CN110950820B - Chlorpromazine derivative, preparation method thereof and chlorpromazine detection reagent - Google Patents
Chlorpromazine derivative, preparation method thereof and chlorpromazine detection reagent Download PDFInfo
- Publication number
- CN110950820B CN110950820B CN201911077366.8A CN201911077366A CN110950820B CN 110950820 B CN110950820 B CN 110950820B CN 201911077366 A CN201911077366 A CN 201911077366A CN 110950820 B CN110950820 B CN 110950820B
- Authority
- CN
- China
- Prior art keywords
- chlorpromazine
- solution
- compound
- buffer solution
- serum albumin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D279/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one sulfur atom as the only ring hetero atoms
- C07D279/10—1,4-Thiazines; Hydrogenated 1,4-thiazines
- C07D279/14—1,4-Thiazines; Hydrogenated 1,4-thiazines condensed with carbocyclic rings or ring systems
- C07D279/18—[b, e]-condensed with two six-membered rings
- C07D279/22—[b, e]-condensed with two six-membered rings with carbon atoms directly attached to the ring nitrogen atom
- C07D279/24—[b, e]-condensed with two six-membered rings with carbon atoms directly attached to the ring nitrogen atom with hydrocarbon radicals, substituted by amino radicals, attached to the ring nitrogen atom
- C07D279/28—[b, e]-condensed with two six-membered rings with carbon atoms directly attached to the ring nitrogen atom with hydrocarbon radicals, substituted by amino radicals, attached to the ring nitrogen atom with other substituents attached to the ring system
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
- G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Food Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a chlorpromazine derivative for detecting the content of chlorpromazine in a human biological sample and a preparation method thereof. The series of anti-chlorpromazine specific antibodies developed by utilizing the novel chlorpromazine derivative can be used for preparing various chlorpromazine immunological test reagents with high sensitivity, strong specificity and good detection effect. The invention also provides a preparation method and a corresponding application method of the 3 chlorpromazine immunoassay reagents. The 3 chlorpromazine immune detection method provided by the invention has the advantages of convenient operation, rapid detection, accurate result, high sensitivity and strong specificity, and can quantitatively detect the chlorpromazine content in human blood samples. Overcomes the defects of complex operation, low automation degree and the like of the chlorpromazine detection method in the prior art, and can effectively guide clinical individuation and reasonable medication.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a chlorpromazine derivative, a preparation method thereof and a chlorpromazine detection reagent.
Background
Chlorpromazine is representative medicine of phenothiazines, is antagonist of central nervous system dopamine receptor, has various pharmacological activities, and has a chemical structural formula shown in formula VIII:
chlorpromazine has the main functions of: treating psychosis, antiemetic, cryoanesthesia, artificial hibernation, treating heart failure, enhancing hypnotic, anesthetic, sedative, improving cardiovascular system function, and treating endocrine diseases. The medicine is easy to be absorbed by oral administration, but the absorption is irregular, and the individuals are quite different. The adverse reactions after taking the medicine are more, and the medicine mainly comprises: dry mouth, blurred vision, epigastric discomfort, debilitation, sleepiness, constipation, palpitations, breast enlargement, obesity, amenorrhea, hypotension, obstructive jaundice, hepatomegaly, extrapyramidal reactions, paralysis agitans syndrome, acute dystonia, akathisia, tardive dyskinesia, rash, contact dermatitis, exfoliative dermatitis, granulocytopenia, asthma, purpura, corneal cloudiness, elevated intraocular pressure, and the like. Therefore, the drug concentration of the patient taking chlorpromazine should be detected periodically.
The traditional chlorpromazine test method mainly comprises the following steps: high Performance Liquid Chromatography (HPLC) and liquid chromatography/tandem mass spectrometry (LC/MS-MS) and the like, which are complex to operate, slow in speed, and high in cost. The anti-chlorpromazine specific series antibody developed by utilizing the novel chlorpromazine derivative can be used for preparing various chlorpromazine immunoassay reagents with high sensitivity, strong specificity and good detection effect, and can effectively overcome the defects of the traditional method. The 3 chlorpromazine detection reagents can quantitatively detect the content of chlorpromazine, and effectively guide clinical individuation and reasonable medication. Compared with the traditional methods such as HPLC, LC/MS-MS and the like, the immunodetection method provided by the invention has the advantages of simple operation, rapid detection, accurate result, low cost and the like, is favorable for large-scale clinical popularization and application in the future, and has good application prospect especially for primary hospitals lacking expensive instruments.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention adopts the following technical scheme:
1. provides a chlorpromazine derivative which is a novel synthetic substance and does not exist in the natural world, and the structural formula of the chlorpromazine derivative is shown as formula I:
2. the synthesis method of the chlorpromazine derivative is different from the conventional synthesis method, has good synthesis effect, and remarkably improves the synthesis efficiency of the chlorpromazine derivative, and the specific synthesis route is as follows:
3. providing a chlorpromazine homogeneous enzyme immunoassay reagent, wherein the detection reagent consists of an R1 reagent and an R2 reagent, the R1 reagent comprises an anti-chlorpromazine specific antibody 1 and an R1 buffer solution, and the R2 reagent comprises a chlorpromazine glucose-6-phosphate dehydrogenase labeled conjugate and an R2 buffer solution; the anti-chlorpromazine specific antibody 1 is an antibody produced after an experimental animal is immunized by chlorpromazine chicken ovalbumin immunogen; the chlorpromazine chicken egg albumin immunogen is formed by coupling the chlorpromazine derivative and chicken egg albumin, and the structural formula of the chlorpromazine chicken egg albumin immunogen is shown as formula II:
the experimental animal is any one of rabbits, goats, mice, sheep, guinea pigs or horses;
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 nicotinamide adenine dinucleotide oxide;
the chlorpromazine glucose-6-phosphate dehydrogenase labeled conjugate is formed by coupling the chlorpromazine derivative and glucose-6-phosphate dehydrogenase; the structural formula is shown in formula III:
the R2 buffer solution is Tris buffer solution containing bovine serum albumin.
4. The preparation method of the chlorpromazine homogeneous enzyme immunoassay reagent comprises the following steps:
(1) Sequentially adding 0.25% bovine serum albumin, 50mmol/L glucose-6-phosphoric acid and 50mmol/L nicotinamide adenine dinucleotide into 50mmol/L Tris buffer solution, stirring and dissolving to prepare an R1 buffer solution, adding an anti-chlorpromazine specific antibody 1 into the R1 buffer solution according to the volume ratio of 1:500-1:5000, uniformly mixing, and regulating the pH to 8.0 by using 6mol/L hydrochloric acid to prepare an R1 reagent;
(2) Adding 0.25% bovine serum albumin into 100mmol/L Tris buffer solution, stirring and dissolving to prepare an R2 buffer solution, adding chlorpromazine glucose-6-phosphate dehydrogenase labeled conjugate into the R2 buffer solution according to the volume ratio of 1:1000-1:8000, uniformly mixing, and regulating the pH value to 7.6 by using 6mol/L hydrochloric acid to prepare an R2 reagent;
the preparation method of the anti-chlorpromazine specific antibody 1 comprises the following steps:
1) Diluting chlorpromazine chicken ovalbumin immunogen to a final concentration of 0.5-5.0mg/mL by using phosphate buffer;
2) Performing immune injection on experimental animals by using a Freund adjuvant method, extracting animal blood after injection for 3-8 times, and separating and purifying antiserum to obtain an anti-chlorpromazine specific antibody 1;
the preparation method of the chlorpromazine chicken egg albumin immunogen comprises the following steps:
(1) dissolving 100-300mg of chicken egg white albumin in 10-100ml of phosphate buffer solution with the concentration of 0.2mol/L and the pH of 8.5 to prepare carrier protein solution;
(2) dissolving 50-500mg of chlorpromazine derivative with 1.0-5.0ml of organic solvent A, activating by a coupling activator, performing coupling reaction with the carrier protein solution prepared in the step (1), and obtaining the chlorpromazine chicken ovalbumin immunogen with immunogenicity through dialysis and purification after the reaction is finished;
the organic solvent A is any one of dimethyl sulfoxide, dimethylformamide, isopropanol, methanol or ethanol;
the preparation method of the chlorpromazine glucose-6-phosphate dehydrogenase labeled conjugate comprises the following steps:
<1> weighing glucose-6-phosphate dehydrogenase, dissolving in phosphate buffer solution with pH of 8.0 at room temperature to make its final concentration 2-6mg/mL, and making into enzyme solution;
<2> dissolving the chlorpromazine derivative with the organic solvent A to a final concentration of 10-50mg/mL, activating with a coupling activator, performing a coupling reaction with the enzyme solution prepared in the step <1>, and purifying by dialysis after the reaction is completed to obtain a chlorpromazine glucose-6-phosphate dehydrogenase labeled conjugate;
the coupling activator is any one of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N' -dicyclohexylcarbodiimide, N-hydroxysuccinimide, tributylamine, glutaraldehyde, a diisocyanate compound or dinitrobenzene dihalide.
5. The application method of the chlorpromazine homogeneous enzyme immunoassay reagent is provided, and comprises the following operation steps:
adding a calibrator and an R1 reagent into a full-automatic biochemical analyzer, uniformly mixing, and incubating for 3-5 minutes at 37 ℃; adding the R2 reagent, uniformly mixing, and after keeping the temperature at 37 ℃ for 5-10 minutes, detecting the main wavelength at 340 nm/the secondary wavelength at 405nm, continuously monitoring the absorbance change rate within 3 minutes, and preparing a calibration curve by a full-automatic biochemical analyzer;
adding a sample to be detected and an R1 reagent into a full-automatic biochemical analyzer, uniformly mixing, and incubating for 3-5 minutes at 37 ℃; adding the R2 reagent, uniformly mixing, and detecting 340nm main wavelength/405 nm secondary wavelength after the temperature is kept constant for 5-10 minutes at 37 ℃, continuously monitoring the absorbance change rate within 3 minutes, and automatically calculating the chlorpromazine content in a sample to be detected by a full-automatic biochemical analyzer according to the calibration curve manufactured in the step (one);
the reagent R1 and the reagent R2 are used according to the volume ratio of 1:1-4:1; the sample to be tested is any one of serum, plasma, urine, saliva, tissue fluid or cerebrospinal fluid.
6. Provided is a chlorpromazine enzyme-linked immunosorbent (ELISA) detection reagent, which comprises: anti-chlorpromazine specific antibody 2, chlorpromazine horseradish peroxidase labeled conjugate and reaction substrate;
the anti-chlorpromazine specific antibody 2 is an antibody produced after an experimental animal is immunized by chlorpromazine bovine serum albumin immunogen; the chlorpromazine bovine serum albumin immunogen is formed by coupling the chlorpromazine derivative and bovine serum albumin, and the structural formula of the chlorpromazine bovine serum albumin immunogen is shown as formula IV:
the experimental animal is any one of rabbits, goats, mice, sheep, guinea pigs or horses;
the chlorpromazine horseradish peroxidase labeled conjugate is formed by coupling the chlorpromazine derivative and horseradish peroxidase; the structural formula is shown in formula V:
the reaction substrate is 3,3', 5' -tetramethyl benzidine;
the preparation method of the anti-chlorpromazine specific antibody 2 comprises the following steps:
A. diluting chlorpromazine bovine serum albumin immunogen to a final concentration of 0.5-5.0mg/mL using phosphate buffer;
B. performing immune injection on experimental animals by using a Freund adjuvant method, extracting animal blood after injection for 3-8 times, and separating and purifying antiserum to obtain an anti-chlorpromazine specific antibody 2;
the preparation method of the chlorpromazine bovine serum albumin immunogen comprises the following steps:
a. dissolving 100-300mg of bovine serum albumin in 10-100ml of phosphate buffer solution with the concentration of 0.2mol/L and the pH of 8.5 to prepare carrier protein solution;
b. dissolving 50-500mg of chlorpromazine derivative with 1.0-5.0ml of organic solvent A, activating by a coupling activator, performing coupling reaction with the carrier protein solution prepared in the step a, and dialyzing and purifying after the reaction is finished to obtain chlorpromazine bovine serum albumin immunogen with immunogenicity;
the organic solvent A is any one of dimethyl sulfoxide, dimethylformamide, isopropanol, methanol or ethanol;
the preparation method of the chlorpromazine horseradish peroxidase labeled conjugate comprises the following steps:
(a) Weighing horseradish peroxidase, dissolving the horseradish peroxidase in a phosphate buffer solution with the pH of 8.0 at room temperature to ensure that the final concentration is 2-6mg/mL, and preparing an enzyme solution;
(b) Dissolving the chlorpromazine derivative by using the organic solvent A to ensure that the final concentration is 10-50mg/mL, activating by using a coupling activator, performing a coupling reaction with the enzyme solution prepared in the step (a), and obtaining the chlorpromazine horseradish peroxidase-labeled conjugate by dialysis and purification after the reaction is finished;
the coupling activator is any one of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N' -dicyclohexylcarbodiimide, N-hydroxysuccinimide, tributylamine, glutaraldehyde, a diisocyanate compound or dinitrobenzene dihalide.
7. The application method of the chlorpromazine ELISA detection reagent comprises the following operation steps:
diluting anti-chlorpromazine specific antibody 2 with phosphate buffer solution according to the proportion of 1:1000-1:20000 to prepare antibody solution, coating the antibody solution on a 96-well enzyme-linked plate according to the dosage of 100 mu L/well, and standing overnight at 4 ℃;
(ii) after 3 washes with phosphate buffer, 200. Mu.L/well of 0.5% bovine serum albumin solution was added, blocked overnight at 4℃and washed 3 times with phosphate buffer;
(iii) adding 20. Mu.L/well of calibrator and sample to be tested;
(iv) adding 100. Mu.L/well of the chlorpromazine horseradish peroxidase-labeled conjugate;
(v) incubating for 30 minutes at room temperature, washing the plate 5 times with phosphate buffer;
(vi) adding 100. Mu.L of 3,3', 5' -tetramethylbenzidine to each well, and incubating for 30 minutes at room temperature;
(vii) 100. Mu.L sulfuric acid at a concentration of 2mol/L was added to each well to terminate the reaction;
(viii) measuring the absorbance at 450nm using an microplate reader;
(ix) preparing a calibration curve according to the light absorption values of the calibrator with different concentrations, and calculating the content of chlorpromazine in the sample to be measured according to the calibration curve and the light absorption value of the sample to be measured;
the sample to be tested is any one of serum, plasma, urine, saliva, tissue fluid or cerebrospinal fluid.
8. Provided is a chlorpromazine latex-enhanced turbidimetric immunoassay reagent comprising: an L1 reagent and an L2 reagent;
the L1 reagent consists of an anti-chlorpromazine specific antibody 3, a buffer solution with pH value of 8.0, bovine serum albumin, sodium chloride, tween-20, glycerol, ethylenediamine tetraacetic acid, a coagulant and a preservative;
the L2 reagent consists of polystyrene latex particles coated by chlorpromazine-human serum albumin complex, buffer solution with pH value of 8.0, bovine serum albumin, sodium chloride, tween-20, glycerol, ethylenediamine tetraacetic acid and preservative;
the anti-chlorpromazine specific antibody 3 is an antibody produced after an experimental animal is immunized by chlorpromazine hemocyanin immunogen; the chlorpromazine hemocyanin immunogen is formed by coupling the chlorpromazine derivative and hemocyanin, and the structural formula of the chlorpromazine hemocyanin immunogen is shown in a formula VI:
the experimental animal is any one of rabbits, goats, mice, sheep, guinea pigs or horses;
the chlorpromazine-human serum albumin complex is formed by coupling the chlorpromazine derivative and human serum albumin, and the structural formula of the chlorpromazine-human serum albumin complex is shown as formula VII:
the diameter of the polystyrene latex particles ranges from 50nm to 250nm;
the buffer solution is any 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 any one of PEG-4000, PEG-6000, PEG-8000 or dextran sodium sulfate;
the preservative is any one of sodium azide, thimerosal, phenol or ethyl mercury sodium thiosulfate.
9. The preparation method of the chlorpromazine latex enhanced turbidimetric immunoassay reagent comprises the following steps:
dissolving 0.5mg/mL of anti-chlorpromazine specific antibody 3 in 50mmol/L of phosphate buffer solution with pH=8.0, then adding 0.5% -2.5% of bovine serum albumin, 0.25% -1.0% of sodium chloride, 0.1% -0.5% of tween-20, 1.0% -5.0% of glycerol, 0.1% -1.0% of ethylenediamine tetraacetic acid, 0.5% -2.5% of PEG-4000 and 0.01% -0.1% of sodium azide, uniformly stirring, and adjusting pH=7.5 to prepare an L1 reagent;
adding 0.5mg of polystyrene latex particles with carboxyl groups on the surface and with the diameter of 150nm into 4.5mL of 0.05mol/L of MES buffer solution with the pH value of 6.2, adding 5mg of carbodiimide, reacting for 1 hour at 37 ℃ to prepare a latex particle solution, diluting 0.5mg of chlorpromazine-human serum albumin complex with 4.5mL of borate buffer solution with the pH value of 0.05mol/L of 9.2, immediately adding the mixture into the latex particle solution, reacting for 12 hours at 37 ℃, adding 1mL of glycine buffer solution with the pH value of 0.1mol/L of 8.5, stirring for 2 hours, centrifuging to remove supernatant after the reaction is finished, washing the precipitate with 10mL of Tris-HCl buffer solution with the pH value of 50mmol/L of 8.0, diluting the mixture with 25mL of glycine buffer solution with the pH value of 50mmol/L of 8.5 to prepare a latex suspension, and finally adding 0.5% -2.5% of bovine serum albumin, 0.25% -1.0% of sodium chloride, 0.5% of Tween and 0.1% -0.0.5% of sodium chloride, stirring for 0% -1.01% -2.0% of tetramine;
the preparation method of the anti-chlorpromazine specific antibody 3 comprises the following steps:
(A) Diluting chlorpromazine hemocyanin immunogen to a final concentration of 0.5-5.0mg/mL by using phosphate buffer;
(B) Performing immune injection on experimental animals by using a Freund adjuvant method, extracting animal blood after injection for 3-8 times, and separating and purifying antiserum to obtain an anti-chlorpromazine specific antibody 3;
the preparation method of the chlorpromazine hemocyanin immunogen comprises the following steps:
< a > dissolving 100-300mg of hemocyanin in 10-100ml of phosphate buffer solution with concentration of 0.2mol/L and pH of 8.5 to prepare carrier protein solution;
dissolving 50-500mg of chlorpromazine derivative with 1.0-5.0ml of organic solvent A, activating by a coupling activator, performing coupling reaction with the carrier protein solution prepared in the step < a >, and obtaining the chlorpromazine hemocyanin immunogen with immunogenicity through dialysis and purification after the reaction is finished;
the organic solvent A is any one of dimethyl sulfoxide, dimethylformamide, isopropanol, methanol or ethanol;
the preparation method of the chlorpromazine-human serum albumin complex comprises the following steps:
diluting 10mg of human serum albumin with 5mL of 0.1mol/L phosphate buffer solution with pH=7.8, adding 100mg of chlorpromazine derivative, adding 50mg of coupling activator, reacting at 4 ℃ for 12 hours, and dialyzing with 0.1mol/L phosphate buffer solution with pH=7.8 at 4 ℃ for 18 hours to obtain chlorpromazine-human serum albumin complex;
the coupling activator is any one of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N' -dicyclohexylcarbodiimide, N-hydroxysuccinimide, tributylamine, glutaraldehyde, a diisocyanate compound or dinitrobenzene dihalide.
The using method of the chlorpromazine latex enhanced turbidimetric immunoassay reagent is basically the same as that of the chlorpromazine homogeneous enzyme immunoassay reagent.
The calibrator consists of chlorpromazine with the concentration of 0ng/ml, 20ng/ml, 40ng/ml, 80ng/ml, 160ng/ml and 320ng/ml respectively, and is composed of 0.1-1.0% of sodium chloride, 0.2-2.0% of bovine serum albumin, 0.25-1.5% of ethylenediamine tetraacetic acid, 0.01% -0.1% of sodium azide and 50mmol/L of Tris-HCl buffer solution with pH=7.2.
The series of anti-chlorpromazine specific antibodies developed by utilizing the novel chlorpromazine derivative can be used for preparing various chlorpromazine immunoassay reagents with high sensitivity, strong specificity and good detection effect. The invention also provides a preparation method and a corresponding application method of the 3 chlorpromazine immunoassay reagents. The 3 chlorpromazine immune detection method provided by the invention has the advantages of convenient operation, rapid detection, accurate result, high sensitivity and strong specificity, and can quantitatively detect the chlorpromazine content in samples such as human serum and plasma. Overcomes the defects of complex operation, low automation degree and the like of the chlorpromazine detection method in the prior art, and can effectively guide clinical individuation and reasonable medication.
Drawings
FIG. 1 is a calibration curve for a chlorpromazine homogeneous enzyme immunoassay reagent;
FIG. 2 is a calibration curve for chlorpromazine ELISA detection reagents;
FIG. 3 is a calibration curve for chlorpromazine latex-enhanced turbidimetric immunoassay reagents.
Detailed Description
The invention will be further described with reference to the accompanying drawings and detailed description, which are simplified schematic illustrations of the basic structure of the invention, which are presented solely by way of illustration, and thus showing only the structures that are relevant to the invention. Unless otherwise indicated, reagents, instruments, equipment, consumables used in the following examples were purchased from regular vendors.
EXAMPLE 1 Synthesis of chlorpromazine derivatives
The chemical structure of chlorpromazine derivative is shown as formula I:
the specific route of the synthetic method of chlorpromazine derivatives is as follows:
the specific synthesis steps are as follows:
1. synthesis of Compound 3
10g of Compound 1 was dissolved in 120mL of DMF, then 5.15-g t-BuOK was added to prepare a reaction solution, then this reaction solution was stirred at 60℃for 1 hour, after completion of stirring, the reaction solution was cooled to 25℃and then 12g of Compound 2 was added, then the mixture was stirred at room temperature overnight, 200mL of purified water was added after completion of stirring, then the reacted solution was extracted with 300mL of DCM, 3 times repeated, the organic phases obtained by the extraction were combined and concentrated, and the concentrated product was purified by a silica gel column (PE: EA=20:1) to give 2.6g of Compound 3 as a yellow solid in 15% yield.
The yellow solid compound 3 was scanned by nuclear magnetic resonance spectroscopy using Bruker Avance III plus MHz and VARIAN MERCURY plus M, using TMS as an internal standard, as follows: 1 H-NMR(400MHz,CDCl 3 ):δ6.84-7.19(m,7H),4.77(brs,1H),3.90(t,2H),3.22(m,2H),1.98(m,2H),1.23(s,9H)。
2. synthesis of Compound 4
10g of Compound 3 was dissolved in 127mL of DMFAfter the addition of 4.88g g t-BuOK and 6.5g CH 3 The reaction solution was prepared, and then this reaction solution was stirred at 60 ℃ for 12 hours, 400mL of purified water was added after the completion of the stirring, and then the reacted solution was extracted with 200mL of DCM, repeated 3 times, the extracted organic phases were combined and concentrated, and the concentrated product was purified by a silica gel column (PE: ea=50:1), to give 8.04g of compound 4 as a yellow oil in 91.3% yield.
The yellow oily compound 4 was scanned by nuclear magnetic resonance spectroscopy using Bruker Avance III plus MHz and VARIAN MERCURY plus M, using TMS as an internal standard, as follows: 1 H-NMR(400MHz,CDCl 3 ):δ6.80-7.14(m,7H),3.82(t,2H),3.31(m,2H),2.78(s,3H),2.01(m,2H),1.40(s,9H)。
3. synthesis of Compound 5
12g of Compound 4 was added to 80mL of a solution of 3mol/L HCl in EA to prepare a reaction solution, which was then stirred at 25℃for 4 hours, and the stirred reaction solution was concentrated under reduced pressure to obtain 8.5g of Compound 5 as a yellow solid in 84.2% yield.
The yellow solid compound 5 was scanned by nuclear magnetic resonance spectroscopy using Bruker Avance III plus MHz and VARIAN MERCURY plus M, using TMS as an internal standard, as follows: 1 H-NMR(400MHz,CDCl 3 ):δ6.80-7.18(m,7H),3.92(t,2H),2.70(t,2H),2.40(s,3H),1.99(m,2H)。
4. synthesis of Compound 7
5.93g of Compound 5, 3.94, g t-BuOK and 4.32g of Compound 6 were added to 75mL of DMF to prepare a reaction solution, which was stirred at 50℃for 3 hours, then 800mL of purified water, 3mL of TEA were added, and the reacted solution was used300mL EA extraction, repeated 3 times, and the extracted organic phases combined, passed through Na 2 SO 4 Dried and concentrated, and the concentrated product was purified by a silica gel column (PE: ea=10:1) to give 4.6g of compound 7 as a yellow solid in 67.6% yield.
The yellow solid compound 7 was scanned by nuclear magnetic resonance spectroscopy using Bruker Avance III plus MHz and VARIAN MERCURY plus M, using TMS as an internal standard, as follows: 1 H-NMR(400MHz,CDCl3):δ6.87-7.15(m,7H),4.15(m,2H),3.91(m,2H),3.21(t,2H),2.63(t,2H),2.35(s,3H),1.94(m,2H),1.27(m,3H)。
5. synthesis of chlorpromazine derivatives
10g of Compound 7 was dissolved in a mixed solvent of 57mL of THF and 22.8mL of purified water, 1.18g of NaOH was then added, the mixture was stirred at room temperature overnight, the pH of the reacted solution was adjusted to 5-6 with 1mol/L of HCI, 20mL of purified water was then added, and the reacted solution was reacted with 100mL of CH 2 Cl 2 The extraction was repeated 3 times, the extracted organic phases were combined and extracted over Na 2 SO 4 Dried and concentrated, and the concentrated product was passed through a silica gel column (CH 2 Cl 2 Meoh=10:1) to give 3.4g of a yellow solid chlorpromazine derivative in 63.9% yield.
Nuclear magnetic resonance spectroscopy scans were performed on the yellow solid chlorpromazine derivative using Bruker Avance III plus MHz and VARIAN MERCURY plus M, using TMS as an internal standard, with the following results: 1 H-NMR(400MHz,DMSO):δ6.98-7.22(m,7H),3.92(t,2H),3.20(s,2H),2.78(t,2H),2.42(s,3H),1.87(m,2H)。
example 2 preparation of chlorpromazine homogeneous enzyme immunoassay reagent
The preparation method of the chlorpromazine homogeneous enzyme immunoassay reagent comprises the following specific steps:
(1) Sequentially adding 0.25% bovine serum albumin, 50mmol/L glucose-6-phosphoric acid and 50mmol/L nicotinamide adenine dinucleotide into 50mmol/L Tris buffer solution, stirring and dissolving to prepare an R1 buffer solution, adding an anti-chlorpromazine specific antibody 1 into the R1 buffer solution according to the volume ratio of 1:1500, uniformly mixing, and regulating the pH value to 8.0 by using 6mol/L hydrochloric acid to prepare an R1 reagent;
(2) Adding 0.25% bovine serum albumin into 100mmol/L Tris buffer solution, stirring and dissolving to prepare an R2 buffer solution, adding chlorpromazine glucose-6-phosphate dehydrogenase marked conjugate into the R2 buffer solution according to the volume ratio of 1:3000, uniformly mixing, and regulating the pH value to 7.6 by using 6mol/L hydrochloric acid to prepare the R2 reagent.
The preparation method of the anti-chlorpromazine specific antibody 1 comprises the following steps:
1) Diluting chlorpromazine chicken ovalbumin immunogen to a final concentration of 2.5mg/mL using phosphate buffer;
2) Injecting the rabbit of the experimental animal by using a Freund adjuvant method, extracting the blood of the rabbit after 5 times of injection, and separating and purifying antiserum to obtain an anti-chlorpromazine specific antibody 1;
the preparation method of the chlorpromazine chicken egg albumin immunogen comprises the following steps:
(1) 200mg of chicken ovalbumin is dissolved in 50ml of phosphate buffer solution with the concentration of 0.2mol/L and the pH value of 8.5 to prepare carrier protein solution;
(2) 300mg of the chlorpromazine derivative described in example 1 was dissolved with 3.0ml of dimethyl sulfoxide, activated by N-hydroxysuccinimide and subjected to a coupling reaction with the carrier protein solution prepared in step (1), and after the reaction was completed, the chlorpromazine chicken ovalbumin immunogen having immunogenicity was obtained by dialysis and purification.
The preparation method of the chlorpromazine glucose-6-phosphate dehydrogenase labeled conjugate comprises the following steps:
<1> weighing glucose-6-phosphate dehydrogenase, dissolving in phosphate buffer solution with pH of 8.0 at room temperature to make its final concentration 4.5mg/mL, and preparing into enzyme solution;
<2> the chlorpromazine derivative described in example 1 was dissolved to a final concentration of 25mg/mL using dimethylformamide, activated by N, N' -dicyclohexylcarbodiimide, and subjected to a coupling reaction with the enzyme solution prepared in step <1>, and after the completion of the reaction, purified by dialysis to obtain a chlorpromazine glucose-6-phosphate dehydrogenase labeled conjugate.
EXAMPLE 3 preparation of chlorpromazine calibrator
Adding chlorpromazine pure powder into 5 parts of Tris-HCl buffer solution with the concentration of 50mmol/L and the pH value of 7.2, stirring and dissolving until the final concentration is 0ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 160ng/mL and 320ng/mL respectively, and then adding sodium chloride with the mass fraction of 0.5%, bovine serum albumin with the mass fraction of 1.0%, ethylenediamine tetraacetic acid with the mass fraction of 0.75% and sodium azide with the mass fraction of 0.05% into each part of solution respectively, and stirring uniformly to obtain chlorpromazine calibrator (6 concentrations).
Example 4 preparation of a calibration Curve for a chlorpromazine homogeneous enzyme immunoassay reagent and quality control experiments
1. Preparing a homogeneous enzyme immunoassay calibration curve:
placing an R1 reagent, an R2 reagent and a calibrator in a full-automatic biochemical analyzer of the Michael BS480, and then setting reaction parameters of the biochemical analyzer, wherein the specific parameters are shown in a table 1; in the actual operation process, the volume ratio of the R1 reagent and the R2 reagent is required 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 1.
TABLE 1 reaction parameters of Micorebs 480 full-automatic biochemical analyzer
2. Quality control experiment:
the chlorpromazine pure product powder is dissolved in methanol to prepare a storage solution with the concentration of 1mg/mL, and the storage solution is diluted in the plasma of healthy people without chlorpromazine, and the final concentration is respectively 0.00, 10.00, 100.00 and 300.00ng/mL to prepare blank, low, medium and high concentration quality control samples. By using the homogeneous enzyme immunoassay method for chlorpromazine, quality control samples are measured, the content of chlorpromazine in each quality control sample is calculated according to the homogeneous enzyme immunoassay calibration curve manufactured in the step 1, the measurement is repeated for 10 times for each quality control sample, and the detection result and data analysis are shown in Table 2 in detail.
TABLE 2 Chloroprozine homogeneous enzyme immunoassay reagent detection results and data analysis
The experimental results show that: CV values of chlorpromazine content in quality control samples with different concentrations are all lower than 5%, recovery rates are all between 95% and 105%, and the accuracy of chlorpromazine content determination in biological samples by the chlorpromazine homogeneous enzyme immunoassay reagent is high, and the result is accurate.
EXAMPLE 5 preparation of key components in chlorpromazine ELISA detection reagents
1. A method for preparing an anti-chlorpromazine specific antibody 2 comprising the steps of:
A. diluting chlorpromazine bovine serum albumin immunogen to a final concentration of 3.0mg/mL using phosphate buffer;
B. performing immune injection on experimental animal sheep by using a Freund adjuvant method, extracting sheep blood after injection for 4 times, and separating and purifying antiserum to obtain an anti-chlorpromazine specific antibody 2;
the preparation method of the chlorpromazine bovine serum albumin immunogen comprises the following steps:
a. dissolving 150mg of bovine serum albumin in 75ml of phosphate buffer solution with the concentration of 0.2mol/L and the pH of 8.5 to prepare a carrier protein solution;
b. dissolving 250mg of chlorpromazine derivative described in example 1 with 2.5ml of dimethylformamide, activating by tributylamine, performing coupling reaction with the carrier protein solution prepared in the step a, and obtaining chlorpromazine bovine serum albumin immunogen with immunogenicity through dialysis and purification after the reaction is finished;
2. the preparation method of the chlorpromazine horseradish peroxidase labeled conjugate comprises the following steps:
(a) Weighing horseradish peroxidase, dissolving the horseradish peroxidase in a phosphate buffer solution with the pH of 8.0 at room temperature to ensure that the final concentration is 3.5mg/mL, and preparing an enzyme solution;
(b) The chlorpromazine derivative described in example 1 was dissolved with isopropyl alcohol to a final concentration of 35mg/mL, activated by N, N' -dicyclohexylcarbodiimide, and subjected to a coupling reaction with the enzyme solution prepared in step (a), and after the reaction was completed, purified by dialysis to obtain a chlorpromazine horseradish peroxidase-labeled conjugate.
EXAMPLE 6 chlorpromazine ELISA detection reagent Performance evaluation experiment
1. Preparation of chlorpromazine ELISA detection calibration curve
Diluting anti-chlorpromazine specific antibody 2 with phosphate buffer solution according to the proportion of 1:10000 to prepare an antibody solution, coating the antibody solution on a 96-well enzyme-linked plate according to the dosage of 100 mu L/well, and standing at 4 ℃ overnight;
(ii) after 3 washes with phosphate buffer, 200. Mu.L/well of 0.5% bovine serum albumin solution was added, blocked overnight at 4℃and washed 3 times with phosphate buffer;
(iii) adding 20. Mu.L/well of calibrator
(iv) adding 100. Mu.L/well of the chlorpromazine horseradish peroxidase-labeled conjugate;
(v) incubating for 30 minutes at room temperature, washing the plate 5 times with phosphate buffer;
(vi) adding 100. Mu.L of 3,3', 5' -tetramethylbenzidine to each well, and incubating for 30 minutes at room temperature;
(vii) 100. Mu.L sulfuric acid at a concentration of 2mol/L was added to each well to terminate the reaction;
(viii) measuring the absorbance at 450nm using an microplate reader;
(ix) a calibration curve is prepared from absorbance values of calibrators of different concentrations, as shown in figure 2.
2. Quality control experiment
The chlorpromazine pure product powder is dissolved in methanol to prepare a storage solution with the concentration of 1mg/mL, and the storage solution is diluted in healthy human blood plasma without chlorpromazine, and the final concentration is respectively 0.00, 15.00, 75.00 and 250.00ng/mL to prepare blank, low, medium and high concentration quality control samples. The absorbance of the blank, low, medium and high concentration quality control samples at 450nm was measured by the chlorpromazine ELISA method. The chlorpromazine content in each quality control sample was calculated by comparing the calibration curve of chlorpromazine ELISA detection shown in FIG. 2, each quality control sample was repeatedly measured 3 times, the recovery rate was calculated according to the measurement result, and the detection data are shown in Table 3.
TABLE 3 evaluation data on the Performance of chlorpromazine ELISA detection reagents
| Quality control sample | Blank space | Low and low | In (a) | High height |
| Sample concentration (ng/mL) | 0.00 | 15.00 | 75.00 | 250.00 |
| Test 1 | 0.00 | 15.35 | 75.25 | 252.35 |
| Test 2 | 0.00 | 15.73 | 74.00 | 263.70 |
| Test 3 | 0.00 | 15.14 | 73.86 | 245.22 |
| Average value (ng/mL) | 0.00 | 15.41 | 74.37 | 253.76 |
| Recovery (%) | / | 102.73 | 99.16 | 101.50 |
The experimental results show that: the recovery rate of chlorpromazine content in samples with different concentrations measured by the chlorpromazine ELISA detection reagent is within the range of 95% -105%, which shows that the accuracy of chlorpromazine content in biological samples measured by the chlorpromazine ELISA detection reagent is higher.
EXAMPLE 7 preparation of chlorpromazine latex-enhanced turbidimetric immunoassay reagent
The preparation method of the chlorpromazine latex enhanced turbidimetric immunoassay reagent comprises the following steps:
dissolving 0.5mg/mL of anti-chlorpromazine specific antibody 3 in 50mmol/L of phosphate buffer solution with pH=8.0, then adding 1.25% of bovine serum albumin, 0.75% of sodium chloride, 0.25% of tween-20, 2.75% of glycerol, 0.5% of ethylenediamine tetraacetic acid, 1.25% of PEG-4000 and 0.03% of sodium azide, uniformly stirring, and adjusting the pH=7.5 to prepare an L1 reagent;
adding 0.5mg of polystyrene latex particles with carboxyl groups on the surface and with the diameter of 150nm into 4.5mL of 0.05mol/L of MES buffer solution with the pH value of 6.2, adding 5mg of carbodiimide, reacting for 1 hour at 37 ℃ to prepare a latex particle solution, diluting 0.5mg of chlorpromazine-human serum albumin complex with 4.5mL of borate buffer solution with the pH value of 0.05mol/L of 9.2, immediately adding the mixture into the latex particle solution, reacting for 12 hours at 37 ℃, adding 1mL of glycine buffer solution with the pH value of 0.1mol/L of 8.5, stirring for 2 hours, centrifuging to remove supernatant after the reaction is finished, washing the precipitate with 10mL of Tris-HCl buffer solution with the pH value of 50mmol/L of 8.0 for 3 times, diluting the mixture with 25mL of glycine buffer solution with the pH value of 50mmol/L of 8.5 to prepare a latex suspension, and finally adding bovine serum albumin with the mass fraction of 1.25%, 0.55% sodium chloride, 0.25% Tween-20%, 3.03% sodium acetate, 0.03% glycerol and 0.03% sodium azide, and a reagent with uniform stirring;
the preparation method of the anti-chlorpromazine specific antibody 3 comprises the following steps:
(A) Diluting chlorpromazine hemocyanin immunogen to a final concentration of 2.5mg/mL using phosphate buffer;
(B) Injecting the experimental animal horses by using a Freund adjuvant method, extracting horse blood after 6 times of injection, and separating and purifying antiserum to obtain anti-chlorpromazine specific antibody 3;
the preparation method of the chlorpromazine hemocyanin immunogen comprises the following steps:
< a > 200mg of hemocyanin was dissolved in 60ml of phosphate buffer with a concentration of 0.2mol/L and a pH of 8.5 to prepare a carrier protein solution;
< b > dissolving 250mg of the above chlorpromazine derivative with 2.5ml of dimethylformamide, activating with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and performing coupling reaction with the carrier protein solution prepared in the step < a >, and purifying by dialysis after the reaction is completed to obtain chlorpromazine hemocyanin immunogen having immunogenicity;
the preparation method of the chlorpromazine-human serum albumin complex comprises the following steps:
10mg of human serum albumin was diluted with 5mL of a phosphate buffer solution having a pH of 0.1 mol/L=7.8, 100mg of the chlorpromazine derivative was added thereto, 50mg of N-hydroxysuccinimide was further added thereto, the mixture was reacted at 4℃for 12 hours, and the mixture was dialyzed at 4℃for 18 hours with a phosphate buffer solution having a pH of 0.1 mol/L=7.8 to obtain a chlorpromazine-human serum albumin complex.
Example 8 preparation of chlorpromazine latex-enhanced turbidimetric immunoassay reagent calibration Curve and quality control experiment
1. Preparing a calibration curve of a latex-enhanced turbidimetric immunoassay reagent:
placing an R1 reagent, an R2 reagent and a calibrator in an Olympus AU480 full-automatic biochemical analyzer, and then setting reaction parameters of the biochemical analyzer, wherein the specific parameters are shown in a table 4; in the actual operation process, the volume ratio of the R1 reagent and the R2 reagent is required to be continuously adjusted, the light measuring point is adjusted at the same time, and finally, a latex enhanced immune turbidimetry detection calibration curve is automatically obtained by a biochemical analyzer, as shown in fig. 3.
TABLE 4 reaction parameters of Olympus AU480 full-automatic Biochemical Analyzer
2. Quality control experiment:
the chlorpromazine pure product powder is dissolved in methanol to prepare a storage solution with the concentration of 1mg/mL, and the storage solution is diluted in the plasma of healthy people without chlorpromazine, and the final concentration is respectively 0.00, 10.00, 100.00 and 300.00ng/mL to prepare blank, low, medium and high concentration quality control samples. And (2) measuring the quality control samples by using the latex-enhanced turbidimetric immunoassay method, calculating the content of chlorpromazine in each quality control sample according to the latex-enhanced turbidimetric immunoassay calibration curve prepared in the step (1), and repeatedly measuring each quality control sample for 10 times, wherein the detection result and the data analysis are shown in Table 5.
TABLE 5 chlorpromazine latex enhanced turbidimetric immunoassay and data analysis
The experimental results show that: CV values of chlorpromazine content in quality control samples with different concentrations are all lower than 5%, recovery rates are all between 95% and 105%, and the accuracy of chlorpromazine content in biological samples measured by the chlorpromazine latex enhanced immunonephelometry detection reagent is high, and the result is accurate.
With the above description of the preferred embodiments according to the present invention as a teaching, a person skilled in the art can make various changes and modifications without departing from the scope of the technical idea of the present invention. The technical scope of the present invention is not limited to the description, but must be determined according to the scope of claims.
Claims (2)
1. A synthesis method of chlorpromazine derivatives, which is characterized in that the specific route of the synthesis method is as follows:
the specific synthesis steps are as follows:
(1) Synthesis of Compound 3: 10g of compound 1 is dissolved in 120mL of DMF, then 5.15-g t-BuOK is added to prepare a reaction solution, then the reaction solution is stirred at 60 ℃ for 1 hour, the reaction solution is cooled to 25 ℃ after stirring is finished, then 12g of compound 2 is added, then the mixture is stirred at room temperature overnight, 200mL of purified water is added after stirring is finished, then the reacted solution is extracted with 300mL of DCM for 3 times, the organic phases obtained by extraction are combined and concentrated, and the concentrated product is purified by a silica gel column (PE: EA=20:1) to obtain compound 3;
(2) Synthesis of Compound 4: 10g of Compound 3 was dissolved in 127mL of DMF and then 4.88g of BuOK and 6.5g of CH were added 3 Preparing a reaction solution I, stirring the reaction solution at 60 ℃ for 12 hours, adding 400mL of purified water after stirring, extracting the reacted solution with 200mL of DCM for 3 times, combining organic phases obtained by extraction, concentrating, and purifying the concentrated product by a silica gel column (PE: EA=50:1) to obtain a compound 4;
(3) Synthesis of Compound 5: adding 12g of compound 4 into 80mL of solution with the concentration of 3mol/L, which is prepared by dissolving HCl in EA, preparing a reaction solution, stirring the reaction solution at 25 ℃ for 4 hours, and concentrating the stirred reaction solution under reduced pressure to obtain a compound 5;
(4) Synthesis of Compound 7: 5.93g of Compound 5, 3.94-g t-BuOK and 4.32g of Compound 6 were added to 75mL of DMF to prepare a reaction solution, which was stirred at 50℃for 3 hours, then 800mL of purified water and 3mL of TEA were added, the reacted solution was extracted with 300mL of EA, repeated 3 times, and the extracted organic phases were combined, and the mixture was purified by Na 2 SO 4 Drying and concentrating, purifying the concentrated product by a silica gel column (PE: ea=10:1) to obtain compound 7;
(5) Synthesis of chlorpromazine derivatives: 10g of Compound 7 was dissolved in a mixed solvent of 57mL of THF and 22.8mL of purified water, 1.18g of NaOH was then added, the mixture was stirred at room temperature overnight, the pH of the reacted solution was adjusted to 5-6 with 1mol/L of HCI, 20mL of purified water was then added, and the reacted solution was reacted with 100mL of CH 2 Cl 2 The extraction was repeated 3 times, the extracted organic phases were combined and extracted over Na 2 SO 4 Dried and concentrated, and the concentrated product was passed through a silica gel column (CH 2 Cl 2 Meoh=10:1) to give chlorpromazine derivatives.
2. The preparation method of the chlorpromazine latex enhanced turbidimetric immunoassay reagent is characterized by comprising the following steps of:
dissolving 0.5mg/mL of anti-chlorpromazine specific antibody 3 in 50mmol/L of phosphate buffer solution with pH=8.0, then adding 0.5% -2.5% of bovine serum albumin, 0.25% -1.0% of sodium chloride, 0.1% -0.5% of tween-20, 1.0% -5.0% of glycerol, 0.1% -1.0% of ethylenediamine tetraacetic acid, 0.5% -2.5% of PEG-4000 and 0.01% -0.1% of sodium azide, uniformly stirring, and adjusting pH=7.5 to prepare an L1 reagent;
adding 0.5mg of polystyrene latex particles with carboxyl groups on the surface and with the diameter of 150nm into 4.5mL of 0.05mol/LpH =6.2 MES buffer solution, adding 5mg of carbodiimide, reacting for 1 hour at 37 ℃ to prepare a latex particle solution, diluting 0.5mg of chlorpromazine-human serum albumin complex with 4.5mL of 0.05mol/L of borate buffer solution with pH=9.2, immediately adding the mixture into the latex particle solution, reacting for 12 hours at 37 ℃, adding 1mL of 0.1mol/LpH =8.5 glycine buffer solution, stirring for 2 hours, centrifuging to remove supernatant after the reaction is finished, washing the precipitate with 10mL of 50mmol/L of Tris-HCl buffer solution with pH=8.0, diluting the mixture with 25mL of 50mmol/L of glycine buffer solution with pH=8.5 to prepare a latex suspension, and finally adding 0.5% -2.5% of bovine serum albumin, 0.25% -1.0% sodium chloride, 0.1% Tween-0.5% and 0.0% -1.0% sodium chloride, stirring for preparing uniform glycerol with the mixture into 0.0% -1.0% -1% of acetic acid;
the preparation method of the anti-chlorpromazine specific antibody 3 comprises the following steps:
(A) Diluting chlorpromazine hemocyanin immunogen to a final concentration of 0.5-5.0mg/mL by using phosphate buffer;
(B) Performing immune injection on experimental animals by using a Freund adjuvant method, extracting animal blood after injection for 3-8 times, and separating and purifying antiserum to obtain an anti-chlorpromazine specific antibody 3;
the preparation method of the chlorpromazine hemocyanin immunogen comprises the following steps:
< a > dissolving 100-300mg of hemocyanin in 10-100ml of phosphate buffer solution with concentration of 0.2mol/L and pH of 8.5 to prepare carrier protein solution;
< b > dissolving 50-500mg of the chlorpromazine derivative as shown in claim 1 in 1.0-5.0ml of organic solvent a, activating by coupling activator and coupling reaction with the carrier protein solution prepared in step < a >, and purifying by dialysis after the reaction is finished to obtain chlorpromazine hemocyanin immunogen with immunogenicity;
the organic solvent A is any one of dimethyl sulfoxide, dimethylformamide, isopropanol, methanol or ethanol;
the preparation method of the chlorpromazine-human serum albumin complex comprises the following steps:
diluting 10mg of human serum albumin with 5ml of 0.1mol/L phosphate buffer solution with pH=7.8, adding 100mg of chlorpromazine derivative shown in claim 1, adding 50mg of coupling activator, reacting at 4 ℃ for 12 hours, and dialyzing at 4 ℃ for 18 hours with 0.1mol/L phosphate buffer solution with pH=7.8 to obtain chlorpromazine-human serum albumin complex;
the coupling activator is any one of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N' -dicyclohexylcarbodiimide, N-hydroxysuccinimide, tributylamine, glutaraldehyde, a diisocyanate compound or dinitrobenzene dihalide;
the synthesis method of the chlorpromazine derivative is as claimed in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911077366.8A CN110950820B (en) | 2019-11-06 | 2019-11-06 | Chlorpromazine derivative, preparation method thereof and chlorpromazine detection reagent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911077366.8A CN110950820B (en) | 2019-11-06 | 2019-11-06 | Chlorpromazine derivative, preparation method thereof and chlorpromazine detection reagent |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110950820A CN110950820A (en) | 2020-04-03 |
| CN110950820B true CN110950820B (en) | 2023-05-02 |
Family
ID=69976120
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911077366.8A Active CN110950820B (en) | 2019-11-06 | 2019-11-06 | Chlorpromazine derivative, preparation method thereof and chlorpromazine detection reagent |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110950820B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112266330B (en) * | 2020-10-15 | 2023-02-24 | 长沙博源医疗科技有限公司 | Methoxyarenol derivative, immunogen, anti-methoxyadrol specific antibody, preparation method and application thereof |
| CN112225672B (en) * | 2020-10-16 | 2023-03-28 | 长沙博源医疗科技有限公司 | Methoxy norepinephrine derivative, immunogen, specific antibody, preparation method and application thereof |
| CN112630430B (en) * | 2020-11-16 | 2021-08-27 | 北京美联泰科生物技术有限公司 | Kit for quantitatively detecting UCHL-1 and application thereof |
| CN114685400B (en) * | 2020-12-25 | 2023-11-10 | 长沙博源医疗科技有限公司 | Key group derivative of aripiprazole, immunogen, anti-aripiprazole specific antibody, and preparation method and application thereof |
| CN114685527B (en) * | 2020-12-25 | 2024-03-05 | 长沙博源医疗科技有限公司 | Olanzapine derivative, immunogen, anti-olanzapine specific antibody, preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101236200A (en) * | 2008-02-06 | 2008-08-06 | 中国计量学院 | Chlorpromazine ELISA reagent kit and its detection method |
| CN102621323A (en) * | 2012-03-30 | 2012-08-01 | 苏州博源医疗科技有限公司 | Method for detecting 6-methylmercaptopurine |
| CN102628868A (en) * | 2011-12-30 | 2012-08-08 | 北京九强生物技术股份有限公司 | Latex enhanced immunoturbidimetry kit for detection of asymmetric dimethylarginine content |
| CN109704954A (en) * | 2018-12-21 | 2019-05-03 | 苏州博源医疗科技有限公司 | 3-(3- hydroxy phenyl) derivative of -3- hydracrylic acid, homogeneous enzyme immunoassay detection reagent and preparation method thereof |
-
2019
- 2019-11-06 CN CN201911077366.8A patent/CN110950820B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101236200A (en) * | 2008-02-06 | 2008-08-06 | 中国计量学院 | Chlorpromazine ELISA reagent kit and its detection method |
| CN102628868A (en) * | 2011-12-30 | 2012-08-08 | 北京九强生物技术股份有限公司 | Latex enhanced immunoturbidimetry kit for detection of asymmetric dimethylarginine content |
| CN102621323A (en) * | 2012-03-30 | 2012-08-01 | 苏州博源医疗科技有限公司 | Method for detecting 6-methylmercaptopurine |
| CN109704954A (en) * | 2018-12-21 | 2019-05-03 | 苏州博源医疗科技有限公司 | 3-(3- hydroxy phenyl) derivative of -3- hydracrylic acid, homogeneous enzyme immunoassay detection reagent and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| "RN807316-54-7";美国化学会;《STN ON THE WEB》;20050103;第3页 * |
| "氯丙嗪抗体制备与酶联免疫吸附测定方法的建立";袁利鹏 等;《现代食品科技》;20190807;第35卷(第08期);第326-327页 * |
| 美国化学会."RN807316-54-7".《STN ON THE WEB》.2005,第1-3页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110950820A (en) | 2020-04-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110950820B (en) | Chlorpromazine derivative, preparation method thereof and chlorpromazine detection reagent | |
| CN110981861B (en) | Clozapine derivative, preparation method thereof and clozapine detection reagent | |
| US10428139B2 (en) | GM hybridoma cell, monoclonal antibody, kit and preparation method and use thereof | |
| WO2014005298A1 (en) | Enzyme-linked immunosorbent assay kit for detecting dinitolmide and use thereof | |
| CN107012128B (en) | Hybridoma cell strain secreting monoclonal antibody against aflatoxin B1 and application thereof | |
| CN110003300B (en) | Derivative of 17-hydroxysteroid, detection reagent and preparation method | |
| CN109704954B (en) | 3- (3-hydroxyphenyl) -3-hydroxypropionic acid derivative, homogeneous enzyme immunoassay reagent and preparation method thereof | |
| CN109824599B (en) | Albendazole hapten as well as preparation method and application thereof | |
| CN103288723B (en) | Sulfonamide hapten and its preparation method and application | |
| US10351830B2 (en) | Conjugates for assays for oxycodone and oxymorphone | |
| CN110357886B (en) | Methotrexate hapten and complete antigen as well as preparation method and application thereof | |
| CN109180768B (en) | Estrone derivative, immunogen, antibody, enzyme-labeled conjugate, detection reagent and preparation method thereof | |
| EP2261259B1 (en) | Immunoassay for N-(3-chlorophenyl)piperazine-based anti-depressants | |
| CN113582923B (en) | Quinaldine hapten, artificial antigen and antibody as well as preparation methods and application thereof | |
| CN111018924B (en) | Daunorubicin derivative, preparation method thereof and daunorubicin detection reagent | |
| CN110981791A (en) | Paraquat derivative, preparation method thereof and paraquat detection reagent | |
| CN110117286B (en) | Heterocyclic amine 8-MeIQx hapten and antibody as well as preparation method and application thereof | |
| CN111825663B (en) | 11-dehydrothromboxane B2 derivative, preparation method thereof and 11-dehydrothromboxane B2 detection reagent | |
| CN111217910B (en) | Monoclonal antibody pair and application thereof in detecting myeloperoxidase protein | |
| CN108205064B (en) | 25OHD3 detection reagent, kit and detection method thereof | |
| CN107083368B (en) | hybridoma cell strains secreting monoclonal antibodies against total aflatoxin and application thereof | |
| CN118084715A (en) | 3-Hydroxy hippuric acid derivative, 3-hydroxy hippuric acid immunodetection reagent and preparation method thereof | |
| CN115181076B (en) | Hapten, antigen, cell strain, antibody, reagent and kit for detecting concentration of aripiprazole and dehydroaripiprazole | |
| CN114685409B (en) | Escitalopram derivative, immunogen, escitalopram-resistant specific antibody, and preparation method and application thereof | |
| CN117069580B (en) | Triclosan hapten as well as preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |