CN112730841A - Immunological detection method of risperidone and/or 9-hydroxy risperidone - Google Patents
Immunological detection method of risperidone and/or 9-hydroxy risperidone Download PDFInfo
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- 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
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- 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
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- 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
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- 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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
Abstract
The application relates to the technical field of immunological detection, and particularly discloses an immunological detection method for risperidone and/or 9-hydroxyrisperidone, which comprises the steps of uniformly mixing a blood sample, magnetic particle working solution containing a conjugate formed by coupling magnetic particles and an antibody, and enzyme-labeled working solution containing a conjugate formed by coupling risperidone, a 9-hydroxyrisperidone hapten and a label, sequentially carrying out incubation reaction and washing, adding an indicator solution for color development, and collecting an optical signal; the structures of risperidone and 9-hydroxyrisperidone haptens are shown as formula I:
antibodies are generated in response to risperidone and the 9-hydroxyrisperidone antigen, which are conjugates formed by coupling risperidone and the 9-hydroxyrisperidone hapten with a carrier protein. The application establishes an immunological detection method with strong specificity and high sensitivity to risperidone and 9-hydroxy risperidone.
Description
Technical Field
The application relates to the technical field of immunological detection, in particular to an immunological detection method of risperidone and/or 9-hydroxy risperidone.
Background
Schizophrenia is a serious, disabling mental disorder. With the increasing social competition, the accelerated pace of life and the change of family structure, people are under greater and greater pressure, and as a result, the mental and health problems are increasingly highlighted. Schizophrenia is the most common disease among psychiatric diseases.
Risperidone is a benzisoxazole derivative with the chemical name of 3- [2- [4- (6-fluoro-1, 2-benzisoxazol-3-yl) -1-piperidinyl ] ethyl ] -6,7,8, 9-tetrahydro-2-methyl-4H-pyrido [1,2- α ] pyrimidin-4-one, and is a novel psychiatric drug. Risperidone targets 5-hydroxytryptamine (5-HT2) and dopamine (D2) receptors, blocking their respective neurotransmitter absorption, thereby effectively improving psychotic symptoms.
The main metabolism of risperidone 9-hydroxyrisperidone has similar pharmacological actions as risperidone, and both of them constitute antipsychotic active ingredients. However, the patients also metabolize inactive metabolites, namely 7-hydroxy risperidone and N-dealkylated risperidone after taking risperidone, and most patients also take sedatives, tricyclic antidepressants, selective serotonin reuptake inhibitors and the like while taking risperidone, which all affect the detection of risperidone and 9-hydroxy risperidone.
At present, the radioimmunoassay and the high performance liquid chromatography are mostly adopted for measuring the blood concentration of risperidone and 9-hydroxy risperidone, but the radioimmunoassay has the defects of high cost, easy radioactive pollution and low instrument popularization rate, and the high performance liquid chromatography needs repeated extraction for many times, has the defects of complex operation, complexity, time and labor waste, low flux and the like, and is not beneficial to clinical popularization.
For example, patent documents with publication dates 2015 07, 01 and application publication number CN104755479A disclose a risperidone and paliperidone hapten, and patent documents with publication dates 2015, 06, 24 and application publication number CN104736565A disclose an antibody against risperidone hapten and its use, which both propose that derivatization is performed at the 9-hydroxy position and the F atom position of the 9-hydroxy risperidone molecule, the prepared antibody has significant cross-reaction with the inactive metabolite 7-hydroxy risperidone, and a detection reagent prepared from the antibody may cause a higher blood concentration of risperidone and paliperidone in a blood sample. For example, patent documents with application publication date of 2018, 08 and 21 and application publication number of CN108431040A disclose an amino acid sequence of a risperidone antibody, but the antibody has serious cross reaction with 7-hydroxyrisperidone which is an inactive metabolite, and the specificity is poor.
Therefore, the establishment of the immunological detection method of risperidone and 9-hydroxy risperidone with strong specificity and high sensitivity has very important significance for formulating an individual dosage scheme of risperidone and evaluating the clinical curative effect and safety of risperidone.
Disclosure of Invention
In order to establish an immunological detection method with strong specificity and high sensitivity on risperidone and 9-hydroxy risperidone, the application provides an immunological detection method for risperidone and/or 9-hydroxy risperidone, which adopts the following technical scheme:
an immunological detection method for risperidone and/or 9-hydroxy risperidone comprises the steps of uniformly mixing a blood sample, magnetic particle working solution containing a conjugate formed by coupling magnetic particles and an antibody and enzyme-labeled working solution containing a conjugate formed by coupling risperidone, a 9-hydroxy risperidone hapten and a label, and adding an indicator solution for color development and collecting an optical signal of 400-550nm after incubation reaction and cleaning are sequentially carried out;
the structures of the risperidone and the 9-hydroxy risperidone hapten are shown as formula I:
the antibody is generated in response to risperidone and a 9-hydroxyrisperidone antigen, which are conjugates formed by coupling risperidone and a 9-hydroxyrisperidone hapten and a carrier protein;
the indicator solution contains an indicator for quantitatively detecting the concentration of the marker.
Preferably, the risperidone and the 9-hydroxy risperidone hapten are prepared by the following method:
(i) carrying out free radical bromination reaction on the compound 1 and N-bromosuccinimide to obtain a compound 2;
(ii) carrying out alkaline hydrolysis reaction on the compound 2 to obtain a compound 3;
(iii) carrying out reductive amination reaction on the compound 3 and benzylamine to obtain a compound 4;
(iv) carrying out condensation reaction on the compound 4 and succinic anhydride to obtain a compound shown in the formula I, wherein the compound shown in the formula I is a hapten of risperidone and 9-hydroxy risperidone;
the specific reaction route is as follows:
wherein the compound 1 is risperidone with the chemical name of 3- [2- [4- (6-fluoro-1, 2-benzisoxazol-3-yl) -1-piperidyl ] ethyl ] -6,7,8, 9-tetrahydro-2-methyl-4H-pyrido [1, 2-alpha ] pyrimidin-4-one;
the chemical name of compound 2 is 3- [2- [4- (6-fluoro-1, 2-benzisoxazol-3-yl) -1-piperidinyl ] ethyl ] -6,7,8, 9-tetrahydro-2-dibromomethyl-4H-pyrido [1, 2-alpha ] pyrimidin-4-one;
the chemical name of compound 3 is 3- [2- [4- (6-fluoro-1, 2-benzisoxazol-3-yl) -1-piperidinyl ] ethyl ] -6,7,8, 9-tetrahydro-2-carbaldehyde-4H-pyrido [1, 2-alpha ] pyrimidin-4-one;
the chemical name of compound 4 is 3- [2- [4- (6-fluoro-1, 2-benzisoxazol-3-yl) -1-piperidinyl ] ethyl ] -6,7,8, 9-tetrahydro-2-methyl-4H-pyrido [1, 2-alpha ] pyrimidin-4-one.
Wherein, in the step (i), the compound 1 and the N-bromosuccinimide react in any proportion to generate a compound 2; in order to improve the reaction yield of the compound 2, the ratio of the amount of the required N-bromosuccinimide substance to the amount of the required compound 1 substance is preferably not less than 1, and more preferably the ratio of the amount of the required N-bromosuccinimide substance to the amount of the required compound 1 substance is not less than 2; in order to save the usage amount of the N-bromosuccinimide, the ratio of the amount of the needed N-bromosuccinimide substance to the amount of the needed compound 1 substance is (2-2.5): 1. Moreover, all the conditions applicable to the free radical bromination reaction of the compound 1 and the N-bromosuccinimide in the related art can be adopted; preferably, the conditions for the radical bromination reaction in step (i) are: the solvent is carbon tetrachloride, the catalyst is benzoyl peroxide, the reaction system is protected from light, and the reaction temperature is 80 ℃.
In step (ii), any conditions applicable to the alkaline hydrolysis reaction of the compound 2 in the related art can be adopted; preferably, the conditions of the alkaline hydrolysis reaction in step (i1) are: the solvent is 1, 4-dioxane, the initiator is lithium hydroxide and water, the reaction temperature is 50 ℃, and the reaction time is 3 hours.
In the step (iii), the compound 3 and benzylamine react in any proportion to generate the compound 4; in order to increase the reaction yield of the compound 4, preferably, the ratio of the amount of the substance of the desired benzylamine to the amount of the substance of the desired compound 3 is not less than 0.5, more preferably, the ratio of the amount of the substance of the desired N-bromosuccinimide to the amount of the substance of the desired compound 3 is (0.5-1.5):1 in order to save the amount of the N-bromosuccinimide used; more preferably, the ratio of the amount of the substance of the desired N-bromosuccinimide to the amount of the substance of the desired compound 3 is 1. Moreover, any conditions applicable to the reductive amination reaction of the compound 3 and benzylamine can be employed in the related art; preferably, the conditions of the reductive amination reaction in step (iii) are: the solvent is methanol, the catalyst is Pd/C catalyst, the hydrogen protection is carried out, the reaction temperature is 80 ℃, and the reaction time is 12 hours.
In step (iv), the compound 4 and succinic anhydride react in any proportion to generate the compound of formula I; in order to improve the reaction yield of the compound shown in the formula I, the ratio of the amount of the needed succinic anhydride to the amount of the needed compound 4 is preferably more than or equal to 0.5, and more preferably, the ratio of the amount of the needed succinic anhydride to the amount of the needed compound 4 is (0.5-1.5): 1; more preferably, the ratio of the amount of the substance of succinic anhydride required to the amount of the substance of compound 4 required is 1. Moreover, any conditions applicable to the condensation reaction of the compound 4 and succinic anhydride can be adopted in the related art; preferably, the conditions for the condensation reaction in step (iv) are: the solvent is acetonitrile, the catalyst is 4-dimethylamino pyridine, the reaction is carried out at room temperature, and the reaction time is 12 h.
Preferably, the conjugate formed by coupling the risperidone and the 9-hydroxy risperidone hapten and the carrier protein is prepared by the following method:
(i) dissolving risperidone and 9-hydroxy risperidone hapten in dimethyl sulfoxide to obtain dimethyl sulfoxide solution of the hapten;
(ii) dissolving a coupling agent in water to obtain an aqueous solution of the coupling agent;
(iii) mixing a dimethyl sulfoxide solution of a hapten and a coupling agent aqueous solution, and reacting at room temperature for 0.5-2 hours to obtain a reaction solution, wherein the weight ratio of risperidone to 9-hydroxy risperidone hapten to coupling agent is 1 (0.5-2);
(iv) dissolving carrier protein in PBS buffer solution to obtain carrier protein solution;
(v) mixing the carrier protein solution with the reaction solution, and stirring at room temperature for 1-3 hours to obtain a reaction mixed solution, wherein the weight ratio of the risperidone, the 9-hydroxy risperidone hapten and the carrier protein is 1 (0.5-2);
(vi) dialyzing the reaction mixed solution against PBS buffer solution to obtain the antigen, wherein the volume ratio of the reaction mixed solution to the PBS buffer solution is 1 (400-600).
Preferably, the coupling agent is selected from 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride.
Preferably, the antibody is prepared by the following method:
(i) selecting a host for antibody production;
(ii) inoculating the host with the conjugate;
(iii) fusing a cell line from the inoculated host with Sp2/0 cells to obtain hybridoma cells producing the antibody;
(iv) the hybridoma cell is prepared from host ascites and purified to obtain the antibody.
Preferably, the carrier protein is selected from any one of bovine serum albumin, chicken ovalbumin, bovine thyroglobulin, human serum albumin and rabbit serum albumin.
Preferably, the magnetic particle working solution is prepared by the following method:
(i) diluting the magnetic particles in MES buffer solution to obtain magnetic particle solution, and adding the antibody into the magnetic particle solution to obtain magnetic particle-antibody mixed solution, wherein the weight ratio of the magnetic particles to the antibody is (25-100): 1;
(ii) adding a coupling agent into the magnetic particle-antibody mixed solution, and carrying out oscillation reaction for 2 hours at room temperature, wherein the weight ratio of the coupling agent to the antibody is (1-3) to 1;
(iii) magnetic absorption is carried out to remove supernatant, TBST buffer solution is added, and reaction is carried out for 1-5h at room temperature;
(iv) and (4) magnetically absorbing to remove supernatant, adding TBST buffer solution to obtain magnetic particle working solution, wherein the concentration of the magnetic particles in the magnetic particle working solution is 0.3-0.5 mg/mL.
Preferably, the enzyme labeling working solution is prepared by the following method:
(i) dissolving a marker in a PBS buffer solution to obtain a marker solution;
(ii) adding the hapten of claim 1 into a marker solution, uniformly mixing, then adding a coupling agent, and uniformly mixing at room temperature for 1-5 h; wherein the weight ratio of the marker, the hapten and the coupling agent is (1.5-3) to 1 (1.5-3);
(iii) and (3) obtaining the enzyme-labeled working solution by adopting a PBS (phosphate buffer solution) dialysis method, wherein the concentration of the marker in the enzyme-labeled working solution is 0.5-2 ug/mL.
Preferably, the marker is alkaline phosphatase and the indicator is a luminescent substrate for quantitative determination of alkaline phosphatase content.
Preferably, the blood sample, the magnetic particle working solution and the enzyme-labeled working solution are uniformly mixed according to the volume ratio of (10-30) to (30-40) to (40-60).
Preferably, the temperature of the incubation reaction is 35-40 ℃, and the time is 3-10 min; more preferably, the incubation reaction is carried out at 37 ℃ for 5 min.
Preferably, the cleaning step specifically comprises: and (4) removing the supernatant by magnetic attraction, adding TBST buffer solution for resuspension, and removing the supernatant by magnetic attraction again.
Preferably, the optical signal is collected by a photomultiplier tube.
In summary, the present application has the following beneficial effects:
the method is characterized by establishing an immunological detection method with strong specificity and high sensitivity on risperidone and 9-hydroxy risperidone, being capable of specifically determining the total concentration of risperidone and 9-hydroxy risperidone, and having the advantages of high sensitivity, simple operation, time saving and labor saving.
Secondly, because risperidone and 9-hydroxy risperidone are small-molecule drugs and have no immunogenicity, risperidone and 9-hydroxy risperidone hapten need to be prepared, and then the risperidone and 9-hydroxy risperidone hapten are used for preparing antibodies. This is due to risperidone and 9-hydroxy risperidone hapten, the derivative site of the risperidone derivative is far from the 7-site and close to the 9-site, and other structures of the risperidone molecule are not changed, and the functional group specific to risperidone is retained. Therefore, risperidone and 9-hydroxy risperidone hapten is used as immunogen and carrier protein to form risperidone and 9-hydroxy risperidone antigen through coupling, and risperidone and 9-hydroxy risperidone antigen stimulate to generate risperidone and 9-hydroxy risperidone antibody, and the risperidone and 9-hydroxy risperidone antibody has the advantages of strong specificity and high sensitivity to risperidone and 9-hydroxy risperidone, and has no cross reaction to inactive metabolite 7-hydroxy risperidone and N-dealkylated risperidone.
The antibody can specifically recognize risperidone and 9-hydroxyrisperidone, and the antibody can simultaneously recognize risperidone and 9-hydroxyrisperidone, and presumably because carrier protein has a certain shielding effect on the 9-position part structure of 9-hydroxyrisperidone, the antibody is very beneficial to the determination of active substances of risperidone, and only one antibody is needed.
The antibody has no cross reaction with 7-hydroxy risperidone and N-dealkylation risperidone, and presumably because the derived sites of risperidone and 9-hydroxy risperidone hapten are far away from the 7 th site, the 7 th site structure can be better exposed, so that the specificity of the prepared antibody is high.
Drawings
FIG. 1 is a correlation analysis of the total concentration of risperidone and 9-hydroxyrisperidone determined by magnetic particle luminescence method established with the antibody provided in the present application and HPLC-MS method.
Detailed Description
The present application will be described in further detail with reference to the following drawings and examples.
Example 1
The structure of the hapten of risperidone and 9-hydroxy risperidone is shown in formula I, and the specific synthetic route of the compound of formula I is as follows:
preparation of compound 2: adding risperidone (4.1g, 0.01mol) into a 250mL single-mouth bottle, adding 150mL of carbon tetrachloride, benzoyl peroxide (0.24g, 0.001mol) and NBS (3.9g, 0.022mol), refluxing the system for 6 hours in a dark state, detecting by TLC, and cooling the system to room temperature. And pouring the reaction solution into 200mL of water for extraction, separating an organic phase, extracting the water phase with dichloromethane for three times, wherein the dosage of dichloromethane is 50mL each time, combining the organic phases, drying with anhydrous sodium sulfate, filtering, and evaporating to dryness to obtain a crude product. The crude product was purified by column chromatography to give 1.8g of compound 2 (yield 32%).
Preparation of compound 3: adding compound 2(1.8g, 3.17mmol), lithium hydroxide (364mg, 15.9mmol) and 50mL of 1, 4-dioxane into a 100mL single-neck bottle, adding 3mL of water, reacting at 50 ℃ for 3 hours, detecting by TLC, and spinning to obtain a crude product. The crude product was purified by column chromatography to give 0.86g of Compound 3 (yield 64%).
Preparation of compound 4: adding compound 3(0.8g, 1.89mmol), benzylamine (200mg, 1.89mmol), Pd/C catalyst (100mg) and 30mL of methanol into a 100mL single-neck bottle, replacing the system with hydrogen for three times, reacting for 12 hours under the protection of hydrogen, detecting by TLC after the reaction is finished, filtering the system, and evaporating the filtrate to dryness to obtain a crude product. The crude product was purified by column chromatography to give 0.27g of Compound 4 (yield 32%).
Preparation of haptens of risperidone and 9-hydroxyrisperidone (compound of formula I): adding the compound 4(230mg, 0.54mmol), succinic anhydride (54mg, 0.54mmol) and DMAP (131mg, 1.08mmol) into a 100mL single-neck flask, adding 30mL acetonitrile, reacting the system at room temperature for 12 hours, detecting the reaction end by TLC, and directly purifying the system by column chromatography to obtain 0.12g of risperidone and hapten of 9-hydroxy risperidone (yield 42%).
Characterization of haptens of risperidone and 9-hydroxyrisperidone:1H-NMR(400MHZ,CDCl3):12.18(S,1H)；9.34(S,1H)；4.70(t,2H)；2.51-2.41(m,4H)；6.97(d,1H)；7.41(m,H)；2.78(m,1H)；7.12(t,1H)；2.26(m,2H)；1.74(m,4H)；1.90(m,2H)；1.77(m,2H)；3.41(t,2H)；3.87(m,2H)；2.06(m,2H)；2.60(m,2H)；2.49(m,2H)；
13C-NMR(400MHZ,CDCl3):166.5,147.1,117.8,164.2,162.0,155.2,146.6,46.8,48.6,96.9,123.5,27.5,115.0,111.0,31.4,30.1,22.7,18.5,173.8,171.9,59.4,44.6,22.4,30.2,29.2；
MS,m/Z 526(M+H)。
example 2
The antigen of risperidone and 9-hydroxy risperidone is prepared by the following specific method:
(i) dissolving hapten of 10mg risperidone and 9-hydroxy risperidone in 1mL of dimethyl sulfoxide to obtain dimethyl sulfoxide solution of the hapten;
(ii) dissolving 10mg of 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride in 100uL of water to obtain an aqueous solution of 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride;
(iii) adding the water solution of 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride into the dimethyl sulfoxide solution of hapten, and reacting at room temperature for 1 hour to obtain a reaction solution;
(iv) dissolving 10mg of bovine serum albumin in 5mL of PBS buffer solution to obtain a bovine serum albumin solution;
(v) mixing the bovine serum albumin solution with the reaction solution, and stirring for 2 hours at room temperature to obtain a reaction mixed solution;
(vi) and (3) dialyzing the reaction mixed solution against PBS buffer solution, wherein the volume ratio of the reaction mixed solution to the PBS buffer solution is 1:500, and repeating the dialysis step for four times to obtain the antigens of the risperidone and the 9-hydroxy risperidone.
It is worth pointing out that the preservation conditions of the antigens of risperidone and 9-hydroxyrisperidone are low-temperature cryopreservation, in particular cryopreservation at-20 ℃.
Example 3
An antibody capable of binding to risperidone and/or 9-hydroxyrisperidone is prepared by the following steps:
(i) selection of hosts for antibody production: wherein, the host can adopt mice, rabbits, goats, sheep and the like, and the mice are adopted as the host in the embodiment;
(ii) vaccination of the host with antigens of risperidone and 9-hydroxyrisperidone: diluting the antigens of risperidone and 9-hydroxy risperidone to 1mg/mL by using normal saline, adding equivalent volume of Freund's complete adjuvant, completely emulsifying, and immunizing the mouse for the first time according to the dose of 0.1 mg/mouse; after four weeks, mixing 1mg of risperidone, 9-hydroxy risperidone antigen and 1mg of Freund's incomplete adjuvant, stirring at 2000rpm/min for 2 hours to complete emulsification, and performing boosting immunization on the mice immunized for the first time according to 0.1 mg/mouse;
(iii) fusing a cell line from an inoculated host with Sp2/0 cells, coating an ELISA 96 pore plate with antigens of risperidone and 9-hydroxyrisperidone, performing titer and competition measurement on the fused cells by adopting an indirect ELISA method and an indirect competition ELISA method respectively, and screening to obtain 3 strains of cells, namely 23H1, 42B2 and 13F2 respectively.
The three cells respectively adopt mouse ascites preparation and Protein A/G affinity purification to obtain antibodies, and an indirect competition ELISA method is adopted to determine the binding capacity of the three antibodies with four micromolecule compounds such as risperidone, 9-hydroxy risperidone, 7-hydroxy risperidone, N-dealkylrisperidone and the like, and the results are shown in Table 1.
TABLE 1
| 23H1 | | 13F2 | |
| Risperidone | |||
| 100％ | 100％ | 100％ | |
| 9-Hydroxyrisperidone | 99％ | 78％ | 95％ |
| 7-Hydroxyrisperidone | 5％ | 1％ | 4％ |
| N-dealkylrisperidone | ＜0.1％ | ＜0.1％ | ＜0.1％ |
As can be seen from Table 1, the antibody produced by 23H1 cells has substantially uniform affinity for risperidone and 9-hydroxyrisperidone, and has very low cross-reactivity with inactive 7-hydroxyrisperidone and N-dealkylrisperidone. The antibody produced by 23H1 cells was selected for use in detecting the concentration of risperidone and 9-hydroxyrisperidone in blood samples.
Example 4
A detection reagent for detecting risperidone and/or 9-hydroxy risperidone comprises magnetic particle working solution, enzyme-labeled working solution and indicator solution.
S1 obtaining of magnetic particle working solution:
s1-1: diluting 50mg Dynal beads M270 COOH magnetic particles in 2mL MES buffer (50mM, pH 6.0) to obtain a magnetic particle solution, and adding 1mg antibody to the magnetic particle solution to obtain a magnetic particle-antibody mixture;
s1-2: adding 2mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride into the magnetic particle-antibody mixed solution, and carrying out shake reaction at room temperature for 2 hours;
s1-3: removing supernatant through magnetic attraction, adding TBST buffer solution to enable the concentration of magnetic particles to reach 0.5mg/mL, and reacting for 2 hours at room temperature;
s1-4: and (4) magnetically absorbing to remove supernatant, adding TBST buffer solution to obtain magnetic particle working solution, wherein the concentration of the magnetic particles in the magnetic particle working solution is 0.4 mg/mL.
S2 obtaining enzyme-labeled working solution:
s2-1: dissolving 1mg of alkaline phosphatase in 1mL of PBS buffer solution to obtain an alkaline phosphatase solution;
s2-2: dissolving 0.5mg of risperidone and hapten of 9-hydroxy risperidone in 0.1mL of dimethyl sulfoxide to obtain a hapten solution;
s2-3: uniformly mixing the alkaline phosphatase solution and the hapten solution, adding 1mg of 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride, and uniformly mixing at room temperature for 2 hours;
s2-4: and (3) obtaining the enzyme-labeled working solution by adopting a PBS (phosphate buffer solution) dialysis method, wherein the concentration of the marker in the enzyme-labeled working solution is 1 ug/mL.
Acquisition of the indicator solution of S3: commercial APS-5 luminescent liquid.
The detection reagent for detecting risperidone and/or 9-hydroxy risperidone can be prepared at present, or the detection reagent for detecting risperidone and/or 9-hydroxy risperidone can be stored at 2-8 ℃ for use and taken at any time; further, the detection reagent for detecting risperidone and/or 9-hydroxy risperidone is prepared into a detection kit, and the detection kit needs to be stored at the temperature of 2-8 ℃.
Example 5
A method for detecting risperidone and/or 9-hydroxy risperidone in a sample comprises the following steps:
uniformly mixing the blood sample, the magnetic particle working solution and the enzyme-labeled working solution in a volume ratio of 20:40:50, incubating and reacting for 5min at 37 ℃, cleaning, adding an indicator solution for color development, and collecting a light signal of 400-550nm by using a photomultiplier tube. The specific steps of cleaning comprise magnetic absorption to remove supernatant, adding TBST buffer solution for heavy suspension, and magnetic absorption again to remove supernatant.
The method for detecting the risperidone and/or 9-hydroxy risperidone in the blood sample and the HPLC-MS method provided by the application are adopted to respectively determine the total concentration of the risperidone and the 9-hydroxy risperidone in the blood sample, and correlation analysis is carried out on the determination results, wherein the analysis results are shown in figure 1.
As can be seen from FIG. 1, the correlation between the total concentration of risperidone and 9-hydroxyrisperidone determined by the magnetic particle luminescence method established based on the antibody provided by the present application and the HPLC-MS method is good, and the clinical requirements are met.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (10)
1. An immunological detection method for risperidone and/or 9-hydroxy risperidone is characterized in that a blood sample, magnetic particle working solution containing a conjugate formed by coupling magnetic particles and an antibody and enzyme-labeled working solution containing a conjugate formed by coupling risperidone, a 9-hydroxy risperidone hapten and a label are uniformly mixed, and after incubation reaction and washing steps are sequentially carried out, indicator solution is added for color development and an optical signal of 400-550nm is collected;
the structures of the risperidone and the 9-hydroxy risperidone hapten are shown as formula I:
the antibody is generated in response to risperidone and a 9-hydroxyrisperidone antigen, which are conjugates formed by coupling risperidone and a 9-hydroxyrisperidone hapten and a carrier protein;
the indicator solution contains an indicator for quantitatively detecting the concentration of the marker.
2. The immunological detection method of claim 1, wherein said risperidone and 9-hydroxyrisperidone antigens are prepared by:
(i) dissolving risperidone and 9-hydroxy risperidone hapten in dimethyl sulfoxide to obtain dimethyl sulfoxide solution of the hapten;
(ii) dissolving a coupling agent in water to obtain an aqueous solution of the coupling agent;
(iii) mixing a dimethyl sulfoxide solution of a hapten and a coupling agent aqueous solution, and reacting at room temperature for 0.5-2 hours to obtain a reaction solution, wherein the weight ratio of risperidone to 9-hydroxy risperidone hapten to coupling agent is 1 (0.5-2);
(iv) dissolving carrier protein in PBS buffer solution to obtain carrier protein solution;
(v) mixing the carrier protein solution with the reaction solution, and stirring at room temperature for 1-3 hours to obtain a reaction mixed solution, wherein the weight ratio of the risperidone, the 9-hydroxy risperidone hapten and the carrier protein is 1 (0.5-2);
(vi) dialyzing the reaction mixed solution against PBS buffer solution to obtain the antigen, wherein the volume ratio of the reaction mixed solution to the PBS buffer solution is 1 (400-600).
3. The immunological detection method of claim 2, wherein said coupling agent is selected from the group consisting of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride.
4. The immunological detection method according to claim 1, wherein the antibody is prepared by a method comprising:
(i) selecting a host for antibody production;
(ii) inoculating the host with the conjugate;
(iii) fusing a cell line from the inoculated host with Sp2/0 cells to obtain hybridoma cells producing the antibody;
(iv) the hybridoma cell is prepared from host ascites and purified to obtain the antibody.
5. The immunological detection method according to claim 1, wherein the carrier protein is selected from any one of bovine serum albumin, chicken ovalbumin, bovine thyroglobulin, human serum albumin and rabbit serum albumin.
6. The immunological detection method according to claim 1, wherein the magnetic microparticle working solution is prepared by a method comprising:
(i) diluting the magnetic particles in MES buffer solution to obtain magnetic particle solution, and adding the antibody into the magnetic particle solution to obtain magnetic particle-antibody mixed solution, wherein the weight ratio of the magnetic particles to the antibody is (25-100): 1;
(ii) adding a coupling agent into the magnetic particle-antibody mixed solution, and carrying out oscillation reaction for 2 hours at room temperature, wherein the weight ratio of the coupling agent to the antibody is (1-3) to 1;
(iii) magnetic absorption is carried out to remove supernatant, TBST buffer solution is added, and reaction is carried out for 1-5h at room temperature;
(iv) and (4) magnetically absorbing to remove supernatant, adding TBST buffer solution to obtain magnetic particle working solution, wherein the concentration of the magnetic particles in the magnetic particle working solution is 0.3-0.5 mg/mL.
7. The immunological detection method according to claim 1, wherein the enzyme-labeled working solution is prepared by a method comprising:
(i) dissolving a marker in a PBS buffer solution to obtain a marker solution;
(ii) adding the hapten of claim 1 into a marker solution, uniformly mixing, then adding a coupling agent, and uniformly mixing at room temperature for 1-5 h; wherein the weight ratio of the marker, the hapten and the coupling agent is (1.5-3) to 1 (1.5-3);
(iii) and (3) obtaining the enzyme-labeled working solution by adopting a PBS (phosphate buffer solution) dialysis method, wherein the concentration of the marker in the enzyme-labeled working solution is 0.5-2 ug/mL.
8. The immunological detection method of claim 1, wherein said marker is alkaline phosphatase and said indicator is a luminescent substrate for quantitative determination of alkaline phosphatase content.
9. The immunological detection method according to claim 1, wherein the blood sample, the magnetic microparticle working solution and the enzyme-labeled working solution are uniformly mixed in a volume ratio of (10-30) to (30-40) to (40-60).
10. The immunological detection method according to claim 1, wherein the incubation reaction is preferably carried out at a temperature of 35 to 40 ℃ for 3 to 10 min.
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