Hybridoma cell strain secreting voriconazole monoclonal antibody and application thereof

Abstract

The present application relates to a kind of secreting voriconazole monoclonal antibody hybridoma cell strain and its application, belong to immune detection technical field.The present application is prepared by screening and obtains a kind of hybridoma cell strain capable of secreting voriconazole monoclonal antibody, the monoclonal antibody secreted by the hybridoma cell strain of the present application has good sensitivity and specificity to voriconazole, specifically, the IC 50 Value of the monoclonal antibody of the present application to voriconazole is 10 ng / mL, and the cross-reactivity of voriconazole structural analog (fluconazole, posaconazole, albaconazole) is less than 10%, so that low concentration of voriconazole can be accurately detected.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to a hybridoma cell line that secretes voriconazole monoclonal antibodies and its applications. Background Technology

[0002] Voriconazole is a second-generation triazole broad-spectrum antifungal drug that exerts its antifungal activity by inhibiting ergosterol synthesis through the inhibition of fungal cytochrome P450-dependent 14α-sterol demethylase. It is a first-line treatment for serious fungal infections such as invasive aspergillosis and candidiasis. Voriconazole is characterized by a narrow therapeutic window, significant individual variability, and chiral properties in clinical applications. Low blood concentrations can easily lead to treatment failure, while excessively high concentrations may cause adverse reactions such as hepatotoxicity and visual disturbances. Therefore, clinical drug monitoring is widely recommended for individualized dosing of voriconazole to ensure efficacy and reduce toxicity risks. Thus, research on voriconazole detection methods is of great significance.

[0003] In recent years, the detection of voriconazole has mainly relied on instrumental analysis. Gui Mingzhu et al. (Correlation Study of Voriconazole Blood Concentration in Children with CYP2C19, CYP2C9 and CYP3A5 Gene Polymorphisms, Pharmaceutical Practice and Service, 2026, 44(02):76-79+107.) used high performance liquid chromatography to detect the trough concentration of voriconazole in blood. The measured drug concentration values ​​ranged from 0.56 to 7.62 μg / mL, and the overall effective rate was 84%. Ni Weijian et al. (Establishment and Clinical Application of Method for Simultaneous Monitoring of Voriconazole, Posaconazole and Itraconazole Blood Concentrations, Chinese Journal of Hospital Pharmacy, 2026, 46(02):132-140. DOI: 10.13286 / j.1001-5213.2026.02.02.) established a method for monitoring the concentration of voriconazole in patients' serum by high performance liquid chromatography. This method showed a linear correlation in the concentration range of 0.202-20.200 μg / mL. These methods are complex to operate and require cumbersome sample processing, making them unsuitable for detecting large batches of samples in actual production. There is an urgent need to establish a simple, highly sensitive, and specific detection method. Enzyme-linked immunosorbent assay (ELISA) is a highly efficient, sensitive, and rapid detection method, and it is increasingly being used in clinical drug monitoring. However, the prerequisite for ELISA detection is the availability of a highly specific and sensitive monoclonal antibody, which is currently lacking in existing technologies. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the lack of a monoclonal antibody with high sensitivity and specificity to voriconazole in the prior art.

[0005] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes voriconazole monoclonal antibodies and its applications. This invention has prepared a hybridoma cell line capable of secreting voriconazole monoclonal antibodies through screening. The monoclonal antibody secreted by this hybridoma cell line exhibits good sensitivity and specificity to voriconazole. Specifically, the monoclonal antibody of this invention has an IC50 value of [missing information - likely related to voriconazole concentration]. 50 With a value of 10 ng / mL and a cross-reactivity of less than 10% with voriconazole structural analogs (fluconazole, posaconazole, and isaconazole), it can accurately detect low concentrations of voriconazole.

[0006] The first objective of this invention is to provide a hybridoma cell line that secretes a voriconazole monoclonal antibody, the hybridoma cell line being proposed to be taxonomically named a monoclonal cell line and having the accession number CGMCC NO.46819.

[0007] Furthermore, the hybridoma cell line is obtained by immunizing animals with a complete antigen prepared from a hapten with the structural formula shown in Formula I:

[0008] .

[0009] Furthermore, the complete antigen is obtained from the hapten coupled with a carrier protein.

[0010] Furthermore, the carrier protein includes bovine serum albumin.

[0011] A second objective of this invention is to provide an application of the above-described hybridoma cell line in the detection of voriconazole.

[0012] A third objective of this invention is to provide a monoclonal antibody secreted by the aforementioned hybridoma cell line.

[0013] A fourth objective of this invention is to provide an application of the above-described monoclonal antibody in the detection of voriconazole.

[0014] A fifth object of the present invention is to provide a voriconazole detection product comprising the above-described monoclonal antibody.

[0015] Furthermore, the test product also includes a coating agent.

[0016] Furthermore, the coating is prepared from a hapten-conjugated carrier protein; the carrier protein includes chicken ovalbumin.

[0017] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0018] The monoclonal antibody secreted by the hybridoma cell line of the present invention exhibits good sensitivity and specificity to voriconazole. Specifically, the monoclonal antibody of the present invention has an IC50 value for voriconazole. 50 With a value of 10 ng / mL and a cross-reactivity of less than 10% with voriconazole analogs (fluconazole, posaconazole, and isaconazole), it can accurately detect low concentrations of voriconazole.

[0019] Preservation of biological materials

[0020] The cell line QJKL, a monoclonal cell line, was deposited on April 24, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46819. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0021] 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.

[0022] Figure 1 This is a standard inhibition curve of the voriconazole monoclonal antibody of the present invention;

[0023] Figure 2 This is the synthetic route for the hapten of the present invention. Detailed Implementation

[0024] 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.

[0025] The culture media involved in the following examples are as follows:

[0026] 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.

[0027] The reagents involved in the following examples are as follows:

[0028] 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.

[0029] Phosphate buffered saline (PBS): 8.00 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, 2.9 g Na2HPO4·12H2O, dissolved in 800 mL pure water, pH adjusted to 7.2-7.4 with NaOH or HCl, and then brought to a final volume of 1000 mL.

[0030] PBST: PBS containing 0.05% Tween 20;

[0031] Antibody dilution buffer: PBS containing 0.1% gelatin;

[0032] TMB colorimetric solution: Solution A: 18.43 g Na2HPO4·12H2O, 9.33 g citric acid, diluted to 1000 mL with pure water; Solution B: 60 mg TMB dissolved in 100 mL ethylene glycol. Mix solutions A and B in a 5:1 ratio to obtain the TMB colorimetric solution, and mix again before use.

[0033] The detection methods involved in the following embodiments are as follows:

[0034] Voriconazole inhibition rate assay: The optimal antigen and antibody concentrations for ic-ELISA were selected using a checkerboard assay. The complete antigen was diluted to 0.3, 0.1, and 0.03 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 0.3, 0.1, and 0.03 μg / mL with antibody dilution buffer. After selecting the optimal operating point, voriconazole standards were diluted to eight concentrations (0, 0.37, 1.11, 3.33, 10, 30, 90, 180 ng / mL). Following the ic-ELISA procedure, the results were plotted using OriginPro 8.5 (see results below). Figure 1 (As shown), the voriconazole standard inhibition curve was obtained, and the IC50 was calculated. 50 .

[0035] Example 1: Synthesis of hapten VOR

[0036] The hapten VOR is obtained by reacting voriconazole technical with succinic anhydride. The synthetic route is as follows: Figure 2 As shown.

[0037] The specific steps are as follows:

[0038] Reaction solution A: Voriconazole (50 mg, 0.14 mmol), succinic anhydride (42 mg, 0.42 mmol), and DMAP (50.4 mg, 0.42 mmol) were weighed and dissolved in pyridine. The reaction was carried out at 60°C for 12 h, followed by extraction with ethyl acetate. After separation, the upper layer was collected and dried under nitrogen to obtain the crude sample.

[0039] The crude sample was purified by recrystallization. The crude sample was dissolved in methanol. After complete dissolution, it was allowed to stand overnight at room temperature, resulting in the precipitation of a brown precipitate. This precipitate was filtered through filter paper, washed 2-3 times with petroleum ether, and dried at 50°C to obtain a brown precipitate (26.8 mg), which was the hapten VOR, with a yield of 53.6%.

[0040] Example 2: Preparation of complete antigen

[0041] The hapten VOR is conjugated to a carrier protein using the carbodiimide method to obtain the voriconazole artificial antigen. Specifically, the hapten VOR prepared in Example 1 is conjugated with bovine serum albumin (BSA) to obtain the complete antigen VOR-EDC-BSA.

[0042] The method for preparing the complete antigen VOR-EDC-BSA is as follows:

[0043] a. Weigh 0.8 mg of the hapten VOR, 1.03 mg of N-hydroxysuccinimide, and 1.73 mg of 1-ethylcarbodiimide hydrochloride prepared in Example 1, and dissolve them in 200 μL of N,N-dimethylformamide (referred to as solution A). Stir the mixture at room temperature for 4-6 h. Weigh 6.0 mg of BSA (the molar ratio of VOR to BSA is 20:1), add 2 mL of carbonate buffer solution (the dissolved BSA protein is referred to as solution B), and add solution A dropwise to solution B at room temperature. Adjust the pH of the mixture to 8-9 with 1 M NaOH solution, and react at room temperature overnight to obtain the conjugate VOR-EDC-BSA.

[0044] b. Dialysis: Cut an 8 cm dialysis bag, boil it in boiling water for 3 minutes and cool it, then store it in deionized water at 4℃ for later use; put the VOR-EDC-BSA conjugate into the dialysis bag and dialyze it in 0.01 mol / L PBS, changing it every 8 hours, and dialyze for 3 days to obtain the complete antigen VOR-EDC-BSA, which should be taken out and stored at -20℃.

[0045] Example 3: Preparation of Immunogen

[0046] The hapten VOR is conjugated to a carrier protein using the carbodiimide method to obtain the voriconazole artificial antigen. Specifically, the hapten VOR prepared in Example 1 is conjugated with chicken ovalbumin OVA to obtain the complete antigen VOR-EDC-OVA.

[0047] The method for preparing the complete antigen VOR-EDC-OVA is as follows:

[0048] a. Weigh 1.8 mg of the hapten VOR, 4.03 mg of N-hydroxysuccinimide, and 6.73 mg of 1-ethylcarbodiimide hydrochloride prepared in Example 1, and dissolve them in 200 μL of N,N-dimethylformamide (referred to as solution A). Stir the mixture at room temperature for 4-6 h. Weigh 6.0 mg of OVA (VOR to OVA molar ratio of 20:1), add 2 mL of carbonate buffer solution (the dissolved OVA protein is referred to as solution B), and add solution A dropwise to solution B at room temperature. Adjust the pH of the mixture to 8-9 with 1 M NaOH solution, and react at room temperature overnight to obtain the conjugate VOR-EDC-OVA.

[0049] b. Dialysis: Cut an 8 cm dialysis bag, boil it in boiling water for 3 minutes and cool it, then store it in deionized water at 4℃ for later use; put the conjugate VOR-EDC-OVA conjugate solution into the dialysis bag and dialyze it in 0.01 mol / L PBS, changing it every 8 hours, and dialyze for 3 days to obtain the complete antigen VOR-EDC-OVA, which should be taken out and stored at -20℃.

[0050] Example 4: Immunization in mice

[0051] For the initial immunization, 100 μg of voriconazole complete antigen was emulsified with an equal volume of complete Freund's adjuvant and administered via multiple subcutaneous injections into the neck and back of BALB / c mice. Four weeks later, a booster immunization was performed with half the dose of complete antigen (50 μg / mouse) emulsified with incomplete Freund's adjuvant. Subsequent booster immunizations were administered at 3-week intervals. For the final sprint immunization, the dose was again halved (25 μg / mouse), and the complete antigen was diluted with physiological saline and administered via intraperitoneal injection. After the third immunization, tail-disconnected blood samples were collected for testing. Serum titers and IC50 values ​​were determined using an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). 50 Choose high-performance ICs 50 Low-grade mice were fused;

[0052] Example 5: Cell Fusion and Screening

[0053] (1) Three days after the sprint immunization, cell fusion was performed according to the conventional PEG 4000 (polyethylene glycol) method. The specific steps are as follows:

[0054] a. Collection of SP2 / 0 tumor cells: 7-10 days before fusion, culture SP2 / 0 tumor cells in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. The required number of SP2 / 0 tumor cells before fusion should be 1-4 x 10⁻⁴ cells. 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. During fusion, tumor cells are collected, suspended in RPMI-1640 basal culture medium, and cell counting is performed.

[0055] b. After euthanizing mice by cervical dislocation, immediately sterilize them in 75% alcohol for about 5 minutes. Aseptically remove the spleen, gently grind it with a syringe tip, and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect 50 mL of the suspension in a sterile centrifuge tube, centrifuge at 1200 r / min for 8 minutes, wash the spleen cells with RPMI-1640 medium, remove any large tissue impurities, and repeat the process three times. After the final centrifugation, dilute the spleen cells to a specific volume, count them, and set aside for later use.

[0056] c. Fusion process (7 min): At min 1, add 1 mL of PEG 4000 dropwise to the cells, gradually increasing the speed. At min 2, allow the centrifuge tube to stand and hold it firmly with both hands. At min 3 and min 4, add 1 mL of RPMI-1640 medium dropwise every 1 min. At min 5 and min 6, add 1 mL of RPMI-1640 medium dropwise every 30 s. At min 7, add 1 mL of RPMI-1640 medium dropwise every 10 s. Then incubate at 37°C for 5 min. Centrifuge at 800 r / min for 10 min, discard the supernatant, gently break up the cells in the centrifuge tube, and add RPMI-1640 selective medium (HAT medium) containing 20% ​​fetal bovine serum and 2% 50×HAT to a 96-well cell plate at a rate of 200 μL / well. Incubate at 37°C in a 5% CO2 incubator.

[0057] (2) Cell screening and cell line establishment: On day 3 after cell fusion, the fused cells were partially replaced with HAT medium; on day 5, the medium was completely replaced with RPMI-1640 transition medium (HT medium) containing 20% ​​fetal bovine serum and 1% 100×HT; on day 7, the cell supernatant was collected for screening. The screening was carried out in two steps: first, positive cell wells were screened using ic-ELISA; second, voriconazole standard was selected, and the inhibitory effect on positive cells was determined using ic-ELISA. Cell wells that showed good inhibition of voriconazole standard were selected, and subcloning was performed using the limiting dilution method. The same method was used for detection seven days later. Subcloning was performed four times according to the above method to finally obtain the voriconazole monoclonal antibody cell line.

[0058] Example 6: Preparation and Identification of Monoclonal Antibodies

[0059] 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 2 × 10⁻⁶ g of paraffin oil. 6 Voriconazole hybridoma cells were used to collect ascites fluid starting on day 7. The ascites fluid was then purified for antibody treatment using the caprylic acid-saturated ammonium sulfate method. Under slightly acidic conditions, caprylic acid precipitates other proteins in the ascites fluid besides IgG immunoglobulins. The precipitate was then discarded by centrifugation. IgG-type monoclonal antibodies were then precipitated with an equal volume of saturated ammonium sulfate solution, centrifuged, and the supernatant was discarded. The antibodies were dissolved in 0.01 M PBS solution (pH 7.4), dialyzed to desalt, and finally the purified monoclonal antibodies were stored at -20°C.

[0060] (1) Coating: The original VOR-EDC-OVA was diluted 3-fold from 1 µg / mL with 0.05M (pH 9.6) carbonate buffer, 100 μL / well, and reacted at 37℃ for 2 h.

[0061] (2) Washing: Pour off the solution in the plate and wash with washing solution 3 times, 3 minutes each time.

[0062] (3) Sealing: After patting dry, add 200 μL / well sealing solution and react at 37℃ for 2 h. Wash and dry for later use.

[0063] (4) Sample addition: The antiserum (antiserum obtained by diluting the blood from the tail of mice with antibody diluent) was serially diluted from 1:1000 and added to each well of the coating at 100 μL / well. The reaction was carried out at 37℃ for 30 min. After thorough washing, HRP-goat anti-mouse IgG diluted at 1:3000 was added at 100 μL / well. The reaction was carried out at 37℃ for 30 min.

[0064] (5) Color development: Take out the microplate, wash it thoroughly, add 100 μL of TMB color development solution to each well, and react at 37°C in the dark for 15 min.

[0065] (6) Termination and measurement: Add 50 μL of stop solution to each well to terminate the reaction, and then measure the OD of each well using a microplate reader. 450 value.

[0066] The IC50 of the monoclonal antibody voriconazole was determined by ic-ELISA. 50 The value was 10 ng / mL, indicating good sensitivity to voriconazole, and it can be used for voriconazole immunoassay.

[0067] Specific experiments

[0068] The IC50 of the monoclonal antibody against voriconazole was determined using an indirect competitive ELISA method. 50 The concentration was 10 ng / mL, and its IC50 for compounds such as fluconazole, posaconazole, and isosorbide dinitrate was verified. 50 and cross-reactivity rate, the cross-reactivity value is calculated as: (IC50 of voriconazole) 50 IC of other compounds 50 () × 100%, as shown in Table 1.

[0069] Table 1. IC50 of voriconazole monoclonal antibody against voriconazole and its cross-links 50 and cross-reactivity rate,

[0070]

[0071] The monoclonal antibody showed 100% cross-linking against voriconazole, while the cross-linking against fluconazole, posaconazole, and isaconazole was less than 1%. This indicates that the monoclonal antibody obtained in this invention has high sensitivity (IC50) to voriconazole. 50It has a value of 10 ng / mL and also exhibits high specificity (cross-reactivity with analogs is less than 1%).

[0072] 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 hybridoma cell line secreting voriconazole monoclonal antibody, characterized in that, The proposed taxonomic name for the hybridoma cell line is monoclonal cell line, and its accession number is CGMCC NO.46819.

2. The hybridoma cell line according to claim 1, characterized in that, The hybridoma cell line was obtained by immunizing animals with a complete antigen prepared from a hapten with the structural formula shown in Formula I: 。 3. The hybridoma cell line according to claim 1, characterized in that, The complete antigen is obtained by conjugating the hapten to a carrier protein.

4. The hybridoma cell line according to claim 1, characterized in that, The carrier protein includes bovine serum albumin.

5. The use of the hybridoma cell line according to any one of claims 1-4 in the detection of voriconazole.

6. A monoclonal antibody secreted by a hybridoma cell line according to any one of claims 1-4.

7. The use of the monoclonal antibody according to claim 6 in the detection of voriconazole.

8. A detection product for voriconazole, characterized in that, The detection product includes the monoclonal antibody as described in claim 6.

9. The testing product according to claim 8, characterized in that, The tested products also include coating agents.

10. The testing product according to claim 9, characterized in that, The coating is prepared by conjugating a hapten with a carrier protein; the carrier protein includes chicken ovalbumin.

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