A matrine hapten, hybridoma cell line, antibody and its application

Abstract

This invention relates to a matrine hapten, a hybridoma cell line, an antibody, and their applications, belonging to the field of food safety immunoassay. The hybridoma cell line BONT, which secretes matrine monoclonal antibodies, has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 18, 2024, with accession number CGMCC No. 45902. The matrine monoclonal antibody secreted by this cell line exhibits good affinity, high specificity, and high sensitivity (IC50). 50 With a concentration of 0.39 ng / mL, it can be used to prepare immunoassay kits for matrine and colloidal gold test strips, establish an immunological detection method for matrine, and provide a powerful detection method for matrine residues in food.

Description

Technical Field

[0001] This invention relates to the field of food safety immunoassay, and in particular to a matrine hapten, a hybridoma cell line, an antibody, and their applications. Background Technology

[0002] Matrine is an alkaloid extracted from the roots, stems, and fruits of the legume *Sophora flavescens* using organic solvents such as ethanol. It belongs to the lupin alkaloid class, a derivative of quinolone alkaloids. Matrine possesses various biological activities, including immunomodulatory, anti-inflammatory, antibacterial, and antitumor effects. It can promote macrophage phagocytosis and enhance T lymphocyte proliferation, thereby improving the body's immunity to fight infection and disease. Furthermore, matrine is a natural plant insecticide, effective against various pests such as cabbage caterpillars, aphids, melon aphids, stem borers, ladybugs, tussock moths, stink bugs on fruit trees, armyworms, wheat midges, and locusts on grain crops. Matrine has contact and stomach poison effects on pests; after application, it paralyzes the central nervous system of pests, leading to suffocation and death. Matrine has a wide target range, inhibits the development of resistance, and remains highly active against already resistant pests. Studies have shown that long-term consumption of food containing residual matrine can cause gastrointestinal discomfort symptoms such as abdominal pain, nausea, and vomiting, as well as dizziness, chest tightness, and difficulty breathing. It may even cause hemolytic reactions and damage liver and kidney function.

[0003] Currently, the main methods for detecting matrine are instrumental detection, with liquid chromatography (LC) and liquid chromatography-tandem mass spectrometry (LC-MS / MS) being the most common. Instrumental detection methods offer high sensitivity and specificity, but they also have drawbacks, such as requiring thorough sample purification, high solvent consumption, expensive equipment, and skilled technicians. Therefore, a rapid and simple method for analyzing matrine residues is needed.

[0004] Enzyme-linked immunosorbent assay (ELISA) is a highly efficient and sensitive detection method. It features simple sample pretreatment, low detection cost, and easy operation, making it suitable for rapid on-site detection of large numbers of samples and widely used in pesticide residue analysis. A prerequisite for ELISA detection is obtaining monoclonal antibodies with high specificity and sensitivity to the target analyte. Therefore, a method for preparing monoclonal antibodies with high specificity and sensitivity to matrine is crucial. The inventors attempted to prepare anti-matrine monoclonal antibodies using hybridoma cells. However, further research is needed on how to prepare haptens and complete antigens, how to induce strong immunity in mice, how to ensure successful secretion of anti-matrine monoclonal antibodies by the prepared hybridoma cell lines, and how to ensure high specificity and sensitivity of the secreted monoclonal antibodies. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a matrine hapten, a hybridoma cell line, an antibody, and their applications.

[0006] This invention is achieved through the following technical solution:

[0007] The first objective of this invention is to provide a matrine hapten, the structural formula of which is:

[0008]

[0009] The second objective of this invention is to provide a method for preparing the matrine hapten, comprising the following steps:

[0010] Matrine hapten was obtained by reacting sophoridine with methyl 4-aminobutyrate hydrochloride in an alkaline solution. The alkaline solution was sodium hydroxide; the reaction was carried out under the condition of stirring at 80°C for 16 hours.

[0011] The second objective of this invention is to provide an artificial matrine antigen, obtained by coupling the matrine hapten with a carrier protein.

[0012] In some embodiments of the present invention, the carrier protein is selected from at least one of chicken ovalbumin, porcine hemocyanin, bovine serum albumin, human serum albumin, lactoferrin, horseradish peroxidase, thyroglobulin, immunoglobulins, and hormones.

[0013] The third objective of this invention is to provide a hybridoma cell line that secretes a matrine monoclonal antibody, characterized in that the hybridoma cell line has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, classified as a monoclonal cell line, with a deposit date of April 18, 2024, and accession number CGMCC No. 45902.

[0014] The fourth objective of this invention is to provide a matrine monoclonal antibody secreted by the hybridoma cell line described above.

[0015] A fifth objective of this invention is to provide a composition comprising the hybridoma cell line and / or the matrine monoclonal antibody.

[0016] The sixth objective of this invention is to provide a matrine immunoassay kit, wherein the matrine immunoassay kit contains the hybridoma cell line or the matrine monoclonal antibody.

[0017] In some embodiments of the present invention, the matrine immunoassay kit further comprises matrine conjugated with a signaling substance or the matrine hapten.

[0018] In some embodiments of the present invention, the matrine immunoassay kit further comprises an enzyme-labeled plate, matrine-coated antigen, matrine standard solution, enzyme-labeled secondary antibody, and substrate reaction solution.

[0019] In some embodiments of the present invention, the matrine-coated antigen has the following structure:

[0020]

[0021] A seventh objective of this invention is to provide a matrine detection colloidal gold test strip, wherein the colloidal gold test strip contains at least one of the aforementioned hybridoma cell line, the aforementioned matrine monoclonal antibody, and the aforementioned composition.

[0022] In some embodiments of the present invention, the matrine detection colloidal gold test strip includes a sample pad, a colloidal gold conjugating pad, a nitrocellulose membrane, and an absorbent pad. A detection line and a control line are sequentially arranged on the nitrocellulose membrane. The colloidal gold conjugating pad is coated with the matrine monoclonal antibody. The detection line is printed from a matrine-coated antigen. The matrine-coated antigen has the following structure:

[0023]

[0024] The eighth objective of this invention is to provide the application of the hybridoma cell line, the matrine monoclonal antibody, the composition, the matrine immunoassay kit, or the matrine detection colloidal gold test strip in the detection of matrine.

[0025] The present invention provides a method for preparing a hybridoma cell line that secretes matrine monoclonal antibodies, comprising the following steps:

[0026] (1) Design and prepare matrine hapten;

[0027] (2) Prepare matrine complete antigen, and prepare Freund's adjuvant and incomplete Freund's adjuvant with the obtained matrine complete antigen;

[0028] (3) The Freund's adjuvant was injected into BALB / c mice via subcutaneous injection on the back for multiple immunizations. The first immunization used complete Freund's adjuvant, and the booster immunization used incomplete Freund's adjuvant.

[0029] (4) Blood was collected from mice that had undergone the above immunization process. The serum immune titer and immunosuppressive ability of the mice were detected by indirect ELISA. Mice with high levels of matrine antibody in their serum were screened to obtain immunity.

[0030] (5) The selected mice were given a final booster immunization with incomplete Freund's adjuvant, and then sprint immunization was performed by intraperitoneal injection. The sprint immunization was performed using matrine complete antigen without Freund's adjuvant.

[0031] (6) The spleen cells and myeloma cells of BALB / c mice after sprint immunization were fused. The fused cells were cultured in a culture medium. Positive cell pores were detected by indirect ELISA. The inhibitory effect of positive cell pores was further determined by indirect competitive ELISA. The positive cell pores with the best inhibition were subcloned by limiting dilution method. Finally, hybridoma cell lines that can secrete matrine monoclonal antibodies were screened out.

[0032] In the above preparation method of the present invention, the interval between the first immunization and the booster immunization in steps (3) and (5) is one month, the interval between booster immunizations is 21 days, and the interval between booster immunization and sprint immunization is 18 to 21 days.

[0033] In the above preparation method of the present invention, the initial immunization dose in steps (3) and (5) is 100 μg / animal, the booster immunization dose is 50 μg / animal, and the sprint immunization dose is 25 μg / animal.

[0034] In the above preparation method of the present invention, the immunization process in steps 3 and 5 includes one initial immunization, four booster immunizations and one sprint immunization.

[0035] In the above preparation method of the present invention, the blood collection in step 4 is performed on the 7th day after the end of the 3rd immunization process.

[0036] In the above preparation method of the present invention, the cell fusion in step 6 is performed 3 days after the end of the sprint immunization.

[0037] In the above preparation method of the present invention, the cell fusion in step 6 is performed by the polyethylene glycol (PEG4000) method.

[0038] In the above preparation method of the present invention, the culture medium in step 6 is RPMI-1640 culture medium.

[0039] In the preparation method of the present invention, the number of subcloning operations in step 6 is 3.

[0040] The beneficial effects of this invention are as follows:

[0041] This invention provides a matrine hapten, a hybridoma cell line, an antibody, and their applications. The invention provides a hybridoma cell line 4F12 that secretes matrine monoclonal antibodies. The matrine monoclonal antibodies secreted by this hybridoma cell line have good affinity, high specificity, and high sensitivity (IC50).50 With a concentration of 0.39 ng / mL, it can be used to prepare immunoassay kits for matrine and colloidal gold test strips, establish an immunological detection method for matrine, and provide a powerful detection method for matrine residues in food.

[0042] Preservation of biological materials:

[0043] A hybridoma cell line, BONT, that secretes a monoclonal antibody against matrine has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. It has been classified as a monoclonal cell line, deposited on April 18, 2024, with accession number CGMCC No. 45902. Attached Figure Description

[0044] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0045] Figure 1 This is the standard inhibition curve of the matrine monoclonal antibody in this invention. Detailed Implementation

[0046] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0047] The culture media involved in the embodiments of the present invention are as follows:

[0048] RPMI-1640 medium (mg / L): L-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cysteine ​​dihydrochloride 65.15, L-glutamic acid 20, glycine 10, L-histidine 15, L-hydroxyproline 20, L-isoleucine 50, L-leucine 50, L-lysine hydrochloride 40, L-methionine 15, L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5, L-tyrosine 23. 19. L-Valine 20. Para-aminobenzoic acid 1. Calcium nitrate 100. Anhydrous magnesium sulfate 48.84. Anhydrous sodium dihydrogen phosphate 676.13. Potassium chloride 400. Sodium chloride 6000. Glucose 2000. Reduced glutathione 1. Phenol red 5. L-glutamine 300. Biotin 0.2. D-calcium pantothenate 0.25. Folic acid 1. I-inositol 35. Nicotinamide 1. Choline chloride 3. Pyridoxine hydrochloride 1. Riboflavin 0.2. Thiamine hydrochloride 1. Vitamin B12 0.005. Sodium bicarbonate 2000.

[0049] The reagents involved in the following examples are as follows:

[0050] Carbonate buffer (CBS): Weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them separately in a small amount of double-distilled water and mix them together. Add double-distilled water to about 800 mL and mix well. Adjust the pH to 9.6 and add double-distilled water to a final volume of 1000 mL. Store at 4°C for later use.

[0051] Phosphate-buffered saline (PBS): 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4, 2.9g Na2HPO4·12H2O, dissolved in 800mL pure water, pH adjusted to 7.2-7.4 with NaOH or HCl, and then brought to a final volume of 1000mL.

[0052] PBST: PBS containing 0.05% Tween 20;

[0053] Antibody dilution buffer: PBS containing 0.1% gelatin;

[0054] TMB colorimetric solution: Solution A: Na₂HPO₄ 4. 12H₂O 18.43g, citric acid 9.33g, diluted to 1000mL with pure water; Solution B: 60mg TMB dissolved in 100mL ethylene glycol. Mix solutions A and B in a 5:1 ratio to obtain the TMB colorimetric solution, mix fresh before use.

[0055] The detection methods involved in the following embodiments are as follows:

[0056] Method for detecting matrine inhibition rate: The optimal antigen and antibody concentrations for ic-ELISA were selected using a checkerboard assay. The antigen was diluted to 0.01 μg / mL, 0.03 μg / mL, 0.1 μg / mL, and 0.3 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 0.01 μg / mL, 0.03 μg / mL, 0.1 μg / mL, and 0.3 μg / mL with antibody dilution buffer. After selecting the optimal operating point, matrine standards were diluted to eight concentrations (0 ng / mL, 0.019 ng / mL, 0.056 ng / mL, 0.167 ng / mL, 0.5 ng / mL, 1.5 ng / mL, 4.5 ng / mL, and 13.5 ng / mL). Following the ic-ELISA procedure, the results were plotted using Origin 2021 (see results shown). Figure 1 As shown), the standard inhibition curve of matrine was obtained, and the IC50 was calculated. 50 .

[0057] Example 1: Preparation of matrine hapten.

[0058] Since small molecules are not immunogenic and cannot stimulate mice to produce an immune response and thus produce antibodies, they need to be conjugated to proteins using protein conjugation technology to acquire immunogenicity. Commonly used active groups in protein conjugation technology include amino, carboxyl, hydroxyl, and thiol groups. In order to obtain a monoclonal antibody that can specifically recognize matrine, it is necessary to design and derive a good hapten. The specific derivation steps are as follows.

[0059]

[0060] Sophoridine (500.0 mg, 2.0 mmol) and methyl 4-aminobutyrate hydrochloride (467.0 mg, 3.0 mmol) were weighed and dissolved in 10 mL of water. Then, 5 mL of NaOH (162.0 mg, 4.1 mmol) aqueous solution was added dropwise to the mixture, and the mixture was stirred at 80 °C for 16 h. After the reaction was completed, the mixture was concentrated under vacuum and purified by high performance liquid chromatography to obtain a white solid compound (matrine hapten).

[0061] Example 2: Synthesis of matrine complete antigen.

[0062] Weigh 4.70 mg of hapten and 4.63 mg of N-hydroxysuccinimide (NHS), dissolve them in 300 μL of N,N-dimethylformamide (DMF), and stir at room temperature for 10 min. Then weigh 7.69 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), dissolve it thoroughly in 100 μL of DMF, and add it to the hapten solution. Stir at room temperature for 6-8 h (referred to as solution A). Take 10 mg of BSA, dilute it to 5 mg / mL with 0.01 M phosphate-buffered saline (PBS) (referred to as solution B), and slowly add solution A dropwise to solution B. React at room temperature overnight. Then dialyze with 0.01 M PBS to remove unreacted small molecule hapten to obtain the complete antigen (as shown in the formula below), and identify it by ultraviolet absorption scanning.

[0063]

[0064] Example 3: Synthesis of matrine-coated antigen.

[0065] The specific steps for synthesizing the coating antigen are as follows: 2.33 mg of hapten and 2.30 mg of N-hydroxysuccinimide (NHS) were dissolved in 300 μL of anhydrous N,N-dimethylformamide (DMF), and the mixture was stirred at room temperature for 10 min to obtain a hapten solution; 3.81 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was dissolved in 100 μL of anhydrous DMF and added to the hapten solution, and the mixture was stirred at room temperature for 6-8 h to obtain solution A; 10 mg of chicken ovalbumin (OVA) was diluted with 2 mL of 0.01 mol / L phosphate buffer (PBS) to obtain solution B; solution A was slowly added dropwise to solution B to obtain a reaction solution; the reaction solution was dialyzed with PBS to remove unreacted small molecule hapten to obtain the coating antigen (as shown in the following formula).

[0066]

[0067] Example 4: Preparation of hybridoma cell lines secreting matrine monoclonal antibodies.

[0068] 1. Acquisition of immunity in animals

[0069] The complete antigen was emulsified with an equal volume of Freund's adjuvant and then administered to BALB / c mice via subcutaneous injection at multiple sites on the back of the neck (except for sprint immunization). The initial immunization used complete Freund's adjuvant at a dose of 100 μg / mouse. For subsequent booster immunizations, incomplete Freund's adjuvant was used at half the dose (50 μg / mouse). For sprint immunizations, no adjuvant was used; the adjuvant was diluted directly with physiological saline and injected intraperitoneally at a dose halved again (25 μg / mouse). The interval between the initial and second booster immunizations was one month, the interval between multiple booster immunizations was 21 days, and the interval between sprint immunizations and the final booster immunization was 18-21 days. The immunization effect in mice was observed using an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), which detected the serum titer and inhibition.

[0070] 2. Cell fusion

[0071] Three days after the sprint immunization, cell fusion was performed using the standard PEG (polyethylene glycol, molecular weight 4000) method, with the following specific steps:

[0072] a. After euthanizing the mouse by tail dislocation and cervical dislocation, immediately disinfect the mouse in 75% alcohol for about 5 minutes. Under aseptic conditions, remove the spleen and grind it moderately with the rubber tip of a syringe and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect the suspension, centrifuge (1200 rpm, 8 minutes), wash the spleen cells three times with RPMI-1640 medium, and after the last centrifugation, dilute the spleen cells to a certain volume, count them, and set them aside for later use.

[0073] b. Collection of SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells are cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. The number of SP2 / 0 tumor cells should reach 1-4 × 10⁶ cells / year before fusion. 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion, tumor cells are collected and suspended in RPMI-1640 basal culture medium for cell counting during fusion.

[0074] c. Fusion process (7 min): Min 1, add 1 mL of PEG to the cells dropwise from slow to fast; Min 2, let stand; Min 3 and Min 4, add 1 mL of RPMI-1640 medium dropwise over 1 min; Min 5 and Min 6, add 2 mL of RPMI-1640 medium dropwise over 1 min; Min 7, add 1 mL of RPMI-1640 medium dropwise every 10 s; then incubate at 37°C for 5 min; centrifuge (800 rpm, 8 min), discard the supernatant, resuspend in RPMI-1640 selection medium containing 20% ​​fetal bovine serum and 2% 50×HAT, add 200 μL / well to a 96-well cell plate, and incubate at 37°C in a 5% CO2 incubator.

[0075] 3. Cell selection and cell line establishment

[0076] On day 3 of cell fusion, the fused cells were partially replaced with RPMI-1640 screening medium. On day 5, the medium was completely replaced with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 1% 100×HT. On day 7, the cell supernatant was collected for screening.

[0077] The screening process consists of two steps: First, positive cell wells are selected using ic-ELISA. Second, matrine is used as a standard, and the inhibitory effect on positive cells is determined using ic-ELISA.

[0078] Cell wells that showed good inhibition of matrine standard were selected, and subcloning was performed using the limiting dilution method. The same method was used for detection seven days later.

[0079] Three subclonings were performed using the method described above, ultimately yielding a cell line capable of secreting monoclonal antibodies that recognize matrine.

[0080] Example 5: Preparation and identification of monoclonal antibodies.

[0081] 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil; 7 days later, each mouse was injected intraperitoneally with 1×10 6 Hybridoma cells were used, and ascites fluid was collected starting from day seven. The ascites fluid was then purified for antibody treatment using the caprylic acid-saturated ammonium sulfate method.

[0082] Under slightly acidic conditions, octanoic acid can precipitate other proteins in the ascites fluid besides IgG immunoglobulin. After centrifugation, the precipitate is discarded. Then, an equal volume of saturated ammonium sulfate solution is used to precipitate IgG-type monoclonal antibodies. After centrifugation, the supernatant is discarded. The antibodies are dissolved in 0.01M PBS solution (pH 7.4), dialyzed to desalt, and finally purified monoclonal antibodies are obtained and stored at -20℃.

[0083] The IC50 of the monoclonal antibody against matrine was determined using an indirect competitive ELISA. 50 The concentration was 0.39 ng / mL, and its cross-reactivity with other analogues was verified, as shown in Table 1. This indicates that it has good sensitivity to matrine and can be used for the immunoassay detection of matrine.

[0084] Table 1. Determination of cross-reactivity of matrine monoclonal antibody 4F12

[0085]

[0086] Example 6: Application of matrine monoclonal antibody.

[0087] The monoclonal antibody prepared from hybridoma cell line 4F12 through in vivo ascites induction was applied to the ELISA assay for matrine. The specific steps are as follows:

[0088] (1) Coat a 96-well microplate with 0.1 μg / mL of the coating stock diluted with carbonate buffer (CBS), 100 μL per well, and coat at 37℃ for 2 h. Then wash the plate three times with PBST washing buffer, 200 μL per well each time, for 3 min each time, and pat dry.

[0089] (2) Block with CBS containing 0.2% gelatin, 200 μL per well, block at 37°C for 2 h, wash the plate three times with PBST washing solution, 200 μL per well each time, 3 min each time, and pat dry;

[0090] (3) Prepare a series of standard solutions using phosphate buffer (PBS). Add 50 μL of the standard solutions to each well of the sealed microplate, and perform three replicates. Then add 50 μL of monoclonal antibody diluted to 0.03 μg / mL to each well. Incubate at 37°C for 0.5 h, then wash and dry the plate.

[0091] (4) Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:3000 with PBS containing 0.1% gelatin to each well, react at 37°C for 0.5 h, then wash and pat dry.

[0092] (5) Add 100 μL of TMB colorimetric solution to each well, develop the color at 37℃ for 15 min, then add 50 μL of 2 M H2SO4 stop solution to each well, and measure the absorbance at 450 nm.

[0093] Using Origin 2021 to plot the standard inhibition curve of matrine (results are shown below). Figure 1 As shown), the IC50 of matrine monoclonal antibody against matrine. 50 The concentration was 0.39 ng / mL, indicating good sensitivity to matrine, and it can be used for the immunoassay of matrine.

[0094] Example 7: Matrine Immunoassay Kit.

[0095] This embodiment provides a matrine immunoassay kit, which includes matrine monoclonal antibody 4F12 prepared in Example 5, an enzyme-linked immunosorbent assay (ELISA) plate, matrine-coated antigen, matrine standard solution, HRP-labeled goat anti-mouse IgG secondary antibody, and TMB chromogenic solution.

[0096] The principle of the immunoassay kit for matrine detection is as follows: The content of matrine in the sample is detected using an indirect competitive ELISA method. The microwells of the ELISA plate are pre-coated with matrine-coated antigen. Matrine standard solution or the sample to be tested, matrine monoclonal antibody, HRP-labeled goat anti-mouse IgG secondary antibody, and TMB chromogenic solution are added to construct a standard inhibition curve for matrine. The content of matrine in the sample is determined based on the standard inhibition curve and the absorbance value of the sample. The detection of matrine residues can be achieved using methods commonly used in this field.

[0097] Example 8: Matrine detection test strip for colloidal gold.

[0098] This embodiment provides a colloidal gold test strip, comprising a sample pad, a colloidal gold conjugate pad, a nitrocellulose membrane, and an absorbent pad. A detection line and a control line are sequentially arranged on the nitrocellulose membrane. The colloidal gold conjugate pad is coated with matrine monoclonal antibody prepared in Example 5. The detection line is printed from matrine-coated antigen. The control line is printed from goat anti-mouse IgG secondary antibody. The assembly method of the colloidal gold test strip can be any method commonly used in the art.

[0099] The principle of colloidal gold test strips for detecting matrine is as follows: It utilizes the indirect competition method to detect the presence of matrine in the sample. If the sample contains matrine, the test line will not develop color, while the control line will. If the sample does not contain matrine, both the test line and the control line will develop color. The detection of matrine can be achieved using methods commonly used in this field.

[0100] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A matrine hapten, characterized in that, The structural formula of the matrine hapten is: 。 2. The method of preparing a hapten of matrine according to claim 1, characterized in that, The method comprises the following steps: The matrine hapten is obtained by mixing sophoridine with 4-aminobutyric acid methyl ester hydrochloride in an alkaline solution.

3. A matrine artificial antigen, characterized in that, The matrine hapten of claim 1 is coupled with a carrier protein to obtain.

4. The matrine artificial antigen according to claim 3, characterized in that, The carrier protein is at least one selected from the group consisting of keyhole limpet hemocyanin, conalbumin, bovine serum albumin, human serum albumin, lactoferrin, horseradish peroxidase, thyroglobulin, immunoglobulin and hormone.

5. A hybridoma cell line secreting a monoclonal antibody to matrine, characterized in that, The hybridoma cell strain of claim 5 is secreted to obtain.

6. A matrine monoclonal antibody, characterized in that, The composition contains the hybridoma cell strain of claim 5 and / or the matrine monoclonal antibody of claim 6.

7. A composition characterized in that, The matrine immunological detection kit contains the hybridoma cell strain of claim 5 or the matrine monoclonal antibody of claim 6.

8. A matrine immunodetection kit, characterized in that, The colloidal gold test strip contains at least one of the hybridoma cell strain of claim 5, the matrine monoclonal antibody of claim 6 and the composition of claim 7.

9. A colloidal gold test strip for detecting matrine, characterized in that, 10. The hybridoma cell strain of claim 5, the matrine monoclonal antibody of claim 6, the composition of claim 7, the matrine immunological detection kit of claim 8 or the matrine colloidal gold test strip of claim 9 are used for detecting matrine; the application does not involve the diagnosis and treatment of diseases. ​

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