Anti-chlorpromazine monoclonal antibodies and uses and products thereof

Abstract

The application provides an anti-chlorpromazine monoclonal antibody and application and product thereof, and relates to the technical field of biology.The anti-chlorpromazine monoclonal antibody provided by the application has a variable region, which comprises a complementarity determining region CDR1-VH with an amino acid sequence as shown in SEQ ID NO.1, a complementarity determining region CDR2-VH with an amino acid sequence as shown in SEQ ID NO.2, a complementarity determining region CDR3-VH with an amino acid sequence as shown in SEQ ID NO.3, a complementarity determining region CDR1-VL with an amino acid sequence as shown in SEQ ID NO.4, a complementarity determining region CDR2-VL with an amino acid sequence as shown in SEQ ID NO.5 and a complementarity determining region CDR3-VL with an amino acid sequence as shown in SEQ ID NO.6.The antibody has good specific binding capacity for chlorpromazine.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an anti-clozapine monoclonal antibody and its applications and products. Background Technology

[0002] Clozapine is a commonly used antipsychotic drug in clinical practice. It belongs to the dibenzodiazepine derivative class of compounds. It improves the positive symptoms of schizophrenia by blocking DA1 receptors on the mesolimbic dopamine pathway and reduces negative symptoms by blocking serotonin receptors in the brain, promoting the disinhibitory release of dopamine. Clozapine is also known as 8-chloro-11(4-methyl-1-piperazinyl)-5H-dibenzo[B,E][1,4]diazepine, with a density of 1.31 g / cm³. 3 Melting point: 182-185℃, Molecular formula: C 18 H 19 ClN4. Appears as a pale yellow crystalline powder, odorless and tasteless. It is readily soluble in chloroform, soluble in ethanol, and practically insoluble in water. Its chemical structural formula is as follows: Figure 1 As shown.

[0003] Clozapine is rapidly and completely absorbed orally; food has no effect on its absorption rate or extent. After absorption, it is rapidly and widely distributed to various tissues. Bioavailability varies considerably among individuals, averaging approximately 50%–60%, and exhibits a first-pass effect in the liver. Peak plasma concentration is reached 3.2 hours (1–4 hours) after administration, with an elimination half-life (t1 / 2β) averaging 9 hours (3.6–14.3 hours). The apparent volume of distribution (Vd) is 4.04–13.78 L / kg, and it has high tissue binding. It is metabolized by the liver, with 80% appearing in urine and feces as metabolites. The main metabolites include N-desmethylclozapine and N-oxide of clozapine. At the same dose and body weight, serum drug concentrations are significantly higher in female patients than in male patients. Smoking accelerates the metabolism of this drug, and renal clearance and metabolism are significantly reduced in the elderly. This drug can be secreted in breast milk and can cross the blood-brain barrier.

[0004] Currently, the detection of clozapine mainly includes methods such as high-performance liquid chromatography-mass spectrometry (HPLC-MS), gas chromatography-mass spectrometry (GC-MS), spectroscopic analysis, capillary electrophoresis, and electrochemical analysis. Most of these methods are limited to laboratory testing, with few being used directly in the field. Achieving on-site detection of clozapine will be of great significance for medical monitoring of clozapine blood concentrations and for solving criminal cases such as suicide and poisoning. Therefore, establishing rapid, sensitive, and accurate detection technologies is essential.

[0005] To address the challenges of large-scale field applications, immunological methods have gained significant attention due to their rapid detection speed, low cost, and high throughput. Some immunological detection methods (such as colloidal gold paper chromatography) also offer advantages such as simple operation and the absence of additional equipment. Therefore, developing high-quality antibodies for clozapine diagnosis has become crucial in determining the future prospects of immunological methods.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The primary objective of this invention is to provide an anti-clozapine monoclonal antibody, which aims to provide a stable and reliable source of raw materials for methods such as colloidal gold immunochromatography for detecting clozapine based on immunological principles, thereby reducing the product's dependence on purchased raw materials and lowering product costs. At the same time, it aims to improve the detection rate and sensitivity of rapid diagnostic products for clozapine, such as gold nanoparticle test strips and ELISA kits, reduce the false positive rate, and enhance the competitiveness of self-produced products.

[0008] A second objective of this invention is to provide a biomaterial.

[0009] A third objective of this invention is to provide the application of the above-mentioned anticlozapine monoclonal antibody in the detection of anticlozapine for non-diagnostic and therapeutic purposes.

[0010] A fourth objective of this invention is to provide a clozapine marker.

[0011] The fifth objective of this invention is to provide a kit for the detection of clozapine.

[0012] In a first aspect, the present invention provides an anti-clozapine monoclonal antibody, wherein the variable region of the anti-clozapine monoclonal antibody comprises: a complementarity-determining region CDR1-VH having an amino acid sequence as shown in SEQ ID NO.1, a complementarity-determining region CDR2-VH having an amino acid sequence as shown in SEQ ID NO.2, a complementarity-determining region CDR3-VH having an amino acid sequence as shown in SEQ ID NO.3, a complementarity-determining region CDR1-VL having an amino acid sequence as shown in SEQ ID NO.4, a complementarity-determining region CDR2-VL having an amino acid sequence as shown in SEQ ID NO.5, and a complementarity-determining region CDR3-VL having an amino acid sequence as shown in SEQ ID NO.6.

[0013] As a further technical solution, the variable region includes a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO.7.

[0014] As a further technical solution, the variable region includes a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO.8.

[0015] As a further technical solution, the anti-clozapine monoclonal antibody is an IgG antibody.

[0016] Secondly, the present invention provides a biomaterial selected from any one of the following:

[0017] a. Nucleotides, said nucleotides comprising nucleotide sequences encoding the above-described anticlonal antibody against clozapine;

[0018] b. A carrier carrying the nucleotides in a;

[0019] c. A cell carrying the nucleotides in a, or containing the vector in b, or expressing the above-mentioned anti-clozapine monoclonal antibody.

[0020] Thirdly, the present invention provides the application of the above-mentioned anti-clozapine monoclonal antibody in the detection of clozapine for non-diagnostic and therapeutic purposes.

[0021] Fourthly, the present invention provides a clozapine marker, comprising the above-mentioned anti-clozapine monoclonal antibody and the marker;

[0022] The anti-clozapine monoclonal antibody and marker were conjugated.

[0023] As a further technical solution, the markers include enzymes, fluorescent molecular markers, fluorescent microspheres, colored microspheres, colloidal gold, biotin, or streptavidin.

[0024] Fifthly, the present invention provides a kit for the detection of clozapine, the kit comprising the above-mentioned anti-clozapine monoclonal antibody or clozapine marker.

[0025] As a further technical solution, the kit includes an immunochromatographic detection kit, an ELISA detection kit, an immunomagnetic microparticle detection kit, an immunofluorescence detection kit, or an immunoblotting detection kit.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This invention utilizes clozapine to immunize Balb / c mice, fusing mouse spleen cells with myeloma cells. Highly specific hybridoma cells are obtained through specific high-throughput screening. Large quantities of mouse ascites are obtained through culture and re-immunization. Subsequent multi-step separation and purification yields a high-purity, high-sensitivity, and high-specificity anti-clozapine monoclonal antibody, anti-Clozapine-mab1, providing the necessary raw material for developing immunoassay strips for clozapine detection. The anti-clozapine monoclonal antibody anti-Clozapine-mab1 provided by this invention can be used for immunological detection such as Western blotting and immunofluorescence. The obtained antibody has been verified to have excellent specific binding ability. Attached Figure Description

[0028] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0029] Figure 1 This is the molecular structural formula of clozapine. Detailed Implementation

[0030] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0031] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0032] The "variable region" or "variable domain" of an antibody refers to the domain at the amino terminus of the antibody's heavy or light chain that recognizes and binds to antigens. The composition and arrangement of the amino acids in this region determine the antibody's specificity in recognizing antigens. The heavy chain variable region can be called "VH," and the light chain variable region can be called "VL." These domains are usually the most variable parts of the antibody and contain antigen-binding sites. The variable regions of both the heavy and light chains consist of three complementarity-determining regions (CDRs) (also known as hypervariable regions) connected by four framework regions (FRs). The CDRs in each chain are held tightly together by the FRs to form the variable region. Typically, the VL / VH variable regions of the heavy and light chains are obtained by connecting the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0033] The term "vector" refers to a nucleic acid delivery vehicle into which nucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material they carry to be expressed in the host cells.

[0034] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. In some embodiments, the vectors of this invention contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0035] In a first aspect, the present invention provides an anti-clozapine monoclonal antibody, wherein the variable region of the anti-clozapine monoclonal antibody comprises: a complementarity-determining region CDR1-VH having an amino acid sequence as shown in SEQ ID NO.1, a complementarity-determining region CDR2-VH having an amino acid sequence as shown in SEQ ID NO.2, a complementarity-determining region CDR3-VH having an amino acid sequence as shown in SEQ ID NO.3, a complementarity-determining region CDR1-VL having an amino acid sequence as shown in SEQ ID NO.4, a complementarity-determining region CDR2-VL having an amino acid sequence as shown in SEQ ID NO.5, and a complementarity-determining region CDR3-VL having an amino acid sequence as shown in SEQ ID NO.6.

[0036] The amino acid sequences of SEQ ID NO.1-SEQ ID NO.6 are shown in Table 1.

[0037] Table 1

[0038]

[0039] In some alternative embodiments, the variable region includes a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO.7.

[0040] QVQLQQPGAELLKPGASVKISCKAIGYTFSSYWIEWVKQRPGHGLEWIGEILPGNGNIKNNEKFKDKATFTADASSNTAYMQLSSLSSEDSAVYFCARWNNGNAYVGHYGVDYWGQGTSVTVSS (SEQ ID NO. 7).

[0041] In some alternative embodiments, the variable region includes a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO. 8.

[0042] QAVVTQESALTTSPGETVTTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLLGDKAALTITGAQTEDEAIYFCALWYSNHLVFGGGTKLTVL (SEQ ID NO. 8).

[0043] In some alternative embodiments, the anticlozapine monoclonal antibody is an IgG antibody.

[0044] Secondly, the present invention provides a biomaterial selected from any one of the following:

[0045] a. Nucleotides, said nucleotides comprising nucleotide sequences encoding the above-described anticlonal antibody against clozapine;

[0046] b. A carrier carrying the nucleotides in a;

[0047] c. A cell carrying the nucleotides in a, or containing the vector in b, or expressing the above-mentioned anti-clozapine monoclonal antibody.

[0048] Thirdly, the present invention provides the application of the above-mentioned anti-clozapine monoclonal antibody in the detection of clozapine for non-diagnostic and therapeutic purposes.

[0049] The anti-clozapine monoclonal antibody provided by this invention can specifically recognize clozapine, and therefore can be used for the detection of clozapine.

[0050] Fourthly, the present invention provides a clozapine marker, comprising the above-mentioned anti-clozapine monoclonal antibody and the marker;

[0051] The anti-clozapine monoclonal antibody and marker were conjugated.

[0052] This marker can be used for the specific labeling of clozapine.

[0053] In some alternative embodiments, the markers include, but are not limited to, enzymes, fluorescent molecular markers, fluorescent microspheres, colored microspheres, colloidal gold, biotin, or streptavidin.

[0054] Fifthly, the present invention provides a kit for the detection of clozapine, the kit comprising the above-mentioned anti-clozapine monoclonal antibody or clozapine marker.

[0055] In some alternative implementations, the kit includes an immunochromatographic assay kit, an ELISA kit, an immunomagnetic microparticle assay kit, an immunofluorescence assay kit, or an immunoblotting assay kit.

[0056] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0057] Example 1

[0058] I. Preparation of Clozapine-BSA Hapten

[0059] 1. Add 100 mg of clozapine to a dry 100 mL single-necked flask, under nitrogen protection, then add 10 mL of pyridine to dissolve the raw material, stirring until the solution is clear. Then add succinic anhydride and reflux for 16 hours. The molar ratio of clozapine to succinic anhydride is 1:1.2-1.5.

[0060] 2. After the reaction was complete, the solvent was concentrated in a 55°C water bath to remove excess solvent. The residue was dissolved in dichloromethane, and silica gel was added and mixed. The solution was then passed through a column. The eluent ratio was DCM (dichloromethane):MeOH (methanol) = 80:1, yielding 110 mg of product.

[0061] 3. Dissolve 50 mg of product 1 in 1 mL of DMF to prepare a 50 mg / mL solution. Add NHS (N-hydroxysuccinimide) and EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) to activate the reaction at room temperature overnight. The molar ratio of product 1 to NHS and EDC is 1:1.2-1.5:1.2-1.5.

[0062] 4. Weigh 100 mg of BSA and dissolve it in 10 mL of PBS buffer to prepare a 10 mg / mL solution. Slowly add the activated small molecule to the protein and react for 8 hours.

[0063] 5. Place the conjugated antigen into a dialysis bag, change the medium every two hours, and change the medium more than six times in total. Collect the sample, measure the concentration with Lowry, and obtain the synthetic antigen clozapine-BSA.

[0064] The chemically synthesized clozapine hapten was used to immunize experimental mice in stages.

[0065] II. Preparation of monoclonal antibody anti-clozapine-mab1.

[0066] Generally, healthy female Balb / c mice aged 6-8 weeks are selected and immunized according to a pre-specified immunization protocol. As an immunogen, BALB / c mice are immunized, and spleen lymphocytes are extracted from successfully immunized mice. These lymphocytes are then fused with mouse myeloma cells SP2 / 0 using cell fusion technology. After two rounds of subclonal selection, a hybridoma cell line stably secreting anti-clozapine monoclonal antibodies is obtained, thus yielding the anti-clozapine monoclonal antibody.

[0067] The chemically synthesized clozapine hapten was used to immunize experimental mice in stages.

[0068] The specific steps of animal immunization experiments include:

[0069] 1. Balb / c mice with the same weight and age were randomly divided into two groups: an aluminum adjuvant group (aluminum hydroxide adjuvant) and a non-aluminum adjuvant group.

[0070] 2. Before the experiment, pre-immune serum was collected from each mouse (pre-immune serum was collected on the fifth day by blood collection through the eyeball, and an appropriate amount of blood was collected to ensure the normal condition of the mice). The collected serum was stored at -80℃.

[0071] 3. Preparation of aluminum adjuvant (aluminum hydroxide adjuvant) group: Before immunization, each antigen was diluted in 75 μL PBS to the corresponding dose (75 μg / mouse) and mixed with alum adjuvant (1 mg / mouse) at a volume ratio of antigen:adjuvant = 3:1 (i.e., 25 μl adjuvant was added to 75 μl of immunogen dilution). The adjuvant was shaken well before use, and the 25 μl adjuvant was slowly added dropwise to the immunogen solution. After thorough mixing of the adjuvant and immunogen dilution, the mixture was allowed to sit for 30 minutes to allow for effective adsorption of the antigen. Subsequent procedures were performed according to the animal immunization experiment procedures.

[0072] 4. Group without aluminum adjuvant: The antigen was diluted in 100 μL PBS to the corresponding dose in the table above (75 μg / mouse), and 100 μL of immunogen was added. Subsequent procedures were carried out according to the animal immunization experiment procedures.

[0073] 5. Subcutaneous injection at 2-week intervals: The experiment was designed with 4 immunizations, but blood was collected from the eyeballs 4 days after each immunization injection. The supernatant of the mice was obtained by centrifugation and the serum titer was tested first. Seven days after the last immunization, the maximum blood volume was collected from the heart, and the supernatant was obtained by centrifugation and stored at -80℃.

[0074] 6. Detect serum titer.

[0075] (1) Three mice were immunized, and the mice were numbered A0, A1 and A2 respectively. After the three immunizations were completed, the serum titer was measured.

[0076] Antigen competition detection was performed using clozapine hapten and clozapine small molecule, respectively. Indirect ELISA and competitive ELISA were performed using clozapine hapten as the coating antigen to detect the serum titer of immunized mice. For the indirect ELISA method, 50 μl of 1 μg / ml coating antigen diluted with coating buffer was added to each well of the ELISA plate. After coating overnight at 4°C, the plate was washed three times with washing buffer (PBST). 200 μl of blocking buffer (5% skim milk powder) was added to each well, and the plate was incubated at 37°C for 2 h. After washing, 50 μl of diluted serum was added to each well, and the plate was incubated at 37°C for 30 min. After washing, 50 μl of goat anti-mouse IgG-HRP solution was added, and the plate was incubated at 37°C for 30 min. After washing, 100 μl of substrate solution was added, and the plate was incubated at 37°C in the dark for 10 min. Finally, 50 μL of 2 mol / L H2SO4 was added to stop the reaction, and the A450 value was read using an ELISA reader. The orbital blood titers of all three mice after the third immunization were >62500.

[0077] The competitive ELISA process is largely the same as the indirect ELISA process, except that blocking buffer is used. After washing the ELISA plate, 50 μl of diluted 200 ng / mL clozapine standard solution is added, followed by 50 μl of diluted serum antibody, to achieve a final concentration of 100 ng / mL. The remaining steps are the same. At a dilution of 1:12500, the competitive detection of 100 ng / mL clozapine can achieve over 80% concentration, allowing for fusion assays.

[0078] The detection data are shown in Table 2 below. The results show that the orbital blood titers of the three mice after the third immunization were all >62500. In the competitive detection of 100 ng / ml small molecules, the inhibition rate was over 70%, indicating good competition.

[0079] Table 2 Serum titer test data

[0080]

[0081] Immunosplenic cells were fused with myeloma cell line SP2 / 0. Fusion cells were screened using HAT selective medium (containing hypoxanthine, aminopterin, and thymine), and ELISA positive screening and subcloning were performed on the fusion cells. Ascites fluid was collected from the selected positive monoclonal cells, and antibodies were purified using a Protein A / G antibody purification column. The purified antibody had an ELISA titer >1:128,000 and a purity >90%.

[0082] III. Enzyme-linked ELISA detection of clozapine binding activity.

[0083] 1. IgG antibody titer detection method

[0084] (1) Base plate coating: Dilute the antigen to 3 μg / ml with coating diluent, add 100 μl of the prepared coating solution to each well, and place in a 4℃ refrigerator for 24 h.

[0085] (2) After 24 hours, remove it from the refrigerator and place it at 37°C for 30 minutes to equilibrate. Then discard the liquid in the hole; wash the hole with washing solution 3 times, 3 minutes each time.

[0086] (3) Blocking enzyme-labeled reaction wells: Add 200 μl of 5% fetal bovine serum to each well and incubate at 37°C for 90 min. After blocking, wash the wells three times with washing buffer for 3 min each time.

[0087] (4) Add the sample to be tested: Dilute the sample according to the required ratio, add 100 μl of the diluted sample to each enzyme-labeled reaction well, and place at 37℃ for 90 min; wash the well with washing solution three times, each time for 3 min.

[0088] (5) Add enzyme-labeled antibody: Add the appropriate concentration of secondary antibody according to the instructions; wash 100 μl per well at 37℃ for 90 min as before.

[0089] (6) Add substrate solution: Add 100 μl of substrate per well and place at 37°C in the dark for 15 to 30 min.

[0090] (7) Termination of reaction: Add 50 μl of termination solution to each well to terminate the reaction, and measure the experimental results within 20 min.

[0091] IV. Detection of the binding activity of monoclonal antibodies to clozapine.

[0092] (1) Cell fusion and clone screening data

[0093] The mice were numbered A0, A1, and A2, and a total of four rounds of fusion were completed.

[0094] A total of 33 positive wells were selected from the A0 mouse fusion screening and subcloning was performed, ultimately resulting in the complete formation of 4 cell lines. In the first subcloning screening, 11 positive clones with OD450 values ​​>2.2 were selected for serial dilution titer testing. A second subcloning screening was performed, completing 2 positive cell lines. A third subcloning screening was then performed on 3 cell lines, completing 2 more positive cell lines.

[0095] A1 mouse fusion screening yielded 111 positive wells, ultimately resulting in the complete formation of 17 cell lines. The first subcloning screening identified 38 positive clones with an OD450 value >2.2. A second subcloning screening further completed 11 cell lines. A third subcloning screening of 22 cell lines completed 3 cell lines. A fourth subcloning screening of 5 cell lines completed 3 cell lines.

[0096] A total of 21 complete cell lines were obtained after four cell fusions.

[0097] (2) Ascites preparation and detection data

[0098] One F1 mouse was injected with each complete cell line. No ascites fluid was produced from the three cell lines. A total of 18 ascites fluid samples were prepared. The potency data of some ascites fluid samples are shown in the table below:

[0099] Table 3. Ascites titer test data

[0100]

[0101] (3) Exploration of antibody purification conditions and detection data

[0102] The ascites fluid was purified using the 3.3% octanoic acid-thiamine precipitation method, yielding a total of 18 antibodies. The titer data for some of these antibodies are shown in the table below:

[0103] Table 4. Data on purified antibody titer detection

[0104]

[0105] Ascites titer and antibody titer tests showed that the monoclonal antibodies from 18 cell lines had good specific binding ability to clozapine antigen. Competitive experiments with small molecule clozapine showed good competition. Therefore, the 18 antibodies were used for testing clozapine colloidal gold products.

[0106] V. Application of monoclonal antibody anti-Clozapine-mab1 in products.

[0107] Eighteen clozapine antibodies were colloidal-gold labeled using an immunochromatographic platform, and then colloidal gold chromatography strips were prepared. The immunochromatographic strips included a sample pad, a conjugation pad, and a detection pad. The detection pad had a test line and a control line. The conjugation pad was coated with a selected anti-Clozapine-mab1 monoclonal antibody labeled with colloidal gold. The test line was coated with clozapine antigen, and the control line was coated with goat anti-mouse IgG antibody. Parallel tests were performed on negative buffer samples and different concentrations of clozapine small molecule standards. When testing negative samples, no clozapine small molecules competed with the clozapine antigen for binding to the gold-labeled anti-Clozapine-mab1 monoclonal antibody. Therefore, the clozapine antigen coated at the test line bound the gold-labeled anti-Clozapine-mab1 monoclonal antibody, resulting in a T-line color development.

[0108] Specifically, the experimental group used 18 clozapine antibodies (1# to 18#) obtained in the above experiment to detect the clozapine standard from Hangzhou Anxu Biotechnology Co., Ltd. The reagents were added, and the results were detected using the ACG1000 (ID-A003) POCT instrument from Hangzhou Anxu Biotechnology Co., Ltd. Antibody #8 was negative and showed a good gradient. Some experimental data are shown in the table below:

[0109] Table 5 Initial Antibody Evaluation Results

[0110]

[0111] Note: G3-G9 represent the color intensity levels of the test strips; higher values ​​indicate a darker color, and + / - indicates a slightly darker or lighter color than that level. A decrease in the value after adding clozapine standard indicates that the small molecule has a competitive effect, meaning the antibody can bind to clozapine.

[0112] The results showed that antibody #8 had a good gradient and could be used in clozapine products, achieving a cut-off value of 350 ng / ml. Stability assessment of antibody #8 was then performed, with three batches of antibody samples prepared. The assessment results are shown in the table below:

[0113] Table 6. Stability assessment results of three batches of antibody #8

[0114]

[0115] Based on the product evaluation results, antibody #8 was named anti-Clozapine-mab1, which can be used in clozapine antigen detection kits.

[0116] VI. Sequence analysis of the V region (VH) of the heavy chain and the V region (VL) of the monoclonal antibody anti-Clozapine-mab1.

[0117] Design primers to amplify the heavy chain V region (VH) and light chain V region (VL) genes.

[0118] Take a hybridoma cell line of anti-Clozapine-mab1 in the logarithmic growth phase (about 107 cells), extract total RNA from the cells according to the instructions of the Trizol RNA extraction kit, use the total RNA as a template to reverse transcribe and synthesize the first strand of cDNA, and use the above amplification product as a template to PCR amplify the VH / VL gene of the antibody.

[0119] The heavy chain VH (approximately 360 bp) and light chain VL (approximately 300 bp) fragments of anti-Clozapine-mab1 were recovered and sent to the company for sequencing.

[0120] Then, the VH / VL gene sequences were analyzed, and the resulting sequences are as follows:

[0121] The variable region sequence of the heavy chain.

[0122] anti-Clozapine-mab1-VH: 372 bp.

[0123] CAGGTTCAGCTGCAGCAGCCTGGAGCTGAACTGCTGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTATTGGCTACACATTCAGTAGCTACTGGATAGAGTGGGTAAAACAGAGGCCTGGACATGGCCTTGAGTGGATAGGAGAGATTCTACCTGGAAATGGTAATATTAAGAATAATGAGAAGTTCAAGGACAAGGCCACATTCACTGCAGATGCATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGTCATCTGAGGACTCTGCCGTCTATTTCTGTGCAAGATGGAACAACGGTAATGCCTACGTGGGTCACTATGGTGTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO.9).

[0124] anti-Clozapine-mab1 protein: 124aa.

[0125] QVQLQQPGAELLKPGASVKISCKAIGYTFSSYWIEWVKQRPGHGLEWIGEILPGNGNIKNNEKFKDKATFTADASSNTAYMQLSSLSSEDSAVYFCARWNNGNAYVGHYGVDYWGQGTSVTVSS (SEQ ID NO.�).

[0126] Variable region sequence of the light chain:

[0127] anti-Clozapine-mab1 LVκ: 327 bp.

[0128] CAAGCCGTGGTGACCCAAGAGAGCGCCCTGACCACAAGCCCCGGCGAGACCGTGACCCTGACCTGCAGAAGCAGCACCGGCCCGTGACCACAAGCAACTACGCCAACTGGGTGCAAGAGAAGCCCGACCACCTGTTCACCGGCCTGATCGGCGGCACCAATAAC AGAGCCCCCGGCGTGCCCGCTAGATTCAGCGGCAGCCTGCTGGGCGACAAGGCCGCCCTGACCATCACCGGCGCTCAGACCGAGGACGAGGCCATCTACTTCTGCGCCCTGTGGTACAGCAACCACCTGGTGTTCGGCGGCGGGACAAAGCTCACCGTGCTG (SEQ ID NO.10).

[0129] anti-Clozapine-mab1 LVκ protein: 109aa.

[0130] QAVVTQESALTTSPGETVTTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLLGDKAALTITGAQTEDEAIYFCALWYSNHLVFGGGTKLTVL (SEQ ID NO. 8).

[0131] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody against clozapine, characterized in that, The variable region of the anti-clozapine monoclonal antibody includes: the complementarity-determining region CDR1-VH of the amino acid sequence shown in SEQ ID NO.1, the complementarity-determining region CDR2-VH of the amino acid sequence shown in SEQ ID NO.2, the complementarity-determining region CDR3-VH of the amino acid sequence shown in SEQ ID NO.3, the complementarity-determining region CDR1-VL of the amino acid sequence shown in SEQ ID NO.4, the complementarity-determining region CDR2-VL of the amino acid sequence shown in SEQ ID NO.5, and the complementarity-determining region CDR3-VL of the amino acid sequence shown in SEQ ID NO.

6.

2. The anti-clozapine monoclonal antibody according to claim 1, characterized in that, The variable region includes the heavy chain variable region VH of the amino acid sequence shown in SEQ ID NO.

7.

3. The anticlozapine monoclonal antibody according to claim 1, characterized in that, The variable region includes the light chain variable region VL of the amino acid sequence shown in SEQ ID NO.

8.

4. The anti-clozapine monoclonal antibody according to claim 1, characterized in that, The anti-clozapine monoclonal antibody is an IgG antibody.

5. A biomaterial, characterized in that, The biomaterial is selected from any one of ac: a. Nucleic acid, wherein the nucleic acid is a nucleotide sequence encoding the anticlonal antibody against clozapine according to any one of claims 1-4; b. A vector carrying the nucleic acid from a; c. A cell carrying the nucleic acid of a, or containing the vector of b, or expressing the anticlozapine monoclonal antibody according to any one of claims 1 to 4.

6. The use of the anti-clozapine monoclonal antibody according to any one of claims 1-4 in the detection of clozapine for non-diagnostic and therapeutic purposes.

7. A clozapine marker, characterized in that, Includes the anticlozapine monoclonal antibody and marker as described in any one of claims 1-4; The anti-clozapine monoclonal antibody and marker were conjugated.

8. The clozapine marker according to claim 7, characterized in that, The label is selected from enzymes, fluorescent molecular labels, fluorescent microspheres, colored microspheres, colloidal gold, biotin, or streptavidin.

9. A kit for the detection of clozapine, characterized in that, The kit comprises the anti-clozapine monoclonal antibody as described in any one of claims 1-4 or the clozapine marker as described in claim 7 or 8.

10. The reagent kit according to claim 9, characterized in that, The kit is selected from immunochromatographic assay kits, ELISA kits, immunomagnetic microparticle assay kits, immunofluorescence assay kits, or immunoblotting assay kits.

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