A trichosanthin monoclonal antibody, its preparation method and application

Abstract

The application belongs to the technical field of food detection, and provides a hybridoma cell strain secreting a trichosanthin monoclonal antibody, named 1B9, and having a preservation number of CCTCC NO: C2026136. The application provides a trichosanthin monoclonal antibody. The application provides a preparation method of the trichosanthin monoclonal antibody, including the following steps: S1. animal immunization; S2. cell fusion and culture; S3. positive clone strain screening; S4. ascites preparation; and S5. purification to obtain the trichosanthin monoclonal antibody. The application provides a trichosanthin immunochromatographic rapid detection test strip and a preparation method thereof. The application provides a trichosanthin rapid detection kit. The mouse immunization in the application adopts a nano-manganese adjuvant, and the antibody produced by the mouse immunized by the nano-manganese adjuvant has higher titer and higher sensitivity than the antibody produced by the mouse immunized by a traditional adjuvant. The test strip provided in the application is convenient to detect, has low detection cost, does not need to be operated by professional personnel, and is suitable for rapid detection on site.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, specifically relating to a triptolide monoclonal antibody, its preparation method, and its application. Background Technology

[0002] In China, there are three main species of Tripterygium wilfordii: Tripterygium wilfordii, *Malus kunmingensis*, and *Tripterygium truncatum*. The chemical composition of Tripterygium wilfordii is quite complex. To date, more than 300 compounds have been extracted from it, mainly sesquiterpenes, diterpenes, triterpenes, and alkaloids, of which more than 100 have been proven to have biological activity. Currently, the most studied compounds are triptolide, triptolide A, triptolide A, triptolide ketone, and triptolide alkaloids, which are the main active ingredients in Tripterygium wilfordii. Among these, triptolide, triptolide A, and triptolide alkaloids are the main toxic components.

[0003] There is currently no standard detection method for triptolide. The methods reported in the literature are mainly liquid chromatography-mass spectrometry (LC-MS / MS). This method relies on large instruments, is complex to operate, requires specialized technicians, has a long detection time, and is costly.

[0004] Immunoassay, based on the principle of antigen-antibody specific binding, is convenient, rapid, and low-cost, making it an ideal technical approach to address the aforementioned needs. Chinese patent CN107698653A discloses a triptolide hapten and its preparation method and application, revealing the molecular structural formula of the triptolide hapten. This invention also discloses the synthesis of a complete antigen using the triptolide hapten, the preparation of polyclonal antibodies, and the development of a detection kit. The triptolide polyclonal antibody and its kit can be used for the detection and analysis of highly toxic substances such as triptolide and triptolide diterpenes in toxic honey, traditional Chinese medicine, and biological matrices. However, in-depth research has revealed the following defects in this patent: First, the antibody's specificity is severely insufficient, leading to poor detection accuracy. The patented preparation of polyclonal antibodies clearly shows in its example data that the polyclonal antiserum exhibits a cross-reactivity rate as high as 270% against triptolide, a key structural analog of triptolide. This means that in actual, complex sample testing, strong false-positive signals are easily generated due to interference from analogs, leading to misjudgment.

[0005] Secondly, it lacks true on-site rapid testing capabilities and has a slow response time. This patented method uses traditional enzyme-linked immunosorbent assay (ELISA), which requires large-scale equipment and the complete process takes more than 2 hours.

[0006] Finally, there is poor batch-to-batch consistency. This patent uses traditional Freund's adjuvant immunization and polyclonal antiserum collection, which cannot meet the requirements of diagnostic reagents for standardization and reproducibility of core raw materials.

[0007] Therefore, existing technologies urgently need to be addressed. Summary of the Invention

[0008] This invention addresses the shortcomings of existing technologies by providing a triptolide monoclonal antibody, its preparation method, and its applications. This invention utilizes monoclonal antibody technology to prepare a highly sensitive and specific triptolide antibody. Based on this antibody, rapid immunochromatographic test strips and kits have been developed, enabling rapid on-site detection of triptolide and effectively solving the problems of complex operation, long processing time, and insufficient antibody specificity and sensitivity in existing technologies.

[0009] To address the above problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a hybridoma cell line that secretes a monoclonal antibody against triptolide, named hybridoma cell line 1B9, with accession number CCTCC NO: C2026136.

[0010] Secondly, the present invention provides a triptolide monoclonal antibody, which is secreted by the hybridoma cell line 1B9 as described above.

[0011] Thirdly, the present invention provides a method for preparing triptolide monoclonal antibody, comprising the following steps: S1. Animal immunization: Mice were immunized by subcutaneous injection of triptolide complete antigen in the abdomen or foot after being mixed with nano-manganese adjuvant; triptolide complete antigen is a conjugate of triptolide hapten and carrier protein. S2. Cell fusion and culture: Spleen cells from immunized mice were fused with mouse myeloma cells SP2 / 0 in the presence of polyethylene glycol. The fused cells were then cultured in a semi-solid culture medium to form cell clones. S3. Screening of positive clones: The triptolide detection antigen was coated; the cells were blocked with blocking solution; the supernatant of the cell culture to be tested was added as the primary antibody and incubated; the enzyme-labeled secondary antibody was added and incubated; the chromogenic solution was added for chromogenic reaction; the reaction was terminated with stop solution; the signal value was detected; and positive hybridoma cell lines were screened out; the positive hybridoma cell line is the hybridoma cell line 1B9 as described in claim 1. S4. Ascites preparation: Positive hybridoma cell lines were inoculated into the peritoneal cavity of mice, ascites was collected, and after centrifugation, the supernatant containing triptolide monoclonal antibody was obtained. S5. Purification: The supernatant of the triptolide monoclonal antibody was subjected to affinity chromatography, and the elution fraction was collected to obtain the purified triptolide monoclonal antibody.

[0012] In the preparation method of triptolide monoclonal antibody as described above, in step S1, the immunogen is diluted with PBS buffer or physiological saline and then mixed with an equal volume of nano-manganese adjuvant. Preferably, the immunogen is diluted with PBS buffer or physiological saline to 1-3 mg / mL. Most preferably, the immunogen is diluted with PBS or physiological saline to 2 mg / mL.

[0013] In the preparation method of triptolide monoclonal antibody as described above, step S5, the specific purification includes the following steps: (1) Pretreatment of ascites samples: Dilute the supernatant of thawed ascites with coupling buffer, centrifuge, and filter; (2) Equilibration: Equilibrate the purification column with coupling buffer; (3) Sample loading: Add the pretreated ascites sample to the purification column; (4) Washing: Pass the purified column through a coupling buffer; (5) Elution: Elute with elution buffer and collect the antibody collection solution; (6) Dialysis: Dialyze the antibody collection solution with PBS buffer, change the solution, and obtain purified triptolide monoclonal antibody.

[0014] Based on the same inventive concept, this invention provides the application of the triptolide monoclonal antibody as described above or the triptolide monoclonal antibody prepared by the preparation method as described above in the preparation of triptolide detection products.

[0015] Based on the same inventive concept, the present invention provides a rapid immunochromatographic test strip for triptolide, wherein the test strip contains triptolide monoclonal antibody as described above or triptolide monoclonal antibody prepared by the preparation method described above.

[0016] The marker in the triptolide immunochromatographic rapid test strip described above is any one of colloidal gold, carboxylated colored microspheres, time-resolved fluorescent microspheres, or fluorescent quantum dot microspheres.

[0017] Based on the same inventive concept, this invention provides a method for preparing the triptolide immunochromatographic rapid test strip as described above, comprising the following steps: attaching a nitrocellulose membrane to a PVC board; spraying goat anti-mouse IgG as a control line and triptolide antigen as a detection line onto the nitrocellulose membrane; spraying a marker containing triptolide monoclonal antibody onto the conjugate pad; sequentially attaching absorbent paper, the conjugate pad, and the sample pad to the PVC board to assemble the test strip. The triptolide antigen is TPL-BSA.

[0018] Based on the same inventive concept, the present invention provides a rapid detection kit for triptolide, comprising triptolide monoclonal antibody as described above or triptolide monoclonal antibody prepared by the preparation method described above.

[0019] Based on the same inventive concept, this invention provides a method for detecting triptolide in a sample, using a triptolide monoclonal antibody as described above or a triptolide monoclonal antibody prepared by the method described above as an antibody to detect the content of triptolide in the sample to be tested. The sample can be one or more of honey, traditional Chinese medicine, or a biological matrix.

[0020] Compared with existing technologies, the effects and advantages of this invention are: 1. The mouse immunization method of this invention uses nano-manganese adjuvant, which, compared with the traditional Freund's adjuvant, can produce a more potent and sensitive triptolide monoclonal antibody. The IC50 of the antibody group with nano-manganese adjuvant is as low as 1.2 ng / mL, while the IC50 of the group with Freund's adjuvant is as high as 4.4 ng / mL or more, with a sensitivity improvement of more than 3 times, providing a high-quality core raw material for the subsequent development of detection products.

[0021] 2. This invention provides a rapid immunochromatographic test strip for triptolide, which, based on the aforementioned high-sensitivity monoclonal antibody, enables rapid on-site detection of triptolide. This test strip requires no professional personnel or large-scale equipment, has a short detection time, and is low-cost, making it suitable for rapid on-site screening of samples such as honey and traditional Chinese medicine. Actual sample testing has verified that for honey samples, the limit of detection (LOD) of the test strip is 10 ng / g, the LOD of the kit is 1.5 ng / g, and the LOD of LC-MS / MS is 3 ng / g, showing good correlation with the LC-MS / MS detection results. Attached Figure Description

[0022] Figure 1 The standard curve for the triptolide detection kit provided in Example 4 of this invention. Detailed Implementation

[0023] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this specification includes any and all combinations of one or more of the associated listed items.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0026] The hybridoma cell line 1B9 described in this invention was deposited at the China Center for Type Culture Collection (CCTCC) on June 25, 2026, with accession number CCTCC NO: C2026136, and the deposit address is Wuhan University, Wuhan, China.

[0027] Example 1: Synthesis of Tripterygium wilfordii (TPL) antigen Weigh 2 mg of triptolide and 4 mg of succinic anhydride and dissolve them in 0.5 mL of anhydrous pyridine. Stir at 50 °C for 16 h. After drying the solvent with nitrogen, dissolve it in 0.5 mL of N,N-dimethylformamide (DMF). Add 4 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and stir at room temperature for 0.5 h. Then add 4 mg of N-hydroxysuccinimide (NHS) and stir at room temperature for 2 h. This is solution A.

[0028] Weigh 10 mg of keyhole hemocyanin (KLH) and dissolve it in 2 mL of phosphate-buffered saline (PBS, 0.01 M, pH 7.4). This is solution B.

[0029] Solution A was added dropwise to solution B, and the reaction was carried out at 25°C for 24 hours. After the reaction, the mixture was dialyzed for 6 hours with PBS (0.01M, pH 7.4) using MD25 dialysis bags (8000-14000D). The dialysate was changed 3 times during the dialysis. The reaction solution was collected after dialysis; this was the triptolide immunogen TPL-KLH. The MD25 dialysis bags (8000-14000D) were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0030] Using the same method, bovine serum albumin (BSA) was used instead of KLH to prepare the TPL-BSA detection antigen.

[0031] Example 2: Preparation of Tripterygium wilfordii monoclonal antibody 2.1 Immunization of BALB / c mice with TPL-KLH In comparison, traditional Freund's adjuvant and nano-manganese adjuvant were used, with each adjuvant immunizing 5 mice. The immunization regimens are as follows: 2.1.1 Freund's adjuvant immunization regimen (1) For the first immunization, 1 mg of Tripterygium wilfordii antigen TPL-KLH was diluted to 2 mg / mL with PBS (0.01 M, pH 7.4). 250 μL of diluted antigen was added to an equal volume of Freund's complete adjuvant, and after vigorous shaking to emulsify, it was injected subcutaneously into the abdomen. A total of 5 mice were injected, with 100 μL injected into each mouse.

[0032] (2) Fourteen days after the first immunization, a second immunization was performed, with 0.5 mg of Tripterygium wilfordii antigen TPL-KLH diluted to 1 mg / mL with physiological saline. 250 μL of diluted antigen was added to an equal volume of Freund's incomplete adjuvant, and after vigorous shaking to emulsify, it was injected subcutaneously into the abdomen. A total of 5 mice were injected, with 100 μL injected into each mouse.

[0033] (3) Fourteen days after the second immunization, a third immunization was performed, with 0.5 mg of Tripterygium wilfordii antigen TPL-KLH diluted to 1 mg / mL with physiological saline. 250 μL of diluted antigen was added to an equal volume of Freund's incomplete adjuvant, and after vigorous shaking to emulsify, it was injected subcutaneously into the abdomen. A total of 5 mice were injected, with 100 μL injected into each mouse.

[0034] (4) Seven days after the third immunization, blood was collected from the eyes of each mouse to test serum titer and binding ability to triptolide standard.

[0035] (5) Sprint immunization: Mice with high serum titers and strong binding ability to triptolide were selected for sprint immunization. Tripterygium antigen TPL-KLH was diluted with physiological saline to 0.5 mg / mL and injected intraperitoneally, with 100 μL injected into each mouse.

[0036] 2.1.2 Nano-manganese adjuvant immunization regimen (1) For the first immunization, 1 mg of Tripterygium wilfordii antigen TPL-KLH was diluted to 2 mg / mL with PBS (0.01 M, pH 7.4). 250 μL of diluted antigen was added to an equal volume of nano-manganese adjuvant, mixed evenly, and then injected subcutaneously into the abdomen of 5 mice, 100 μL per mouse.

[0037] (2) Fourteen days after the first immunization, a second immunization was performed, with 0.5 mg of Tripterygium wilfordii antigen TPL-KLH diluted to 1 mg / mL with physiological saline. 250 μL of diluted antigen was added to an equal volume of nano-manganese adjuvant, mixed thoroughly, and injected subcutaneously into the abdomen. A total of 5 mice were injected, with 100 μL injected into each mouse.

[0038] (3) Fourteen days after the second immunization, a third immunization was performed, with 0.5 mg of Tripterygium wilfordii antigen TPL-KLH diluted to 1 mg / mL with physiological saline. 250 μL of diluted antigen was added to an equal volume of nano-manganese adjuvant, mixed evenly, and injected subcutaneously into the abdomen. A total of 5 mice were injected, with 100 μL injected into each mouse.

[0039] (4) Seven days after the third immunization, blood was collected from the eyes of each mouse to test serum titer and binding ability to triptolide standard.

[0040] (5) Sprint immunization: Mice with high serum titers and strong binding ability to triptolide were selected for sprint immunization. Tripterygium antigen TPL-KLH was diluted with physiological saline to 0.5 mg / mL and injected intraperitoneally, with 100 μL injected into each mouse.

[0041] 2.2 Cell Fusion Seven days after the sprint immunization, the spleens of mice were harvested for cell fusion.

[0042] (1) Take one mouse myeloma cell line (hereinafter referred to as "SP2 / 0"), revive and passage it, count the cells and passage it proportionally for 3 days.

[0043] (2) SP2 / 0 cell preparation: Gently pipette the well-conditioned SP2 / 0 cells off the culture dish and transfer them to a 50mL centrifuge tube.

[0044] (3) Preparation of mouse thymus feeder layer: Take the thymus of the mouse and crush it, transfer it to a 15mL centrifuge tube, and add 1mL of HAT medium for later use. HAT supplement (Hybri-Max™ HAT medium supplement (50×), Sigma-Aldrich, catalog number H0262).

[0045] (4) Pour a small amount of serum-free DMEM medium (purchased from Sangon Biotech (Shanghai) Co., Ltd., E600003, high glucose) into a petri dish, and place the cell sieve and syringe core in the petri dish. After euthanizing the mice by removing their eyeballs and collecting blood, immerse them in 75% alcohol for 5 min. Under sterile conditions, remove the mouse spleen, place it on the cell sieve, and gently crush the spleen thoroughly with the syringe core. Then, aspirate the crushed cells into a centrifuge tube containing SP2 / 0 cells. The mass ratio of spleen cells to SP2 / 0 cells is 3:1. Centrifuge at 1200 rpm for 5 min at room temperature.

[0046] (5) Discard the supernatant after centrifugation, gently mix the cells with serum-free DMEM medium, and centrifuge again at 1200 rpm for 5 min at room temperature.

[0047] (6) Discard the supernatant after centrifugation, gently tap the bottom of the centrifuge tube to fully suspend the cells, then place the centrifuge tube in a 37°C water bath and slowly add 1 mL of polyethylene glycol (PEG4000) within 1 min. After standing for 1 min, slowly add 2 mL of serum-free DMEM medium within 2 min, and then slowly add 8 mL of serum-free DMEM medium within 2 min. Centrifuge at 1000 rpm for 5 min at room temperature.

[0048] (7) Discard the supernatant, add 10 mL of fetal bovine serum, and carefully mix the cells. Add the prepared thymocytes, then add 25 mL of sterile semi-solid culture medium (MC). Mix thoroughly and pour into 20 cell culture dishes, 2 mL per dish, and place in a humidified box. Incubate at 37°C in 5% CO2. The semi-solid culture medium (MC) is IMDM medium containing 2% sodium carboxymethyl cellulose by mass. IMDM medium was purchased from Sigma-Aldrich, catalog number I7633.

[0049] (8) Around day 10, begin selecting clones. At this point, the cell clusters are visible to the naked eye and can be clearly distinguished under a microscope. Select 10 × 92 cell clones and culture them in 96-well cell culture plates (pre-coated with 25% HT medium containing mouse thymocytes, 50 μL / well). Change the medium once on day 2. By day 4, the cells have filled the 96-well plate. At this point, use indirect ELISA to detect antibodies in the supernatant of the culture medium. HT supplement (Hybri-Max™ HT medium supplement (50×), Sigma-Aldrich, catalog number H0137).

[0050] 2.3 Screening of positive clones (1) Dilute TPL-BSA with coating buffer (carbonate buffer, CBS, 50mM, pH 9.6) to a concentration of 1μg / mL. Add 100μL / well to a 96-well microplate for coating. After coating overnight at 4℃, wash three times with PBST washing buffer (containing 0.05% Tween-20).

[0051] (2) Block with blocking buffer (5% skim milk powder / PBS), 200 μL / well, incubate at 37°C for 2 h, and then wash 3 times with washing buffer. The blocking buffer is 5% skim milk powder PBS buffer.

[0052] (3) Add 100 μL of primary antibody (cell culture supernatant in 96-well plate), negative control (SP2 / 0 cell culture supernatant), blank control (PBS solution, 0.01M, pH 7.4), and positive control (PBS buffer diluted 1000 times with positive serum) to the corresponding wells. After incubation at 37°C for 45 min, wash the plate three times with washing buffer.

[0053] (4) Add 100 μL / well of enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP) diluted 1000 times with PBS, incubate at 37°C for 45 min, and then wash 3 times with plate washing solution.

[0054] (5) Add 100 μL of colorimetric reagent (TMB) per well, and the color development time is about 10-15 min.

[0055] (6) Add 50 μL of stop solution (0.2 M sulfuric acid aqueous solution) per well to terminate the reaction.

[0056] (7) Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader and record and save the data.

[0057] A positive hybridoma cell line had an OD450 value 2.1 times that of the negative control. For clones that tested positive in the first screening, further culture was followed by a second screening using the same method. Immunization with Freund's adjuvant yielded three positive hybridoma cell lines, clone numbers 2A6, 2H12, and 3F9. Immunization with nano-manganese adjuvant yielded three more positive hybridoma cell lines, clone numbers 1B9, 2C9, and 6D6.

[0058] 2.4 Ascites preparation (1) Adjust the temperature of the constant temperature water bath to 37℃.

[0059] (2) Take the frozen hybridoma cell line out of the liquid nitrogen tank and immediately put it into 37°C warm water and shake it quickly until it is completely thawed.

[0060] (3) Transfer the cell cryopreservation suspension to a centrifuge tube, add 10 mL of DMEM medium containing 20% ​​newborn calf serum, and gently pipette to mix.

[0061] (4) Centrifuge the cell suspension at 1000 rpm for 5 min at room temperature and discard the supernatant.

[0062] (5) Add DMEM medium containing 10% fetal bovine serum to the cell pellet, gently pipette to mix, and then transfer to a cell culture dish. Incubate at 37°C in a cell culture incubator.

[0063] (6) Culture the cells to the logarithmic growth phase, wash with PBS solution and count the cells.

[0064] (7) One week before inoculation, mice were injected with wax oil via intraperitoneal injection, 500 μL / Balb / c mice.

[0065] (8) Cells were inoculated via intraperitoneal injection, with each mouse receiving 1 mL (5 × 10⁶ cells). 5 -10×10 5 (units / mL).

[0066] (9) One week later, collect the ascites fluid, centrifuge at 5000 rpm for 10 min, collect the supernatant and store at -20℃.

[0067] 2.5 Antibody Purification (1) Pretreatment of ascites samples: The supernatant of the thawed ascites was diluted 3 times with coupling buffer (20mM sodium phosphate solution, pH7.0), centrifuged at 1200rpm and 4℃ for 10min, and then filtered with a 0.22um filter membrane to remove fat, cell debris and small particulate matter.

[0068] (2) Equilibration: Equilibrate the column with 10 times the column volume of the protein G antibody purification column using coupling buffer, maintaining a flow rate of 1 mL / min. The protein G antibody purification column was purchased from Sigma-Aldrich.

[0069] (3) Sample loading: Add 5 mL of pretreated ascites sample to the purification column, collect the effluent, and keep the flow rate at 1 mL / min.

[0070] (4) Washing: Pass the column through 5 column volumes of coupling buffer at a flow rate of 1 mL / min.

[0071] (5) Elution: Elute the antibody with 5 column volumes of elution buffer (0.1M glycine-hydrochloric acid solution, pH 3.0), collect it in a centrifuge tube, maintain a flow rate of 1 mL / min, and then immediately adjust the pH of the collected solution to 7.0 with 1M pH 9.0 Tris-HCl buffer.

[0072] (6) Dialysis: The antibody was dialyzed with 0.01M PBS buffer for 6 hours, and the buffer was changed 3 times to obtain purified TPL antibody.

[0073] The purified antibodies were tested for titer and sensitivity. The antibody titer results are shown in Table 1. The antibody sensitivity results are shown in Table 2.

[0074] Table 1. Antibody titer test results

[0075] As shown in Table 1, under the same antibody concentration, the antibody signal prepared with nano-adjuvant was higher than that with Freund's adjuvant, indicating that the antibody titers prepared with nano-adjuvant were significantly higher than those prepared with Freund's adjuvant. At an antibody concentration of 0.32 μg / mL, the OD450 value of the 1B9 antibody was 1.726, while the highest value for the corresponding concentration in the Freund's adjuvant group was only 1.236. The antibody signal in the nano-adjuvant group was significantly higher than that in the Freund's adjuvant group, showing a higher titer.

[0076] Table 2 Antibody sensitivity test results

[0077] Table 2 shows that the IC50 values ​​of the antibodies prepared with the three Freund's adjuvants were 4 ng / mL, 4.4 ng / mL, and 4.3 ng / mL, respectively, while the IC50 values ​​of the three nano-manganese adjuvant antibodies were 1.2, 2.0, and 1.7 ng / mL, respectively. Therefore, the nano-adjuvant antibodies showed significantly higher sensitivity than the Freund's adjuvant antibodies. In particular, the IC50 of the antibody secreted by hybridoma cell line 1B9 was as low as 1.2 ng / mL. The antibody secreted by hybridoma cell line 1B9 was selected for subsequent kit and test strip studies.

[0078] Example 3: Preparation of a rapid test strip for triptolide 3.1 Colloidal gold-labeled antibodies (1) Preparation of colloidal gold Measure 100 mL of ultrapure water and add it to a clean 250 mL Erlenmeyer flask. Place the flask on the heating plate of a magnetic stirrer, add the stir bar, and turn on the stirring knob to start stirring. Use a pipette to add 2 mL of a 1% (w / w) chloroauric acid aqueous solution to the 100 mL ultrapure water. Turn on the heating knob and heat to boiling. Quickly add 2 mL of a 2% (w / w) trisodium citrate aqueous solution. The solution will change from gray to black within 2 minutes, and finally turn red. Heat and stir for 10 minutes, then turn off the heating knob and stir until it reaches room temperature. Add ultrapure water to bring the volume to 200 mL and store away from light.

[0079] (2) Preparation of gold-labeled antibodies (colloidal gold qualitative test strips) ① Take three clean 1.5mL centrifuge tubes and add 1mL of colloidal gold to each tube.

[0080] ② Add 10µL of 0.2M potassium carbonate to each container and mix well.

[0081] ③ Add 15µg of TPL antibody secreted by hybridoma cell line 1B9, mix well, and react at room temperature for 30min.

[0082] ④ Add 10µL of 20% BSA, mix well, and react at room temperature for 30 min.

[0083] ⑤ Centrifuge at 13000 rpm for 40 min, discard the supernatant, and reconstitute the precipitate with 1 / 10 volume of reconstitution solution for later use. The reconstitution solution is 0.01M pH 7.4 PBS buffer with 5% trehalose, 1% BSA, 0.5% Tween 20, and 0.05% ProClin 300 added.

[0084] 3.2 Microsphere-labeled antibodies Optional materials include carboxylated colored microspheres, time-resolved fluorescent microspheres, and fluorescent quantum dot microspheres, for quantitative fluorescence detection. This embodiment uses time-resolved fluorescent microspheres. The time-resolved fluorescent microspheres were purchased from Boyue Biotechnology Co., Ltd., catalog number EU0200C, 200nm, 1% solid content.

[0085] (1) Prepare 10 mg / mL NHS and 10 mg / mL EDC immediately before use: Solution A: 10 mg / mL N-hydroxysuccinimide (NHS) / MES solution; Solution B: 10 mg / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) / MES solution.

[0086] (2) Add 100 μL of microspheres to a 2 mL centrifuge tube, then add 900 μL of 50 mM pH 6.0 MES buffer, and vortex for 5 s to mix evenly; (3) Add 10 μL of solution A to the microsphere suspension, mix for 5 s, then add 10 μL of solution B, mix again for 5 s, and shake at room temperature for 30 min. (4) Centrifuge at 14000 r / min for 20 min, discard the supernatant, add 1 mL of borate buffer, sonicate to disperse evenly, and set aside for use; (5) After the above 1 mL of activated microsphere suspension is dispersed by ultrasonication, 10 μg of TPL antibody secreted by hybridoma cell line 1B9 is added, vortexed for 5 s to mix evenly, and shaken at room temperature for 60 min to react. (6) Add 100 μL of 10% BSA aqueous solution, shake well at room temperature and block the reaction for 30 min; (7) Centrifuge at 13000 r / min for 15 min. Add 1 mL of microsphere preservation solution to the centrifuged microspheres, mix well, and store in the dark for later use. The microsphere preservation solution is 0.01 M pH 7.4 PBS buffer with 10% trehalose, 1% BSA, 0.5% Tween 20, and 0.05% ProClin 300 added.

[0087] 3.3 Test strip assembly (1) Cut the nitrocellulose membrane into 30cm strips, attach them to the PVC board, and equilibrate for at least 30 minutes at 25°C and 50% humidity.

[0088] (2) Scratching membrane ① Line C: Goat anti-mouse IgG was diluted with 10mM, pH 7.4 PBS buffer to a concentration of 0.5mg / mL and then sprayed onto a nitrocellulose membrane using a scrubbing tool at a distance of 5mm from the absorbent paper. This is the quality control line.

[0089] ②T line: Tripterygium wilfordii antigen TPL-BSA was diluted with 10mM, pH 7.4 PBS to a concentration of 0.2mg / mL. The solution was then sprayed onto a nitrocellulose membrane using a membrane scrubbing instrument at a distance of 10mm from the absorbent paper. This is the detection line.

[0090] The prepared film was dried at 37°C for 16 hours.

[0091] (3) Gold spraying / microsphere spraying ① Spraying instrument parameter settings: spray volume 8μL / cm, spray length 295mm; ② Colloidal gold or microspheres labeled with triptolide monoclonal antibody were placed in an oven at 37°C and dried for 24 hours.

[0092] (4) Test strip assembly ① Apply absorbent paper: Peel off the top film, apply absorbent paper to the top, and press the absorbent paper 2mm over the NC film; ② Gold-plated pad / microsphere pad: Press the gold pad or microsphere pad onto the NC film 2mm thick; ③ Apply sample pad: Peel off the bottom film, apply the sample pad to the bottom, and press the gold pad or microsphere pad 2mm over the sample pad; ④Strip cutting: Cutting with a strip cutting machine to a width of 3.5mm; ⑤ Packing the test strips: Place the cut test strips into the test strips and then seal them in an aluminum foil bag for storage.

[0093] Example 4: Preparation of a rapid detection kit for triptolide. (1) Preparation of enzyme-linked immunosorbent assay (ELISA) plate for triptolide detection ① Preparation of coating solution: Dilute the triptolide antigen TPL-BSA with 0.05 M carbonate buffer (CBS, pH 9.6) to 1 μg / mL and mix thoroughly. ② Coating: Add coating solution to the microplate, 100 μL per well, and incubate at 4℃ for 16 h; ③ Washing the plate: Take the microplate out of the refrigerator at 4°C, add 300 μL of PBST washing buffer to each well, let it stand for 3 min and then discard the liquid. Repeat 3 times. After washing, shake off the liquid in the well and pat dry with absorbent paper. ④ Blocking: Add 200 μL of blocking solution to each well and let stand at room temperature for 2 hours; the blocking solution in this step is 0.01 M pH 7.4 PBS buffer with 2% BSA added.

[0094] ⑤ Drying: Discard the sealing liquid inside the hole, pat dry, and then dry at 37°C with forced air for 2 hours; ⑥ Seal the plate: Seal the plate with a sealed bag.

[0095] (2) Optimization of Tripterygium wilfordii antibody concentration and enzyme-labeled secondary antibody concentration The concentration of triptolide antibody and the concentration of enzyme-labeled secondary antibody were optimized using the checkerboard method.

[0096] Tripterygium wilfordii antibody (1B9) and enzyme-labeled secondary antibody were prepared at concentrations of 0.025, 0.05, 0.1, 0.2, and 0.4 μg / mL using PBST, and the checkerboard assay was employed. 50 μL of PBST, 50 μL of diluted antibody, and 50 μL of diluted enzyme-labeled secondary antibody were added to each well, and incubated at 25°C for 30 min. The liquid was then discarded, and the wells were washed three times with 300 μL of PBST wash buffer each time. After washing, the liquid in the wells was discarded, and the wells were patted dry with absorbent paper. 100 μL of TMB substrate was added to each well, and the wells were incubated at 25°C for 10 min. Finally, 50 μL of stop solution (0.2 M sulfuric acid aqueous solution) was added, and the readings were taken at 450 nm using a microplate reader. The concentrations corresponding to a detection signal (OD450) of 2 ± 0.2 were selected as the usage concentrations of the antibody and enzyme-labeled antibody. The results are shown in Table 3.

[0097] Table 3. Optimization of Tripterygium wilfordii antibody concentration and enzyme-labeled secondary antibody concentration

[0098] The optimal working concentrations were selected based on the antibody concentration and enzyme-labeled antibody concentration corresponding to an OD450 signal of approximately 2.0. The results showed that the optimal antibody concentration was 0.025 μg / mL and the optimal enzyme-labeled antibody concentration was 0.05 μg / mL.

[0099] (3) Establishment of the standard curve for the triptolide detection kit A standard curve for triptolide detection was established using a monoclonal antibody against triptolide secreted by hybridoma cell line 1B9 (hereinafter referred to as "1B9 antibody"). The specific experimental method is as follows: ① Dilute TPL-BSA with coating buffer to a concentration of 1 μg / mL, add 100 μL to each well of a 96-well microplate for coating, coat at 4℃ for 12 h, and then wash the plate 3 times with PBST washing buffer.

[0100] ② Add 200 μL of blocking buffer to each well, incubate at 37°C for 2 hours, and then wash the plate three times with washing buffer. The blocking buffer is 0.01 M, pH 7.4 PBS buffer with 2% BSA added.

[0101] ③ Dilute the purified 1B9 antibody to 0.025 μg / mL with PBS buffer. Dilute the triptolide standard to concentrations of 0.025, 0.05, 0.1, 0.2, and 0.4 ng / mL with PBS buffer, respectively. Add 50 μl of 1B9 antibody or triptolide standard solution to the corresponding wells, incubate at 37°C for 30 min, and wash three times with washing buffer.

[0102] ④ Dilute the enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP) with PBS buffer to a concentration of 0.05 μg / mL. Add 100 μL of the diluted enzyme-labeled secondary antibody to each well, incubate at 37°C for 30 min, and then wash three times with washing buffer.

[0103] ⑤ Add the colorimetric reagent, 100 μL / well, and develop for approximately 10 minutes. The colorimetric reagent was purchased from Huzhou Yingchuang Biotechnology Co., Ltd. as a single-component TMB colorimetric reagent I.

[0104] ⑥ Add stop solution (0.2M sulfuric acid aqueous solution), 50 μL / well, to terminate the reaction.

[0105] ⑦ Measure the absorbance at 450nm using an ELISA reader and record and save the data.

[0106] Standard curves are shown in Table 4 and Figure 1 As shown.

[0107] Table 4 Standard Curve Data

[0108] As the concentration of triptolide standard increased, the OD450 value gradually decreased, exhibiting a good dose-response relationship. A double logarithmic regression equation was fitted with the logarithm of the standard concentration on the x-axis and the logarithm of the OD450 value on the y-axis, yielding the standard curve equation: y = 0.13229 - 1.69168x, R² = 0.9987. x represents the logarithm of the triptolide standard concentration (lgC), and y represents the logarithm of the measured OD450 value (lg OD450). Based on this curve, the half-maximal inhibitory concentration (IC50) was calculated to be 1.2 ng / mL, with a detection range of 0.3–24.3 ng / mL.

[0109] (4) Comparison of sample detection results and their correlation with instrument detection results Negative honey samples were used, and different concentrations of triptolide standard were added. The results were analyzed using the test strip method, kit method, and the method outlined in this application (BJS 202108 Determination of Triptolide in Honey), and the accuracy of the results was compared. The results are shown in Table 5.

[0110] Table 5. Results of test strip, reagent kit, LC-MS / MS, and recovery rate detection.

[0111] In the table, – indicates negative, +- indicates suspected positive, and + indicates positive.

[0112] Table 5 shows that for honey samples, the limit of detection (LOD) of the test strips is 10 ng / g, the LOD of the kit is 1.5 ng / g, and the LOD of LC-MS / MS is 3 ng / g. The recoveries of both the kit and LC-MS / MS methods range from 90% to 120%, indicating a high degree of consistency between the kit and LC-MS / MS methods. Furthermore, the kit exhibits higher sensitivity than LC-MS / MS. While the test strip method is less sensitive than the kit, it offers advantages such as ease of operation and the elimination of the need for specialized personnel.

[0113] It should be noted that the specific embodiments are merely representative examples of the present invention, and the technical solution of the present invention is obviously not limited to the above embodiments, and there can be many variations. Those skilled in the art who obtain the present invention based on its explicit disclosure or without objection from the written description should consider it to be within the scope of protection of this patent.

Claims

1. A hybridoma cell line secreting a monoclonal antibody against triptolide, characterized in that, It was named hybridoma cell line 1B9, with accession number CCTCC NO: C2026136.

2. A triptolide monoclonal antibody, characterized in that, The triptolide monoclonal antibody is secreted by the hybridoma cell line 1B9 as described in claim 1.

3. A method for preparing triptolide monoclonal antibody, characterized in that, Includes the following steps: S1. Animal immunization: Using triptolide complete antigen as an immunogen, mice were immunized by mixing it with nano-manganese adjuvant; S2. Cell fusion and culture: Spleen cells from immunized mice were fused with mouse myeloma cells SP2 / 0 in the presence of polyethylene glycol. The fused cells were then cultured to form cell clones. S3. Screening of positive clones: The triptolide detection antigen was coated; the cells were blocked with blocking solution; the supernatant of the cell culture to be tested was added as the primary antibody and incubated; the enzyme-labeled secondary antibody was added and incubated; the chromogenic solution was added for chromogenic reaction; the reaction was terminated with stop solution; the signal value was detected; and positive hybridoma cell lines were screened out; the positive hybridoma cell line is the hybridoma cell line 1B9 as described in claim 1. S4. Ascites preparation: Positive hybridoma cell lines were inoculated into the peritoneal cavity of mice, ascites was collected, and after centrifugation, the supernatant containing triptolide monoclonal antibody was obtained. S5. Purification: The supernatant of the triptolide monoclonal antibody was subjected to affinity chromatography, and the elution fraction was collected to obtain the purified triptolide monoclonal antibody.

4. The method for preparing triptolide monoclonal antibody according to claim 3, characterized in that, In step S1, the immunogen is diluted with PBS or physiological saline and then mixed with an equal volume of nano-manganese adjuvant.

5. The method for preparing triptolide monoclonal antibody according to claim 4, characterized in that, In step S5, the specific purification includes the following steps: (1) Pretreatment of ascites samples: Dilute the thawed ascites supernatant with coupling buffer, centrifuge, and filter; (2) Equilibration: Equilibrate the purification column with coupling buffer; (3) Sample loading: Add the pretreated ascites sample to the purification column; (4) Washing: Pass the purified column through a coupling buffer; (5) Elution: Elute with elution buffer and collect the antibody collection solution; (6) Dialysis: Dialyze the antibody collection solution with PBS buffer, change the solution, and obtain purified triptolide monoclonal antibody.

6. The application of the triptolide monoclonal antibody as described in claim 2 or the triptolide monoclonal antibody prepared by the preparation method as described in claims 3-5 in the preparation of triptolide detection products.

7. A rapid immunochromatographic test strip for triptolide, characterized in that, The test strip contains the triptolide monoclonal antibody as described in claim 2 or the triptolide monoclonal antibody prepared by the preparation method described in claims 3-5.

8. A method for preparing a rapid immunochromatographic test strip for triptolide as described in claim 7, characterized in that, The procedure includes the following steps: attaching a nitrocellulose membrane to a PVC board; spraying goat anti-mouse IgG as a control line and triptolide antigen as a detection line onto the nitrocellulose membrane; spraying a marker containing triptolide monoclonal antibody onto the conjugate pad; and sequentially attaching absorbent paper, the conjugate pad, and the sample pad to the PVC board to assemble the test strip.

9. A rapid detection kit for triptolide, characterized in that, It contains the triptolide monoclonal antibody as described in claim 2 or the triptolide monoclonal antibody prepared by the preparation method as described in claims 3-5.

10. A method for detecting triptolide in a sample, characterized in that, The content of triptolide in the sample to be tested can be detected using the triptolide monoclonal antibody as described in claim 2 or the triptolide monoclonal antibody prepared by the preparation method as described in claims 3-5.

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