CN116444647A - Clozapine complete antigen and antibody, and preparation method and application thereof - Google Patents
Clozapine complete antigen and antibody, and preparation method and application thereof Download PDFInfo
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- CN116444647A CN116444647A CN202211632837.9A CN202211632837A CN116444647A CN 116444647 A CN116444647 A CN 116444647A CN 202211632837 A CN202211632837 A CN 202211632837A CN 116444647 A CN116444647 A CN 116444647A
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a clozapine complete antigen and application thereof in preparation of clozapine antibodies and detection by an immunoassay. The structure of the clozapine complete antigen is shown as formula 1. The invention also discloses an anti-clozapine 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 the clozapine analogue. The invention discloses an ELISA method and a fluorescence immunochromatography method for detecting clozapine analogues, wherein the fluorescence immunochromatography method is simple to operate, short in detection time, low in cost, high in specificity and good in repeatability.
Description
Technical Field
The invention relates to detection of psychotic drugs. In particular, the invention relates to a clozapine complete antigen, a clozapine monoclonal antibody obtained by using the complete antigen and application thereof in detecting clozapine analogues by using a fluorescent immunochromatography method.
Background
Clozapine (CLZ) is one of the most potent antipsychotics, and causes few extrapyramidal side effects compared to all antipsychotics commonly used clinically. Its appearance has been about 30 years old, and has been popular in the early days, but falls to the valley in 1970 after it causes granulocytopenia. At the beginning of the 90 s, the traditional Chinese medicine composition has better curative effect on acute and chronic psychosis and is widely used in clinic again. Clozapine has a strong antipsychotic effect but the extrapyramidal response is lighter, indicating that the antipsychotic effect and the extrapyramidal response can be separated.
The blood concentration of clozapine with the same dosage has larger individual difference, and the maximum difference of the blood concentration is 94 times. The medicine has a plurality of influencing factors including food, smoking, sex, coffee, medicine interaction, administration route and the like, and has certain help for guiding reasonable application of clozapine, improving curative effect and preventing adverse reaction and toxic reaction. The adverse reactions caused by clozapine are more and mainly represented as follows: 1) The traditional Chinese medicine composition has strong sedative effect and more anticholinergic adverse reactions, and is commonly characterized by dizziness, weakness, somnolence, hyperhidrosis, salivation, nausea, vomiting, dry mouth, constipation, postural hypotension and tachycardia; 2) Common appetite and weight gain; 3) An abnormal change in the electrocardiogram may be caused. Can cause electroencephalogram changes or seizures; 4) Can also cause increased blood glucose; 5) Serious adverse reactions are granulocytopenia and secondary infections; 6) Can cause urinary incontinence or central system disorder, and should be used with or without caution in clinic. Therefore, clinical medication must be based on patient condition, paying attention to the individuation principle of drug dosage, and adjusting the dosage of clozapine according to blood concentration. In summary, in order to further optimize and normalize the use of clozapine in different populations, adjusting the dosing regimen according to the pathophysiological condition of the patient, it is necessary to monitor the blood level of clozapine (therapeutic drug monitoring, TDM) for a specific population.
In the Chinese pharmacopoeia, liquid chromatography is used for measuring clozapine, and the current detection of clozapine also comprises a liquid chromatography-mass spectrometry (HPLC-MS), a gas chromatography-mass spectrometry (GC-MS), an electrochemical analysis detection method and the like, so that 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
The first technical object of the present invention is to provide a clozapine complete antigen capable of solving the above technical problems.
The second technical aim of the invention is to provide a preparation method of clozapine complete antigen which can solve the technical problems.
A third technical object of the present invention is to provide a clozapine monoclonal antibody obtained by using the complete antigen, which solves the above-mentioned problems.
A fourth technical object of the present invention is to provide an application of a clozapine monoclonal antibody obtained by using the complete antigen in detecting clozapine analogues by using a fluorescence immunochromatography.
The first technical object of the present invention is achieved by the following technical scheme:
a complete antigen having a structure represented by formula 1:
wherein, protein is a Protein carrier;
in a preferred embodiment, the protein carrier is any one protein selected from the group consisting of: bovine Serum Albumin (BSA), ovalbumin (OVA), keyhole Limpet Hemocyanin (KLH), human Serum Albumin (HSA), and artificially synthesized Polylysine (PLL), etc.
In a preferred embodiment, the protein carrier is Bovine Serum Albumin (BSA), or Keyhole Limpet Hemocyanin (KLH).
The second technical purpose of the invention is realized by the following technical proposal:
a method of preparing the complete antigen, the method comprising the steps of:
(1) Ligating clozapine hapten to Bovine Serum Albumin (BSA) or Keyhole Limpet Hemocyanin (KLH)) to produce the complete antigen of claim 1;
(a) The chlorzapine derivative is activated and reacted 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 6 hours;
(b) And (3) adding the solution A obtained in the step (a) into a BSA/KLH solution, and reacting overnight at room temperature to obtain the clozapine-BSA/KLH conjugate, namely the clozapine complete antigen.
Use of said complete antigen for the preparation of a specific monoclonal antibody to clozapine.
The third technical object of the present invention is achieved by the following technical scheme:
a monoclonal antibody that specifically binds clozapine.
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, wuhan, university of Wuhan), and the preservation number is CCTCC NO: C2020225.
In another preferred embodiment, the monoclonal antibody has a sensitivity of 3.70ng/mL for detecting clozapine analogues.
In another preferred embodiment, the monoclonal antibody does not bind other psychotropic drugs.
In another preferred example, the other psychotherapeutic agent is clonazepam, risperidone, quetiapine, aripiprazole, olanzapine, sodium valproate, paroxetine.
Preferably, the hybridoma cell line of the monoclonal antibody is a mouse hybridoma cell line which is preserved by China center for type culture collection (CCTCC, china, the university of Wuhan, and the university of Wuhan) and has the preservation number of CCTCC NO: C2020225.
The fourth technical object of the present invention is achieved by the following technical scheme:
use of a monoclonal antibody for the preparation of a reagent, test card or kit for detecting clozapine 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 clozapine 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 clozapine 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 invention, the detection method is a fluorescence detection method.
A fluorescent immunochromatographic assay card for detecting a clozapine 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 clozapine fluorescent 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 clozapine fluorescent immunochromatography detection card is completely arranged in the lower cover, and the detection window and the sample adding window correspond to a sample adding part, a quality control belt and a detection belt on the clozapine fluorescent immunochromatography detection card respectively.
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 a clozapine analog, said kit comprising:
(a) The clozapine fluorescent immunochromatographic assay card of claim 9;
(b) A clozapine detection assay matched with the clozapine fluorescent immunochromatographic assay card of claim 9;
(c) Instructions for the use of the clozapine detection kit to detect clozapine 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 Fluor647, CF TM 647. TRITC (Rhodamine), etc.
In a preferred embodiment, the solvent portion of the assay solution is phosphate buffer containing bovine serum albumin.
It is understood that within the scope of the present invention, the above-described technical features of the present invention 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 an ultraviolet scan profile of clozapine immunogen and coating antigen;
FIG. 2 shows a schematic diagram of the structure of a clozapine 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 a clozapine test card configuration 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': clozapine project label; 10': a bar code identification area;
FIG. 3 shows a standard curve pattern of clozapine fluorescent immunochromatographic assay;
figure 4 shows a standard curve profile of clozapine ELISA assay.
Detailed Description
The inventor synthesizes a clozapine derivative serving as a hapten through long-term and intensive research, connects the clozapine derivative with a proper protein carrier to generate a complete antigen, uses the complete antigen as an immunogen to immunize a Balb/C mouse, fuses spleen cells of the Balb/C mouse with myeloma SP20 cells of the mouse to obtain a hybridoma cell strain capable of specifically secreting an anti-clozapine monoclonal antibody, further prepares and purifies the anti-clozapine monoclonal antibody to obtain the clozapine monoclonal antibody, and then prepares a clozapine immunodetection card with high sensitivity and specificity by using the complete antigen and the clozapine antibody. On this basis, the present invention 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 present inventors have derivatised clozapine to prepare the hapten of the present invention. The terms "hapten", "clozapine hapten" or "clozapine derivative" as used herein all refer to a clozapine derivative derived from a compound having the following structural formula 2:
the clozapine hapten of the invention can be prepared by the following method:
(a) The structural formula of the clozapine is as follows:
(b) Reacting the clozapine structure with succinic anhydride to obtain the hapten of the invention.
In a preferred embodiment, the conditions of step (b) are as follows: the reaction temperature is 55 to 80 ℃, preferably 60 to 70 ℃, more preferably 65 ℃; the reaction time is 2 to 5 hours, preferably 3 to 4 hours, more preferably 3.5 hours; the reaction solvent is methanol, pyridine or tetrahydrofuran, preferably methanol or pyridine, more preferably pyridine.
In a specific embodiment, the clozapine hapten is prepared as shown in the following equation:
finish the process
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 the clozapine hapten of the invention 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 structure of the clozapine complete antigen is shown as formula 1:
among them, protein is a Protein carrier, and Bovine Serum Albumin (BSA) or Keyhole Limpet Hemocyanin (KLH) is preferable in the present invention.
The conditions for the connection of the clozapine hapten and the protein carrier are as follows: the clozapine hapten is conjugated to Bovine Serum Albumin (BSA) or Keyhole Limpet Hemocyanin (KLH) to produce the 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 20-28 ℃, preferably 23-28 ℃, more preferably 25 ℃; 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 5 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 invention may be prepared by various techniques known to those skilled in the art. For example, the complete antigens of the invention 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 11 months and 27 days in 2020, wherein the preservation number is CCTCC NO: C2020225; the classification is named: hybridoma cell line C214241.
In a specific embodiment, the monoclonal antibodies of the invention 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 invention has a sensitivity of 3.70ng/mL for detecting clozapine analogues. The monoclonal antibodies of the invention do not cross-react with a carrier protein of clozapine complete antigen, such as BSA or KLH. Further, the monoclonal antibodies of the invention are also not conjugated to other psychotherapeutic agents, including but not limited to clonazepam, risperidone, quetiapine, aripiprazole, olanzapine, sodium valproate, paroxetine.
Detection kit
The detection kit of the invention refers to a kit which contains the monoclonal antibody of the invention and can be used for detecting the clozapine analogue. The kit can comprise a container, instructions for use, buffers, immunoassays, etc., as needed.
The test kit of the present invention 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 invention is described using a test card as an example in the examples, but it should not be construed that the kit of the present invention is limited to the test card.
In a specific embodiment, the fluorescent immunochromatographic assay card for detecting a clozapine analog of the present invention 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 invention, and the quality control belt is coated with rabbit antigen IgG.
In a preferred embodiment, the clozapine fluorescent immunochromatography detection card further comprises a card box, 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 clozapine fluorescent immunochromatography detection card is completely arranged in the lower cover, and the detection window and the sample adding window correspond to a sample adding part, a quality control belt and a detection belt on the clozapine fluorescent immunochromatography detection card respectively. 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 invention also provides a detection kit for detecting the clozapine analogue, which is provided with:
(a) The clozapine fluorescence immunochromatography detection card;
(b) The clozapine analogue detection analysis solution is matched with the clozapine fluorescence immunochromatography detection card;
(c) Instructions for detecting a clozapine analog using the clozapine 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 Fluor647, 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 clozapine complete antigen
The clozapine complete antigen is applied to antibody preparation, and the antibody is a monoclonal antibody or a polyclonal antibody; the corresponding antibody prepared from the clozapine complete antigen is applied to various immunological detection fields for detecting the content of clozapine analogues, including but not limited to the immunological detection fields such as ELISA, chemiluminescence, colloidal gold method, fluorescence immunochromatography and the like.
The 'application of the clozapine complete antigen to antibody preparation' refers to the preparation of anti-clozapine polyclonal antibodies and monoclonal antibodies by utilizing the clozapine complete antigen and utilizing 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 'application of the clozapine complete antigen in the field of clozapine immunodetection' refers to the establishment of various immunodetection methods for detecting the content of the clozapine analogue by using corresponding antibodies prepared from the clozapine 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 method for detecting the clozapine analogue immune detection not only comprises the specific quantity detection, but also comprises semi-quantitative detection and various qualitative detection methods based on immunological detection.
In specific embodiments, the invention takes the preparation of specific monoclonal antibodies by immunized mice as an example, and uses ELISA and fluorescent immunochromatography as specific examples to illustrate the application of the clozapine complete antigen in the clozapine immunology detection.
The invention has the advantages that:
1. the invention discloses a general structure of a clozapine hapten derivative for the first time and a derivatization method of the hapten derivative, namely: the method is characterized in that the conventional clozapine is taken as a raw material, a multi-carbon chain carboxyl is added at the secondary amino end of the clozapine to complete derivatization, so that the core structure of the clozapine is reserved to the maximum extent, and the method is used for preparing specific clozapine antibodies;
2. the invention discloses a structure of a clozapine complete (artificial) antigen and a preparation method thereof for the first time;
3. the invention discloses the application of the clozapine complete antigen in the fields of clozapine antibody preparation and immunological detection for the first time, and provides a reliable method for promoting clinical detection of the concentration of clozapine blood;
4. the monoclonal antibody can detect clozapine with high sensitivity and is not combined with other psychotropic therapeutic drugs;
5. the clozapine detection kit can simply and rapidly detect the content of clozapine in a biological sample on site.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. 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 clozapine hapten
The preparation method takes clozapine (CAS: 5786-21-0) as a raw material and adopts a one-step reaction, and the synthetic route is as follows:
clozapine (600 mg,1.84 mmol) was weighed out and dissolved with pyridine (30 ml) at room temperature, and succinic anhydride (276 mg,2.76 mmol) dissolved with pyridine (10 ml) was added thereto. The mixture was stirred at 65℃for 5 hours, then 1mol/L sodium hydroxide solution (10 ml) was added to the mixture, and stirring was continued for five minutes. After the reaction mixture was washed with ethyl acetate (55 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 the clozapine derivative (365 mg, yield: 46.5%) as a white solid.
Preparation of clozapine complete antigen (immunogen and coating antigen)
The clozapine derivative (hapten) obtained in example 1 was coupled to Bovine Serum Albumin (BSA) and Keyhole Limpet Hemocyanin (KLH) by the carbodiimide method, respectively. The specific coupling method is as follows:
40mg of the chlorzapine derivative compound was weighed, dissolved in 4ml of DMF to give a final concentration of 10mg/ml, 200. Mu.L of the compound was mixed with 12. Mu.L of EDC (100 mg/ml), 20ul of NHS (50 mg/ml) was 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 4.5mg/ml KLH 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 5.1mg/ml and 4.3mg/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 KLH (keyhole limpet hemocyanin).
Comparison of the results of uv scanning peaks for clozapine artificial antigen (immunogen and coating) as shown in figure 1, the peak of the conjugate was distinguished from the peaks of BSA and OVA, indicating successful conjugation.
Comparison of the results of UV scanning peaks for clozapine artificial antigen (immunogen and coating) as shown in FIG. 2, the peaks of the conjugate were distinguished from the peaks of BSA and KLH, indicating successful conjugation.
EXAMPLE 2 preparation of monoclonal antibodies Using clozapine complete antigen
1. Immunization of animals
The clozapine immunogen obtained in example 1 was diluted to 0.5mg/mL, 500. Mu.L of the 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 position. 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-clozapine antibodies were determined. The antibody titer of the mice after four immunizations was 1:256,000 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 national academy of medical science) with splenocytes, 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 10mL of serum-free culture medium preheated at 37 ℃ is added dropwise, and the mixture is kept stand for 5min. 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. Purifying by affinity chromatography (Protein G Resin affinity purification) to obtain high-purity monocgramThe amount of the monoclonal antibody and the protein was 6mg.
EXAMPLE 3 immunoassay Using clozapine 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 clozapine fluorescent immunochromatography test paper card
a. The prepared clozapine coating antigen (clozapine-BSA) and rabbit antigen IgG are diluted to appropriate concentrations (0.4-3.0 mg/mL) with coating buffer (phosphate buffer), respectively. Uniformly spraying diluted clozapine-BSA 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 of 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) Clozapine fluorescence immunochromatography test paper card detection principle
The competition method is adopted for detection, and the clozapine antigen in the sample and the clozapine antigen (coating antigen) on the detection line (T line) compete for binding with the fluorescent-labeled anti-clozapine antibody in the 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; on the contrary, when the concentration of the clozapine antigen in the sample is higher, the T/C value is smaller. Therefore, the higher the clozapine 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 clozapine analogues by fluorescence immunochromatography
The standard clozapine was added to the blank serum and the serum was prepared to have concentration gradients of 1000, 800, 600, 400, 200, 100, 50, 25, 0ng/mL 9, and diluted 5-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 =99.72ng/mL。
TABLE 1 fluorescent immunochromatography for detecting clozapine samples at different concentrations
TABLE 2 equation (four parameters) for inhibition curves
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 the T/C values of X-2X sd corresponding to clozapine concentration values in the standard curve of fig. 3 as shown in table 3.
TABLE 3 repeated detection of samples of 0ng/mL clozapine by fluorescence immunochromatography
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 3.70ng/mL, i.e. a sensitivity of 3.70ng/mL.
6) Fluorescent immunochromatography for detecting precision deviation of clozapine
The established clozapine detection system is used for detecting clozapine standard substances with the concentration of 80ng/mL, 400ng/mL and 800ng/mL respectively, the detection is repeated for 10 times, and the precision (CV) of detecting the low-medium-high concentration clozapine is calculated. Table 4 shows the results of the precision of measuring the concentration of clozapine.
TABLE 4 repeated detection of 80, 400, 800ng/mL clozapine standard results by fluorescence immunochromatography
7) Accuracy deviation of detecting clozapine by fluorescent immunochromatography
After the standard (5 mg/mL) is diluted to 8000, 4000 and 1000ng/mL by buffer solution, 10 mu L of different concentration standard is added into 90 mu L of low concentration enterprise internal reference, and the detection is carried out according to the detection steps, and each sample is repeated 5 times. Table 5 is the accuracy result.
TABLE 5 repeated detection of clozapine standard results by fluorescence immunochromatography
8) Cross reaction of clozapine fluorescent 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 clozapine 50 Value comparison calculates the cross-reaction rate. The calculation formula is as follows: cross reaction Rate= (IC) 50 clozapine /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 |
Clonazepam | ≦0.1% |
Risperidone | ≦0.1% |
Quetiapine | ≦0.1% |
Aripiprazole | ≦0.1% |
Olanzapine | ≦0.1% |
Sodium valproate | ≦0.1% |
Paroxetine | ≦0.1% |
ELISA quantitative determination of clozapine
1) ELISA detection standard curve establishment
The prepared clozapine-coated antigen (clozapine-BSA) 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 ℃,5% BSA blocked for 3h, 200. Mu.L/well. Washing for 3 times and 5 min/time; the standard clozapine was added to the blank plasma to prepare 8 concentration gradients of 1000, 800, 600, 400, 200, 100, 50, 0ng/mL, and diluted 5-fold with 0.01MPBS, respectively. Respectively adding 50 mu L of clozapine with different concentrations and 50 mu L of clozapine antibody into the microwells, and incubating at 37 ℃ for 1h; after 3 washes, HRP-labeled secondary antibody was added and incubated for 1h (100. Mu.L/well), and after 3 washes, color development solution was added, and the reaction was allowed to proceed at room temperature for 15min in the dark, and stop solution was added for reading (450 nm). Table 7 shows the absorbance results of the ELISA test standard curve, and the equation corresponding to the curve in FIG. 4 is shown in Table 8
TABLE 7 ELISA method for detecting clozapine at different concentrations
TABLE 8 equation (four parameters) for inhibition curves
2) Sensitivity of ELISA for detecting clozapine
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 the OD value of X-2X sd versus clozapine concentration value in the standard curve of fig. 4.
TABLE 9 repeated ELISA method for measuring results of samples of 0ng/mL clozapine
Substituting the absorbance value of X-2X sd in the data of table 9 as y value into the equation corresponding to the standard curve of fig. 4 results in a concentration value of 3.31ng/mL, i.e., a sensitivity of 3.31ng/mL.
3) ELISA method for detecting precision and accuracy deviation of clozapine
The standard substances of clozapine with the concentration of 100ng/mL and 600ng/mL are detected by using an established ELISA detection system respectively, the detection is repeated for 15 times, and the precision (CV) for detecting the clozapine with the 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 10 results of repeated ELISA tests for 100, 600ng/mL clozapine standards
4) ELISA method for detecting cross reaction of clozapine
The cross reaction results are consistent with those of the detection of clozapine by fluorescence immunochromatography.
All documents mentioned in this application are incorporated by reference 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 claims appended hereto.
Claims (10)
1. A complete antigen, characterized in that: 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 artificially synthesized Polylysine (PLL), etc.
2. A method of preparing the complete antigen of claim 1, the method comprising the steps of:
(1) Ligating clozapine hapten to Bovine Serum Albumin (BSA) or Keyhole Limpet Hemocyanin (KLH)) to produce the complete antigen of claim 1;
(a) The chlorzapine derivative is activated and reacted 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 6 hours;
(b) And (3) adding the solution A obtained in the step (a) into a BSA/KLH solution, and reacting overnight at room temperature to obtain the clozapine-BSA/KLH conjugate, namely the clozapine complete antigen.
3. Use of the complete antigen of claim 1 for the preparation of a specific monoclonal antibody to clozapine.
4. A monoclonal antibody that specifically binds clozapine.
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) with the preservation number of C2020225.
6. 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. C2020225, preserved by the chinese collection of typical cultures (CCTCC, chinese, wuhan, university of wuhan).
7. Use of the monoclonal antibody of claim 4 or 5 for preparing a reagent, a test card or a kit for detecting clozapine in a sample;
the sample is a biological sample, preferably a plasma sample.
8. A method of detecting the presence or absence of clozapine 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 clozapine in the sample;
the monoclonal antibody carries a detectable label;
the markers are selected from the group consisting of: colloidal gold labels, colored labels or fluorescent labels;
the detection method is a fluorescence detection method.
9. A fluorescent immunochromatographic assay card for detecting a clozapine analog, said assay card comprising 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 clozapine fluorescent immunochromatography detection card also 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 clozapine fluorescent 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 clozapine fluorescent immunochromatography detection card.
10. A test kit for detecting a clozapine analog, said kit comprising:
(a) The clozapine fluorescent immunochromatographic assay card of claim 9;
(b) A clozapine detection assay matched with the clozapine fluorescent immunochromatographic assay card of claim 9;
(c) Instructions for the use of the clozapine detection kit to detect clozapine 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 comprises FITC (Fluorescein), alexa Fluor647 and CF TM 647、TRITC(Rhodamine);
The solvent part of the detection analysis solution is phosphate buffer solution containing bovine serum albumin.
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CN110950808A (en) * | 2019-10-30 | 2020-04-03 | 杭州博拓生物科技股份有限公司 | Clozapine artificial antigen and preparation method thereof |
CN110981861A (en) * | 2019-12-18 | 2020-04-10 | 苏州博源医疗科技有限公司 | Clozapine derivative, preparation method thereof and clozapine detection reagent |
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CN103781915A (en) * | 2011-05-24 | 2014-05-07 | 萨拉戴克斯生物医学公司 | Clozapine immunoassay |
CN110950808A (en) * | 2019-10-30 | 2020-04-03 | 杭州博拓生物科技股份有限公司 | Clozapine artificial antigen and preparation method thereof |
CN110981861A (en) * | 2019-12-18 | 2020-04-10 | 苏州博源医疗科技有限公司 | Clozapine derivative, preparation method thereof and clozapine detection reagent |
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