Hybridoma cell strain secreting isavuconazole monoclonal antibody and application thereof

Abstract

The invention relates to a hybridoma cell strain secreting an isavuconazole monoclonal antibody and application of the hybridoma cell strain, and belongs to the technical field of immunodetection. The monoclonal antibody secreted by the hybridoma cell strain has good sensitivity and specificity to the isavuconazole, the IC50 value of the monoclonal antibody to the isavuconazole is 13.317 ng / mL, and the IC50 value of the monoclonal antibody to the structural analogue of the isavuconazole is 13.317 ng / mL. According to the present invention, the low-concentration isavuconazole, such as fluconazole, ketoconazole, posaconazole, clotrimazole, miconazole, econazole, voriconazole, bifenconazole, luliconazole and sertaconazole, does not have the cross reaction, and the cross reaction rate is less than 1%, such that the low-concentration isavuconazole can be accurately detected.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunoassay, in particular to a hybridoma cell strain secreting isavuconazole monoclonal antibody and application thereof. BACKGROUND

[0002] Isavuconazole, CAS No. 241479-67-4, molecular weight 437.47, is a new triazole antifungal drug with broad-spectrum antifungal activity, mainly used for the treatment of severe invasive fungal infections. Such infections are serious, with high mortality, and the patients are mostly immunocompromised individuals (such as transplant patients, chemotherapy patients). The drug needs to maintain sufficient blood concentration to effectively inhibit fungal growth, otherwise it may lead to infection progression or recurrence. Therefore, timely detection of drug concentration in the blood of patients can directly reflect the effective dose in the body, and guide the adjustment of the administration scheme.

[0003] Currently, the main methods for determining the content of isavuconazole include ultra-performance liquid chromatography-mass spectrometry (UPLC-MS / MS), gas chromatography-tandem mass spectrometry (GC-MS / MS) and high-performance liquid chromatography (HPLC-UV). These methods require professional equipment and operators, and the detection cost is high, which is difficult to meet the needs of instant and batch detection. The enzyme-linked immunoassay (ELISA) technology provides a new solution for isavuconazole detection. This technology can realize trace detection of isavuconazole with high sensitivity and fast detection speed, and the operation is relatively simple, which greatly improves the flexibility and timeliness of detection, and is expected to solve the problems of traditional detection methods and promote the development of isavuconazole detection technology, providing more efficient technical support for drug residue detection. The premise of using enzyme-linked immunoassay to detect isavuconazole is to obtain a monoclonal antibody that can accurately detect isavuconazole. Therefore, it is crucial to find a monoclonal antibody that can accurately detect isavuconazole, and there is no monoclonal antibody for isavuconazole in the prior art. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to overcome the lack of a monoclonal antibody for detecting isavuconazole in the prior art.

[0005] To solve the above technical problems, the present application provides a hybridoma cell strain secreting isavuconazole monoclonal antibody and application thereof. The monoclonal antibody secreted by the hybridoma cell strain of the present application has good sensitivity and specificity to isavuconazole, and the IC 50 value of the monoclonal antibody to isavuconazole is 13.317 ng / mL, and there is no cross reaction to structural analogues of isavuconazole, such as fluconazole, ketoconazole, posaconazole, clotrimazole, miconazole, econazole, voriconazole, bifonazole, luliconazole and saperconazole, so that the isavuconazole can be accurately detected at low concentration.

[0006] The first object of the present application is to provide a hybridoma cell strain, which is deposited in China General Microbiological Culture Collection Center on April 17, 2025, and classified as a monoclonal cell strain, and the deposit address is No. 3, Beichen West Road, Beijing City, and the deposit number is CGMCC No. 46504.

[0007] Further, the hybridoma cell strain is obtained by immunizing animals with a complete antigen prepared from a hapten, wherein the structure of the hapten is shown as formula I:

[0008]

[0009] Further, the complete antigen is obtained by coupling the hapten with a carrier protein.

[0010] Further, the carrier protein includes keyhole limpet hemocyanin.

[0011] The second object of the present application is to provide an application of the above-mentioned hybridoma cell strain in detecting asexconazole.

[0012] The third object of the present application is to provide a monoclonal antibody secreted by the above-mentioned hybridoma cell strain.

[0013] The fourth object of the present application is to provide an application of the above-mentioned monoclonal antibody in detecting asexconazole.

[0014] The fifth object of the present application is to provide a detection product of asexconazole, which includes the above-mentioned monoclonal antibody.

[0015] Further, the detection product includes a coating antigen.

[0016] Further, the coating antigen is prepared by coupling a hapten with a carrier protein, wherein the carrier protein includes chicken egg white albumin.

[0017] The beneficial effects of the present application are as follows:

[0018] The present application first isolates a hybridoma cell strain capable of accurately detecting low-concentration asexconazole. Specifically, the monoclonal antibody secreted by the hybridoma cell strain of the present application has good sensitivity and specificity to asexconazole, wherein the IC 50 value of the monoclonal antibody to asexconazole is 13.317 ng / mL, and there is no cross reaction to structural analogues of asexconazole, such as fluconazole, ketoconazole, posaconazole, clotrimazole, miconazole, econazole, voriconazole, bifonazole, luliconazole and saperconazole, so that low-concentration asexconazole can be accurately detected.

[0019] Biological material preservation

[0020] The monoclonal cell line MCPA was deposited on April 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC), classified as a monoclonal cell line, with accession number CGMCC No. 46504, and the deposit address is No. 3, No. 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, wherein...

[0022] Figure 1 This is the standard curve of the inhibition of esaconazole by the esaconazole monoclonal antibody of the present invention. Detailed Implementation

[0023] 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.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[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 solution (CBS): Take Na2CO3 1.59 g, NaHCO3 2.93 g, respectively, dissolve in a small amount of double distilled water, mix, add double distilled water to about 800 mL, mix well, adjust the pH value to 9.6, add double distilled water to constant volume to 1000 mL, store at 4℃ for standby;

[0029] Phosphate buffer solution (PBS): 8.00 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, 2.9 g Na2HPO4·12H2O, dissolved in 800 mL of pure water, adjusted to pH 7.2-7.4 with NaOH or HCl, and constant volume to 1000 mL with ultrapure water;

[0030] Washing solution (PBST): 0.5 mL of Tween-20 was added to 1000 mL of 0.01 mol / L pH 7.4 PBS solution;

[0031] PBST: PBS containing 0.05% Tween-20;

[0032] Antibody diluent: washing solution containing 0.1% gelatin;

[0033] TMB developing solution: A solution: Na2HPO4·12H2O 18.43 g, citric acid 9.33 g, constant volume to 1000 mL with pure water; B solution: 60 mg TMB dissolved in 100 mL ethylene glycol. A and B solutions are mixed in a volume ratio of 5:1 to obtain TMB developing solution, which is mixed immediately before use.

[0034] The detection method involved in the following examples is as follows:

[0035] Azelastine inhibition rate detection method: The most appropriate antigen and antibody concentration in ic-ELISA is selected by chessboard test. The antigen is diluted to 1, 0.3, 0.1 and 0.03 μg / mL with carbonate buffer solution (CBS), and the antibody is diluted to 1, 0.3, 0.1 and 0.03 μg / mL with antibody diluent. After selecting the best working point, azelastine standard is diluted to 300, 100, 33.33, 11.11, 3.70, 1.23, and 0.41 ng / mL, etc. According to the icELISA operation steps, finally use Origin 2024 to draw the graph, and obtain the azelastine standard inhibition curve, and calculate the IC 50 .

[0036] Example 1: Preparation of hapten

[0037] 200 mg of azelastine was dissolved in an organic solvent, 2 M NaOH was added, heated at 70℃ water bath for 3 h, then washed with deionized water and dried to obtain yellow oil, which was azelastine hapten ISKZ-COOH, and the reaction equation was as follows:

[0038]

[0039] Example 2: Preparation of complete antigen

[0040] Take 11.8 mg of aliskiren hapten ISKZ-COOH, dissolve in 300 μL of DMF, add 6.2 mg of N-hydroxysuccinimide under stirring at room temperature, react for 15 min, then add 11.9 mg of 1-ethylcarbodiimide hydrochloride, react for 6 h at room temperature, and the obtained mixture is referred to as A liquid; then take 5 mg of keyhole limpet hemocyanin (KLH) and dissolve in 2 mL of carbonate buffer, referred to as B liquid; slowly drop A liquid into B liquid under stirring at room temperature, react for 24 h, dialyze with PBS for 3 days, and the conjugate ISKZ-COOH-KLH is obtained, which is stored at -20 °C for later use.

[0041] Example 3: Preparation of coating antigen

[0042] Take 5.2 mg of the prepared ISKZ-COOH, dissolve in 300 μL of DMF, add 3.7 mg of N-hydroxysuccinimide under stirring at room temperature, react for 15 min, then add 6.7 mg of 1-ethylcarbodiimide hydrochloride, react for 4 h, and the obtained mixture is referred to as A liquid; then take 5 mg of chicken egg albumin (OVA) and dissolve in 2 mL of carbonate buffer, referred to as B liquid; slowly drop A liquid into B liquid under stirring at room temperature, react for 12 h, dialyze with 0.01 mol / L PBS for 3 days, and the conjugate ISKZ-COOH-OVA is obtained, which is stored at -20 °C for later use.

[0043] Example 4: Preparation of hybridoma cell strain

[0044] 1. Immunization of mice: healthy 6-8 week old BALB / c mice are selected for immunization. The aliskiren immunogen is mixed with an equal amount of Freund's adjuvant and emulsified, and then subcutaneously injected into the back of the BALB / c mice. Complete Freund's adjuvant is used for the first immunization, and incomplete Freund's adjuvant is used thereafter. The interval between the first immunization and the second booster immunization is 28 days, and the interval between multiple booster immunizations is 21 days. Blood is collected 7 days after the third immunization (5 μL of mouse tail blood + 995 μL of antibody diluent = antiserum), and the mouse serum titer and inhibition are determined using icELISA. Mice with high titer and good inhibition are selected, and a boost immunization is performed 21 days after the fifth immunization, which is intraperitoneally injected, and the dose is required to be halved and free of any adjuvant.

[0045] 2. Cell fusion: three days after the boost immunization, cell fusion is performed according to the conventional PEG (polyethylene glycol, molecular weight 1500) method, and the specific steps are as follows:

[0046] a, After the mice were sacrificed by enucleation and cervical dislocation, they were immediately disinfected in 75% alcohol for 5 minutes. The spleen was removed under sterile conditions, and the spleen cells were obtained by gently grinding the spleen with a syringe rubber head and passing through a 200-mesh cell sieve. The spleen cell suspension was collected and centrifuged (1200 rpm, 8 min). The spleen cells were washed three times with RPMI-1640 medium, and after the last centrifugation, the spleen cells were diluted to a certain volume, counted, and stored for use;

[0047] b, Collection of mouse myeloma SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. Before fusion, the number of SP2 / 0 tumor cells was required to reach (1-4) x 10 7 , to ensure that the SP2 / 0 tumor cells were in the logarithmic growth phase before fusion. At the time of fusion, the tumor cells were collected and suspended in RPMI-1640 base medium for cell counting;

[0048] c, 7 min of fusion process. In the first minute, 1 mL of PEG 1500 was added to the cells from slow to fast; in the second minute, it was left alone. In the third and fourth minutes, 1 mL of RPMI-1640 medium was added within 1 min; in the fifth and sixth minutes, 2 mL of RPMI-1640 medium was added within 1 min; in the seventh minute, 1 mL of RPMI-1640 medium was added every 10 seconds. Then 37°C incubation for 5-8 min. Centrifugation (800 rpm, 8 min), discard the supernatant, resuspend in RPMI-1640 screening medium containing 20% fetal bovine serum, 2% 50x HAT, add 200 μL / well to a 96-well cell plate, and incubate in a 37°C, 5% CO2 incubator.

[0049] 3, Cell screening and cell strain establishment: On the third day of cell fusion, the fusion cells were subjected to RPMI-1640 screening medium semi-replacement, and on the fifth day, they were subjected to full replacement with RPMI-1640 transition medium containing 20% fetal bovine serum and 1% 100x HT. On the seventh day, the cell supernatant was collected for screening. The screening was divided into two steps: first, positive cell wells were screened out using icELISA, and second, icELISA was used to determine the inhibition effect of positive cells using azaconazole as a standard. Cell wells with good inhibition of azaconazole standard were selected, and subcloning was performed using limited dilution. The same method was used for detection, repeated three times, and a cell strain was obtained.

[0050] Example 5: Preparation and identification of monoclonal antibodies

[0051] Take 6-8 weeks old BALB / c mice, each mouse intraperitoneal injection of sterile paraffin oil 0.5 mL; 7 days after each mouse intraperitoneal injection of 1 x 10 6 Hybridoma cells, starting from the 7th day, collect ascites, and purify the ascites by caprylic acid-ammonium sulfate method. In the partial acid condition, n-octanoic acid can precipitate other impurities in the ascites except IgG immunoglobulin, then centrifugal, discard the precipitate; then use an equal amount of saturated ammonium sulfate solution to precipitate IgG type monoclonal antibody, centrifugal, discard the supernatant, dissolve with 0.01M PBS solution (pH 7.4) after, dialysis desalination, finally get the purified monoclonal antibody stored at -20℃.

[0052] Using icELISA method, the IC 50 of monoclonal antibody to azaconazole is 13.317 ng / mL, and the IC 50 and cross reactivity of functional analogues are verified, and the cross reactivity of related fungicides is less than 1%, wherein the cross reactivity = (IC 50 of azaconazole / IC 50 of analogues) x 100%, according to the cross reactivity value, it can be seen that the antibody has high sensitivity and specificity to azaconazole, and the specific data is shown in Table 1.

[0053] Table 1 Sensitivity and specificity of monoclonal antibody to azaconazole

[0054] IC 50 (ng / mL) Cross-reactivity (%) Albaconazole 13.317 100 Fluconazole >100 <1 Ketoconazole >100 <1 Posaconazole >100 <1 Clotrimazole >100 <1 Miconazole >100 <1 Econazole >100 <1 Voriconazole >100 <1 Bifonazole >100 <1 Luliconazole >100 <1 Sertaconazole >100 <1

[0055] The monoclonal antibody is applied to the azaconazole addition recovery test, and the specific steps are as follows:

[0056] a, coating: the coating original azaconazole-OVA is diluted by 100 μL / well from 1 μg / mL, 37℃ reaction for 2h;

[0057] b, washing: pour out the solution in the plate, and wash with washing solution for 3 times, 3min each time;

[0058] c, blocking: after drying, add 200 μL / well of blocking solution, 37℃ reaction for 2h. After washing, dry and reserve;

[0059] d, sample adding: dilute the antiserum (after taking blood from the tail of mice, dilute the corresponding multiple with the antibody diluent, which is the antiserum) by 100 μL / well from 1:1000, 37℃ reaction for 30min; after washing, add 100 μL / well of HRP-goat anti-mouse IgG diluted by 1:3000, 37℃ reaction for 30min;

[0060] e. Color development: After the enzyme-labeled plate was taken out and washed thoroughly, 100 μL of TMB color developing solution was added to each well, and the reaction was carried out at 37°C for 15 min in the dark;

[0061] f. Termination and determination: 50 μL of termination solution was added to each well to terminate the reaction, and then the OD value of each well was determined by an enzyme-labeled instrument. 450

[0062] Urine and blood were selected as the samples to be tested, and the samples to be tested were subjected to standard addition treatment. The addition recovery of aliskiren in urine and blood was detected by liquid chromatography tandem mass spectrometry (LC-MS / MS), as shown in Table 2. The average recovery of LC-MS / MS was 92.9-109.2%, and the relative standard deviation (RSD) was 2.45-6.56%; the determination recovery of indirect competitive ELISA was 97.4-105.5%, and the RSD was 2.42-6.67%, indicating that the method for detecting aliskiren in samples based on aliskiren monoclonal antibody was relatively accurate, and could be used for the immune analysis detection of aliskiren residues in blood plasma and urine.

[0063] Table 2 Addition recovery of monoclonal antibody to aliskiren in urine and blood plasma

[0064]

[0065] Obviously, the above examples are only examples for clearly illustrating, and are not limited to the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. All the embodiments do not need to be exhausted, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.​

Claims

1. A hybridoma cell line, characterized in that, The hybridoma cell strain is named monoclonal cell strain, and the preservation number is CGMCC No. 46504.

2. The hybridoma cell line according to claim 1, characterized in that, The hybridoma cell strain is obtained by immunizing animals with a complete antigen prepared from a hapten, wherein the structure of the hapten is shown as formula I:

3. The hybridoma cell line according to claim 2, characterized in that, The complete antigen is obtained by coupling the hapten with a carrier protein.

4. The hybridoma cell line according to claim 3, characterized in that, The carrier protein includes keyhole limpet hemocyanin.

5. The hybridoma cell strain according to any one of claims 1-4, for use in detecting alisabemide.

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

7. The monoclonal antibody according to claim 6, for use in detecting alisabemide.

8. An assay product for the detection of alischanzole, characterized by The detection product includes the monoclonal antibody according to claim 6.

9. The detection product of claim 8, wherein, The detection product further includes a coating agent.

10. The test product of claim 9, wherein, The coating agent is prepared by coupling a hapten with a carrier protein, wherein the carrier protein includes chicken egg white albumin.

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