CN116813745A - Olanzapine complete antigen and antibody, and preparation methods and applications thereof - Google Patents

Olanzapine complete antigen and antibody, and preparation methods and applications thereof Download PDF

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Publication number
CN116813745A
CN116813745A CN202310190849.9A CN202310190849A CN116813745A CN 116813745 A CN116813745 A CN 116813745A CN 202310190849 A CN202310190849 A CN 202310190849A CN 116813745 A CN116813745 A CN 116813745A
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China
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olanzapine
detection
monoclonal antibody
card
complete antigen
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Inventor
李忠鹏
何扬锦
陈颖
费晓旭
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Zhejiang Quasi Policy Biological Technology Co ltd
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Zhejiang Quasi Policy Biological Technology Co ltd
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    • 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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/77—Ovalbumin
    • 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
    • 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
    • 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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • 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
    • 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

Abstract

The application discloses an olanzapine complete antigen and application thereof in olanzapine antibody preparation and immunodetection. The olanzapine complete antigen has a structure shown in formula 1:formula 1; the application also discloses an anti-olanzapine monoclonal antibody prepared by the complete antigen, a hybridoma cell for producing the monoclonal antibody, and a detection card and a detection kit for detecting olanzapine analogues. The application discloses an ELISA method and a fluorescence immunochromatography method for detecting olanzapine analogues, wherein the fluorescence immunochromatography method has the advantages of simple operation, short detection time, low cost, high specificity and good repeatability.

Description

Olanzapine complete antigen and antibody, and preparation methods and applications thereof
Technical Field
The application relates to detection of psychotropic drugs. In particular, the application relates to olanzapine complete antigen, an olanzapine monoclonal antibody obtained by using the complete antigen and application thereof in detection of olanzapine analogues by using a fluorescent immunochromatography method.
Background
Atypical antipsychotics Olanzapine (Olanzapine, trade name repropril) have a chemical structure which belongs to the class of thiophenedinitrogen and have a pharmacological effect similar to that of clozapine, and since they are put on the market by us gift company 10 in 1996, they can effectively improve the positive and negative symptoms of schizophrenia, have no serious side effects of granulocytopenia caused by clozapine, and are not obvious and have received attention. Olanzapine is taken orally and absorbed fast and completely, food has no influence on the absorption rate and degree, the olanzapine is rapidly and widely distributed to various tissues after absorption, the individual difference of bioavailability is large, the serum medicine concentration of female patients is obviously higher than that of male patients under the condition of the same dosage and certain weight, smoking can accelerate the metabolism of the olanzapine, and the renal clearance rate and metabolism are obviously reduced in the elderly. Patients take olanzapine with the same dosage, the individual difference is larger, and the clearance rate of different individuals to the medicine is 4-10 times of variation. According to literature data, olanzapine treatment is optimally carried out in a range of 20-40ng/mL blood concentration within 12 hours, and adverse reaction is obviously increased when the blood concentration exceeds 80 ng/mL. Important manifestations of overdose include be delirious death, spasticity, coma, suspected MMS, respiratory depression, respiratory negligence, hypertension or hypotension, arrhythmia (incidence of less than 2% in excess), and cardiopulmonary function inhibition, among others. The clinical doctor can comprehensively consider the administration dosage according to the gender and age of the patient, and realize individual administration.
Currently, olanzapine is mainly detected by a liquid chromatography-mass spectrometry (HPLC-MS), and the instrument is expensive, and the operation is complex and time-consuming. The fluorescent quantitative immunochromatography technology is a novel quantitative detection technology combining the advantages of the immunofluorescence technology and the traditional immunochromatography technology. The technology has flexible and simple operation, lower cost and short reaction time, and can realize timely quantitative detection. The medicine taking device can adjust the medicine taking of a patient in time for improving the data support, achieves the aim of individual medicine taking, ensures the curative effect and ensures the medicine taking safety.
Disclosure of Invention
An olanzapine complete antigen having a structure represented by formula 1:
；
formula 1;
wherein, protein is a Protein carrier;
the protein carrier is any one protein selected from the following groups: bovine Serum Albumin (BSA), ovalbumin (OVA), keyhole Limpet Hemocyanin (KLH), human Serum Albumin (HSA) and synthetic Polylysine (PLL).
In a preferred embodiment, the protein carrier is Bovine Serum Albumin (BSA) or Ovalbumin (OVA).
A method of preparing the olanzapine complete antigen of claim 1 comprising the steps of:
(1) Ligating olanzapine hapten to Bovine Serum Albumin (BSA) or Ovalbumin (OVA) to produce the complete antigen of claim 1;
(a) Enabling olanzapine derivative to be subjected to activation reaction with N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide (NHS) to obtain a reactant A solution, wherein the activation reaction temperature is room temperature, and the reaction time is 2 hours;
(b) And (3) adding the solution A obtained in the step (a) into a BSA/OVA solution, and reacting overnight at room temperature to obtain the olanzapine-BSA/OVA conjugate, namely the olanzapine complete antigen.
Use of said olanzapine complete antigen for the preparation of a specific monoclonal antibody to olanzapine.
A monoclonal antibody that specifically binds olanzapine.
Preferably, the monoclonal antibody is produced by a mouse hybridoma cell line; the hybridoma cell line is preserved by China center for type culture collection (CCTCC, china, chinese, university of Wuhan, and the university of Wuhan), and the preservation number is CCTCCNO: C2022383. The classification is named: hybridoma cell line ZCSW02.
In another preferred embodiment, the monoclonal antibody has a sensitivity of 5.17ng/mL for detecting olanzapine analog.
In another preferred embodiment, the monoclonal antibody does not bind other psychotropic drugs.
In another preferred example, the other psychotherapeutic agent is lithium carbonate, carbamazepine, sodium valproate, clozapine.
A hybridoma cell line producing the monoclonal antibody of claim 4 or 5, which is a mouse hybridoma cell line with a preservation number of CCTCC No. C2022383, preserved by the chinese collection of typical cultures (CCTCC, chinese, wuhan, university of wuhan).
The use of said monoclonal antibody for the preparation of a reagent, a test card or a kit for the detection of olanzapine in a sample.
In a preferred embodiment, the sample is a biological sample, preferably a plasma sample.
A method of detecting the presence or absence of olanzapine in a biological sample, said method comprising the steps of:
(a) Contacting the biological sample with the monoclonal antibody of claim 4 or 5;
(b) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of olanzapine in the sample.
In a preferred embodiment, the monoclonal antibody carries a detectable label; preferably, the marker is selected from the group consisting of: colloidal gold labels, colored labels or fluorescent labels.
In a preferred embodiment of the present application, the detection method is a fluorescence detection method.
A fluorescent immunochromatographic assay card for detecting olanzapine analog, said assay card comprising a substrate; a liquid-absorbing member; a detection section; the sample adding component is characterized in that the detecting component is fixed on a substrate, a quality control belt and a detecting belt are arranged in the middle of the detecting component, the liquid absorbing component and the sample adding component are fixed at two ends of the detecting component in a partially overlapped mode, wherein the detecting belt is coated with the complete antigen as claimed in claim 1, and the quality control belt is coated with rabbit antigen IgG.
In a preferred embodiment, the olanzapine fluorescence immunochromatography detection card further comprises a card box, the card box is composed of a lower cover and an upper cover, the upper cover is provided with a sample adding window and a detection window, the olanzapine fluorescence immunochromatography detection card is completely arranged in the lower cover, and the detection window and the sample adding window respectively correspond to a sample adding part, a quality control belt and a detection belt on the olanzapine fluorescence immunochromatography detection card.
In a preferred embodiment, the upper cover is further provided with a product numbering area; a bar code identification area.
In a preferred embodiment, the substrate is a dark rigid substrate; black PVC substrates are preferred.
In a preferred embodiment, the detection member is a nitrocellulose membrane.
In a preferred embodiment, the sample application member is a glass fiber.
In a preferred embodiment, the absorbent member is absorbent paper.
A test kit for detecting olanzapine analogues, said kit comprising:
(a) The olanzapine fluorescent immunochromatographic assay card of claim 9;
(b) An olanzapine detection assay solution mated with the olanzapine fluorescent immunochromatographic assay card of claim 9;
(c) Instructions for use of the olanzapine detection kit to detect olanzapine analogues;
wherein the detection analyte is a detection analyte comprising the fluorescent-labeled monoclonal antibody of claim 4 or 5 and an anti-rabbit IgG antibody.
In another preferred embodiment, the fluorescent dye for labeling in the detection assay solution includes, but is not limited to, FITC (Fluorescein), alexa Fluor 647, CF TM 647. TRITC (Rhodamine), etc.
In a preferred embodiment, the solvent portion of the assay solution is a phosphate buffer containing BSA.
It is understood that within the scope of the present application, the above-described technical features of the present application and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows UV scan patterns of olanzapine immunogen and coating antigen;
FIG. 2 shows a schematic diagram of the structure of an olanzapine fluorescent immunochromatographic assay card; wherein:
FIG. 2A shows a test card configuration without a plastic card; 1: a black PVC substrate; 2: absorbent paper: 3: a nitrocellulose membrane; 4: glass fibers; 5: a quality control line (C line); 6: a detection line strip (T line);
FIG. 2B shows an olanzapine detection card structure with a plastic cartridge; 1': a lower cover; 2': an upper cover; 3': a sample application window; 4': glass fibers; 5': a detection window; 6': a quality control line (C line); 7': a detection line strip (T line); 8': a nitrocellulose membrane; 9': olanzapine project label; 10': a bar code identification area;
FIG. 3 shows a standard curve pattern of olanzapine fluorescent immunochromatographic assay;
figure 4 shows a standard curve profile of olanzapine ELISA assay.
Detailed Description
The inventor synthesizes olanzapine complete antigen through long-term and intensive research, uses the olanzapine complete antigen as immunogen to immunize Balb/C mice, fuses spleen cells of the Balb/C mice with myeloma SP20 cells of the mice to obtain hybridoma cell strains capable of specifically secreting an anti-olanzapine monoclonal antibody, further prepares and purifies the olanzapine monoclonal antibody, and then prepares an olanzapine immunodetection card with high sensitivity and specificity by using the complete antigen and the olanzapine antibody. On this basis, the present application has been completed.
Hapten
Some small molecule substances, such as clozapine, have a low molecular weight and are not capable of inducing an immune response alone, i.e. are not immunogenic, but are immunogenic when cross-linked or conjugated to a carrier such as a macromolecular protein or a non-antigenic polylysine, and induce an immune response. These small molecule substances can bind to the response effect products, are antigenic, are only immunoreactive, are not immunogenic, and are also called incomplete antigens.
Thus, in order to prepare the complete antigen of clozapine, the inventors have derivatised olanzapine to prepare the hapten of the application. The terms "hapten", "olanzapine hapten" or "olanzapine derivative" as used herein all refer to a derivative of olanzapine derived from the following structural formula 2:
olanzapine hapten of the present application can be prepared by the following method:
(a) Olanzapine has the structural formula:
(b) The olanzapine structure reacts with succinic anhydride to obtain the hapten of the application.
In a preferred embodiment, the conditions of step (b) are as follows: the reaction temperature is 20-40 ℃, preferably 30-40 ℃, more preferably 37 ℃; the reaction time is 2 to 5 hours, preferably 3 to 4 hours, more preferably 3 hours; the reaction solvent is methanol, pyridine, tetrahydrofuran, preferably methanol, pyridine, more preferably methanol.
In a specific embodiment, olanzapine hapten is prepared as shown in the following equation:
complete antigen
Substances having immunogenicity and immunoreactivity, called complete antigens (complete antigens), such as most proteins, bacteria, viruses, bacterial exotoxins, animal serum, etc. The complete antigen can stimulate the organism to produce antibody or sensitized lymphocyte and can also generate specific binding reaction with the complete antigen in vivo and in vitro.
Typically, haptens need to be coupled covalently or with macromolecules such as Bovine Serum Albumin (BSA), ovalbumin (OVA) or hemocyanin (KLH) to become complete antigens that are both immunoreactive and immunogenic.
The term "complete antigen" as used herein refers to the product of olanzapine hapten of the application in combination with a suitable protein carrier. As used herein, the term "protein carrier" refers to any immunologically acceptable protein for forming complete antigens, including but not limited to: bovine Serum Albumin (BSA), or Ovalbumin (OVA) is preferred, such as Bovine Serum Albumin (BSA), ovalbumin (KLH), human Serum Albumin (HSA), and synthetic Polylysine (PLL).
The olanzapine complete antigen has a structure shown in formula 1:
1 (1)
Among them, protein is a Protein carrier, and Bovine Serum Albumin (BSA) or Ovalbumin (OVA) is preferable in the present application.
The conditions for the connection of olanzapine hapten to protein carrier are as follows: olanzapine hapten is conjugated to Bovine Serum Albumin (BSA) or Ovalbumin (OVA) to produce complete antigen.
(a) In a preferred embodiment, the conditions for linking the hapten obtained to the protein carrier are as follows: the reaction temperature is 20-35 ℃, preferably 25-30 ℃, more preferably 28 ℃; the reaction pH is 7.0 to 8.0, preferably 7.2 to 7.6, more preferably 7.5; the reaction time is 2 to 8 hours, preferably 4 to 6 hours, more preferably 4 hours.
Preparation of monoclonal antibodies
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homologous antibodies, i.e., the individual antibodies comprising the population are identical except for the possible presence of a small number of spontaneous mutations. Thus, the modifier "monoclonal" refers to a mixture of antibodies whose properties are not discrete.
Antibodies of the application may be prepared by various techniques known to those skilled in the art. For example, the complete antigens of the application may be administered to animals to induce monoclonal antibody production. For monoclonal antibodies, hybridoma technology can be used to prepare (see Kohler et al, nature 256;495, 1975; kohler et al, eur. J. Immunol. 6:511, 1976; kohler et al, eur. J. Immunol. 6:292, 1976; hammerling et al, in Monoclonal Antibodies and T Cell Hybridomas, elsevier, N.Y., 1981) or can be prepared using recombinant DNA methods (U.S. Pat. No. 4,816,567).
Representative myeloma cells are those which fuse efficiently, support stable high levels of antibody production by the antibody-producing cell of choice, and are sensitive to the medium (HAT medium matrix), including myeloma cell lines, such as murine myeloma cell lines, including those derived from MOPC-21 and MPC-11 mouse tumors (available from Salk Institute Cell Distribution Center, san diego, california, usa) and SP-2, NZ0 or X63-Ag8-653 cells (available from American Type cμ Lture Collection, rocyvale, maryland, usa). Human myeloma and mouse-human hybrid myeloma cell lines have also been described for the production of human monoclonal antibodies [ Kozbor, j. Immunol.,133:3001 (1984); techniques and applications for the production of monoclonal antibodies by Brodeur et al (Monoclonal Antibodies Production Techniques and Applications), pages 51-63 (Marcel Dekker, inc., new York, 1987) ].
The culture medium in which the hybridoma cells are grown is analyzed to detect the production of monoclonal antibodies having the desired specificity, such as by an in vitro binding assay, e.g., an enzyme-linked immunosorbent assay (ELISA) or a Radioimmunoassay (RIA). The location of cells expressing the antibody can be detected by FACS. The hybridoma clones can then be subcloned by limiting dilution steps (subcloned) and grown by standard methods (Goding, monoclonal antibody (Monoclonal Antibodies): principles and practices (Principles and Practice), academic Press (1986) pages 59-103). Suitable media for this purpose include, for example, DMEM or RPMI-1640 medium. In addition, hybridoma cells can grow as ascites tumors in animals.
Monoclonal antibodies secreted by the subclones are suitably isolated from culture medium, ascites fluid or serum by conventional immunoglobulin purification procedures such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
The monoclonal antibody is produced by a mouse hybridoma cell line which is preserved in China center for type culture collection (CCTCC, china, the university of Wuhan, and the university of Wuhan) at the 1 month 11 year 2023, wherein the preservation number is CCTCC NO: C2022383; the classification is named: hybridoma cell line ZCSW02.
In a specific embodiment, the monoclonal antibodies of the application bear a detectable label. More preferably, the marker is selected from the group consisting of: colloidal gold labels, colored labels or fluorescent labels.
In a specific embodiment, the monoclonal antibody of the application has a sensitivity of 5.17ng/mL for detecting olanzapine analogues. The monoclonal antibodies of the application do not cross-react with the carrier protein of olanzapine complete antigen, e.g. BSA or OVA. Further, the monoclonal antibodies of the application also do not bind other psychotherapeutic agents, including but not limited to lithium carbonate, carbamazepine, sodium valproate, clozapine.
Detection kit
The detection kit of the application refers to a kit which contains the monoclonal antibody of the application and can be used for detecting olanzapine analogues. The kit can comprise a container, instructions for use, buffers, immunoassays, etc., as needed.
The test kit of the present application may take various forms, such as a test card, a test kit containing various reagents required for the test, and the like. The kit of the present application is described using a test card as an example in the examples, but it should not be construed that the kit of the present application is limited to the test card.
In a specific embodiment, the fluorescent immunochromatographic assay card for detecting olanzapine analog of the present application comprises a substrate; a liquid-absorbing member; a detection section; and a loading component; the detection part is fixed on the substrate, the quality control belt and the detection belt are arranged in the middle of the detection part, the liquid absorbing part and the sample adding part are fixed at two ends of the detection part in a partially overlapped mode, wherein the detection belt is coated with the complete antigen of the application, and the quality control belt is coated with rabbit antigen IgG.
In a preferred embodiment, the olanzapine fluorescence immunochromatography detection card further comprises a card box, wherein the card box comprises a lower cover and an upper cover, the upper cover is provided with a sample adding window and a detection window, the olanzapine fluorescence immunochromatography detection card is completely arranged in the lower cover, and the detection window and the sample adding window respectively correspond to a sample adding part, a quality control belt and a detection belt on the olanzapine fluorescence immunochromatography detection card. The upper cover can be also provided with a product numbering area; a bar code identification area. The substrate may be a dark colored rigid substrate; black PVC substrates are preferred. The detection member may be a nitrocellulose membrane. The loading member may be a glass fiber. The absorbent member may be absorbent paper.
By "partially overlapping secured" as used herein is meant that two adjacent components form a certain overlap region, rather than a complete overlap in which one component is fully contained within the other component, and the two components are secured by the overlap region. The manner of fixation may be selected autonomously by the person skilled in the art, for example by means of gluing or the like.
On the basis of the detection card, the application also provides a detection kit for detecting olanzapine analogues, which is provided with:
(a) The olanzapine fluorescence immunochromatography detection card;
(b) Detecting and analyzing liquid of olanzapine analogue matched with the olanzapine fluorescence immunochromatography detection card;
(c) Instructions for detecting olanzapine analogs using said olanzapine analog detection kit;
wherein the detection analyte is a detection analyte containing the fluorescent-labeled monoclonal antibody and the anti-rabbit IgG antibody.
The fluorescent markers can be selected autonomously by those skilled in the art as desired, including but not limited to FITC (Fluorescein), alexa Fluor 647, CF TM 647. TRITC (Rhodamine), etc.
In a preferred embodiment, the solvent portion of the detection assay is a phosphate buffer containing BSA.
Immunodetection application of olanzapine complete antigen
The olanzapine complete antigen of the application is applied to antibody preparation, and the antibody is a monoclonal antibody or a polyclonal antibody; the olanzapine complete antigen of the application is used for preparing corresponding antibodies in various immunological detection fields for detecting the content of olanzapine analogues, including but not limited to ELISA, chemiluminescence method, colloidal gold method, fluorescence immunochromatography method and other immunological detection fields.
The 'olanzapine complete antigen applied to antibody preparation' refers to the preparation of an anti-olanzapine polyclonal antibody and a monoclonal antibody by utilizing the olanzapine complete antigen and the complete antigen to deimmunize experimental animals; the laboratory animals should not be construed as pure mice in the embodiments, and should include, but are not limited to: mice, rats, rabbits, goats, sheep, horses, donkeys, chickens, dogs, and other laboratory animals.
The ' olanzapine complete antigen ' applied to the field of detection of olanzapine immunodetection ' refers to an immunodetection method for establishing various detection of the content of olanzapine analogues by using corresponding antibodies prepared from the olanzapine complete antigen as immunodetection raw materials. The immunodetection field comprises, but is not limited to, ELISA, chemiluminescence, colloidal gold, fluorescence immunochromatography and other immunological detection methods; the immunological detection method for detecting olanzapine analogues not only comprises the detection of specified quantity, but also comprises semi-quantitative detection and various qualitative detection methods based on immunological detection.
In specific embodiments, the application takes the preparation of specific monoclonal antibodies by immunized mice as an example, and takes ELISA and fluorescence immunochromatography as specific examples to illustrate the application of olanzapine complete antigen in olanzapine immunological detection.
The application has the advantages that:
1. the application discloses a structure of olanzapine complete (artificial) antigen and a preparation method thereof for the first time;
2. the application discloses the application of olanzapine complete antigen in the fields of olanzapine antibody preparation and immunological detection for the first time, and provides a reliable method for promoting clinical olanzapine blood concentration detection;
3. the monoclonal antibody can detect olanzapine analogues with high sensitivity and is not combined with other psychotropic therapeutic drugs;
4. the olanzapine analogue detection kit can simply and quickly detect the olanzapine analogue on site.
The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.
Examples
Example 1 preparation of olanzapine hapten
Olanzapine (CAS: 132539-06-1) is used as a raw material and prepared through a one-step reaction, and the synthetic route is as follows:
olanzapine (312 mg,1.0 mmol) was weighed out and dissolved in absolute methanol (35 ml) at room temperature, and succinic anhydride (150 mg,1.5 mmol) dissolved in absolute methanol (15 ml) was added thereto. The mixture was stirred at 37℃for 2 hours, then 1mol/L sodium hydroxide solution (15 ml) was added to the mixture, and stirring was continued for five minutes. After the reaction mixture was washed with ethyl acetate (80 ml. Times.3), the aqueous phase was removed and the pH of the aqueous phase was adjusted to 5 with hydrochloric acid (6 mol/L). The precipitate was collected by filtration, washed with ice water and finally dried in vacuo to give olanzapine derivative (234 mg, yield: 59.4%) as a yellow solid.
Preparation of olanzapine complete antigen (immunogen and coating antigen)
The olanzapine derivative (hapten) obtained in example 1 was coupled to Bovine Serum Albumin (BSA) and Ovalbumin (OVA) by EDC. The specific coupling method is as follows:
22mg of olanzapine derivative compound was weighed, dissolved in 2.2ml of DMF to give a final concentration of 10mg/ml, 200. Mu.L thereof was mixed with 10. Mu.L of EDC (100 mg/ml), 15. Mu.L of NHS (50 mg/ml) was further added thereto, and the mixture was uniformly mixed and reacted under stirring for 2 hours.
The above reaction mixture was centrifuged (1600 rmp), added to 1ml of a 6mg/ml BSA solution (or 1ml of a 4.5 mg/ml OVA solution) and reacted for 2 to 4 hours at room temperature under stirring, and then dialyzed with phosphate buffer for 4 times, and changed for 12 hours. The dialysate was collected and the concentrations of immunogen and coating antigen were determined to be 3.4mg/ml and 2.7mg/ml using Quick Start Bradford Protein Assay Kit from BIO-RAD, inc. The structural formula of the obtained clozapine artificial antigen (immunogen and coating antigen) is shown as follows, wherein "Protein" is BSA (bovine serum albumin) or OVA (ovalbumin).
Comparison of the results of the UV scanning peaks for olanzapine artificial antigen (immunogen and coating) as shown in FIG. 1, the peak of the conjugate was distinguished from the BSA and OVA peaks indicating successful conjugation.
EXAMPLE 2 preparation of monoclonal antibodies Using olanzapine complete antigen
1. Immunization of animals
Olanzapine immunogen obtained in example 1 was diluted to 0.5mg/mL, 500. Mu.L of immunogen was mixed with an equal volume of Freund's complete adjuvant, emulsified completely, and BALB/c mice (Shanghai Laek laboratory animal Co., ltd.) were immunized and injected at the groin site. The first immunization was with complete Freund's adjuvant followed by incomplete Freund's adjuvant. One week after the fourth immunization, the orbit was bled, serum was isolated, and the titers of anti-olanzapine antibodies were measured. The antibody titer of the mice after four immunizations was 1:512,000 as measured by ELISA.
2. Cell fusion and screening
Four immunized mice were boosted again by intraperitoneal injection of about 100 μg of immunogen, and after 3 days, the spleens of the mice were taken for fusion. Mixing SP2/0 cells (Nanjing medical science college) with spleen cells, adding serum-free culture solution (Hyclone SH30022.018 DMEM (High Glucose)), centrifuging (1500 rpm,3 min), collecting precipitated cells, dropwise adding 1mL 50% polyethylene glycol 4000, and standing for 90 seconds. Then, the serum-free culture medium 10 mL, which had been preheated at 37℃was added dropwise, and allowed to stand for 5 minutes. After fusion, the cell suspension was centrifuged (1000 rpm,3 min), and the whole culture was inoculated into 96-well plates with feeder cells, 2X 10 per well 4 /mL myeloma cells. Placing at 37deg.C, 5% CO 2 Culturing in an incubator for two days, and adding 2 XHAT complete culture solution to make the final concentration in the well 1 XHAT. When the hybridoma cell colony grows to 1/10-1/5 area of the hole bottom, the ELISA method is used for screening the fusion cell antibody positive hole.
3. Ascites production and antibody purification
BALB/c mice were given intraperitoneal injections of 0.5mL of paraffin oil, and after 7 days, intraperitoneal injections of 0.5mL of 1X 10 6 Positive hybridoma cells. And (3) observing the growth condition of the mice, wherein abdominal bulge is visible about 7 days, and collecting ascites in time. The monoclonal antibody with high purity is obtained by purification through an affinity chromatography technology (Protein G Resin affinity purification), and the Protein amount is 4mg.
EXAMPLE 3 immunoassay Using olanzapine complete antigen
1. Fluorescent immunochromatographic assay
1) Preparation of detection analysis liquid
a. The monoclonal antibodies and anti-rabbit IgG antibodies obtained in example 2 were each fluorescently labeled (Hangzhou Kogyo Biotechnology Co., ltd.);
b. the fluorescence-labeled antibody was diluted with a phosphate buffer containing BSA to prepare a detection assay solution.
2) Preparation of olanzapine fluorescent immunochromatography test paper card
a. The olanzapine coating antigen (olanzapine-OVA) and rabbit antigen IgG prepared were diluted to appropriate concentrations (0.4-3.0 mg/mL) with coating buffer (phosphate buffer), respectively. Uniformly spraying diluted olanzapine-OVA and rabbit antigen IgG on a nitrocellulose membrane (respectively forming a detection line and a quality control line) at the temperature of 25+/-5 ℃, drying for about 1.5-2 hours under the humidity condition of 12% -30%, and drying and preserving for later use;
b. and c, respectively and sequentially pasting the coated nitrocellulose membrane, the glass fiber paper and the absorbent paper obtained in the step a on the black PVC substrate to form a detection card (shown in figure 2A), and cutting the detection card into proper widths according to requirements.
c. And c, loading the detection card obtained in the step B into a lower cover of the card box, and covering an upper cover to form the complete detection card with the card box (shown in fig. 2B).
3) Detection of
And (3) uniformly mixing 60 mu L of diluted sample with 60 mu L of detection analysis liquid, taking 100 mu L of sample adding window added with a detection card, reacting for 15-20 min, detecting by using a FCR fluorescence immunoassay analyzer (Suzhou and Mich precision instruments Co., ltd.), and comparing and displaying a detection result according to the ratio of a T line signal value to a C line signal value (T/C value) of the sample and a built-in standard curve.
4) Olanzapine fluorescence immunochromatography test paper card detection principle
The competition method is adopted for detection, and olanzapine antigen in the sample and olanzapine antigen (coating antigen) on a detection line (T line) compete for binding with fluorescent-labeled anti-olanzapine antibody in detection analysis liquid. When the concentration of the antigen in the sample is lower, the more fluorescent antibodies are combined on the detection line, and further the stronger the fluorescent signal on the detection line is, so that the larger the ratio (T/C value) of the fluorescent signal of the detection line (T line) to the fluorescent signal of the quality control line (C line) is; conversely, the T/C value is smaller when the olanzapine antigen concentration in the sample is higher. Therefore, the higher the olanzapine content in the sample, the lower the T/C value. And comparing the T/C value with a built-in standard curve and displaying a detection result.
5) Sensitivity and standard curve for detecting olanzapine analogues by fluorescence immunochromatography
Olanzapine standard was added to the blank serum and the serum was prepared to have concentration gradients of 400, 200, 100, 50, 25, 12.5, and 0ng/mL of 7, and diluted 10-fold with 0.9% NaCl, respectively. The above series of concentration samples were tested according to the above test procedure, each sample was repeated 3 times, the test results of the test are shown in table 1, and a standard curve (four parameters) is drawn according to the data of table 1 with the concentrations on the abscissa and the T/C values on the ordinate, as shown in fig. 3. The corresponding equation of the curve in FIG. 3 is shown in Table 2, and IC is calculated 50 =51.7 ng/mL, true IC 50 =5.71ng/mL。
TABLE 1 fluorescent immunochromatography detection of olanzapine samples at different concentrations
Olanzapine concentration-ng/mL T/C value
0 11.96
12.5 9.02
25 7.39
50 6.02
100 4.62
200 3.42
400 2.26
TABLE 2 equation (four parameters) for inhibition curves
Four parameter Logistic curve fitting
Equation y= (A-D)/[ 1+ (x/C)/(B)]+D let x be the concentration and y be the T/C value
A = 11.98008
B = 0.76420
C = 46.12140
D = 0.48522
r^2 = 0.99902
Samples of 0ng/mL were repeatedly tested 10 times, and the mean (X), standard Deviation (SD) and precision (CV) of the T/C values were calculated, respectively. The sensitivity was calculated by calculating the T/C values for X-2X sd as shown in table 3 for olanzapine concentrations in the standard curve of fig. 3.
TABLE 3 repeated detection of olanzapine sample at 0ng/mL by fluorescence immunochromatography
Detecting repetition times 10 repeat T/C values of 0ng/mL
1 9.95
2 10.98
3 10.82
4 9.45
5 9.87
6 10.19
7 9.52
8 9.53
9 10.87
10 10.4
Average value (X) 10.16
Standard Deviation (SD) 0.59
X-2*SD 8.98
The T/C value of X-2X sd in the data of table 3 was substituted as y value into the equation corresponding to the standard curve of fig. 3 to a concentration value of 11.81ng/mL, i.e. a sensitivity of 11.81ng/mL.
6) Method for detecting precision deviation of olanzapine by fluorescent immunochromatography
The concentrations of 20ng/mL, 80ng/mL and the detection system of olanzapine are respectively utilized
200ng/mL olanzapine standard, each repeated 10 times, calculate the precision (CV) of detecting low, medium and high concentrations of olanzapine. Table 4 shows the results of the precision of measuring olanzapine concentrations.
TABLE 4 repeated detection of 20, 80, 200ng/mL olanzapine standard results by fluorescence immunochromatography
Concentration (ng/mL) 20 80 200
1 22.32 80.27 196.04
2 21.09 84.37 202.39
3 22.66 80.90 211.63
4 19.64 78.87 223.43
5 18.09 84.43 182.19
6 16.52 82.67 235.72
7 22.89 78.77 182.85
8 17.63 74.08 203.76
9 21.17 74.65 190.38
10 20.61 77.77 213.47
Mean value of 21.07 79.89 193.24
SD 2.23 3.61 17.34
CV% 10.6 4.5 9.0
Deviation% 5.4 -0.1 -3.4
7) Accuracy deviation of olanzapine detection by fluorescence immunochromatography
After the standard (5 mg/mL) was diluted to 2000, 1000, 400ng/mL with buffer solution, 10. Mu.L of each of the different concentration standard was added to 90. Mu.L of the low concentration enterprise internal reference, and the detection was performed according to the above-described detection procedure, and each sample was repeated 5 times. Table 5 is the accuracy result.
TABLE 5 repeated detection of olanzapine standard results by fluorescence immunochromatography
8) Cross reaction of olanzapine fluorescence immunochromatography detection system
Preparing 3 common clinical related medicines into samples with different gradient concentrations by using blank mixed serum respectively, performing fluorescence immunochromatography detection, and calculating IC (integrated circuit) 50 IC with olanzapine 50 Value comparison calculates the cross-reaction rate. The calculation formula is as follows: cross reaction Rate= (IC) 50 olanzapine /IC 50 clinical related drugs ) The results of the cross-reactivity are shown in Table 6:
TABLE 6 fluorescence immunochromatography detection of Cross-reaction results of clinically relevant drugs
Clinically relevant medicine Cross reaction rate
Lithium carbonate ≦0.1%
Carbamazepine (Kamahalanobis) ≦0.1%
Sodium valproate ≦0.1%
Clozapine ≦0.1%
2. ELISA quantitative detection of olanzapine
1) ELISA detection standard curve establishment
The prepared olanzapine-coated antigen (olanzapine-OVA) was diluted to 1-2. Mu.g/mL with carbonic acid buffer (0.05M, pH 9.6), coated in 96-well plates, 100. Mu.L/well, overnight at 4℃and blocked with 5% BSA for 3 h, 200. Mu.L/well. Washing for 3 times and 5 min/time; olanzapine standard was added to the blank plasma in concentration gradients of 600, 400, 200, 100, 50, 25, 12.5, 0ng/mL7, and diluted 10-fold with 0.01MPBS, respectively. Respectively adding 50 mu L of olanzapine with different concentrations and 50 mu L of olanzapine antibody into the microwells, and incubating at 37 ℃ for 1h; after 3 washes, HRP-labeled secondary antibody was added and incubated 1h (100 μl/well), washed 3 times, color development was added, reacted at room temperature in the dark for 15 min, and stop solution read (450 nm) was added. Table 8 shows the absorbance results of the ELISA test standard curve, and the equation corresponding to the curve in FIG. 4 is shown in Table 9
TABLE 8 ELISA method for detecting olanzapine at different concentrations
Olanzapine concentration-ng/mL OD value
0 2.257
12.5 1.703
25 1.395
50 1.137
100 0.789
200 0.613
400 0.379
TABLE 9 equation (four parameters) for inhibition curves
Four parameter Logistic curve fitting
Equation y= (A-D)/[ 1+ (x/C)/(B)]+D let x be the concentration and y be the OD value
A = 2.25915
B = 0.82193
C = 43.49989
D = 0.09473
r^2 = 0.99847
2) Sensitivity of ELISA for detecting olanzapine
The negative plasma samples were repeatedly tested 10 times, and the mean (X), standard Deviation (SD) and precision (CV) of ELISA absorbance values were calculated, respectively. The sensitivity was calculated by OD values of X-2X sd corresponding to olanzapine concentration values in the standard curve of fig. 4.
TABLE 10 repeated ELISA method for measuring results of olanzapine sample at 0ng/mL
Detecting repetition times 10 repeated OD values of 0ng/mL
1 2.361
2 2.173
3 2.488
4 2.587
5 2.151
6 2.023
7 2.330
8 2.459
9 2.436
10 2.028
Average value (X) 2.304
Standard Deviation (SD) 0.199
X-2*SD 1.906
The absorbance of X-2X sd in the data of table 9 was substituted as y value into the equation corresponding to the standard curve of fig. 4 to a concentration value of 5.95ng/mL, i.e., a sensitivity of 5.95ng/mL.
3) ELISA method for detecting precision and accuracy deviation of olanzapine
Olanzapine standard substances with the concentration of 40ng/mL and 180ng/mL are detected by using an established ELISA detection system respectively, the detection is repeated for 15 times, and the precision (CV) for detecting olanzapine with low concentration and the accuracy deviation of the detection are calculated. The results show that the precision of the high concentration and the low concentration is less than 15%, and the accuracy deviation is less than 15%.
TABLE 11 results of repeated ELISA assays for 40, 180ng/mL olanzapine standards
Concentration (ng/mL) 40.00 180.00
1 41.09 170.68
2 41.3 177.79
3 41.34 184.65
4 45.16 184.07
5 38.03 174.24
6 40.02 195.19
7 45.47 183.53
8 40.34 171.97
9 40.57 180.25
10 39.72 183.25
11 44.71 190.12
12 44.66 181.2
13 43.76 181.88
14 40.65 180.73
15 37.02 184.25
Average value (X) 41.59 181.59
SD 2.61 6.38
CV% 6.3 3.5
Deviation% 4.0 0.9
4) ELISA method for detecting cross reaction of olanzapine
The cross-reaction results are consistent with those of olanzapine detected by fluorescent immunochromatography.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (10)

1. An olanzapine complete antigen characterized by: the complete antigen has a structure represented by formula 1:
；
wherein, protein is a Protein carrier;
the protein carrier is any one protein selected from the following groups: bovine Serum Albumin (BSA), ovalbumin (OVA), keyhole Limpet Hemocyanin (KLH), human Serum Albumin (HSA) and synthetic Polylysine (PLL).
2. A method of preparing the olanzapine complete antigen of claim 1 characterized by: the method comprises the steps of:
(1) Ligating olanzapine hapten to Bovine Serum Albumin (BSA) or Ovalbumin (OVA) to produce the complete antigen of claim 1;
enabling olanzapine derivative to be subjected to activation reaction with N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide (NHS) to obtain a reactant A solution, wherein the activation reaction temperature is room temperature, and the reaction time is 2 hours;
and (3) adding the solution A obtained in the step (a) into a BSA/OVA solution, and reacting overnight at room temperature to obtain the olanzapine-BSA/OVA conjugate, namely the olanzapine complete antigen.
3. Use of the olanzapine complete antigen as defined in claim 1 wherein: which is used for preparing a specific monoclonal antibody of olanzapine.
4. A monoclonal antibody, characterized in that: the monoclonal antibody specifically binds olanzapine.
5. The monoclonal antibody of claim 4, wherein the monoclonal antibody is produced by a mouse hybridoma cell line; the hybridoma cell line is preserved by China center for type culture collection (CCTCC, china, chinese, university of Wuhan, and the university of Wuhan), and the preservation number is CCTCC NO: C2022383.
6. A hybridoma cell line producing the monoclonal antibody of claim 4 or 5, characterized in that: the hybridoma cell line is a mouse hybridoma cell line which is preserved by China center for type culture collection (CCTCC, china, chinese, university of Wuhan) and has the preservation number of CCTCC NO: C2022383.
7. Use of a monoclonal antibody according to claim 4 or 5, characterized in that: which is used for preparing a reagent, a detection card or a kit for detecting olanzapine in a sample.
8. A method for detecting the presence or absence of olanzapine in a biological sample, characterized by: the method comprises the steps of:
(a) Contacting the biological sample with the monoclonal antibody of claim 4 or 5;
(b) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of olanzapine in the sample;
wherein the monoclonal antibody carries a detectable label;
the markers are selected from the group consisting of: colloidal gold labels, colored labels or fluorescent labels.
9. A fluorescent immunochromatographic assay card for detecting olanzapine analogues, characterized in that: the test card includes a substrate; a liquid-absorbing member; a detection section; the sample adding component is characterized in that the detection component is fixed on a substrate, a quality control belt and a detection belt are arranged in the middle of the detection component, the liquid absorbing component and the sample adding component are fixed at two ends of the detection component in a partially overlapped mode, wherein the detection belt is coated with the complete antigen as set forth in claim 1, and the quality control belt is coated with rabbit antigen IgG;
the olanzapine fluorescence immunochromatography detection card also comprises a card box, wherein the card box consists of a lower cover and an upper cover, the upper cover is provided with a sample adding window and a detection window, the olanzapine fluorescence immunochromatography detection card is completely arranged in the lower cover, and the detection window and the sample adding window respectively correspond to a sample adding part, a quality control belt and a detection belt on the olanzapine fluorescence immunochromatography detection card.
10. A test kit for detecting olanzapine analogues, characterized in that: the kit is provided with:
(a) The olanzapine fluorescent immunochromatographic assay card of claim 9;
(b) An olanzapine detection assay solution mated with the olanzapine fluorescent immunochromatographic assay card of claim 9;
(c) Instructions for use of the olanzapine detection kit to detect olanzapine analogues;
wherein the detection analyte is a detection analyte comprising the fluorescent-labeled monoclonal antibody of claim 4 or 5 and an anti-rabbit IgG antibody;
the fluorescent dye for labeling in the detection analysis liquid includes, but is not limited to, FITC (Fluorescein), alexa Fluor 647, CF TM 647、TRITC (Rhodamine)。
CN202310190849.9A 2023-03-02 2023-03-02 Olanzapine complete antigen and antibody, and preparation methods and applications thereof Pending CN116813745A (en)

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