoms and can also reduce negative symptoms. An 85% improvement of the negative symptoms of schizophrenia is a direct therapeutic effect of olanzapine; ② treating affective disorder. Olanzapine is effective in treating bipolar disorder, including some patients who do not respond well to lithium salts, anticonvulsants, and typical antipsychotics; ③ treating mental disorder accompanied by senile dementia; fourthly, obsessive-compulsive disorder is treated; treating somatoform disorder; sixthly, treating anorexia nervosa. However, it is affected by various factors, such as age, administration mode, smoking, coffee, etc., and when it is used in combination with other drugs, it is easy to generate clinically significant drug interactions, resulting in changes in pharmacological actions and toxic and side effects. It is crucial to monitor clozapine levels in serum in a timely manner and to adjust the therapeutic dose in a timely manner. In addition, measurement of clozapine plasma levels is very useful in cases where patient compliance is difficult to judge, therapeutic dose is ineffective and pharmacokinetic drug interactions may exist. Therefore, the individual administration scheme can be formulated and adjusted by monitoring the concentration of clozapine in the blood of the administration patient, the curative effect is improved, and the administration safety is guaranteed.
The blood concentration detection method of olanzapine comprises various methods such as liquid chromatography combined tandem mass spectrometry (LC-MS/MS), High Performance Liquid Chromatography (HPLC), reverse high performance liquid chromatography (RP-HPLC), high performance liquid chromatography-ultraviolet detection (HPLC-UV), high performance liquid chromatography-electrochemical method (HPLC-ECD), gas chromatography-mass spectrometry (GC-MS), full-automatic two-dimensional liquid chromatography (2D-LC/UV) and the like. The high performance liquid chromatography has the characteristics of accuracy, sensitivity and low price; but the measuring time is longer and the detection concentration is limited; the method uses buffer solution for the mobile phase, so that the operation is relatively complicated; the automatic monitoring is difficult to realize, and the large-scale use of the kit in clinical detection is restricted. The liquid chromatography-tandem mass spectrometry can greatly simplify the purification process of a sample, the result is not interfered by olanzapine metabolite, the linear range is wide, the lower limit of quantification is low, the requirement and the cost on the test are high, the pretreatment is complex, and the condition is not easy to control.
Therefore, olanzapine detection products which are wide in linear range, high in sensitivity, high in accuracy, high in precision, short in detection time, simple in sample processing, high in instrument automation degree and capable of continuously detecting multiple samples are lacking in the market at present.
Disclosure of Invention
In order to overcome the disadvantages of the prior art, a first object of the present invention is to provide a olanzapine derivative which is a novel synthetic compound that does not exist in nature.
The first purpose of the invention is realized by adopting the following technical scheme: an olanzapine derivative, which has a structural formula shown in a formula I:
Figure 100002_DEST_PATH_IMAGE001
formula I.
The second purpose of the invention is to provide a synthetic method of the olanzapine derivative, which is different from the conventional synthetic method, has good synthetic effect and obviously improves the synthetic efficiency of the olanzapine derivative.
The second purpose of the invention is realized by adopting the following technical scheme: a process for the synthesis of olanzapine derivatives of the formula I as shown above, the process of reaction is shown in the following formula:
Figure 100002_DEST_PATH_IMAGE002
；
specifically, the reaction process comprises the following steps:
(A1) synthesis of Compound 2:
Figure 100002_DEST_PATH_IMAGE004
compound 1 (6 g, 26 mmol) and piperazine (18 g, 0.19 mol) were added to butanol (100 mL) at room temperature to prepare a reaction mixture, and the reaction mixture was stirred under heating at 125 ℃ for 18 hours. After the reaction was complete, the reaction mixture was concentrated in vacuo. The residue obtained by the concentration was purified by a flash chromatography (methanol: dichloromethane 0-10%) to obtain compound 2.
(A2) Synthesis of Compound 3:
Figure DEST_PATH_IMAGE006
compound 2 (6 g, 20 mmol), tert-butyl 5-bromovalerate (5.7 g, 24 mmol), K2CO3(4.2 g, 28 mmol) and KI (0.3 g, 2 mmol) were added to acetonitrile (100 mL) at room temperature to prepare a reaction mixture. The reaction mixture was stirred at room temperature for 18 hours. After the reaction was complete, the reaction mixture was concentrated in vacuo. The residue obtained by the concentration was purified by a flash chromatography (methanol: dichloromethane 0-5%) to obtain compound 3.
(A3) Synthesis of olanzapine derivatives:
Figure DEST_PATH_IMAGE007
compound 3 (5 g, 11 mmol) and trifluoroacetic acid (5 mL) were dissolved together in DCM (45 mL) to prepare a reaction mixture, and the reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction mixture was concentrated in vacuo. The residue obtained by concentration was purified by a flash chromatography (methanol: dichloromethane 0-5%) to obtain olanzapine derivative.
The third purpose of the invention is to provide an olanzapine immunogen.
The third purpose of the invention is realized by adopting the following technical scheme: an olanzapine immunogen formed by connecting an olanzapine derivative shown in a structural formula I and a carrier protein, wherein the structural formula of the olanzapine immunogen is shown in a formula II:
Figure DEST_PATH_IMAGE008
a formula II;
the carrier protein is recombinant sheep serum albumin, and further, the amino acid sequence of the recombinant sheep serum albumin is shown in a sequence table SEQ ID NO: 1 is shown.
The amino acid sequence (SEQ ID NO: 1) of the recombinant sheep serum albumin is specifically as follows:
MKWVTFISLLLKFSSAYSRGVFRRDTHKSEIAHRFNDLGEENFQGLVLIAFSQYLKQCPFDEHVKLVKELTEFAKTCVADESHAGKDKSLHTLFGDELCKVATLRETYGDMADKCEKQEPERNECFLNHKDKSPDLPKLKPEPDTLCAEFKADEKKFWGKYLYEVARKHPYFYAPELLYYANKYNGVFQECKQAEDKGACLLPKIDAMREKVLASSARQRLKCASIQKFGERALKAWSVARLSQKFPKADFTDVTKIVTDLTKVHKECCHGDKLECADDRADLAKYICDHQDALKSKLKECKDKPVLEKSHCIAEVDKDAVPENLPPLTADFAEDKEVCKNYQEAKDVFLGSFLYEYSKRHPEYAVSVKLRLAKEYEATLEDCCAKEDPHACYKTVFDKLKHLVDEPQNLIKKNCELFEKHGEYGFQNALIVKYTRKAPQVSTPTLVEISRSLGKVGTKCKAKPESERMPCTEDYLSLILNRLCVLHEKTPVSEKVTKKCTESLVNRRPCFSDLTLDETYVPKPFDEKFKTFHADICTLPDTEKQIKKQTALVELKKHKPKATDEQLKTVMENFVAFVDKCCAADKKEGCFVLEGPKLVASTQAALA
a fourth object of the invention is to provide a process for the preparation of an immunogen of olanzapine as described above.
The fourth purpose of the invention is realized by adopting the following technical scheme: a process for the preparation of an immunogen of olanzapine as described above comprising the steps of:
(B1) preparation of carrier protein solution: dissolving the recombinant sheep serum albumin in a phosphate buffer solution to obtain a carrier protein solution;
(B2) preparation of olanzapine derivative solution: mixing the olanzapine derivative shown in the structural formula I with dimethylformamide, ethanol, a potassium phosphate buffer solution, 1-ethyl-3- (-3-dimethylaminopropyl) carbodiimide and N-hydroxy thiosuccinimide, and stirring for dissolving to obtain an olanzapine derivative solution;
(B3) synthesis of olanzapine immunogen: and (4) adding the olanzapine derivative solution obtained in the step (B2) into the carrier protein solution obtained in the step (B1), stirring for reaction, and dialyzing for purification to obtain an olanzapine immunogen.
Specifically, the preparation method of the olanzapine immunogen comprises the following steps:
(b1) preparation of carrier protein solution: dissolving the recombinant sheep serum albumin in 0.35mol/L potassium phosphate buffer (pH = 8.5) to obtain a carrier protein solution, wherein the final concentration of the recombinant sheep serum albumin is 5.0 mg/mL;
(b2) preparation of olanzapine derivative solution: mixing 250.0mg of the above olanzapine derivative, 7.5mL of dimethylformamide, 7.5mL of ethanol, 15.0mL of potassium phosphate buffer (10.0 mmol/L, pH = 8.0), 150.0mg of 1-ethyl-3- (-3-dimethylaminopropyl) carbodiimide, and 90.0mg of N-hydroxythiosuccinimide, and dissolving and reacting them with stirring for 3 hours to obtain an olanzapine derivative solution;
(b3) synthesis of olanzapine immunogen: dropwise adding the olanzapine derivative solution obtained in the step (b 2) into the carrier protein solution obtained in the step (b 1), stirring overnight at-4 ℃, and dialyzing to purify the olanzapine immunogen.
A fifth object of the invention is to provide an anti-olanzapine specific antibody.
The fifth purpose of the invention is realized by adopting the following technical scheme: an anti-olanzapine specific antibody is a specific antibody obtained after injection of the olanzapine immunogen to a test animal, wherein the test animal is one of rabbit, goat, sheep, mouse, rat, guinea pig or horse.
A sixth object of the present invention is to provide a method for preparing the anti-olanzapine specific antibody as described above.
The sixth purpose of the invention is realized by adopting the following technical scheme: a method for preparing an anti-olanzapine specific antibody as described above, comprising the steps of:
(C1) diluting the olanzapine immunogen with a phosphate buffer solution to obtain an olanzapine artificial antigen solution, mixing the olanzapine artificial antigen solution with an equal amount of Freund's complete adjuvant, and performing multi-point injection on the experimental animal;
(C2) after 3-6 weeks, mixing the same olanzapine artificial antigen solution with an equal amount of Freund incomplete adjuvant, performing multipoint injection on the experimental animal, and then injecting once every 3-6 weeks for 3-10 times in total;
(C3) and (4) taking blood from the experimental animal subjected to injection in the step (C2), separating and purifying to obtain the anti-olanzapine specific antibody.
Specifically, the preparation method of the anti-olanzapine specific antibody comprises the following steps:
(c1) diluting the olanzapine immunogen with 0.15mol/L sodium phosphate buffer solution (pH = 7.0) to a final concentration of 3.5mg/mL to obtain an artificial antigen solution, mixing the 3.0mL artificial antigen solution with an equal amount of Freund's complete adjuvant, and performing multi-point injection on the experimental animal rabbit;
(c2) after 4 weeks, carrying out multi-point injection on the experimental animal rabbit by using 3.0mL of the same artificial antigen solution and an equivalent amount of Freund's incomplete adjuvant, and then carrying out injection once every 5 weeks for 6 times in total;
(c3) and (c 2) blood is taken from the rabbit of the experimental animal which is injected in the step (c 2), and the blood is separated and purified to obtain the anti-olanzapine specific antibody.
A seventh object of the invention is to provide the use of an anti-olanzapine specific antibody as described above.
The seventh purpose of the invention is realized by adopting the following technical scheme: the application of the anti-olanzapine specific antibody is characterized in that the anti-olanzapine specific antibody is used for preparing an olanzapine detection reagent, and the olanzapine detection reagent comprises an olanzapine homogeneous enzyme immunoassay reagent and an olanzapine latex enhanced immunoturbidimetry detection reagent.
Preferably, the application of the anti-olanzapine specific antibody is characterized in that the olanzapine homogeneous enzyme immunoassay reagent consists of an R1 reagent and an R2 reagent, the R1 reagent comprises the anti-olanzapine specific antibody and an R1 buffer solution, and the R2 reagent comprises an olanzapine glucose-6-phosphate dehydrogenase labeled conjugate and an R2 buffer solution;
the R1 buffer solution contains an enzyme substrate, coenzyme, bovine serum albumin and Tris buffer solution, wherein the enzyme substrate is glucose-6-phosphate, and the coenzyme is oxidized nicotinamide adenine dinucleotide;
the olanzapine glucose-6-phosphate dehydrogenase labeled conjugate is formed by coupling olanzapine derivatives shown in the structural formula I and glucose-6-phosphate dehydrogenase; the structural formula is shown as formula III:
Figure DEST_PATH_IMAGE009
formula III;
the R2 buffer solution is Tris buffer solution containing bovine serum albumin.
Specifically, the preparation method of the olanzapine homogeneous enzyme immunoassay reagent comprises the following steps:
(D1) adding 250.0mg of bovine serum albumin, 250.0mg of glucose-6-phosphate and 50.0mg of oxidized nicotinamide adenine dinucleotide into 250mL of Tris buffer (50 mmol/L, pH = 8.5) in sequence, stirring and dissolving to prepare an R1 buffer solution, adding an anti-olanzapine specific antibody into the R1 buffer solution according to the volume ratio of 1: 1000, uniformly mixing, and adjusting the pH to 7.6 by using 1.0 mol/L hydrochloric acid to prepare an R1 reagent;
(D2) 250.0mg of bovine serum albumin was added to 250mL of Tris buffer (100 mmol/L, pH = 8.7) and dissolved with stirring to prepare an R2 buffer, and then the olanzapine glucose-6-phosphate dehydrogenase labeled conjugate was added to the above R2 buffer at a volume ratio of 1: 1000 and mixed, and then pH was adjusted to 8.0 with 1.0 mol/L hydrochloric acid to prepare a reagent R2.
The preparation method of the olanzapine glucose-6-phosphate dehydrogenase labeled conjugate comprises the following steps:
(E1) weighing 20.0 mg of glucose-6-phosphate dehydrogenase with activity unit of 200KU, dissolving the glucose-6-phosphate dehydrogenase in 50.0mL of sodium phosphate (100 mmol/L, pH = 8.0) buffer solution at room temperature, adding 150.0mg of reduced nicotinamide adenine dinucleotide, 75.0 mg of glucose-6-phosphate and 0.75 mL of carbitol, dropwise adding 2.5 mL of dimethyl sulfoxide, and stirring for dissolving to obtain a glucose-6-phosphate dehydrogenase solution;
(E2) weighing 15.0 mg of olanzapine derivative shown in the structural formula I in an anhydrous state, dissolving the olanzapine derivative into 500.0 muL of dimethylformamide, cooling the solution to 0 ℃, adding 4.5 muL of tributylamine, 2.5 muL of isobutyl chloroformate and 3.5 muL of L N, N' -dicyclohexylcarbodiimide, and stirring for 45 minutes at 0 ℃ to obtain an olanzapine derivative activation solution;
(E3) dropwise adding the olanzapine derivative activating solution into a glucose-6-phosphate dehydrogenase solution, stirring and reacting for 12 hours at the temperature of-4 ℃, and purifying by a G-25 gel chromatographic column after the reaction is finished to obtain an olanzapine glucose-6-phosphate dehydrogenase labeled conjugate.
Preferably, in the application of the anti-olanzapine specific antibody, the olanzapine latex enhanced immunoturbidimetry detection reagent consists of an L1 reagent and an L2 reagent;
the L1 reagent consists of the anti-olanzapine specific antibody, a buffer solution with the pH =8.0, bovine serum albumin, sodium chloride, Tween-20, glycerol, ethylene diamine tetraacetic acid, a coagulant and a preservative;
the L2 reagent consists of olanzapine-bovine serum albumin complex coated polystyrene latex particles, buffer solution with pH =8.0, bovine serum albumin, sodium chloride, Tween-20, glycerol, ethylene diamine tetraacetic acid and a preservative;
the olanzapine-bovine serum albumin complex is formed by coupling olanzapine derivatives shown in the structural formula I and bovine serum albumin, and the structural formula is shown in the formula IV:
Figure DEST_PATH_IMAGE010
a formula IV;
the diameter range of the polystyrene latex particles is 50-250 nm;
the buffer solution is one of phosphate buffer solution, glycine buffer solution, MES buffer solution, borate buffer solution, Tris-HCl buffer solution or barbital buffer solution;
the coagulant is one of PEG-4000, PEG-6000, PEG-8000 or dextran sodium sulfate;
the preservative is one of sodium azide, thimerosal, phenol or ethylmercuric sodium thiosulfate.
Specifically, the preparation method of the olanzapine latex enhanced immunoturbidimetric assay reagent comprises the following steps:
(F1) dissolving 5.0mL of an anti-olanzapine-specific antibody in 250.0mL of potassium phosphate buffer (50.0 mmol/L pH = 8.0), adding 100.0mg of bovine serum albumin, 25.0mg of sodium chloride, 250.0. mu.L of Tween-20, 250.0. mu.L of glycerol, 100.0. mu.L of ethylenediaminetetraacetic acid, 150.0. mu.L of PEG-4000 and 5.0mg of sodium azide, stirring the mixture uniformly, and adjusting the pH =7.3 to prepare a reagent L1;
(F2) adding 1.5mg of polystyrene latex particles with carboxyl groups on the surface and a diameter of 125nm into 15.0mL of MES buffer (50.0 mmol/L, pH = 7.0), then adding 5.0mg of carbodiimide, reacting at 25 ℃ for 3 hours to prepare a latex particle solution, diluting 1.2mg of olanzapine-bovine serum albumin complex with 7.5mL of borate buffer (50.0 mmol/L, pH = 9.2), immediately adding to the latex particle solution, reacting at 41 ℃ for 18 hours, then adding 3.0mL of glycine buffer (100.0 mmol/L, pH = 8.0), stirring for 3 hours, centrifuging after the reaction is terminated to remove the supernatant, washing the precipitate with 20.0mL of Tris-HCl buffer (50.0 mmol/L, pH = 8.0) for 3 times, then diluting the emulsion suspension with 50.0mL of glycine buffer (50.0 mmol/L, pH = 8.6) to form a gel, and finally adding 100.0mg of bovine serum albumin, 25.0mg of sodium chloride, 250.0 mu L of tween-20, 250.0 mu L of glycerol, 100.0 mu L of ethylenediamine tetraacetic acid and 5.0mg of sodium azide by mass fraction, and uniformly stirring to prepare the L2 reagent.
The preparation method of the olanzapine-bovine serum albumin complex comprises the following steps:
10.0mg of bovine serum albumin was diluted with 7.5mL of sodium phosphate buffer (100.0 mmol/L, pH = 7.5), then 100.0mg of olanzapine derivative represented by the above formula I was added, 50.0mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide was further added, and the mixture was reacted at 0 ℃ for 10 hours, followed by dialysis against 100.0mL of phosphate buffer (100.0 mmol/L, pH = 7.5) at-4 ℃ for 12 hours to obtain olanzapine-bovine serum albumin complex.
Compared with the prior art, the invention has the beneficial effects that:
1. the olanzapine derivative and the synthesis method thereof designed by the invention are targeted new design and research and do not exist in the prior art;
2. the recombinant sheep serum albumin obtained by genetic engineering transformation and the olanzapine derivative are coupled to obtain the olanzapine immunogen, the coupling efficiency is high, and the immunogenicity of the olanzapine immunogen is obviously improved. The anti-olanzapine specific antibody prepared by the olanzapine immunogen has strong specificity and high sensitivity, and does not have any cross reaction with 100 common medicines, so the anti-olanzapine specific antibody can be used for preparing an olanzapine detection reagent with higher accuracy, precision, sensitivity and specificity.
3. The two olanzapine detection reagents can realize high-flux and rapid detection of olanzapine on a full-automatic biochemical analyzer, can simultaneously detect a plurality of samples, have the advantages of simple and convenient operation, high sensitivity, strong specificity, accurate result and the like, can effectively reduce the olanzapine detection cost, and are favorable for clinical popularization and use.
Drawings
FIG. 1 is a standard curve for ELISA detection of olanzapine of example 4;
FIG. 2 is a calibration curve of the homogeneous enzyme immunoassay reagent for olanzapine of example 8;
FIG. 3 is a calibration curve of the olanzapine latex enhanced immunoturbidimetric assay reagent of example 10.
Detailed Description
The present invention will be further described with reference to the accompanying drawings, which are simplified schematic drawings illustrating only the basic structure of the present invention and showing only the constitution related to the present invention, and detailed embodiments. Unless otherwise specified, reagents, instruments, equipment, consumables used in the following examples are all available from a regular distributor.
Example 1: synthesis of olanzapine derivatives
Olanzapine derivatives are synthesized by the following synthetic route:
Figure 75344DEST_PATH_IMAGE002
；
the specific synthetic steps are as follows:
(1) synthesis of Compound 2:
Figure DEST_PATH_IMAGE004A
compound 1 (6 g, 26 mmol) and piperazine (18 g, 0.19 mol) were added to butanol (100 mL) at room temperature to prepare a reaction mixture, and the reaction mixture was stirred under heating at 125 ℃ for 18 hours. After the reaction was complete, the reaction mixture was concentrated in vacuo. The residue obtained by the concentration was purified by a flash chromatography (methanol: dichloromethane 0-10%) to obtain 6 g of compound 2 as a yellow solid.
(2) Synthesis of Compound 3:
Figure DEST_PATH_IMAGE006A
compound 2 (6 g, 20 mmol), tert-butyl 5-bromovalerate (5.7 g, 24 mmol), K2CO3(4.2 g, 28 mmol) and KI (0.3 g, 2 mmol) were added to acetonitrile (100 mL) at room temperature to prepare a reaction mixture. The reaction mixture was stirred at room temperature for 18 hours. After the reaction was complete, the reaction mixture was concentrated in vacuo. The residue obtained from the concentration was purified by flash chromatography (methanol: dichloromethane 0-5%) to obtain 5 g of compound 3 as a yellow solid.
(3) Synthesis of olanzapine derivatives:
Figure 182365DEST_PATH_IMAGE007
compound 3 (5 g, 11 mmol) and trifluoroacetic acid (5 mL) were dissolved together in DCM (45 mL) to prepare a reaction mixture, and the reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction mixture was concentrated in vacuo. The residue obtained by the concentration was purified by a flash chromatography (methanol: dichloromethane 0-5%) to obtain 2.5 g of olanzapine derivative.
Example 2: preparation of olanzapine immunogen
The preparation method of the olanzapine immunogen comprises the following specific steps:
(1) preparation of carrier protein solution: dissolving the recombinant sheep serum albumin in 0.35mol/L potassium phosphate buffer (pH = 8.5) to obtain a carrier protein solution, wherein the final concentration of the recombinant sheep serum albumin is 5.0 mg/mL;
(2) preparation of olanzapine derivative solution: mixing 250.0mg of the above olanzapine derivative, 7.5mL of dimethylformamide, 7.5mL of ethanol, 15.0mL of potassium phosphate buffer (10.0 mmol/L, pH = 8.0), 150.0mg of 1-ethyl-3- (-3-dimethylaminopropyl) carbodiimide, and 90.0mg of N-hydroxythiosuccinimide, and dissolving and reacting them with stirring for 3 hours to obtain an olanzapine derivative solution;
(3) synthesis of olanzapine immunogen: and (3) dropwise adding the olanzapine derivative solution obtained in the step (2) into the carrier protein solution obtained in the step (1), stirring overnight at-4 ℃, and dialyzing and purifying to obtain an olanzapine immunogen.
Example 3: preparation of anti-olanzapine specific antibody
The preparation method of the anti-olanzapine specific antibody comprises the following specific steps:
(1) diluting the olanzapine immunogen with 0.15mol/L sodium phosphate buffer solution (pH = 7.0) to a final concentration of 3.5mg/mL to obtain an artificial antigen solution, mixing the 3.0mL artificial antigen solution with an equal amount of Freund's complete adjuvant, and performing multi-point injection on the experimental animal rabbit;
(2) after 4 weeks, carrying out multi-point injection on the experimental animal rabbit by using 3.0mL of the same artificial antigen solution and an equivalent amount of Freund's incomplete adjuvant, and then carrying out injection once every 5 weeks for 6 times in total;
(3) and (3) taking blood from the experimental animal rabbit subjected to injection in the step (2), separating and purifying to obtain the anti-olanzapine specific antibody.
Example 4: ELISA method for testing performance of anti-olanzapine specific antibody
1. Establishment of standard curve for ELISA detection of olanzapine:
(1) preparation of a standard substance:
a1 mg/mL stock solution was prepared by dissolving pure olanzapine powder (purchased from Sigma) in methanol. The stock solutions were diluted sequentially with ELISA buffer to 100.00ng/mL, 50.00ng/mL, 25.00ng/mL, 12.50ng/mL, 6.25ng/mL, 0.00ng/mL of standard solution. Wherein, the ELISA buffer solution is prepared by 50.0mmol/L Tris buffer solution, 1.5 percent of NaCl by mass fraction and 0.25 percent of BSA by volume fraction.
(2) A standard curve was prepared using ELISA assay for olanzapine:
the anti-olanzapine specific antibody prepared in example 3 was diluted with potassium phosphate buffer (50.0 mmol/L, pH = 8.0) to a final concentration of 1: 10000, coated at 100 μ L/well on a 96-well enzyme-linked plate, and left at 4 ℃ for 18 hours; after the 96-well enzyme-linked plate coated with the anti-olanzapine-specific antibody was washed 3 times with potassium phosphate buffer, 200. mu.L/well of 0.5% volume fraction BSA solution was added and allowed to stand at 4 ℃ for 12 hours. Then, the mixture was washed 3 times with potassium phosphate buffer, and 20. mu.L/well of a standard solution was added. Then adding HRP-olanzapine conjugate with the working concentration of 100 mu L/hole; after incubation for 30min at room temperature, washing the plate for 5 times by using a potassium phosphate buffer solution; then 100. mu.L of TMB substrate was added to each well and incubated at room temperature for 30 min. Then, 100. mu.L of a stop solution (2.0 mol/L sulfuric acid) was added to each well. Absorbance at 450nm was measured using a microplate reader. The standard curve was prepared by calibration based on the absorbance at 450nm corresponding to each standard solution, and the results are shown in FIG. 1.
2. And (3) detecting the olanzapine content in the sample to be detected:
(1) preparing a sample to be tested:
the preparation method comprises the following steps: olanzapine pure powder (purchased from Sigma) was dissolved in methanol solution to make a 1.0mg/mL stock solution, and this stock solution was diluted in blank plasma to final concentrations of 0.00ng/mL, 12.50ng/mL, 25.00ng/mL, 50.00ng/mL to form blank, low, medium, and high concentration plasma samples, respectively. The blank plasma is healthy human plasma without olanzapine.
(2) The test method comprises the following steps:
and (3) testing the absorbance values of the blank, low, medium and high concentration plasma samples at 450nm by using the ELISA test method for olanzapine and replacing the blank, low, medium and high concentration plasma samples with the standard solution.
(3) And (3) testing results:
the olanzapine content in each sample was calculated and 3 duplicate wells were performed for each sample in comparison to the standard curve for ELISA test of olanzapine shown in figure 1, and the recovery was calculated based on the actual amount of olanzapine in the above samples, with the results shown in table 1.
TABLE 1 ELISA test results for olanzapine
Plasma sample Blank space Low value Median value of High value
Sample concentration (ng/mL) 0.00 12.50 25.00 50.00
Measurement 1 0.00 12.73 25.17 50.40
Measurement 2 0.00 12.51 24.87 49.88
Measurement 3 0.00 12.36 25.03 51.00
Mean value (ng/mL) 0.00 12.53 25.02 50.43
Recovery (%) - 100.27 100.09 100.85
From the results in table 1, it can be seen that: the recovery rate of olanzapine in samples with different concentrations is high and is 97-103% when the ELISA detection method of the olanzapine specific antibody is used for detecting the olanzapine in the samples, and the detection method is high in sensitivity and high in accuracy of detection results.
Example 5: interference test of 100 common drugs
Selecting 100 common medicines as interferents to perform interference test, preparing 100 pure medicine powders into a solution with the concentration of 100.0 mug/mL as an interferent sample to be tested, and detecting the concentration of the corresponding interferents by adopting the ELISA detection method of the embodiment 4, wherein the names and detection results of the 100 common medicines are detailed in the table 2.
TABLE 2 common drug interference test results
Serial number Name of Compound Actual detection value (ng/mL) Serial number Name of the Compound Actual detection value (ng/mL)
1 Aspirin 0.00 2 Phenylpropanolamine 0.00
3 Beta-phenylethylamines 0.00 4 Procaine amides 0.00
5 Amphetamines 0.00 6 Procaine 0.00
7 Ampicillin 0.00 8 Quinidine (quinidine) 0.00
9 Methotrexate (M) azepine 0.00 10 Zuomei acid 0.00
11 Chlorpromazine 0.00 12 Phenylephrine 0.00
13 Cloladronic acid 0.00 14 Cinnamyl Aikening 0.00
15 Dimethylphenoxy heptanoic acid 0.00 16 Ecgonine 0.00
17 Fenoprofen 0.00 18 Dixiyang 0.00
19 Methylamphetamine 0.00 20 Cotinine 0.00
21 Gentisic acid 0.00 22 Atenolol 0.00
23 Gemfibrozil 0.00 24 Xinde' an medicine 0.00
25 Hydrocodone 0.00 26 Phenylethylpiperidinone 0.00
27 Ibuprofen 0.00 28 Phenylbutyrophenones 0.00
29 Imipramine 0.00 30 Lysergic acid diethylamide 0.00
31 Diamino diphenyl sulfone 0.00 32 Cannabinol 0.00
33 Naproxen 0.00 34 Loperamide 0.00
35 Hydrochlorothiazide 0.00 36 Yikeshuling 0.00
37 Pertipidine 0.00 38 Phenylalanine (phenylalanine) 0.00
39 Allyl oxymorphone 0.00 40 Fluoxetine hydrochloride 0.00
41 Ephedrine 0.00 42 Salbutamol 0.00
43 Nicotinamide 0.00 44 Penicillin 0.00
45 Methylamirfurthion 0.00 46 Methyldiethanolamine 0.00
47 Pregabal 0.00 48 Dimethylene dioxyamphetamine 0.00
49 Methylenedioxyamphetamine 0.00 50 Doxylamine succinate 0.00
51 Tetrahydrocannabinol 0.00 52 Nalbuphine 0.00
53 Nystatin 0.00 54 Normorphine 0.00
55 Acetylmorphine 0.00 56 Oxycodone 0.00
57 Benzphetamine 0.00 58 Ketamin 0.00
59 Promethazine 0.00 60 Diphenhydramine 0.00
61 Aspartame 0.00 62 Phenylbutylamine 0.00
63 Aripiprazole 0.00 64 Fluconazole 0.00
65 Clozapine 0.00 66 Furosemide 0.00
67 Escitalopram 0.00 68 Gabapentin 0.00
69 Imatinib 0.00 70 Warfarin 0.00
71 Lamotrigine 0.00 72 Rosuvastatin 0.00
73 Linezolid 0.00 74 Acetaminophen 0.00
75 Risperidone 0.00 76 Sulpiride 0.00
77 Sertraline 0.00 78 Fluvoxamine 0.00
79 Topiramate 0.00 80 Fluoxetine 0.00
81 Venlafaxine 0.00 82 Ziprasidone derivatives 0.00
83 Voriconazole 0.00 84 Haloperidol 0.00
85 Levetiracetam 0.00 86 Imipenem 0.00
87 Oxcarbazepine 0.00 88 Axintinib 0.00
89 Zonisamide 0.00 90 Peprazole pani 0.00
91 Amitriptyline 0.00 92 Regorafenib 0.00
93 Chlorpromazine 0.00 94 Isoniazid 0.00
95 Medicine for treating multiple anxiety 0.00 96 Rifampicin 0.00
97 Paroxetine 0.00 98 Levofloxacin 0.00
99 Chloromycetin 0.00 100 Moxifloxacin hydrate 0.00
The measurement results show that: the concentration of the corresponding interferents is detected by the ELISA detection method of example 4, and the actual detection values of the 100 common drugs are all 0.00 ng/mL. Therefore, the anti-olanzapine specific antibody has stronger antigen recognition specificity and has no cross reaction with 100 common medicines.
Example 6: preparation of olanzapine homogeneous enzyme immunoassay reagent
The preparation method of the olanzapine homogeneous enzyme immunoassay reagent comprises the following specific steps:
(1) adding 250.0mg of bovine serum albumin, 250.0mg of glucose-6-phosphate and 50.0mg of oxidized nicotinamide adenine dinucleotide into 250mL of Tris buffer (50 mmol/L, pH = 8.5) in sequence, stirring and dissolving to prepare an R1 buffer solution, adding an anti-olanzapine specific antibody into the R1 buffer solution according to the volume ratio of 1: 1000, uniformly mixing, and adjusting the pH to 7.6 by using 1.0 mol/L hydrochloric acid to prepare an R1 reagent;
(2) 250.0mg of bovine serum albumin was added to 250mL of Tris buffer (100 mmol/L, pH = 8.7) and dissolved with stirring to prepare an R2 buffer, and then the olanzapine glucose-6-phosphate dehydrogenase labeled conjugate was added to the above R2 buffer at a volume ratio of 1: 1000 and mixed, and then pH was adjusted to 8.0 with 1.0 mol/L hydrochloric acid to prepare a reagent R2.
The preparation method of the olanzapine glucose-6-phosphate dehydrogenase labeled conjugate comprises the following steps:
(1) weighing 20.0 mg of glucose-6-phosphate dehydrogenase with activity unit of 200KU, dissolving the glucose-6-phosphate dehydrogenase in 50.0mL of sodium phosphate (100 mmol/L, pH = 8.0) buffer solution at room temperature, adding 150.0mg of reduced nicotinamide adenine dinucleotide, 75.0 mg of glucose-6-phosphate and 0.75 mL of carbitol, dropwise adding 2.5 mL of dimethyl sulfoxide, and stirring for dissolving to obtain a glucose-6-phosphate dehydrogenase solution;
(2) weighing 15.0 mg of olanzapine derivative synthesized in example 1 in an anhydrous state, dissolving the olanzapine derivative in 500.0 muL of dimethylformamide, cooling the solution to 0 ℃, adding 4.5 muL of tributylamine, 2.5 muL of isobutyl chloroformate and 3.5 muL of L N, N' -dicyclohexylcarbodiimide, and stirring for 45 minutes at 0 ℃ to obtain an olanzapine derivative activation solution;
(3) dropwise adding the olanzapine derivative activating solution into a glucose-6-phosphate dehydrogenase solution, stirring and reacting for 12 hours at the temperature of-4 ℃, and purifying by a G-25 gel chromatographic column after the reaction is finished to obtain an olanzapine glucose-6-phosphate dehydrogenase labeled conjugate.
Example 7: preparation of olanzapine calibrator and quality control product
(1) Preparation of a calibrator: adding olanzapine pure product powder into 6 parts of Tris-HCl buffer solution with the concentration of 50.0mmol/L and the pH =7.2 respectively, stirring and dissolving until the final concentration is 0.00ng/mL, 6.25ng/mL, 12.50ng/mL, 25.00ng/mL, 50.00ng/mL and 100.00ng/mL respectively, then adding sodium chloride with the mass fraction of 0.5%, bovine serum albumin with the mass fraction of 1.0%, ethylene diamine tetraacetic acid with the mass fraction of 0.75% and sodium azide with the mass fraction of 0.05% into each part of solution, and stirring uniformly to obtain an olanzapine calibrator (with 6 concentrations).
(2) Preparing a quality control product: respectively adding olanzapine pure powder into 4 parts of Tris-HCl buffer solution with the concentration of 50.0mmol/L and the pH =7.2, stirring and dissolving until the final concentrations are 0.00ng/mL, 12.50ng/mL, 25.00ng/mL and 50.00ng/mL respectively, then respectively adding sodium chloride with the mass fraction of 0.5%, bovine serum albumin with the mass fraction of 1.0%, ethylenediaminetetraacetic acid with the mass fraction of 0.75% and sodium azide with the mass fraction of 0.05% into each solution, and uniformly stirring to obtain the olanzapine quality control product (with the concentration of 4).
Example 8: preparation of olanzapine homogeneous enzyme immunoassay reagent calibration curve and quality control experiment
1. Preparing an olanzapine homogeneous enzyme immunoassay calibration curve:
placing an R1 reagent, an R2 reagent and a calibrator into a Merrill BS480 full-automatic biochemical analyzer, and then setting reaction parameters of the biochemical analyzer, wherein the specific parameters are detailed in Table 3; in the actual operation process, the volume ratio of the R1 reagent to the R2 reagent needs to be continuously adjusted, the light measuring point is adjusted at the same time, and finally, a homogeneous enzyme immunoassay calibration curve is automatically obtained by a biochemical analyzer, as shown in figure 2.
TABLE 3 Merrill BS480 full-automatic biochemical analyzer reaction parameter settings
Name of item Olanzapine
R1 reagent 160.0µL
R2 reagent 40.0µL
Sample size 10.0µL
Calibration method Two-point end point method
Dominant wavelength 340nm
Sub-wavelength 405nm
Reaction time
10 minutes
Incubation time 8 minutes
Reaction direction Rise up
Results ng/mL
Accuracy of results 0.01
Fitting method Line graph
Concentration of calibrator 0.00ng/mL、6.25ng/mL、12.50ng/mL、25.00ng/mL、50.00ng/mL、100.00ng/mL
2. Quality control quality detection experiment:
and (3) measuring the quality control products by using the olanzapine homogeneous enzyme immunoassay method, calculating the content of olanzapine in each quality control product according to the homogeneous enzyme immunoassay calibration curve prepared in the step (1), repeatedly measuring each quality control product for 10 times, and analyzing the detection results and data as shown in table 4.
TABLE 4 detection results and data analysis of olanzapine homogeneous enzyme immunoassay reagent
Quality control product Blank space Low value Median value High value
Concentration (ng/mL) 0.00 12.50 25.00 50.00
Test 1 0.00 12.52 25.87 50.50
Test 2 0.00 12.46 26.04 50.45
Test 3 0.00 12.67 25.03 51.00
Test 4 0.00 12.60 25.15 49.74
Test 5 0.00 12.40 25.26 48.95
Test 6 0.00 12.50 24.75 49.98
Test 7 0.00 12.59 24.92 50.01
Test 8 0.00 12.70 24.55 50.10
Test 9 0.00 12.71 25.41 50.24
Test 10 0.00 12.39 24.77 49.79
Mean value (ng/mL) 0.00 12.55 25.18 50.08
Standard Deviation (SD) / 0.12 0.48 0.55
Precision (CV%) / 0.94 1.93 1.09
Recovery (%) / 100.43 100.70 100.15
The experimental results show that: the CV values of olanzapine content in quality control products with different concentrations are lower than 5%, and the recovery rates are between 95% and 105%, which shows that the olanzapine homogeneous enzyme immunoassay reagent has high precision and accurate results in measuring the olanzapine content in biological samples.
Example 9: preparation of olanzapine latex enhanced immunoturbidimetry detection reagent
The preparation method of the olanzapine latex enhanced immunoturbidimetric assay reagent comprises the following steps:
(F1) dissolving 5.0mL of an anti-olanzapine-specific antibody in 250.0mL of potassium phosphate buffer (50.0 mmol/L pH = 8.0), adding 100.0mg of bovine serum albumin, 25.0mg of sodium chloride, 250.0. mu.L of Tween-20, 250.0. mu.L of glycerol, 100.0. mu.L of ethylenediaminetetraacetic acid, 150.0. mu.L of PEG-4000 and 5.0mg of sodium azide, stirring the mixture uniformly, and adjusting the pH =7.3 to prepare a reagent L1;
(F2) adding 1.5mg of polystyrene latex particles with carboxyl groups on the surface and a diameter of 125nm into 15.0mL of MES buffer (50.0 mmol/L, pH = 7.0), then adding 5.0mg of carbodiimide, reacting at 25 ℃ for 3 hours to prepare a latex particle solution, further adding 1.2mg of olanzapine-bovine serum albumin complex diluted with 7.5mL of borate buffer (50.0 mmol/L, pH = 9.2), immediately adding to the above latex particle solution, reacting at 41 ℃ for 18 hours, then adding 3.0mL of glycine buffer (100.0 mmol/L, pH = 8.0), stirring for 3 hours, centrifuging after the reaction is terminated to remove the supernatant, washing the precipitate with 20.0mL of HCl-buffer (50.0 mmol/L, pH = 8.0) for 3 times, then diluting the Tris suspension with 50.0mL of glycine buffer (50.0 mmol/L, pH = 8.6) to prepare a gel, and finally adding 100.0mg of bovine serum albumin, 25.0mg of sodium chloride, 250.0 mu L of tween-20, 250.0 mu L of glycerol, 100.0 mu L of ethylenediamine tetraacetic acid and 5.0mg of sodium azide by mass fraction, and uniformly stirring to prepare the L2 reagent.
The preparation method of the olanzapine-bovine serum albumin complex comprises the following steps:
10.0mg of bovine serum albumin was diluted with 7.5mL of sodium phosphate buffer (100.0 mmol/L, pH = 7.5), then 100.0mg of olanzapine derivative synthesized in example 1 was added, 50.0mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide was further added, and the mixture was reacted at 0 ℃ for 10 hours, followed by dialysis against 100.0mL of phosphate buffer (100.0 mmol/L, pH = 7.5) at-4 ℃ for 12 hours to obtain olanzapine-bovine serum albumin complex.
Example 10: preparation of olanzapine latex enhanced immunoturbidimetry detection reagent calibration curve and quality control experiment
1. Preparing a calibration curve of the olanzapine latex enhanced immunoturbidimetry detection reagent:
putting an L1 reagent, an L2 reagent and a calibrator into an olympus AU480 full-automatic biochemical analyzer, and setting reaction parameters of the biochemical analyzer, wherein the specific parameters are detailed in a table 5; in the actual operation process, the volume ratio of the L1 reagent to the L2 reagent needs to be continuously adjusted, the light measuring point is adjusted at the same time, and finally, a latex enhanced immunoturbidimetric assay calibration curve is automatically obtained by a biochemical analyzer, as shown in FIG. 3.
TABLE 5 fully automatic Biochemical Analyzer reaction parameters of Orlinbas AU480
Name of item Olanzapine
L1 reagent 160.0µL
L2 reagent 40.0µL
Sample size 10.0µL
Calibration method Two-point end point method
Dominant wavelength 570nm
Sub-wavelength 412nm
Reaction time
10 minutes
Incubation time 5 minutes
Reaction direction Descend
Results ng/mL
Accuracy of results 0.01
Fitting method Logit-log 4P
Concentration of calibrator 0.00ng/mL、6.25ng/mL、12.50ng/mL、25.00ng/mL、50.00ng/mL、100.00ng/mL
2. Quality control quality detection experiment:
and (3) determining the quality control products by using the latex enhanced turbidimetric immunoassay method, calculating the content of olanzapine in each quality control product according to the latex enhanced turbidimetric immunoassay calibration curve prepared in the step (1), repeatedly determining each quality control product for 10 times, and analyzing the detection results and data as shown in table 6.
TABLE 6 detection results and data analysis of olanzapine latex enhanced immunoturbidimetric reagent
Quality control product Blank space Low value Median value High value
Concentration (ng/mL) 0.00 12.50 25.00 50.00
Test 1 0.00 12.55 25.11 49.59
Test 2 0.00 12.57 24.98 50.45
Test 3 0.00 12.70 24.73 52.10
Test 4 0.00 12.50 25.87 49.43
Test 5 0.00 12.54 26.00 50.81
Test 6 0.00 12.32 25.03 49.49
Test 7 0.00 12.46 25.04 50.52
Test 8 0.00 12.38 24.59 49.70
Test 9 0.00 12.62 24.86 51.03
Test 10 0.00 12.66 24.90 50.20
Mean value (ng/mL) 0.00 12.53 25.11 50.33
Standard Deviation (SD) / 0.12 0.46 0.84
Precision (CV%) / 0.95 1.84 1.67
Recovery (%) / 100.24 100.44 100.66
The experimental results show that: the CV values of olanzapine content in quality control products with different concentrations are lower than 5%, and the recovery rates are between 95% and 105%, which shows that the olanzapine latex enhanced immunoturbidimetry detection reagent has high precision and accurate results in measuring the olanzapine content in a biological sample.
Various other changes and modifications to the above-described embodiments and concepts will become apparent to those skilled in the art, and all such changes and modifications are intended to be included within the scope of the present invention as defined in the appended claims.
Sequence listing
<110> Suzhou Boyuan medical science and technology Co., Ltd
<120> olanzapine derivative, immunogen, anti-olanzapine specific antibody, preparation method and application thereof
<130> 2020.12.13
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 607
<212> PRT
<213> Artificial Synthesis (Artificial Sequence)
<400> 1
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Leu Lys Phe Ser Ser Ala
1 5 10 15
Tyr Ser Arg Gly Val Phe Arg Arg Asp Thr His Lys Ser Glu Ile Ala
20 25 30
His Arg Phe Asn Asp Leu Gly Glu Glu Asn Phe Gln Gly Leu Val Leu
35 40 45
Ile Ala Phe Ser Gln Tyr Leu Lys Gln Cys Pro Phe Asp Glu His Val
50 55 60
Lys Leu Val Lys Glu Leu Thr Glu Phe Ala Lys Thr Cys Val Ala Asp
65 70 75 80
Glu Ser His Ala Gly Lys Asp Lys Ser Leu His Thr Leu Phe Gly Asp
85 90 95
Glu Leu Cys Lys Val Ala Thr Leu Arg Glu Thr Tyr Gly Asp Met Ala
100 105 110
Asp Lys Cys Glu Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Asn
115 120 125
His Lys Asp Lys Ser Pro Asp Leu Pro Lys Leu Lys Pro Glu Pro Asp
130 135 140
Thr Leu Cys Ala Glu Phe Lys Ala Asp Glu Lys Lys Phe Trp Gly Lys
145 150 155 160
Tyr Leu Tyr Glu Val Ala Arg Lys His Pro Tyr Phe Tyr Ala Pro Glu
165 170 175
Leu Leu Tyr Tyr Ala Asn Lys Tyr Asn Gly Val Phe Gln Glu Cys Lys
180 185 190
Gln Ala Glu Asp Lys Gly Ala Cys Leu Leu Pro Lys Ile Asp Ala Met
195 200 205
Arg Glu Lys Val Leu Ala Ser Ser Ala Arg Gln Arg Leu Lys Cys Ala
210 215 220
Ser Ile Gln Lys Phe Gly Glu Arg Ala Leu Lys Ala Trp Ser Val Ala
225 230 235 240
Arg Leu Ser Gln Lys Phe Pro Lys Ala Asp Phe Thr Asp Val Thr Lys
245 250 255
Ile Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys His Gly Asp
260 265 270
Lys Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys
275 280 285
Asp His Gln Asp Ala Leu Lys Ser Lys Leu Lys Glu Cys Lys Asp Lys
290 295 300
Pro Val Leu Glu Lys Ser His Cys Ile Ala Glu Val Asp Lys Asp Ala
305 310 315 320
Val Pro Glu Asn Leu Pro Pro Leu Thr Ala Asp Phe Ala Glu Asp Lys
325 330 335
Glu Val Cys Lys Asn Tyr Gln Glu Ala Lys Asp Val Phe Leu Gly Ser
340 345 350
Phe Leu Tyr Glu Tyr Ser Lys Arg His Pro Glu Tyr Ala Val Ser Val
355 360 365
Lys Leu Arg Leu Ala Lys Glu Tyr Glu Ala Thr Leu Glu Asp Cys Cys
370 375 380
Ala Lys Glu Asp Pro His Ala Cys Tyr Lys Thr Val Phe Asp Lys Leu
385 390 395 400
Lys His Leu Val Asp Glu Pro Gln Asn Leu Ile Lys Lys Asn Cys Glu
405 410 415
Leu Phe Glu Lys His Gly Glu Tyr Gly Phe Gln Asn Ala Leu Ile Val
420 425 430
Lys Tyr Thr Arg Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu
435 440 445
Ile Ser Arg Ser Leu Gly Lys Val Gly Thr Lys Cys Lys Ala Lys Pro
450 455 460
Glu Ser Glu Arg Met Pro Cys Thr Glu Asp Tyr Leu Ser Leu Ile Leu
465 470 475 480
Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Val
485 490 495
Thr Lys Lys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser
500 505 510
Asp Leu Thr Leu Asp Glu Thr Tyr Val Pro Lys Pro Phe Asp Glu Lys
515 520 525
Phe Lys Thr Phe His Ala Asp Ile Cys Thr Leu Pro Asp Thr Glu Lys
530 535 540
Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Lys Lys His Lys Pro
545 550 555 560
Lys Ala Thr Asp Glu Gln Leu Lys Thr Val Met Glu Asn Phe Val Ala
565 570 575
Phe Val Asp Lys Cys Cys Ala Ala Asp Lys Lys Glu Gly Cys Phe Val
580 585 590
Leu Glu Gly Pro Lys Leu Val Ala Ser Thr Gln Ala Ala Leu Ala
595 600 605

Claims (10)

1. An olanzapine derivative, which is characterized in that the structural formula is shown as the formula I:
Figure DEST_PATH_IMAGE001
formula I.
2. A process for the synthesis of olanzapine derivatives according to claim 1, wherein said process is represented by the formula:
Figure DEST_PATH_IMAGE002
。
3. an immunogen of olanzapine, wherein said immunogen of olanzapine is formed by linking an olanzapine derivative of claim 1 to a carrier protein, and the structural formula of said immunogen is represented by formula ii:
Figure DEST_PATH_IMAGE003
formula II;
wherein the carrier protein is recombinant sheep serum albumin.
4. The olanzapine immunogen of claim 3, wherein the amino acid sequence of the recombinant sheep serum albumin is as set forth in SEQ ID NO: 1 is shown.
5. A process for the preparation of an immunogen of olanzapine according to any one of claims 3-4, comprising the steps of:
(B1) preparation of carrier protein solution: dissolving recombinant sheep serum albumin as defined in any one of claims 3 to 4 in a phosphate buffer to obtain a carrier protein solution;
(B2) preparation of olanzapine derivative solution: mixing the olanzapine derivative according to claim 1 with dimethylformamide, ethanol, potassium phosphate buffer, 1-ethyl-3- (-3-dimethylaminopropyl) carbodiimide and N-hydroxythiosuccinimide, and dissolving with stirring to obtain an olanzapine derivative solution;
(B3) synthesis of olanzapine immunogen: and (4) adding the olanzapine derivative solution obtained in the step (B2) into the carrier protein solution obtained in the step (B1), stirring for reaction, and dialyzing for purification to obtain an olanzapine immunogen.
6. An antibody specific to olanzapine, which is obtained by injecting the olanzapine immunogen of any one of claims 3 to 4 into a test animal, wherein the test animal is one of rabbit, goat, sheep, mouse, rat, guinea pig or horse.
7. A method of preparing the antibody specific for olanzapine according to claim 6, comprising the steps of:
(C1) diluting olanzapine immunogen according to any one of claims 3-4 with phosphate buffer to obtain olanzapine artificial antigen solution, mixing the olanzapine artificial antigen solution with equal amount of Freund's complete adjuvant, and performing multi-point injection on experimental animal as described in claim 6;
(C2) after 3-6 weeks, mixing the same olanzapine artificial antigen solution with an equal amount of Freund incomplete adjuvant, performing multipoint injection on the experimental animal, and then injecting once every 3-6 weeks for 3-10 times in total;
(C3) and (4) taking blood from the experimental animal subjected to injection in the step (C2), separating and purifying to obtain the anti-olanzapine specific antibody.
8. The use of an antibody specific for olanzapine according to any one of claims 6-7, wherein said antibody specific for olanzapine is used for preparing an olanzapine detection reagent comprising an olanzapine homogeneous enzyme immunoassay reagent and an olanzapine latex enhanced immunoturbidimetric assay reagent.
9. The use of an antibody specific for olanzapine according to claim 8, characterized in that said olanzapine homogeneous enzyme immunoassay reagent consists of R1 reagent and R2 reagent, said R1 reagent comprises the anti-olanzapine specific antibody according to any one of claims 6 to 7 and R1 buffer, said R2 reagent comprises olanzapine glucose-6-phosphate dehydrogenase labeled conjugate and R2 buffer;
the R1 buffer solution contains an enzyme substrate, coenzyme, bovine serum albumin and a Tris buffer solution, wherein the enzyme substrate is glucose-6-phosphate, and the coenzyme is oxidized nicotinamide adenine dinucleotide;
the olanzapine glucose-6-phosphate dehydrogenase labeled conjugate is formed by coupling olanzapine derivative and glucose-6-phosphate dehydrogenase according to claim 1; the structural formula is shown as formula III:
Figure DEST_PATH_IMAGE004
formula III;
the R2 buffer solution is Tris buffer solution containing bovine serum albumin.
10. The use of an antibody specific for olanzapine according to claim 8, wherein said olanzapine latex enhanced immunoturbidimetry detection reagent consists of L1 reagent and L2 reagent;
the L1 reagent consists of the anti-olanzapine specific antibody of any one of claims 6-7, a buffer at pH =8.0, bovine serum albumin, sodium chloride, tween-20, glycerol, ethylenediaminetetraacetic acid, a coagulant, and a preservative;
the L2 reagent consists of olanzapine-bovine serum albumin complex coated polystyrene latex particles, buffer solution with pH =8.0, bovine serum albumin, sodium chloride, Tween-20, glycerol, ethylene diamine tetraacetic acid and a preservative;
the olanzapine-bovine serum albumin complex is formed by coupling olanzapine derivatives disclosed by claim 1 with bovine serum albumin, and the structural formula of the olanzapine-bovine serum albumin complex is shown as a formula IV:
Figure DEST_PATH_IMAGE005
a formula IV;
the diameter range of the polystyrene latex particles is 50-250 nm;
the buffer solution is one of phosphate buffer solution, glycine buffer solution, MES buffer solution, borate buffer solution, Tris-HCl buffer solution or barbital buffer solution;
the coagulant is one of PEG-4000, PEG-6000, PEG-8000 or dextran sodium sulfate;
the preservative is one of sodium azide, thimerosal, phenol or ethylmercuric sodium thiosulfate.
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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050080265A1 (en) * 2001-10-16 2005-04-14 Edgar Dale M. Treatment of CNS disorders using CNS target modulators
US20070043021A1 (en) * 2005-08-11 2007-02-22 Michael Solomon Olanzapine analogs and methods of use thereof
CN101636182A (en) * 2006-10-25 2010-01-27 雷蒙特亚特特拉维夫大学有限公司 Novel psychotropic agents having glutamate NMDA activity
WO2014031587A1 (en) * 2012-08-21 2014-02-27 Janssen Pharmaceutica Nv Haptens of olanzipine
CN103848847A (en) * 2012-12-04 2014-06-11 广东东阳光药业有限公司 Improved method for preparing olanzapine and crystal form II thereof
CN104755093A (en) * 2012-08-21 2015-07-01 奥索临床诊断有限公司 Antibodies to olanzapine haptens and use thereof
CN110938082A (en) * 2019-10-30 2020-03-31 杭州博拓生物科技股份有限公司 Olanzapine artificial antigen and preparation method thereof
CN110950820A (en) * 2019-11-06 2020-04-03 苏州博源医疗科技有限公司 Chlorpromazine derivative, preparation method thereof and chlorpromazine detection reagent

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050080265A1 (en) * 2001-10-16 2005-04-14 Edgar Dale M. Treatment of CNS disorders using CNS target modulators
US20070043021A1 (en) * 2005-08-11 2007-02-22 Michael Solomon Olanzapine analogs and methods of use thereof
CN101636182A (en) * 2006-10-25 2010-01-27 雷蒙特亚特特拉维夫大学有限公司 Novel psychotropic agents having glutamate NMDA activity
WO2014031587A1 (en) * 2012-08-21 2014-02-27 Janssen Pharmaceutica Nv Haptens of olanzipine
CN104755093A (en) * 2012-08-21 2015-07-01 奥索临床诊断有限公司 Antibodies to olanzapine haptens and use thereof
CN103848847A (en) * 2012-12-04 2014-06-11 广东东阳光药业有限公司 Improved method for preparing olanzapine and crystal form II thereof
CN110938082A (en) * 2019-10-30 2020-03-31 杭州博拓生物科技股份有限公司 Olanzapine artificial antigen and preparation method thereof
CN110950820A (en) * 2019-11-06 2020-04-03 苏州博源医疗科技有限公司 Chlorpromazine derivative, preparation method thereof and chlorpromazine detection reagent

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BUJACZ,A.ET AL.: "Chain A, Serum albumin，PDB: 4LUF_A", 《GENPEPT》, pages 1 - 3 *
SAMUEL C. MARTIN ET AL.: "Aminoquinoline-Rhodium(II) Conjugates as Src-Family SH3 Ligands", 《ACS MED. CHEM. LETT.》, vol. 10, pages 1382 - 31 *
李文标等: "高效液相色谱电化学检测法测定血清奥氮平浓度", 《药物分析杂志》, vol. 20, no. 05, pages 304 - 307 *

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