CN116354904A - Quetiapine derivative hapten, antigen, anti-quetiapine antibody and application thereof - Google Patents
Quetiapine derivative hapten, antigen, anti-quetiapine antibody and application thereof Download PDFInfo
- Publication number
- CN116354904A CN116354904A CN202310456651.0A CN202310456651A CN116354904A CN 116354904 A CN116354904 A CN 116354904A CN 202310456651 A CN202310456651 A CN 202310456651A CN 116354904 A CN116354904 A CN 116354904A
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- Prior art keywords
- quetiapine
- antibody
- derivative
- hapten
- enzyme
- 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.)
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to a quetiapine derivative hapten, an antigen, an anti-quetiapine antibody and application thereof. The derivative site of the quetiapine derivative hapten is positioned in various inactive metabolites of quetiapine and quetiapine: n-dealkylated quetiapine, O-dealkylated quetiapine, 7 hydroxy quetiapine and sulfoxide quetiapine on a common characteristic structure, so that the prepared anti-quetiapine antibody can specifically identify quetiapine and has no obvious cross reaction with various metabolites. Furthermore, the anti-quetiapine antibody secreted by the hybridoma cell C-KLP-12H2 can recognize quetiapine with high sensitivity and specificity, does not have cross reaction with olanzapine, aripiprazole, risperidone, N-dealkylquetiapine, O-dealkylquetiapine, 7-hydroxyquetiapine and sulfoxide quetiapine, and can further establish a method for accurately monitoring the blood and urine concentration of quetiapine.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a quetiapine derivative hapten, an antigen, an anti-quetiapine antibody and application thereof.
Background
Quetiapine (quetiapine) is an atypical antipsychotic of the dibenzothiazepine type, mainly acts on receptors such as central dopamine and 5-hydroxytryptamine, and has remarkable effects on positive and negative symptoms of schizophrenia. Because it is not easy to cause extrapyramidal reaction and has good tolerance, it has been widely used in clinic in recent years. The "monitoring of blood concentration in psychiatric department (TDM) consensus guide in 2011" indicates that quetiapine has the characteristics of narrow therapeutic window, large individual difference, and the TDM recommendation is classified into 2 class recommendation classes. The clinical monitoring of the quetiapine blood concentration can ensure the safety and effectiveness of treatment. However, it has been reported that adverse reactions including orthostatic hypotension and abnormal liver function enzymes occur in patients taking quetiapine, and that cases of toxic coma are reported to occur due to administration of quetiapine in a large dose. Therefore, it is imperative to establish a method for detecting the blood concentration of quetiapine. In addition, studies have shown that quetiapine concentration in urine can also be an effective monitoring means for pharmacokinetic studies and monitoring of in vivo drug concentrations.
However, current quetiapine blood concentration monitoring has little development in domestic clinical practice, and is related to complicated quetiapine blood concentration detection methods, lack of consciousness of blood concentration monitoring by people, interference measurement of various metabolites of quetiapine and the like. The detection analysis of quetiapine mainly comprises an HPLC-UV method, an HPLC and an LC-MS method, but the HPLC method has longer analysis time and large interference of endogenous substances, and is difficult to meet the requirements of clinical examination on high efficiency and high accuracy, the HPLC-UV method has insufficient sensitivity, the LC-MS method has expensive equipment and low flux, and the requirements of clinical high-flux, rapid and accurate detection cannot be met.
Immunological detection is one of the common methods for detecting in vitro samples, and the premise of establishing high-sensitivity and high-specificity quetiapine immunological detection is to obtain high-quality anti-quetiapine antibodies, and further relies on the design and preparation of quetiapine artificial antigens. Chinese patent CN110763799a, publication No. 2020, 2 and 07, discloses a method for measuring quetiapine by high performance liquid chromatography, chinese patent CN109900820a, publication No. 2019, 6 and 18, discloses a method for measuring quetiapine in blood by high performance liquid chromatography-mass spectrometry, but the methods in both patents require complex pretreatment and specialized operators. Chinese patent CN111808046a, publication date 2020, 10-23, discloses a quetiapine artificial hapten, an artificial antigen, and preparation methods and applications thereof. However, this patent only provides a method for preparing quetiapine antigen, and does not provide sensitivity and specificity to the corresponding antibody. Chinese patent CN107266432a, publication No. 2017, 10, 20, discloses quetiapine-derived hapten for immunoassay, and the mating antibody only discloses sensitivity performance, and no specific performance. Chinese patent CN110054693a, publication No. 2019, 7, 26, discloses antibodies to quetiapine-derived hapten and uses thereof, and the corresponding established detection methods are satisfactory in sensitivity, and evaluate cross-reactions to other psychotic drugs risperidone, quetiapine, aripiprazole in terms of specificity, but do not analyze cross-reactions with quetiapine metabolites.
In view of the above, there is currently a lack of anti-quetiapine antibodies with high sensitivity and simultaneously very good specificity.
Disclosure of Invention
The invention aims to provide a quetiapine derivative hapten, an antigen, an anti-quetiapine antibody and application thereof, wherein the antibody obtained by immunization of the quetiapine derivative hapten can specifically identify quetiapine, has no cross reaction with an inactive metabolite and has higher sensitivity.
The invention provides a quetiapine derivative hapten, which has a structure shown in a formula I:
formula I.
The invention also provides a quetiapine artificial antigen, which comprises carrier protein and quetiapine derivative hapten according to the technical scheme; the quetiapine derivative hapten and the carrier protein are covalently coupled through a chemical cross-linking agent.
Preferably, the carrier protein comprises bovine serum albumin, chicken ovalbumin, bovine thyroglobulin, human serum albumin or rabbit serum albumin.
Preferably, the chemical crosslinking agent comprises 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.
The invention also provides a hybridoma cell C-KLP-12H2 obtained by the quetiapine artificial antigen immune screening in the technical scheme, and the preservation number of the hybridoma cell C-KLP-12H2 is CGMCC No.45340.
The invention also provides an anti-quetiapine antibody which is obtained by secreting the hybridoma cell C-KLP-12H2 according to the technical scheme.
Preferably, the anti-quetiapine antibody comprises a monoclonal antibody or a polyclonal antibody.
The invention also provides application of the anti-quetiapine antibody in immunodetection of quetiapine blood concentration or preparation of a kit and/or a reagent for immunodetection of quetiapine blood concentration.
The invention also provides a quetiapine homogeneous enzyme immunoassay kit which comprises a reagent R1 and a reagent R2;
the reagent R1 comprises the following components in concentration: 20mM NAD,30mM glucose-6-phosphate, 55mM Tris buffer with pH value of 8.0 and 0.01wt.% to 0.1wt.% of the anti-quetiapine antibody according to the above technical scheme;
the reagent R2 comprises the following components in concentration: 0.01wt.% to 0.1wt.% quetiapine derivative-glucose-6-phosphate dehydrogenase enzyme-labeled conjugate and 0.1M Tris buffer at pH 8.5;
the quetiapine derivative-glucose-6-phosphate dehydrogenase enzyme-labeled conjugate is obtained by coupling 6-phosphate glucose dehydrogenase labels with the quetiapine derivative hapten according to the technical scheme.
The invention also provides a method for detecting the blood concentration of quetiapine based on a magnetic particle chemiluminescence method, which comprises the following steps:
mixing and incubating a sample to be detected, a magnetic particle working solution and an enzyme-labeled working solution, cleaning, adding a luminescent substrate for reaction and color development, measuring a luminescent value RLU, substituting a standard four-parameter equation, and calculating the concentration of quetiapine in the sample to be detected;
the magnetic particle working solution is obtained by labeling the anti-quetiapine antibody in the technical scheme by magnetic particles;
the enzyme-labeled working solution is obtained by enzyme-labeling the quetiapine derivative hapten in the technical scheme.
The beneficial effects are that:
the invention provides a quetiapine derivative hapten, which has a structure shown in a formula I:
formula I.
The derivative site of the quetiapine derivative hapten is positioned on the N atom of 1, 4-thiazaalkane, and the derivative site is positioned on various inactive metabolites of quetiapine and quetiapine: n-dealkylated quetiapine, O-dealkylated quetiapine, 7 hydroxy quetiapine and sulfoxide quetiapine are on the common characteristic structure, and further the characteristics of different compounds are shown, so that the prepared anti-quetiapine antibody can specifically identify the quetiapine and has no obvious cross reaction with various metabolites.
The invention also provides a hybridoma cell C-KLP-12H2, and the anti-quetiapine antibody secreted by the hybridoma cell C-KLP-12H2 can identify quetiapine and olanzapine, aripiprazole, risperidone, N-dealkylquetiapine, O-dealkylquetiapine, 7-hydroxyquetiapine and sulfoxide quetiapine with high sensitivity and specificity, and has no cross reaction, so that a method for accurately monitoring the blood and urine concentration of the quetiapine can be established.
Biological material preservation information
The strain is classified and named as hybridoma cell strain, and is preserved in China general microbiological culture Collection center (China Committee for culture Collection) with a preservation date of 2022, 12 months and 26 days, a preservation address of North Chenxi Lu No. 1, 3 of the Chaoyang district of Beijing city, a preservation number of CGMCC No.45340, and a cell number of C-KLP-12H2.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a preparation route diagram of quetiapine-derived hapten from quetiapine in example 1 of the present invention;
FIG. 2 is a preparation route diagram of the first intermediate prepared from quetiapine in example 1 of the present invention;
FIG. 3 is a preparation route diagram of the preparation of intermediate two from intermediate one in example 1 of the present invention;
FIG. 4 is a preparation route diagram of the preparation of intermediate III from intermediate II in example 1 of the present invention;
FIG. 5 is a preparation scheme for preparing quetiapine-derived hapten from intermediate three in example 1 of the present invention;
FIG. 6 is a nuclear magnetic resonance hydrogen spectrum of quetiapine-derived hapten of example 1 of the present invention;
FIG. 7 is a graph of quetiapine calibration by magnetic particle luminescence method in example 5 of the present invention;
FIG. 8 is a graph of the correlation analysis of quetiapine homogeneous enzyme test-free kit and HPLC-MS method in example 6 of the present invention.
Detailed Description
The invention provides a quetiapine derivative hapten, which has a structure shown in a formula I:
formula I.
The derivative site of the quetiapine derivative hapten is positioned in various inactive metabolites of quetiapine and quetiapine: n-dealkylated quetiapine, O-dealkylated quetiapine, 7 hydroxy quetiapine and sulfoxide quetiapine on a common characteristic structure, so that the prepared anti-quetiapine antibody can specifically identify quetiapine and has no obvious cross reaction with various metabolites.
The preparation method of the quetiapine derivative hapten preferably comprises the following steps:
mixing quetiapine, imidazole, tert-butyl dimethyl chlorosilane and N, N-dimethylformamide, and performing hydroxyl protection to obtain an intermediate I;
mixing the intermediate I, N-dimethylformamide, sodium hydride and monomethyl succinate acyl chloride, and carrying out condensation reaction to obtain a condensation reaction liquid;
mixing and layering the condensation reaction liquid and water, and extracting the obtained first aqueous phase with ethyl acetate to obtain an intermediate II;
mixing the intermediate II, tetrahydrofuran and tetrabutylammonium fluoride-tetrahydrofuran solution, and performing dehydroxy protection to obtain an intermediate III;
mixing the intermediate III, methanol and sodium hydroxide solution, performing hydrolysis reaction, and extracting the obtained second aqueous phase with ethyl acetate to obtain a third aqueous phase which is an aqueous solution containing the quetiapine derivative hapten.
In the present invention, quetiapine, imidazole, t-butyldimethylchlorosilane and N, N-dimethylformamide are preferably mixed to perform hydroxyl protection. The mass ratio of quetiapine to imidazole is preferably 1.1-1.2: 0.32 to 0.36, more preferably 1.15:0.34; the mass ratio of quetiapine to tert-butyl dimethyl chlorosilane is preferably 1.1.1-1.2: 0.44 to 0.46, more preferably 1.15:0.45; the ratio of the mass of quetiapine to the volume of N, N-dimethylformamide is preferably 1.1-1.2 g:15 to 25mL, more preferably 1.15g:20mL. The temperature of the hydroxyl group protection according to the present invention is preferably room temperature. The time for protecting the hydroxyl group is not particularly limited in the present invention, so long as the reaction is complete. The present invention preferably employs thin layer chromatography to determine whether the reaction is complete.
After the hydroxyl is protected, the invention preferably mixes the obtained hydroxyl protection reaction liquid with water, filters and dries the obtained mixture to obtain an intermediate I; the structural formula of the intermediate I is shown as a formula II:
formula II.
The specific steps of the filtering and drying are not particularly limited in the present invention, and steps and parameters well known to those skilled in the art may be adopted.
After the intermediate I is obtained, the invention preferably mixes the intermediate I, N-dimethylformamide, sodium hydride and monomethyl succinate acyl chloride, and obtains condensation reaction liquid through condensation reaction. The invention preferably mixes the intermediate I with N, N-dimethylformamide, then cools to 0 ℃ and then mixes with sodium hydride and succinic acid monomethyl chloride. The mass ratio of the intermediate I to the N, N-dimethylformamide is preferably 0.78-0.82 g:25 to 35mL, more preferably 0.8g:30mL; the mass ratio of the intermediate I to the sodium hydride is preferably 7.95-8.05: 0.95 to 1.05, more preferably 8:1, a step of; the mass ratio of the intermediate I to the succinic acid monomethyl chloride is preferably 7.95-8.05: 2.95 to 3.05, more preferably 8:3. the temperature of the condensation reaction is preferably room temperature, and the time is preferably 10-14 h, more preferably 12h.
After the condensation reaction liquid is obtained, the condensation reaction liquid is preferably mixed with water for layering, and the obtained first aqueous phase is extracted by ethyl acetate to obtain an ethyl acetate phase. The water of the invention is preferably ice water; the mass ratio of the water volume to the first intermediate is preferably 100mL:0.8g. The number of extraction times in the present invention is preferably 2 to 4, more preferably 3. The ethyl acetate phase of the present invention is preferably obtained by combining ethyl acetate phases obtained by 3 extractions.
After the ethyl acetate phase is obtained, the ethyl acetate phase is preferably backwashed by adopting saturated sodium chloride solution, and the obtained organic phase is dried, filtered and evaporated to dryness, so that an intermediate II is obtained by column chromatography; the structural formula of the intermediate II is shown in a formula III:
formula III.
The drying is preferably carried out by using a 5A molecular sieve; the column chromatography was performed using a C18 column.
After the intermediate II is obtained, the intermediate II, tetrahydrofuran and tetrabutylammonium fluoride-tetrahydrofuran solution are preferably mixed for dehydroxy protection. The volume ratio of the mass of the intermediate II to the tetrahydrofuran is preferably 0.18-0.22 g:25 to 35mL, more preferably 0.2g:30mL; the mass ratio of the intermediate II to the tetrabutylammonium fluoride-tetrahydrofuran solution is preferably 0.18-0.22 g:4 to 6mL, more preferably 0.2g:5mL; the concentration of the tetrabutylammonium fluoride-tetrahydrofuran solution is 1mol/L. The temperature of the dehydroxylation protection according to the present invention is preferably room temperature. The time for the dehydroxylation protection is not particularly limited in the present invention, as long as the reaction is complete.
After the dehydroxylation protection, the solvent in the obtained dehydroxylation protection liquid is preferably evaporated to dryness, and the remainder is mixed with water to obtain a solid-liquid mixture. The mass ratio of the volume of water to the second intermediate is preferably 100mL:0.2g.
After the solid-liquid mixture is obtained, the solid-liquid mixture is preferably filtered, and the obtained filter cake is dried to obtain an intermediate III; the structural formula of the intermediate III is shown as formula IV:
formula IV.
The specific steps of the filtration and drying are not particularly limited in the present invention, and may be steps well known to those skilled in the art.
After the intermediate III is obtained, the intermediate III, methanol and sodium hydroxide solution are mixed, and a second water phase is obtained through hydrolysis reaction. The mass ratio of the intermediate III to the methanol is preferably 0.09-0.11 g:45 to 55mL, more preferably 0.1g:50mL; the mass ratio of the intermediate III to the sodium hydroxide solution is preferably 0.09-0.11 g: 8-12 mL, more preferably 0.1g:10mL; the concentration of the sodium hydroxide solution is preferably 0.8 to 1.2N, more preferably 1N. The temperature of the hydrolysis reaction is preferably room temperature, and the time is preferably 12-16 h, more preferably 14h.
After the second aqueous phase is obtained, the present invention preferably uses ethyl acetate to extract the second aqueous phase to obtain a third aqueous phase. The number of times of extraction in the invention is preferably 2 to 4 times, more preferably 3 times. The third aqueous phase is preferably obtained by mixing aqueous phases obtained by 3 times of extraction.
After the third water phase is obtained, the pH value of the third water phase is preferably regulated to 3, the obtained mixture is filtered, and the obtained filter cake is dried to obtain the quetiapine derivative hapten. The pH value of the third water phase is preferably adjusted by using 3.6% of diluted hydrochloric acid by mass fraction. The filtering and drying operation is not particularly limited in the present invention, and conventional operations in the art may be adopted.
The invention also provides a quetiapine artificial antigen, which comprises carrier protein and quetiapine derivative hapten according to the technical scheme; the quetiapine derivative hapten and the carrier protein are covalently coupled through a chemical cross-linking agent.
The quetiapine artificial antigen of the present invention preferably comprises a quetiapine immunogen or a quetiapine coating antigen, more preferably a quetiapine immunogen.
The carrier protein of the present invention preferably comprises bovine serum albumin, chicken ovalbumin, bovine thyroglobulin, human serum albumin or rabbit serum albumin, more preferably bovine serum albumin or bovine thyroglobulin. The chemical coupling agent of the present invention preferably comprises 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride. The amino group on the carrier protein is coupled with the carboxyl group on the quetiapine derivative hapten, and an amide bond is formed through condensation reaction. The mol ratio of the quetiapine derivative hapten to the carrier protein is (10-100): 1, more preferably 50:1.
the preparation method of the quetiapine artificial antigen preferably comprises the following steps:
mixing and dissolving the quetiapine derivative hapten and dimethyl sulfoxide, and mixing and reacting the obtained quetiapine derivative hapten solution with a coupling agent aqueous solution to obtain a solution A;
mixing carrier protein with PBS buffer solution to obtain carrier protein solution;
mixing the solution A with a carrier protein solution for reaction to obtain a mixed solution containing the quetiapine artificial antigen;
and dialyzing the mixed solution containing the quetiapine artificial antigen by using a PBS buffer solution, wherein the obtained retention solution comprises the quetiapine artificial antigen.
The quetiapine derivative hapten is preferably mixed and dissolved with dimethyl sulfoxide, and the obtained quetiapine derivative hapten solution is mixed and reacted with a coupling agent aqueous solution to obtain a solution A. The mass concentration of the quetiapine derivative hapten in the quetiapine derivative hapten solution is preferably 10mg/mL. The mass concentration of the coupling agent in the aqueous solution of the coupling agent is preferably 0.05-0.2 mg/mu L, and more preferably 0.1 mg/mu L. The temperature of the reaction is preferably room temperature, and the reaction time is preferably 0.2-2 h, more preferably 1h.
The present invention preferably mixes the carrier protein with PBS buffer to obtain a carrier protein solution. The mass concentration of the carrier protein in the carrier protein solution is preferably 10-30 mg/mL, more preferably 20mg/mL.
After the solution A and the carrier protein solution are obtained, the solution A and the carrier protein solution are preferably mixed for reaction to obtain the mixed solution containing the quetiapine artificial antigen. Stirring is carried out during the reaction, and the temperature of stirring is preferably room temperature; the stirring time is preferably 0.2 to 2 hours, more preferably 2 hours. The stirring process is not particularly limited, and conventional stirring processes in the art can be adopted.
After the mixed solution containing the quetiapine artificial antigen is obtained, the invention preferably utilizes PBS buffer solution to dialyze the mixed solution containing the quetiapine artificial antigen, and the obtained retention solution comprises the quetiapine artificial antigen. The molecular weight cut-off for the dialysis according to the invention is preferably 7000Da. The number of times of dialysis in the present invention is preferably 2 to 8 times, more preferably 4 times.
The invention also provides a hybridoma cell C-KLP-12H2 obtained by the quetiapine artificial antigen immune screening in the technical scheme, and the preservation number of the hybridoma cell C-KLP-12H2 is CGMCC No.45340.
The hybridoma cell C-KLP-12H2 is preferably prepared by adopting a hybridoma cell fusion technology, particularly preferably, quetiapine artificial antigen is mixed with an adjuvant to immunize animals, the obtained spleen cells are fused with Sp2/0 cells, and the obtained hybridoma is subjected to titer and competition measurement to obtain the hybridoma cell C-KLP-12H2. The animals of the present invention preferably include, but are not limited to, mice, rabbits, goats, and sheep.
The invention also provides an anti-quetiapine antibody which is obtained by secreting the hybridoma cell C-KLP-12H2 according to the technical scheme. The anti-quetiapine antibody of the present invention preferably comprises a monoclonal antibody or a polyclonal antibody, more preferably a monoclonal antibody, and even more preferably an anti-quetiapine antibody C-KLP-12H2. The anti-quetiapine antibody has high sensitivity recognition capability to quetiapine and high specificity.
Based on the advantages, the invention also provides application of the anti-quetiapine antibody in immunodetection of quetiapine blood concentration for non-disease diagnosis purpose or preparation of a kit and/or a reagent for immunodetection of quetiapine blood concentration. The immunodetection method of the present invention preferably comprises one or more of enzyme-linked immunosorbent assay, magnetic bead chemiluminescence method, homogeneous enzyme method, fluorescence immunochromatography method and colloidal gold immunoassay method, and more preferably comprises magnetic bead chemiluminescence method or homogeneous enzyme method.
The invention also preferably provides a quetiapine homogeneous enzyme immunoassay kit which comprises a reagent R1 and a reagent R2; the reagent R1 comprises the following components in concentration: 20mM NAD,30mM glucose-6-phosphate, 55mM Tris buffer with pH value of 8.0 and 0.01wt.% to 0.1wt.% of the anti-quetiapine antibody according to the above technical scheme; the reagent R2 comprises the following components in concentration: 0.01wt.% to 0.1wt.% quetiapine derivative-glucose-6-phosphate dehydrogenase (G6 PDH) enzyme-labeled conjugate and 0.1M Tris buffer at pH 8.5; the quetiapine derivative-glucose-6-phosphate dehydrogenase enzyme-labeled conjugate is obtained by coupling 6-phosphate glucose dehydrogenase labels with the quetiapine derivative hapten according to the technical scheme.
The concentration of the anti-quetiapine antibody in the present invention is preferably 0.05wt.%. The concentration of quetiapine derivative-glucose-6-phosphate dehydrogenase (G6 PDH) enzyme-labeled conjugate of the present invention is preferably 0.05wt.%.
The preparation method of the quetiapine derivative-glucose-6-phosphate dehydrogenase (G6 PDH) enzyme-labeled conjugate preferably comprises the following steps: dissolving glucose-6-phosphate dehydrogenase in PBS buffer solution to obtain glucose-6-phosphate dehydrogenase PBS solution;
dissolving the quetiapine derivative hapten in dimethyl sulfoxide to obtain a quetiapine derivative hapten dimethyl sulfoxide solution.
Mixing the glucose-6-phosphate dehydrogenase PBS solution, the quetiapine derivative hapten dimethyl sulfoxide solution and the carbodiimide (EDC), and standing for reaction to obtain the quetiapine derivative-glucose-6-phosphate dehydrogenase (G6 PDH) enzyme-labeled conjugate in the mixed solution.
In the present invention, the volume ratio of the enzyme activity of the glucose-6-phosphate dehydrogenase to the PBS buffer is preferably 600U:1mL; the pH of the PBS buffer is preferably 7.4. The ratio of the mass of the quetiapine derivative hapten to the volume of dimethyl sulfoxide is preferably 0.5mg: 100. Mu.L.
The volume of the PBS solution of the glucose-6-phosphate dehydrogenase and the volume of the dimethyl sulfoxide solution of the quetiapine derivative hapten are preferably 1mL, and the mass of the carbodiimide (EDC) is as follows: 100 μl:1mg. The temperature of the standing reaction is preferably room temperature; the time is preferably 0.5 to 3 hours, more preferably 2 hours.
In the present invention, the method of using the kit preferably comprises the steps of: mixing and incubating a reagent R1, a reagent R2 and a sample to be detected, and determining OD 340 Substituting the quetiapine into a standard curve to obtain the concentration of quetiapine in the sample to be detected. In the invention, the sample to be tested is preferably mixed with the reagent R1 for first incubation, and then mixed with the reagent R2 for second incubation. The time of the first incubation is preferably 2-6 min, more preferably 5min, and the temperature is preferably 37 ℃; the second incubation time is preferably 2-6 min, more preferably 5min, and the temperature is preferably 37 ℃. The standard curve of the invention is preferably: y=1.0068x+31.992, r 2 =0.9929。
The invention also provides a quetiapine blood concentration detection method based on the magnetic particle chemiluminescence method for non-disease diagnosis purpose, which comprises the following steps:
mixing and incubating a sample to be detected, a magnetic particle working solution and an enzyme-labeled working solution, cleaning, adding a luminescent substrate corresponding to the enzyme for reaction and color development, measuring a luminescent value RLU, substituting a standard four-parameter equation, and calculating the quetiapine blood concentration in the sample to be detected; the magnetic particle working solution is obtained by labeling the anti-quetiapine antibody in the technical scheme by magnetic particles; the enzyme-labeled working solution is obtained by enzyme-labeling the quetiapine derivative hapten in the technical scheme.
In the invention, the volume ratio of the sample to be detected, the magnetic particle working solution of the anti-quetiapine antibody and the enzyme-labeled working solution of the quetiapine derivative hapten marked by enzyme is 1:4:5. the temperature of the incubation is preferably 36-38 ℃, more preferably 37 ℃. The incubation time is preferably 4-6 min, more preferably 5min. The cleaning solution is preferably 20mM Tris solution containing 0.9% NaCl by mass and 0.1% TW20 by volume concentration, and the pH value is 8.0. The luminescent substrate is preferably AMPPD luminescent liquid. The wavelength of the measured luminescence value RLU is preferably 400-600 nm.
In the invention, the standard four-parameter equation is shown in formula (1): y= (A-D)/[ 1+ (X/C) B]+d equation (1), wherein: a=30769642.7844191, b=1.0268648739658, c=7.770684590935, d= -12138.7431763364; correlation coefficient R 2 :0.99999. the standard four parameter equation of the present invention is preferably fitted with quetiapine calibrators at concentrations of 0, 50, 200, 500, 1500 and 3000ng/mL, the quetiapine calibrators preferably being obtained in a calf serum configuration.
The sample to be tested according to the present invention preferably comprises urine or blood.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
A preparation method of quetiapine derivative hapten comprises the following steps of:
1) 1.15g of quetiapine and 0.34g of imidazole are added into a reaction bottle, 20mL of N, N-Dimethylformamide (DMF) and 450mg of tertiary butyl dimethyl chlorosilane are added for reaction at room temperature overnight, the reaction is detected by Thin Layer Chromatography (TLC), after the reaction system is cooled down, the mixture is added into 100mL of water, solids are separated out, the mixture is filtered, and a filter cake is dried to obtain an intermediate I, wherein the preparation process is shown in figure 2.
2) Adding 0.8g of the intermediate I prepared in the step 1) into a reaction bottle, adding 30mL of DMF, cooling to 0 ℃, adding 10mg of sodium hydride, adding 300mg of monomethyl succinate acyl chloride, reacting the mixed system at room temperature for 12 hours, detecting the end of the reaction by TLC, adding the system into 100mL of ice water, extracting the obtained water phase with ethyl acetate three times, 40mL each time, combining the organic phases, backwashing the organic phases once by using a saturated sodium chloride solution, drying the organic phases, filtering, evaporating to dryness, and purifying by column chromatography to obtain an intermediate II, wherein the preparation process is shown in figure 3.
3) Adding 0.2g of the intermediate II prepared in the step 2) into a reaction bottle, adding 30mL of tetrahydrofuran and 5mL of tetrabutylammonium fluoride-tetrahydrofuran solution with the concentration of 1mg/mL, reacting the mixed system at room temperature overnight, detecting the reaction by TLC, adding the system into 100mL of water after evaporating to dryness, separating out solids, filtering, drying a filter cake to obtain an intermediate III, and preparing the intermediate III, wherein the preparation process is shown in figure 4.
4) Adding 0.1g of the intermediate III prepared in the step 3) into a reaction bottle, adding 50mL of methanol, dissolving 10mL of 1N NaOH, reacting overnight at room temperature, evaporating the system the next day, extracting the water phase with ethyl acetate three times, 30mL each time, adjusting the pH value of the water phase to 3, separating out solids, filtering, and drying the filter cake to obtain the quetiapine derivative hapten, wherein the preparation process is shown in figure 5.
The nuclear magnetic resonance hydrogen spectrum identification result of the quetiapine derivative hapten is shown in fig. 6, and the specific result is as follows:
1HNMR(400MHz,DMSO):7.39-7.36(m,1H);7.34-7.21(m,2H);7.15-6.91(m,2H);6.85-6.71(m,3H);3.78-3.57(m,7H);3.56-3.38(m,6H);2.44-2.37(m,4H);2.34-2.21(m,3H)。
example 2
The preparation of quetiapine immunogen comprises the following specific steps:
1. taking 10mg of quetiapine hapten derivative prepared in example 1, and adding 1mL of DMSO to dissolve completely;
2. 10mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is weighed and dissolved in 100 mu L of water, and then added into the solution obtained in the step 1, and reacted for 1h at room temperature to obtain solution A;
3. weighing 20mg of bovine thyroglobulin and dissolving the bovine thyroglobulin in 5mL of PBS buffer solution to obtain solution B;
4. mixing the solution A and the solution B, and stirring for 2 hours at room temperature;
5. the solution obtained by mixing solution A and solution B was dialyzed 4 times against PBS buffer using a dialysis bag having a molecular weight cutoff of 7000Da, and the remaining solution was frozen at-20 ℃.
Example 3
The preparation of quetiapine coating antigen comprises the following specific steps:
1. taking 10mg of quetiapine hapten derivative prepared in example 1, and adding 1mL of DMSO to dissolve completely;
2. 10mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is weighed and dissolved in 100 mu L of water, and then added into the solution obtained in the step 1, and reacted for 1h at room temperature to obtain solution A;
3. weighing 20mg of bovine serum albumin and dissolving the bovine serum albumin in 5mL of PBS buffer solution to obtain solution B;
4. mixing the solution A and the solution B, and stirring for 2 hours at room temperature;
5. the solution obtained by mixing solution A and solution B was dialyzed 4 times against PBS buffer using a dialysis bag having a molecular weight cutoff of 7000Da, and the remaining solution was frozen at-20 ℃.
Example 4
The preparation of quetiapine monoclonal antibody comprises the following steps:
quetiapine mab was prepared using techniques well known in the art using classical hybridoma cell fusion techniques.
Diluting the quetiapine artificial immunogen prepared in the example 2 to 1mg/mL by using PBS buffer solution, adding an equal volume of Freund's complete adjuvant, emulsifying completely, and performing primary immunization on mice according to a dosage of 0.1 mg/mouse;
after 4 weeks of interval, 1mg of quetiapine immunogen prepared in example 2 was mixed and emulsified with Freund's incomplete adjuvant in equal volumes, and the mice were immunized a second time at a dose of 0.1 mg/dose;
spleen cells were fused with Sp2/0 cells, titers and competition assays were performed on the fused cells using quetiapine-bovine thyroglobulin 1. Mu.g/mL coated ELISA 96-well plates, 2 cells (C-KLP-12H 2, C-KLP-5B 7) were obtained by screening, and the results of indirect competition ELISA detection of cell supernatants are shown in Table 1.
TABLE 1 Indirect competition ELISA detection results of cell supernatants
From table 1, it can be derived that: the C-KLP-12H2 clone showed good sensitivity to quetiapine and excellent specificity, without cross-reactivity to various metabolites. Is suitable for establishing a drug concentration monitoring method. Therefore, the ascites of the C-KLP-12H2 mouse is prepared, protein A/G is purified and used for establishing a subsequent quetiapine drug concentration monitoring method, and the C-KLP-12H2 hybridoma cell strain is preserved with the preservation number of CGMCC No.45340.
Example 5
The method for establishing the detection method of the quetiapine blood concentration by the magnetic particle chemiluminescence method comprises the following steps:
1) 50mg Dynal beads M280 Tosyl magnetic beads are diluted in 2mL 50mM BB pH 8.0 buffer solution, 1mg of quetiapine monoclonal antibody C-KLP-12H2 prepared in example 4 is added, and after being mixed uniformly, the mixture is subjected to shaking reaction at 37 ℃ for 8 hours.
2) The supernatant was removed by magnetic attraction, and TBST (50 mM Tris,0.9% NaCl,0.1% TW20, pH 7.4) was added and reacted at 37℃for 12 hours.
3) Removing supernatant by magnetic attraction, adding TBST, diluting to 0.4mg/mL, naming as magnetic particle working solution, and preserving at 2-8deg.C for use.
4) 1mg ALP was dissolved in 1mL PBS, 0.5mg quetiapine derivative hapten (dissolved in 100. Mu.L DMSO) was added, mixed well, 1mg EDC solid was added, and mixed well for 2h at room temperature. Dialysis into PBS, 50mM MES 0.9% NaCl 5mg/mL BSA 1mM MgCl 2 The pH value is 6.7, diluted to 1 mug/mL and named as enzyme-labeled working solution.
5) The reaction procedure: 10 mu L of sample and 40 mu L of magnetic particle liquid working solution prepared in the step 3) and 50 mu L of enzyme-labeled working solution prepared in the step 4), incubating at 37 ℃ for 5min, cleaning, and adding AMPPD luminescent liquid for color development.
6) Calibration standards for quetiapine at various concentrations of 0, 50, 200, 500, 1500 and 3000ng/mL were prepared using a calf serum matrix, and the standard curves prepared are shown in table 2 and fig. 7, wherein X represents concentration and Y represents signal value in table 2 and fig. 7, and the right end point value on the abscissa of fig. 7 is 3000 instead of 300, since the pictures are not shown completely, and are described herein; and determining the precision and the lowest detection limit of the detection method, wherein the result is shown in a table 3, and the data in the table 3 are obtained by measuring quality control products QC1 and QC2 and calculating CV 10 times each; loB is a negative sample measured 20 times and the average +2SD is calculated as the detection limit.
TABLE 2 calibration curve data for quetiapine plasma concentration detection method by magnetic particle chemiluminescence method
TABLE 3 precision and sensitivity of detection method of quetiapine blood concentration by magnetic particle chemiluminescence method
From table 3, it can be derived that: the magnetic particle luminescence method quetiapine determination reagent prepared by the C-KLP-12H2 antibody has high sensitivity and meets clinical requirements.
Example 6
Preparation of quetiapine average phase enzyme immunoassay reagent
The quetiapine average phase enzyme immunoassay reagent comprises two separately arranged reagents, and specifically comprises the following steps:
preparation of reagent R1: 20mM NAD,30mM glucose-6-phosphate (G6P), 55mM Tris buffer pH 8.0 were dissolved to prepare a homogeneous enzyme substrate; to the above homogeneous enzyme substrate was added 0.05% of the anti-quetiapine monoclonal antibody C-KLP-12H2.
Preparation of reagent R2: 0.05% of the quetiapine derivative-G6 PDH enzyme-labeled conjugate prepared was added to a Tris buffer of pH8.5 at 0.1M, and the specific ratio in this example was 0.05%.
Wherein, the quetiapine derivative-G6 PDH enzyme-labeled conjugate is prepared by dissolving 600U G6PDH in 1mL PBS, adding 0.5mg quetiapine derivative-ALP (dissolved in 100 mu L DMSO), mixing uniformly, adding 1mg EDC solid, mixing uniformly at room temperature for 2h. Dialyzed into PBS, diluted to 5U/mL with 100mM Tris 0.9%NaCl 5mg/mL BSA pH8.5, and designated as R2 working solution.
Test procedure for 20. Mu.L sample +50. Mu. L R1 for 5min followed by addition of 50. Mu. L R2 for 5min and determination of OD 340 The rate of rise of the wavelength was set to Hitachi 7170.
Clinical 40 examples of quetiapine urine concentration samples are selected, and compared with a homogeneous enzyme method constructed by the invention and an LC-MS method, the results are shown in figure 8:
as can be seen from FIG. 8, the homogeneous enzyme method established based on the antibody prepared by the present invention has good correlation between the total quetiapine content and LC-MS method, R 2 0.9929, which meets the clinical requirements.
From the above embodiments it can be derived that: the quetiapine derivative hapten provided by the invention can be used for preparing a specific antibody which has high sensitivity and good specificity and can effectively identify quetiapine.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. A quetiapine derivative hapten has a structure shown in a formula I:
formula I.
2. A quetiapine artificial antigen comprising a carrier protein and the quetiapine-derived hapten of claim 1; the quetiapine derivative hapten and the carrier protein are covalently coupled through a chemical cross-linking agent.
3. The quetiapine artificial antigen of claim 2, wherein the carrier protein comprises bovine serum albumin, chicken ovalbumin, bovine thyroglobulin, human serum albumin or rabbit serum albumin.
4. A quetiapine artificial antigen according to claim 2 or 3, characterised in that the chemical cross-linking agent comprises 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.
5. A hybridoma cell C-KLP-12H2 obtained by immunoscreening the quetiapine artificial antigen of any one of claims 2-4, wherein the hybridoma cell C-KLP-12H2 has a preservation number of CGMCC No.45340.
6. An anti-quetiapine antibody obtained by secretion of the hybridoma cell C-KLP-12H2 according to claim 5.
7. The anti-quetiapine antibody of claim 6, wherein the anti-quetiapine antibody comprises a monoclonal antibody or a polyclonal antibody.
8. Use of the anti-quetiapine antibody of claim 6 in the immunoassay of quetiapine blood concentration or in the preparation of a kit and/or reagent for the immunoassay of quetiapine blood concentration.
9. A quetiapine homogeneous enzyme immunoassay kit, which is characterized by comprising a reagent R1 and a reagent R2;
the reagent R1 comprises the following components in concentration: 20mM NAD,30mM glucose-6-phosphate, 55mM Tris buffer pH 8.0 and 0.01-0.1 wt.% of the anti-quetiapine antibody of claim 6 or 7;
the reagent R2 comprises the following components in concentration: 0.01wt.% to 0.1wt.% quetiapine derivative-glucose-6-phosphate dehydrogenase enzyme-labeled conjugate and 0.1M Tris buffer at pH 8.5;
the quetiapine derivative-glucose-6-phosphate dehydrogenase enzyme-labeled conjugate is obtained by coupling a 6-phosphate glucose dehydrogenase label and the quetiapine derivative hapten of claim 1.
10. A method for detecting quetiapine blood concentration based on a magnetic particle chemiluminescence method, which is characterized by comprising the following steps:
mixing and incubating a sample to be detected, a magnetic particle working solution and an enzyme-labeled working solution, cleaning, adding a luminescent substrate for reaction and color development, measuring a luminescent value RLU, substituting a standard four-parameter equation, and calculating the concentration of quetiapine in the sample to be detected;
the magnetic particle working solution is obtained by labeling the anti-quetiapine antibody of claim 6 or 7 with magnetic particles;
the enzyme-labeled working solution is obtained by enzyme-labeling the quetiapine derivative hapten according to claim 1.
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