Lamotrigine complete antigen and antibody and preparation method and application thereof

Abstract

The present invention discloses a complete lamotrigine antigen and its use in the preparation of lamotrigine antibodies and immunoassay detection. The structure of the complete lamotrigine antigen is shown in Formula 1. The present invention also discloses anti-lamotrigine monoclonal antibodies prepared using the complete antigen, hybridoma cells producing the monoclonal antibodies, and detection cards and kits for detecting lamotrigine analogs. The present invention discloses an ELISA method and a fluorescent immunochromatographic method for detecting lamotrigine analogs. The fluorescent immunochromatographic method is simple to operate, has a short detection time, low cost, high specificity, and good reproducibility.

Description

Technical Field

[0001] The present invention relates to the detection of psychotropic drugs; in particular, the present invention relates to a complete lamotrigine antigen, a lamotrigine monoclonal antibody obtained by using the complete antigen, and its application in the detection of lamotrigine analogs by fluorescent immunochromatography. Background Art

[0002] The development of lamotrigine (LTG) was one of the earliest attempts at rational antiepileptic drug (AED) development. LTG was first synthesized in the 1970s as part of a rational approach to discovering new AEDs, based on the observation that many existing AEDs are folate antagonists. However, as a phenyltriazine, lamotrigine is chemically unrelated to any existing AEDs. In March 1993, a Food and Drug Administration (FDA) advisory committee recommended LTG for marketing in the United States. Compared with classic antiepileptic drugs, lamotrigine offers advantages such as good tolerability, minimal enzyme induction and inhibition, and reduced toxicity during pregnancy. Lamotrigine is rapidly and completely absorbed from the intestine, with oral bioavailability approaching 100%. Its half-life ranges from 24.1 to 35 hours, but this can be altered by enzyme-inducing and -inhibiting drugs. Patient weight, age, concomitant medications, and genetic polymorphisms can influence the pharmacokinetics of lamotrigine, resulting in significant inter-individual variability. A literature search of lamotrigine adverse reactions from the China Hospital Knowledge Database from 1994 to 2010 revealed that LTG-induced adverse reactions include skin damage, damage to the blood system, damage to the central nervous system, digestive system adverse reactions, autoimmune diseases, and teratogenicity. In summary, to further optimize and standardize the use of lamotrigine in different populations and adjust dosing regimens based on the patient's pathophysiological conditions, it is necessary to conduct lamotrigine plasma concentration monitoring (TDM) in specific populations.

[0003] Currently, lamotrigine testing primarily involves liquid chromatography coupled with mass spectrometry (HPLC-MS) and high-performance liquid chromatography (HPLC). These methods are expensive, complex, and time-consuming. Fluorescence quantitative immunochromatography (FQIC) is a novel quantitative detection technology that combines the advantages of immunofluorescence and traditional immunochromatography. This technology is flexible and easy to operate, low-cost, and has a short reaction time, enabling timely quantitative detection. This provides physicians with enhanced data support for adjusting patient medications, enabling personalized medication administration and ensuring both efficacy and safety. Summary of the Invention

[0004] The technical purpose of the present invention is to overcome the above technical problems and provide a complete lamotrigine antigen with simple operation, short detection time, low cost, high specificity and good repeatability, obtain lamotrigine monoclonal antibodies using the complete antigen and use thereof in the detection of lamotrigine analogs by fluorescent immunochromatography.

[0005] The technical objectives of the present invention are achieved through the following technical solutions:

[0006] A complete lamotrigine antigen, wherein the complete antigen has a structure shown in Formula 1:

[0007]

[0008] Among them, Protein is a protein carrier.

[0009] A method for preparing the complete lamotrigine antigen according to claim 1, comprising the steps of:

[0010] (1) linking the lamotrigine hapten to bovine serum albumin (BSA) or ovalbumin (OVA) to obtain the complete lamotrigine antigen;

[0011] (a) dissolving lamotrigine hapten in N,N-dimethylformamide (DMF) to form solution A;

[0012] (b) adding solution A obtained in step (a) and 25-25% glutaraldehyde to the BSA / OVA solution and reacting at room temperature for 1.5-2.5 hours to obtain a lamotrigine-BSA / OVA conjugate, i.e., a complete lamotrigine antigen.

[0013] A use of a complete lamotrigine antigen for preparing a specific monoclonal antibody against lamotrigine.

[0014] A monoclonal antibody that specifically binds to lamotrigine; the monoclonal antibody is produced by a mouse hybridoma cell line.

[0015] A monoclonal antibody, wherein the hybridoma cell line is deposited by China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China) with a deposit number of CCTCC NO: C2022381.

[0016] A hybridoma cell line of the monoclonal antibody, wherein the hybridoma cell line is a mouse hybridoma cell line deposited by China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China) with the deposit number CCTCC NO: C2022381.

[0017] In another preferred embodiment, the sensitivity of the monoclonal antibody for detecting lamotrigine analogs is 1.5 ng / mL.

[0018] In another preferred embodiment, the monoclonal antibody does not bind to other psychotropic drugs.

[0019] In another preferred embodiment, the other psychotropic drugs are sodium valproate, topiramate, clozapine, carbamazepine, and oxcarbazepine.

[0020] A use of the monoclonal antibody is to prepare a reagent, a detection card or a kit for detecting lamotrigine in a sample.

[0021] A method for detecting whether lamotrigine is present in a biological sample, the method comprising the steps of:

[0022] (a) contacting the biological sample with the monoclonal antibody;

[0023] (b) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of lamotrigine in the sample;

[0024] Wherein, the monoclonal antibody carries a detectable marker; the marker is selected from the following group: colloidal gold marker, colored marker or fluorescent marker; the detection method is fluorescence detection method.

[0025] A fluorescent immunochromatographic test card for detecting lamotrigine analogs, comprising a substrate; a liquid aspirating component; a detection component; and a sample loading component, characterized in that the detection component is fixed on the substrate, a quality control tape and a detection tape are arranged in the middle of the detection component, and the liquid aspirating component and the sample loading component are fixed at both ends of the detection component in a partially overlapping manner, wherein the detection tape is coated with the complete antigen according to claim 1, and the quality control tape is coated with rabbit antigen IgG; the lamotrigine fluorescent immunochromatographic test card also includes a card box, which is composed of a lower cover and an upper cover, the upper cover being provided with a sample loading window and a detection window, the lamotrigine fluorescent immunochromatographic test card being completely arranged in the lower cover, and the detection window and the sample loading window respectively corresponding to the sample loading component, the quality control tape, and the detection tape on the lamotrigine fluorescent immunochromatographic test card.

[0026] In a preferred embodiment, the upper cover is further provided with a product number area and a bar code identification area.

[0027] In a preferred embodiment, the substrate is a dark hard substrate, preferably a black PVC substrate.

[0028] In a preferred example, the detection component is a nitrocellulose membrane.

[0029] In a preferred example, the sample adding component is glass fiber.

[0030] In a preferred embodiment, the liquid-absorbing component is absorbent paper.

[0031] A detection kit for detecting lamotrigine analogs, wherein the kit contains:

[0032] (a) the lamotrigine fluorescent immunochromatographic test card;

[0033] (b) a lamotrigine detection analysis solution that is compatible with the lamotrigine fluorescent immunochromatographic test card;

[0034] (c) instructions for use of the lamotrigine detection kit for detecting lamotrigine analogs;

[0035] Wherein, the detection and analysis liquid is a detection and analysis liquid containing the fluorescently labeled monoclonal antibody according to claim 4 or 5 and anti-rabbit IgG antibody;

[0036] The fluorescent dyes used for labeling in the detection analysis solution include but are not limited to Alexa Fluor 647, CF TM 647, TRITC (Rhodamine), etc.

[0037] In a preferred embodiment, the solvent portion of the detection analysis solution is a phosphate buffer containing BSA.

[0038] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 UV scanning patterns of the lamotrigine immunogen and coating are shown;

[0040] FIG2 shows a schematic diagram of the structure of a lamotrigine fluorescent immunochromatographic test card; wherein:

[0041] Figure 2A Shows the structure of the test card without plastic card; 1: black PVC substrate; 2: absorbent paper; 3: nitrocellulose membrane; 4: glass fiber; 5: quality control line (C line); 6: test line (T line);

[0042] Figure 2B The structure of the lamotrigine test card with a plastic card box is shown; 1': lower cover; 2': upper cover; 3': sample loading window; 4': glass fiber; 5': detection window; 6': quality control line (C line); 7': detection line (T line); 8': nitrocellulose membrane; 9': lamotrigine project logo; 10': barcode identification area;

[0043] Figure 3 The standard curve profile of the lamotrigine fluorescent immunochromatographic assay is shown;

[0044] Figure 4 The standard curve of the lamotrigine ELISA assay is shown. DETAILED DESCRIPTION

[0045] After extensive and in-depth research, the inventors synthesized a complete lamotrigine antigen. Using this antigen as an immunogen, they immunized Balb / C mice, fused their spleen cells with mouse myeloma SP20 cells, and obtained a hybridoma cell line that specifically secreted anti-lamotrigine monoclonal antibodies. Lamotrigine monoclonal antibodies were then prepared and purified. Subsequently, the complete antigen and lamotrigine antibodies were used to prepare a highly sensitive and specific lamotrigine immunoassay card. Based on this, the present invention was completed.

[0046] Complete antigen

[0047] Substances that are both immunogenic and immunoreactive are called complete antigens, such as most proteins, bacteria, viruses, bacterial exotoxins, and animal sera. Complete antigens can stimulate the body to produce antibodies or sensitize lymphocytes and can also undergo specific binding reactions with them in vivo and in vitro.

[0048] Usually, haptens need to be coupled to macromolecules such as bovine serum albumin (BSA), ovalbumin (OVA) or hemocyanin (KLH) or covalently bonded to become complete antigens that are both immunoreactive and immunogenic.

[0049] As used herein, the term "complete antigen" refers to the product of the lamotrigine hapten of the present invention combined with an appropriate protein carrier. As used herein, the term "protein carrier" refers to any immunologically acceptable protein used to form a complete antigen, including but not limited to bovine serum albumin, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin (HSA), and synthetic polylysine (PLL), preferably bovine serum albumin (BSA) or ovalbumin (OVA).

[0050] The structure of the complete lamotrigine antigen of the present invention is shown in Formula 1:

[0051]

[0052] Here, Protein is a protein carrier, and in the present invention, preferably bovine serum albumin (BSA) or ovalbumin (OVA).

[0053] The conditions for linking the lamotrigine hapten to the protein carrier are as follows: the lamotrigine hapten is linked to bovine serum albumin (BSA) or ovalbumin (OVA) to prepare a complete antigen.

[0054] (a) Lamotrigine hapten is dissolved in N,N-dimethylformamide (DMF) to form solution A.

[0055] (b) In a preferred embodiment, the conditions for connecting liquid A to the protein carrier are as follows: the reaction temperature is 20-28°C, preferably 23-28°C, more preferably 25°C; the reaction pH is 7.0-8.0, preferably 7.2-7.6, more preferably 7.5; and the reaction time is 1-5 hours, preferably 2-4 hours, more preferably 4 hours.

[0056] Preparation of monoclonal antibodies

[0057] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible spontaneous mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0058] The antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, the complete antigen of the present invention can be administered to animals to induce the production of monoclonal antibodies. Monoclonal antibodies can be prepared using hybridoma technology (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, NY, 1981) or can be prepared using recombinant DNA methods (U.S. Patent No. 4,816,567).

[0059] Representative myeloma cells are those that fuse efficiently, support stable high-level production of the antibody by the selected antibody-producing cells, and are sensitive to a medium (HAT medium medium), including myeloma cell lines, such as murine myeloma cell lines, including myeloma cell lines derived from MOPC-21 and MPC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, California, USA), and SP-2, NZ0, or X63-Ag8-653 cells (available from the American Type Culture Collection, Rockville, Maryland, USA). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibodies Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)].

[0060] The culture medium in which the hybridoma cells are grown is analyzed to detect the production of monoclonal antibodies with the desired specificity, such as by in vitro binding assays, for example, enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). The location of cells expressing the antibody can be detected using FACS. The hybridoma clones can then be subcloned by limiting dilution steps and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986) 59-103 pages). Suitable culture media for this purpose include, for example, DMEM or RPMI-1640 culture medium. In addition, hybridoma cells can be grown as ascites tumors in animals.

[0061] The monoclonal antibodies secreted by the subclones are suitably isolated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0062] The monoclonal antibody of the present invention is produced by a mouse hybridoma cell line, which was deposited in the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China) on January 11, 2023, with the deposit number CCTCCNO: C2022381; the classification name is: hybridoma cell line ZCSW01.

[0063] In a specific embodiment, the monoclonal antibody of the present invention carries a detectable label. More preferably, the label is selected from the group consisting of colloidal gold labels, colored labels, and fluorescent labels.

[0064] In a specific embodiment, the monoclonal antibody of the present invention has a sensitivity of 0.08 μg / mL for detecting lamotrigine analogs. The monoclonal antibody of the present invention does not cross-react with carrier proteins of the complete lamotrigine antigen, such as BSA or OVA. Furthermore, the monoclonal antibody of the present invention does not bind to other psychotropic drugs, including but not limited to sodium valproate, topiramate, clozapine, carbamazepine, and oxcarbazepine.

[0065] Detection kit

[0066] The detection kit of the present invention refers to a kit containing the monoclonal antibody of the present invention and can be used for the detection of lamotrigine analogs. The kit can include a container, instructions for use, a buffer, an immunoadjuvant, etc. as needed.

[0067] The detection kit of the present invention can be in various forms, such as a detection card, a test box containing various test reagents, etc. In the examples, the detection card is used as an example to illustrate the kit of the present invention, but it should not be understood that the kit of the present invention is limited to the detection card.

[0068] In a specific embodiment, the fluorescent immunochromatographic test card for detecting lamotrigine analogs of the present invention includes a substrate; a liquid aspiration component; a detection component; and a sample loading component; wherein the detection component is fixed on the substrate, a quality control tape and a detection tape are arranged in the middle of the detection component, and the liquid aspiration component and the sample loading component are fixed at both ends of the detection component in a partially overlapping manner, wherein the detection tape is coated with the complete antigen of the present invention, and the quality control tape is coated with rabbit antigen IgG.

[0069] In a preferred embodiment, the lamotrigine fluorescent immunochromatographic test card of the present invention further includes a card box, which is composed of a lower cover and an upper cover. The upper cover is provided with a sample loading window and a detection window. The lamotrigine fluorescent immunochromatographic test card is completely disposed in the lower cover. The detection window and the sample loading window correspond to the sample loading component, the quality control strip, and the detection strip on the lamotrigine fluorescent immunochromatographic test card, respectively. A product number area and a barcode identification area may also be provided on the upper cover. The substrate may be a dark hard substrate; preferably a black PVC substrate. The detection component may be a nitrocellulose membrane. The sample loading component may be glass fiber. The liquid absorbing component may be absorbent paper.

[0070] The term "partially overlapping" as used herein means that two adjacent components form a certain overlapping area, rather than a complete overlap where one component is completely contained within the other, and that the two components are secured via this overlapping area. The securing method may be selected by those skilled in the art, such as by bonding.

[0071] On the basis of the above-mentioned detection card, the present invention also provides a detection kit for detecting lamotrigine analogs, which contains:

[0072] (a) the above-mentioned lamotrigine fluorescent immunochromatographic test card;

[0073] (b) a lamotrigine analogue detection analysis solution that is compatible with the lamotrigine fluorescence immunochromatographic test card;

[0074] (c) instructions for use of the lamotrigine analog detection kit for detecting lamotrigine analogs;

[0075] Wherein, the detection and analysis liquid is a detection and analysis liquid containing the fluorescently labeled monoclonal antibody of the present invention and anti-rabbit IgG antibody.

[0076] Those skilled in the art can select fluorescent labels according to their needs, including but not limited to FITC (Fluorescein), Alexa Fluor 647, CFTM647, TRITC (Rhodamine) and CAL Fluor (R) Red 610.

[0077] In a preferred embodiment, the solvent portion of the detection analysis solution is a phosphate buffer containing BSA.

[0078] Application of immunoassay for complete lamotrigine antigen

[0079] The complete lamotrigine antigen of the present invention is used for antibody preparation, and the antibody should be a monoclonal antibody or a polyclonal antibody; the corresponding antibody prepared from the complete lamotrigine antigen of the present invention is used in various immunological detection fields for detecting the content of lamotrigine analogs, including but not limited to ELISA, chemiluminescence, colloidal gold method and fluorescent immunochromatography.

[0080] The above-mentioned "lamotrigine complete antigen is used for antibody preparation" refers to the use of the lamotrigine complete antigen of the present invention to immunize experimental animals with the complete antigen to prepare anti-lamotrigine polyclonal antibodies and monoclonal antibodies; the experimental animals should not be understood as simple mice in the specific embodiment, and should include but are not limited to: mice, rats, rabbits, goats, sheep, horses, donkeys, chickens, dogs and other experimental animals.

[0081] The aforementioned "lamotrigine complete antigen applied to lamotrigine immunoassays" refers to the use of corresponding antibodies prepared from lamotrigine complete antigen as immunoassay raw materials to establish various immunoassay methods for detecting the content of lamotrigine analogs. This immunoassay field includes, but is not limited to, immunological detection methods such as ELISA, chemiluminescence, colloidal gold, and fluorescent immunochromatography. The immunoassay methods for detecting lamotrigine analogs include not only quantitative detection but also semi-quantitative and various qualitative detection methods based on immunoassays.

[0082] In a specific embodiment, the present invention takes the preparation of specific monoclonal antibodies by immunizing mice as an example, and uses ELISA and fluorescent immunochromatography as specific examples to illustrate the application of complete lamotrigine antigen in the immunological detection of lamotrigine.

[0083] Advantages of the present invention:

[0084] 1. The present invention discloses for the first time the structure of a complete (artificial) lamotrigine antigen and its preparation method;

[0085] 2. This invention discloses for the first time the application of complete lamotrigine antigen in the preparation of lamotrigine antibodies and immunological detection, providing a reliable method for clinical lamotrigine blood concentration detection;

[0086] 3. The monoclonal antibody of the present invention can detect lamotrigine analogs with high sensitivity and does not bind to other psychotropic drugs;

[0087] 4. The lamotrigine analog detection kit of the present invention can simply and quickly detect lamotrigine analogs on site.

[0088] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0089] Example

[0090] Example 1. Preparation of Lamotrigine Complete Antigen (Immunogen and Coating Gen)

[0091] Lamotrigine (hapten) was conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) respectively by EDC method. The specific conjugation method is as follows:

[0092] Weigh 6 mg of lamotrigine and dissolve it in 300 μL of N,N-dimethylformamide (DMF). Add 10 mg / mL of OVA or BSA, and then add 18 μL of 30% glutaraldehyde dropwise. Incubate at room temperature in the dark for 2-3 hours. Dialyze the solution against phosphate buffered saline for 2-3 days, changing the dialysate 2-3 times daily. Collect the dialysate by centrifugation to prepare lamotrigine-BSA / OVA. The molar ratio of lamotrigine to BSA / OVA is controlled at 35:1.

[0093] The structural formula of the obtained lamotrigine artificial antigen (immunogen and coating agent) is shown below, wherein "Protein" is BSA (bovine serum albumin) or OVA (ovalbumin).

[0094]

[0095] The results of UV scanning peaks of lamotrigine artificial antigens (immunogen and coating) are compared as follows Figure 1 As shown, the peak of the conjugate is different from the peaks of BSA and OVA, indicating successful conjugation.

[0096] Example 2. Preparation of monoclonal antibodies using lamotrigine complete antigen

[0097] 1. Immunization of Animals

[0098] The lamotrigine immunogen obtained in Example 1 was diluted to 0.5 mg / mL. 500 μL of the immunogen was mixed with an equal volume of complete Freund's adjuvant, emulsified, and used to immunize BALB / c mice (Shanghai Slake Laboratory Animal Co., Ltd.) by inguinal injection. Complete Freund's adjuvant was used for the first immunization, followed by incomplete Freund's adjuvant. One week after the fourth immunization, orbital blood was collected, serum was isolated, and the anti-lamotrigine antibody titer was measured. ELISA assay showed that the antibody titer of the mice after four immunizations was 1:256,000.

[0099] 2. Cell Fusion and Screening

[0100] After four immunizations, mice were given a booster immunization by intraperitoneal injection of about 100 μg of immunogen. Three days later, the spleen of the mice was taken for fusion. SP2 / 0 cells (Nanjing Academy of Military Medical Sciences) were mixed with spleen cells, added with serum-free culture medium (Hyclone SH30022.018 DMEM (High Glucose)), centrifuged (1500 rpm, 3 min), the precipitated cells were taken, 1 mL of 50% polyethylene glycol 4000 was added dropwise, and allowed to stand for 90 seconds. Then 10 mL of serum-free culture medium preheated at 37°C was added dropwise and allowed to stand for 5 min. After fusion, the cell suspension was centrifuged (1000 rpm, 3 min) and inoculated with complete culture medium into a 96-well plate with feeder cells, with 2×10 cells per well. 4 / mL myeloma cells. Culture in a 37°C, 5% CO2 incubator for two days. Add 2×HAT complete culture medium to a final concentration of 1×HAT in the wells. When hybridoma colonies have grown to 1 / 10-1 / 5 of the well bottom area, screen for fusion cell antibody-positive wells using ELISA.

[0101] 3. Ascites Preparation and Antibody Purification

[0102] BALB / c mice were intraperitoneally injected with 0.5 mL of paraffin oil. Seven days later, 0.5 mL of 1×10 6 Positive hybridoma cells. Observe the growth of the mice. If abdominal swelling is visible around day 7, collect ascites promptly. Purify the monoclonal antibody using affinity chromatography (Protein G Resin affinity purification) to obtain a high-purity protein of 6 mg.

[0103] Example 3. Immunoassay using complete lamotrigine antigen

[0104] 1. Fluorescence immunochromatographic detection

[0105] 1) Preparation of assay solution

[0106] a. Fluorescently labeled monoclonal antibodies and anti-rabbit IgG antibodies obtained in Example 2 (Hangzhou Qitai Biotechnology Co., Ltd.);

[0107] b. Dilute the fluorescently labeled antibody with phosphate buffer containing BSA to prepare a detection analysis solution.

[0108] 2) Preparation of Lamotrigine Fluorescent Immunochromatographic Test Card

[0109] a. Dilute the prepared lamotrigine coating agent (lamotrigine-OVA) and rabbit antigen IgG to appropriate concentrations (0.4-3.0 mg / mL) using coating buffer (phosphate buffer). Spray the diluted lamotrigine-OVA and rabbit antigen IgG evenly onto a nitrocellulose membrane at 25±5°C (forming the test and control lines, respectively). Dry the membrane at 12%-30% humidity for approximately 1.5-2 hours and store dry until ready for use.

[0110] b. The nitrocellulose membrane, glass fiber paper and absorbent paper obtained in step a are sequentially pasted on the black PVC substrate to form a test card (eg Figure 2A Cut to appropriate width as needed.

[0111] c. Place the test card obtained in step b into the lower cover of the card box and close the upper cover to form a complete test card with a card box (such as Figure 2B shown).

[0112] 3) Detection

[0113] Take 60 μl of diluted sample and mix it evenly with 60 μl of detection analysis solution, take 100 μL and add it to the sample loading window of the detection card. After reacting for 15 to 20 minutes, use FCR fluorescence immunoassay analyzer (Suzhou Hemai Precision Instrument Co., Ltd.) to detect it. The test result is displayed based on the comparison of the T line signal value and C line signal ratio (T / C value) of the sample with the built-in standard curve.

[0114] 4) Lamotrigine Fluorescent Immunochromatographic Test Card Principle

[0115] Using a competitive assay, the lamotrigine antigen in the sample competes with the lamotrigine antigen (coating agent) on the test line (T line) for binding to the fluorescently labeled anti-lamotrigine antibody in the assay solution. When the antigen concentration in the sample is lower, more fluorescent antibodies bind to the test line, and the fluorescent signal on the test line is stronger. Therefore, the ratio (T / C value) of the fluorescent signal on the test line (T line) to the fluorescent signal on the control line (C line) is greater. Conversely, when the lamotrigine antigen concentration in the sample is higher, the T / C value is lower. Therefore, the higher the lamotrigine content in the sample, the lower the T / C value. The test results are displayed based on the comparison of the T / C value with the built-in standard curve.

[0116] 5) Sensitivity and standard curve of fluorescent immunochromatographic assay for lamotrigine analogs

[0117] Lamotrigine standard was added to blank serum to prepare 9 concentration gradients of 20, 16, 12, 6, 3, 1.5, 0.75, 0.38, and 0 ng / mL, and then diluted 40 times with 0.9% NaCl. The above series of concentration samples were tested according to the above test steps, with each sample repeated 3 times. The test results are shown in Table 1. Based on the data in Table 1, a standard curve (four parameters) was drawn with concentration as the horizontal axis and T / C value as the vertical axis. Figure 3 shown. Figure 3 The equation corresponding to the middle curve is shown in Table 2, and IC is calculated 50 =1.08 ng / mL, true IC 50 =27ng / mL.

[0118] Table 1 Detection of lamotrigine samples with different concentrations by fluorescence immunochromatography

[0119]

[0120] Table 2 Equation corresponding to the inhibition curve (four parameters)

[0121]

[0122]

[0123] Repeat the test 10 times for the 0 ng / mL sample and calculate the mean (X), standard deviation (SD) and precision (CV) of the T / C values. The sensitivity is calculated as X-2*SD T / C value. Figure 3 The corresponding lamotrigine concentration values ​​in the standard curve are shown in Table 3.

[0124] Table 3. Results of repeated testing of 0 ng / mL lamotrigine samples by fluorescence immunochromatography

[0125]

[0126] Substitute the T / C value of X-2*SD in the data of Table 3 as the y value Figure 3 The concentration value in the equation corresponding to the standard curve is 1.5 ng / mL, that is, the sensitivity is 1.5 ng / mL.

[0127] 6) Precision deviation of lamotrigine detection by fluorescence immunochromatography

[0128] The established lamotrigine assay system was used to test lamotrigine standards at concentrations of 2 ng / mL, 8 ng / mL, and 12 ng / mL, with 10 replicates for each assay. The precision (CV) for low, medium, and high lamotrigine concentrations was calculated. Table 4 shows the precision results for high and low lamotrigine concentrations.

[0129] Table 4. Results of repeated testing of 2, 8, and 12 ng / mL lamotrigine standards by fluorescence immunochromatography

[0130]

[0131] 7) Accuracy deviation of fluorescent immunochromatographic assay for lamotrigine

[0132] After diluting the standard (5 mg / mL) to 100 ng / mL with buffer, 10 μL of each standard at different concentrations was added to 90 μL of a low-concentration in-house reference. Testing was performed according to the above test procedures, with each sample repeated five times. Table 5 shows the accuracy results.

[0133] Table 5. Results of repeated testing of lamotrigine standard by fluorescence immunochromatography

[0134]

[0135]

[0136] 8) Cross-reactivity of the lamotrigine fluorescence immunochromatographic assay system

[0137] Three common clinically used drugs were prepared into samples with different gradient concentrations using blank mixed serum, and fluorescent immunochromatographic detection was performed to calculate their IC 50 IC with lamotrigine 50 The cross-reaction rate was calculated by comparing the values. Calculation formula: Cross-reaction rate = (IC 50拉莫三嗪 / IC 50临床用相关药物 )%,cross-reaction rate results are shown in Table 6:

[0138] Table 6. Cross-reaction results of clinically relevant drugs detected by fluorescence immunochromatography

[0139] Clinically related drugs Cross-reactivity rate Sodium valproate ≦0.1％ Topiramate ≦0.1％ Clozapine ≦0.1％ Carbamazepine ≦0.1％ Oxcarbazepine ≦0.1％

[0140] 2. ELISA quantitative detection of lamotrigine

[0141] 1) Establishment of ELISA standard curve

[0142] The prepared lamotrigine coating precursor (lamotrigine-OVA) was diluted to 1-2 μg / mL in carbonate buffer (0.05 M, pH 9.6) and coated onto a 96-well plate at 100 μL / well. The plate was incubated overnight at 4°C, blocked with 5% BSA for 3 hours, and then washed three times, 5 minutes each time. Lamotrigine standards were spiked into blank plasma to prepare nine concentrations: 20, 16, 12, 6, 3, 1.5, 0.75, 0.38, and 0 ng / mL. Each concentration was then diluted 40-fold in 0.01 M PBS. A 50 μL mixture of lamotrigine at various concentrations and a 50 μL lamotrigine antibody was added to the microwells and incubated at 37°C for 1 hour. After three washes, HRP-conjugated secondary antibody was added (100 μL / well) and incubated for 1 hour. After three washes, the plate was washed three times, and the colorimetric solution was added. The plate was reacted in the dark for 15 minutes at room temperature. The plate was then read at 450 nm with a stop buffer. Table 8 shows the absorbance value results of the standard curve of ELISA detection. Figure 4 The equation corresponding to the middle curve is shown in Table 9

[0143] Table 8. ELISA for the detection of different concentrations of lamotrigine

[0144]

[0145]

[0146] Table 9. Equations corresponding to the inhibition curve (four parameters)

[0147]

[0148] 2) Sensitivity of ELISA for lamotrigine detection

[0149] The negative plasma samples were tested 10 times, and the mean (X), standard deviation (SD) and precision (CV) of the ELISA absorbance values ​​were calculated. The sensitivity was calculated as X-2*SD OD value in Figure 4 The corresponding lamotrigine concentration value in the standard curve.

[0150] Table 10. Results of repeated ELISA testing of 0 ng / mL lamotrigine samples

[0151]

[0152]

[0153] Substitute the absorbance value of X-2*SD in the data of Table 9 as the y value Figure 4 The concentration value obtained in the equation corresponding to the standard curve is 0.9 ng / mL, that is, the sensitivity is 0.9 ng / mL.

[0154] 3) Precision and accuracy deviation of lamotrigine detection by ELISA

[0155] Using an established ELISA assay system, lamotrigine standards at concentrations of 2 ng / mL and 12 ng / mL were tested 10 times each. The precision (CV) and accuracy deviation (A / D) of the assays for both high and low lamotrigine concentrations were calculated. The results showed that both the precision and A / D were less than 15% for both high and low concentrations, and less than 15% for both.

[0156] Table 11. Results of repeated ELISA tests of 2 and 12 ng / mL lamotrigine standards

[0157]

[0158] 4) ELISA method to detect cross-reactivity of lamotrigine

[0159] The cross-reaction results were consistent with those of lamotrigine detection by fluorescent immunochromatography.

[0160] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A monoclonal antibody, characterized in that The monoclonal antibody specifically binds to lamotrigine; the monoclonal antibody is produced by a mouse hybridoma cell line; the hybridoma cell line is deposited by the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China) with a deposit number of CCTCC NO: C2022381.

2. A hybridoma cell line, characterized in that The hybridoma cell line is a mouse hybridoma cell line preserved by China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China) with the preservation number of CCTCC NO: C2022381.

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